| 1. |
- Jansson, Erik, 1984, et al.
(författare)
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Effect of cholesterol depletion on the pore dilation of TRPV1
- 2013
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Ingår i: Molecular Pain. - : SAGE Publications. - 1744-8069. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.
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| 2. |
- Ahemaiti, Aikeremu, 1984, et al.
(författare)
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A multifunctional pipette for localized drug administration to brain slices
- 2013
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Ingår i: Journal of Neuroscience Methods. - : Elsevier BV. - 0165-0270 .- 1872-678X. ; 219:2, s. 292-296
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a superfusion method utilizing an open-volume microfluidic device for administration of pharmacologically active substances to selected areas in brain slices with high spatio-temporal resolution. The method consists of a hydrodynamically confined flow of the active chemical compound, which locally stimulates neurons in brain slices, applied in conjunction with electrophysiological recording techniques to analyze the response. The microfluidic device, which is a novel free-standing multifunctional pipette, allows diverse superfusion experiments, such as testing the effects of different concentrations of drugs or drug candidates on neurons in different cell layers with high positional accuracy, affecting only a small number of cells. We demonstrate herein the use of the method with electrophysiological recordings of pyramidal cells in hippocampal and prefrontal cortex brain slices from rats, determine the dependence of electric responses on the distance of the superfusion device from the recording site, document a multifold gain in solution exchange time as compared to whole slice perfusion, and show that the device is able to store and deliver up to four solutions in a series. Localized solution delivery by means of open-volume microfluidic technology also reduces reagent consumption and tissue culture expenses significantly, while allowing more data to be collected from a single tissue slice, thus reducing the number of laboratory animals to be sacrificed for a study. (C) 2013 Elsevier B.V. All rights reserved.
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| 3. |
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| 4. |
- Ahemaiti, Aikeremu, 1984
(författare)
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Microfluidics based techniques for electrophysiological studies of cells
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis work investigates the application of microfluidics to perform electrophysiological studies on cells, including investigations of the effect of cholesterol on the dynamic ion permeability of TRPV1 ion channels, and the application of a microfluidic device, the multifunctional pipette, in electrophysiological studies on brain slices. In the first part of this thesis, Chinese hamster ovary (CHO) cells overexpressing the TRPV1 ion channel were used in a dynamic ion permeability study, where the activation properties of the TRPV1 ion channel were investigated using the patch clamp technique after depletion of membrane cholesterol. The dynaflow system, an open-volume multichannel microfluidic system, and the multifunctional pipette, a freestanding microfluidic device utilizing hydrodynamically confined flow for spatially confined solution exchange, were used to deliver chemical stimuli exclusively to the patched cell. The result showed that the depletion of membrane cholesterol impaired the dynamic permeability of large cations in TRPV1 in low calcium solutions. The second project focused on the application of the multifunctional pipette in neuropharmacological studies of the brain slices. We developed an experimental setup, performed feasibility studies, characterized the device performance and compared it with common superfusion techniques, using extra- and intracellular electrophysiological recordings of pyramidal cells in hippocampal and prefrontal cortex brain slices from rats. The multifunctional pipette was used in these experiments for highly localized delivery of the competitive AMPA receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) to selected locations on the slices. By applying multifunctional pipette, we achieved a multifold gain in solution exchange time and more efficient drug delivery compared to whole slice perfusion. The amount of drugs required in the microfluidics-supported experiments was by several orders of magnitude smaller. The multifunctional pipette enabled selective perfusion of a single dendritic layer in the CA1 region of hippocampus with CNQX, without affecting other layers in this region.
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| 5. |
- Ahemaiti, Aikeremu, 1984, et al.
(författare)
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Spatial characterization of a multifunctional pipette for drug delivery in hippocampal brain slices
- 2015
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Ingår i: Journal of Neuroscience Methods. - : Elsevier BV. - 0165-0270 .- 1872-678X. ; 241, s. 132-136
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Tidskriftsartikel (refereegranskat)abstract
- Background: Among the various fluidic control technologies, microfluidic devices are becoming powerful tools for pharmacological studies using brain slices, since these devices overcome traditional limitations of conventional submerged slice chambers, leading to better spatiotemporal control over delivery of drugs to specific regions in the slices. However, microfluidic devices are not yet fully optimized for such studies. New method: We have recently developed a multifunctional pipette (MFP), a free standing hydrodynamically confined microfluidic device, which provides improved spatiotemporal control over drug delivery to biological tissues. Results: We demonstrate herein the ability of the MFP to selectively perfuse one dendritic layer in the CA1 region of hippocampus with CNQX, an AMPA receptor antagonist, while not affecting the other layers in this region. Our experiments also illustrate the essential role of hydrodynamic confinement in sharpening the spatial selectivity in brain slice experiments. Concentration-response measurements revealed that the ability of the MFP to control local drug concentration is comparable with that of whole slice perfusion, while in comparison the required amounts of active compounds can be reduced by several orders of magnitude. Comparison with existing method: The multifunctional pipette is applied with an angle, which, compared to other hydrodynamically confined microfluidic devices, provides more accessible space for other probing and imaging techniques. Conclusions: Using the MFP it will be possible to study selected regions of brain slices, integrated with various imaging and probing techniques, without affecting the other parts of the slices.
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| 6. |
- Franck, Marina Christina Mikaela, et al.
