From decision to action: Detailed modelling of frog tadpoles reveals neuronal mechanisms of decision-making and reproduces unpredictable swimming movements in response to sensory signals

How does the brain process sensory stimuli, and decide whether to initiate locomotor behaviour? To investigate this question we develop two whole body computer models of a tadpole. The “Central Nervous System” (CNS) model uses evidence from whole-cell recording to define 2000 neurons in 12 classes to study how sensory signals from the skin initiate and stop swimming. In response to skin stimulation, it generates realistic sensory pathway spiking and shows how hindbrain sensory memory populations on each side can compete to initiate reticulospinal neuron firing and start swimming. The 3-D “Virtual Tadpole” (VT) biomechanical model with realistic muscle innervation, body flexion, body-water interaction, and movement is then used to evaluate if motor nerve outputs from the CNS model can produce swimming-like movements in a volume of “water”. We find that the whole tadpole VT model generates reliable and realistic swimming. Combining these two models opens new perspectives for experiments.

Nanchung and Inactive define pore properties of the native auditory transduction channel in Drosophila

Auditory transduction is mediated by chordotonal (Cho) neurons in Drosophila larvae, but the molecular identity of the mechanotransduction (MET) channel is elusive. Here, we established a whole-cell recording system of Cho neurons and showed that two transient receptor potential vanilloid (TRPV) channels, Nanchung (NAN) and Inactive (IAV), are essential for MET currents in Cho neurons. NAN and IAV form active ion channels when expressed simultaneously in S2 cells. Point mutations in the pore region of NAN-IAV change the reversal potential of the MET currents. Particularly, residues 857 through 990 in the IAV carboxyl terminus regulate the kinetics of MET currents in Cho neurons. In addition, TRPN channel NompC contributes to the adaptation of auditory transduction currents independent of its ion-conduction function. These results indicate that NAN-IAV, rather than NompC, functions as essential pore-forming subunits of the native auditory transduction channel in Drosophila and provide insights into the gating mechanism of MET currents in Cho neurons.

ROMK channels are inhibited in the aldosterone-sensitive distal nephron (ASDN) of renal-tubule Nedd4-2 deficient mice.

We used whole-cell-recording to examine renal-outer-medullary-K+ channel (ROMK or Kir1.1) and epithelial-Na+-Channel (ENaC) in late-distal-convoluted-tubule (DCT2)/initial-connecting-tubule (iCNT) and in the cortical-collecting-duct (CCD) of kidney-tubule-specific Nedd4-2 knockout mice (Ks-Nedd4-2-KO) and floxed-Nedd4l mice (control). TPNQ-sensitive K+ currents (ROMK) were smaller in both DCT2/iCNT and CCD of Ks-Nedd4-2-KO mice on normal diet than control mice. Neither high-dietary-salt-intake (HS) nor low-dietary-salt-intake (LS) had a significant effect on ROMK activity in the DCT2/iCNT and CCD of control and Ks-Nedd4-2-KO mice. In contrast, high-dietary-K+-intake (HK) increased while low-dietary-K+-intake (LK) decreased TPNQ-sensitive K+ currents in floxed-Nedd4l mice. However, effects of dietary-K+ intake on ROMK channel activity were absent in Ks-Nedd4-2-KO mice since neither HK nor LK significantly affected TPNQ-sensitive K+ currents in DCT2/iCNT and CCD. Moreover. TPNQ-sensitive K+ currents in DCT2/iCNT and the CCD of Ks-Nedd4-2-KO mice on HK were similar to the control mice on LK. Amiloride-sensitive Na+ currents in DCT2/iCNT and CCD were significantly higher in Ks-Nedd4-2-KO mice than floxed-Nedd4l mice on normal-K+-diet. HK increased ENaC activity of DCT2/iCNT only in the control mice but HK stimulated ENaC of the CCD in both control and Ks-Nedd4-2-KO mice. Moreover, HK-induced increase in amiloride-sensitive Na+-currents was larger in Ks-Nedd4-2-KO mice than the control mice. Deletion of Nedd4-2 increased WNK1 expression and abolished the HK-induced inhibition of WNK1. We conclude that deletion of Nedd4-2 increases ENaC activity but decreases ROMK activity in aldosterone-sensitive distal nephron (ASDN) and that HK fails to stimulate ROMK but robustly increases ENaC activity in the CCD of Nedd4-2-deficeint mice.

