The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in a subset of primary afferent neurones where it is activated by a variety of pungent and chemically reactive compounds such as allyl isothiocyanate or cinnamaldehyde. This voltage- dependent channel is activated through covalent modification of cytoplasmic cysteines and, from the cytoplasmic side, is also critically regulated by calcium ions. Both, amino (N-) and carboxyl (C-) termini have been shown to be involved in these processes. Using electrophysiological and molecular-biology techniques, we explored the role of specific cytoplasmic domains in the activation of TRPA1. By measuring chemically-, voltage-, and calcium-activated membrane TRPA1-mediated currents, we identified highly conserved serine and threonine residues along the N-terminal ankyrin repeat domain, mutation of which strongly affected responses of the channel. In addition, using C-terminally truncated construct previously reported to be involved in calcium regulation, we present a new finding that the distal C-terminal tail contributes to voltage-dependent activation of TRPA1.

In order to understand how the brain functions, under normal as well as pathological conditions, it is important to study the mechanisms underlying information integration. Depending on the nature of an input arriving at a synapse, different strategies may be used by the neuron to integrate and respond to the input. Naturally, if a short train of high-frequency synaptic input arrives, it may be beneficial for the neuron to be equipped with a fast mechanism that is highly sensitive to inputs on a short time scale. If, on the contrary, inputs arriving with low frequency are to be processed, it may be necessary for the neuron to possess slow mechanisms of integration. For example, in certain working memory tasks (e. g. delay-match-to-sample), sensory inputs may arrive separated by silent intervals in the range of seconds, and the subject should respond if the current input is identical to the preceeding input. It has been suggested that single neurons, due to intrinsic mechanisms outlasting the duration of input, may be able to perform such calculations. In this work, I have studied a mechanism thought to be particularly important in supporting the integration of low-frequency synaptic inputs. It is mediated by a cascade of events that starts with activation of group I metabotropic glutamate receptors (mGlu1/5), and ends with a membrane depolarization caused by a current that is mediated by canonical transient receptor potential (TRPC) ion channels. This current, denoted ITRPC, is the focus of this thesis. A specific objective of this thesis is to study the role of ITRPC in the integration of synaptic inputs arriving at a low frequency, < 10 Hz. Our hypothesis is that, in contrast to the well-studied, rapidly decaying AMPA and NMDA currents, ITRPC is well-suited for supporting temporal summation of such synaptic input. The reason for choosing this range of frequencies is that neurons often communicate with signals (spikes) around 8 Hz, as shown by single-unit recordings in behaving animals. This is true for several regions of the brain, including the entorhinal cortex (EC) which is known to play a key role in producing working memory function and enabling long-term memory formation in the hippocampus. Although there is strong evidence suggesting that ITRPC is important for neuronal communication, I have not encountered a systematic study of how this current contributes to synaptic integration. Since it is difficult to directly measure the electrical activity in dendritic branches using experimental techniques, I use computational modeling for this purpose. I implemented the components necessary for studying ITRPC, including a detailed model of extrasynaptic glutamate concentration, mGlu1/5 dynamics and the TRPC channel itself. I tuned the model to replicate electrophysiological in vitro data from pyramidal neurons of the rodent EC, provided by our experimental collaborator. Since we were interested in the role of ITRPC in temporal summation, a specific aim was to study how its decay time constant (τdecay) is affected by synaptic stimulus parameters. The hypothesis described above is supported by our simulation results, as we show that synaptic inputs arriving at frequencies as low as 3 - 4 Hz can be effectively summed. We also show that τdecay increases with increasing stimulus duration and frequency, and that it is linearly dependent on the maximal glutamate concentration. Under some circumstances it was problematic to directly measure τdecay, and we then used a pair-pulse paradigm to get an indirect estimate of τdecay. I am not aware of any computational model work taking into account the synaptically evoked ITRPC current, prior to the current study, and believe that it is the first of its kind. We suggest that ITRPC is important for slow synaptic integration, not only in the EC, but in several cortical and subcortical regions that contain mGlu1/5 and TRPC subunits, such as the prefrontal cortex. I will argue that this is further supported by studies using pharmacological blockers as well as studies on genetically modified animals. / QC 20101005

Spinal cord stimulation (SCS) is used to reduce angina that accompanies cardiac ischemia, but little is known about the molecular mechanisms mediating this effect. We studied the expression of SP, neurokinin-1 (NK-1) receptor, and transient receptor potential vanilloid type 1 (TRPV1) mRNA in the rat spinal cord at thoracic 4 (T4), cervical 2 (C2) and caudal brain stem by RT-PCR during intermittent occlusion of the left anterior descending coronary artery (CoAO), during sustained SCS by itself at the C2 spinal segment, and during sustained SCS plus intermittent CoAO. Only SP mRNA was increased significantly in T4 and brainstem during CoAO, while SCS decreased the mRNA levels of SP, NK-1 and TRPV1 significantly in T4 and the brainstem. SCS attenuated the increase of SP and TRPV1 mRNA levels at T4 level induced by intermittent CoAO when the stimulation was applied prior to the initiation of the cardiac ischemia. These results support the role for SP as a putative neurotransmitter for the myocardial ischemia-sensitive afferent neuron signal to the spinal level. They suggest that modification of the ischemic cardiac nociceptive afferent signal by SCS involves a change in SP and TRPV1 expression.

