scholarly journals Imaging Striatal Dopamine Release Using a Non-Genetically Encoded Near-Infrared Fluorescent Catecholamine Nanosensor

2018 ◽  
Author(s):  
Abraham G. Beyene ◽  
Kristen Delevich ◽  
Jackson Travis Del Bonis-O’Donnell ◽  
David J. Piekarski ◽  
Wan Chen Lin ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Near Infrared ◽  
Critical Role ◽  
Brain Slices ◽  
Biological Tissues ◽  
Single Wall Carbon Nanotubes ◽  
Fluorescence Signal ◽  
Optogenetic Stimulation ◽  
Optical Tools ◽  
Stimulation Of

AbstractNeuromodulation plays a critical role in brain function in both health and disease. New optical tools, and their validation in biological tissues, are needed that can image neuromodulation with high spatial and temporal resolution, which will add an important new dimension of information to neuroscience research. Here, we demonstrate the use of a catecholamine nanosensor with fluorescent emission in the 1000-1300 nm near-infrared window to measure dopamine transmission in ex vivo brain slices. These near-infrared catecholamine nanosensors (nIRCats) represent a broader class of nanosensors that can be synthesized from non-covalent conjugation of single wall carbon nanotubes (SWNT) with single strand oligonucleotides. We show that nIRCats can be used to detect catecholamine efflux in brain tissue driven by both electrical stimulation or optogenetic stimulation. Spatial analysis of electrically-evoked signals revealed dynamic regions of interest approximately 2 microns in size in which transients scaled with simulation intensity. Optogenetic stimulation of dopaminergic terminals produced similar transients, whereas optogenetic stimulation of glutamatergic terminals showed no effect on nIRCat signal. Bath application of nomifensine prolonged nIRCat fluorescence signal, consistent with reuptake blockade of dopamine. We further show that the chemically synthetic molecular recognition elements of nIRCats permit measurement of dopamine dynamics in the presence of dopamine receptor agonists and antagonists. These nIRCat nanosensors may be advantageous for future use because i) they do not require virus delivery, gene delivery, or protein expression, ii) their near-infrared fluorescence facilitates imaging in optically scattering brain tissue and is compatible for use in conjunction with other optical neuroscience tool sets, iii) the broad availability of unique near-infrared colors have the potential for simultaneous detection of multiple neurochemical signals, and iv) they are compatible with pharmacology. Together, these data suggest nIRCats and other nanosensors of this class can serve as versatile new optical tools to report dynamics of extracellular neuromodulation in the brain.

scholarly journals Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor

2019 ◽  
Vol 5 (7) ◽  
pp. eaaw3108 ◽  
Author(s):  
Abraham G. Beyene ◽  
Kristen Delevich ◽  
Jackson Travis Del Bonis-O’Donnell ◽  
David J. Piekarski ◽  
Wan Chen Lin ◽  
...  
Keyword(s):  
Brain Function ◽  
Dopamine Release ◽  
Near Infrared ◽  
Critical Role ◽  
Infrared Range ◽  
Optical Tools ◽  
Initial Release ◽  
Health And Disease ◽  
D2 Autoreceptor ◽  
Near Infrared Range

Neuromodulation plays a critical role in brain function in both health and disease, and new tools that capture neuromodulation with high spatial and temporal resolution are needed. Here, we introduce a synthetic catecholamine nanosensor with fluorescent emission in the near infrared range (1000–1300 nm), near infrared catecholamine nanosensor (nIRCat). We demonstrate that nIRCats can be used to measure electrically and optogenetically evoked dopamine release in brain tissue, revealing hotspots with a median size of 2 µm. We also demonstrated that nIRCats are compatible with dopamine pharmacology and show D2 autoreceptor modulation of evoked dopamine release, which varied as a function of initial release magnitude at different hotspots. Together, our data demonstrate that nIRCats and other nanosensors of this class can serve as versatile synthetic optical tools to monitor neuromodulatory neurotransmitter release with high spatial resolution.

