Rahul Kaushik

Neurons are the fundamental units of cognitive processing. The prevailing view holds that neurons are input-output units that fire action potentials only upon receiving sufficient synaptic input. Here, we demonstrate that this may not be the sole operational mode of neurons in vivo during cognitive tasks, by showing that hippocampal place cells actively maintain firing and preserve spatial information through molecularly defined intrinsic mechanisms. Using in vitro and in vivo recordings coupled with molecular manipulation in mice, we reveal that hippocampal persistent firing during spatial working memory is supported by individual neurons via TRPC4 ion channels. Despite the general belief that persistent activity maintains working memory content, we demonstrate that persistent firing instead maintains immediate spatial representations, potentially supporting the encoding of spatial information to guide behavior. These findings position individual neurons as active contributors to information retention, extending their traditional role as passive input-output units and potentially broadening our understanding of neural computation.

The condensed form of the neural extracellular matrix (ECM), known as perineuronal nets (PNNs), is predominantly associated with parvalbumin-expressing (PV+) interneurons in the cortex and hippocampus. PNNs are enriched in lecticans, including neurocan (Ncan). Genetic variation in the human Ncan gene has been linked to alterations in hippocampus-dependent memory, structural differences in the prefrontal cortex (PFC), and increased risk for schizophrenia and bipolar disorder. However, the specific contribution of Ncan to PFC-dependent cognitive functions and synaptic organization remains incompletely understood. Here, we investigated the consequences of region-specific reduction of Ncan in the infralimbic cortex of mice, a region considered homologous to the dorsomedial part of the human medial PFC. Using intracortical adeno-associated virus delivery of shRNA targeting Ncan, we examined PNN-associated markers, synaptic organization, and PFC-dependent behaviors. Ncan knockdown was associated with reduced Wisteria floribunda agglutinin (WFA) labeling of PNNs surrounding PV+ interneurons, suggesting alterations in PNN composition. Behaviorally, mice with reduced Ncan expression exhibited mild but consistent impairments in medial PFC–dependent temporal order recognition memory and in aspects of reversal spatial learning in a labyrinth task. At the cellular level, immunohistochemical analyses revealed a reduction in markers associated with perisomatic GABAergic innervation of PV+ interneurons, together with an increase in the density of vGLUT1-immunopositive puncta in the surrounding neuropil. These structural changes were not observed in constitutive Ncan knockout mice, suggesting the presence of compensatory mechanisms in this model. Together, our findings highlight a functional role of Ncan in supporting perisomatic GABAergic innervation, temporal order recognition memory and cognitive flexibility, important cognitive resources impaired in neuropsychiatric disorders.

The neural extracellular matrix (ECM) accumulates in the form of perineuronal nets (PNNs), particularly around fast-spiking GABAergic interneurons in the cortex and hippocampus, but also in association with the axon initial segments (AIS) and nodes of Ranvier. Increasing evidence highlights the role of Neurocan (Ncan), a brain-specific component of ECM, in the pathophysiology of neuropsychiatric disorders like bipolar disorder and schizophrenia. Ncan localizes at PNNs, nodes of Ranvier and the AIS, highlighting its potential role in regulation of axonal excitability. Here, we used knockdown and knockout approaches in mouse primary cortical neurons in combination with immunocytochemistry, western blotting and electrophysiological techniques to characterize the role of Ncan in the organization of PNNs and AIS and in the regulation of neuronal activity. We found that reduced Ncan levels led to remodeling of PNNs around neurons via upregulated Aggrecan mRNA and protein levels, increased expression of activity-dependent c-Fos and FosB genes and elevated spontaneous synaptic activity. The latter correlated with increased levels of Ankyrin-G in the AIS particularly in excitatory neurons, and with the elevated expression of Na _v 1.6 channels. Our results suggest that Ncan regulate expression of key proteins in PNNs and AISs and provide new insights into its role in the fine-tuning of neuronal functions.