No description available.

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Le réseau neuronal de l’hippocampe joue un rôle central dans la mémoire en modifiant de façon durable l’efficacité de ses synapses. Dans les interneurones de la couche oriens/alveus (O/A), l’induction de la potentialisation à long terme (PLT) requiert les courants postsynaptiques excitateurs évoqués par les récepteurs métabotropes du glutamate de sous-type 1a (CPSEmGluR1a) et l’entrée subséquente de Ca2+ via des canaux de la famille des transient receptor potential (TRP). Le but de ce projet était d’identifier les canaux TRP responsables des CPSEmGluR1a et d’explorer les mécanismes moléculaires régulant leur ouverture. Nous avons déterminé par des enregistrements électrophysiologiques que les CPSEmGluR1a étaient spécifiques aux interneurones O/A et qu’ils étaient indépendants de la phospholipase C. Nous avons ensuite examiné l’expression des TRPC et leur interaction avec mGluR1a par les techniques de RT-PCR, d’immunofluorescence et de co-immunoprécipitation. Nos résultats montrent que TRPC1 et mGluR1a s’associent dans l’hippocampe et que ces deux protéines sont présentes dans les dendrites des interneurones O/A. En revanche, TRPC4 ne semble s’associer à mGluR1a qu’en système recombinant et leur colocalisation paraît limitée au corps cellulaire. Finalement, nous avons procédé à des enregistrements d’interneurones dans lesquels l’expression des TRPC a été sélectivement supprimée par la transfection d’ARN interférant et avons ainsi démontré que TRPC1, mais non TRPC4, est une sous-unité obligatoire du canal responsable des CPSEmGluR1a. Ces travaux ont permis de mieux comprendre les mécanismes moléculaires à la base de la transmission synaptique des interneurones O/A et de mettre en évidence un rôle potentiel de TRPC1 dans la PLT. / The hippocampal neuronal network plays a crucial role in memory by producing long lasting changes in the efficacy of its synapses. In interneurons of stratum oriens/alveus (O/A), long term potentiation (LTP) induction requires metabotropic glutamate receptor subtype 1a (mGluR1a)-evoked excitatory postsynaptic currents (EPSCs) and subsequent Ca2+ entry through transient receptor potential (TRP) channels. The objectives of this project were to identify the TRP channels that mediate mGluR1a-evoked EPSCs and to explore molecular mechanisms that underlie their activation. Electrophysiological recordings showed that mGluR1a-evoked EPSCs were specifically observed in O/A interneurons and they were phospholipase C-independent. We then examined TRPC expression and their interaction with mGluR1a by RT-PCR, immunofluorescence and co-immunoprecipitation techniques. Our results show that TRPC1 and mGluR1a associate in hippocampus and that both proteins have overlapping distributions in dendrites of O/A interneurons. In contrast, TRPC4 seems to associate with mGluR1a only in recombinant system and their co-localization appears to be limited to the cell body. Finally, we performed recordings of interneurons in which TRPC expression was selectively suppressed by small interfering RNAs and we found that TRPC1, but not TRPC4, is an obligatory subunit of the channel that mediate mGluR1a-evoked EPSCs. This work brought new insight on molecular mechanisms underlying synaptic transmission of O/A interneurons and uncovered a potential role for TRPC1 in LTP.

