Comparative study between radiofrequency-induced and muscimol-induced inhibition of cultured networks of cortical neuron

Previous studies have shown that spontaneously active cultured networks of cortical neuron grown planar microelectrode arrays are sensitive to radiofrequency (RF) fields and exhibit an inhibitory response more pronounced as the exposure time and power increase. To better understand the mechanism behind the observed effects, we aimed at identifying similarities and differences between the inhibitory effect of RF fields (continuous wave, 1800 MHz) to the γ-aminobutyric acid type A (GABAA) receptor agonist muscimol (MU). Inhibition of the network bursting activity in response to RF exposure became apparent at an SAR level of 28.6 W/kg and co-occurred with an elevation of the culture medium temperature of ~1°C. Exposure to RF fields preferentially inhibits bursting over spiking activity and exerts fewer constraints on neural network bursting synchrony, differentiating it from a pharmacological inhibition with MU. Network rebound excitation, a phenomenon relying on the intrinsic properties of cortical neurons, was observed following the removal of tonic hyperpolarization after washout of MU but not in response to cessation of RF exposure. This implies that hyperpolarization is not the main driving force mediating the inhibitory effects of RF fields. At the level of single neurons, network inhibition induced by MU and RF fields occurred with reduced action potential (AP) half-width. As changes in AP waveform strongly influence efficacy of synaptic transmission, the narrowing effect on AP seen under RF exposure might contribute to reducing network bursting activity. By pointing only to a partial overlap between the inhibitory hallmarks of these two forms of inhibition, our data suggest that the inhibitory mechanisms of the action of RF fields differ from the ones mediated by the activation of GABAA receptors

Morphological and electrophysiological characterization of lamina IIi PKCγ-interneurons within the medullary dorsal horn of adult rats.

L'allodynie mécanique est un symptôme cardinal des douleurs persistantes. Elle est due à l’activation de circuits, habituellement bloqués, des couches superficielles de la corne dorsale spinale ou du sous-noyau caudal du trijumeau (Sp5C), par lesquels les afférences mécaniques à bas seuil peuvent accéder aux neurones nociceptifs de projection de la couche I. Un élément déterminant de ces circuits est une classe d’interneurones excitateurs de la couche II interne (IIi) exprimant l'isoforme gamma de la protéine kinase C (PKCγ), et recevant des afférences des mécanorecepteurs à bas seuil. La modulation de l’inhibition tonique de ces interneurones PKCγ contribue à l’apparition de l’allodynie mécanique. Cependant la morphologie, les propriétés électrophysiologiques et les caractéristiques des afférences excitatrices et inhibitrices de ces interneurones PKCγ ne sont toujours pas connues. Utilisant des techniques d’électrophysiologie (enregistrements patch-clamp) et d'immunohistochimie sur tranches de Sp5C, nous avons caractérisé les propriétés des interneurones PKCγ de la couche IIi du Sp5C chez le rat adulte et comparé ces propriétés avec celles d’interneurones voisins n’exprimant pas la PKCγ.Cette étude révèle que l’arborisation neuritique des interneurones PKCγ s’étend largement au sein de la couche IIi, et peut se prolonger du coté dorsal jusqu’à la couche II externe, sans jamais atteindre la couche I. En outre, en fonction de cette extension neuritique, au moins deux sous-populations d'interneurones PKCγ peuvent être dissociées – centrales et radiales – qui s’avèrent être aussi fonctionnellement différentes. Comparés aux autres neurones non-PKCγ de la conche IIi, les interneurones PKCγ, dans leur ensemble, présentent un seuil de déclenchement des potentiels d’action plus bas et, souvent associée, plus fréquemment une réponse tonique à un courant dépolarisant, indiquant ainsi qu’ils sont plus facilement excitables. Cependant, ils reçoivent inversement une excitation synaptique plus faible. Quant aux afférences inhibitrices, la plupart des interneurones PKCγ expriment des synapses mixtes associant récepteurs GABAAergiques (GABAAR) et récepteurs glycinergiques (GlyR). Seul un petit nombre d’entre eux exprime des synapses uniquement GABAAR ou GlyR. Pourtant, tous les interneurones PKCγ reçoivent non seulement des mIPSCs mixtes GABAAR-GlyR, mais aussi des mIPSCs uniquement GABAAR ou uniquement GlyR.Mechanical allodynia, a cardinal symptom of persistent pain, is associated with the unmasking of usually blocked local circuits within the superficial spinal or medullary dorsal horn (MDH), through which low-threshold mechanical inputs can gain access to the lamina I nociceptive output neurons. Key determinants of these circuits are lamina II (IIi) excitatory interneurons that selectively concentrate the gamma isoform of protein kinase C (PKCγ) and receive low-threshold mechanical receptor (LTMR) inputs. Tonic inhibition of PKCγ interneurons is thought to gate circuits underlying mechanical allodynia. However, the morphology, electrophysiological properties and excitatory and inhibitory synaptic inputs on these PKCγ interneurons are still unknown. Using whole-cell patch-clamp recordings and immunohistochemical techniques in slices of adult rat MDH, we characterized these lamina IIi PKCγ interneurons and compared them with neighboring non-PKCγ interneurons. Our results reveal that the neurites of PKCγ interneurons arborize extensively within lamina IIi, can spread dorsally into lamina IIo, but never reach lamina I. In addition, according to cell bodies and the orientation and extent of dendritic arbors, at least two morphologically different classes of PKCγ interneurons can be identified – central and radial – which appear to be also functionally different. Compared with neighboring lamina IIi non-PKCγ interneurons, PKCγ interneurons exhibit a lower threshold for action potentials, consistent with a more frequent tonic spike discharge to depolarizing step current, indicating that they are more excitable than other lamina IIi neurons. On the other hand, they receive a weaker excitatory synaptic drive. According to inhibitory inputs, most PKCγ interneurons display mixed-GABAA (GABAAR) and glycine (GlyR) receptor synapses with only very few of them displaying also GABAAR-alone or GlyR-alone synapses. Interestingly, all PKCγ interneurons exhibit mixed GABAAR–GlyR as well as GABAAR-only and GlyR-only mIPSCs. Altogether, this study indicates that PKCγ interneurons within lamina IIi of MDH are different from other lamina IIi neighboring neurons according to morphology, electrophysiological properties and synaptic inputs. This is consistent with their specific role in the gating of dorsally directed circuits within the MDH underlying mechanical allodynia. Moreover, we have identified two morphological and functional subclasses of PKCγ interneurons which might thus differently contribute to this gating

