Reuniens thalamus recruits recurrent excitation in medial prefrontal cortex.

Medial prefrontal cortex (mPFC) and hippocampus are critical for memory retrieval, decision making and emotional regulation. While ventral CA1 (vCA1) shows direct and reciprocal connections with mPFC, dorsal CA1 (dCA1) forms indirect pathways to mPFC, notably via the thalamic Reuniens nucleus (Re). Neuroanatomical tracing has documented structural connectivity of this indirect pathway through Re however, its functional operation is largely unexplored. Here we used in vivo and in vitro electrophysiology along with optogenetics to address this question. Whole-cell patch-clamp recordings in acute mouse brain slices revealed both monosynaptic excitatory responses and disynaptic feedforward inhibition for both Re-mPFC and Re-dCA1 pathways. However, we also identified a novel biphasic excitation of mPFC by Re, but not dCA1. These early monosynaptic and late recurrent components are in marked contrast to the primarily feedforward inhibition characteristic of thalamic inputs to neocortex. Local field potential recordings in mPFC brain slices revealed that this biphasic excitation propagates throughout all cortical lamina, with the late excitation specifically enhanced by GABAAR blockade. In vivo Neuropixels recordings in head-fixed awake mice revealed a similar biphasic excitation of mPFC units by Re activation. In summary, Re output produces recurrent feed-forward excitation within mPFC suggesting a potent amplification system in the Re-mPFC network. This may facilitate amplification of dCA1->mPFC signals for which Re acts as the primary conduit, as there is little direct connectivity. In addition, the capacity of mPFC neurons to fire bursts of action potentials in response to Re input suggests that these synapses have a high gain. Significance statement: The interactions between medial prefrontal cortex and hippocampus are crucial for memory formation and retrieval. Yet, it is still poorly understood how the functional connectivity of direct and indirect pathways underlies these functions. This research explores the synaptic connectivity of the indirect pathway through the Reuniens nucleus of the thalamus using electrophysiological recordings and optogenetic manipulations. The study found that Reuniens stimulation recruits recurrent and long-lasting activity in mPFC - a phenomenon not previously recorded. This recurrent activity might create a temporal window ideal for coincidence detection and be an underlying mechanism for memory formation and retrieval.

Atlas of the aging mouse brain reveals white matter as vulnerable foci.

Aging is the key risk factor for cognitive decline, yet the molecular changes underlying brain aging remain poorly understood. Here, we conducted spatiotemporal RNA sequencing of the mouse brain, profiling 1,076 samples from 15 regions across 7 ages and 2 rejuvenation interventions. Our analysis identified a brain-wide gene signature of aging in glial cells, which exhibited spatially defined changes in magnitude. By integrating spatial and single-nucleus transcriptomics, we found that glial aging was particularly accelerated in white matter compared with cortical regions, whereas specialized neuronal populations showed region-specific expression changes. Rejuvenation interventions, including young plasma injection and dietary restriction, exhibited distinct effects on gene expression in specific brain regions. Furthermore, we discovered differential gene expression patterns associated with three human neurodegenerative diseases, highlighting the importance of regional aging as a potential modulator of disease. Our findings identify molecular foci of brain aging, providing a foundation to target age-related cognitive decline.

A CMOS-based highly scalable flexible neural electrode interface.

Perception, thoughts, and actions are encoded by the coordinated activity of large neuronal populations spread over large areas. However, existing electrophysiological devices are limited by their scalability in capturing this cortex-wide activity. Here, we developed an electrode connector based on an ultra-conformable thin-film electrode array that self-assembles onto silicon microelectrode arrays enabling multithousand channel counts at a millimeter scale. The interconnects are formed using microfabricated electrode pads suspended by thin support arms, termed Flex2Chip. Capillary-assisted assembly drives the pads to deform toward the chip surface, and van der Waals forces maintain this deformation, establishing Ohmic contact. Flex2Chip arrays successfully measured extracellular action potentials ex vivo and resolved micrometer scale seizure propagation trajectories in epileptic mice. We find that seizure dynamics in absence epilepsy in the Scn8a+/- model do not have constant propagation trajectories.

Prefrontal PV interneurons facilitate attention and are linked to attentional dysfunction in a mouse model of absence epilepsy.

