Elvira de la Peña

Specificity of cold thermotransduction is determined by differential ionic channel expression

Sensations of cold are mediated by specific thermoreceptor nerve endings excited by low temperature and menthol. Here we identify a population of cold-sensitive cultured mouse trigeminal ganglion neurons with a unique set of biophysical properties. Their impulse activity during cooling and menthol application was similar to that of cold thermoreceptor fibers in vivo. We show that cooling closes a background K+ channel, causing depolarization and firing that is limited by the slower reduction of a cationic inward current (Ih). In cold-insensitive neurons, firing is prevented by a slow, transient, 4-AP-sensitive K+ current (IKD) that acts as an excitability brake. In addition, pharmacological blockade of IKD induced thermosensitivity in cold-insensitive neurons, a finding that may explain cold allodynia in neuropathic pain. These results suggest that cold sensitivity is not associated to a specific transduction molecule but instead results from a favorable blend of ionic channels expressed in a small subset of sensory neurons.

Attenuation of thermal nociception and hyperalgesia by VR1 blockers

Vanilloid receptor subunit 1 (VR1) appears to play a critical role in the transduction of noxious chemical and thermal stimuli by sensory nerve endings in peripheral tissues. Thus, VR1 antagonists are useful compounds to unravel the contribution of this receptor to pain perception, as well as to induce analgesia. We have used a combinatorial approach to identify new, nonpeptidic channel blockers of VR1. Screening of a library of trimers of N-alkylglycines resulted in the identification of two molecules referred to as DD161515 {N-[2-(2-(N-methylpyrrolidinyl)ethyl]glycyl]-[N-[2,4-dichlorophenethyl]glycyl]-N-(2,4-dichlorophenethyl)glycinamide} and DD191515 {[N-[3-(N,N-diethylamino)propyl]glycyl]-[N-[2,4-dichlorophenethyl]glycyl]-N-(2,4-dichlorophenethyl)glycinamide} that selectively block VR1 channel activity with micromolar efficacy, rivaling that characteristic of vanilloid-related inhibitors. These compounds appear to be noncompetitive VR1 antagonists that recognize a receptor site distinct from that of capsaicin. Intraperitoneal administration of both trialkylglycines into mice significantly attenuated thermal nociception as measured in the hot plate test. It is noteworthy that these compounds eliminated pain and neurogenic inflammation evoked by intradermal injection of capsaicin into the animal hindpaw, as well as the thermal hyperalgesia induced by tissue irritation with nitrogen mustard. In contrast, responses to mechanical stimuli were not modified by either compound. Modulation of sensory nerve fibers excitability appears to underlie the peptoid analgesic activity. Collectively, these results indicate that blockade of VR1 activity attenuates chemical and thermal nociception and hyperalgesia, supporting the tenet that this ionotropic receptor contributes to chemical and thermal sensitivity and pain perception in vivo. These trialkylglycine-based, noncompetitive VR1 antagonists may likely be developed into analgesics to treat inflammatory pain.

Variable Threshold of Trigeminal Cold-Thermosensitive Neurons Is Determined by a Balance between TRPM8 and Kv1 Potassium Channels

Molecular determinants of threshold differences among cold thermoreceptors are unknown. Here we show that such differences correlate with the relative expression of I KD, a current dependent on Shaker-like Kv1 channels that acts as an excitability brake, and I TRPM8, a cold-activated excitatory current. Neurons responding to small temperature changes have high functional expression of TRPM8 (transient receptor potential cation channel, subfamily M, member 8) and low expression of I KD. In contrast, neurons activated by lower temperatures have a lower expression of TRPM8 and a prominent I KD. Otherwise, both subpopulations have nearly identical membrane and firing properties, suggesting that they belong to the same neuronal pool. Blockade of I KD shifts the threshold of cold-sensitive neurons to higher temperatures and augments cold-evoked nocifensive responses in mice. Similar behavioral effects of I KD blockade were observed in TRPA1−/− mice. Moreover, only a small percentage of trigeminal cold-sensitive neurons were activated by TRPA1 agonists, suggesting that TRPA1 does not play a major role in the detection of low temperatures by uninjured somatic cold-specific thermosensory neurons under physiological conditions. Collectively, these findings suggest that innocuous cooling sensations and cold discomfort are signaled by specific low- and high-threshold cold thermoreceptor neurons, differing primarily in their relative expression of two ion channels having antagonistic effects on neuronal excitability. Thus, although TRPM8 appears to function as a critical cold sensor in the majority of peripheral sensory neurons, the expression of Kv1 channels in the same terminals seem to play an important role in the peripheral gating of cold-evoked discomfort and pain.

