Neurons In Cortical Layers Research Articles

Local stimulation of pyramidal neurons in deep cortical layers of anesthetized rats enhances cortical visual information processing

In the primary visual cortex area V1 activation of inhibitory interneurons, which provide negative feedback for excitatory pyramidal neurons, can improve visual response reliability and orientation selectivity. Moreover, optogenetic activation of one class of interneurons, parvalbumin (PV) positive cells, reduces the receptive field (RF) width. These data suggest that in V1 the negative feedback improves visual information processing. However, according to information theory, noise can limit information content in a signal, and to the best of our knowledge, in V1 signal-to-noise ratio (SNR) has never been estimated following either pyramidal or inhibitory neuron activation. Therefore, we optogenetically activated pyramidal or PV neurons in the deep layers of cortical area V1 and measured the SNR and RF area in nearby pyramidal neurons. Activation of pyramidal or PV neurons increased the SNR by 267% and 318%, respectively, and reduced the RF area to 60.1% and 77.5%, respectively, of that of the control. A simple integrate-and-fire neuron model demonstrated that an improved SNR and a reduced RF area can increase the amount of information encoded by neurons. We conclude that in V1 activation of pyramidal neurons improves visual information processing since the location of the visual stimulus can be pinpointed more accurately (via a reduced RF area), and more information is encoded by neurons (due to increased SNR).

Molecular pathology, developmental changes and synaptic dysfunction in (pre-) symptomatic human C9ORF72-ALS/FTD cerebral organoids

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD, even before symptom onset. Because these early disease phenotypes remain incompletely understood, we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients, presymptomatic C9ORF72-HRE (C9-HRE) carriers, and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition, single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids, including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further, molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly, organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology, whereas the extent to which more downstream cellular defects, as found in C9-ALS/FTD models, were detected varied for the different presymptomatic C9-HRE cases. Together, these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies.

Integrative analysis of long isoform sequencing and functional data identifies distinct cortical layer neuronal subtypes derived from human iPSCs.

Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated nonneuronal cells in the culture as compared with those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, β tubulin, and Synapsin I) and whole cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo.NEW & NOTEWORTHY Low differentiation efficiency is one of the major drawbacks of the existing protocols to differentiate iPSCs into neuronal lineages. Here, we present time-efficient and robust approach of neuronal differentiation leading to the generation of functional brain units, cortical layer neurons. We found iPSCs cultured in Essential 8 media (E8M) resulted in neuronal differentiation without the signs of growth of spontaneously differentiated cells in culture at any point in 35 days compared with Stemflex media (SFM).

Ash1 l loss-of-function results in structural birth defects and altered cortical development

Abstract The histone methyltransferase ASH1L plays a crucial role in regulating gene expression across various organ systems during development, yet its role in brain development remains largely unexplored. Over 130 individuals with autism harbour heterozygous loss-of-function ASH1L variants, and population studies confirm it as a high-risk autism gene. Previous studies on Ash1 l deficient mice have reported autistic-like behaviours and provided insights into the underlying neuropathophysiology. In this study, we used mice with a cre-inducible deletion of Ash1 l exon 4, which results in a frame shift and premature stop codon (p.V1693Afs*2). Our investigation evaluated the impact of Ash1 l loss-of-function on survival and craniofacial skeletal development. Using a tamoxifen-inducible cre strain, we targeted Ash1 l knockout early in cortical development (Emx1-Cre-ERT2; e10.5). Immunohistochemistry was utilized to assess cortical lamination, while EdU incorporation aided in birthdating cortical neurons. Additionally, single-cell RNA sequencing was employed to compare cortical cell populations and identify genes with differential expression. At e18.5, the proportion of homozygous Ash1 l germline knockout embryos appeared normal; however, no live Ash1 l null pups were present at birth (e18.5: n = 77, P = 0.90; p0: n = 41, P = 0.00095). Notably, Ash1l-/- exhibited shortened nasal bones (n = 31, P = 0.017). In the cortical-specific knockout model, SATB2 neurons showed increased numbers (n = 6/genotype, P = 0.0001) and were distributed through the cortical plate. Birthdating revealed generation of ectopically placed deep layer neurons that express SATB2 (e13.5 injection: n = 4/genotype, P = 0.0126). Single cell RNA sequencing revealed significant differences in gene expression between control and mutant upper layer neurons, leading to distinct clustering. Pseudotime analysis indicated that the mutant cluster followed an altered cell differentiation trajectory. This study underscores the essential role of Ash1 l in postnatal survival and normal craniofacial development. In the cortex, ASH1L exerts broad effects on gene expression and is indispensable for determining the fate of upper layer cortical neurons. These findings provide valuable insights into the potential mechanisms of ASH1L neuropathology, shedding light on its significance in neurodevelopmental disorders like autism.

