Glial Control of Cortical Neuronal Circuit Maturation and Plasticity.

The brain is a highly adaptable organ that is molded by experience throughout life. Although the field of neuroscience has historically focused on intrinsic neuronal mechanisms of plasticity, there is growing evidence that multiple glial populations regulate the timing and extent of neuronal plasticity, particularly over the course of development. This review highlights recent discoveries on the role of glial cells in the establishment of cortical circuits and the regulation of experience-dependent neuronal plasticity during critical periods of neurodevelopment. These studies provide strong evidence that neuronal circuit maturation and plasticity are non-cell autonomous processes that require both glial-neuronal and glial-glial cross talk to proceed. We conclude by discussing open questions that will continue to guide research in this nascent field.

Glial cells undergo rapid changes following acute chemogenetic manipulation of cortical layer 5 projection neurons.

Bidirectional communication between neurons and glial cells is crucial to establishing and maintaining normal brain function. Some of these interactions are activity-dependent, yet it remains largely unexplored how acute changes in neuronal activity affect glial-to-neuron and neuron-to-glial dynamics. Here, we use excitatory and inhibitory designer receptors exclusively activated by designer drugs (DREADD) to study the effects of acute chemogenetic manipulations of a subpopulation of layer 5 cortical projection and dentate gyrus neurons in adult (Rbp4Cre) mouse brains. We show that acute chemogenetic neuronal activation reduces synaptic density, and increases microglia and astrocyte reactivity, but does not affect parvalbumin (PV+) neurons, only perineuronal nets (PNN). Conversely, acute silencing increases synaptic density and decreases glial reactivity. We show fast glial response upon clozapine-N-oxide (CNO) administration in cortical and subcortical regions. Together, our work provides evidence of fast, activity-dependent, bidirectional interactions between neurons and glial cells.

Lateral/caudal ganglionic eminence makes limited contribution to cortical oligodendrocytes.

The emergence of myelinating oligodendrocytes represents a pivotal developmental milestone in vertebrates, given their capacity to ensheath axons and facilitate the swift conduction of action potentials. It is widely accepted that cortical oligodendrocyte progenitor cells (OPCs) arise from medial ganglionic eminence (MGE), lateral/caudal ganglionic eminence (LGE/CGE), and cortical radial glial cells (RGCs). Here, we used two different fate mapping strategies to challenge the established notion that the LGE generates cortical OPCs. Furthermore, we used a Cre/loxP-dependent exclusion strategy to reveal that the LGE/CGE does not give rise to cortical OPCs. Additionally, we showed that specifically eliminating MGE-derived OPCs leads to a significant reduction of cortical OPCs. Together, our findings indicate that the LGE does not generate cortical OPCs, contrary to previous beliefs. These findings provide a new view of the developmental origins of cortical OPCs and a valuable foundation for future research on both normal development and oligodendrocyte-related disease.

Sub-threshold neuronal activity and the dynamical regime of cerebral cortex.

Cortical neurons exhibit temporally irregular spiking patterns and heterogeneous firing rates. These features arise in model circuits operating in a 'fluctuation-driven regime', in which fluctuations in membrane potentials emerge from the network dynamics. However, it is still debated whether the cortex operates in such a regime. We evaluated the fluctuation-driven hypothesis by analyzing spiking and sub-threshold membrane potentials of neurons in the frontal cortex of mice performing a decision-making task. We showed that while standard fluctuation-driven models successfully account for spiking statistics, they fall short in capturing the heterogeneity in sub-threshold activity. This limitation is an inevitable outcome of bombarding single-compartment neurons with a large number of pre-synaptic inputs, thereby clamping the voltage of all neurons to more or less the same average voltage. To address this, we effectively incorporated dendritic morphology into the standard models. Inclusion of dendritic morphology in the neuronal models increased neuronal selectivity and reduced error trials, suggesting a functional role for dendrites during decision-making. Our work suggests that, during decision-making, cortical neurons in high-order cortical areas operate in a fluctuation-driven regime.

