A model of human neural networks reveals NPTX2 pathology in ALS and FTLD.

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1, which involve human-specific mechanisms2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors6. Single-cell transcriptomics and comparison to independent neural stem cells7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids8. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.

Presynaptic Kainate Receptors onto Somatostatin Interneurons Are Recruited by Activity throughout Development and Contribute to Cortical Sensory Adaptation.

Somatostatin (SST) interneurons produce delayed inhibition because of the short-term facilitation of their excitatory inputs created by the expression of metabotropic glutamate receptor 7 (mGluR7) and presynaptic GluK2-containing kainate receptors (GluK2-KARs). Using mice of both sexes, we find that as synaptic facilitation at layer (L)2/3 SST cell inputs increases during the first few postnatal weeks, so does GluK2-KAR expression. Removal of sensory input by whisker trimming does not affect mGluR7 but prevents the emergence of presynaptic GluK2-KARs, which can be restored by allowing whisker regrowth or by acute calmodulin activation. Conversely, late trimming or acute inhibition of Ca2+/calmodulin-dependent protein kinase II is sufficient to reduce GluK2-KAR activity. This developmental and activity-dependent regulation also produces a specific reduction of L4 GluK2-KARs that advances in parallel with the maturation of sensory processing in L2/3. Finally, we find that removal of both GluK2-KARs and mGluR7 from the synapse eliminates short-term facilitation and reduces sensory adaptation to repetitive stimuli, first in L4 of somatosensory cortex, then later in development in L2/3. The dynamic regulation of presynaptic GluK2-KARs potentially allows for flexible scaling of late inhibition and sensory adaptation.SIGNIFICANCE STATEMENT Excitatory synapses onto somatostatin (SST) interneurons express presynaptic, calcium-permeable kainate receptors containing the GluK2 subunit (GluK2-KARs), activated by high-frequency activity. In this study we find that their presence on L2/3 SST synapses in the barrel cortex is not based on a hardwired genetic program but instead is regulated by sensory activity, in contrast to that of mGluR7. Thus, in addition to standard synaptic potentiation and depression mechanisms, excitatory synapses onto SST neurons undergo an activity-dependent presynaptic modulation that uses GluK2-KARs. Further, we present evidence that loss of the frequency-dependent synaptic components (both GluK2-KARs and mGluR7 via Elfn1 deletion) contributes to a decrease in the sensory adaptation commonly seen on repetitive stimulus presentation.

Identification of a Developmental Switch in Information Transfer between Whisker S1 and S2 Cortex in Mice.

The whiskers of rodents are a key sensory organ that provides critical tactile information for animal navigation and object exploration throughout life. Previous work has explored the developmental sensory-driven activation of the primary sensory cortex processing whisker information (wS1), also called barrel cortex. This body of work has shown that the barrel cortex is already activated by sensory stimuli during the first postnatal week. However, it is currently unknown when over the course of development these stimuli begin being processed by higher-order cortical areas, such as secondary whisker somatosensory area (wS2). Here we investigate the developmental engagement of wS2 by whisker stimuli and the emergence of corticocortical communication from wS1 to wS2. Using in vivo wide-field imaging and multielectrode recordings in control and conditional KO mice of either sex with thalamocortical innervation defects, we find that wS1 and wS2 are able to process bottom-up information coming from the thalamus from birth. We also identify that it is only at the end of the first postnatal week that wS1 begins to provide functional excitation into wS2, switching to more inhibitory actions after the second postnatal week. Therefore, we have uncovered a developmental window when information transfer between wS1 and wS2 reaches mature function.SIGNIFICANCE STATEMENT At the end of the first postnatal week, the primary whisker somatosensory area starts providing excitatory input to the secondary whisker somatosensory area 2. This excitatory drive weakens during the second postnatal week and switches to inhibition in the adult.

Postmitotic Prox1 Expression Controls the Final Specification of Cortical VIP Interneuron Subtypes.

