Oxytocin Modifies the Excitability and the Action Potential Shape of the Hippocampal CA1 GABAergic Interneurons.

Oxytocin (OT) is a neuropeptide that modulates social-related behavior and cognition in the central nervous system of mammals. In the CA1 area of the hippocampus, the indirect effects of the OT on the pyramidal neurons and their role in information processing have been elucidated. However, limited data are available concerning the direct modulation exerted by OT on the CA1 interneurons (INs) expressing the oxytocin receptor (OTR). Here, we demonstrated that TGOT (Thr4,Gly7-oxytocin), a selective OTR agonist, affects not only the membrane potential and the firing frequency but also the neuronal excitability and the shape of the action potentials (APs) of these INs in mice. Furthermore, we constructed linear mixed-effects models (LMMs) to unravel the dependencies between the AP parameters and the firing frequency, also considering how TGOT can interact with them to strengthen or weaken these influences. Our analyses indicate that OT regulates the functionality of the CA1 GABAergic INs through different and independent mechanisms. Specifically, the increase in neuronal firing rate can be attributed to the depolarizing effect on the membrane potential and the related enhancement in cellular excitability by the peptide. In contrast, the significant changes in the AP shape are directly linked to oxytocinergic modulation. Importantly, these alterations in AP shape are not associated with the TGOT-induced increase in neuronal firing rate, being themselves critical for signal processing.

Role of Na+/Ca2+ Exchanger (NCX) in Glioblastoma Cell Migration (In Vitro).

Glioblastoma (GBM) is the most malignant form of primary brain tumor. It is characterized by the presence of highly invasive cancer cells infiltrating the brain by hijacking neuronal mechanisms and interacting with non-neuronal cell types, such as astrocytes and endothelial cells. To enter the interstitial space of the brain parenchyma, GBM cells significantly shrink their volume and extend the invadopodia and lamellipodia by modulating their membrane conductance repertoire. However, the changes in the compartment-specific ionic dynamics involved in this process are still not fully understood. Here, using noninvasive perforated patch-clamp and live imaging approaches on various GBM cell lines during a wound-healing assay, we demonstrate that the sodium-calcium exchanger (NCX) is highly expressed in the lamellipodia compartment, is functionally active during GBM cell migration, and correlates with the overexpression of large conductance K+ channel (BK) potassium channels. Furthermore, a NCX blockade impairs lamellipodia formation and maintenance, as well as GBM cell migration. In conclusion, the functional expression of the NCX in the lamellipodia of GBM cells at the migrating front is a conditio sine qua non for the invasion strategy of these malignant cells and thus represents a potential target for brain tumor treatment.

Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington's disease.

Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors.

Neuromodulatory functions exerted by oxytocin on different populations of hippocampal neurons in rodents.

Oxytocin (OT) is a neuropeptide widely known for its peripheral hormonal effects (i.e., parturition and lactation) and central neuromodulatory functions, related especially to social behavior and social, spatial, and episodic memory. The hippocampus is a key structure for these functions, it is innervated by oxytocinergic fibers, and contains OT receptors (OTRs). The hippocampal OTR distribution is not homogeneous among its subregions and types of neuronal cells, reflecting the specificity of oxytocin's modulatory action. In this review, we describe the most recent discoveries in OT/OTR signaling in the hippocampus, focusing primarily on the electrophysiological oxytocinergic modulation of the OTR-expressing hippocampal neurons. We then look at the effect this modulation has on the balance of excitation/inhibition and synaptic plasticity in each hippocampal subregion. Additionally, we review OTR downstream signaling, which underlies the OT effects observed in different types of hippocampal neuron. Overall, this review comprehensively summarizes the advancements in unraveling the neuromodulatory functions exerted by OT on specific hippocampal networks.

In vitro-derived medium spiny neurons recapitulate human striatal development and complexity at single-cell resolution.

Stem cell engineering of striatal medium spiny neurons (MSNs) is a promising strategy to understand diseases affecting the striatum and for cell-replacement therapies in different neurological diseases. Protocols to generate cells from human pluripotent stem cells (PSCs) are scarce and how well they recapitulate the endogenous fetal cells remains poorly understood. We have developed a protocol that modulates cell seeding density and exposure to specific morphogens that generates authentic and functional D1- and D2-MSNs with a high degree of reproducibility in 25 days of differentiation. Single-cell RNA sequencing (scRNA-seq) shows that our cells can mimic the cell-fate acquisition steps observed in vivo in terms of cell type composition, gene expression, and signaling pathways. Finally, by modulating the midkine pathway we show that we can increase the yield of MSNs. We expect that this protocol will help decode pathogenesis factors in striatal diseases and eventually facilitate cell-replacement therapies for Huntington's disease (HD).