Phosphorylation of the DNA damage repair factor 53BP1 by ATM kinase controls neurodevelopmental programs in cortical brain organoids.

53BP1 is a well-established DNA damage repair factor that has recently emerged to critically regulate gene expression for tumor suppression and neural development. However, its precise function and regulatory mechanisms remain unclear. Here, we showed that phosphorylation of 53BP1 at serine 25 by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical brain organoids. Dynamic phosphorylation of 53BP1-serine 25 controls 53BP1 target genes governing neuronal differentiation and function, cellular response to stress, and apoptosis. Mechanistically, ATM and RNF168 govern 53BP1's binding to gene loci to directly affect gene regulation, especially at genes for neuronal differentiation and maturation. 53BP1 serine 25 phosphorylation effectively impedes its binding to bivalent or H3K27me3-occupied promoters, especially at genes regulating H3K4 methylation, neuronal functions, and cell proliferation. Beyond 53BP1, ATM-dependent phosphorylation displays wide-ranging effects, regulating factors in neuronal differentiation, cytoskeleton, p53 regulation, as well as key signaling pathways such as ATM, BDNF, and WNT during cortical organoid differentiation. Together, our data suggest that the interplay between 53BP1 and ATM orchestrates essential genetic programs for cell morphogenesis, tissue organization, and developmental pathways crucial for human cortical development.

Distinct codes for environment structure and symmetry in postrhinal and retrosplenial cortices.

Complex sensory information arrives in the brain from an animal's first-person ('egocentric') perspective. However, animals can efficiently navigate as if referencing map-like ('allocentric') representations. The postrhinal (POR) and retrosplenial (RSC) cortices are thought to mediate between sensory input and internal maps, combining egocentric representations of physical cues with allocentric head direction (HD) information. Here we show that neurons in the POR and RSC of female Long-Evans rats are tuned to distinct but complementary aspects of local space. Egocentric bearing (EB) cells recorded in square and L-shaped environments reveal that RSC cells encode local geometric features, while POR cells encode a more global account of boundary geometry. Additionally, POR HD cells can incorporate egocentric information to fire in two opposite directions with two oppositely placed identical visual landmarks, while only a subset of RSC HD cells possess this property. Entorhinal grid and HD cells exhibit consistently allocentric spatial firing properties. These results reveal significant regional differences in the neural encoding of spatial reference frames.

TRPM4 inhibition slows neuritogenesis progression of cortical neurons.

TRPM4 is a non-selective cation channel activated by intracellular Ca2+ but only permeable to monovalent cations, its activation regulates membrane potential and intracellular calcium. This channel participates in the migration and adhesion of non-excitable cells and forms an integral part of the focal adhesion complex. In neurons, TRPM4 expression starts before birth and its function at this stage is not clear, but it may function in processes such as neurite development. Here we investigate the role of TRPM4 in neuritogenesis. We found that neurons at DIV 0 express TRPM4, the inhibition of TRPM4 using 9-Ph reduces neurite number and slows the progression of neurite development, keeping neurons in stage 1. The genetic suppression of TRPM4 using an shRNA at later stages (DIV2) reduces neurite length. Conversely, at DIV 0, TRPM4 inhibition augments the Cch-induced Ca2 + i increase, altering the calcium homeostasis. Together, these results show that TRPM4 participates in progression of neurite development and suggest a critical role of the calcium modulation during this stage of neuronal development.

Near-infrared laser diode mitigates Aß1-42-induced neurodegeneration in cortical neurons.