Subpopulations of PKCγ interneurons within the medullary dorsal horn revealed by electrophysiologic and morphologic approach

International audienceMechanical allodynia, a cardinal symptom of persistent pain, is associated with the unmasking of usually blocked local circuits within the superficial spinal or medullary dorsal horn (MDH) through which low-threshold mechanical inputs can gain access to the lamina I nociceptive output neurons. Specific interneurons located within inner lamina II (IIi) and expressing the gamma isoform of protein kinase C (PKCγ⁺) have been shown to be key elements for such circuits. However, their morphologic and electrophysiologic features are still unknown. Using whole-cell patch-clamp recordings and immunohistochemical techniques in slices of adult rat MDH, we characterized such lamina IIi PKCγ⁺ interneurons and compared them with neighboring PKCγ⁻ interneurons. Our results reveal that PKCγ⁺ interneurons display very specific activity and response properties. Compared with PKCγ⁻ interneurons, they exhibit a smaller membrane input resistance and rheobase, leading to a lower threshold for action potentials. Consistently, more than half of PKCγ⁺ interneurons respond with tonic firing to step current. They also receive a weaker excitatory synaptic drive. Most PKCγ⁺ interneurons express Ih currents. The neurites of PKCγ⁺ interneurons arborize extensively within lamina IIi, can spread dorsally into lamina IIo, but never reach lamina I. In addition, at least 2 morphologically and functionally different subpopulations of PKCγ⁺ interneurons can be identified: central and radial PKCγ⁺ interneurons. The former exhibit a lower membrane input resistance, rheobase and, thus, action potential threshold, and less PKCγ⁺ immunoreactivity than the latter. These 2 subpopulations might thus differently contribute to the gating of dorsally directed circuits within the MDH underlying mechanical allodynia

Microdosimetry of Multi Electrodes Array in an RF Exposure System for In vitro Real-Time Recordings

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GABA_A and glycine receptor-mediated inhibitory synaptic transmission onto adult rat lamina IIi PKCγ-interneurons: pharmacological but not anatomical specialization

Mechanical allodynia (pain to normally innocuous tactile stimuli) is a widespread symptom of inflammatory and neuropathic pain. Spinal or medullary dorsal horn (SDH or MDH) circuits mediating tactile sensation and pain need to interact in order to evoke mechanical allodynia. PKCγ-expressing (PKCγ ) interneurons and inhibitory controls within SDH/MDH inner lamina II (II ) are pivotal in connecting touch and pain circuits. However, the relative contribution of GABA and glycine to PKCγ interneuron inhibition remains unknown. We characterized inhibitory inputs onto PKCγ interneurons by combining electrophysiology to record spontaneous and miniature IPSCs (sIPSCs, mIPSCs) and immunohistochemical detection of GABA Rα2 and GlyRα1 subunits in adult rat MDH. While GlyR-only- and GABA R-only-mediated mIPSCs/sIPSCs are predominantly recorded from PKCγ interneurons, immunohistochemistry reveals that ~80% of their inhibitory synapses possess both GABA Rα2 and GlyRα1. Moreover, nearly all inhibitory boutons at gephyrin-expressing synapses on these cells contain glutamate decarboxylase and are therefore GABAergic, with around half possessing the neuronal glycine transporter (GlyT2) and therefore being glycinergic. Thus, while GABA and glycine are presumably co-released and GABA Rs and GlyRs are present at most inhibitory synapses on PKCγ interneurons, these interneurons exhibit almost exclusively GABA R-only and GlyR-only quantal postsynaptic inhibitory currents, suggesting a pharmacological specialization of their inhibitory synapses

