The emerging role of rapid corticosteroid actions on excitatory and inhibitory synaptic signaling in the brain.

Over the past two decades, there has been increasing evidence for the importance of rapid-onset actions of corticosteroid hormones in the brain. Here, we highlight the distinct rapid corticosteroid actions that regulate excitatory and inhibitory synaptic transmission in the hypothalamus, the hippocampus, basolateral amygdala, and prefrontal cortex. The receptors that mediate rapid corticosteroid actions are located at or close to the plasma membrane, though many of the receptor characteristics remain unresolved. Rapid-onset corticosteroid effects play a role in fast neuroendocrine feedback as well as in higher brain functions, including increased aggression and anxiety, and impaired memory retrieval. The rapid non-genomic corticosteroid actions precede and complement slow-onset, long-lasting transcriptional actions of the steroids. Both rapid and slow corticosteroid actions appear to be indispensable to adapt to a continuously changing environment, and their imbalance can increase an individual's susceptibility to psychopathology.

Autoantibody pathogenicity in a multifocal motor neuropathy induced pluripotent stem cell-derived model.

OBJECTIVE: We investigated the pathogenicity of immunoglobulin M (IgM) anti-GM1 antibodies in serum from patients with multifocal motor neuropathy (MMN) using human induced pluripotent stem cell (iPSC)-derived motor neurons (MNs). METHODS: iPSCs were generated from fibroblasts and differentiated into MNs. We studied the binding of IgM to MNs, their complement-activating properties, and effects on structural integrity using fluorescence and electron microscopy. Live cell imaging was used to study effects of antibody binding on MNs in the presence and absence of complement. RESULTS: IgM antibody binding to MNs was detected using sera from MMN patients with and without detectable anti-GM1 IgM antibody titers in enzyme-linked immunosorbent assay, but not with sera from (disease) controls. Competition and depletion experiments showed that antibodies specifically bound to GM1 on iPSC-derived MNs. Binding of these antibodies disrupted calcium homeostasis by both complement-dependent and complement-independent pathways. MNs showed marked axonal damage after complement activation, and reduced antibody pathogenicity following treatment with immunoglobulin preparations. INTERPRETATION: Our data provide evidence for the pathogenicity of anti-GM1 IgM antibodies in MMN patients and link their presence to the clinical characteristics of axonal damage and immunoglobulin responsiveness. This iPSC-derived disease model will facilitate diagnosis, studies on autoantibody pathogenicity, drug development, and screening in immune-mediated neuropathies. Ann Neurol 2016;80:71-88.

Ultradian corticosterone pulses balance glutamatergic transmission and synaptic plasticity.

The rodent adrenal hormone corticosterone (CORT) reaches the brain in hourly ultradian pulses, with a steep rise in amplitude before awakening. The impact of a single CORT pulse on glutamatergic transmission is well documented, but it remains poorly understood how consecutive pulses impact on glutamate receptor trafficking and synaptic plasticity. By using high-resolution imaging and electrophysiological approaches, we report that a single pulse of CORT to hippocampal networks causes synaptic enrichment of glutamate receptors and increased responses to spontaneously released glutamatergic vesicles, collectively abrogating the ability to subsequently induce synaptic long-term potentiation. Strikingly, a second pulse of CORT one hour after the first--mimicking ultradian pulses--completely normalizes all aspects of glutamate transmission investigated, restoring the plastic range of the synapse. The effect of the second pulse is precisely timed and depends on a nongenomic glucocorticoid receptor-dependent pathway. This normalizing effect through a sequence of CORT pulses--as seen around awakening--may ensure that hippocampal glutamatergic synapses remain fully responsive and able to encode new stress-related information when daily activities start.

Unraveling the time domains of corticosteroid hormone influences on brain activity: rapid, slow, and chronic modes.