Alzheimer's disease, a prevalent neurodegenerative condition primarily affecting older adults, remains incurable. Its principle pathological hallmark is the accelerated accumulation of amyloid ß (Aß) protein. This study investigates the potential of photobiomodulation using near infrared light to counteract Aß1-42-induced synaptic degeneration and neurotoxicity. We focused on the effect of 808 nm near-infrared laser diode (LD) on Aß1-42 cytotoxicity in primary cultured cortical neurons. We assessed cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, observing substantial benefits from LD irradiation with a power of 10 mW and a dose of 30 J. Cells exposed to Aß1-42 exhibited morphological changes indicative of synaptic damage and a significant decrease in the number of postsynaptic density protein-95 (PSD-95) contacts, which were significantly improved with near-infrared LD therapy. Furthermore, this therapy reduced Aß and phosphorylated tau (P-tau) protein accumulation. Additionally, near-infrared LD irradiation substantially lessened the Aß1-42-induced rise in glial fibrillary acid protein (GFAP) and ionized calcium-binding adaptor molecule 1 (IBA1) in astrocytes and microglia. Remarkably, near-infrared LD irradiation effectively inhibited phosphorylation of key proteins involved in Aß1-42-induced necroptosis, namely Receptor-interacting protein kinase-3 (RIP3) and Mixed Lineage Kinase domain-Like protein (MLKL). Our findings suggest that near-infrared LD treatment significantly reduces neurodegeneration by reducing glial overactivation and neuronal necroptosis triggered by Aß1-42. Thus, near-infrared LD treatment emerges as a promising approach for slowing or treating Alzheimer's disease, offering new avenues in its management.

Protein Kinase C-Delta Mediates Cell Cycle Reentry and Apoptosis Induced by Amyloid-Beta Peptide in Post-Mitotic Cortical Neurons.

Amyloid-beta peptide (Aß) is a neurotoxic constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. The detailed mechanisms by which protein kinase C-delta (PKCδ) contributes to Aß toxicity is not yet entirely understood. Using fully differentiated primary rat cortical neurons, we found that inhibition of Aß25-35-induced PKCδ increased cell viability with restoration of neuronal morphology. Using cyclin D1, proliferating cell nuclear antigen (PCNA), and histone H3 phosphorylated at Ser-10 (p-Histone H3) as the respective markers for the G1-, S-, and G2/M-phases, PKCδ inhibition mitigated cell cycle reentry (CCR) and subsequent caspase-3 cleavage induced by both Aß25-35 and Aß1-42 in the post-mitotic cortical neurons. Upstream of PKCδ, signal transducers and activators of transcription (STAT)-3 mediated PKCδ induction, CCR, and caspase-3 cleavage upon Aß exposure. Downstream of PKCδ, aberrant neuronal CCR was triggered by overactivating cyclin-dependent kinase-5 (CDK5) via calpain2-dependent p35 cleavage into p25. Finally, PKCδ and CDK5 also contributed to Aß25-35 induction of p53-upregulated modulator of apoptosis (PUMA) in cortical neurons. Together, we demonstrated that, in the post-mitotic neurons exposed to Aßs, STAT3-dependent PKCδ expression triggers calpain2-mediated p35 cleavage into p25 to overactivate CDK5, thus leading to aberrant CCR, PUMA induction, caspase-3 cleavage, and ultimately apoptosis.

DNA methylation controls stemness of astrocytes in health and ischaemia.

Astrocytes are the most abundant cell type in the mammalian brain and provide structural and metabolic support to neurons, regulate synapses and become reactive after injury and disease. However, a small subset of astrocytes settles in specialized areas of the adult brain where these astrocytes instead actively generate differentiated neuronal and glial progeny and are therefore referred to as neural stem cells1-3. Common parenchymal astrocytes and quiescent neural stem cells share similar transcriptomes despite their very distinct functions4-6. Thus, how stem cell activity is molecularly encoded remains unknown. Here we examine the transcriptome, chromatin accessibility and methylome of neural stem cells and their progeny, and of astrocytes from the striatum and cortex in the healthy and ischaemic adult mouse brain. We identify distinct methylation profiles associated with either astrocyte or stem cell function. Stem cell function is mediated by methylation of astrocyte genes and demethylation of stem cell genes that are expressed later. Ischaemic injury to the brain induces gain of stemness in striatal astrocytes7. We show that this response involves reprogramming the astrocyte methylome to a stem cell methylome and is absent if the de novo methyltransferase DNMT3A is missing. Overall, we unveil DNA methylation as a promising target for regenerative medicine.

