GABA
/glycine-mediated neuronal inhibition critically depends on intracellular chloride (Cl
) concentration which is mainly regulated by the K
-Cl
co-transporter 2 (KCC2) in the adult central nervous ...system (CNS). KCC2 heterogeneity thus affects information processing across CNS areas. Here, we uncover a gradient in Cl
extrusion capacity across the superficial dorsal horn (SDH) of the spinal cord (laminae I-II: LI-LII), which remains concealed under low Cl
load. Under high Cl
load or heightened synaptic drive, lower Cl
extrusion is unveiled in LI, as expected from the gradient in KCC2 expression found across the SDH. Blocking TrkB receptors increases KCC2 in LI, pointing to differential constitutive TrkB activation across laminae. Higher Cl
lability in LI results in rapidly collapsing inhibition, and a form of activity-dependent synaptic plasticity expressed as a continuous facilitation of excitatory responses. The higher metaplasticity in LI as compared to LII differentially affects sensitization to thermal and mechanical input. Thus, inconspicuous heterogeneity of Cl
extrusion across laminae critically shapes plasticity for selective nociceptive modalities.
•Updating von Frey methodology to apply monofilaments more efficiently.•What the test measures: theoretical considerations.•Practical usage of von Frey's hairs in the laboratory ...environment.•Tightening sensory threshold estimates from raw data.•Statistics of (group) threshold estimation.
Here, we reconsider the status quo in testing mechanical sensitivity with von Frey's hairs. The aim is to improve paw withdrawal estimates by integrating current psychometric theory, and to maximise the clinical relevance and statistical power of mechanosensory models. A wealth of research into human tactile stimulus perception may be extended to the quantification of laboratory animal behaviour. We start by reviewing each step of the test, from its design and application through to data analysis. Filament range is assessed as a whole; possible test designs are compared; techniques of filament application to mice and rats are considered; curve fitting software is introduced; possibilities for data pooling and curve fitting are evaluated. A rational update of classical methods in line with recent advances in psychometrics and supported by open source software is expected to improve data homogeneity, and Reduce and Refine animal use in accord with the ‘3R’ principles.
Analysis of the interplay between cell proliferation and death has been greatly advantaged by the development of CNS slice preparations. In slices, interactions between neurons and neurons and the ...glial cells are fundamentally preserved in a fashion close to the in vivo situation. In parallel, these preparations offer the possibility of an easy experimental manipulation. Two main types of slices are currently in use: the acute slices, which are short living preparations where the major functions of the intact brain (including neurogenesis) are maintained, and the organotypic cultures, where the maturation and plasticity of neuronal circuitries in relation to naturally occurring neuronal death and/or experimental insults can be followed over several weeks in vitro. We will discuss here the main advantages/disadvantages linked to the use of CNS slices for histological analysis of neuronal proliferation and death, as well as the main findings obtained in the most popular types of preparations, i.e. the cortical, hippocampal, cerebellar and retinal slices.
C-nociceptors (C-Ncs) and non-nociceptive C-low threshold mechanoreceptors (C-LTMRs) are two subpopulations of small unmyelinated non-peptidergic C-type neurons of the dorsal root ganglia (DRGs) with ...central projections displaying a specific pattern of termination in the spinal cord dorsal horn. Although these two subpopulations exist in several animals, remarkable neurochemical differences occur between mammals, particularly rat/humans from one side and mouse from the other. Mouse is widely investigated by transcriptomics. Therefore, we here studied the immunocytochemistry of murine C-type DRG neurons and their central terminals in spinal lamina II at light and electron microscopic levels. We used a panel of markers for peptidergic (CGRP), non-peptidergic (IB4), nociceptive (TRPV1), non-nociceptive (VGLUT3) C-type neurons and two strains of transgenic mice: the TAFA4
Venus
knock-in mouse to localize the TAFA4
+
C-LTMRs, and a genetically engineered
ginip
mouse that allows an inducible and tissue-specific ablation of the DRG neurons expressing GINIP, a key modulator of GABA
B
R-mediated analgesia. We confirmed that IB4 and TAFA4 did not coexist in small non-peptidergic C-type DRG neurons and separately tagged the C-Ncs and the C-LTMRs. We then showed that TRPV1 was expressed in only about 7% of the IB4
+
non-peptidergic C-Ncs and their type Ia glomerular terminals within lamina II. Notably, the selective ablation of GINIP did not affect these neurons, whereas it reduced IB4 labeling in the medial part of lamina II and the density of C-LTMRs glomerular terminals to about one half throughout the entire lamina. We discuss the significance of these findings for interspecies differences and functional relevance.