Absence seizures are characterized by brief periods of unconsciousness accompanied by lapses in motor function that can occur hundreds of times throughout the day. Outside of these frequent moments of unconsciousness, approximately a third of people living with the disorder experience treatment-resistant attention impairments. Convergent evidence suggests prefrontal cortex (PFC) dysfunction may underlie attention impairments in affected patients. To examine this, we use a combination of slice physiology, fiber photometry, electrocorticography (ECoG), optogenetics, and behavior in the Scn8a+/-mouse model of absence epilepsy. Attention function was measured using a novel visual attention task where a light cue that varied in duration predicted the location of a food reward. In Scn8a+/-mice, we find altered parvalbumin interneuron (PVIN) output in the medial PFC (mPFC) in vitro and PVIN hypoactivity along with reductions in gamma power during cue presentation in vivo. This was associated with poorer attention performance in Scn8a+/-mice that could be rescued by gamma-frequency optogenetic stimulation of PVINs. This highlights cue-related PVIN activity as an important mechanism for attention and suggests PVINs may represent a therapeutic target for cognitive comorbidities in absence epilepsy.

People who experience absence seizures may go through brief lapses in consciousness hundreds of times a day. They also often have difficulties engaging and remaining focused on a task, which can severely limit their ability to study, work and go through their day-to-day life. These impairments in attention persist even when medication puts a stop to the seizures, suggesting that they are not directly linked to the epileptic episodes. In fact, recent work has indicated that these deficits may be caused instead by alterations in the activity of the prefrontal cortex, the brain area which helps to regulate attention and impulsivity. However, the exact nature of these changes remains unclear, making it difficult to design treatments that could improve patients' quality of life. To explore this question, Ferguson et al. developed a new behavioral test that allowed them to measure the attention levels of mice genetically engineered to have absence seizures. The experiments confirmed that these animals had impaired attention even when brain activity recordings showed that they were not experiencing seizures. Further work revealed that poor performance on the behavioral test was linked to decreased activity in parvalbumin interneurons, a group of cells in the prefrontal cortex which can inhibit many other types of neurons. In mutant mice, this change was associated with alterations in network activity broadly in the cortex, including in electrical patterns which are linked to cognitive processes. Promisingly, increasing the activity of the interneurons during the attention task improved performance, suggesting that this type of cell could represent a therapeutic target for attention deficit in absence epilepsy.

Loss of Rai1 enhances hippocampal excitability and epileptogenesis in mouse models of Smith-Magenis syndrome.

Hyperexcitability of brain circuits is a common feature of autism spectrum disorders (ASDs). Genetic deletion of a chromatin-binding protein, retinoic acid induced 1 (RAI1), causes Smith-Magenis syndrome (SMS). SMS is a syndromic ASD associated with intellectual disability, autistic features, maladaptive behaviors, overt seizures, and abnormal electroencephalogram (EEG) patterns. The molecular and neural mechanisms underlying abnormal brain activity in SMS remain unclear. Here we show that panneural Rai1 deletions in mice result in increased seizure susceptibility and prolonged hippocampal seizure duration in vivo and increased dentate gyrus population spikes ex vivo. Brain-wide mapping of neuronal activity pinpointed selective cell types within the limbic system, including the hippocampal dentate gyrus granule cells (dGCs) that are hyperactivated by chemoconvulsant administration or sensory experience in Rai1-deficient brains. Deletion of Rai1 from glutamatergic neurons, but not from gamma-aminobutyric acidergic (GABAergic) neurons, was responsible for increased seizure susceptibility. Deleting Rai1 from the Emx1Cre-lineage glutamatergic neurons resulted in abnormal dGC properties, including increased excitatory synaptic transmission and increased intrinsic excitability. Our work uncovers the mechanism of neuronal hyperexcitability in SMS by identifying Rai1 as a negative regulator of dGC intrinsic and synaptic excitability.

Maturation and circuit integration of transplanted human cortical organoids.

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

Maladaptive myelination promotes generalized epilepsy progression.

Activity-dependent myelination can fine-tune neural network dynamics. Conversely, aberrant neuronal activity, as occurs in disorders of recurrent seizures (epilepsy), could promote maladaptive myelination, contributing to pathogenesis. In this study, we tested the hypothesis that activity-dependent myelination resulting from absence seizures, which manifest as frequent behavioral arrests with generalized electroencephalography (EEG) spike-wave discharges, promote thalamocortical network hypersynchrony and contribute to epilepsy progression. We found increased oligodendrogenesis and myelination specifically within the seizure network in two models of generalized epilepsy with absence seizures (Wag/Rij rats and Scn8a+/mut mice), evident only after epilepsy onset. Aberrant myelination was prevented by pharmacological seizure inhibition in Wag/Rij rats. Blocking activity-dependent myelination decreased seizure burden over time and reduced ictal synchrony as assessed by EEG coherence. These findings indicate that activity-dependent myelination driven by absence seizures contributes to epilepsy progression; maladaptive myelination may be pathogenic in some forms of epilepsy and other neurological diseases.