Le réseau neuronal de l’hippocampe joue un rôle central dans la mémoire en modifiant de façon durable l’efficacité de ses synapses. Dans les interneurones de la couche oriens/alveus (O/A), l’induction de la potentialisation à long terme (PLT) requiert les courants postsynaptiques excitateurs évoqués par les récepteurs métabotropes du glutamate de sous-type 1a (CPSEmGluR1a) et l’entrée subséquente de Ca2+ via des canaux de la famille des transient receptor potential (TRP). Le but de ce projet était d’identifier les canaux TRP responsables des CPSEmGluR1a et d’explorer les mécanismes moléculaires régulant leur ouverture. Nous avons déterminé par des enregistrements électrophysiologiques que les CPSEmGluR1a étaient spécifiques aux interneurones O/A et qu’ils étaient indépendants de la phospholipase C. Nous avons ensuite examiné l’expression des TRPC et leur interaction avec mGluR1a par les techniques de RT-PCR, d’immunofluorescence et de co-immunoprécipitation. Nos résultats montrent que TRPC1 et mGluR1a s’associent dans l’hippocampe et que ces deux protéines sont présentes dans les dendrites des interneurones O/A. En revanche, TRPC4 ne semble s’associer à mGluR1a qu’en système recombinant et leur colocalisation paraît limitée au corps cellulaire. Finalement, nous avons procédé à des enregistrements d’interneurones dans lesquels l’expression des TRPC a été sélectivement supprimée par la transfection d’ARN interférant et avons ainsi démontré que TRPC1, mais non TRPC4, est une sous-unité obligatoire du canal responsable des CPSEmGluR1a. Ces travaux ont permis de mieux comprendre les mécanismes moléculaires à la base de la transmission synaptique des interneurones O/A et de mettre en évidence un rôle potentiel de TRPC1 dans la PLT. / The hippocampal neuronal network plays a crucial role in memory by producing long lasting changes in the efficacy of its synapses. In interneurons of stratum oriens/alveus (O/A), long term potentiation (LTP) induction requires metabotropic glutamate receptor subtype 1a (mGluR1a)-evoked excitatory postsynaptic currents (EPSCs) and subsequent Ca2+ entry through transient receptor potential (TRP) channels. The objectives of this project were to identify the TRP channels that mediate mGluR1a-evoked EPSCs and to explore molecular mechanisms that underlie their activation. Electrophysiological recordings showed that mGluR1a-evoked EPSCs were specifically observed in O/A interneurons and they were phospholipase C-independent. We then examined TRPC expression and their interaction with mGluR1a by RT-PCR, immunofluorescence and co-immunoprecipitation techniques. Our results show that TRPC1 and mGluR1a associate in hippocampus and that both proteins have overlapping distributions in dendrites of O/A interneurons. In contrast, TRPC4 seems to associate with mGluR1a only in recombinant system and their co-localization appears to be limited to the cell body. Finally, we performed recordings of interneurons in which TRPC expression was selectively suppressed by small interfering RNAs and we found that TRPC1, but not TRPC4, is an obligatory subunit of the channel that mediate mGluR1a-evoked EPSCs. This work brought new insight on molecular mechanisms underlying synaptic transmission of O/A interneurons and uncovered a potential role for TRPC1 in LTP.

