| 1. |
- Avaliani, Natalia, et al.
(author)
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Optogenetics reveal delayed afferent synaptogenesis on grafted human induced pluripotent stem cell-derived neural progenitors.
- 2014
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In: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 32:12, s. 3088-3098
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Journal article (peer-reviewed)abstract
- Reprogramming of somatic cells into pluripotency stem cell state have opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSC), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for six weeks, lt-NES cell-derived neurons displayed neuronal properties such as TTX-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the It-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time-point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, e.g. such as epilepsy. Stem Cells 2014.
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| 2. |
- Berglind, Fredrik, et al.
(author)
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Optogenetic inhibition of chemically induced hypersynchronized bursting in mice.
- 2014
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In: Neurobiology of Disease. - : Elsevier BV. - 0969-9961. ; 65, s. 133-141
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Journal article (peer-reviewed)abstract
- Synchronized activity is common during various physiological operations but can culminate in seizures and consequently in epilepsy in pathological hyperexcitable conditions in the brain. Many types of seizures are not possible to control and impose significant disability for patients with epilepsy. Such intractable epilepsy cases are often associated with degeneration of inhibitory interneurons in the cortical areas resulting in impaired inhibitory drive onto the principal neurons. Recently emerging optogenetic technique has been proposed as an alternative approach to control such seizures but whether it may be effective in situations where inhibitory processes in the brain are compromised has not been addressed. Here we used pharmacological and optogenetic techniques to block inhibitory neurotransmission and induce epileptiform activity in vitro and in vivo. We demonstrate that NpHR-based optogenetic hyperpolarization and thereby inactivation of a principal neuronal population in the hippocampus is effectively attenuating seizure activity caused by disconnected network inhibition both in vitro and in vivo. Our data suggest that epileptiform activity in the hippocampus caused by impaired inhibition may be controlled by optogenetic silencing of principal neurons and potentially can be developed as an alternative treatment for epilepsy.
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| 3. |
- Kokaia, Merab, et al.
(author)
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An optogenetic approach in epilepsy.
- 2013
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In: Neuropharmacology. - : Elsevier BV. - 1873-7064 .- 0028-3908. ; 69:Jun 12, s. 89-95
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Research review (peer-reviewed)abstract
- Optogenetic tools comprise a variety of different light-sensitive proteins from single-cell organisms that can be expressed in mammalian neurons and effectively control their excitability. Two main classes of optogenetic tools allow to either depolarize or hyperpolarize, and respectively generate or inhibit action potentials in selective populations of neurons. This opens unprecedented possibilities for delineating the role of certain neuronal populations in brain processing and diseases. Moreover, optogenetics may be considered for developing potential treatment strategies for brain diseases, particularly for excitability disorders such as epilepsy. Expression of the inhibitory halorhodopsin NpHR in hippocampal principal cells has been recently used as a tool to effectively control chemically and electrically induced epileptiform activity in slice preparations, and to reduce in vivo spiking induced by tetanus toxin injection in the motor cortex. In this review we give a comprehensive summary of what has been achieved so far in the field of epilepsy using optogenetics, and discuss some of the possible strategies that could be envisaged in the future. We also point out some of the challenges and pitfalls in relation to possible outcomes of using optogenetics for controlling network excitability, and associated brain diseases. This article is part of a Special Issue entitled 'Epilepsy'.
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| 4. |
- Krook-Magnuson, Esther, et al.
(author)
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How Might Novel Technologies Such as Optogenetics Lead to Better Treatments in Epilepsy?
- 2014
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In: Issues in Clinical Epileptology: A View From the Bench. - Dordrecht : Springer Netherlands. - 0065-2598. - 9789401789141 ; 813, s. 319-336
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Book chapter (peer-reviewed)abstract
- Recent technological advances open exciting avenues for improving the understanding of mechanisms in a broad range of epilepsies. This chapter focuses on the development of optogenetics and on-demand technologies for the study of epilepsy and the control of seizures. Optogenetics is a technique which, through cell-type selective expression of light-sensitive proteins called opsins, allows temporally precise control via light delivery of specific populations of neurons. Therefore, it is now possible not only to record interictal and ictal neuronal activity, but also to test causality and identify potential new therapeutic approaches. We first discuss the benefits and caveats to using optogenetic approaches and recent advances in optogenetics related tools. We then turn to the use of optogenetics, including on-demand optogenetics in the study of epilepsies, which highlights the powerful potential of optogenetics for epilepsy research.