The condensed form of neural extracellular matrix (ECM), perineuronal nets (PNNs), is predominantly associated with parvalbumin-expressing (PV+) interneurons in the cortex and hippocampus. PNNs are enriched in several lecticans, including neurocan (Ncan). A polymorphism in the human Ncan gene has been associated with alterations in hippocampus-dependent memory function, variation of prefrontal cortex structure, and a higher risk for schizophrenia or bipolar disorder. Ncan knockout (KO) mice show related behavioral abnormalities, such as hyperactivity. Here we focused on studying how dysregulation of Ncan specifically in the mPFC may affect cognitive and synaptic functions. Intracortical adeno-associated virus (AAV) delivery was used to express shRNA against Ncan. Analysis of PNNs in Ncan shRNA-injected mice revealed a reduction in PNNs labelling by Wisteria floribunda agglutinin (WFA) around PV+ interneurons. Reduced Ncan expression resulted in a loss of the mPFC-dependent temporal order recognition and impairment of reversal spatial learning in a labyrinth (dry maze) task. As a potential synaptic substrate of these cognitive abnormalities, we report a robust reduction in the perisomatic GABAergic innervation of PV+ cells in Ncan KO and Ncan shRNA-injected mice. We also observed an increase in the density of vGLUT1-immunopositive synaptic puncta in the neuropil of Ncan shRNA-injected mice, which was, however, compensated in Ncan KO mice. Thus, our findings highlight a functional role of Ncan in supporting perisomatic GABAergic inhibition, temporal order recognition memory and cognitive flexibility, as one of the important cognitive resources depleted in neuropsychiatric disorders. Contribution to the field: In this study, we asked if the extracellular matrix proteoglycan neurocan (Ncan) plays a functional role in the prefrontal cortex (PFC) of mice. Using viral delivery and expression of shRNA to knock down the expression of Ncan in the PFC, we provide evidence that neuronal Ncan is essential for the maintenance of perineuronal nets enveloping perisomatic interneurons by influencing the expression of glycoepitopes stained with Wisteria floribunda agglutinin and by modulating mRNA expression levels of other PNNs constituents. At the behavioral level, the knockdown of Ncan in mPFC impaired the temporal order recognition memory and consolidation/retrieval of spatial memories after reversal learning in the dry maze task. At the synaptic level, we found that Ncan knockdown reduced perisomatic GABAergic innervation of perisomatic interneurons and increased the density of vGLUT1+ excitatory presynaptic terminals in the neuropil of the PFC. Moreover, knockdown of Ncan changed the expression levels of several genes involved in activity-dependent synaptic remodeling. In summary, we conclude that neuronal Ncan is essential for multiple cognitive flexibility-related synaptic and cognitive functions in the PFC.

Summary: The brain extracellular matrix (ECM) assembles around neurons and synapses, and is thought to change only rarely, through proteolysis and renewed protein synthesis. We report here an alternative ECM remodeling mechanism, based on the recycling of ECM molecules. We found that a key ECM protein, Tenascin-R, is frequently endocytosed, and later resurfaces, preferentially near synapses. The TNR molecules complete this cycle within ∼3 days, in an activity-dependent fashion.

Objective: Leucine‐rich glioma‐inactivated 1 (LGI1) is a secreted transsynaptic protein that interacts presynaptically with Kv1.1 potassium channels and a disintegrin and metalloprotease (ADAM) protein 23, and postsynaptically influences α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors through a direct link with the ADAM22 cell adhesion protein. Haploinsufficiency of LGI1 or autoantibodies directed against LGI1 are associated with human epilepsy, generating the hypothesis that a subacute reduction of LGI1 is sufficient to increase network excitability.Methods: We tested this hypothesis in ex vivo hippocampal slices and in neuronal cultures, by subacutely reducing LGI1 expression with shRNA.Results: Injection of shRNA‐LGI1 in the hippocampus increased dentate granule cell excitability and low‐frequency facilitation of mossy fibers to CA3 pyramidal cell neurotransmission. Application of the Kv1 family blocker, α‐dendrotoxin, occluded this effect, implicating the involvement of Kv1.1. This subacute reduction of LGI1 was also sufficient to increase neuronal network activity in neuronal primary culture.Significance: These results indicate that a subacute reduction in LGI1 potentiates neuronal excitability and short‐term synaptic plasticity, and increases neuronal network excitability, opening new avenues for the treatment of limbic encephalitis and temporal lobe epilepsies.

