Publications by authors named "Michael E Hildebrand"

21 Publications

  • Page 1 of 1

Advances and Barriers in Understanding Presynaptic -Methyl--Aspartate Receptors in Spinal Pain Processing.

Front Mol Neurosci 2022 31;15:864502. Epub 2022 Mar 31.

Department of Neuroscience, Carleton University, Ottawa, ON, Canada.

For decades, -methyl--aspartate (NMDA) receptors have been known to play a critical role in the modulation of both acute and chronic pain. Of particular interest are NMDA receptors expressed in the superficial dorsal horn (SDH) of the spinal cord, which houses the nociceptive processing circuits of the spinal cord. In the SDH, NMDA receptors undergo potentiation and increases in the trafficking of receptors to the synapse, both of which contribute to increases in excitability and plastic increases in nociceptive output from the SDH to the brain. Research efforts have primarily focused on postsynaptic NMDA receptors, despite findings that presynaptic NMDA receptors can undergo similar plastic changes to their postsynaptic counterparts. Recent technological advances have been pivotal in the discovery of mechanisms of plastic changes in presynaptic NMDA receptors within the SDH. Here, we highlight these recent advances in the understanding of presynaptic NMDA receptor physiology and their modulation in models of chronic pain. We discuss the role of specific NMDA receptor subunits in presynaptic membranes of nociceptive afferents and local SDH interneurons, including their modulation across pain modalities. Furthermore, we discuss how barriers such as lack of sex-inclusive research and differences in neurodevelopmental timepoints have complicated investigations into the roles of NMDA receptors in pathological pain states. A more complete understanding of presynaptic NMDA receptor function and modulation across pain states is needed to shed light on potential new therapeutic treatments for chronic pain.
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http://dx.doi.org/10.3389/fnmol.2022.864502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9008455PMC
March 2022

Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain.

Brain 2022 04;145(3):1124-1138

Department of Neuroscience, Carleton University, K1S 5B6 Ontario, Canada.

The prevalence and severity of many chronic pain syndromes differ across sex, and recent studies have identified differences in immune signalling within spinal nociceptive circuits as a potential mediator. Although it has been proposed that sex-specific pain mechanisms converge once they reach neurons within the superficial dorsal horn, direct investigations using rodent and human preclinical pain models have been lacking. Here, we discovered that in the Freund's adjuvant in vivo model of inflammatory pain, where both male and female rats display tactile allodynia, a pathological coupling between KCC2-dependent disinhibition and N-methyl-D-aspartate receptor (NMDAR) potentiation within superficial dorsal horn neurons was observed in male but not female rats. Unlike males, the neuroimmune mediator brain-derived neurotrophic factor (BDNF) failed to downregulate inhibitory signalling elements (KCC2 and STEP61) and upregulate excitatory elements (pFyn, GluN2B and pGluN2B) in female rats, resulting in no effect of ex vivo brain-derived neurotrophic factor on synaptic NMDAR responses in female lamina I neurons. Importantly, this sex difference in spinal pain processing was conserved from rodents to humans. As in rodents, ex vivo spinal treatment with BDNF downregulated markers of disinhibition and upregulated markers of facilitated excitation in superficial dorsal horn neurons from male but not female human organ donors. Ovariectomy in female rats recapitulated the male pathological pain neuronal phenotype, with BDNF driving a coupling between disinhibition and NMDAR potentiation in adult lamina I neurons following the prepubescent elimination of sex hormones in females. This discovery of sexual dimorphism in a central neuronal mechanism of chronic pain across species provides a foundational step towards a better understanding and treatment for pain in both sexes.
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http://dx.doi.org/10.1093/brain/awab408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9050559PMC
April 2022

South-to-south mentoring as a vehicle for implementing sustainable health security in Africa.

One Health Outlook 2021 Oct 6;3(1):20. Epub 2021 Oct 6.

International Federation of Biosafety Associations (IFBA), Ottawa, Canada.

Background: While sustainability has become a universal precept in the development of global health security systems, supporting policies often lack mechanisms to drive policies into regular practice. 'On-paper' norms and regulations are to a great extent upheld by frontline workers who often lack the opportunity to communicate their first-hand experiences to decisionmakers; their role is an often overlooked, yet crucial, aspect of a sustainable global health security landscape. Initiatives and programs developing transdisciplinary professional skills support the increased bidirectional dialogue between these frontline workers and key policy- and decisionmakers which may sustainably narrow the gap between global health security policy design and implementation.