Brain cells are continuously exposed to corticosteroid hormones, although the levels vary (e.g., after stress). Corticosteroids alter neural activity via two receptor types, mineralocorticoid (MR) and glucocorticoid receptors (GR). These receptors regulate gene transcription but also, as we now know, act nongenomically. Via nongenomic pathways, MRs enhance and GRs suppress neural activity. In the hypothalamus, inhibitory GR effects contribute to negative feedback regulation of the stress axis. Nongenomic MR actions are also important extrahypothalamically and help organisms to immediately select an appropriate response strategy. Via genomic mechanisms, corticosteroid actions in the basolateral amygdala and ventral-most part of the cornu ammonis 1 hippocampal area are generally excitatory, providing an extended window for encoding of emotional aspects of a stressful event. GRs in hippocampal and prefrontal pyramidal cells increase surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and strengthen glutamatergic signaling through pathways partly overlapping with those involved in long-term potentiation. This raises the threshold for subsequent induction of synaptic potentiation and promotes long-term depression. Synapses activated during stress are thus presumably strengthened but protected against excitatory inputs reaching the cells later. This restores higher cognitive control and promotes, for example, consolidation of stress-related contextual information. When an organism experiences stress early in life or repeatedly in adulthood, the ability to induce synaptic potentiation is strongly reduced and the likelihood to induce depression enhanced, even under rest. Treatment with antiglucocorticoids can ameliorate cellular effects after chronic stress and thus provide an interesting lead for treatment of stress-related disorders.

Complex housing in adulthood state-dependently affects the excitation-inhibition balance in the infralimbic prefrontal cortex of male C57Bl/6 mice.

The prefrontal cortex (PFC) plays an important role in social behavior and is sensitive to stressful circumstances. Challenging life conditions might change PFC function and put individuals at risk for maladaptive social behavior. The excitation-inhibition (EI) balance of prefrontal neurons appears to play a crucial role in this process. Here, we examined how a challenging life condition in C57BL/6JolaHsd mice, i.e. group-housing 6 mice in a complex environment for 10 days in adulthood, changes the EI-balance of infralimbic prefrontal neurons in layer 2/3, compared to standard pair-housing. Slices were prepared from "undisturbed" mice, i.e. the first mouse taken from the cage, or mice taken ∼15 min later, who were mildly aroused after removal of the first mouse. We observed a housing-condition by arousal-state interaction, with in the complex housing group an elevated EI-balance in undisturbed and reduced EI-balance in mildly aroused animals, while no differences were observed in standard housed animals. The change was explained by a shift in mIPSC and mEPSC frequency, while amplitudes remained unaffected. Female mice showed no housing-by-state interaction, but a main effect of housing was found for mIPSCs, with a higher frequency in complex- versus standard-housed females. No effects were observed in males who were complex-housed from a young age onwards. Explorative investigations support a potential mediating role of corticosterone in housing effects on the EI-balance of males. We argue that taking the arousal state of individuals into account is necessary to better understand the consequences of exposure to challenging life conditions for prefrontal function.

ATAXIN-2 intermediate-length polyglutamine expansions elicit ALS-associated metabolic and immune phenotypes.

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules, neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43Tg-ALS mice, ATXN2-Q33 causes reduced motor function, NMJ alterations, neuron degeneration and altered in vitro stress granule dynamics. Furthermore, gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together, these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy.

Metaplasticity of amygdalar responses to the stress hormone corticosterone.

High levels of corticosteroids (as circulate after stress) quickly and reversibly enhance hippocampal glutamatergic transmission via nongenomic actions requiring mineralocorticoid receptors. Subsequently, the hormone slowly and long-lastingly normalizes hippocampal cell function, through nuclear glucocorticoid receptors. Here we describe a rapid mineralocorticoid receptor-dependent enhancement of glutamatergic transmission in basolateral amygdala neurons. Contrary to the hippocampus, this rapid enhancement is long-lasting, potentially allowing an extended window for encoding of emotional aspects during stressful events. Importantly, the long-lasting change in state of amygdala neurons greatly affects the responsiveness to subsequent surges of corticosterone, revealing a quick suppression of glutamatergic transmission, which requires the glucocorticoid receptor. Responses of basolateral amygdala neurons to the stress hormone corticosterone can thus switch from excitatory to inhibitory, depending on the recent stress history of the organism.

Corticosterone rapidly reduces glutamatergic but not GABAergic transmission in the infralimbic prefrontal cortex of male mice.