Key points
Tymothy syndrome (TS) is a multisystem disorder featuring cardiac arrhythmias, autism and adrenal gland dysfunction that originates from a de novo point mutation in the gene encoding the ...Cav1.2 (CACNA1C) L‐type channel.
To study the role of Cav1.2 channel signals in autism, the autistic TS2‐neo mouse has been generated bearing the G406R point‐mutation associated with TS type‐2.
Using heterozygous TS2‐neo mice, we report that the G406R mutation reduces the rate of inactivation and shifts leftward the activation and inactivation of L‐type channels, causing marked increase of resting Ca2+ influx (‘window’ Ca2+ current).
The increased ‘window current’ causes marked reduction of NaV channel density, switches normal tonic firing to abnormal burst firing, reduces mitochondrial metabolism, induces cell swelling and decreases catecholamine release.
Overnight incubations with nifedipine rescue NaV channel density, normal firing and the quantity of catecholamine released. We provide evidence that chromaffin cell malfunction derives from altered Cav1.2 channel gating.
L‐type voltage‐gated calcium (Cav1) channels have a key role in long‐term synaptic plasticity, sensory transduction, muscle contraction and hormone release. A point mutation in the gene encoding Cav1.2 (CACNA1C) causes Tymothy syndrome (TS), a multisystem disorder featuring cardiac arrhythmias, autism spectrum disorder (ASD) and adrenal gland dysfunction. In the more severe type‐2 form (TS2), the missense mutation G406R is on exon 8 coding for the IS6‐helix of the Cav1.2 channel. The mutation causes reduced inactivation and induces autism. How this occurs and how Cav1.2 gating‐changes alter cell excitability, neuronal firing and hormone release on a molecular basis is still largely unknown. Here, using the TS2‐neo mouse model of TS we show that the G406R mutation altered excitability and reduced secretory activity in adrenal chromaffin cells (CCs). Specifically, the TS2 mutation reduced the rate of voltage‐dependent inactivation and shifted leftward the activation and steady‐state inactivation of L‐type channels. This markedly increased the resting ‘window’ Ca2+ current that caused an increased percentage of CCs undergoing abnormal action potential (AP) burst firing, cell swelling, reduced mitochondrial metabolism and decreased catecholamine release. The increased ‘window’ Ca2+ current caused also decreased NaV channel density and increased steady‐state inactivation, which contributed to the increased abnormal burst firing. Overnight incubation with the L‐type channel blocker nifedipine rescued the normal AP firing of CCs, the density of functioning NaV channels and their steady‐state inactivation. We provide evidence that CC malfunction derives from the altered Cav1.2 channel gating and that dihydropyridines are potential therapeutics for ASD.
Key points
Tymothy syndrome (TS) is a multisystem disorder featuring cardiac arrhythmias, autism and adrenal gland dysfunction that originates from a de novo point mutation in the gene encoding the Cav1.2 (CACNA1C) L‐type channel.
To study the role of Cav1.2 channel signals in autism, the autistic TS2‐neo mouse has been generated bearing the G406R point‐mutation associated with TS type‐2.
Using heterozygous TS2‐neo mice, we report that the G406R mutation reduces the rate of inactivation and shifts leftward the activation and inactivation of L‐type channels, causing marked increase of resting Ca2+ influx (‘window’ Ca2+ current).
The increased ‘window current’ causes marked reduction of NaV channel density, switches normal tonic firing to abnormal burst firing, reduces mitochondrial metabolism, induces cell swelling and decreases catecholamine release.
