Single-photon optogenetics enables precise, cell-type-specific modulation of neuronal circuits, making it a crucial tool in neuroscience. Its miniaturization in the form of fully implantable wide-field stimulator arrays enables long-term interrogation of cortical circuits and bears promise for brain-machine interfaces for sensory and motor function restoration. However, achieving selective activation of functional cortical representations poses a challenge, as studies show that targeted optogenetic stimulation results in activity spread beyond one functional domain. While recurrent network mechanisms contribute to activity spread, here we demonstrate with detailed simulations of isolated pyramidal neurons from cats of unknown sex that already neuron morphology causes a complex spread of optogenetic activity at the scale of one cortical column. Since the shape of a neuron impacts its optogenetic response, we find that a single stimulator at the cortical surface recruits a complex spatial distribution of neurons that can be inhomogeneous and vary with stimulation intensity and neuronal morphology across layers. We explore strategies to enhance stimulation precision, finding that optimizing stimulator optics may offer more significant improvements than the preferentially somatic expression of the opsin through genetic targeting. Our results indicate that, with the right optical setup, single-photon optogenetics can precisely activate isolated neurons at the scale of functional cortical domains spanning several hundred micrometers.
- MeSH
- Cats MeSH
- Models, Neurological MeSH
- Cerebral Cortex physiology cytology MeSH
- Neurons physiology MeSH
- Optogenetics * methods MeSH
- Pyramidal Cells physiology MeSH
- Photic Stimulation methods MeSH
- Animals MeSH
- Check Tag
- Cats MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.
Schizophrenia research has increased in recent decades and focused more on its neural basis. Decision-making and cognitive flexibility are the main cognitive functions that are impaired and considered schizophrenia endophenotypes. Cognitive impairment was recently connected with altered functions of N-methyl-d-aspartate (NMDAR) glutamatergic receptors, which increased cortical activity. Selective NMDAR antagonists, such as MK-801, have been used to model cognitive inflexibility in schizophrenia. Decreased GABAergic inhibitory activity has been shown elsewhere with enhanced cortical activity. This imbalance in the excitatory/inhibitory may reduce the entrainment of prefrontal gamma and hippocampal theta rhythms and result in gamma/theta band de-synchronization. The current study established an acute MK-801 administration model of schizophrenia-like cognitive inflexibility in rats and used the attentional set-shifting task in which rats learned to switch/reverse the relevant rule. During the task, we used in vivo optogenetic stimulations of parvalbumin-positive interneurons at specific light pulses in the prefrontal cortex and ventral hippocampus. The first experiments showed that acute dizocilpine in rats produced schizophrenia-like cognitive inflexibility. The second set of experiments demonstrated that specific optogenetic stimulation at specific frequencies of parvalbumin-positive interneurons in the prefrontal cortex and ventral hippocampus rescued the cognitive flexibility rats that received acute MK-801. These findings advance our knowledge of the pivotal role of parvalbumin interneurons in schizophrenia-like cognitive impairment and may guide further research on this severe psychiatric disorder.