Correlation between the number of interstitial neurons of the white matter and number of neurons within cortical layers: Histological analyses in postnatal macaque.

We have examined the number and distribution of NeuN-immunoreactive cortical white matter interstitial cells (WMICs) and compared them to the neurons in layers 1-6 across the overlying cortex in coronal sections from postnatal macaques. The data have been gathered from over 300 selected regions at gyral crowns, at sulci, and at linear regions of the cortex where we also determined cortical layer thicknesses: standard thicknesses and tangential thicknesses. Cortical thicknesses and cell numbers showed variability according to gyral, linear, or sulcal regions. In spite of these variations, our standardized cell numbers in layers 1 to 6b and interstitial cells underlying layer 6b-white matter boundary have shown a consistent correlation between the number of WMICs and the number of layer 5 and 6a cortical neurons on all cortical regions studied: for each WMIC, there are on the order of five cortical neurons in layer 5 and approximately three cortical neurons in layer 6a, irrespective of the origins of the selected cortical area or whether they are from gyral, linear, or sulcal regions. We propose that the number of interstitial neurons in the postnatal macaque cortex is correlated to the density of neurons within layers 5 and 6a and, from a clinical perspective, the change in density or distribution of interstitial neurons in schizophrenia or epilepsy may in fact be linked to the number of layers 5 and 6a neurons.

Silicate Microfiber Scaffolds Support the Formation and Expansion of the Cortical Neuronal Layer of Cerebral Organoids With a Sheet-Like Configuration.

Cerebral organoids (COs) are derived from human-induced pluripotent stem cells in vitro and mimic the features of the human fetal brain. The development of COs is largely dependent on "self-organization" mechanisms, in which differentiating cells committed to cortical cells autonomously organize into the cerebral cortex-like tissue. However, extrinsic manipulation of their morphology, including size and thickness, remains challenging. In this study, we discovered that silicate microfiber scaffolds could support the formation of cortical neuronal layers and successfully generated cortical neuronal layers, which are 9 times thicker than conventional COs, in 70 days. These cortical neurons in the silicate microfiber layer were differentiated in a fetal brain-like lamination pattern. While these cellular characteristics such as cortical neurons and neural stem/progenitor cells were like those of conventional COs, the cortical neuronal layers were greatly thickened in sheet-like configuration. Moreover, the cortical neurons in the scaffolds showed spontaneous electrical activity. We concluded that silicate microfiber scaffolds support the formation of the cortical neuronal layers of COs without disturbing self-organization-driven corticogenesis. The extrinsic manipulation of the formation of the cortical neuronal layers of COs may be useful for the research of developmental mechanisms or pathogenesis of the human cerebral cortex, particularly for the development of regenerative therapy and bioengineering.