Doctor of Philosophy(PhD) / Two recently identified transient receptor potential (TRP) channels, TRPM8 and TRPA1, have been proposed to play an important role in mammalian cool and cold peripheral sensory transduction. When expressed in cell-lines the cloned TRPM8 and TRPA1 receptors have distinct pharmacological and temperature response characteristics. Although these receptors are also transported to the central terminals of primary afferents, little is known about their centrally mediated actions. In this thesis, I use an in vitro electrophysiological approach to investigate the dorsal horn processing of cool afferent modalities and the role of TRP ion channels. The results of this thesis provide further information on thermal processing, indicate direction for further research and suggest possible therapeutic targets for the management of abnormal cold sensory processing. Initial experiments demonstrate that the cooling agents and known TRPM8 and TRPA1 agonists, menthol and icilin, inhibit primary afferent evoked excitatory postsynaptic currents (EPSCs) in rat spinal cord dorsal horn neurons. In addition, temperature reduction, menthol and icilin increase the frequency of miniature EPSCs without affecting amplitude distribution or kinetics. Little or no direct postsynaptic effect on dorsal horn neurons, GABAergic or glycinergic transmission was found. In combination, these observations demonstrate that temperature reduction, menthol and icilin act presynaptically to increase the probability of glutamate release from primary afferent fibres. Further examination of the changes in glutamatergic synaptic transmission induced by temperature reduction, menthol and icilin reveals a subset of neurons sensitive to innocuous cool (< 29 oC) and low concentrations of icilin (3-10 µM) which closely match the temperature activation and pharmacological profile of TRPM8. In addition, the majority of lamina I and II neurons displayed characteristics partly consistent with TRPA1-activation, including a concentration-dependent response to icilin and blockade by ruthenium red. The present experiments did not allow thermal characterisation of these TRPA1-like responses. Together these observations indicate that the effects of menthol and icilin on glutamatergic synaptic transmission in the superficial dorsal horn are mediated by TRPM8 and possibly by TRPA1. Examination of the anatomical location of neurons activated by temperature reduction, menthol, icilin and capsaicin allowed the central termination pattern of thermoreceptive primary afferent fibres with specific TRP-like response characteristics to be determined. TRPM8-like presynaptic activation was confined to a subpopulation of neurons located in lamina I and outer lamina II, while the majority of neurons throughout laminae I and II received inputs sensitive to menthol, high concentrations of icilin and capsaicin. These findings suggest that innocuous cool sensation projects to a specific subpopulation of superficial dorsal horn neurons unlike other modalities (mediated by TRPV1, possibly TRPA1 and other receptors), which non-selectively engage circuits within the entire superficial dorsal horn. No morphological specificity was identified for recovered neurons after electrophysiological characterisation. Finally, mu-opioids were shown to inhibit basal glutamatergic synaptic transmission as well as menthol- and icilin-induced transmission in the superficial dorsal horn. Of particular interest, delta-opioids selectively inhibited icilin-induced synaptic transmission within the same location. The selective effect of delta-opioids suggests a possible role in modulating receptors activated by icilin (TRPM8 and TRPA1). Overall, this thesis provides further evidence that TRPM8 is responsible for the transduction of innocuous cold sensation in mammals and is a potential therapeutic target in humans with cold hyperaesthesia secondary to abnormal thermal processing. The use of delta-opioid agonists warrants further investigation in cold hypersensitivity states and potentially other forms of pain.

Doctor of Philosophy(PhD) / Two recently identified transient receptor potential (TRP) channels, TRPM8 and TRPA1, have been proposed to play an important role in mammalian cool and cold peripheral sensory transduction. When expressed in cell-lines the cloned TRPM8 and TRPA1 receptors have distinct pharmacological and temperature response characteristics. Although these receptors are also transported to the central terminals of primary afferents, little is known about their centrally mediated actions. In this thesis, I use an in vitro electrophysiological approach to investigate the dorsal horn processing of cool afferent modalities and the role of TRP ion channels. The results of this thesis provide further information on thermal processing, indicate direction for further research and suggest possible therapeutic targets for the management of abnormal cold sensory processing. Initial experiments demonstrate that the cooling agents and known TRPM8 and TRPA1 agonists, menthol and icilin, inhibit primary afferent evoked excitatory postsynaptic currents (EPSCs) in rat spinal cord dorsal horn neurons. In addition, temperature reduction, menthol and icilin increase the frequency of miniature EPSCs without affecting amplitude distribution or