Rapid non-genomic effects of corticosteroid hormones, affecting glutamatergic and GABAergic transmission, have been described for many limbic structures in the rodent brain. These rapid effects appear to be region specific. It is not always clear which (or even whether) corticosteroid receptor -the glucocorticoid receptor (GR) or mineralocorticoid receptor (MR)- initiate these rapid effects. In the hippocampus and amygdala membrane-associated MR, but also membrane-associated GR (in amygdala), are involved. Other studies indicate that the rapid modulation may be induced by transactivation of kinases, or other receptors, like the G-protein coupled estrogen receptor (GPER) which was recently found to bind the mineralocorticoid aldosterone. In the current study we explored, in young adult male C57Bl6 mice, possible rapid effects of corticosterone on layer 2/3 infralimbic-prefrontal cortex (IL-PFC) neurons. We show that corticosterone, via non-genomic MR activation, reduces the mEPSC -but does not affect mIPSC- frequency; we observed no effect on mEPSC or mIPSC amplitude. As a result, overall spontaneous activity in the IL-PFC is suppressed. A potential role of GPER cannot be excluded, since G-15, an antagonist of GPER, also prevented the rapid effects of corticosterone.

Acceleration of GABA-switch after early life stress changes mouse prefrontal glutamatergic transmission.

Early life stress (ELS) alters the excitation-inhibition-balance (EI-balance) in various rodent brain areas and may be responsible for behavioral impairment later in life. The EI-balance is (amongst others) influenced by the switch of GABAergic transmission from excitatory to inhibitory, the so-called "GABA-switch". Here, we investigated how ELS affects the GABA-switch in mouse infralimbic Prefrontal Cortex layer 2/3 neurons, using the limited-nesting-and-bedding model. In ELS mice, the GABA-switch occurred already between postnatal day (P) 6 and P9, as opposed to P15-P21 in controls. This was associated with increased expression of the inward chloride transporter NKCC1, compared to the outward chloride transporter KCC2, both of which are important for the intracellular chloride concentration and, hence, the GABA reversal potential (Erev). Chloride transporters are not only important for regulating chloride concentration postsynaptically, but also presynaptically. Depending on the Erev of GABA, presynaptic GABAA receptor stimulation causes a depolarization or hyperpolarization, and thereby enhanced or reduced fusion of glutamate vesicles respectively, in turn changing the frequency of miniature postsynaptic currents (mEPSCs). In accordance, bumetanide, a blocker of NKCC1, shifted the Erev GABA towards more hyperpolarized levels in P9 control mice and reduced the mEPSC frequency. Other modulators of chloride transporters, e.g. VU0463271 (a KCC2 antagonist) and aldosterone -which increases NKCC1 expression-did not affect postsynaptic Erev in ELS P9 mice, but did increase the mEPSC frequency. We conclude that the mouse GABA-switch is accelerated after ELS, affecting both the pre- and postsynaptic chloride homeostasis, the former altering glutamatergic transmission. This may considerably affect brain development.

Using SuperClomeleon to Measure Changes in Intracellular Chloride during Development and after Early Life Stress.

Intraneuronal chloride concentrations ([Cl-]i) decrease during development resulting in a shift from depolarizing to hyperpolarizing GABA responses via chloride-permeable GABAA receptors. This GABA shift plays a pivotal role in postnatal brain development, and can be strongly influenced by early life experience. Here, we assessed the applicability of the recently developed fluorescent SuperClomeleon (SClm) sensor to examine changes in [Cl-]i using two-photon microscopy in brain slices. We used SClm mice of both sexes to monitor the developmental decrease in neuronal chloride levels in organotypic hippocampal cultures. We could discern a clear reduction in [Cl-]i between day in vitro (DIV)3 and DIV9 (equivalent to the second postnatal week in vivo) and a further decrease in some cells until DIV22. In addition, we assessed alterations in [Cl-]i in the medial prefrontal cortex (mPFC) of postnatal day (P)9 male SClm mouse pups after early life stress (ELS). ELS was induced by limiting nesting material between P2 and P9. ELS induced a shift toward higher (i.e., immature) chloride levels in layer 2/3 cells in the mPFC. Although conversion from SClm fluorescence to absolute chloride concentrations proved difficult, our study underscores that the SClm sensor is a powerful tool to measure physiological changes in [Cl-]i in brain slices.