Long-term exposure to cadmium disrupts neurodevelopment in mature cerebral organoids

Cadmium (Cd) is a pervasive environmental pollutant. Increasing evidence suggests that Cd exposure during pregnancy can induce adverse neurodevelopmental outcomes. However, due to the limitations of neural cell and animal models, it is challenging to study the developmental neurotoxicity and underlying toxicity mechanism of long-term exposure to environmental pollutants during human brain development. In this study, chronic Cd exposure was performed in human mature cerebral organoids for 49 or 77 days. Our study found that prolonged exposure to Cd resulted in the inhibition of cerebral organoid growth and the disruption of neural differentiation and cortical layer organization. These potential consequences of chronic Cd exposure may include impaired GFAP expression, a reduction in SOX2+ neuronal progenitor cells, an increase in TUJ1+ immature neurons, as well as an initial increase and a subsequent decrease in both TBR2+ intermediate progenitors and CTIP2+ deep layer cortical neurons. Transcriptomic analyses revealed that long-term exposure to Cd disrupted zinc and copper ion homeostasis through excessive synthesis of metallothionein and disturbed synaptogenesis, as evidenced by inhibited postsynaptic protein. Our study employed mature cerebral organoids to evaluate the developmental neurotoxicity induced by long-term Cd exposure.

Differential expression of 5mC epigenomic regulators in Alzheimer’s disease

AbstractBackgroundEpigenetic processes are molecular mechanisms that contribute to the pathogenesis of neurodegenerative diseases. 5‐methylcytosine (5mC) and the oxidised state, 5‐formilcytosine (5fC), are known transcriptional regulators moderated by sets of writer, reader, and eraser effector proteins. We propose that gene expression variation in DNA effector proteins may contribute to Alzheimer’s Disease (AD) neuropathology. We also aimed to characterize the distribution of 5fC modifications across brain regions and within cellular compartments to better understand the role of 5mC/5fC modifications in AD.MethodsRNA sequencing data for 17 effector proteins obtained from the Aging, Dementia and Traumatic Brain Injury Study was analysed to assess variation in abundance of 5mC writers, readers, and erasers. Gene expression data were compared across four brain regions in 51 AD and 56 healthy control samples, and between Braak and CERAD neuropathological scales. In addition, we examined the presence and distribution of 5fC by immunofluorescence (IF) across four brain regions in sections from healthy individuals.ResultsTranscripts of DNA methylation writers DNMT1, DNMT3A and DNMT3B were found increased in the AD cohort across 3 brain regions, whilst the reader UHRF1 mRNA was decreased in the same assessment. GADD45B and AICDA, DNA methylation erasers, showed changes in mRNA abundance across Braak and CERAD neuropathological load groupings. IF analysis indicated 5fC modification to be highly abundant in dentate gyrus and within the cytoplasm of hippocampal mossy cells and of neurons in cortical layer III. Within neuronal cells, we observed 5fC partially colocalizing with mitochondria especially within hippocampal neurons.ConclusionOur findings indicate that changes in 5mC/5hmC/5fC effector protein expression are associated with AD and its neuropathology. The characterisation of 5fC localisation in human brain indicates high abundance within the cytoplasm of hippocampal cells suggesting that 5fC may be involved in pre‐ and post‐transcriptional control of memory processing pathways and, when disrupted, may lead to neurodegenerative disease.

An Ultraflexible Electrode Array for Large-Scale Chronic Recording in the Nonhuman Primate Brain.

Single-unit (SU) recording in nonhuman primates (NHPs) is indispensible in the quest of how the brain works, yet electrodes currently used for the NHP brain are limited in signal longevity, stability, and spatial coverage. Using new structural materials, microfabrication, and penetration techniques, we develop a mechanically robust ultraflexible, 1 µm thin electrode array (MERF) that enables pial penetration and high-density, large-scale, and chronic recording of neurons along both vertical and horizontal cortical axes in the nonhuman primate brain. Recording from three monkeys yields 2,913 SUs from 1,065 functional recording channels (up to 240 days), with some SUs tracked for up to 2 months. Recording from the primary visual cortex (V1) reveals that neurons with similar orientation preferences for visual stimuli exhibited higher spike correlation. Furthermore, simultaneously recorded neurons in different cortical layers of the primary motor cortex (M1) show preferential firing for hand movements of different directions. Finally, it is shown that a linear decoder trained with neuronal spiking activity across M1 layers during monkey's hand movements can be used to achieve on-line control of cursor movement. Thus, the MERF electrode array offers a new tool for basic neuroscience studies and brain-machine interface (BMI) applications in the primate brain.