Bidirectional shifts of TRPM8 channel gating by temperature and chemical agents modulate the cold sensitivity of mammalian thermoreceptors

TRPM8, a member of the melastatin subfamily of transient receptor potential (TRP) cation channels, is activated by voltage, low temperatures and cooling compounds. These properties and its restricted expression to small sensory neurons have made it the ion channel with the most advocated role in cold transduction. Recent work suggests that activation of TRPM8 by cold and menthol takes place through shifts in its voltage‐activation curve, which cause the channel to open at physiological membrane potentials. By contrast, little is known about the actions of inhibitors on the function of TRPM8. We investigated the chemical and thermal modulation of TRPM8 in transfected HEK293 cells and in cold‐sensitive primary sensory neurons. We show that cold‐evoked TRPM8 responses are effectively suppressed by inhibitor compounds SKF96365, 4‐(3‐chloro‐pyridin‐2‐yl)‐piperazine‐1‐carboxylic acid (4‐tert‐butyl‐phenyl)‐amide (BCTC) and 1,10‐phenanthroline. These antagonists exert their effect by shifting the voltage dependence of TRPM8 activation towards more positive potentials. An opposite shift towards more negative potentials is achieved by the agonist menthol. Functionally, the bidirectional shift in channel gating translates into a change in the apparent temperature threshold of TRPM8‐expressing cells. Accordingly, in the presence of the antagonist compounds, the apparent response‐threshold temperature of TRPM8 is displaced towards colder temperatures, whereas menthol sensitizes the response, shifting the threshold in the opposite direction. Co‐application of agonists and antagonists produces predictable cancellation of these effects, suggesting the convergence on a common molecular process. The potential for half maximal activation of TRPM8 activation by cold was ∼140 mV more negative in native channels compared to recombinant channels, with a much higher open probability at negative membrane potentials in the former. In functional terms, this difference translates into a shift in the apparent temperature threshold for activation towards higher temperatures for native currents. This difference in voltage‐dependence readily explains the high threshold temperatures characteristic of many cold thermoreceptors. The modulation of TRPM8 activity by different chemical agents unveils an important flexibility in the temperature–response curve of TRPM8 channels and cold thermoreceptors.

