Astrocyte-secreted neurocan controls inhibitory synapse formation and function.

Astrocytes strongly promote the formation and maturation of synapses by secreted proteins. Several astrocyte-secreted synaptogenic proteins controlling excitatory synapse development were identified; however, those that induce inhibitory synaptogenesis remain elusive. Here, we identify neurocan as an astrocyte-secreted inhibitory synaptogenic protein. After secretion from astrocytes, neurocan is cleaved into N- and C-terminal fragments. We found that these fragments have distinct localizations in the extracellular matrix. The neurocan C-terminal fragment localizes to synapses and controls cortical inhibitory synapse formation and function. Neurocan knockout mice lacking the whole protein or only its C-terminal synaptogenic domain have reduced inhibitory synapse numbers and function. Through super-resolution microscopy, in vivo proximity labeling by secreted TurboID, and astrocyte-specific rescue approaches, we discovered that the synaptogenic domain of neurocan localizes to somatostatin-positive inhibitory synapses and strongly regulates their formation. Together, our results unveil a mechanism through which astrocytes control circuit-specific inhibitory synapse development in the mammalian brain.

Training-induced circuit-specific excitatory synaptogenesis in mice is required for effort control.

Synaptogenesis is essential for circuit development; however, it is unknown whether it is critical for the establishment and performance of goal-directed voluntary behaviors. Here, we show that operant conditioning via lever-press for food reward training in mice induces excitatory synapse formation onto a subset of anterior cingulate cortex neurons projecting to the dorsomedial striatum (ACC→DMS). Training-induced synaptogenesis is controlled by the Gabapentin/Thrombospondin receptor α2δ-1, which is an essential neuronal protein for proper intracortical excitatory synaptogenesis. Using germline and conditional knockout mice, we found that deletion of α2δ-1 in the adult ACC→DMS circuit diminishes training-induced excitatory synaptogenesis. Surprisingly, this manipulation does not impact learning but results in a significant increase in effort exertion without affecting sensitivity to reward value or changing contingencies. Bidirectional optogenetic manipulation of ACC→DMS neurons rescues or phenocopies the behaviors of the α2δ-1 cKO mice, highlighting the importance of synaptogenesis within this cortico-striatal circuit in regulating effort exertion.

Astrocyte-Secreted Neurocan Controls Inhibitory Synapse Formation and Function.

Astrocytes strongly promote the formation and maturation of synapses by secreted proteins. To date, several astrocyte-secreted synaptogenic proteins controlling different stages of excitatory synapse development have been identified. However, the identities of astrocytic signals that induce inhibitory synapse formation remain elusive. Here, through a combination of in vitro and in vivo experiments, we identified Neurocan as an astrocyte-secreted inhibitory synaptogenic protein. Neurocan is a chondroitin sulfate proteoglycan that is best known as a protein localized to the perineuronal nets. However, Neurocan is cleaved into two after secretion from astrocytes. We found that the resulting N- and C-terminal fragments have distinct localizations in the extracellular matrix. While the N-terminal fragment remains associated with perineuronal nets, the Neurocan C-terminal fragment localizes to synapses and specifically controls cortical inhibitory synapse formation and function. Neurocan knockout mice lacking the whole protein or only its C-terminal synaptogenic region have reduced inhibitory synapse numbers and function. Through super-resolution microscopy and in vivo proximity labeling by secreted TurboID, we discovered that the synaptogenic domain of Neurocan localizes to somatostatin-positive inhibitory synapses and strongly regulates their formation. Together, our results unveil a mechanism through which astrocytes control circuit-specific inhibitory synapse development in the mammalian brain.

Elucidating a locus coeruleus-dentate gyrus dopamine pathway for operant reinforcement.

Animals can learn to repeat behaviors to earn desired rewards, a process commonly known as reinforcement learning. While previous work has implicated the ascending dopaminergic projections to the basal ganglia in reinforcement learning, little is known about the role of the hippocampus. Here, we report that a specific population of hippocampal neurons and their dopaminergic innervation contribute to operant self-stimulation. These neurons are located in the dentate gyrus, receive dopaminergic projections from the locus coeruleus, and express D1 dopamine receptors. Activation of D1 + dentate neurons is sufficient for self-stimulation: mice will press a lever to earn optogenetic activation of these neurons. A similar effect is also observed with selective activation of the locus coeruleus projections to the dentate gyrus, and blocked by D1 receptor antagonism. Calcium imaging of D1 + dentate neurons revealed significant activity at the time of action selection, but not during passive reward delivery. These results reveal the role of dopaminergic innervation of the dentate gyrus in supporting operant reinforcement.

The role of astrocyte structural plasticity in regulating neural circuit function and behavior.