Membrane Resonance in Pyramidal and GABAergic Neurons of the Mouse Perirhinal Cortex.

The perirhinal cortex (PRC) is a polymodal associative region of the temporal lobe that works as a gateway between cortical areas and hippocampus. In recent years, an increasing interest arose in the role played by the PRC in learning and memory processes, such as object recognition memory, in contrast with certain forms of hippocampus-dependent spatial and episodic memory. The integrative properties of the PRC should provide all necessary resources to select and enhance the information to be propagated to and from the hippocampus. Among these properties, we explore in this paper the ability of the PRC neurons to amplify the output voltage to current input at selected frequencies, known as membrane resonance. Within cerebral circuits the resonance of a neuron operates as a filter toward inputs signals at certain frequencies to coordinate network activity in the brain by affecting the rate of neuronal firing and the precision of spike timing. Furthermore, the ability of the PRC neurons to resonate could have a fundamental role in generating subthreshold oscillations and in the selection of cortical inputs directed to the hippocampus. Here, performing whole-cell patch-clamp recordings from perirhinal pyramidal neurons and GABAergic interneurons of GAD67-GFP+ mice, we found, for the first time, that the majority of PRC neurons are resonant at their resting potential, with a resonance frequency of 0.5-1.5 Hz at 23°C and of 1.5-2.8 Hz at 36°C. In the presence of ZD7288 (blocker of HCN channels) resonance was abolished in both pyramidal neurons and interneurons, suggesting that Ih current is critically involved in resonance generation. Otherwise, application of TTx (voltage-dependent Na+ channel blocker) attenuates the resonance in pyramidal neurons but not in interneurons, suggesting that only in pyramidal neurons the persistent sodium current has an amplifying effect. These experimental results have also been confirmed by a computational model. From a functional point of view, the resonance in the PRC would affect the reverberating activity between neocortex and hippocampus, especially during slow wave sleep, and could be involved in the redistribution and strengthening of memory representation in cortical regions.

SREBP2 gene therapy targeting striatal astrocytes ameliorates Huntington's disease phenotypes.

Brain cholesterol is produced mainly by astrocytes and is important for neuronal function. Its biosynthesis is severely reduced in mouse models of Huntington's disease. One possible mechanism is a diminished nuclear translocation of the transcription factor sterol regulatory element-binding protein 2 (SREBP2) and, consequently, reduced activation of SREBP2-controlled genes in the cholesterol biosynthesis pathway. Here we evaluated the efficacy of a gene therapy based on the unilateral intra-striatal injection of a recombinant adeno-associated virus 2/5 (AAV2/5) targeting astrocytes specifically and carrying the transcriptionally active N-terminal fragment of human SREBP2 (hSREBP2). Robust hSREBP2 expression in striatal glial cells in R6/2 Huntington's disease mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed dopamine receptor D2 (Drd2) transcript levels decline, cleared mutant huntingtin aggregates and attenuated behavioural deficits. We conclude that glial SREBP2 participates in Huntington's disease brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of the disease.

Striatal infusion of cholesterol promotes dose-dependent behavioral benefits and exerts disease-modifying effects in Huntington's disease mice.

A variety of pathophysiological mechanisms are implicated in Huntington's disease (HD). Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from pre-symptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. In addition, systemic injection of cholesterol-loaded brain-permeable nanoparticles ameliorates synaptic and cognitive function in a transgenic mouse model of HD. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of exogenous cholesterol in the HD brain, we employed osmotic mini-pumps to infuse three escalating doses of cholesterol directly into the striatum of HD mice in a continuous and rate-controlled manner. All tested doses prevented cognitive decline, while amelioration of disease-related motor defects was dose-dependent. In parallel, we found morphological and functional recovery of synaptic transmission involving both excitatory and inhibitory synapses of striatal medium spiny neurons. The treatment also enhanced endogenous cholesterol biosynthesis and clearance of mutant Huntingtin aggregates. These results indicate that cholesterol infusion to the striatum can exert a dose-dependent, disease-modifying effect and may be therapeutically relevant in HD.

Stem Cell-Derived Human Striatal Progenitors Innervate Striatal Targets and Alleviate Sensorimotor Deficit in a Rat Model of Huntington Disease.