Diversified expression of gamma-protocadherins regulates synaptic specificity in the mouse neocortex

Synaptic specificity is the basis of forming neural microcircuits. However, how a neuron chooses which neurons out of many potentials to form synapses remains largely unknown. Here we identified that the diversified expression of clustered protocadherin γs (cPCDHγs) plays an essential role in regulating such specificity. Our 5-prime end single-cell sequencing data revealed the diversified expression pattern of cPCDHγs in neocortical neurons. Whole-cell recording of neuron pairs in developing mouse brain slices showed that knocking out PCDHγs significantly increased the local connection rate of nearby pyramidal neurons. By contrast, neurons overexpressing the same group of clustered PCDHγ isoforms through in utero electroporation dramatically decreased their synaptic connectivity. Finally and more importantly, decreasing the similarity level of PCDHγ isoforms over-expressed in neuron pairs through sequential in utero electroporation led to a progressive elevation of synaptic connectivity. Our observations provide strong evidence to support that the existence of diversely expressed cPCDHγs allows a neuron to choose which neurons not to form a synapse, rather than choosing which neurons to make synapses.

Reversible Slow Desensitization of the Acid-Sensing Ion Channel (ASIC) 1a

Among the proton-activated channels of the ASIC family, ASIC1a exhibits a specific tachyphylaxis phenomenon in the form of a progressive decrease in the response amplitude during a series of activations. This process is well known, but its mechanism is poorly understood. Here, we demonstrated a partial reversibility of this effect by long-term whole-cell recording of CHO cells transfected with rASIC1a cDNA. Long but infrequent acidifications provided the same recovery time course as short acidifications of the same frequency. Steady-state desensitization is not related to the slow desensitization and attenuates the development of the slow desensitization. Consequently, we found that drugs, which facilitate ASIC1a activation (e.g., amitriptyline), cause an enhancement of slow desensitization, while inhibition of ASIC1a by 9-aminoacridine attenuates the slow desensitization. In summary, for influences of vastly different origin, including increase of calcium concentration, different pH conditions, and action of modulating drugs, we found a correlation between the effect on response amplitude and on development of slow desensitization. Thus, our results prove that a slow desensitization requires the open ion-permeable state.

Cannabidiol Selectively Binds to the Voltage-Gated Sodium Channel Nav1.4 in Its Slow-Inactivated State and Inhibits Sodium Current

Cannabidiol (CBD), one of the cannabinoids from the cannabis plant, can relieve the myotonia resulting from sodium channelopathy, which manifests as repetitive discharges of muscle membrane. We investigated the binding kinetics of CBD to Nav1.4 channels on the muscle membrane. The binding affinity of CBD to the channel was evaluated using whole-cell recording. The CDOCKER program was employed to model CBD docking onto the Nav1.4 channel to determine its binding sites. Our results revealed no differential inhibition of sodium current by CBD when the channels were in activation or fast inactivation status. However, differential inhibition was observed with a dose-dependent manner after a prolonged period of depolarization, leaving the channel in a slow-inactivated state. Moreover, CBD binds selectively to the slow-inactivated state with a significantly faster binding kinetics (>64,000 M−1 s−1) and a higher affinity (Kd of fast inactivation vs. slow-inactivation: >117.42 μM vs. 51.48 μM), compared to the fast inactivation state. Five proposed CBD binding sites in a bundle crossing region of the Nav1.4 channels pore was identified as Val793, Leu794, Phe797, and Cys759 in domain I/S6, and Ile1279 in domain II/S6. Our findings imply that CBD favorably binds to the Nav1.4 channel in its slow-inactivated state.

Microglial Activation of GLP-1R Signaling in Neuropathic Pain Promotes Gene Expression Adaption Involved in Inflammatory Responses