Non-genomic steroid signaling through the mineralocorticoid receptor: Involvement of a membrane-associated receptor?

Corticosteroid receptors in the mammalian brain mediate genomic as well as non-genomic actions. Although receptors mediating genomic actions were already cloned 35 years ago, it remains unclear whether the same molecules are responsible for the non-genomic actions or that the latter involve a separate class of receptors. Here we focus on one type of corticosteroid receptors, i.e. the mineralocorticoid receptor (MR). We summarize some of the known properties and the current insight in the localization of the MR in peripheral cells and neurons, especially in relation to non-genomic signaling. Previous studies from our own and other labs provided evidence that MRs mediating non-genomic actions are identical to the ones involved in genomic signaling, but may be translocated to the plasma cell membrane instead of the nucleus. With fixed cell imaging and live cell imaging techniques we tried to visualize these presumed membrane-associated MRs, using antibodies or overexpression of MR-GFP in COS7 and hippocampal cultured neurons. Despite the physiological evidence for MR location in or close to the cell membrane, we could not convincingly visualize membrane localization of endogenous MRs or GFP-MR molecules. However, we did find punctae of labeled antibodies intracellularly, which might indicate transactivating spots of MR near the membrane. We also found some evidence for trafficking of MR via beta-arrestins. In beta-arrestin knockout mice, we didn't observe metaplasticity in the basolateral amygdala anymore, indicating that internalization of MRs could play a role during corticosterone activation. Furthermore, we speculate that membrane-associated MRs could act indirectly via activating other membrane located structures like e.g. GPER and/or receptor tyrosine kinases.

Systemic Injection of Aged Blood Plasma in Adult C57BL/6 Mice Induces Neurophysiological Impairments in the Hippocampal CA1.

BACKGROUND: Aging is characterized by systemic alterations and forms an important risk factor for Alzheimer's disease (AD). Recently, it has been indicated that blood-borne factors present in the systemic milieu contribute to the aging process. Exposing young mice to aged blood plasma results in impaired neurogenesis and synaptic plasticity in the dentate gyrus, as well as impaired cognition. Vice versa, treating aged mice with young blood plasma rescues impairments associated with aging. OBJECTIVE: Whether blood-borne factors are sufficient to drive impairments outside the dentate gyrus, how they impact neurophysiology, and how the functional outcome compares to impairments found in mouse models for AD is still unclear. METHODS: Here, we treated adult mice with blood plasma from aged mice and assessed neurophysiological parameters in the hippocampal CA1. RESULTS: Mice treated with aged blood plasma show significantly impaired levels of long-term potentiation (LTP), similar to those present in APP/PS1 mice. These impaired levels of LTP in plasma-treated mice are associated with alterations in basic properties of glutamatergic transmission and the enhanced activity of voltage-gated Ca2+ channels. CONCLUSION: Together, the data presented in this study show that blood-borne factors are sufficient to drive neurophysiological impairments in the hippocampal CA1.

Spinal Muscular Atrophy Patient iPSC-Derived Motor Neurons Display Altered Proteomes at Early Stages of Differentiation.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by loss of motor neurons (MN) in the spinal cord leading to progressive muscle atrophy and weakness. SMA is caused by mutations in the survival motor neuron 1 (SMN1) gene, resulting in reduced levels of survival motor neuron (SMN) protein. The mechanisms that link SMN deficiency to selective motor neuron dysfunction in SMA remain largely unknown. We present here, for the first time, a comprehensive quantitative TMT-10plex proteomics analysis that covers the development of induced pluripotent stem cell-derived MNs from both healthy individuals and SMA patients. We show that the proteomes of SMA samples segregate from controls already at early stages of neuronal differentiation. The altered proteomic signature in SMA MNs is associated with mRNA splicing, ribonucleoprotein biogenesis, organelle organization, cellular biogenesis, and metabolic processes. We highlight several known SMN-binding partners and evaluate their expression changes during MN differentiation. In addition, we compared our study to human and mouse in vivo proteomic studies revealing distinct and similar signatures. Altogether, our work provides a comprehensive resource of molecular events during early stages of MN differentiation, containing potentially therapeutically interesting protein expression profiles for SMA.