Throughout development, neuronal identity is controlled by key transcription factors that determine the unique properties of a cell. During embryogenesis, the transcription factor Prox1 regulates VIP-positive cortical interneuron migration, survival, and connectivity. Here, we explore the role of Prox1 as a regulator of genetic programs that guide the final specification of VIP interneuron subtypes in early postnatal life. Synaptic in vitro electrophysiology in male and female mice shows that postnatal Prox1 removal differentially affects the dynamics of excitatory inputs onto VIP bipolar and multipolar subtypes. RNA sequencing reveals that one of the downstream targets of Prox1 is the postsynaptic protein Elfn1, a constitutive regulator of presynaptic release probability. Further genetic, pharmacological, and electrophysiological experiments demonstrate that removing Prox1 reduces Elfn1 function in VIP multipolar but not in bipolar cells. Finally, overexpression experiments and analysis of native Elfn1 mRNA expression reveal that Elfn1 levels are differentially controlled at the post-transcriptional stage. Thus, in addition to activity-dependent processes that contribute to the developmental trajectory of VIP cells, genetic programs engaged by Prox1 control the final differentiation of multipolar and bipolar subtypes.SIGNIFICANCE STATEMENT The transcription factor Prox1 generates functional diversification of cortical VIP interneuron subtypes in early postnatal life, thus expanding the inhibitory repertoire of the cortex.

The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue.

Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample ( www.mesospim.org ).

Neuronal activity is required for the development of specific cortical interneuron subtypes.

Electrical activity has been shown to regulate development in a variety of species and in various structures, including the retina, spinal cord and cortex. Within the mammalian cortex specifically, the development of dendrites and commissural axons in pyramidal cells is activity-dependent. However, little is known about the developmental role of activity in the other major cortical population of neurons, the GABA-producing interneurons. These neurons are morphologically and functionally heterogeneous and efforts over the past decade have focused on determining the mechanisms that contribute to this diversity. It was recently discovered that 30% of all cortical interneurons arise from a relatively novel source within the ventral telencephalon, the caudal ganglionic eminence (CGE). Owing to their late birth date, these interneurons populate the cortex only after the majority of other interneurons and pyramidal cells are already in place and have started to functionally integrate. Here we demonstrate in mice that for CGE-derived reelin (Re)-positive and calretinin (Cr)-positive (but not vasoactive intestinal peptide (VIP)-positive) interneurons, activity is essential before postnatal day 3 for correct migration, and that after postnatal day 3, glutamate-mediated activity controls the development of their axons and dendrites. Furthermore, we show that the engulfment and cell motility 1 gene (Elmo1), a target of the transcription factor distal-less homeobox 1 (Dlx1), is selectively expressed in Re(+) and Cr(+) interneurons and is both necessary and sufficient for activity-dependent interneuron migration. Our findings reveal a selective requirement for activity in shaping the cortical integration of specific neuronal subtypes.

Prox1 Regulates the Subtype-Specific Development of Caudal Ganglionic Eminence-Derived GABAergic Cortical Interneurons.

Neurogliaform (RELN+) and bipolar (VIP+) GABAergic interneurons of the mammalian cerebral cortex provide critical inhibition locally within the superficial layers. While these subtypes are known to originate from the embryonic caudal ganglionic eminence (CGE), the specific genetic programs that direct their positioning, maturation, and integration into the cortical network have not been elucidated. Here, we report that in mice expression of the transcription factor Prox1 is selectively maintained in postmitotic CGE-derived cortical interneuron precursors and that loss of Prox1 impairs the integration of these cells into superficial layers. Moreover, Prox1 differentially regulates the postnatal maturation of each specific subtype originating from the CGE (RELN, Calb2/VIP, and VIP). Interestingly, Prox1 promotes the maturation of CGE-derived interneuron subtypes through intrinsic differentiation programs that operate in tandem with extrinsically driven neuronal activity-dependent pathways. Thus Prox1 represents the first identified transcription factor specifically required for the embryonic and postnatal acquisition of CGE-derived cortical interneuron properties. SIGNIFICANCE STATEMENT: Despite the recognition that 30% of GABAergic cortical interneurons originate from the caudal ganglionic eminence (CGE), to date, a specific transcriptional program that selectively regulates the development of these populations has not yet been identified. Moreover, while CGE-derived interneurons display unique patterns of tangential and radial migration and preferentially populate the superficial layers of the cortex, identification of a molecular program that controls these events is lacking.Here, we demonstrate that the homeodomain transcription factor Prox1 is expressed in postmitotic CGE-derived cortical interneuron precursors and is maintained into adulthood. We found that Prox1 function is differentially required during both embryonic and postnatal stages of development to direct the migration, differentiation, circuit integration, and maintenance programs within distinct subtypes of CGE-derived interneurons.