In the advanced stages of Alzheimer's disease (AD), microglia are transformed to an activated phenotype with thickened and retracted processes, migrate to the site of amyloid‐beta (Aβ) plaques, and proliferate. In the early stages of AD, it is still poorly understood whether the microglial function is altered and which factors may regulate these changes. Here, we focused on studying microglia in the retrosplenial cortex (RSC) in 3‐ to 4‐month‐old 5xFAD mice as a transgenic mouse model of AD. At this age, there are neither Aβ plaques, nor activation of microglia, nor dysregulation in the expression of genes encoding major extracellular matrix (ECM) molecules or extracellular proteases in the RSC. Still, histochemical evaluation of the fine structure of neural ECM revealed increased levels of Wisteria floribunda agglutinin labeling in holes of perineuronal nets and changes in the perimeter of ECM barriers around the holes in 5xFAD mice. Two‐photon vital microscopy demonstrated normal morphology and resting motility of microglia but strongly diminished number of microglial cells that migrated to the photolesion site in 5xFAD mice. Enzymatic digestion of ECM by chondroitinase ABC (ChABC) ameliorated this defect. Accordingly, the characterization of cell surface markers by flow cytometry demonstrated altered expression of microglial CD45. Moreover, ChABC treatment reduced the invasion of myeloid‐derived mononuclear cells into the RSC of 5xFAD mice. Hence, the migration of both microglia and myeloid cells is altered during the early stages of amyloidosis and can be restored at least partially by the attenuation of the ECM.

Synthetic excitatory synaptic organizer: The human brain contains trillions of synapses within a vast network of neurons. Synapse remodeling is essential to ensure the efficient reception and integration of external stimuli and to store and retrieve information. Building and remodeling of synapses occurs throughout life under the control of synaptic organizer proteins. Errors in this process can lead to neuropsychiatric or neurological disorders. Suzuki et al. combined structural elements of natural synaptic organizers to develop an artificial version called CPTX, which has different binding properties (see the Perspective by Salinas). CPTX could act as a molecular bridge to reconnect neurons and restore excitatory synaptic function in animal models of cerebellar ataxia, familial Alzheimer's disease, and spinal cord injury. The findings illustrate how structure-guided approaches can help to repair neuronal circuits. Science , this issue p. eabb4853 ; see also p. 1052

Perineuronal nets (PNNs) represent a highly condensed specialized form of brain extracellular matrix (ECM) enwrapping mostly parvalbumin‐positive interneurons in the brain in a mesh‐like fashion. PNNs not only regulate the onset and completion of the critical period during postnatal brain development, control cell excitability, and synaptic transmission but are also implicated in several brain disorders including schizophrenia. Holes in the perineuronal nets, harboring the synaptic contacts, along with hole‐surrounding ECM barrier can be viewed as PNN compartmentalization units that might determine the properties of synapses and heterosynaptic communication. In this study, we developed a novel open‐source script for Fiji (ImageJ) to semi‐automatically quantify structural alterations of PNNs such as the number of PNN units, area, mean intensity of PNN marker expression in 2D and 3D, shape parameters of PNN units in the ketamine‐treated Sprague–Dawley rat model of schizophrenia using high‐resolution confocal microscopic images. We discovered that the mean intensity of ECM within PNN units is inversely correlated with the area and the perimeter of the PNN holes. The intensity, size, and shape of PNN units proved to be three major principal factors to describe their variability. Ketamine‐treated rats had more numerous but smaller and less circular PNN units than control rats. These parameters allowed to correctly classify individual PNNs as derived from control or ketamine‐treated groups with ≈85% reliability. Thus, the proposed multidimensional analysis of PNN units provided a robust and comprehensive morphometric fingerprinting of fine ECM structure abnormalities in the experimental model of schizophrenia.