Overnight incubations with nifedipine rescue NaV channel density, normal firing and the quantity of catecholamine released. We provide evidence that chromaffin cell malfunction derives from altered Cav1.2 channel gating.
Presynaptic GABAB receptors (GABABRs) are highly expressed in dorsal root ganglion neurons and spinal cord dorsal horn. GABABRs located in superficial dorsal horn play an important antinociceptive ...role, by acting at both pre- and postsynaptic sites. GABABRs expressed in deep dorsal horn could be involved in the processing of touch sensation and possibly in the generation of tactile allodynia in chronic pain. The objective of this study was to characterize the morphological and functional properties of GABABRs expressed on Aβ fibers projecting to lamina III/IV and to understand their role in modulating excitatory synaptic transmission. We performed high-resolution electron microscopic analysis, showing that GABAB2 subunit is expressed on 71.9% of terminals in rat lamina III-IV. These terminals were engaged in axodendritic synapses and, for the 46%, also expressed glutamate immunoreactivity. Monosynaptic excitatory postsynaptic currents, evoked by Aβ fiber stimulation and recorded from lamina III/IV neurons in spinal cord slices, were strongly depressed by application of baclofen (0.1–2.5 µM), acting as a presynaptic modulator. Application of the GABABR antagonist CGP 55845 caused, in a subpopulation of neurons, the potentiation of the first of two excitatory postsynaptic currents recorded with the paired-pulse protocol, showing that GABABRs are endogenously activated. A decrease in the paired-pulse ratio accompanied the effect of CGP 55845, implying the involvement of presynaptic GABABRs. CGP 55845 facilitated only the first excitatory postsynaptic current also during a train of four consecutive stimuli applied to Aβ fibers. These results suggest that GABABRs tonically inhibit glutamate release from Aβ fibers at a subset of synapses in deep dorsal horn. This modulation specifically affects only the early phase of synaptic excitation in lamina III-IV neurons.
C-low-threshold mechanoreceptors (C-LTMRs) are sensory neurons that, beyond conveying pleasant touch, modulate nociceptive transmission within the spinal cord. However, pain alleviation by C-LTMRs ...remains poorly understood. Here, we show that the C-LTMR-derived TAFA4 chemokine induces a reinforcement of inhibitory synaptic transmission within spinal networks, which consequently depresses local excitatory synapses and impairs synaptic transmission from high-threshold C-fibers. In animals with inflammation induced by Freund’s complete adjuvant, TAFA4 decreases the noxious stimulus-induced neuronal responses recorded in vivo and alleviates mechanical pain. Both effects are blocked by antagonists of GABAergic transmission. Furthermore, TAFA4 promotes microglial retraction in inflammation and increases the number of inhibitory synapses on lamina IIi somata. Altogether, these results demonstrate GABAergic interneurons to be the first integration relay for C-LTMRs and highlight a tight interplay between sensory neurons, microglial cells, and spinal interneurons, which fine-tunes inhibitory activity and nociceptive transmission in pathological conditions.
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•C-LTMR neurons contact GABAergic neurons•TAFA4, a chemokine secreted by C-LMTR, promotes microglial process retraction•TAFA4 enables restoration of inhibitory inputs to spinal neurons•TAFA4 attenuates nociceptive transmission in the spinal cord
TAFA4 is a recently discovered chemokine secreted by C-low-threshold mechanoreceptors (C-LTMRs). Kambrun et al. describe the interplay between C-LTMRs, spinal GABAergic neurons, and microglial cells that is responsible for the mechanical pain alleviation induced by TAFA4 by inflammation.