kinetics. Little or no direct postsynaptic effect on dorsal horn neurons, GABAergic or glycinergic transmission was found. In combination, these observations demonstrate that temperature reduction, menthol and icilin act presynaptically to increase the probability of glutamate release from primary afferent fibres. Further examination of the changes in glutamatergic synaptic transmission induced by temperature reduction, menthol and icilin reveals a subset of neurons sensitive to innocuous cool (< 29 oC) and low concentrations of icilin (3-10 µM) which closely match the temperature activation and pharmacological profile of TRPM8. In addition, the majority of lamina I and II neurons displayed characteristics partly consistent with TRPA1-activation, including a concentration-dependent response to icilin and blockade by ruthenium red. The present experiments did not allow thermal characterisation of these TRPA1-like responses. Together these observations indicate that the effects of menthol and icilin on glutamatergic synaptic transmission in the superficial dorsal horn are mediated by TRPM8 and possibly by TRPA1. Examination of the anatomical location of neurons activated by temperature reduction, menthol, icilin and capsaicin allowed the central termination pattern of thermoreceptive primary afferent fibres with specific TRP-like response characteristics to be determined. TRPM8-like presynaptic activation was confined to a subpopulation of neurons located in lamina I and outer lamina II, while the majority of neurons throughout laminae I and II received inputs sensitive to menthol, high concentrations of icilin and capsaicin. These findings suggest that innocuous cool sensation projects to a specific subpopulation of superficial dorsal horn neurons unlike other modalities (mediated by TRPV1, possibly TRPA1 and other receptors), which non-selectively engage circuits within the entire superficial dorsal horn. No morphological specificity was identified for recovered neurons after electrophysiological characterisation. Finally, mu-opioids were shown to inhibit basal glutamatergic synaptic transmission as well as menthol- and icilin-induced transmission in the superficial dorsal horn. Of particular interest, delta-opioids selectively inhibited icilin-induced synaptic transmission within the same location. The selective effect of delta-opioids suggests a possible role in modulating receptors activated by icilin (TRPM8 and TRPA1). Overall, this thesis provides further evidence that TRPM8 is responsible for the transduction of innocuous cold sensation in mammals and is a potential therapeutic target in humans with cold hyperaesthesia secondary to abnormal thermal processing. The use of delta-opioid agonists warrants further investigation in cold hypersensitivity states and potentially other forms of pain.

Diabetes mellitus is a complex disease characterised by chronic hyperglycaemia due to compromised insulin synthesis and secretion, or decreased tissue sensitivity to insulin, if not all three conditions. Endothelial dysfunction is a common complication in diabetes in which endothelium-dependent vasodilation is impaired. The aim of this study was to examine the involvement of TRPV4 in diabetes endothelial dysfunction. Male Charles River Wistar rats (350-450 g) were injected with 65mg/kg streptozotocin (STZ) intraperitoneally. STZ-injected rats were compared with naïve rats (not injected with STZ) or control rats (injected with 10ml/kg of 20mM citrate buffer, pH 4.0-4.5), if not both. Rats with blood glucose concentrations greater than 16mmol/L were considered to be diabetic. As the results revealed, STZ-diabetic rats showed significant endothelial dysfunction characterised by impaired muscarinic-induced vasodilation, as well as significant impairment in TRPV4-induced vasodilation in aortic rings and mesenteric arteries. Furthermore, STZ-diabetic primary aortic endothelial cells (ECs) showed a significant reduction in TRPV4-induced intracellular calcium ([Ca2+]i) elevation. TRPV4, endothelial nitric oxide synthase (eNOS), and caveolin-1 (CAV-1) were also significantly downregulated in STZ-diabetic primary aortic ECs and were later significantly restored by in vitro insulin treatment. Methylglyoxal (MGO) was significantly elevated in STZ-diabetic rat serum, and nondiabetic aortic rings incubated with MGO (100μM) for 12 hours showed significant endothelial dysfunction. Moreover, nondiabetic primary aortic ECs treated with MGO (100μM) for 5 days showed significant TRPV4 downregulation and significant suppression of 4-α-PDD-induced [Ca2+]i elevation, which was later restored by L-arginine (100μM) co-incubation. Incubating nondiabetic aortic rings with MGO (100μM) for 2 hours induced a spontaneous loss of noradrenaline-induced contractility persistence. Moreover, MGO induced significant [Ca2+]i elevation in Chinese hamster ovary cells expressing rat TRPM8 channels (rTRPM8), which was significantly inhibited by AMTB (1-5μM). Taken together, TRPV4, CAV-1, and eNOS can form a functional complex that is downregulated in STZ-diabetic aortic ECs and restored by insulin treatment. MGO elevation might furthermore contribute to diabetes endothelial dysfunction and TRPV4 downregulation. By contrast, MGO induced the loss of contractility persistence, possibly due to MGO's acting as a TRPM8 agonist.