066 Noradrenaline and acetylcholine inhibit sleep-promoting neurons of ventrolateral preoptic area through a local GABAergic circuit

SLEEP ◽  
2021 ◽  
Vol 44 (Supplement_2) ◽  
pp. A27-A28
Author(s):  
Roberto De Luca ◽  
Stefano Nardone ◽  
Lin Zhu ◽  
Elda Arrigoni
Keyword(s):  
Muscarinic Receptors ◽  
Inhibitory Action ◽  
Preoptic Area ◽  
Brain Slices ◽  
Inhibitory Effect ◽  
Gabaergic Neurons ◽  
Optogenetic Stimulation ◽  
Direct Inhibitory Effect ◽  
Afferent Inputs ◽  
Stimulation Of

Abstract Introduction The ventrolateral preoptic (VLPO) nucleus is a key area involved in the initiation and maintenance of sleep. During wakefulness, sleep-promoting galanin neurons in the VLPO are directly inhibited by arousal signals including noradrenaline and acetylcholine. We have found that while these neurotransmitters directly inhibit VLPO galanin neurons, they also activate GABAergic neurons in the VLPO that do not express galanin. We propose that when activated by monoaminergic and cholinergic inputs, these local VLPO GABAergic neurons provide an additional inhibition of the VLPO galanin sleep-promoting neurons. We tested this model in brain slices in mice. Methods We studied VLPO galanin neurons in mouse brain slices using patch-clamp recordings. We recorded from fluorescently labeled VLPO galanin neurons following the injection of a cre-dependent AAV encoding for mCherry, into the VLPO of Gal-cre mice. For the optogenetic studies we expressed channelrhodopsin-2 (ChR-2) in VLPO VGAT neurons and mCherry in galanin neurons by injecting a flp-dependent and a cre-dependent AAV encoding respectively for ChR2 and mCherry into the VLPO of VGAT-flp::Gal-cre mice. We photo-stimulated local GABAergic neurons and recorded from labeled VLPO galanin neurons. Noradrenaline, carbachol and receptor antagonists were bath-applied. Results Noradrenaline and carbachol inhibited VLPO galanin neurons by alpha-2 and muscarinic receptors and these effects were maintained in the presence of tetrodotoxin (TTX) indicating, as previously proposed, a direct inhibitory effect of noradrenaline and carbachol on VLPO galanin neurons. In addition, both noradrenaline and carbachol increased the frequency of spontaneous inhibitory post-synaptic currents (sIPSCs) of VLPO galanin neurons, suggesting an additional inhibitory action on VLPO galanin neurons. Finally, optogenetic stimulation of local VLPO GABAergic neurons produced short latency, TTX-resistant, opto-evoked IPSCs in VLPO galanin neurons. Both noradrenaline and carbachol increased the amplitude of these opto-evoked IPSCs by the activation of alpha-1 and muscarinic receptors. Conclusion Our results demonstrate that noradrenaline and acetylcholine inhibit VLPO galanin neurons directly and indirectly. Both noradrenaline and acetylcholine increase GABAergic afferent inputs to VLPO galanin neurons by activating local GABAergic neurons. We propose that during wakefulness this feedforward inhibition provides additional inhibition of VLPO galanin sleep-promoting neurons. Support (if any) NS091126 and HL149630

scholarly journals Optogenetic stimulation of long-range inputs and functional characterization of connectivity in patch-clamp recordings in mouse brain slices

2018 ◽  
Author(s):  
Louis Richevaux ◽  
Louise Schenberg ◽  
Mathieu Beraneck ◽  
Desdemona Fricker
Keyword(s):  
Patch Clamp ◽  
Long Range ◽  
Brain Slices ◽  
Cell Type ◽  
Patch Clamp Recording ◽  
Axon Terminals ◽  
Optogenetic Stimulation ◽  
Cell Type Specific ◽  
The Brain ◽  
Stimulation Of