Swelling-activated calcium signalling in cultured mouse primary sensory neurons

The effects of hypo‐osmotic membrane stretch on intracellular calcium concentration ([Ca2+]i), cell volume and cellular excitability were investigated in cultured mouse primary sensory trigeminal neurons. Hypotonic solutions (15–45%) led to rapid cell swelling in all neurons. Swelling was accompanied by dose‐dependent elevations in [Ca2+]i in a large fraction of neurons. Responses could be classified into three categories. (i) In 57% of the neurons [Ca2+]i responses had a slow rise time and were generally of small amplitude. (ii) In 21% of the neurons, responses had a faster rise and were larger in amplitude. (iii) The remaining cells (22%) did not show [Ca2+]i responses to hypo‐osmotic stretch. Slow and fast [Ca2+]i changes were observed in trigeminal neurons of different sizes with variable responses to capsaicin (0.5 µm). The swelling‐induced [Ca2+]i responses were not abolished after depletion of intracellular Ca2+ stores with cyclopiazonic acid or preincubation in thapsigargin, but were suppressed in the absence of external Ca2+. They were strongly attenuated by extracellular nickel and gadolinium. Hypotonic stimulation led to a decrease in input resistance and to membrane potential depolarization. Under voltage‐clamp, the [Ca2+]i elevation produced by hypotonic stimulation was accompanied by the development of an inward current and a conductance increase. The time course and amplitude of the [Ca2+]i response to hypo‐osmotic stimulation showed a close correlation with electrophysiological properties of the neurons. Fast [Ca2+]i responses were characteristic of trigeminal neurons with short duration action potentials and marked inward rectification. These findings suggest that hypo‐osmotic stimulation activates several Ca2+‐influx pathways, including Gd3+‐sensitive stretch‐activated ion channels, in a large fraction of trigeminal ganglion neurons. Opening of voltage‐gated Ca2+ channels also contributes to the response. The pattern and rate of Ca2+ influx may be correlated with functional subtypes of sensory neurons.

The contribution of TRPM8 channels to cold sensing in mammalian neurones

Different classes of ion channels have been implicated in sensing cold temperatures at mammalian thermoreceptor nerve endings. A major candidate is TRPM8, a non‐selective cation channel of the transient receptor potential family, activated by menthol and low temperatures. We investigated the role of TRPM8 in cold sensing during transient expression in mouse cultured hippocampal neurones, a tissue that lacks endogenous expression of thermosensitive TRPs. In the absence of synaptic input, control hippocampal neurones were not excited by cooling. In contrast, all TRPM8‐transfected hippocampal neurones were excited by cooling and menthol. However, in comparison to cold‐sensitive trigeminal sensory neurones, hippocampal neurones exhibited much lower threshold temperatures, requiring temperatures below 27°C to fire action potentials. These results directly demonstrate that expression of TRPM8 in mammalian neurones induces cold sensing, albeit at lower temperatures than native TRPM8‐expressing neurones, suggesting the presence of additional modulatory mechanisms in the cold response of sensory neurones.

Characteristics and physiological role of hyperpolarization activated currents in mouse cold thermoreceptors

Hyperpolarization‐activated currents (Ih) are mediated by the expression of combinations of hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channel subunits (HCN1–4). These cation currents are key regulators of cellular excitability in the heart and many neurons in the nervous system. Subunit composition determines the gating properties and cAMP sensitivity of native Ih currents. We investigated the functional properties of Ih in adult mouse cold thermoreceptor neurons from the trigeminal ganglion, identified by their high sensitivity to moderate cooling and responsiveness to menthol. All cultured cold‐sensitive (CS) neurons expressed a fast activating Ih, which was fully blocked by extracellular Cs+ or ZD7288 and had biophysical properties consistent with those of heteromeric HCN1–HCN2 channels. In CS neurons from HCN1(−/−) animals, Ih was greatly reduced but not abolished. We find that Ih activity is not essential for the transduction of cold stimuli in CS neurons. Nevertheless, Ih has the potential to shape the excitability of CS neurons. First, Ih blockade caused a membrane hyperpolarization in CS neurons of about 5 mV. Furthermore, impedance power analysis showed that all CS neurons had a prominent subthreshold membrane resonance in the 5–7 Hz range, completely abolished upon blockade of Ih and absent in HCN1 null mice. This frequency range matches the spontaneous firing frequency of cold thermoreceptor terminals in vivo. Behavioural responses to cooling were reduced in HCN1 null mice and after peripheral pharmacological blockade of Ih with ZD7288, suggesting that Ih plays an important role in peripheral sensitivity to cold.

