Molecular and cellular mechanisms of temperature reception by primary afferents

Primary afferent neurons (PANs) provide an organism interface with the environment by conveying to the CNS information about temperature, mechanical and other challenges. Temperature perception is of interest as the underlying mechanisms remain largely unknown because of restrictions of biomedical experiments. Computer models provide a complementary tool to overcome some restrictions. We combined experimental and simulation studies to disclose biophysical mechanisms, by which thermosensitive PANs convert temperatures into generator potentials and the action potential (AP) firing

Mechanisms of posttetanic potentiation and its possible role in maturation of the calyx of Held synapse

In dieser Arbeit wurde eine bisher übersehene Form der synaptischen Kurzzeit-Plastizität am Heldschen Calyx, einer glutamatergen Synapse im auditorischen Hirnstamm, indentrifiziert.Stimulation der afferenten Fasern, welche die präsynaptische Calyces bilden, mit hochfrequenten Stimulationsreizzügen (100 Hz für 1 8 s), induzierte eine robuste Potenzierung der exzitatorischen postsynaptischen Ströme (EPSCs, excitatory postsynaptic currents), welche synaptischer Depression der EPSCs während des Reizzuges folgte. Diese transiente Potenzierung der synaptischen Transmission an der Heldschen Calyx-Synapse teilte mehrere Eigenschaften mit post-tetanischer Potenzierung (PTP), welche bereits an anderen Synapsen beschrieben wurde. Während des Maximums der post-tetanischen Potenzierung beobachteten wir eine Verdopplung der synaptischen Übertragungsstärke. Die Abfallszeitkonstante der post-tetanischen Potenzierung, welche von der Länge des Stimulationsreizzugs abhing, lag zwischen zehn Sekunden und einer Minute. Eine unveränderte Miniatur-EPSC-Amplitude während des PTP-Maximums, sowie der Befund der Unterdrückung von PTP durch präsynaptische Ganzzellableitungen, deuteten auf einen präsynaptischen Ursprung von PTP hin.Im Vergleich zu einer älteren Altersgruppe (postnataler Tag 4 14, P9 P14), konnte PTP leichter in Synapsen junger Ratten (P4 7) induziert werden. Dies führte zu der Annahme, dass PTP während der Entwicklung reguliert werden könnte, und dass sie eine Rolle während der Ausbildung bzw. Reifung der Heldschen Calyx spielen könnte. Als nächstes erforschten wir mögliche Mechanismen, die PTP zu Grunde liegen könnten. Der erste Befund war, dass PTP primär durch eine Zunahme der Freisetzungswahrscheinlichkeit von Vesikeln des Reservoirs an freisetzungskompetenten präsynaptischen Vesikeln ( readily releasable pool , RRP) vermittelt wird. Gleichzeitig fanden wir keine Veränderung in der Größe des RRPs. Zweitens observierten wir, dass eine überraschend geringe Erhöhung (~ 80 nM während des PTP-Maximums) des residualen, räumlich gemittelten, präsynaptischen Ca2+-Signals ([Ca2+]i) für die Induktion von PTP verantwortlich war. Die große Potenz der geringen [Ca2+]i-Erhöhung während PTP, und der Befund der Unterdrückung von PTP durch präsynaptische Ganzzellableitungen (welche von einem schnelleren Abfall des [Ca2+]i-Signals begleitet wurde) legten nahe, dass zusätzlich zu Ca2+ weitere präsynaptische Botenstoffe (messengers) in PTP involviert sein könnten. Deshalb testeten wir die Proteinkinase C/Munc-13-Transduktionskaskade. Durch pharmakologische Manipulation konnten wir feststellen, dass Proteinkinase C eine Rolle in der Induktion von PTP am Heldschen Calyx spielt.Folglich ist der Heldsche Calyx eine sehr plastische Synapse, welche mehrere Kurzzeit-Plastizitätsformen aufweist, die denen anderer Synapsen im zentralen Nervensystem sehr ähneln. Die Möglichkeit direkter präsynatischer Ableitungen an dieser Synapse werden in Zukunft ein besseres Verständnis der Mechanismen von Kurzzeit-Potenzierung ermöglichen

Suppression of bursting activity of the hippocampal granular neuron by the hypothermal deactivation of TRP-channels: a model study