Methods: The International Federation of Biosafety Associations' (IFBA) Global Mentorship Program recruits biosafety and biosecurity champions across Africa to provide local peer mentorship to developing professionals in their geographic region. Mentors and mentees complete structured one year program cycles, where they are provided with written overviews of monthly discussion topics, and attend optional virtual interactive activities. Feedback from African participants of the 2019-2020 program cycle was collected using a virtual Exit Survey, where aspects of program impact and structure were assessed.

Results: Following its initial call for applications, the IFBA Global Mentorship Program received considerable interest from professionals across the African continent, particularly in East and North Africa. The pilot program cycle matched a total of 62 individuals from an array of professional disciplines across several regions, 40 of which were located on the African continent. The resulting mentorship pairs shared knowledge, skills, and experiences towards translating policy objectives to action on the front lines. Mentorship pairs embraced multidisciplinary approaches to harmonize health security strategies across the human and animal health sectors. South-to-South mentorship therefore provided mentees with locally relevant support critical to translation of best technical practices to local capacity and work.

Conclusion: The IFBA's South-to-South Global Mentorship Program has demonstrated its ability to form crucial links between frontline biosafety professionals, laboratory workers, and policy- and decision-makers across several implicated sectors. By supporting regionally relevant peer mentorship programs, the gap between health security policy development and implementation may be narrowed.
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http://dx.doi.org/10.1186/s42522-021-00050-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8492092PMC
October 2021

Ion Channels and Electrophysiological Properties of Astrocytes: Implications for Emergent Stimulation Technologies.

Front Cell Neurosci 2021 20;15:644126. Epub 2021 May 20.

Department of Neuroscience, Carleton University, Ottawa, ON, Canada.

Astrocytes comprise a heterogeneous cell population characterized by distinct morphologies, protein expression and function. Unlike neurons, astrocytes do not generate action potentials, however, they are electrically dynamic cells with extensive electrophysiological heterogeneity and diversity. Astrocytes are hyperpolarized cells with low membrane resistance. They are heavily involved in the modulation of K and express an array of different voltage-dependent and voltage-independent channels to help with this ion regulation. In addition to these K channels, astrocytes also express several different types of Na channels; intracellular Na signaling in astrocytes has been linked to some of their functional properties. The physiological hallmark of astrocytes is their extensive intracellular Ca signaling cascades, which vary at the regional, subregional, and cellular levels. In this review article, we highlight the physiological properties of astrocytes and the implications for their function and influence of network and synaptic activity. Furthermore, we discuss the implications of these differences in the context of optogenetic and DREADD experiments and consider whether these tools represent physiologically relevant techniques for the interrogation of astrocyte function.
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http://dx.doi.org/10.3389/fncel.2021.644126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173131PMC
May 2021

The T-type calcium channel antagonist, Z944, reduces spinal excitability and pain hypersensitivity.

Br J Pharmacol 2021 09 22;178(17):3517-3532. Epub 2021 May 22.

Department of Neuroscience, Carleton University, Ottawa, Ontario, Canada.

Background And Purpose: T-type voltage-gated calcium channels are an emerging therapeutic target for neurological disorders including epilepsy and pain. Inhibition of T-type channels reduces the excitability of peripheral nociceptive sensory neurons and reverses pain hypersensitivity in male rodent pain models. However, administration of peripherally restricted T-type antagonists failed to show efficacy in multiple clinical and preclinical pain trials, suggesting that inhibition of peripheral T-type channels alone may be insufficient for pain relief.

Experimental Approach: We utilized the selective and CNS-penetrant T-type channel antagonist, Z944, in electrophysiological, calcium imaging and behavioural paradigms to determine its effect on lamina I neuron excitability and inflammatory pain behaviours.

Key Results: Voltage-clamp recordings from lamina I spinal neurons of adult rats revealed that approximately 80% of neurons possess a low threshold T-type current, which was blocked by Z944. Due to this highly prevalent T-type current, Z944 potently blocked action-potential evoked somatic and dendritic calcium transients in lamina I neurons. Moreover, application of Z944 to spinal cord slices attenuated action potential firing rates in over half of laminae I/II neurons. Finally, we found that intraperitoneal injection of Z944 (1-10 mg·kg ) dose-dependently reversed mechanical allodynia in the complete Freund's adjuvant model of persistent inflammatory pain, with a similar magnitude and time course of analgesic effects between male and female rats.