A nasal chemosensation-dependent critical window for somatosensory development.

Nasal chemosensation is considered the evolutionarily oldest mammalian sense and, together with somatosensation, is crucial for neonatal well-being before auditory and visual pathways start engaging the brain. Using anatomical and functional approaches in mice, we reveal that odor-driven activity propagates to a large part of the cortex during the first postnatal week and enhances whisker-evoked activation of primary whisker somatosensory cortex (wS1). This effect disappears in adult animals, in line with the loss of excitatory connectivity from olfactory cortex to wS1. By performing neonatal odor deprivation, followed by electrophysiological and behavioral work in adult animals, we identify a key transient regulation of nasal chemosensory information necessary for the development of wS1 sensory-driven dynamics and somatosensation. Our work uncovers a cross-modal critical window for nasal chemosensation-dependent somatosensory functional maturation.

Interfacing Aptamer-Modified Nanopipettes with Neuronal Media and Ex Vivo Brain Tissue.

Aptamer-functionalized biosensors exhibit high selectivity for monitoring neurotransmitters in complex environments. We translated nanoscale aptamer-modified nanopipette sensors to detect endogenous dopamine release in vitro and ex vivo. These sensors employ quartz nanopipettes with nanoscale pores (ca. 10 nm diameter) that are functionalized with aptamers that enable the selective capture of dopamine through target-specific conformational changes. The dynamic behavior of aptamer structures upon dopamine binding leads to the rearrangement of surface charge within the nanopore, resulting in measurable changes in ionic current. To assess sensor performance in real time, we designed a fluidic platform to characterize the temporal dynamics of nanopipette sensors. We then conducted differential biosensing by deploying control sensors modified with nonspecific DNA alongside dopamine-specific sensors in biological milieu. Our results confirm the functionality of aptamer-modified nanopipettes for direct measurements in undiluted complex fluids, specifically in the culture media of human-induced pluripotent stem cell-derived dopaminergic neurons. Moreover, sensor implantation and repeated measurements in acute brain slices was possible, likely owing to the protected sensing area inside nanoscale DNA-filled orifices, minimizing exposure to nonspecific interferents and preventing clogging. Further, differential recordings of endogenous dopamine released through electrical stimulation in the dorsolateral striatum demonstrate the potential of aptamer-modified nanopipettes for ex vivo recordings with unprecedented spatial resolution and reduced tissue damage.

Developmental Cajal-Retzius cell death contributes to the maturation of layer 1 cortical inhibition and somatosensory processing.

The role of developmental cell death in the formation of brain circuits is not well understood. Cajal-Retzius cells constitute a major transient neuronal population in the mammalian neocortex, which largely disappears at the time of postnatal somatosensory maturation. In this study, we used mouse genetics, anatomical, functional, and behavioral approaches to explore the impact of the early postnatal death of Cajal-Retzius cells in the maturation of the cortical circuit. We find that before their death, Cajal-Retzius cells mainly receive inputs from layer 1 neurons, which can only develop their mature connectivity onto layer 2/3 pyramidal cells after Cajal-Retzius cells disappear. This developmental connectivity progression from layer 1 GABAergic to layer 2/3 pyramidal cells regulates sensory-driven inhibition within, and more so, across cortical columns. Here we show that Cajal-Retzius cell death prevention leads to layer 2/3 hyper-excitability, delayed learning and reduced performance in a multi-whisker-dependent texture discrimination task.

The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation.

Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.

The Beautiful Brain: communicating fundamental neuroscience through masterpieces of art.