Knowledge of cell type specific synaptic connectivity is a crucial prerequisite for understanding brain wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional, electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light sensitive ion channels allows to selectively stimulate axons coming from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterodorsal thalamic nuclei, and also in other subcortical or cortical areas throughout the brain. Here we describe a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the anterodorsal thalamus, followed by photostimulation of their axon terminals in hippocampal slices. In combination with whole-cell patch clamp recording from a postsynaptically connected presubicular neuron, photostimulation of thalamic axons allows the detection of functional synaptic connections, their pharmacological characterization, and the evaluation of their strength in the brain slice preparation. We demonstrate that axons originating in the anterodorsal thalamus ramify densely in presubicular layers 1 and 3. The photostimulation of Chronos expressing thalamic axon terminals in presubiculum initiates short latency postsynaptic responses in a presubicular layer3 neuron, indicating a monosynaptic connection. In addition, biocytin filling of the recorded neuron and posthoc revelation confirms the layer localization and pyramidal morphology of the postsynaptic neuron. Taken together, the optogenetic stimulation of long-range inputs in ex vivo brain slices is a useful method to determine the cell-type specific functional connectivity from distant brain regions.

scholarly journals Simultaneous Ca 2+ Imaging and Optogenetic Stimulation of Cortical Astrocytes in Adult Murine Brain Slices

2020 ◽  
Vol 94 (1) ◽  
Author(s):  
Lakshmini Balachandar ◽  
Karla A. Montejo ◽  
Eleane Castano ◽  
Melissa Perez ◽  
Carolina Moncion ◽  
...  
Keyword(s):  
Brain Slices ◽  
Optogenetic Stimulation ◽  
Cortical Astrocytes ◽  
Murine Brain ◽  
Stimulation Of

074 Basal Forebrain GABAergic Neurons Promote Arousal by Disinhibiting the Orexin Neurons via Local GABAergic Interneurons

SLEEP ◽  
2021 ◽  
Vol 44 (Supplement_2) ◽  
pp. A31-A31
Author(s):  
Michela Cristofolini ◽  
Roberto De Luca ◽  
Anne Venner ◽  
Loris Ferrari ◽  
Kevin Grace ◽  
...  
Keyword(s):  
Basal Forebrain ◽  
Viral Vector ◽  
Brain Slices ◽  
Nrem Sleep ◽  
Gabaergic Neurons ◽  
Gabaergic Interneurons ◽  
Optogenetic Stimulation ◽  
Stimulation Of

Abstract Introduction Optogenetic and chemogenetic studies have shown that activation of basal forebrain (BF) GABAergic neurons rapidly wakes up mice from non-REM (NREM) sleep. These wake-promoting responses have been attributed to BF GABAergic neurons projecting to the cerebral cortex and more specifically to the inhibition of cortical fast-spiking interneurons. Tracing studies have however found that BF GABAergic neurons also densely innervate the lateral hypothalamus (LH) perifornical area, although the role of this pathway in behavioral state control remains mostly unexplored. Methods We conducted in vivo and in vitro optogenetic studies. We selectively expressed channelrhodopsin-2 (ChR2) in BF GABAergic neurons by injecting a cre-dependent viral vector encoding for ChR2 into the BF of VGAT-cre mice. We photostimulated the BF GABAergic input to the LH with optical fibers placed into the LH of EEG instrumented mice. For in vitro recordings we expressed ChR2 in BF GABAergic neurons and we fluorescently labeled orexin or LH GABAergic neurons. We recorded in brain slices from identified orexin neurons or GABA neurons while photostimulating the BF GABAergic input. Results Optogenetic stimulation of the BF GABAergic fibers in the LH produced rapid arousals from NREM sleep. The same stimulation however did not wake up the mice if they were in REM sleep. We conducted additional studies in brain slices to identify the postsynaptic neurons in the LH targeted by the BF GABAergic input. We found that while optogenetic stimulation of the BF GABAergic input did not produce opto-evoked synaptic responses in the orexin neurons, it produced short-latency opto-evoked inhibitory postsynaptic currents (IPSCs) in LH GABAergic neurons. These opto-evoked IPSCs were GABAA receptor-mediated and were maintained in tetrodotoxin (TTX) indicating monosynaptic connectivity. We have previously found that orexin neurons are inhibited by local LH GABAergic neurons. Our hypothesis is that these local GABAergic interneurons are the target of the BF GABAergic arousal input. Conclusion BF GABAergic neurons drive arousal through projections to the LH. We propose that this arousal response is due to the inhibition of local GABAergic interneurons which in turn disinhibit the LH wake-promoting neurons including the orexin neurons. Support (if any) NS091126 and HL149630

scholarly journals Computational modeling of the photon transport, tissue heating, and cytochrome C oxidase absorption during transcranial near-infrared stimulation

