TRPA1 expression and its functional activation in rodent cortex

TRPA1 is a non-selective cation channel involved in pain sensation and neurogenic inflammation. Although TRPA1 is well established in a number of organs including the nervous system, its presence and function in the mammalian cortex remains unclear. Here, we demonstrate the expression of TRPA1 in rodent somatosensory cortex through immunostaining and investigate its functional activation by whole-cell electrophysiology, Ca2+ imaging and two-photon photoswitching. Application of TRPA1 agonist (AITC) and antagonist (HC-030031) produced significant modulation of activity in layer 5 (L5) pyramidal neurons in both rats and mice; AITC increased intracellular Ca2+ concentrations and depolarized neurons, and both effects were blocked by HC-030031. These modulations were absent in the TRPA1 knockout mice. Next, we used optovin, a reversible photoactive molecule, to activate TRPA1 in individual L5 neurons of rat cortex. Optical control of activity was established by applying a tightly focused femtosecond-pulsed laser to optovin-loaded neurons. Light application depolarized neurons (n = 17) with the maximal effect observed at λ = 720 nm. Involvement of TRPA1 was further confirmed by repeating the experiment in the presence of HC-030031, which diminished the light modulation. These results demonstrate the presence of TRPA1 in L5 pyramidal neurons and introduce a highly specific approach to further understand its functional significance.The experiments were supported by an Australian Research Council (ARC) Discovery Project (DP130101364), an ARC Future Fellowship (E.A.) and the ARC Centre of Excellence for Integrative Brain Function (CE140100007)

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Enhanced sensory coding in mouse vibrissal and visual cortex through TRPA1

Raw data and codes for manuscript: Kheradpezhouh, E., Tang, M.F., Mattingley, J.B., Arabzadeh, E. Enhanced sensory coding in mouse vibrissal and visual cortex through TRPA1. Cell Report

The role of TRPM2 channels in oxidative stress-induced liver damage.

The increased production of highly reactive oxygen and nitrogen species plays a significant role in development of a number of liver disorders associated with hepatocellular death and impaired cell regeneration. Liver injury induced by drug toxicity, ischemia-reperfusion, excessive alcohol consumption and different types of viral hepatitis is in large part mediated by oxidative stress. Liver damage due to oxidative stress induced by drugs, including acetaminophen, accounts for 5% of all hospital admissions and for almost half of all acute liver failures. One of the features of hepatocellular death mediated by oxidative stress is Ca²⁺ overload due its release from intracellular organelles and activation of ion channels on the plasma membrane. Ca²⁺ is fundamental for normal cellular functioning. Ca²⁺ signalling, mediated by the rise in free cytoplasmic Ca²⁺ concentration ([Ca²⁺]c)[c subscript], regulates many cellular events. However, a sustained rise in [Ca²⁺]c [c subscript] can be detrimental, leading to mitochondrial dysfunction and cell death through apoptosis and necrosis. Although it is well recognised that Ca²⁺ plays a significant role in oxidative stress-induced liver damage, the molecular identities of the ion channels that provide a pathway for Ca²⁺ entry in hepatocytes remain unidentified. One of the potential candidates that could be responsible for such Ca²⁺ entry pathway in hepatocytes is Transient Receptor Potential Melastatin 2 (TRPM2) channel. TRPM2 is a non-selective cation channel permeable to Na⁺ and Ca²⁺. The main physiological activator of TRPM2 channel is ADP-ribose, which binding to NUDT9-H motif in the TRPM2 C-terminus leads to the opening of the channel pore. It is known that oxidative stress promotes generation and release of ADPR from mitochondria and nuclei into the cytoplasmic space, thus promoting activation of TRPM2-mediated Ca²⁺ entry. In this thesis, we hypothesised that oxidative stress-induced Ca²⁺ entry in hepatocytes is mediated by TRPM2 channels, and used acetaminophen overdose as a model of oxidative stress-induced liver damage. We show that hepatocytes express long isoform of TRPM2, which mediates ADPR- and H₂O₂-induced Ca²⁺ entry and the cation current in these cells. Furthermore, we show that TRPM2 channels are activated in hepatocytes treated with high concentrations of acetaminophen and are responsible for Ca²⁺ overload in acetaminophen-induced liver toxicity. Experiments using TRPM2 KO mice provide first evidence of a pivotal role of TRPM2 channels in acetaminophen-induced liver injury, showing that lack of TRPM2 expression largely protects liver from acetaminophen overdose. An important finding that TRPM2 channels translocate from intracellular compartments to the plasma membrane provides explanation for a slow development of Ca²⁺ entry in response to H₂O₂ and acetaminophen. Finally, we show that substances previously known to protect liver from acetaminophen-induced damage are, in fact, inhibitors of TRPM2 current. Chlorpromazine, an antipsychotic drug, reversibly blocks TRPM2 channel pore, and curcumin, a chemical found in common spice, potently blocks activation of TRPM2 current by ADPR. The results presented in this thesis provide a fundamental knowledge about the role of TRPM2 channels in oxidative stress-induced liver injury, but also open a new chapter in search for the new drugs and drug targets for the treatment of a number of oxidative stress-related liver pathologies.Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 201