(författare)
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Urocortin3-expressing neurons in sensory transmission
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Urocortin 3 (UCN3) is a neuropeptide involved in mechanosensation and stress regulation, and Ucn3-Cre neurons have been assigned a role in mechanical itch. Here, we show that Ucn3 marks a population of excitatory neurons in the mouse dorsal horn, divided into two non-overlapping subpopulations expressing protein kinase C g or calretinin/calbindin 2. Chemogenetic activation of spinal Ucn3-Cre neurons evoked a targeted biting/licking behavior towards the corresponding dermatome. Genetic deletion of vesicular glutamate transporter 2 (VGLUT2) in Ucn3-Cre neurons removed the phenotype, showing that the biting/licking behavior is VGLUT2-dependent. Conditional deletion of VGLUT2 did not affect acute thermal or mechanical withdrawal responses, nor thermal withdrawal responses after nerve growth factor-induced hypersensitivity or the prurifensive response to 48/80 or von Frey stimuli applied in nape. Instead, we found that a group of spinal Ucn3 neurons were activated in response to artificial scratching or 48/80-induced itch. Electrophysiological experiments showed that spinal Ucn3 neurons received both glycinergic and GABAergic tonic inhibition, and monosynaptic inputs from both Aβ and C fibers, which could be confirmed by rabies tracing. Spinal Ucn3/Ucn3-Cre neurons thus represent a mechanically sensitive population with several roles in the itch-scratch cycle.
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| 7. |
- Freitag, Fabio B., et al.
(författare)
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Targeting barrel field spiny stellate cells using a vesicular monoaminergic transporter 2-Cre mouse line
- 2021
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Rodent primary somatosensory cortex (S1) is organized in defined layers, where layer IV serves as the main target for thalamocortical projections. Serotoninergic signaling is important for the organization of thalamocortical projections and consequently proper barrel field development in rodents, and the vesicular monoamine transporter 2 (VMAT2) can be detected locally in layer IV S1 cortical neurons in mice as old as P10, but the identity of the Vmat2-expressing neurons is unknown. We here show that Vmat2 mRNA and also Vmat2-Cre recombinase are still expressed in adult mice in a sub-population of the S1 cortical neurons in the barrel field. The Vmat2-Cre cells showed a homogenous intrinsically bursting firing pattern determined by whole-cell patch-clamp, localized radial densely spinous basal dendritic trees and almost exclusively lack of apical dendrite, indicative of layer IV spiny stellate cells. Single cell mRNA sequencing analysis showed that S1 cortical Vmat2-Cre;tdTomato cells express the layer IV marker Rorb and mainly cluster with layer IV neurons, and RNAscope analysis revealed that adult Vmat2-Cre neurons express Vmat2 and vesicular glutamate transporter 1 (Vglut1) and Vglut2 mRNA to a high extent. In conclusion, our analysis shows that cortical Vmat2 expression is mainly confined to layer IV neurons with morphological, electrophysiological and transcriptional characteristics indicative of spiny stellate cells.
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| 8. |
- Weman, Hannah M., 1993-, et al.
(författare)
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Spinal Glycine Receptor Alpha 3 Cells Communicate Sensations of Chemical Itch in Hairy Skin
- 2024
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Ingår i: Journal of Neuroscience. - : Society for Neuroscience. - 0270-6474 .- 1529-2401. ; 44:19
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Tidskriftsartikel (refereegranskat)abstract
- Glycinergic neurons regulate nociceptive and pruriceptive signaling in the spinal cord, but the identity and role of the glycineregulated neurons are not fully known. Herein, we have characterized spinal glycine receptor alpha 3 (Glra3) subunit-expressing neurons in Glra3-Cre female and male mice. Glra3-Cre(+) neurons express Glra3, are located mainly in laminae III-VI, and respond to glycine. Chemogenetic activation of spinal Glra3-Cre(+) neurons induced biting/licking, stomping, and guarding behaviors, indicative of both a nociceptive and pruriceptive role for this population. Chemogenetic inhibition did not affect mechanical or thermal responses but reduced behaviors evoked by compound 48/80 and chloroquine, revealing a pruriceptive role for these neurons. Spinal cells activated by compound 48/80 or chloroquine express Glra3, further supporting the phenotype. Retrograde tracing revealed that spinal Glra3-Cre(+) neurons receive input from afferents associated with pain and itch, and dorsal root stimulation validated the monosynaptic input. In conclusion, these results show that spinal Glra3(+) neurons contribute to acute communication of compound 48/80- and chloroquine-induced itch in hairy skin.
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| 9. |
- Weman, Hannah M., 1993-, et al.
(författare)
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Spinal glycine receptor alpha 3 subunit-expressing cells transmit the sensations of compound 48/80 and chloroquine
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Glycinergic neurons regulate nociceptive and pruriceptive signaling in the spinal cord, but the identity and role of the glycine-regulated neurons are not fully known. Herein, we have characterized spinal glycine receptor alpha 3 (Glra3) subunit-expressing neurons in a Glra3-Cre mouse line. Glra3-Cre neurons express Glra3, are located mainly in laminae III-VI and respond to glycine. Chemogenetic activation of spinal Glra3-Cre neurons induced biting/licking, stamping and guarding behaviors, while inhibition reduced behaviors evoked by compound 48/80 and chloroquine, revealing a pruriceptive role for this population. Spinal cells activated by compound 48/80 or chloroquine express Glra3, further supporting the phenotype. Retrograde tracing revealed that spinal Glra3-Cre neurons receive input mainly from NF200(+), IB4(+), TRKA(+), CGRP(+) or SST(+) primary afferents. Dorsal root stimulation validated the monosynaptic input from primary afferents, which was mainly comprised of C-fiber or C-fiber and Aα∕β input. Conclusively, these results show that spinal Glra3 neurons contribute to acute transmission of compound 48/80 and chloroquine-induced itch.
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