2019 ◽  
Author(s):  
Mahasweta Bhattacharya ◽  
Anirban Dutta
Keyword(s):  
Cytochrome C Oxidase ◽  
Brain Tissue ◽  
Spectral Region ◽  
Temperature Increase ◽  
Near Infrared ◽  
Cortical Excitability ◽  
Tissue Heating ◽  
Photothermal Effects ◽  
The Brain ◽  
Stimulation Of

AbstractTranscranial near-infrared stimulation (tNIRS) has been proposed as a tool to modulate cortical excitability. However, the underlying mechanisms are not clear where the heating effects on the brain tissue needs investigation due to increased near-infrared (NIR) absorption by water and fat. Moreover, the risk of localized heating of tissues (including the skin) during optical stimulation of the brain tissue is a concern. The challenge in estimating localized tissue heating is due to the light interaction with the tissues’ constituents, which is dependent on the combination ratio of the scattering and absorption properties of the constituent. Here, apart from tissue heating that can modulate the cortical excitability (“photothermal effects”); the other mechanism reported in the literature is the stimulation of the mitochondria in the cells which are active in the adenosine triphosphate (ATP) synthesis. In the mitochondrial respiratory chain, the complex IV, also named as the cytochrome c oxidase(CCO), is the unit four with three copper atoms. The absorption peaks of CCO are in the visible (420-450nm and 600-700nm) and the near-infrared (760-980nm) spectral region which have been shown to be promising for low level light therapy (LLLT), also known as “photobiomodulation”. While much higher CCO absorption peaks in the visible spectrum can be used for the photobiomodulation of the skin, 810nm has been proposed for the non-invasive brain stimulation (using tNIRS) due to the optical window in the NIR spectral region. In this article, we applied a computational approach to delineate the “photothermal effects” from the “photobiomodulation,” i.e., to estimate the amount of light absorbed individually by each chromophore in the brain tissue (with constant scattering) and the related tissue heating. Photon migration simulations were performed for motor cortex tNIRS based on a prior work that used a 500mW cm−2 light source placed on the scalp. We simulated photon migration at 630nm and 700nm (red spectral region) and 810nm (near-infrared spectral region). We found a temperature increase in the scalp below 0.25 ° C and a minimal temperature increase in the gray matter less than 0.04 ° C at 810nm. Similar heating was found for 630nm and 700nm used for LLLT, so photothermal effects are postulated to be unlikely in the brain tissue.

scholarly journals Optogenetic stimulation of the liver-projecting melanocortinergic pathway promotes hepatic glucose production

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Eunjin Kwon ◽  
Hye-Young Joung ◽  
Shun-Mei Liu ◽  
Streamson C. Chua ◽  
Gary J. Schwartz ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Receptor Gene ◽  
Critical Role ◽  
Neuron Activity ◽  
Motor Nucleus ◽  
Glucose Levels ◽  
Optogenetic Stimulation ◽  
Blood Glucose Levels ◽  
Melanocortin 4 Receptor Gene ◽  
Stimulation Of

AbstractThe central melanocortin system plays a fundamental role in the control of feeding and body weight. Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) also regulate overall glucose homeostasis via insulin-dependent and -independent pathways. Here, we report that a subset of ARC POMC neurons innervate the liver via preganglionic parasympathetic acetylcholine (ACh) neurons in the dorsal motor nucleus of the vagus (DMV). Optogenetic stimulation of this liver-projecting melanocortinergic pathway elevates blood glucose levels that is associated with increased expression of hepatic gluconeogenic enzymes in female and male mice. Pharmacological blockade and knockdown of the melanocortin-4 receptor gene in the DMV abolish this stimulation-induced effect. Activation of melanocortin-4 receptors inhibits DMV cholinergic neurons and optogenetic inhibition of liver-projecting parasympathetic cholinergic fibers increases blood glucose levels. This elevated blood glucose is not due to altered pancreatic hormone release. Interestingly, insulin-induced hypoglycemia increases ARC POMC neuron activity. Hence, this liver-projecting melanocortinergic circuit that we identified may play a critical role in the counterregulatory response to hypoglycemia.