On the basis of a comparison of these observations, it can be assumed that hypothermic suppression of the hippocampal neuronal bursting by deactivation of thermo-sensitive TRP channels can be one of the mechanisms of the therapeutic effect of hypothermia

Mathematical Model of the Calcium-Dependent Chloride Current in a Smooth Muscle Cell

Employing the Hodgkin-Huxley formalism, we have developed a mathematical model of the calciumdependent chloride current on the basis of published experimental data concerning the kinetics of such current in cells of different types. The obtained results are destined for further use in a currently developed model of a smooth muscle cell of the bladder detrusor. A feature of the simulated current is the presence of two components with common kinetics of calcium-dependent activation and different (fast and slow) kinetics of voltage-dependent activation. In computational experiments performed with the use of a protocol of stepwise clamp of the membrane potential or the intracellular calcium concentration ([Са2+]i), static and dynamic dependences of the current on the membrane potential and [Са2+]i (the current-voltage and current-concentration relations, IVs and ICs, respectively) were obtained; analogous dependences of the kinetic variables of calcium- and voltage-dependent activation of the current were also plotted. The obtained characteristics of the simulated current were close to those of the prototype currents. The following properties were typical of the current: (i) the outward rectification, (ii) enhancement of the rectification effect with increase in the [Са2+]i, and (iii) a higher sensitivity to [Са2+]i deviations from the basal level (manifested in greater ratios of the current/concentration increments) within the range <1 μM, as compared to that within the range of higher concentrations

Impact of the Ratio of Metabotropic and Ionotropic Components of Parasympathetic Action on the Excitability of a Urinary Bladder Smooth Muscle Cell: a Simulation Study

On a computer model of a smooth muscle cell (SMC) of the urinary bladder detrusor (UBD) having a corresponding set of ion channels and intracellular signaling mechanisms, we investigated the influence of ionotropic (purine, P) and metabotropic (muscarinic, M) components of the parasympathetic stimulus on the membrane potential of the cell and Ca2+ concentration inside it ([Ca2+]i). The P and M components of the stimulus were simulated, respectively, by the increasing conductivity of P2X receptor channels of the SMC membrane (GP2X) and the permeability of calcium channels of the sarcoplasmic reticulum activated by inositol triphosphate (PIP3), considering that IP3 is the end product of the metabotropic chain starting from the M3 cholinergic receptors. The GP2X and PIP3 values, latent periods (LPs) of their activation, and relations of the above parameters were chosen in such a mode that application of a single stimulus evoked the SMC response with the P and M components close to those of the prototype. The normal magnitude and LP of the M component of the concentration response (calcium transient) were significantly greater than the respective parameters of the P component; the M component was accompanied by generation of an action potential (AP) with after-processes analogous to those of the prototype. A decrease in the PIP3 simulating a deficiency of M3 receptors observed under a few pathological conditions led to a decrease in the electric and concentration SMC responses, down to full elimination of AP generation and changes in [Са2+]i. Under such conditions, a significant increase in the GP2X could provide a [Са2+]i increase to a nearly normal level. Using paired parasympathetic stimulation with different interstimulus intervals, ∆Т, allowed us to obtain a situation where the M response to the first stimulus (M1) was preceded by the P response to the second stimulus (P2) with a short adjustable interval. The use of such stimulation with certain values of the ∆Т and conductivity of purinergic channels GP2X can compensate for the attenuation of the M component, due to interaction of the latter with the P component caused by the second stimulus. Thus, pathological attenuation of the M component of the parasympathetic stimulation effect can be compensated in clinical practice (at least partly) by applying purinomimetics and/or paired stimulation

Phorbol esters modulate spontaneous and Ca2+-evoked transmitter release via acting on both munc13 and protein kinase C

