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 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 (K(2P)), 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 K(2P) 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.

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 approximately 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.

The mechanisms whereby deposition of monosodium urate (MSU) crystals in gout activates nociceptors to induce joint pain are incompletely understood. We tried to reproduce the signs of painful gouty arthritis, injecting into the knee joint of rats suspensions containing amorphous or triclinic, needle MSU crystals. The magnitude of MSU-induced inflammation and pain behavior signs were correlated with the changes in firing frequency of spontaneous and movement-evoked nerve impulse activity recorded in single knee joint nociceptor saphenous nerve fibers. Joint swelling, mechanical and cold allodynia, and hyperalgesia appeared 3 hours after joint injection of MSU crystals. In parallel, spontaneous and movement-evoked joint nociceptor impulse activity raised significantly. Solutions containing amorphous or needle-shaped MSU crystals had similar inflammatory and electrophysiological effects. Intra-articular injection of hyaluronan (HA, Synvisc), a high-MW glycosaminoglycan present in the synovial fluid with analgesic effects in osteoarthritis, significantly reduced MSU-induced behavioral signs of pain and decreased the enhanced joint nociceptor activity. Our results support the interpretation that pain and nociceptor activation are not triggered by direct mechanical stimulation of nociceptors by MSU crystals, but are primarily caused by the release of excitatory mediators by inflammatory cells activated by MSU crystals. Intra-articular HA decreased behavioral and electrophysiological signs of pain, possibly through its viscoelastic filtering effect on the mechanical forces acting over sensitized joint sensory endings and probably also by a direct interaction of HA molecules with the transducing channels expressed in joint nociceptor terminals.

TRPM8 is a non-selective cation channel expressed in primary sensory neurons and other tissues, including the prostate and urothelium. Its participation in different physiological and pathological processes such as thermoregulation, pain, itch, inflammation and cancer has been widely described, making it a promising target for therapeutic approaches. The detection and quantification of TRPM8 seems crucial for advancing the knowledge of the mechanisms underlying its role in these pathophysiological conditions. Antibody-based techniques are commonly used for protein detection and quantification, although their performance with many ion channels, including TRPM8, is suboptimal. Thus, the search for reliable antibodies is of utmost importance. In this study, we characterized the performance of six TRPM8 commercial antibodies in three immunodetection techniques: Western blot, immunocytochemistry and immunohistochemistry. Different outcomes were obtained for the tested antibodies; two of them proved to be successful in detecting TRPM8 in the three approaches while, in the conditions tested, the other four were acceptable only for specific techniques. Considering our results, we offer some insight into the usefulness of these antibodies for the detection of TRPM8 depending on the methodology of choice.

Chemotherapy-induced peripheral neuropathy is a frequent, disabling side effect of anticancer drugs. Oxaliplatin, a platinum compound used in the treatment of advanced colorectal cancer, often leads to a form of chemotherapy-induced peripheral neuropathy characterized by mechanical and cold hypersensitivity. Current therapies for chemotherapy-induced peripheral neuropathy are ineffective, often leading to the cessation of treatment. Transient receptor potential ankyrin 1 (TRPA1) is a polymodal, non-selective cation-permeable channel expressed in nociceptors, activated by physical stimuli and cellular stress products. TRPA1 has been linked to the establishment of chemotherapy-induced peripheral neuropathy and other painful neuropathic conditions. Sigma-1 receptor is an endoplasmic reticulum chaperone known to modulate the function of many ion channels and receptors. Sigma-1 receptor antagonist, a highly selective antagonist of Sigma-1 receptor, has shown effectiveness in a phase II clinical trial for oxaliplatin chemotherapy-induced peripheral neuropathy. However, the mechanisms involved in the beneficial effects of Sigma-1 receptor antagonist are little understood. We combined biochemical and biophysical (i.e. intermolecular Förster resonance energy transfer) techniques to demonstrate the interaction between Sigma-1 receptor and human TRPA1. Pharmacological antagonism of Sigma-1R impaired the formation of this molecular complex and the trafficking of functional TRPA1 to the plasma membrane. Using patch-clamp electrophysiological recordings we found that antagonists of Sigma-1 receptor, including Sigma-1 receptor antagonist, exert a marked inhibition on plasma membrane expression and function of human TRPA1 channels. In TRPA1-expressing mouse sensory neurons, Sigma-1 receptor antagonists reduced inward currents and the firing of actions potentials in response to TRPA1 agonists. Finally, in a mouse experimental model of oxaliplatin neuropathy, systemic treatment with a Sigma-1 receptor antagonists prevented the development of painful symptoms by a mechanism involving TRPA1. In summary, the modulation of TRPA1 channels by Sigma-1 receptor antagonists suggests a new strategy for the prevention and treatment of chemotherapy-induced peripheral neuropathy and could inform the development of novel therapeutics for neuropathic pain.

Food addiction is linked to obesity and eating disorders and is characterized by a loss of behavioral control and compulsive food intake. Here, using a food addiction mouse model, we report that the lack of cannabinoid type-1 receptor in dorsal telencephalic glutamatergic neurons prevents the development of food addiction-like behavior, which is associated with enhanced synaptic excitatory transmission in the medial prefrontal cortex (mPFC) and in the nucleus accumbens (NAc). In contrast, chemogenetic inhibition of neuronal activity in the mPFC-NAc pathway induces compulsive food seeking. Transcriptomic analysis and genetic manipulation identified that increased dopamine D2 receptor expression in the mPFC-NAc pathway promotes the addiction-like phenotype. Our study unravels a new neurobiological mechanism underlying resilience and vulnerability to the development of food addiction, which could pave the way towards novel and efficient interventions for this disorder.

Hyperpolarization-activated currents (I(h)) 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 I(h) currents. We investigated the functional properties of I(h) 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 I(h), 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, I(h) was greatly reduced but not abolished. We find that I(h) activity is not essential for the transduction of cold stimuli in CS neurons. Nevertheless, I(h) has the potential to shape the excitability of CS neurons. First, I(h) 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 I(h) 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 I(h) with ZD7288, suggesting that I(h) plays an important role in peripheral sensitivity to cold.

LIS1 is one of principal genes related with Type I lissencephaly, a severe human brain malformation characterized by abnormal neuronal migration in the cortex. The LIS1 gene encodes a brain-specific 45kDa non-catalytic subunit of platelet-activating factor (PAF) acetylhydrolase-1b (PAFAH1b), an enzyme that inactivates the PAF. We have studied the role of Lis1 using a Lis1/sLis1 murine model, which has deleted the first coding exon from Lis1 gene. Homozygous mice are not viable but heterozygous have shown a delayed corticogenesis and neuronal dysplasia, with enhanced cortical excitability. Lis1/sLis1 embryos also exhibited a delay of cortical innervation by the thalamocortical fibers. We have explored in Lis1/sLis1 mice anomalies in forebrain cholinergic neuron development, which migrate from pallium to subpallium, and functionally represent the main cholinergic input to the cerebral cortex, modulating cortical activity and facilitating attention, learning, and memory. We hypothesized that primary migration anomalies and/or disorganized cortex could affect cholinergic projections from the basal forebrain and septum in Lis1/sLis1 mouse. To accomplish our objective we have first studied basal forebrain neurons in Lis1/sLis1 mice during development, and described structural and hodological differences between wild-type and Lis1/sLis1 embryos. In addition, septohippocampal projections showed altered development in mutant embryos. Basal forebrain abnormalities could contribute to hippocampal excitability anomalies secondary to Lis1 mutations and may explain the cognitive symptoms associated to cortical displasia-related mental diseases and epileptogenic syndromes.

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 degrees 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.