Huntington disease (HD) is an inherited late-onset neurological disorder characterized by progressive neuronal loss and disruption of cortical and basal ganglia circuits. Cell replacement using human embryonic stem cells may offer the opportunity to repair the damaged circuits and significantly ameliorate disease conditions. Here, we showed that in-vitro-differentiated human striatal progenitors undergo maturation and integrate into host circuits upon intra-striatal transplantation in a rat model of HD. By combining graft-specific immunohistochemistry, rabies virus-mediated synaptic tracing, and ex vivo electrophysiology, we showed that grafts can extend projections to the appropriate target structures, including the globus pallidus, the subthalamic nucleus, and the substantia nigra, and receive synaptic contact from both host and graft cells with 6.6 ± 1.6 inputs cell per transplanted neuron. We have also shown that transplants elicited a significant improvement in sensory-motor tasks up to 2 months post-transplant further supporting the therapeutic potential of this approach.

Oxytocin Increases Phasic and Tonic GABAergic Transmission in CA1 Region of Mouse Hippocampus.

Oxytocin is a neuropeptide that plays important peripheral and central neuromodulatory functions. Our data show that, following activation of oxytocin receptors (OtRs) with the selective agonist TGOT (Thr4,Gly7-oxytocin), a significant increase in frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSC) occurred in hippocampal CA1 pyramidal neurons (PYR) in mice. TGOT affected also sIPSC deactivation kinetics, suggesting the involvement of perisynaptic GABAA receptors (GABAARs) as well. By contrast, TGOT did not cause significant changes in frequency, amplitude or deactivation kinetics of miniature IPSC, suggesting that the effects elicited by the agonist are strictly dependent on the firing activity of presynaptic neurons. Moreover, TGOT was able to modulate tonic GABAergic current mediated by extrasynaptic GABAARs expressed by PYRs. Consistently, at spike threshold TGOT induced in most PYRs a significant membrane hyperpolarization and a decrease in firing rate. The source of increased inhibition onto PYRs was represented by stuttering fast-spiking GABAergic interneurons (INs) that directly respond to TGOT with a depolarization and an increase in their firing rate. One putative ionic mechanism underlying this effect could be represented by OtR activation-induced up-modulation of L-type Ca2+ channels. In conclusion, our results indicate that oxytocin can influence the activity of a subclass of hippocampal GABAergic INs and therefore regulate the operational modes of the downstream PYRs by increasing phasic and tonic GABAergic transmission in CA1 region of mouse hippocampus.

Inhibiting pathologically active ADAM10 rescues synaptic and cognitive decline in Huntington's disease.

A disintegrine and metalloproteinase 10 (ADAM10) is implicated in synaptic function through its interaction with postsynaptic receptors and adhesion molecules. Here, we report that levels of active ADAM10 are increased in Huntington's disease (HD) mouse cortices and striata and in human postmortem caudate. We show that, in the presence of polyglutamine-expanded (polyQ-expanded) huntingtin (HTT), ADAM10 accumulates at the postsynaptic densities (PSDs) and causes excessive cleavage of the synaptic protein N-cadherin (N-CAD). This aberrant phenotype is also detected in neurons from HD patients where it can be reverted by selective silencing of mutant HTT. Consistently, ex vivo delivery of an ADAM10 synthetic inhibitor reduces N-CAD proteolysis and corrects electrophysiological alterations in striatal medium-sized spiny neurons (MSNs) of 2 HD mouse models. Moreover, we show that heterozygous conditional deletion of ADAM10 or delivery of a competitive TAT-Pro-ADAM10709-729 peptide in R6/2 mice prevents N-CAD proteolysis and ameliorates cognitive deficits in the mice. Reduction in synapse loss was also found in R6/2 mice conditionally deleted for ADAM10. Taken together, these results point to a detrimental role of hyperactive ADAM10 at the HD synapse and provide preclinical evidence of the therapeutic potential of ADAM10 inhibition in HD.

Restoration of brain circulation and cellular functions hours post-mortem.

The brains of humans and other mammals are highly vulnerable to interruptions in blood flow and decreases in oxygen levels. Here we describe the restoration and maintenance of microcirculation and molecular and cellular functions of the intact pig brain under ex vivo normothermic conditions up to four hours post-mortem. We have developed an extracorporeal pulsatile-perfusion system and a haemoglobin-based, acellular, non-coagulative, echogenic, and cytoprotective perfusate that promotes recovery from anoxia, reduces reperfusion injury, prevents oedema, and metabolically supports the energy requirements of the brain. With this system, we observed preservation of cytoarchitecture; attenuation of cell death; and restoration of vascular dilatory and glial inflammatory responses, spontaneous synaptic activity, and active cerebral metabolism in the absence of global electrocorticographic activity. These findings demonstrate that under appropriate conditions the isolated, intact large mammalian brain possesses an underappreciated capacity for restoration of microcirculation and molecular and cellular activity after a prolonged post-mortem interval.