Lrig1 regulates cell fate specification of glutamatergic neurons via FGF-driven Jak2/Stat3 signaling in cortical progenitors.

The cell-intrinsic mechanisms underlying the decision of a stem/progenitor cell to either proliferate or differentiate remain incompletely understood. Here, we identify the transmembrane protein Lrig1 as a physiological homeostatic regulator of FGF2-driven proliferation and self-renewal of neural progenitors at early-to-mid embryonic stages of cortical development. We show that Lrig1 is expressed in cortical progenitors (CPs), and its ablation caused expansion and increased proliferation of radial/apical progenitors and of neurogenic transit-amplifying Tbr2+ intermediate progenitors. Notably, our findings identify a previously unreported EGF-independent mechanism through which Lrig1 negatively regulates neural progenitor proliferation by modulating the FGF2-induced IL6/Jak2/Stat3 pathway, a molecular cascade that plays a pivotal role in the generation and maintenance of CPs. Consistently, Lrig1 knockout mice showed a significant increase in the density of pyramidal glutamatergic neurons placed in superficial layers 2 and 3 of the postnatal neocortex. Together, these results support a model in which Lrig1 regulates cortical neurogenesis by influencing the cycling activity of a set of progenitors that are temporally specified to produce upper layer glutamatergic neurons.

Dysregulated Purinergic Signalling in Fragile X Syndrome Cortical Astrocytes.

The symptoms of fragile X syndrome (FXS), caused by a single gene mutation to Fmr1, have been increasingly linked to disordered astrocyte signalling within the cerebral cortex. We have recently demonstrated that the purinergic signalling pathway, which utilizes nucleoside triphosphates and their metabolites to facilitate bidirectional glial and glial-neuronal interactions, is upregulated in cortical astrocytes derived from the Fmr1 knockout (KO) mouse model of FXS. Heightened Fmr1 KO P2Y purinergic receptor levels were correlated with prolonged intracellular calcium release, elevated synaptogenic protein secretion, and hyperactivity of developing circuits. However, due to the relative lack of sensitive and reproducible quantification methods available for measuring purines and pyrimidines, determining the abundance of these factors in Fmr1 KO astrocytes was limited. We therefore developed a hydrophilic interaction liquid chromatography protocol coupled with mass spectrometry to compare the abundance of intracellular and extracellular purinergic molecules between wildtype and Fmr1 KO mouse astrocytes. Significant differences in the concentrations of UDP, ATP, AMP, and adenosine intracellular stores were found within Fmr1 KO astrocytes relative to WT. The extracellular level of adenosine was also significantly elevated in Fmr1 KO astrocyte-conditioned media in comparison to media collected from WT astrocytes. Glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase, which facilitates ligand breakdown following synaptic release, was also elevated in Fmr1 KO astrocyte cultures. Together, these differences demonstrated further dysregulation of the purinergic signalling system within Fmr1 KO cortical astrocytes, potentially leading to significant alterations in FXS purinergic receptor activation and cellular pathology.

Differential stability of task variable representations in retrosplenial cortex.

Cortical neurons store information across different timescales, from seconds to years. Although information stability is variable across regions, it can vary within a region as well. Association areas are known to multiplex behaviorally relevant variables, but the stability of their representations is not well understood. Here, we longitudinally recorded the activity of neuronal populations in the mouse retrosplenial cortex (RSC) during the performance of a context-choice association task. We found that the activity of neurons exhibits different levels of stability across days. Using linear classifiers, we quantified the stability of three task-relevant variables. We find that RSC representations of context and trial outcome display higher stability than motor choice, both at the single cell and population levels. Together, our findings show an important characteristic of association areas, where diverse streams of information are stored with varying levels of stability, which may balance representational reliability and flexibility according to behavioral demands.