Response dynamics of rat barrel cortex neurons to repeated sensory stimulation

Neuronal adaptation is a common feature observed at various stages of sensory processing. Here, we quantified the time course of adaptation in rat somatosensory cortex. Under urethane anesthesia, we juxta-cellularly recorded single neurons (n = 147) while applying a series of whisker deflections at various frequencies (2–32 Hz). For ~90% of neurons, the response per unit of time decreased with frequency. The degree of adaptation increased along the train of deflections and was strongest at the highest frequency. However, a subset of neurons showed facilitation producing higher responses to subsequent deflections. The response latency to consecutive deflections increased both for neurons that exhibited adaptation and for those that exhibited response facilitation. Histological reconstruction of neurons (n = 45) did not reveal a systematic relationship between adaptation profiles and cell types. In addition to the periodic stimuli, we applied a temporally irregular train of deflections with a mean frequency of 8 Hz. For 70% of neurons, the response to the irregular stimulus was greater than that of the 8 Hz regular. This increased response to irregular stimulation was positively correlated with the degree of adaptation. Altogether, our findings demonstrate high levels of diversity among cortical neurons, with a proportion of neurons showing facilitation at specific temporal intervals

A protocol for simultaneous in vivo juxtacellular electrophysiology and local pharmacological manipulation in mouse cortex

Here, we describe a protocol to simultaneously record and label single cortical neurons in vivo under local application of a chemical such as a receptor agonist. This protocol provides a useful tool to investigate how the chemical of interest affects the processing of sensory information by cortical neurons. The juxtacellular labeling allows identification of the cell type and morphology of the recorded neurons. We draw examples to show pharmacological modulations in encoding of vibrotactile stimuli in the mouse primary somatosensory cortex

Localized two-photon photoswitching of Optovin in rat cortical neurons

Photoswitching molecules are widely used to control neuronal activity by activation of endogenous cellular channels and receptors. Optovin is a photoswitching molecule that targets the cation channel transient receptor potential ankyrin 1 (TRPA1). In zebrafish, bulk single-photon photoswitching of Optovin produced behavioural responses associated with TRPA1 activation. We recently found that TRPA1 is widely expressed in the rodent brain particularly in cortical pyramidal neurons and demonstrated its functional activation via TRPA1 agonists and by two-photon (2P) photoswitching of Optovin in the soma. Here, we exploit the highly localized 2P photoswitching of Optovin to activate TRPA1 along different dendritic and axonal regions of individual pyramidal neurons in the rodent cortex. The technique facilitates measurement of isolated responses when light is directed to the axon and along various dendritic domains of L5 pyramidal neurons. Using this technique, we show that 2P photoswitching of Optovin results in higher levels of depolarization along the base of the apical trunk compared to any other location in the neuron.This work was supported by the Australian Research Council (ARC) Discovery Projects (DP130101364, DP170100908 and DP140101555), the National Health and Medical Research Council (NHMRC) Project Grants (PG1124411 and PG1105944) and the ARC Centre of Excellence for Integrative Brain Function (CE140100007)