Brain circuits undergo substantial structural changes during development, driven by the formation, stabilization, and elimination of synapses. Synaptic connections continue to undergo experience-dependent structural rearrangements throughout life, which are postulated to underlie learning and memory. Astrocytes, a major glial cell type in the brain, are physically in contact with synaptic circuits through their structural ensheathment of synapses. Astrocytes strongly contribute to the remodeling of synaptic structures in healthy and diseased central nervous systems by regulating synaptic connectivity and behaviors. However, whether structural plasticity of astrocytes is involved in their critical functions at the synapse is unknown. This review will discuss the emerging evidence linking astrocytic structural plasticity to synaptic circuit remodeling and regulation of behaviors. Moreover, we will survey possible molecular and cellular mechanisms regulating the structural plasticity of astrocytes and their non-cell-autonomous effects on neuronal plasticity. Finally, we will discuss how astrocyte morphological changes in different physiological states and disease conditions contribute to neuronal circuit function and dysfunction.

3D Free-Standing Ordered Graphene Network Geometrically Regulates Neuronal Growth and Network Formation.

The control of cell-microenvironment interactions plays a pivotal role in constructing specific scaffolds for tissue engineering. Here, we fabricated a 3D free-standing ordered graphene (3D-OG) network with a precisely defined pattern. When primary cortical cells are cultured on 3D-OG scaffolds, they form well-defined 3D connections. Astrocytes have a more ramified shape similar to that seen in vivo because of the nanosized ripples and wrinkles on the surface of graphene skeleton. Neurons have axons and dendrites aligned along the graphene skeleton, allowing the formation of neuronal networks with highly controlled connections. Neuronal networks have higher electrical activity with functional signaling over a long distance along the graphene skeleton. Our study, for the first time, investigated the geometrical cues on ordered neuronal growth and network formation with the support of graphene in 3D, which therefore advanced the development of customized scaffolds for brain-machine interfaces or neuroprosthetic devices.

Foxg1 Upregulation Enhances Neocortical Activity.

Foxg1 is an ancient transcription factor gene orchestrating a number of neurodevelopmental processes taking place in the rostral brain. In this study, we investigated its impact on neocortical activity. We found that mice overexpressing Foxg1 in neocortical pyramidal cells displayed an electroencephalography (EEG) with increased spike frequency and were more prone to kainic acid (KA)-induced seizures. Consistently, primary cultures of neocortical neurons gain-of-function for Foxg1 were hyperactive and hypersynchronized. That reflected an unbalanced expression of key genes encoding for ion channels, gamma aminobutyric acid and glutamate receptors, and was likely exacerbated by a pronounced interneuron depletion. We also detected a transient Foxg1 upregulation ignited in turn by neuronal activity and mediated by immediate early genes. Based on this, we propose that even small changes of Foxg1 levels may result in a profound impact on pyramidal cell activity, an issue relevant to neuronal physiology and neurological aberrancies associated to FOXG1 copy number variations.

A Fully 3D Interconnected Graphene-Carbon Nanotube Web Allows the Study of Glioma Infiltration in Bioengineered 3D Cortex-Like Networks.

Currently available 3D assemblies based on carbon nanotubes (CNTs) lag far behind their 2D CNT-based bricks and require major improvements for biological applications. By using Fe nanoparticles confined to the interlamination of graphite as catalyst, a fully 3D interconnected CNT web is obtained through the pores of graphene foam (GCNT web) by in situ chemical vapor deposition. This 3D GCNT web has a thickness up to 1.5 mm and a completely geometric, mechanical and electrical interconnectivity. Dissociated cortical cells cultured inside the GCNT web form a functional 3D cortex-like network exhibiting a spontaneous electrical activity that is closer to what is observed in vivo. By coculturing and fluorescently labeling glioma and healthy cortical cells with different colors, a new in vitro model is obtained to investigate malignant glioma infiltration. This model allows the 3D trajectories and velocity distribution of individual infiltrating glioma to be reconstructed with an unprecedented precision. The model is cost effective and allows a quantitative and rigorous screening of anticancer drugs. The fully 3D interconnected GCNT web is biocompatible and is an ideal tool to study 3D biological processes in vitro representing a pivotal step toward precise and personalized medicine.

The role of dimensionality in neuronal network dynamics.

Recent results from network theory show that complexity affects several dynamical properties of networks that favor synchronization. Here we show that synchronization in 2D and 3D neuronal networks is significantly different. Using dissociated hippocampal neurons we compared properties of cultures grown on a flat 2D substrates with those formed on 3D graphene foam scaffolds. Both 2D and 3D cultures had comparable glia to neuron ratio and the percentage of GABAergic inhibitory neurons. 3D cultures because of their dimension have many connections among distant neurons leading to small-world networks and their characteristic dynamics. After one week, calcium imaging revealed moderately synchronous activity in 2D networks, but the degree of synchrony of 3D networks was higher and had two regimes: a highly synchronized (HS) and a moderately synchronized (MS) regime. The HS regime was never observed in 2D networks. During the MS regime, neuronal assemblies in synchrony changed with time as observed in mammalian brains. After two weeks, the degree of synchrony in 3D networks decreased, as observed in vivo. These results show that dimensionality determines properties of neuronal networks and that several features of brain dynamics are a consequence of its 3D topology.