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

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.Fil: Lodola, Francesco. Università degli Studi di Milano; ItaliaFil: de Giusti, Verónica Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Maniezzi, Claudia. Università degli Studi di Milano; ItaliaFil: Martone, Daniele. Università degli Studi di Milano; ItaliaFil: Stadiotti, Ilaria. Università degli Studi di Milano; ItaliaFil: Sommariva, Elena. Università degli Studi di Milano; ItaliaFil: Maione, Angela Serena. Università degli Studi di Milano; Itali

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.Centro de Investigaciones Cardiovasculare

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 (V-m), andmodulation of intracellular Ca2+ dynamics. Finally, cell contractility was analyzed using a custom MATLAB algorithm. Photostimulation of intramembrane Ziapin2 causes a transient V-m 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

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 modulates GABAA receptor-mediated inhibition onto CA1 pyramidal neurons in mouse

Oxytocin (OT) is a neuropeptide that exerts different peripheral and central actions. I aimed at characterizing the neuromodulatory effects of OT in the hippocampus. Electrophysiological experiments were performed on mouse brain slices using the whole-cell patch-clamp technique on pyramidal neurons (PYR) and GABAergic interneurons (INs) located in CA1 stratum pyramidale. The effect of TGOT (Thr4,Gly7-oxytocin), a selective OT receptor (OTR) agonist, was first evaluated on spontaneous inhibitory postsynaptic currents (sIPSC) recorded from PYRs in Otr+/+ mice. TGOT caused a significant decrease in the sIPSC interval and an increase in the sIPSC amplitude; it also caused an increase in the sIPSC time constant of decay: this suggests the involvement of GABAA receptors (GABAAR) located in a perisynaptic position that deactivate slower than synaptic receptors, generating slower sIPSCs. The TGOT-mediated effects were dependent on the activation of OTRs, being abolished by the OTR antagonist SSR126768A; furthermore, TGOT didn’t modulate sIPSCs in Otr-/- mice. Then, we recorded the miniature inhibitory postsynaptic currents (mIPSC), isolated by applying tetrodotoxin, a voltage-gated Na+ channel blocker, to prevent action potential firing in the presynaptic terminal. TGOT was not able to modulate the mIPSC interval, amplitude and kinetics of decay, indicating that the effects elicited by the agonist are dependent on the firing activity of the presynaptic neuron. After having clarified the action of TGOT on ‘phasic’ inhibitory transmission, elicited by synaptic and perisynaptic GABAARs, we enquire if the peptide could influence ‘tonic’ currents, mediated by extrasynaptic GABAARs. First, we demonstrated the presence of tonic currents by measuring the ‘baseline holding current’ required to clamp PYRs at a given potential, in control conditions and during the application of the GABAAR antagonist bicuculline: we observed an inward shift in the ‘baseline holding current’ in the presence of bicuculline, consistent with the abolition of tonic currents. Then, we found that TGOT was able to increase tonic currents, causing an outward shift in the ‘baseline holding current’. Subsequently, we tried to understand the source of that TGOT-mediated increased inhibition, finding that TGOT depolarized mainly the stuttering fast-spiking INs. The same depolarization was observed in the presence of synaptic blockers, suggesting that the effect is due to a direct binding to OTRs. Indeed, the perfusion of the OTR antagonist completely abolished the depolarization. We tried to investigate the ionic mechanism underlying the TGOT-induced depolarization. We tested the putative involvement of a Ca2+ current by using nifedipine, a selective L-type channel blocker. Actually, in the majority of INs examined, nifedipine was able to abolish the depolarization elicited by TGOT. Finally, we investigated the effect of TGOT on the membrane potential of PYRs. Most of them, examined at their spike threshold, became hyperpolarized by TGOT and their firing rate was significantly decreased. The hyperpolarizing response was completely abolished by the blockade of GABAARs, indicating that the effect requires the activation of extrasynaptic GABAARs that mediate a prolonged (or tonic) hyperpolarizing current. The TGOT-mediated hyperpolarization caused a reduction in cell excitability, altering the capability of PYRs to generate action potentials in response to depolarizing current steps. This was evident in the firing rate-to-injected current (F-I) relationship that was shifted to the right during perfusion of TGOT. The gain (i.e., the slope) of the curve was not influenced by TGOT. This behavior indicates an increase in tonically active inhibitory currents that lead to a persistent reduction in the input resistance and therefore in cell excitability

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 SREBP-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. 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 Drd2 transcript levels decline, cleared mutant Huntingtin aggregates and attenuated behavioral 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

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

none14The 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.openBinini, Noemi; Talpo, Francesca; Spaiardi, Paolo; Maniezzi, Claudia; Pedrazzoli, Matteo; Raffin, Francesca; Mattiello, Niccolò; Castagno, Antonio N; Masetto, Sergio; Yanagawa, Yuchio; Dickson, Clayton T; Ramat, Stefano; Toselli, Mauro; Biella, Gerardo RosarioBinini, Noemi; Talpo, Francesca; Spaiardi, Paolo; Maniezzi, Claudia; Pedrazzoli, Matteo; Raffin, Francesca; Mattiello, Niccolò; Castagno, Antonio N; Masetto, Sergio; Yanagawa, Yuchio; Dickson, Clayton T; Ramat, Stefano; Toselli, Mauro; Biella, Gerardo Rosari

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.</p