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| 5. |
- Ledri, Marco, et al.
(author)
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Altered profile of basket cell afferent synapses in hyper-excitable dentate gyrus revealed by optogenetic and two-pathway stimulations.
- 2012
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In: European Journal of Neuroscience. - : Wiley. - 1460-9568 .- 0953-816X. ; 36:1, s. 1971-1983
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Journal article (peer-reviewed)abstract
- Cholecystokinin (CCK-) positive basket cells form a distinct class of inhibitory GABAergic interneurons, proposed to act as fine-tuning devices of hippocampal gamma-frequency (30-90 Hz) oscillations, which can convert into higher frequency seizure activity. Therefore, CCK-basket cells may play an important role in regulation of hyper-excitability and seizures in the hippocampus. In normal conditions, the endogenous excitability regulator neuropeptide Y (NPY) has been shown to modulate afferent inputs onto dentate gyrus CCK-basket cells, providing a possible novel mechanism for excitability control in the hippocampus. Using GAD65-GFP mice for CCK-basket cell identification, and whole-cell patch-clamp recordings, we explored whether the effect of NPY on afferent synapses to CCK-basket cells is modified in the hyper-excitable dentate gyrus. To induce a hyper-excitable state, recurrent seizures were evoked by electrical stimulation of the hippocampus using the well-characterized rapid kindling protocol. The frequency of spontaneous and miniature excitatory and inhibitory post-synaptic currents recorded in CCK-basket cells was decreased by NPY. The excitatory post-synaptic currents evoked in CCK-basket cells by optogenetic activation of principal neurons were also decreased in amplitude. Interestingly, we observed an increased proportion of spontaneous inhibitory post-synaptic currents with slower rise times, indicating that NPY may inhibit gamma aminobutyric acid release preferentially in peri-somatic synapses. These findings indicate that increased levels and release of NPY observed after seizures can modulate afferent inputs to CCK-basket cells, and therefore alter their impact on the oscillatory network activity and excitability in the hippocampus.
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| 6. |
- Ledri, Marco
(author)
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Endogenous modulators of hyperexcitability in epilepsy: electrophysiological and optogenetic delineation of neuropeptide Y mechanisms in interneurons
- 2012
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Doctoral thesis (other academic/artistic)abstract
- Epilepsy is one of the most common neurological disorders worldwide, affecting 1% of the general population, and is characterized by a predisposition for the generation of epileptic seizures. Despite having several different aetiologies, a common underlying cause of epilepsy seems to be an acquired imbalance between excitatory and inhibitory circuits in the brain, which leads to hyperexcitability and appearance of seizures. Current treatment relies on the use of antiepileptic drugs (AEDs), but these only treat the symptoms, not affect the causes of the disease, and trigger undesirable side effects because of their systemic administration. Recent advancements in drug discovery have led to the development of new AEDs that are better tolerated and with improved pharmacokinetics, but 30-40% of all patients with epilepsy, and particularly those with temporal lobe epilepsy (TLE) remain resistant to the treatment. Thus, there is an urgent need for developing new antiepileptic treatment strategies. In the last years, research on novel antiepileptic treatments has identified several endogenous molecules as potential new targets for therapeutic intervention. Among these, neuropeptide Y (NPY ) seems to be a particularly promising target, as it plays an important role in controlling neuronal excitability in different brain areas, including the hippocampus. Indeed, overexpression of NPY via gene therapy approaches in animal models of epilepsy has profound effects on seizure generation and suppression, providing proof of principle evidence that such approach could be successfully used to reduce and control seizures. The actions of NPY are mediated by various receptors, and their activation predominantly causes suppression of glutamatergic synaptic transmission, which leads to decreased excitability. However, little is known about the effect NPY has on GABAergic inhibitory cell populations, and NPY mechanisms of action have to be carefully determined if such an approach could be used in humans. There are several different subtypes of inhibitory cell populations in all cortical areas, and each of them serve a different function with distinct roles in controlling network activity. Perisomatic-targeting interneurons comprise those inhibitory cell types that form synapses onto the perisomatic region of target cells, an area including the cell soma, proximal dendrites and axon initial segment. Thanks to the strategic location of their targets, perisomatic interneurons are particularly suited to control the output of large numbers of excitatory principal cells, with major impact on the network excitability. Two main subclasses, called basket cells, make up the majority of perisomatic interneurons, and their classification is based on the expression of either the neuropeptide Cholecystokinin (CCK) or the calcium binding protein Parvalbumin (PV). PV-basket cells are thought to be important for the generation of gamma-frequency oscillations, while CCK- basket cells are proposed to modulate this activity. Since gamma oscillations can convert into higher frequency epileptiform activity, and NPY strongly modulates network excitability, this thesis aimed to investigate the effects of NPY on CCK- and PV- basket cells, to understand if actions of NPY on perisomatic interneurons could contribute to its seizure-suppressant effects. Using transgenic mice, electrophysiological and optogenetic techniques, the evidence provided in this thesis demonstrates that NPY strongly modulates excitatory and inhibitory incoming synaptic transmission onto CCK-basket cells, but does not directly affect PV cell output onto principal cells. These effects could alter the way CCK-basket cells react to network activity, and have potential impacts on network excitability. In addition, we show that hyperexcitability enhances GABAergic output from PV cells, uncovering a potential mechanism that could increase principal cell synchrony and contribute to the generation of seizures.