Precise spatiotemporal control of voltage-gated sodium channels by photocaged saxitoxin.

Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.

NF1 mutation drives neuronal activity-dependent initiation of optic glioma.

Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours1. Although the role of neurons in tumour progression has previously been demonstrated2, the importance of neuronal activity to tumour initiation is less clear-particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with the neurofibromatosis 1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood3,4, raising  the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis 1 tumour suppressor gene (Nf1)5 to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin 3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome.

Long-term maturation of human cortical organoids matches key early postnatal transitions.

Human stem-cell-derived models provide the promise of accelerating our understanding of brain disorders, but not knowing whether they possess the ability to mature beyond mid- to late-fetal stages potentially limits their utility. We leveraged a directed differentiation protocol to comprehensively assess maturation in vitro. Based on genome-wide analysis of the epigenetic clock and transcriptomics, as well as RNA editing, we observe that three-dimensional human cortical organoids reach postnatal stages between 250 and 300 days, a timeline paralleling in vivo development. We demonstrate the presence of several known developmental milestones, including switches in the histone deacetylase complex and NMDA receptor subunits, which we confirm at the protein and physiological levels. These results suggest that important components of an intrinsic in vivo developmental program persist in vitro. We further map neurodevelopmental and neurodegenerative disease risk genes onto in vitro gene expression trajectories to provide a resource and webtool (Gene Expression in Cortical Organoids, GECO) to guide disease modeling.

Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel.

CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.

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

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

Nonlinearities between inhibition and T-type calcium channel activity bidirectionally regulate thalamic oscillations.

Absence seizures result from 3 to 5 Hz generalized thalamocortical oscillations that depend on highly regulated inhibitory neurotransmission in the thalamus. Efficient reuptake of the inhibitory neurotransmitter GABA is essential, and reuptake failure worsens human seizures. Here, we show that blocking GABA transporters (GATs) in acute rat brain slices containing key parts of the thalamocortical seizure network modulates epileptiform activity. As expected, we found that blocking either GAT1 or GAT3 prolonged oscillations. However, blocking both GATs unexpectedly suppressed oscillations. Integrating experimental observations into single-neuron and network-level computational models shows how a non-linear dependence of T-type calcium channel gating on GABAB receptor activity regulates network oscillations. Receptor activity that is either too brief or too protracted fails to sufficiently open T-type channels necessary for sustaining oscillations. Only within a narrow range does prolonging GABAB receptor activity promote channel opening and intensify oscillations. These results have implications for therapeutics that modulate inhibition kinetics.

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

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

Reliability of human cortical organoid generation.

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

Shank and Zinc Mediate an AMPA Receptor Subunit Switch in Developing Neurons.

During development, pyramidal neurons undergo dynamic regulation of AMPA receptor (AMPAR) subunit composition and density to help drive synaptic plasticity and maturation. These normal developmental changes in AMPARs are particularly vulnerable to risk factors for Autism Spectrum Disorders (ASDs), which include loss or mutations of synaptic proteins and environmental insults, such as dietary zinc deficiency. Here, we show how Shank2 and Shank3 mediate a zinc-dependent regulation of AMPAR function and subunit switch from GluA2-lacking to GluA2-containing AMPARs. Over development, we found a concomitant increase in Shank2 and Shank3 with GluA2 at synapses, implicating these molecules as potential players in AMPAR maturation. Since Shank activation and function require zinc, we next studied whether neuronal activity regulated postsynaptic zinc at glutamatergic synapses. Zinc was found to increase transiently and reversibly with neuronal depolarization at synapses, which could affect Shank and AMPAR localization and activity. Elevated zinc induced multiple functional changes in AMPAR, indicative of a subunit switch. Specifically, zinc lengthened the decay time of AMPAR-mediated synaptic currents and reduced their inward rectification in young hippocampal neurons. Mechanistically, both Shank2 and Shank3 were necessary for the zinc-sensitive enhancement of AMPAR-mediated synaptic transmission and act in concert to promote removal of GluA1 while enhancing recruitment of GluA2 at pre-existing Shank puncta. These findings highlight a cooperative local dynamic regulation of AMPAR subunit switch controlled by zinc signaling through Shank2 and Shank3 to shape the biophysical properties of developing glutamatergic synapses. Given the zinc sensitivity of young neurons and its dependence on Shank2 and Shank3, genetic mutations and/or environmental insults during early development could impair synaptic maturation and circuit formation that underlie ASD etiology.

Assembly of functionally integrated human forebrain spheroids.

The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.