The Beautiful Brain is a documentary web series that breaks down borders between science and art. Five episodes retrace in a simple, but visually effective, way five key steps of brain development, using awe-inspiring masterpieces of art as analogies. This unconventional series focuses on fundamental research in neuroscience, the communication of which is not always easy and straightforward. In this article, we share our experience of how we tried to overcome the difficulties of communicating fundamental science to the lay audience. Moreover, we give insights into the path we followed to create The Beautiful Brain, with the hope that our experience may be an inspiration to other basic scientists who wish to communicate their own research.

Adamtsl3 mediates DCC signaling to selectively promote GABAergic synapse function.

The molecular code that controls synapse formation and maintenance in vivo has remained quite sparse. Here, we identify that the secreted protein Adamtsl3 functions as critical hippocampal synapse organizer acting through the transmembrane receptor DCC (deleted in colorectal cancer). Traditionally, DCC function has been associated with glutamatergic synaptogenesis and plasticity in response to Netrin-1 signaling. We demonstrate that early post-natal deletion of Adamtsl3 in neurons impairs DCC protein expression, causing reduced density of both glutamatergic and GABAergic synapses. Adult deletion of Adamtsl3 in either GABAergic or glutamatergic neurons does not interfere with DCC-Netrin-1 function at glutamatergic synapses but controls DCC signaling at GABAergic synapses. The Adamtsl3-DCC signaling unit is further essential for activity-dependent adaptations at GABAergic synapses, involving DCC phosphorylation and Src kinase activation. These findings might be particularly relevant for schizophrenia because genetic variants in Adamtsl3 and DCC have been independently linked with schizophrenia in patients.

Sparse postnatal labeling and quantification of superficial cortical cell synapses in the mouse neocortex.

We present a strategy for measuring the density of presynaptic boutons of superficial neuronal cells in the mouse neocortex. First, we show how to sparsely label individual postmitotic cells by neonatal pial-surface electroporation. Then, we present a custom-made code that allows quantification of the density of presynaptic boutons along axonal processes. Although we applied this strategy to somatostatin-positive cells, a major population of cortical interneurons, the same approach can be adjusted to target other types of neuronal cells. For complete details on the use and execution of this protocol, please refer to Gesuita et al. (2022).1.

An interactive time series image analysis software for dendritic spines.

Live fluorescence imaging has demonstrated the dynamic nature of dendritic spines, with changes in shape occurring both during development and in response to activity. The structure of a dendritic spine correlates with its functional efficacy. Learning and memory studies have shown that a great deal of the information stored by a neuron is contained in the synapses. High precision tracking of synaptic structures can give hints about the dynamic nature of memory and help us understand how memories evolve both in biological and artificial neural networks. Experiments that aim to investigate the dynamics behind the structural changes of dendritic spines require the collection and analysis of large time-series datasets. In this paper, we present an open-source software called SpineS for automatic longitudinal structural analysis of dendritic spines with additional features for manual intervention to ensure optimal analysis. We have tested the algorithm on in-vitro, in-vivo, and simulated datasets to demonstrate its performance in a wide range of possible experimental scenarios.

Microglia contribute to the postnatal development of cortical somatostatin-positive inhibitory cells and to whisker-evoked cortical activity.

Microglia play a key role in shaping the formation and refinement of the excitatory network of the brain. However, less is known about whether and how they organize the development of distinct inhibitory networks. We find that microglia are essential for the proper development of somatostatin-positive (SST+) cell synapses during the second postnatal week. We further identify a pair of molecules that act antagonistically to one another in the organization of SST+ cell axonal elaboration. Whereas CX3CL1 acts to suppress axonal growth and complexity, CXCL12 promotes it. Assessing the functional importance of microglia in the development of cortical activity, we find that a whisker stimulation paradigm that drives SST+ cell activation leads to reduced cortical spiking in brains depleted of microglia. Collectively, our data demonstrate an important role of microglia in regulating the development of SST+ cell output early in life.

Expression of COUP-TFII nuclear receptor in restricted GABAergic neuronal populations in the adult rat hippocampus.