scholarly journals Chemogenetic stimulation of mouse central amygdala corticotropin-releasing factor neurons: Effects on cellular and behavioral correlates of alcohol dependence

2020 ◽  
Author(s):  
Max Kreifeldt ◽  
Melissa A Herman ◽  
Harpreet Sidhu ◽  
Giovana C de Macedo ◽  
Roxana Shahryari ◽  
...  
Keyword(s):  
Alcohol Withdrawal ◽  
Alcohol Intake ◽  
Anterior Commissure ◽  
Critical Role ◽  
Brain Slices ◽  
Receptor Activation ◽  
Corticotropin Releasing Factor ◽  
Central Nucleus ◽  
Crf Neurons ◽  
Stimulation Of

AbstractBackgroundCorticotropin-releasing factor (CRF) signaling in the central nucleus of the amygdala (CeA) plays a critical role in rodent models of excessive alcohol drinking. However, the source of CRF acting in the CeA during alcohol withdrawal remains to be identified. In the present study, we hypothesized that CeA CRF interneurons may represent a behaviorally relevant source of CRF to the CeA increasing motivation for alcohol via negative reinforcement.MethodsWe tested this hypothesis in male mice and used chemogenetics to stimulate CeA CRF neurons in vitro and in vivo.ResultsWe first observed that Crh mRNA expression in the anterior part of the mouse CeA, at the junction with the interstitial nucleus of the posterior limb of the anterior commissure, correlates positively with alcohol intake in C57BL/6J males with a history of chronic binge drinking. We then found that chemogenetic activation of CeA CRF neurons in Crh-IRES-Cre mouse brain slices increases gamma-aminobutyric acid (GABA) release in the medial CeA in part via CRF1 receptor activation, indicating local CRF release. While chemogenetic stimulation of CeA CRF neurons exacerbated novelty-induced feeding suppression, as seen in C57BL/6J males withdrawn from chronic intermittent alcohol inhalation, it had no effect on voluntary alcohol consumption, following either acute or chronic manipulation.ConclusionsAltogether, these findings indicate that hyperactivity of CeA CRF neurons may contribute to elevated CeA GABA levels and negative affect during alcohol withdrawal but is not sufficient to drive alcohol intake escalation in dependent mice.

scholarly journals Voltage-sensitive dye delivery through the blood brain barrier using adenosine receptor agonist Regadenoson

2018 ◽  
Author(s):  
Rebecca W. Pak ◽  
Jeeun Kang ◽  
Heather Valentine ◽  
Leslie M. Loew ◽  
Daniel L.J. Thorek ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Blood Brain Barrier ◽  
Near Infrared ◽  
Brain Activity ◽  
Brain Barrier ◽  
Fluorescence Signal ◽  
Voltage Sensitive Dye ◽  
Blood Brain ◽  
Adenosine Receptor Agonist ◽  
The Brain

AbstractOptical imaging of brain activity has mostly employed genetically manipulated mice, which cannot be translated to clinical human usage. Observation of brain activity directly is challenging due to difficulty in delivering dyes and other agents through the blood brain barrier (BBB). Using fluorescence imaging, we have demonstrated the feasibility of delivering the near-infrared voltage-sensitive dye (VSD) IR-780 perchlorate to the brain tissue through pharmacological techniques, via an adenosine agonist (Regadenoson). Comparison of VSD fluorescence of mouse brains without and with Regadenoson showed significantly increased residence time of the fluorescence signal in the latter case, indicative of VSD diffusion into the brain tissue. Dose and timing of Regadenoson were varied to optimize BBB permeability for VSD delivery.

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