- MeSH
- Dizocilpine Maleate * pharmacology MeSH
- Hippocampus metabolism MeSH
- Interneurons metabolism MeSH
- Cognition MeSH
- Rats MeSH
- Optogenetics MeSH
- Parvalbumins metabolism MeSH
- Prefrontal Cortex metabolism MeSH
- Receptors, N-Methyl-D-Aspartate metabolism MeSH
- Schizophrenia * MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The development of painful paclitaxel-induced peripheral neuropathy (PIPN) represents a major dose-limiting side effect of paclitaxel chemotherapy. Here we report a promising effect of duvelisib (Copiktra), a novel FDA-approved PI3Kδ/γ isoform-specific inhibitor, in preventing paclitaxel-induced pain-like behavior and pronociceptive signaling in DRGs and spinal cord dorsal horn (SCDH) in rat and mouse model of PIPN. Duvelisib blocked the development of mechanical hyperalgesia in both males and females. Moreover, duvelisib prevented paclitaxel-induced sensitization of TRPV1 receptors, and increased PI3K/Akt signaling in small-diameter DRG neurons and an increase of CD68+ cells within DRGs. Specific optogenetic stimulation of inhibitory neurons combined with patch-clamp recording revealed that duvelisib inhibited paclitaxel-induced weakening of inhibitory, mainly glycinergic control on SCDH excitatory neurons. Enhanced excitatory and reduced inhibitory neurotransmission in the SCDH following PIPN was also alleviated by duvelisib application. In summary, duvelisib showed a promising ability to prevent neuropathic pain in PIPN. The potential use of our findings in human medicine may be augmented by the fact that duvelisib is an FDA-approved drug with known side effects.SIGNIFICANCE STATEMENT We show that duvelisib, a novel FDA-approved PI3Kδ/γ isoform-specific inhibitor, prevents the development of paclitaxel-induced pain-like behavior in males and females and prevents pronociceptive signaling in DRGs and spinal cord dorsal horn in rat and mouse model of paclitaxel-induced peripheral neuropathy.
- MeSH
- Pain MeSH
- Phosphatidylinositol 3-Kinases MeSH
- Antineoplastic Agents, Phytogenic * pharmacology MeSH
- Hyperalgesia chemically induced drug therapy prevention & control MeSH
- Isoquinolines MeSH
- Rats MeSH
- Mice MeSH
- Peripheral Nervous System Diseases MeSH
- Neuralgia * chemically induced drug therapy prevention & control MeSH
- Paclitaxel adverse effects MeSH
- Purines MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Epilepsy is a complex disorder affecting the central nervous system and is characterised by spontaneously recurring seizures (SRSs). Epileptic patients undergo symptomatic pharmacological treatments, however, in 30% of cases, they are ineffective, mostly in patients with temporal lobe epilepsy. Therefore, there is a need for developing novel treatment strategies. Transplantation of cells releasing γ-aminobutyric acid (GABA) could be used to counteract the imbalance between excitation and inhibition within epileptic neuronal networks. We generated GABAergic interneuron precursors from human embryonic stem cells (hESCs) and grafted them in the hippocampi of rats developing chronic SRSs after kainic acid-induced status epilepticus. Using whole-cell patch-clamp recordings, we characterised the maturation of the grafted cells into functional GABAergic interneurons in the host brain, and we confirmed the presence of functional inhibitory synaptic connections from grafted cells onto the host neurons. Moreover, optogenetic stimulation of grafted hESC-derived interneurons reduced the rate of epileptiform discharges in vitro. We also observed decreased SRS frequency and total time spent in SRSs in these animals in vivo as compared to non-grafted controls. These data represent a proof-of-concept that hESC-derived GABAergic neurons can exert a therapeutic effect on epileptic animals presumably through establishing inhibitory synapses with host neurons.