Sudden Unexpected Death in Epilepsy and Respiratory Defects in a Mouse Model of DEPDC5-Related Epilepsy.

DEPDC5 is a common causative gene in familial focal epilepsy with or without malformations of cortical development. Its pathogenic variants also confer a significantly higher risk for sudden unexpected death in epilepsy (SUDEP), providing opportunities to investigate the pathophysiology intersecting neurodevelopment, epilepsy, and cardiorespiratory function. There is an urgent need to gain a mechanistic understanding of DEPDC5-related epilepsy and SUDEP, identify biomarkers for patients at high risk, and develop preventive interventions. Depdc5 was specifically deleted in excitatory or inhibitory neurons in the mouse brain to determine neuronal subtypes that drive epileptogenesis and SUDEP. Electroencephalogram (EEG), cardiac, and respiratory recordings were performed to determine cardiorespiratory phenotypes associated with SUDEP. Baseline respiratory function and the response to hypoxia challenge were also studied in these mice. Depdc5 deletion in excitatory neurons in cortical layer 5 and dentate gyrus caused frequent generalized tonic-clonic seizures and SUDEP in young adult mice, but Depdc5 deletion in cortical interneurons did not. EEG suppression immediately following ictal offset was observed in fatal and non-fatal seizures, but low amplitude rhythmic theta frequency activity was lost only in fatal seizures. In addition, these mice developed baseline respiratory dysfunction prior to SUDEP, during which ictal apnea occurred long before terminal cardiac asystole. Depdc5 deletion in excitatory neurons is sufficient to cause DEPDC5-related epilepsy and SUDEP. Ictal apnea and respiratory dysregulation play critical roles in SUDEP. Our study also provides a novel mouse model to investigate the underlying mechanisms of DEPDC5-related epilepsy and SUDEP. ANN NEUROL 2023;94:812-824.

Lmo4 synergizes with Fezf2 to promote direct in vivo reprogramming of upper layer cortical neurons and cortical glia towards deep-layer neuron identities.

In vivo direct neuronal reprogramming relies on the implementation of an exogenous transcriptional program allowing to achieve conversion of a particular neuronal or glial cell type towards a new identity. The transcription factor (TF) Fezf2 is known for its role in neuronal subtype specification of deep-layer (DL) subcortical projection neurons. High ectopic Fezf2 expression in mice can convert both upper-layer (UL) and striatal projection neurons into a corticofugal fate, even if at low efficiency. In this study, we show that Fezf2 synergizes with the nuclear co-adaptor Lmo4 to further enhance reprogramming of UL cortical pyramidal neurons into DL corticofugal neurons, at both embryonic and early postnatal stages. Reprogrammed neurons express DL molecular markers and project toward subcerebral targets, including thalamus, cerebral peduncle (CP), and spinal cord (SC). We also show that co-expression of Fezf2 with the reprogramming factors Neurog2 and Bcl2 in early postnatal mouse glia promotes glia-to-neuron conversion with partial hallmarks of DL neurons and with Lmo4 promoting further morphological complexity. These data support a novel role for Lmo4 in synergizing with Fezf2 during direct lineage conversion in vivo.

Cell-type specific defects in PTEN-mutant cortical organoids converge on abnormal circuit activity.

De novo heterozygous loss-of-function mutations in phosphatase and tensin homolog (PTEN) are strongly associated with autism spectrum disorders; however, it is unclear how heterozygous mutations in this gene affect different cell types during human brain development and how these effects vary across individuals. Here, we used human cortical organoids from different donors to identify cell-type specific developmental events that are affected by heterozygous mutations in PTEN. We profiled individual organoids by single-cell RNA-seq, proteomics and spatial transcriptomics and revealed abnormalities in developmental timing in human outer radial glia progenitors and deep-layer cortical projection neurons, which varied with the donor genetic background. Calcium imaging in intact organoids showed that both accelerated and delayed neuronal development phenotypes resulted in similar abnormal activity of local circuits, irrespective of genetic background. The work reveals donor-dependent, cell-type specific developmental phenotypes of PTEN heterozygosity that later converge on disrupted neuronal activity.

