Spinal Glycine Receptor Alpha 3 Cells Communicate Sensations of Chemical Itch in Hairy Skin.

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 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.

A deep-dive into fictive locomotion - a strategy to probe cellular activity during speed transitions in fictively swimming zebrafish larvae.

Fictive locomotion is frequently used to study locomotor output in paralyzed animals. We have evaluated the character of swim episodes elicited by different strategies in zebrafish. Motor output was measured on both sides of a body segment using electrodes and a pipeline for synchronizing stimulation and recording, denoising data and peak-finding was developed. The optomotor response generated swims most equivalent to spontaneous activity, while electrical stimulation and NMDA application caused various artefacts. Our optimal settings, optomotor stimulation using 5-day-old larvae, were combined with calcium imaging and optogenetics to validate the setup's utility. Expression of GCaMP5G by the mnx1 promoter allowed correlation of calcium traces of dozens of motor neurons to the fictive locomotor output. Activation of motor neurons through channelrhodopsin produced aberrant locomotor episodes. This strategy can be used to investigate novel neuronal populations in a high-throughput manner to reveal their role in shaping motor output. This article has an associated First Person interview with the first author of the paper.

Targeting barrel field spiny stellate cells using a vesicular monoaminergic transporter 2-Cre mouse line.

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.

Differentiation of Human Embryonic Stem Cells into Neuron, Cholinergic, and Glial Cells.

Human embryonic stem cells (hESCs) are pluripotent cells, capable of differentiation into different cellular lineages given the opportunity. Derived from the inner cell mass of blastocysts in early embryonic development, the cell self-renewal ability makes them a great tool for regenerative medicine, and there are different protocols available for maintaining hESCs in their undifferentiated state. In addition, protocols for differentiation into functional human neural stem cells (hNSCs), which have the potential for further differentiation into various neural cell types, are available. However, many protocols are time-consuming and complex and do not always fit for purpose. In this study, we carefully combined, optimized, and developed protocols for differentiation of hESCs into adherent monolayer hNSCs over a short period of time, with the possibility of both expansion and freezing. Moreover, the method details further differentiation into neurons, cholinergic neurons, and glial cells in a simple, single step by step protocol. We performed immunocytochemistry, qPCR, and electrophysiology to examine the expression profile and characteristics of the cells to verify cell lineage. Using presented protocols, the creation of neuronal cultures, cholinergic neurons, and a mixed culture of astrocytes and oligodendrocytes can be completed within a three-week time period.

Spinal gastrin releasing peptide receptor expressing interneurons are controlled by local phasic and tonic inhibition.

Dorsal horn gastrin-releasing peptide receptor (GRPR) neurons have a central role in itch transmission. Itch signaling has been suggested to be controlled by an inhibitory network in the spinal dorsal horn, as increased scratching behavior can be induced by pharmacological disinhibition or ablation of inhibitory interneurons, but the direct influence of the inhibitory tone on the GRPR neurons in the itch pathway have not been explored. Here we have investigated spinal GRPR neurons through in vitro and bioinformatical analysis. Electrophysiological recordings revealed that GRPR neurons receive local spontaneous excitatory inputs transmitted by glutamate and inhibitory inputs by glycine and GABA, which were transmitted either by separate glycinergic and GABAergic synapses or by glycine and GABA co-releasing synapses. Additionally, all GRPR neurons received both glycine- and GABA-induced tonic currents. The findings show a complex inhibitory network, composed of synaptic and tonic currents that gates the excitability of GRPR neurons, which provides direct evidence for the existence of an inhibitory tone controlling spontaneous discharge in an itch-related neuronal network in the spinal cord. Finally, calcium imaging revealed increased levels of neuronal activity in Grpr-Cre neurons upon application of somatostatin, which provides direct in vitro evidence for disinhibition of these dorsal horn interneurons.

Liraglutide modulates GABAergic signaling in rat hippocampal CA3 pyramidal neurons predominantly by presynaptic mechanism.

BACKGROUND: γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain where it regulates activity of neuronal networks. The receptor for glucagon-like peptide-1 (GLP-1) is expressed in the hippocampus, which is the center for memory and learning. In this study we examined effects of liraglutide, a GLP-1 analog, on GABA signaling in CA3 hippocampal pyramidal neurons. METHODS: We used patch-clamp electrophysiology to record synaptic and tonic GABA-activated currents in CA3 pyramidal neurons in rat hippocampal brain slices. RESULTS: We examined the effects of liraglutide on the neurons at concentrations ranging from one nM to one µM. Significant changes of the spontaneous inhibitory postsynaptic currents (sIPSCs) were only recorded with 100 nM liraglutide and then in just ≈50% of the neurons tested at this concentration. In neurons affected by liraglutide both the sIPSC frequency and the most probable amplitudes increased. When the action potential firing was inhibited by tetrodotoxin (TTX) the frequency and amplitude of IPSCs in TTX and in TTX plus 100 nM liraglutide were similar. CONCLUSIONS: The results demonstrate that liraglutide regulation of GABA signaling of CA3 pyramidal neurons is predominantly presynaptic and more limited than has been observed for GLP-1 and exendin-4 in hippocampal neurons.

Spatial characterization of a multifunctional pipette for drug delivery in hippocampal brain slices.

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.

A multifunctional pipette for localized drug administration to brain slices.

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.

Effect of cholesterol depletion on the pore dilation of TRPV1.

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.