Background. Neuropathic pain is a common chronic pain, which is related to hypersensitivity to stimulus and greatly affects the quality of life of patients. Maladaptive gene changes and molecular signaling underlie the sensitization of nociceptive pathways. We previously found that the activation of microglial glucagon-like peptide 1 receptor (GLP-1R) could potently relieve formalin-, bone cancer-, peripheral nerve injury-, and diabetes-induced pain hypersensitivity. So far, little is known about how the gene profile changes upon the activation of GLP-1R signaling in the pathophysiology of neuropathic pain. Methods. Spinal nerve ligation (SNL) was performed to induce neuropathic pain in rats. Mechanical allodynia was assessed using von Frey filaments. The expression of IL-10, β-endorphin, and μ-opioid receptor (MOR) was examined by real-time quantitative polymerase chain reaction (qPCR) and whole-cell recording. Measurements of cellular excitability of the substantia gelatinosa (SG) neurons by whole-cell recording were carried out. R packages of differential gene expression analysis based on the negative binomial distribution (DESeq2) and weighted correlation network analysis (WGCNA) were used to analyze differential gene expression and the correlated modules among GLP-1R clusters in neuropathic pain. Results. The GLP-1R agonist, exenatide, has an antiallodynic effect on neuropathic pain, which could be reversed by intrathecal injections of the microglial inhibitor minocycline. Furthermore, differential gene expression analysis (WGCNA) indicated that intrathecal injections of exenatide could reverse the abnormal expression of 591 genes in the spinal dorsal horn induced by nerve injury. WGCNA revealed 58 modules with a close relationship between the microglial GLP-1R pathway and features of nerve injuries, including pain, ligation, paw withdrawal latency (PWL), and anxiety. The brown module was identified as the highest correlated module, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that inflammatory responses were most correlated with PWL. To further unravel the changes of hyperalgesia-related neuronal electrophysiological activity mediated by microglia GLP-1 receptors, whole-cell recording identified that MOR agonism stimulated a robust outward current in the sham groups compared with the spinal nerve ligation (SNL) groups. This inhibitory effect on the SNL group was more sensitive than that of the sham group after bath application of β-endorphin. Conclusions. Our results further confirmed that the GLP-1R pathway is involved in alleviating pain hypersensitivity mediated by spinal microglia activation, and inflammatory responses were the most correlated pathway associated with PWL changes in response to exenatide treatment. We found that the identification of gene regulation in response to GLP-1R activation is an effective strategy for identifying new therapeutic targets for neuropathic pain. Investigation for the activation of spinal microglial GLP-1R which might ameliorate inflammatory responses through gene expression and structural changes is providing a potential biomarker in pain management.

From libraries to wayfinding, waylosing, and symbolism

After demonstrating that a building is a system of systems, we examine the symbolism of certain libraries. A cognitive account of wayfinding uses the Seattle Public Library to analyze getting lost in buildings—which we contrast with waylosing as in exploration. Cognitive maps in the brain represent places and the means to find one’s way between them. Different “worlds” each have their own, modeled as a world graph (WG) with distinctive places represented by nodes, and paths represented by edges. Complementing this, a locometric map represents locomotor effort in getting from one place to another. Single-cell recording from rat hippocampus reveals place cells whose activity correlates with the place in which the animal finds itself. However, “place” here corresponds to location on a locometric map, rather than distinctive places of WG nodes. The taxon affordance model (TAM), models how one navigates without a cognitive map. Several brain regions are involved, but not hippocampus. The world graph model (WGM) makes essential use of the hippocampus in coordination with brain regions processing the relevant WG. Finally, we contrast symbolic form in buildings with the use of explicit signage. Oscar Niemeyer’s Brasilia Cathedral exemplifies how architects may achieve novel symbolic forms.

Deletion of renal Nedd4-2 abolishes the effect of high K+ intake (HK) on Kir4.1/Kir5.1 and NCC activity in the distal convoluted tubule.

High-dietary K+ (HK) intake inhibits the basolateral Kir4.1/Kir5.1 activity in the distal convoluted tubule (DCT) and HK-induced inhibition of Kir4.1/Kir5.1 is essential for HK-induced inhibition of Na-Cl cotransporter (NCC). We now examine whether Nedd4-2-deletion compromises the effect of HK on basolateral Kir4.1/Kir5.1 and NCC in the DCT. Single- channel-recording and whole-cell-recording showed that neither HK decreased nor low-dietary-K+ (LK) increased the basolateral Kir4.1/Kir5.1 activity of the DCT in kidney-tubule-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. In contrast, HK inhibited and LK increased Kir4.1/Kir5.1 activity in the control mice (Nedd4lflox/flox). Also, HK-intake decreased the negativity of K+-current (IK) reversal potential in the DCT (depolarization) only in the control mice but not in Ks-Nedd4-2 KO mice. Renal clearance experiments showed that HK-intake decreased while LK intake increased hydrochlorothiazide (HCTZ)-induced renal Na+ excretion only in the control mice but this effect was absent in Ks-Nedd4-2 KO mice. Western blot also demonstrated that HK-induced inhibition of phosphor-NCC (pNCC at Thr53) and total NCC (tNCC) was observed only in the control but not in Ks-Nedd4-2 KO mice. Furthermore, the expression of all three subunits of epithelia-Na+-channel (ENaC) in the Ks-Nedd4-2 KO mice on HK was higher than in the control mice. Thus, plasma K+ concentrations were similar between Nedd4lflox/flox and Ks-Nedd4-2 KO mice on HK for 7 days despite high NCC expression. We conclude that Nedd4-2 plays a role in regulating HK-induced inhibition of Kir4.1/Kir5.1 and NCC in the DCT.