The COUP-TFII nuclear receptor, also known as NR2F2, is expressed in the developing ventral telencephalon and modulates the tangential migration of a set of subpallial neuronal progenitors during forebrain development. Little information is available about its expression patterns in the adult brain. We have identified the cell populations expressing COUP-TFII and the contribution of some of them to network activity in vivo. Expression of COUP-TFII by hippocampal pyramidal and dentate granule cells, as well as neurons in the neocortex, formed a gradient increasing from undetectable in the dorsal to very strong in the ventral sectors. In the dorsal hippocampal CA1 area, COUP-TFII was restricted to GABAergic interneurons and expressed in several, largely nonoverlapping neuronal populations. Immunoreactivity was present in calretinin-, neuronal nitric oxide synthase-, and reelin-expressing cells, as well as in subsets of cholecystokinin- or calbindin-expressing or radiatum-retrohippocampally projecting GABAergic cells, but not in parvalbumin- and/or somatostatin-expressing interneurons. In vivo recording and juxtacellular labeling of COUP-TFII-expressing cells revealed neurogliaform cells, basket cells in stratum radiatum and tachykinin-expressing radiatum dentate innervating interneurons, identified by their axodendritic distributions. They showed cell type-selective phase-locked firing to the theta rhythm but no activation during sharp wave/ripple oscillations. These basket cells in stratum radiatum and neurogliaform cells fired at the peak of theta oscillations detected extracellularly in stratum pyramidale, unlike previously reported ivy cells, which fired at the trough. The characterization of COUP-TFII-expressing neurons suggests that this developmentally important transcription factor plays cell type-specific role(s) in the adult hippocampus.

Slow GABA transient and receptor desensitization shape synaptic responses evoked by hippocampal neurogliaform cells.

The kinetics of GABAergic synaptic currents can vary by an order of magnitude depending on the cell type. The neurogliaform cell (NGFC) has recently been identified as a key generator of slow GABA(A) receptor-mediated volume transmission in the isocortex. However, the mechanisms underlying slow GABA(A) receptor-mediated IPSCs and their use-dependent plasticity remain unknown. Here, we provide experimental and modeling data showing that hippocampal NGFCs generate an unusually prolonged (tens of milliseconds) but low-concentration (micromolar range) GABA transient, which is responsible for the slow response kinetics and which leads to a robust desensitization of postsynaptic GABA(A) receptors. This strongly contributes to the use-dependent synaptic depression elicited by various patterns of NGFC activity including the one detected during theta network oscillations in vivo. Synaptic depression mediated by NGFCs is likely to play an important modulatory role in the feedforward inhibition of CA1 pyramidal cells provided by the entorhinal cortex.

Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.

By combining an inducible genetic fate mapping strategy with electrophysiological analysis, we have systematically characterized the populations of cortical GABAergic interneurons that originate from the caudal ganglionic eminence (CGE). Interestingly, compared with medial ganglionic eminence (MGE)-derived cortical interneuron populations, the initiation [embryonic day 12.5 (E12.5)] and peak production (E16.5) of interneurons from this embryonic structure occurs 3 d later in development. Moreover, unlike either pyramidal cells or MGE-derived cortical interneurons, CGE-derived interneurons do not integrate into the cortex in an inside-out manner but preferentially (75%) occupy superficial cortical layers independent of birthdate. In contrast to previous estimates, CGE-derived interneurons are both considerably greater in number (approximately 30% of all cortical interneurons) and diversity (comprised by at least nine distinct subtypes). Furthermore, we found that a large proportion of CGE-derived interneurons, including the neurogliaform subtype, express the glycoprotein Reelin. In fact, most CGE-derived cortical interneurons express either Reelin or vasoactive intestinal polypeptide. Thus, in conjunction with previous studies, we have now determined the spatial and temporal origins of the vast majority of cortical interneuron subtypes.

A 'Marginal' tale: the development of the neocortical layer 1.

The development of neocortical layer 1 is a very dynamic process and the scene of multiple transient events, with Cajal-Retzius cell death being one of the most characteristic ones. Layer 1 is also the route of migration for a substantial number of GABAergic interneurons during embryogenesis and where some of which will ultimately remain in the adult. The two cell types, together with a diverse set of incoming axons and dendrites, create an early circuit that will dramatically change in structure and function in the adult cortex to give prominence to inhibition. Through the engagement of a diverse set of GABAergic inhibitory cells by bottom-up and top-down inputs, adult layer 1 becomes a powerful computational platform for the neocortex.