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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 humanNcangene 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 byWisteria floribundaagglutinin (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 fieldIn 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 ofNcanin 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 withWisteria floribundaagglutinin 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.

Our previous studies demonstrated that enzymatic removal of highly sulfated heparan sulfates with heparinase 1 impaired axonal excitability and reduced expression of ankyrin G at the axon initial segments in the CA1 region of the hippocampus ex vivo, impaired context discrimination in vivo, and elevated Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity in vitro. Here, we show that in vivo delivery of heparinase 1 in the CA1 region of the hippocampus elevated autophosphorylation of CaMKII 24 hours after injection in mice. Patch-clamp recording in CA1 neurons revealed no significant heparinase effects on the amplitude or frequency of miniature excitatory and inhibitory postsynaptic currents, while the threshold for action potential generation was increased and fewer spikes were generated in response to current injection. Delivery of heparinase on the next day after contextual fear conditioning induced context overgeneralization 24 hours after injection. Co-administration of heparinase with the CaMKII inhibitor (autocamtide-2-related inhibitory peptide) rescued neuronal excitability and expression of ankyrin G at the axon initial segment. It also restored context discrimination, suggesting a key role of CaMKII in neuronal signaling downstream of heparan sulfate proteoglycans and highlighting a link between impaired CA1 pyramidal cell excitability and context generalization during recall of contextual memories.

Entorhinal cortex (EC) is one of the first cerebral regions affected in the early phase of Alzheimer's disease (AD). Soluble forms of amyloid beta (Aβ) impair synaptic transmission in experimental AD models. Protein kinase Mζ (PKMζ) is an atypical persistently active protein kinase C, known to maintain long term synaptic plasticity and memory, but its role in AD has not yet been described. We examined effect of PKMζ overexpression on the late long-term potentiation (L-LTP) in the dentate gyrus (DG) following EC amyloidopathy. Oligomeric Aβ 1-42 (oAβ) or vehicle was bilaterally microinjected into the EC of the male Wistar rats. After 1 week, 2 µL of lentiviral vector (~108 TU/mL) encoding PKMζ genome was injected into the DG. One week later, synaptic responses and the LTP persistence were assessed in DG of freely moving animals during 90 minutes to 7 days period. Novel object recognition, passive avoidance and spatial memories were also tested. In rats with EC amyloidopathy, LTP was induced with less amplitude compared to the control group, and extinguished after 24 h. PKMζ overexpression in DG augmented synaptic responses (PS-LTP amplitudes) and maintained LTP over 1 week. PKMζ ameliorated recognition and memory deficits in rats with EC amyloidopathy. Microinjection of PKMζ inhibitor, zeta inhibitory peptide, into the DG abolished the boosting effect of PKMζ on synaptic activity and memory performance. PKMζ-dependent pathway could be a potential therapeutic target to combat synaptic failure and memory deficit in the early phase of AD.

“This is a pre-print of an article published in journal of Cellular and Molecular Neurobiology. The final authenticated version is available online at: https://doi.org/10.1007/s10571-020-01032-9"; International audience; Rabies virus (RABV) is a neurotropic virus exclusively infecting neurons in the central nervous system. RABV encodes five proteins. Among them, the viral glycoprotein (RVG) plays a key role in viral entry into neurons and rabies pathogenesis. It was shown that the nature of the C-terminus of the RABV G protein, which possesses a PDZ-binding motif (PBM), modulates the virulence of the RABV strain. The neuronal protein partners recruited by this PBM may alter host cell function. This study was conducted to investigate the effect of RVG on synaptic function in the hippocampal dentate gyrus (DG) of rat. Two μl (108 T.U./ml) of the lentiviral vector containing RVG gene was injected into the DG of rat hippocampus. After 2 weeks, the rat’s brain was cross-sectioned and RVG-expressing cells were detected by fluorescent microscopy. Hippocampal synaptic activity of the infected rats was then examined by recording the local field potentials from DG after stimulation of the perforant pathway. Short-term synaptic plasticity was also assessed by double pulse stimulation. Expression of RVG in DG increased long-term potentiation population spikes (LTP-PS), whereas no facilitation of LTP-PS was found in neurons expressing δRVG (deleted PBM). Furthermore, RVG and δRVG strengthened paired-pulse facilitation. Heterosynaptic long-term depression (LTD) in the DG was significantly blocked in RVG-expressing group compared to the control group. This blockade was dependent to PBM motif as rats expressing δRVG in the DG-expressed LTD comparable to the RVG group. Our data demonstrate that RVG expression facilitates both short- and long-term synaptic plasticity in the DG indicating that it may involve both pre- and postsynaptic mechanisms to alter synaptic function. Further studies are needed to elucidate the underlying mechanisms.

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.