The role of superficial and deep layers in the generation of high frequency oscillations and interictal epileptiform discharges in the human cortex

Describing intracortical laminar organization of interictal epileptiform discharges (IED) and high frequency oscillations (HFOs), also known as ripples. Defining the frequency limits of slow and fast ripples. We recorded potential gradients with laminar multielectrode arrays (LME) for current source density (CSD) and multi-unit activity (MUA) analysis of interictal epileptiform discharges IEDs and HFOs in the neocortex and mesial temporal lobe of focal epilepsy patients. IEDs were observed in 20/29, while ripples only in 9/29 patients. Ripples were all detected within the seizure onset zone (SOZ). Compared to hippocampal HFOs, neocortical ripples proved to be longer, lower in frequency and amplitude, and presented non-uniform cycles. A subset of ripples (≈ 50%) co-occurred with IEDs, while IEDs were shown to contain variable high-frequency activity, even below HFO detection threshold. The limit between slow and fast ripples was defined at 150 Hz, while IEDs’ high frequency components form clusters separated at 185 Hz. CSD analysis of IEDs and ripples revealed an alternating sink-source pair in the supragranular cortical layers, although fast ripple CSD appeared lower and engaged a wider cortical domain than slow ripples MUA analysis suggested a possible role of infragranularly located neural populations in ripple and IED generation. Laminar distribution of peak frequencies derived from HFOs and IEDs, respectively, showed that supragranular layers were dominated by slower (< 150 Hz) components. Our findings suggest that cortical slow ripples are generated primarily in upper layers while fast ripples and associated MUA in deeper layers. The dissociation of macro- and microdomains suggests that microelectrode recordings may be more selective for SOZ-linked ripples. We found a complex interplay between neural activity in the neocortical laminae during ripple and IED formation. We observed a potential leading role of cortical neurons in deeper layers, suggesting a refined utilization of LMEs in SOZ localization.

Hindlimb muscle representations in mouse motor cortex defined by viral tracing.

Descending pathways from the cortex to the spinal cord are involved in the control of natural movement. Although mice are widely used to study the neurobiology of movement and as models of neurodegenerative disease, an understanding of motor cortical organization is lacking, particularly for hindlimb muscles. In this study, we used the retrograde transneuronal transport of rabies virus to compare the organization of descending cortical projections to fast- and slow-twitch hindlimb muscles surrounding the ankle joint in mice. Although the initial stage of virus transport from the soleus muscle (predominantly slow-twitch) appeared to be more rapid than that associated with the tibialis anterior muscle (predominantly fast-twitch), the rate of further transport of virus to cortical projection neurons in layer V was equivalent for the two injected muscles. After appropriate survival times, dense concentrations of layer V projection neurons were identified in three cortical areas: the primary motor cortex (M1), secondary motor cortex (M2), and primary somatosensory cortex (S1). The origin of the cortical projections to each of the two injected muscles overlapped almost entirely within these cortical areas. This organization suggests that cortical projection neurons maintain a high degree of specificity; that is, even when cortical projection neurons are closely located, each neuron could have a distinct functional role (controlling fast- versus slow-twitch and/or extensor versus flexor muscles). Our results represent an important addition to the understanding of the mouse motor system and lay the foundation for future studies investigating the mechanisms underlying motor system dysfunction and degeneration in diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy.

Primary somatosensory cortex bidirectionally modulates sensory gain and nociceptive behavior in a layer-specific manner

The primary somatosensory cortex (S1) is a hub for body sensation of both innocuous and noxious signals, yet its role in somatosensation versus pain is debated. Despite known contributions of S1 to sensory gain modulation, its causal involvement in subjective sensory experiences remains elusive. Here, in mouse S1, we reveal the involvement of cortical output neurons in layers 5 (L5) and 6 (L6) in the perception of innocuous and noxious somatosensory signals. We find that L6 activation can drive aversive hypersensitivity and spontaneous nocifensive behavior. Linking behavior to neuronal mechanisms, we find that L6 enhances thalamic somatosensory responses, and in parallel, strongly suppresses L5 neurons. Directly suppressing L5 reproduced the pronociceptive phenotype induced by L6 activation, suggesting an anti-nociceptive function for L5 output. Indeed, L5 activation reduced sensory sensitivity and reversed inflammatory allodynia. Together, these findings reveal a layer-specific and bidirectional role for S1 in modulating subjective sensory experiences.