The BDNF-ERK/MAPK axis reduces phosphatase and actin regulator1, 2 and 3 (PHACTR1, 2 and 3) mRNA expressions in cortical neurons.

Actin rearrangement and phosphorylation-dephosphorylation in the nervous system contribute to plastic alteration of neuronal structure and function. Phosphatase and actin regulator (PHACTR) family members are actin- and protein phosphatase 1 (PP1)-binding proteins. Because some family members act as regulators of neuronal morphology, studying the regulatory mechanisms of PHACTR is valuable for understanding the basis of neuronal circuit formation. Although expression patterns of PHACTR family molecules (PHACTR1-4) vary across distinct brain areas, little is known about the extracellular ligands that influence their mRNA levels. In this study, we focused on an important neurotrophin, brain-derived neurotrophic factor (BDNF), and examined its effect on mRNA expression of PHACTR family member in cortical neurons. PHACTR1-3, but not PHACTR4, were affected by stimulation of primary cultured cortical neurons with BDNF; namely, sustained downregulation of their mRNA levels was observed. The observed downregulation was blocked by an inhibitor of the extracellular signal-regulated protein kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, U0126, suggesting that ERK/MAPK plays an inhibitory role for gene induction of PHACTR1-3. These findings aid the elucidation of how BDNF regulates actin- and PP1-related neuronal functions.

GET73 modulates lipopolysaccharide- and ethanol-induced increase in cytokine/chemokine levels in primary cultures of microglia of rat cerebral cortex.

BACKGROUND: - Alcohol-induced pro-inflammatory activation might influence cellular and synaptic pathology, thus contributing to the behavioral phenotypes associated with alcohol use disorders. In the present study, the possible anti-inflammatory properties of N-[(4-trifluoromethyl)-benzyl]4-methoxybutyramide (GET73), a promising therapeutic agent for alcohol use disorder treatment, were evaluated in primary cultures of rat cortical microglia. METHODS: - Primary cultures of cerebral cortex microglial cells were treated with 100 ng/ml lipopolysaccharide (LPS; 8 h, 37 °C) or 75 mM ethanol (EtOH; 4 days, 37 °C) alone or in the presence of GET73 (1-30 µM). At the end of the incubation period, multiparametric quantification of cytokines/chemokines was performed by using the xMAP technology and Luminex platform. Furthermore, cultured microglial cell viability following the treatment with EtOH and GET73, alone or in combination, has been measured by a colorimetric assay (i.e. MTT assay). RESULTS: - GET73 (10 and 30 µM) partially or fully prevented the LPS-induced increase of IL-6, IL-1ß, RANTES/CCL5 protein and MCP-1/CCL2 levels. On the contrary, GET73 failed to attenuate the TNF-α level increase induced by LPS. Furthermore, GET73 treatment (10-30 µM) significantly attenuated or prevented the EtOH-induced increase of TNF-α, IL-6, IL-1ß and MCP-1/CCL2 levels. Finally, at all the concentrations tested (1-30 µM), the GET73 treatment did not alter the EtOH-induced reduction of microglial cell viability. CONCLUSIONS: - The current results provide the first in vitro evidence of GET73 protective properties against EtOH-induced neuroinflammation. These data add more information on the complex and multifactorial profile of action of the compound, further supporting the significance of developing GET73 as a therapeutic tool for the treatment of individuals with alcohol use disorders.

The effects of light emitting diodes on mitochondrial function and cellular viability of M-1 cell and mouse CD1 brain cortex neurons.