The growth factors BDNF and GDNF are gaining more and more attention as modulators of synaptic transmission in the mature central nervous system (CNS). The two molecules undergo a regulated secretion ...in neurons and may be anterogradely transported to terminals where they can positively or negatively modulate fast synaptic transmission. There is today a wide consensus on the role of BDNF as a pro-nociceptive modulator, as the neurotrophin has an important part in the initiation and maintenance of inflammatory, chronic, and/or neuropathic pain at the peripheral and central level. At the spinal level, BDNF intervenes in the regulation of chloride equilibrium potential, decreases the excitatory synaptic drive to inhibitory neurons, with complex changes in GABAergic/glycinergic synaptic transmission, and increases excitatory transmission in the superficial dorsal horn. Differently from BDNF, the role of GDNF still remains to be unraveled in full. This review resumes the current literature on the interplay between BDNF and GDNF in the regulation of nociceptive neurotransmission in the superficial dorsal horn of the spinal cord. We will first discuss the circuitries involved in such a regulation, as well as the reciprocal interactions between the two factors in nociceptive pathways. The development of small molecules specifically targeting BDNF, GDNF and/or downstream effectors is opening new perspectives for investigating these neurotrophic factors as modulators of nociceptive transmission and chronic pain. Therefore, we will finally consider the molecules of (potential) pharmacological relevance for tackling normal and pathological pain.
Choroid plexuses (ChPs) are intraventricular structures mainly composed by specialized epithelial cells interconnected by tight junctions that establish the blood-cerebrospinal fluid (CSF) barrier. ...ChPs are essential to produce CSF and transport solutes from and into the brain. Deterioration of ChP function and morphology has been correlated to worsening of neurodegenerative disorders. We here map morpho-functional changes in the ChP epithelial cells during healthy aging, starting from young adult to 2-years old mice.
We used a multi-tiered approach, including transmission electron microscopy (TEM), immunohistochemistry, RT-qPCR, Western Blot and 2-photon microscopy (2-PM) at multiple timepoints ranging from young adult to 2-years old mice.
We identified distinct morpho-functional modifications in epithelial cells of ChP starting from 8 to 12 months of age, which mostly remained stable up to 2 years. These changes include flattening of the epithelium, reduction of microvilli length and an augmentation of interrupted tight junctions. We also found a decrease in mitochondria density together with elongation of mitochondria in older mice. Morphological mitochondrial rearrangements were accompanied by increased superoxide levels, decreased membrane potential and decreased mitochondrial motility in aged mice. Interestingly, most of the age-related changes were not accompanied by modification of protein and/or gene expression levels and aged mitochondria effectively responded to acute pharmacological stressful stimuli.
Our study suggests a long-term progression of multiple morpho-functional features of the mouse choroid plexus epithelium during adulthood followed by structural remodeling during the aging process. These findings can lead to a better understanding on how functional and morphological rearrangements of ChP are correlated during aging.
C-LTMRs are known to convey affective aspects of touch and to modulate injury-induced pain in humans and mice. However, a role for these neurons in temperature sensation has been suggested, but ...not fully demonstrated. Here, we report that deletion of C-low-threshold mechanoreceptor (C-LTMR)-expressed bhlha9 causes impaired thermotaxis behavior and exacerbated formalin-evoked pain in male, but not female, mice. Positive modulators of GABAA receptors failed to relieve inflammatory formalin pain and failed to decrease the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) selectively in bhlha9 knockout (KO) males. This could be explained by a drastic change in the GABA content of lamina II inner inhibitory interneurons contacting C-LTMR central terminals. Finally, C-LTMR-specific deep RNA sequencing revealed more genes differentially expressed in male than in female bhlha9 KO C-LTMRs. Our data consolidate the role of C-LTMRs in modulation of formalin pain and provide in vivo evidence of their role in the discriminative aspects of temperature sensation.
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•BHLHA9 function in C-LTMRs is sexually dimorphic•Loss of BHLHA9 impaired formalin-evoked pain selectively in males•BHLHA9 in C-LTMRs is required for fine discriminative aspects of temperature in males•Loss of BHLHA9 non-cell-autonomously impairs ionotropic GABAergic signaling
Bohic et al. demonstrate that mice lacking the transcription factor BHLHA9 exhibit impaired thermotaxis and formalin-evoked pain. BHLHA9 mediates these effects by modulating spinal GABAergic signaling. The data consolidate the role of C-LTRMs in pain sensation and provide in vivo evidence of C-LTRMs in the discriminative aspects of temperature sensation.
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