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| 7. |
- Ledri, Marco, et al.
(author)
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Global Optogenetic Activation of Inhibitory Interneurons during Epileptiform Activity.
- 2014
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In: The Journal of Neuroscience. - 1529-2401. ; 34:9, s. 3364-3377
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Journal article (peer-reviewed)abstract
- Optogenetic techniques provide powerful tools for bidirectional control of neuronal activity and investigating alterations occurring in excitability disorders, such as epilepsy. In particular, the possibility to specifically activate by light-determined interneuron populations expressing channelrhodopsin-2 provides an unprecedented opportunity of exploring their contribution to physiological and pathological network activity. There are several subclasses of interneurons in cortical areas with different functional connectivity to the principal neurons (e.g., targeting their perisomatic or dendritic compartments). Therefore, one could optogenetically activate specific or a mixed population of interneurons and dissect their selective or concerted inhibitory action on principal cells. We chose to explore a conceptually novel strategy involving simultaneous activation of mixed populations of interneurons by optogenetics and study their impact on ongoing epileptiform activity in mouse acute hippocampal slices. Here we demonstrate that such approach results in a brief initial action potential discharge in CA3 pyramidal neurons, followed by prolonged suppression of ongoing epileptiform activity during light exposure. Such sequence of events was caused by massive light-induced release of GABA from ChR2-expressing interneurons. The inhibition of epileptiform activity was less pronounced if only parvalbumin- or somatostatin-expressing interneurons were activated by light. Our data suggest that global optogenetic activation of mixed interneuron populations is a more effective approach for development of novel therapeutic strategies for epilepsy, but the initial action potential generation in principal neurons needs to be taken in consideration.
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| 8. |
- Ledri, Marco, et al.
(author)
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Tuning afferent synapses of hippocampal interneurons by neuropeptide Y.
- 2011
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In: Hippocampus. - : Wiley. - 1050-9631. ; 21, s. 198-211
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Journal article (peer-reviewed)abstract
- Cholecystokinin (CCK)-expressing basket cells encompass a subclass of inhibitory GABAergic interneurons that regulate memory-forming oscillatory network activity of the hippocampal formation in accordance to the emotional and motivational state of the animal, conveyed onto these cells by respective extrahippocampal afferents. Various excitatory and inhibitory afferent and efferent synapses of the hippocampal CCK basket cells express serotoninergic, cholinergic, cannabinoid, and benzodiazepine sensitive receptors, all contributing to their functional plasticity. We explored whether CCK basket cells are modulated by neuropeptide Y (NPY), one of the major local neuropeptides that strongly inhibits hippocampal excitability and has significant effect on its memory function. Here, using GAD65-GFP transgenic mice for prospective identification of CCK basket cells and whole-cell patch-clamp recordings, we show for the first time that excitatory and inhibitory inputs onto CCK basket cells in the dentate gyrus of the hippocampus are modulated by NPY through activation of NPY Y2 receptors. The frequency of spontaneous and miniature EPSCs, as well as the amplitudes of stimulation-evoked EPSCs were decreased. Similarly, the frequency of both spontaneous and miniature IPSCs, and the amplitudes of stimulation-evoked IPSCs were decreased after NPY application. Most of the effects of NPY could be attributed to a presynaptic site of action. Our data provide the first evidence that the excitatory and inhibitory inputs onto the CCK basket cells could be modulated by local levels of NPY, and may change the way these cells process extrahippocampal afferent information, influencing hippocampal function and its network excitability during normal and pathological oscillatory activities. (c) 2009 Wiley-Liss, Inc.
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