Understanding changes in the expression of genes involved in regulating various components of the neural extracellular matrix (ECM) during aging can provide an insight into aging-associated decline in synaptic and cognitive functions. Hence, in this study, we compared the expression levels of ECM-related genes in the hippocampus of young, aged and very aged mice. ECM gene expression was downregulated, despite the accumulation of ECM proteoglycans during aging. The most robustly downregulated gene was carbohydrate sulfotransferase 3 (

Understanding changes in the expression of genes involved in regulating various components of the neural extracellular matrix (ECM) during aging can provide an insight into aging-associated decline in synaptic and cognitive functions. Hence, in this study, we compared the expression levels of ECM-related genes in the hippocampus of young, aged and very aged mice. ECM gene expression was downregulated, despite the accumulation of ECM proteoglycans during aging. The most robustly downregulated gene was carbohydrate sulfotransferase 3 (Chst3), the enzyme responsible for the chondroitin 6-sulfation (C6S) of proteoglycans. Further analysis of epigenetic mechanisms revealed a decrease in H3K4me3, three methyl groups at the lysine 4 on the histone H3 proteins, associated with the promoter region of the Chst3 gene, resulting in the downregulation of Chst3 expression in non-neuronal cells. Cluster analysis revealed that the expression of lecticans—substrates of CHST3—is tightly co-regulated with this enzyme. These changes in ECM-related genes were accompanied by an age-confounded decline in cognitive performance. Despite the co-directional impairment in cognitive function and average Chst3 expression in the studied age groups, at the individual level we found a negative correlation between mRNA levels of Chst3 and cognitive performance within the very aged group. An analysis of correlations between the expression of ECM-related genes and cognitive performance in novel object versus novel location recognition tasks revealed an apparent trade-off in the positive gene effects in one task at the expense of another. Further analysis revealed that, despite the reduction in the Chst3 mRNA, the expression of CHST3 protein is increased in glial cells but not in neurons, which, however, does not lead to changes in the absolute level of C6S and even results in the decrease in C6S in perineuronal, perisynaptic and periaxonal ECM relative to the elevated expression of its protein carrier versican.

Medial temporal lobe epilepsy (MTLE) is among the most common and most drug-resistant types of epilepsies associated with remodeling of the trisynaptic circuit of the hippocampus. The cornu ammonis (CA)3 region, as the “pacemaker” of the circuit, and CA3 → CA1 synapse (Schaffer collaterals) are potential targets for suppression of MTLE. We examined optogenetic manipulation of CA3 neurons in controlling the perforant pathway kindled seizures. One week after implantation of stimulating electrodes in perforant pathway, a recording electrode in CA1, and an optic fiber in CA3, rats underwent rapid kindling procedure. A lentivector with capability to move in retrograde monosynaptic direction and to insert the gene of red light sensitive opsin Jaws in neurons was injected into CA1 of the kindled rats. One week later, the kindled rats were stimulated at afterdischarge (AD) threshold under red light illumination to CA3; and duration of AD (ADD), generalized seizures (S5D), and total seizure behavior (SD) were recorded. Encoding Jaws in CA1, CA3, and entorhinal neuronal cells of the vector injected rats was verified by immunohistochemistry. More than 90% of CA1, CA3, and entorhinal neurons of the counted sections expressed Jaws. Red light (625 nm) illumination to CA3 of the kindled rats expressing Jaws entirely suppressed generalized seizures and significantly diminished ADD and SD. Encoding the light-sensitive chloride pump Jaws in the CA3, is an efficient optogenetic strategy to stop perforant pathway kindled seizures.

Background: A human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector (LV) pseudotyped by a variant of rabies envelope glycoprotein, FUG-B2, has previously been prepared and used in transfection of hippocampal CA1 ("Cornu Ammonis" area 1) neurons. This study aimed to verify reactive gliosis and neuronal damage after injection of the vector into the rat hippocampus. Methods: HEK 293T cells were transfected with transfer (fck-Jaws-GFP-ER2), envelope (FUG-B2), and packaging (pMDLg/pRRE, pRSV-Rev) plasmids, and the vector was injected into CA1 of the rat hippocampus. After one week, transduction efficiency, and the number of neuronal and astroglial cells were determined in CA1 and CA3 by double staining of the brain slices. Results: Hippocampal cells were successfully transfected as 92.7% of CA1 and 95.8% of CA3 neuronal cells expressed GFP. The frequency of neuronal and astroglial cells in CA1 and CA3 of the vector-injected rats remained unchanged compared to those in the control and the saline-injected rats. Furthermore, no morphological change was found in hippocampal astrocytes and neuronal cells. Conclusion: The HIV-1-based LV pseudotyped by FUG-B2 is safe and does not cause neuroinflammation and neuronal loss once directly delivered into the rat hippocampus.