- MeSH
- gamma-Aminobutyric Acid metabolism MeSH
- Hippocampus metabolism pathology MeSH
- Interneurons cytology metabolism MeSH
- Stem Cells cytology metabolism MeSH
- Rats MeSH
- Cells, Cultured MeSH
- Kainic Acid adverse effects MeSH
- Humans MeSH
- Disease Models, Animal MeSH
- Recurrence MeSH
- Status Epilepticus chemically induced metabolism pathology therapy MeSH
- Stem Cell Transplantation methods MeSH
- Seizures chemically induced metabolism pathology therapy MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- MeSH
- Electric Stimulation MeSH
- Genetic Therapy MeSH
- Genetic Diseases, Inborn MeSH
- Clinical Trials as Topic MeSH
- Stem Cells MeSH
- Humans MeSH
- Retinal Diseases * genetics therapy MeSH
- Neuroprotective Agents MeSH
- Optogenetics MeSH
- Prostheses and Implants MeSH
- Retina metabolism MeSH
- Therapeutics * trends MeSH
- Vitamin A administration & dosage therapeutic use MeSH
- Research MeSH
- Check Tag
- Humans MeSH
Two-Photon Processor (TPP) is a versatile, ready-to-use, and freely available software package in MATLAB to process data from in vivo two-photon calcium imaging. TPP includes routines to search for cell bodies in full-frame (Search for Neural Cells Accelerated; SeNeCA) and line-scan acquisition, routines for calcium signal calculations, filtering, spike-mining, and routines to construct parametric fields. Searching for somata in artificial in vivo data, our algorithm achieved better performance than human annotators. SeNeCA copes well with uneven background brightness and in-plane motion artifacts, the major problems in simple segmentation methods. In the fast mode, artificial in vivo images with a resolution of 256 × 256 pixels containing ≈ 100 neurons can be processed at a rate up to 175 frames per second (tested on Intel i7, 8 threads, magnetic hard disk drive). This speed of a segmentation algorithm could bring new possibilities into the field of in vivo optophysiology. With such a short latency (down to 5-6 ms on an ordinary personal computer) and using some contemporary optogenetic tools, it will allow experiments in which a control program can continuously evaluate the occurrence of a particular spatial pattern of activity (a possible correlate of memory or cognition) and subsequently inhibit/stimulate the entire area of the circuit or inhibit/stimulate a different part of the neuronal system. TPP will be freely available on our public web site. Similar all-in-one and freely available software has not yet been published.
- MeSH
- Algorithms * MeSH
- Aniline Compounds analysis MeSH
- Fluoresceins analysis MeSH
- Fluorescent Dyes MeSH
- Microscopy, Fluorescence, Multiphoton methods MeSH
- Cerebral Cortex chemistry MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Software * MeSH
- Calcium analysis MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Článek seznamuje stručně se současnými možnostmi léčby dystrofií sítnice. V počínajících stadiích chorob mají své uplatnění medikamentózní léčba zahrnující vitaminoterapii a farmakologickou léčbu, elektrostimulace a genová terapie. Zkoumána je účinnost neuroprotektivních substancí aplikovaných prostřednictvím technologie tzv. „opouzdřených buněk“ a systémové imunoterapie. V pokročilých stadiích sítnicových chorob spojených se ztrátou fotoreceptorů se používají elektronické sítnicové protézy a ověřuje se možnost použití kmenových buněk, transplantace buněk a tkání a optogenetické léčby.
There is a brief review of actual treatment possibilities of retinal dystrophies in this article. Vitaminotherapy, electrostimulation and gene therapy are used in beginning stages of disorders. The efficiency of neuroprotective substances that are applied intravitreally using encapsulated cell technology is examined, as well as influence of systemic immunotherapy. In advanced stages of retinal diseases that are connected with loss of photoreceptors are used electronic retinal prostheses. Effectivity of treatment with stem cells, transplantation of cells and tissues and optogenetic therapy are evaluated.
- MeSH
- Electric Stimulation MeSH
- Genetic Therapy MeSH
- Genetic Diseases, Inborn MeSH
- Clinical Trials as Topic MeSH
- Stem Cells MeSH
- Humans MeSH
- Retinal Diseases * genetics therapy MeSH
- Neuroprotective Agents MeSH
- Optogenetics MeSH
- Prostheses and Implants MeSH
- Retina metabolism MeSH
- Therapeutics * trends MeSH
- Vitamin A administration & dosage therapeutic use MeSH
- Research MeSH
- Check Tag
- Humans MeSH
- Keywords
- mozkové okruhy,
- MeSH
- Behavior, Animal MeSH
- Diptera MeSH
- Electric Stimulation MeSH
- Genetic Phenomena MeSH
- Brain Mapping * methods MeSH
- Brain * physiology MeSH
- Neural Conduction physiology genetics MeSH
- Neurons physiology MeSH
- Optogenetics * MeSH
- Signal Transduction MeSH
- Photic Stimulation MeSH
- Research MeSH
- Voltage-Sensitive Dye Imaging MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