The coming out of the brain mineralocorticoid receptor.

Corticosteroids - secreted after stress - have profound effects on brain and behavior. These effects are mediated by mineralocorticoid and glucocorticoid receptors, which are abundantly expressed in limbic neurons. The role of mineralocorticoid receptors in higher brain functions has never been well understood. Here we argue that the recently discovered low-affinity membrane version of the mineralocorticoid receptor contributes to the initial phase of the stress reaction; this is complemented by the glucocorticoid receptor which terminates the stress response. This concept may explain why human carriers of a mineralocorticoid receptor gene variant display enhanced neuroendocrine and autonomic responsiveness to a psychological stressor.

Age-dependent shift in spontaneous excitation-inhibition balance of infralimbic prefrontal layer II/III neurons is accelerated by early life stress, independent of forebrain mineralocorticoid receptor expression.

In this study we tested the hypothesis i) that age-dependent shifts in the excitation-inhibition balance of prefrontal neurons are accelerated by early life stress, a risk factor for the etiology of many psychiatric disorders; and if so, ii) that this process is exacerbated by genetic forebrain-specific downregulation of the mineralocorticoid receptor, a receptor that was earlier found to be a protective factor for negative effects of early life stress in both rodents and humans. In agreement with the literature, an age-dependent downregulation of the excitation-inhibition balance was found both with regard to spontaneous and evoked synaptic currents. The age-dependent shift in spontaneous excitatory relative to inhibitory currents was significantly accelerated by early life stress, but this was not exacerbated by reduction in mineralocorticoid receptor expression. The age-dependent changes in the excitation-inhibition balance were mirrored by similar changes in receptor subunit expression and morphological alterations, particularly in spine density, which could thus potentially contribute to the functional changes. However, none of these parameters displayed acceleration by early life stress, nor depended on mineralocorticoid receptor expression. We conclude that, in agreement with the hypothesis, early life stress accelerates the developmental shift of the excitation-inhibition balance but, contrary to expectation, there is no evidence for a putative protective role of the mineralocorticoid receptor in this system. In view of the modest effect of early life stress on the excitation-inhibition balance, alternative mechanisms potentially underlying the development of psychiatric disorders should be further explored.

Corticosteroid hormones in the central stress response: quick-and-slow.

Recent evidence shows that corticosteroid hormones exert rapid non-genomic effects on neurons in the hypothalamus and the hippocampal CA1 region. The latter depend on classical mineralocorticoid receptors which are accessible from the outside of the plasma membrane and display a 10-fold lower affinity for corticosterone than the nuclear version involved in neuroprotection. Consequently, this 'membrane' receptor could play an important role while corticosteroid levels are high, i.e. during the initial phase of the stress response. We propose that during this phase corticosterone promotes hippocampal excitability and amplifies the effect of other stress hormones. These permissive non-genomic effects may contribute to fast behavioral effects and encoding of stress-related information. The fast effects are complemented by slower glucocorticoid receptor-mediated effects which facilitate suppression of temporary raised excitability, recovery from the stressful experience and storage of information for future use.

Effects of corticosterone and the beta-agonist isoproterenol on glutamate receptor-mediated synaptic currents in the rat basolateral amygdala.

Behavioral and field potential studies suggest that--shortly after stress--noradrenaline and corticosterone interact to affect the function of basolateral amygdala (BLA) neurons. Here, we tested, at the single-cell level, to what extent alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated and N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses of identified BLA neurons are affected by relatively low concentrations of the beta-agonist isoproterenol, how this is influenced by concomitant application of corticosterone, and how isoproterenol effects are influenced by corticosterone given several hours in advance. We observed that isoproterenol concentration-dependently enhances AMPA receptor-mediated (but not NMDA receptor-mediated) responses; near-maximal effects were induced by 1 microm isoproterenol. Corticosterone alone did not rapidly affect AMPA and NMDA-mediated responses. NMDA-mediated responses were also not affected by the hormone in a delayed manner; AMPA-mediated responses were slowly suppressed by corticosterone, but only with high stimulation intensities. If corticosterone was co-applied with isoproterenol (0.4 or 1 microm), facilitation of AMPA-mediated responses was comparable to that seen with isoproterenol alone. However, if corticosterone was applied several hours in advance of the beta-agonist, the effect of 0.4 microm isoproterenol on AMPA-mediated responses was reduced. This supports the notion that, in the BLA, isoproterenol facilitates synaptic transmission, a process that can be suppressed by corticosterone in a slow manner. Overall, the data suggest that, despite the previously reported ability of corticosterone to cause long-term increases in excitability in the BLA, the hormone still retains some capacity to slowly exert a normalizing action on local activity.