Hyaluronan modulates TRPV1 channel opening, reducing peripheral nociceptor activity and pain

Hyaluronan (HA) is present in the extracellular matrix of all body tissues, including synovial fluid in joints, in which it behaves as a filter that buffers transmission of mechanical forces to nociceptor nerve endings thereby reducing pain. Using recombinant systems, mouse-cultured dorsal root ganglia (DRG) neurons and in vivo experiments, we found that HA also modulates polymodal transient receptor potential vanilloid subtype 1 (TRPV1) channels. HA diminishes heat, pH and capsaicin (CAP) responses, thus reducing the opening probability of the channel by stabilizing its closed state. Accordingly, in DRG neurons, HA decreases TRPV1-mediated impulse firing and channel sensitization by bradykinin. Moreover, subcutaneous HA injection in mice reduces heat and capsaicin nocifensive responses, whereas the intra-articular injection of HA in rats decreases capsaicin joint nociceptor fibres discharge. Collectively, these results indicate that extracellular HA reduces the excitability of the ubiquitous TRPV1 channel, thereby lowering impulse activity in the peripheral nociceptor endings underlying pain.

The Influence of Cold Temperature on Cellular Excitability of Hippocampal Networks

The hippocampus plays an important role in short term memory, learning and spatial navigation. A characteristic feature of the hippocampal region is its expression of different electrical population rhythms and activities during different brain states. Physiological fluctuations in brain temperature affect the activity patterns in hippocampus, but the underlying cellular mechanisms are poorly understood. In this work, we investigated the thermal modulation of hippocampal activity at the cellular network level. Primary cell cultures of mouse E17 hippocampus displayed robust network activation upon light cooling of the extracellular solution from baseline physiological temperatures. The activity generated was dependent on action potential firing and excitatory glutamatergic synaptic transmission. Involvement of thermosensitive channels from the transient receptor potential (TRP) family in network activation by temperature changes was ruled out, whereas pharmacological and immunochemical experiments strongly pointed towards the involvement of temperature-sensitive two-pore-domain potassium channels (K2P), TREK/TRAAK family. In hippocampal slices we could show an increase in evoked and spontaneous synaptic activity produced by mild cooling in the physiological range that was prevented by chloroform, a K2P channel opener. We propose that cold-induced closure of background TREK/TRAAK family channels increases the excitability of some hippocampal neurons, acting as a temperature-sensitive gate of network activation. Our findings in the hippocampus open the possibility that small temperature variations in the brain in vivo, associated with metabolism or blood flow oscillations, act as a switch mechanism of neuronal activity and determination of firing patterns through regulation of thermosensitive background potassium channel activity.

Participation of low‐threshold calcium spikes in excitatory synaptic transmission in guinea pig medial frontal cortex

We studied the activation of low‐threshold calcium spikes (LTS) by excitatory postsynaptic potentials in pyramidal neurons from guinea pig medial frontal cortex with intracellular recording. We used extracellular bicuculline and phaclofen and intracellular QX‐314 to block inhibitory synaptic potentials and sodium currents. Postsynaptic potentials were evoked by stimulation of layer I. We found that large (> 10–15 mV) excitatory synaptic potentials evoked from membrane potentials more negative than −75 mV were able to trigger LTS. The activation of LTS resulted in an increase of the rising slope or amplitude of the synaptic potentials depending on the size of the excitatory postsynaptic potential (EPSP). We used 100 μm NiCl2 to confirm the presence of LTS as part of the EPSPs. The N‐methyl‐d‐aspartate (NMDA) and non‐NMDA components of the excitatory synaptic potentials were isolated using (±)2‐amino‐5‐phosphonovaleric acid (APV; 50 μm) or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 20 μm); both components could, independently, trigger an LTS. With recordings made with K+ acetate‐filled electrodes, we show that the activation of LTS was critical to allow excitatory synaptic potentials to reach the threshold of action potential firing; also, this amplification of synaptic responses produced the firing of more than a single action potential by the postsynaptic cell. These results demonstrate that in cortical pyramidal neurons the activation of low‐threshold calcium spikes results in the amplification of synaptic responses.