Diacylglycerol (DAG) and phorbol esters strongly potentiate transmitter release at synapses by activating protein kinase C (PKC) and members of the Munc13 family of presynaptic vesicle priming proteins. This PKC/Munc13 pathway has emerged as a crucial regulator of release probability during various forms of activity-dependent enhancement of release. Here, we investigated the relative roles of PKC and Munc13-1 in the phorbol ester potentiation of evoked and spontaneous transmitter release at the calyx of Held synapse. The phorbol ester phorbol 12,13-dibutyrate (1 mu M) potentiated the frequency of miniature EPSCs, and the amplitudes of evoked EPSCs with a similar time course. Preincubating slices with the PKC blocker Ro31-82200 reduced the potentiation, mainly by affecting a late phase of the phorbol ester potentiation. The Ro31-8220-insensitive potentiation was most likely mediated by Munc13-1, because in organotypic slices of Munc13-1H567K knock-in mice, in which DAG binding to Munc13-1 is abolished, the potentiation of spontaneous release by phorbol ester was strongly suppressed. Using direct presynaptic depolarizations in paired recordings, we show that the phorbol ester potentiation does not go along with an increase in the number of readily releasable vesicles, despite an increase in the cumulative EPSC amplitude during 100 Hz stimulation trains. Our data indicate that activation of Munc13 and PKC both contribute to an enhancement of the fusion probability of readily releasable vesicles. Thus, docked and readily releasable vesicles are a substrate for modulation via intracellular second-messenger pathways that act via Munc13 and PKC

Biophysical Mechanism of Parasympathetic Excitation of Urinary Bladder Smooth Muscle Cells: a Simulation Study

Using the Hodgkin-Huxley formalism, we developed a computer model of a smooth muscle cell (SMC) of the urinary bladder detrusor; the model included the main types of ion channels and pumps, as well as intracellular calcium regulatory mechanisms inherent in the prototype cell. The biophysical mechanisms of generation of action potentials (APs) necessary for initiation of muscle contraction and those of calcium transients in response to parasympathetic activation of metabotropic М2/М3-cholinoreceptors and co-activation of Р2Х-purinoreceptors were investigated. The simulated SMC in response to a depolarizing current pulse generated an AP that was, by a number of indices, similar to real APs and was also accompanied by a transient elevation of the intracellular calcium concentration. We demonstrated a possibility of generation of such APs in response to a transient increase in the conductivity of channels of calcium-dependent chloride current accompanied by increase in the conductivity of channels associated with Р2Х-receptors (the conductivity ratio was 95 to 5 % and similar to that in the prototype). For the AP generation, temporal relations of the processes of increases in the mentioned conductances simulating the final effect of activation of М2/М3- and Р2Х-receptors were significant. These results obtained on the rather simplified model allow researchers to use the latter as an appropriate starting point for the development of more detailed models (in particular, those representing cascades of metabolic reactions triggered by a parasympathetic action)

Inflammatory Mediators Increase Nav1.9 Current and Excitability in Nociceptors through a Coincident Detection Mechanism

Altered function of Na+ channels is responsible for increased hyperexcitability of primary afferent neurons that may underlie pathological pain states. Recent evidence suggests that the Nav1.9 subunit is implicated in inflammatory but not acute pain. However, the contribution of Nav1.9 channels to the cellular events underlying nociceptor hyperexcitability is still unknown, and there remains much uncertainty as to the biophysical properties of Nav1.9 current and its modulation by inflammatory mediators. Here, we use gene targeting strategy and computer modeling to identify Nav1.9 channel current signature and its impact on nociceptors' firing patterns. Recordings using internal fluoride in small DRG neurons from wild-type and Nav1.9-null mutant mice demonstrated that Nav1.9 subunits carry the TTX-resistant “persistent” Na+ current called NaN. Nav1.9−/− nociceptors showed no significant change in the properties of the slowly inactivating TTX-resistant SNS/Nav1.8 current. The loss in Nav1.9-mediated Na+ currents was associated with the inability of small DRG neurons to generate a large variety of electrophysiological behaviors, including subthreshold regenerative depolarizations, plateau potentials, active hyperpolarizing responses, oscillatory bursting discharges, and bistable membrane behaviors. We further investigated, using CsCl- and KCl-based pipette solutions, whether G-protein signaling pathways and inflammatory mediators upregulate the NaN/Nav1.9 current. Bradykinin, ATP, histamine, prostaglandin-E2, and norepinephrine, applied separately at maximal concentrations, all failed to modulate the Nav1.9 current. However, when applied conjointly as a soup of inflammatory mediators they rapidly potentiated Nav1.9 channel activity, generating subthreshold amplification and increased excitability. We conclude that Nav1.9 channel, the molecular correlate of the NaN current, is potentiated by the concerted action of inflammatory mediators that may contribute to nociceptors' hyperexcitability during peripheral inflammation