Circadian and Ultradian Variations in Corticosterone Level Influence Functioning of the Male Mouse Basolateral Amygdala.

The hypothalamic-pituitary-adrenal axis involves timed signaling between the hypothalamus, pituitary, and adrenal glands and back to the brain, causing an inherently oscillating system. Corticosteroids such as corticosterone (CORT) are secreted in a circadian rhythm, characterized by low and high levels at the start of the inactive and active phases, respectively. The circadian rhythm overarches ultradian CORT pulses, with approximate 1-hour interpulse intervals. We examined the physiological relevance of pulsatile CORT exposure for neurons of the basolateral amygdala (BLA), an area important for fear learning. We first applied four pulses of equal, high CORT concentration and measured the frequency of miniature excitatory postsynaptic currents (mEPSCs) reflecting spontaneous glutamate signaling. BLA neurons responded differently to each pulse, showing "metaplasticity," extending earlier studies. Next, we mimicked the progression of the inactive and active phases by four CORT pulses of increasing and decreasing concentrations, respectively. CORT pulses of increasing concentration were necessary and sufficient to gradually increase baseline (between-pulse) mEPSC frequency during the mimicked inactive phase, whereas the opposite was seen with decreasing CORT levels during the mimicked active phase. To study the relevance of changed glutamate transmission on behavior, mice were tested in tone-cued fear conditioning during the active or inactive phase. Animals tested at the inactive compared with the active phase showed efficient fear learning; this was also observed when animals tested during the active phase were treated with the CORT synthesis blocker metyrapone. This suggests that natural CORT rhythms influence electrical activity in the BLA, possibly contributing to altered behavioral function.

Stress-induced changes in hippocampal function.

Exposure of an organism to stress leads to activation of the sympatho-adrenomedullary system and the hypothalamo-pituitary-adrenal axis. Consequently, levels of noradrenaline, peptides like vasopressin and CRH, and corticosteroid hormones in the brain rise. These hormones affect brain function at those sites where receptors are enriched, like the hippocampus, lateral septum, amygdala nuclei, and prefrontal cortex. During the initial phase of the stress response, when hormone levels are high, these compounds mostly enhance excitability and promote long-term potentiation. Later on, when hormone levels have subsided but gene-mediated effects of corticosteroids start to appear, the excitability is normalized to the pre-stress level, in the CA1 hippocampal area, but possibly less so in the dentate gyrus and amygdala. A disturbed balance between these early and late phases of the stress response as well as a shift toward the relative contribution of the dentate/amygdala pathways may explain why the normal restorative capacity fails in vulnerable people experiencing a life-threatening situation, which could contribute to the development of PTSD.

Microglia innately develop within cerebral organoids.

Cerebral organoids are 3D stem cell-derived models that can be utilized to study the human brain. The current consensus is that cerebral organoids consist of cells derived from the neuroectodermal lineage. This limits their value and applicability, as mesodermal-derived microglia are important players in neural development and disease. Remarkably, here we show that microglia can innately develop within a cerebral organoid model and display their characteristic ramified morphology. The transcriptome and response to inflammatory stimulation of these organoid-grown microglia closely mimic the transcriptome and response of adult microglia acutely isolated from post mortem human brain tissue. In addition, organoid-grown microglia mediate phagocytosis and synaptic material is detected inside them. In all, our study characterizes a microglia-containing organoid model that represents a valuable tool for studying the interplay between microglia, macroglia, and neurons in human brain development and disease.