Modelling novelty detection in the thalamocortical loop.

In complex natural environments, sensory systems are constantly exposed to a large stream of inputs. Novel or rare stimuli, which are often associated with behaviorally important events, are typically processed differently than the steady sensory background, which has less relevance. Neural signatures of such differential processing, commonly referred to as novelty detection, have been identified on the level of EEG recordings as mismatch negativity (MMN) and on the level of single neurons as stimulus-specific adaptation (SSA). Here, we propose a multi-scale recurrent network with synaptic depression to explain how novelty detection can arise in the whisker-related part of the somatosensory thalamocortical loop. The "minimalistic" architecture and dynamics of the model presume that neurons in cortical layer 6 adapt, via synaptic depression, specifically to a frequently presented stimulus, resulting in reduced population activity in the corresponding cortical column when compared with the population activity evoked by a rare stimulus. This difference in population activity is then projected from the cortex to the thalamus and amplified through the interaction between neurons of the primary and reticular nuclei of the thalamus, resulting in rhythmic oscillations. These differentially activated thalamic oscillations are forwarded to cortical layer 4 as a late secondary response that is specific to rare stimuli that violate a particular stimulus pattern. Model results show a strong analogy between this late single neuron activity and EEG-based mismatch negativity in terms of their common sensitivity to presentation context and timescales of response latency, as observed experimentally. Our results indicate that adaptation in L6 can establish the thalamocortical dynamics that produce signatures of SSA and MMN and suggest a mechanistic model of novelty detection that could generalize to other sensory modalities.

Long-range connectome of pyramidal neurons in the sensorimotor cortex

The neocortex mediates information processing through highly organized circuitry that contains various neuron types. Distinct populations of projection neurons in different cortical regions and layers make specific connections and participate in distinct physiological functions. Herein, with the fluorescence micro-optical sectioning tomography (fMOST) and transgenetic mice that targeted intratelencephalic (IT) and pyramidal tract (PT) neurons at specific layers, we dissected the long-range connectome of pyramidal neurons in six subregions of the sensorimtor cortex. The distribution of the input neurons indicated that IT and PT neurons in the same region received information from similar regions, while the neurons in different subregions received from the preferred neuron populations. Both the input and projection areas of these six subregions showed the transverse and longitudinal correspondence in the cortico-cortical, cortico-thalamic, and cortico-striatal circuits, which indicated that the connections were topologically organized. This study provides a comprehensive resource on the anatomical connections of cortical circuits.

Enhanced Radiation-Sparing Effects of Ultra-High Dose Rate Proton Radiation (FLASH-RT) in a Human Induced Pluripotent Stem Cell-Derived Cerebral Organoid Model

