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.

Early consequences of the phospholamban mutation PLN-R14del+/- in a transgenic mouse model.

AIMS: The heterozygous phospholamban (PLN) mutation R14del (PLN R14del+/- ) is associated with a severe arrhythmogenic cardiomyopathy (ACM) developing in the adult. "Superinhibition" of SERCA2a by PLN R14del is widely assumed to underlie the pathogenesis, but alternative mechanisms such abnormal energy metabolism have also been reported. This work aims to (1) to evaluate Ca2+ dynamics and energy metabolism in a transgenic (TG) mouse model of the mutation prior to cardiomyopathy development; (2) to test whether they are causally connected. METHODS: Ca2+ dynamics, energy metabolism parameters, reporters of mitochondrial integrity, energy, and redox homeostasis were measured in ventricular myocytes of 8-12 weeks-old, phenotypically silent, TG mice. Mutation effects were compared to pharmacological PLN antagonism and analyzed during modulation of sarcoplasmic reticulum (SR) and cytosolic Ca2+ compartments. Transcripts and proteins of relevant signaling pathways were evaluated. RESULTS: The mutation was characterized by hyperdynamic Ca2+ handling, compatible with a loss of SERCA2a inhibition by PLN. All components of energy metabolism were depressed; myocyte energy charge was preserved under quiescence but reduced during stimulation. Cytosolic Ca2+ buffering or SERCA2a blockade reduced O2 consumption with larger effect in the mutant. Signaling changes suggest cellular adaptation to perturbed Ca2+ dynamics and response to stress. CONCLUSIONS: (1) PLN R14del+/- loses its ability to inhibit SERCA2a, which argues against SERCA2a superinhibition as a pathogenetic mechanism; (2) depressed energy metabolism, its enhanced dependency on Ca2+ and activation of signaling responses point to an early involvement of metabolic stress in the pathogenesis of this ACM model.

Optical modulation of excitation-contraction coupling in human-induced pluripotent stem cell-derived cardiomyocytes.

Non-genetic photostimulation is a novel and rapidly growing multidisciplinary field that aims to induce light-sensitivity in living systems by exploiting exogeneous phototransducers. Here, we propose an intramembrane photoswitch, based on an azobenzene derivative (Ziapin2), for optical pacing of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The light-mediated stimulation process has been studied by applying several techniques to detect the effect on the cell properties. In particular, we recorded changes in membrane capacitance, in membrane potential (Vm), and modulation of intracellular Ca2+ dynamics. Finally, cell contractility was analyzed using a custom MATLAB algorithm. Photostimulation of intramembrane Ziapin2 causes a transient Vm hyperpolarization followed by a delayed depolarization and action potential firing. The observed initial electrical modulation nicely correlates with changes in Ca2+ dynamics and contraction rate. This work represents the proof of principle that Ziapin2 can modulate electrical activity and contractility in hiPSC-CMs, opening up a future development in cardiac physiology.

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.

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.

NMDA receptors elicit flux-independent intracellular Ca2+ signals via metabotropic glutamate receptors and flux-dependent nitric oxide release in human brain microvascular endothelial cells.

The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-d-aspartate (NMDA) receptors (NMDARs) to mediate extracellular Ca2+ entry and stimulate neuronal nitric oxide (NO) synthase to release NO and trigger neurovascular coupling (NVC). Neuronal and glial NMDARs may also operate in a flux-independent manner, although it is unclear whether their non-ionotropic mode of action is involved in NVC. Recently, endothelial NMDARs were found to trigger Ca2+-dependent NO production and induce NVC, but the underlying mode of signaling remains elusive. Herein, we report that GluN1 protein, as well as GluN2C and GluN3B transcripts and proteins, were expressed and that NMDA did not elicit inward currents, but induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in the human brain microvascular endothelial cell line, hCMEC/D3. A multidisciplinary approach, including live cell imaging, whole-cell patch-clamp recordings, pharmacological manipulation and gene targeting, revealed that NMDARs increase the [Ca2+]i in a flux-independent manner in hCMEC/D3 cells. The Ca2+ response to NMDA was triggered by endogenous Ca2+ release from the endoplasmic reticulum and the lysosomal Ca2+ stores and sustained by store-operated Ca2+ entry. Unexpectedly, pharmacological and genetic blockade of mGluR1 and mGluR5 dramatically impaired NMDARs-mediated Ca2+ signals. These findings indicate that NMDARs may increase the endothelial [Ca2+]i in a flux-independent manner via group 1 mGluRs. However, imaging of DAF-FM fluorescence revealed that NMDARs may also induce Ca2+-dependent NO release by signaling in a flux-dependent manner. These findings, therefore, shed novel light on the mechanisms whereby brain microvascular endothelium decodes glutamatergic signaling and regulates NVC.

Modeling Cardiomyopathies in a Dish: State-of-the-Art and Novel Perspectives on hiPSC-Derived Cardiomyocytes Maturation.

The stem cell technology and the induced pluripotent stem cells (iPSCs) production represent an excellent alternative tool to study cardiomyopathies, which overcome the limitations associated with primary cardiomyocytes (CMs) access and manipulation. CMs from human iPSCs (hiPSC-CMs) are genetically identical to patient primary cells of origin, with the main electrophysiological and mechanical features of CMs. The key issue to be solved is to achieve a degree of structural and functional maturity typical of adult CMs. In this perspective, we will focus on the main differences between fetal-like hiPSC-CMs and adult CMs. A viewpoint is given on the different approaches used to improve hiPSC-CMs maturity, spanning from long-term culture to complex engineered heart tissue. Further, we outline limitations and future developments needed in cardiomyopathy disease modeling.

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.