Human neuroepithelial stem cell regional specificity enables spinal cord repair through a relay circuit.

Traumatic spinal cord injury results in persistent disability due to disconnection of surviving neural elements. Neural stem cell transplantation has been proposed as a therapeutic option, but optimal cell type and mechanistic aspects remain poorly defined. Here, we describe robust engraftment into lesioned immunodeficient mice of human neuroepithelial stem cells derived from the developing spinal cord and maintained in self-renewing adherent conditions for long periods. Extensive elongation of both graft and host axons occurs. Improved functional recovery after transplantation depends on neural relay function through the grafted neurons, requires the matching of neural identity to the anatomical site of injury, and is accompanied by expression of specific marker proteins. Thus, human neuroepithelial stem cells may provide an anatomically specific relay function for spinal cord injury recovery.

Loss of Either Rac1 or Rac3 GTPase Differentially Affects the Behavior of Mutant Mice and the Development of Functional GABAergic Networks.

Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons.

Molecular and functional definition of the developing human striatum.

The complexity of the human brain derives from the intricate interplay of molecular instructions during development. Here we systematically investigated gene expression changes in the prenatal human striatum and cerebral cortex during development from post-conception weeks 2 to 20. We identified tissue-specific gene coexpression networks, differentially expressed genes and a minimal set of bimodal genes, including those encoding transcription factors, that distinguished striatal from neocortical identities. Unexpected differences from mouse striatal development were discovered. We monitored 36 determinants at the protein level, revealing regional domains of expression and their refinement, during striatal development. We electrophysiologically profiled human striatal neurons differentiated in vitro and determined their refined molecular and functional properties. These results provide a resource and opportunity to gain global understanding of how transcriptional and functional processes converge to specify human striatal and neocortical neurons during development.

Rac1 and rac3 GTPases control synergistically the development of cortical and hippocampal GABAergic interneurons.

The intracellular mechanisms driving postmitotic development of cortical γ-aminobutyric acid (GABA)ergic interneurons are poorly understood. We have addressed the function of Rac GTPases in cortical and hippocampal interneuron development. Developing neurons express both Rac1 and Rac3. Previous work has shown that Rac1 ablation does not affect the development of migrating cortical interneurons. Analysis of mice with double deletion of Rac1 and Rac3 shows that these GTPases are required during postmitotic interneuron development. The number of parvalbumin-positive cells was affected in the hippocampus and cortex of double knockout mice. Rac depletion also influences the maturation of interneurons that reach their destination, with reduction of inhibitory synapses in both hippocampal CA1 and cortical pyramidal cells. The decreased number of cortical migrating interneurons and their altered morphology indicate a role of Rac1 and Rac3 in regulating the motility of cortical interneurons, thus interfering with their final localization. While electrophysiological passive and active properties of pyramidal neurons including membrane capacity, resting potential, and spike amplitude and duration were normal, these cells showed reduced spontaneous inhibitory currents and increased excitability. Our results show that Rac1 and Rac3 contribute synergistically to postmitotic development of specific populations of GABAergic cells, suggesting that these proteins regulate their migration and differentiation.

Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32+ medium-sized spiny neurons.

Medium-sized spiny neurons (MSNs) are the only neostriatum projection neurons, and their degeneration underlies some of the clinical features of Huntington's disease. Using knowledge of human developmental biology and exposure to key neurodevelopmental molecules, human pluripotent stem (hPS) cells were induced to differentiate into MSNs. In a feeder-free adherent culture, ventral telencephalic specification is induced by BMP/TGFß inhibition and subsequent SHH/DKK1 treatment. The emerging FOXG1(+)/GSX2(+) telencephalic progenitors are then terminally differentiated, resulting in the systematic line-independent generation of FOXP1(+)/FOXP2(+)/CTIP2(+)/calbindin(+)/DARPP-32(+) MSNs. Similar to mature MSNs, these neurons carry dopamine and A2a receptors, elicit a typical firing pattern and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32(+) neurons, leading to a restoration of apomorphine-induced rotation behavior. In summary, hPS cells can be efficiently driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method that represents a platform for in vitro human developmental neurobiology studies and drug screening approaches.