<h3>Purpose/Objective(s)</h3> Superior normal tissue sparing with similar antitumoral effectiveness has been observed in preclinical animal models following delivery of radiation at ultra-high dose rates (FLASH-RT), relative to conventional dose rates (CONV-RT). Clinical application of FLASH-RT could reduce devastating radiation-related side effects for patients with aggressive brain tumors such as glioblastoma; however, the FLASH effect must be established in representative models of human systems before its implementation in the clinic. Our objective is to measure relevant response outcomes to proton FLASH-RT using advanced cerebral organoid (CO) models of normal and diseased human brain tissue, correspondingly generated with human induced pluripotent stem cells and patient-derived glioma stem-like cells (GSCs). <h3>Materials/Methods</h3> Mature COs were irradiated to 9 Gy using conventional (CONV, 0.2 Gy/s) and FLASH (100 Gy/s) dose rates using a proton beam and prepared for histological analysis at 30 days post-irradiation. Spatial distribution of hypoxia was visualized in COs by incubating for 2 hours with pimonidazole prior to preparation for histologic analysis. Image analysis was performed with QuPath. Lactate dehydrogenase (LDH) release, a marker of cell death, was sequentially quantified. COs implanted with GSCs expressing a luciferase reporter were co-cultured for 14 days and irradiated as above. Tumor cell proliferation was tracked via luciferase expression. <h3>Results</h3> At 2- and 4-days post-irradiation, there was an increase in LDH release for CONV versus FLASH-radiated COs (p<0.01, p<0.01 respectively) which normalized after 9 days. At 30 days, when specific cell populations in FLASH-radiated COs were histologically compared to CONV-radiated COs, there were higher numbers of proliferating neural progenitor cells (SOX2+/Ki67+, p<0.01), mature neurons (NeuN+, p=0.03), and deep cortical surface layer neurons (SATB2+, p=0.01). The spatial distribution of neural progenitor cells and mature neurons overlapped with hypoxic regions of the CO (pimonidazole+). A trend towards lower expression of activated microglia (IBA1+/CD68+) in FLASH-irradiated COs compared to CONV-radiated COs was noted (p=0.12). A 48.6% versus 51.2% reduction in tumor proliferation in FLASH versus CONV-irradiated GSC-laden-COs was observed at 4d post-irradiation (p=0.98). <h3>Conclusion</h3> We report preliminary findings of normal tissue toxicity reduction and iso-effective tumor response in a human-derived brain model following proton FLASH-RT. Potential protective effects following FLASH-RT to be further validated include preservation of specific cell populations within hypoxic niches including progenitor and differentiated neurons with a corresponding reduction in chronic neuroinflammation.

Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells

Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling.

0027 Reciprocal Modulation of Cortical Excitatory and Inhibitory Synapses by Wake-Sleep Homeostatic State

Abstract Introduction A widely debated function of sleep involves a homeostatic program of down-regulation of excitatory synaptic strength following an overall increase during the preceding waking period, preserving however the previously existing synaptic weights associated with newly acquired memories. We tested this hypothesis by applying thorough statistical analysis of parameters of excitatory and inhibitory miniature postsynaptic currents (mEPSC/mIPSC) recorded ex vivo in mouse cortical pyramidal neurons at three characteristic wake/sleep stages. Methods Cingulate cortex coronal slices were obtained at fixed Zeitgeber time (ZT6, to control for circadian clock) from control C57BL6 (MEF2C f/f) mice subjected to 6h acute sleep deprivation (SD), recovery sleep =4hSD+2h(RS), or 6h control sleep (CS). mEPSCs and mIPSCs were recorded from functionally identified whole-cell patch-clamped pyramidal neurons in cortical layer 2/3 (L2/3). Statistical analysis of frequencies, amplitudes, and charge transfer rates of mEPSCs and mIPSCs was done using non-parametric Kruskal-Wallis multiple comparison test and K-means clustering test. Results mEPSC frequency (F) and charge transfer (CT) were significantly reduced for RS and CS compared to SD (F: -57%, -47%; CT: -64%, -55%). mEPSC amplitude (A) was significantly reduced for CS compared to SD (-15%). Two-centroid clustering test revealed that analyzed parameters of F, A and CT for SD condition were approximately evenly split between upper and lower range clusters, while the same parameters for RS and CS conditions revealed a pronounced redistribution (&amp;gt;75% lower-, &amp;lt;25% upper ranges). Wake/sleep state related changes of mIPSC parameters showed opposite pattern compared to excitatory synapses. All three parameters were increased in RS vs. SD (F: +63%, A: +7%, CT: +42%) and this difference reached significance levels in CS vs. SD (F: +88%, A: +24%, CT: +109%). Clustering analysis of mIPSC parameters revealed mostly stable distribution pattern between upper and lower ranges for all wake/sleep states. Conclusion Significant changes in excitatory/inhibitory balance in the frontal cortex is part of the homeostatic response upon transition from wakefulness to various phases of sleep. The excitatory component prevails during wakefulness, while the inhibitory component peaks during sleep. Support (If Any) NIH grant R01 NS103422 to RWG.