The invention of Light Emitting Diode (LED) revolutionized energy-efficient illumination, but concerns persist regarding the potential harm of blue light to our eyes. In this study, we scrutinized the impact of LED light characteristics on eyes using two cell types: M-1 (rich in mitochondria) and CD-1 (neuronal). Variations in color rendering index (CRI) and correlated color temperature (CCT) were investigated, alongside exposure durations ranging from 0 to 24 hours. The findings illuminated the potential benefits of high-quality LED lighting, characterized by a high CRI and low CCT, which emits a greater proportion of red light. This form of lighting was associated with enhanced cell proliferation, elevated ATP levels, and reduced oxidative stress. In contrast, LEDs with low CRI and high CCT exhibited adverse effects, diminishing cell viability and increasing oxidative stress. These results suggest that high-quality LED lighting may have neuroprotective potential as a treatment option, such as for retinal ganglion cells.

Reconsidering Neurogenetic Indication in the Human Brain: Broad Expression of Doublecortin Transcript in the Hippocampal and Cortical Cell Populations.

INTRODUCTION: Neurogenesis in the adult brain may play an important role in memory and cognition; however, knowledge of neurogenic markers in the human brain remains limited. We compared the single-nucleus transcriptome of the hippocampus with that of other cortical regions to identify hippocampus-specific neurogenic markers. METHODS: We analyzed 26,189 nuclei from four human brains collected within 16 h of death. Clustering and annotation were performed to examine differential expression, gene ontology, and intercellular communication. DCX expression was validated by ddPCR. RESULTS: Immature markers such as DCX, CALB2, NES, SOX2, PAX6, DPYSL3, and TUBB3 were expressed in both hippocampus and prefrontal cortex, with higher levels in the prefrontal cortex. ddPCR confirmed higher expression of DCX in the prefrontal cortex. DCX was involved in both neurogenesis and neuroprotection pathways. CONCLUSION: Neurogenic markers are not definitive indicators of adult neurogenesis as their roles are more complex than previously understood.

SYNGAP1 deficiency disrupts synaptic neoteny in xenotransplanted human cortical neurons in vivo.

Human brain ontogeny is characterized by a considerably prolonged neotenic development of cortical neurons and circuits. Neoteny is thought to be essential for the acquisition of advanced cognitive functions, which are typically altered in intellectual disability (ID) and autism spectrum disorders (ASDs). Human neuronal neoteny could be disrupted in some forms of ID and/or ASDs, but this has never been tested. Here, we use xenotransplantation of human cortical neurons into the mouse brain to model SYNGAP1 haploinsufficiency, one of the most prevalent genetic causes of ID/ASDs. We find that SYNGAP1-deficient human neurons display strong acceleration of morphological and functional synaptic formation and maturation alongside disrupted synaptic plasticity. At the circuit level, SYNGAP1-haploinsufficient neurons display precocious acquisition of responsiveness to visual stimulation months ahead of time. Our findings indicate that SYNGAP1 is required cell autonomously for human neuronal neoteny, providing novel links between human-specific developmental mechanisms and ID/ASDs.

Protocol for generating human cortical organoids enriched in outer radial glia by guided differentiation.

The generation of human pluripotent stem cell (hPSC)-derived brain organoids is continuously refined, enhancing their reproducibility and complexity. Here, we present a guided differentiation protocol for generating cortical forebrain organoids and cortico-pericyte (CP)assembloids composed of a robust outer radial glia (oRG) population and an expanded outer subventricular zone (oSVZ). We describe the steps to generate hPSC-derived cortical organoids (COs), cortical pericytes, and CP assembloids. Moreover, we outline the procedures to characterize the organoids by immunostaining and to perform single-cell dissociation. For complete details on the use and execution of this protocol, please refer to Walsh et al.1.

Early neuronal inhibition sculpts adult cortical interhemispheric connectivity.

The maturation of cerebral cortical networks during early life involves a major reorganization of long-range axonal connections. In a recent study, Bragg-Gonzalo, Aguilera, et al. discovered that in mice, the interhemispheric connections sent by S1L4 callosal projection neurons are pruned via the tight control of their ipsilateral synaptic integration, which relies on the early activity of specific interneurons.