Dosimetry of Microelectrodes Array Chips for Electrophysiological Studies Under Simultaneous Radio Frequency Exposures

Studying the response of neuronal networks to radio frequency (RF) signals requires the use of a specific device capable of accessing and simultaneously recording neuronal activity during electromagnetic fields (EMF) exposure. In this study, a microelectrode array (MEA) that records the spontaneous activity of neurons is coupled to an open transverse electromagnetic (TEM) cell that propagates EMF. We characterize this system both numerically and experimentally at 1.8 GHz. Two MEA versions were compared, for the first time, to determine the impact of their design dissimilarities on the response to EMF. Macroscopic and microscopic measurements using, respectively, a fiber-optic probe and a temperature-dependent fluorescent dye (Rhodamine-B) were carried out. Results indicate that one MEA shows more stability toward the changes of the surrounding environment compared to the other MEA. Using a fiber-optic thermometer, the measured specific absorption rate (SAR) probe value in the center of the more stable MEA was 5.5 ± 2.3 W/kg. Using a Rhod-B microdosimetry technique, the measured SAR value at the level of the MEA electrodes was 7.0 ± 1.04 W/kg. SAR values are normalized per 1 W incident power. Due to the additional metallic planes and a smaller chip aperture, this new recording chip is steadier in terms of SAR and temperature stability allowing high exposure homogeneity as required during biological experiments. A typical neuronal activity recording under EMF exposure is reported

Comparative study between radiofrequency-induced and muscimol-induced inhibition of cul-3 tured networks of cortical neuron

Previous studies have shown that spontaneously active cultured networks of cortical neuron grown planar microelectrode arrays are sensitive to radiofrequency (RF) fields and exhibit an inhibitory response more pronounced as the exposure time and power increase. To better understand the mechanism behind the observed effects, we aimed at identifying similarities and differences between the inhibitory effect of RF fields (continuous wave, 1800 MHz) to the γ-aminobutyric acid type A (GABAA) receptor agonist muscimol (MU). Inhibition of the network bursting activity in response to RF exposure became apparent at an SAR level of 28.6 W/kg and co-occurred with an elevation of the culture medium temperature of ~1 °C. Exposure to RF fields preferentially inhibits bursting over spiking activity and exerts fewer constraints on neural network bursting synchrony, differentiating it from a pharmacological inhibition with MU. Network rebound excitation, a phenomenon relying on the intrinsic properties of cortical neurons, was observed following the removal of tonic hyperpolarization after washout of MU but not in response to cessation of RF exposure. This implies that hyperpolarization is not the main driving force mediating the inhibitory effects of RF fields. At the level of single neurons, network inhibition induced by MU and RF fields occurred with reduced action potential (AP) half-width. As changes in AP waveform strongly influence efficacy of synaptic transmission, the narrowing effect on AP seen under RF exposure might contribute to reducing network bursting activity. By pointing only to a partial overlap between the inhibitory hallmarks of these two forms of inhibition, our data suggest that the inhibitory mechanisms of the action of RF fields differ from the ones mediated by the activation of GABAA receptors

Decreased spontaneous electrical activity in neuronal networks exposed to radiofrequency 1800 MHz signals

International audienceThe rapid development of wireless communications has raised questions about their potential health risks. So far, the only identified biological effects of radiofrequency fields (RF) are known to be caused by heating. but the issue of potential nonthermal biological effects, especially on the central nervous system (CNS), remains open. We previously reported a decrease in the firing and bursting rates of neuronal cultures exposed to a Global System for Mobile (GSM) RF field at 1,800 MHz for 3 min (Moretti D. Garenne A, Haro E, Poulleier de Gannes F. Lagroye I, Leveque P, Veyret B. Lewis N. Bioelectromagnetics 34 571-578, 2013). The aim of the present work was to assess the dose-response relationship for this effect and also to identify a potential differential response elicited by pulse-modulated GSM and continuous-wave (CW) RF fields. Spontaneous bursting activity of neuronal cultures from rat embryonic cortices was recorded using 60-electrode multielectrode arrays (MEAs). At 17-28 days in vitro, the neuronal cultures were subjected to 15-min RF exposures, at specific absorption rates (SAR) ranging from 0.01 to 9.2 W/kg. Both GSM and CW signals elicited a clear decrease in bursting rate during the RF exposure phase. This effect became more marked with increasing SAR and lasted even beyond the end of exposure for the highest SAR levels. Moreover, the amplitude of the effect was greater with the GSM signal. Altogether. our experimental findings provide evidence for dose-dependent effects of RF signals on the bursting rate of neuronal cultures and suggest that part of the mechanism is nonthermal. NEW and NOTEWORTHY In this study, we investigated the effects of some radiofrequency (RF) exposure parameters on the electrical activity of neuronal cultures. We detected a clear decrease in bursting activity, dependent on exposure duration. The amplitude of this effect increased with the specific absorption rate (SAR) level and was greater with Global System for Mobile signal than with continuous-wave signal, at the same average SAR. Our experiment provides unique evidence of a decrease in electrical activity of cortical neuronal cultures during RF exposure