Conclusion And Implications: T-type calcium channels critically shape the excitability of lamina I pain processing neurons and inhibition of these channels by the clinical stage antagonist Z944 potently reverses pain hypersensitivity across sexes.
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http://dx.doi.org/10.1111/bph.15498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8453510PMC
September 2021

Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats.

Channels (Austin) 2021 12;15(1):179-192

Department of Neuroscience, Carleton University , Ottawa, Canada.

N-methyl-D-aspartate receptors (NMDARs) are excitatory ionotropic glutamate receptors expressed throughout the CNS, including in the spinal dorsal horn. The GluN2 subtypes of NMDAR subunit, which include GluN2A, GluN2B, and GluN2D in the dorsal horn, confer NMDARs with structural and functional variability, enabling heterogeneity in synaptic transmission and plasticity. Despite essential roles for NMDARs in physiological and pathological pain processing, the distribution and function of these specific GluN2 isoforms across dorsal horn laminae remain poorly understood. Surprisingly, there is a complete lack of knowledge of GluN2 expression in female rodents. We, therefore, investigated the relative expression of specific GluN2 variants in the dorsal horn of lumbar (L4/L5) spinal cord from both male and female rats. In order to detect synaptic GluN2 isoforms, we used pepsin antigen-retrieval to unmask these highly cross-linked protein complexes. We found that GluN2B and GluN2D are preferentially localized to the pain-processing superficial regions of the dorsal horn in males, while only GluN2B is predominantly localized to the superficial dorsal horn of female rats. The GluN2A subunit is diffusely localized to neuropil throughout the dorsal horn of both males and females, while GluN2B and GluN2D immunolabelling are found both in the neuropil and on the soma of dorsal horn neurons. Finally, we identified an unexpected enhanced expression of GluN2B in the medial division of the superficial dorsal horn, but in males only. These sex-specific localization patterns of GluN2-NMDAR subunits across dorsal horn laminae have significant implications for the understanding of divergent spinal mechanisms of pain processing.
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http://dx.doi.org/10.1080/19336950.2020.1871205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849732PMC
December 2021

Conserved contributions of NMDA receptor subtypes to synaptic responses in lamina II spinal neurons across early postnatal development.

Mol Brain 2020 03 5;13(1):31. Epub 2020 Mar 5.

Department of Neuroscience, Carleton University, Ottawa, ON, Canada.

NMDA receptors are heteromeric complexes that contribute to excitatory synaptic transmission and plasticity. The presence of specific variants of GluN2 subunits in these complexes enables diversity in NMDA receptor function and regulation. At brain synapses, there is a switch from slow GluN2B-mediated NMDA receptors to faster GluN2A-dominated NMDA receptors as well as an increase in the ratio of AMPA to NMDA receptors during early postnatal development. This glutamate receptor switch is observed across brain regions and is critical for synaptic maturation, circuit development, and associative learning. However, whether a similar receptor subunit switch occurs within pain processing neurons in the developing spinal cord remains untested. To investigate this, we performed whole-cell patch clamp recordings of excitatory synaptic responses from lamina II dorsal horn neurons of one to three week-old rats. We found that GluN2B and GluN2A both prominently contribute to NMDA receptor responses at neonatal lamina II synapses, with a small contribution from GluN2D as well. Surprisingly, we found that this molecular identity of NMDA receptor responses as well as the relative contribution of AMPA receptors versus NMDA receptors did not change at lamina II synapses across early postnatal development (P7 to P21). The lack of a developmental switch and persistence of slow-decaying GluN2B- and GluN2D-mediated synaptic responses throughout neuronal maturation in the dorsal horn has implications for understanding both the regulation of synaptic glutamatergic receptors as well as spinal mechanisms of pain processing.
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http://dx.doi.org/10.1186/s13041-020-00566-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057509PMC
March 2020

Loss of STEP61 couples disinhibition to N-methyl-d-aspartate receptor potentiation in rodent and human spinal pain processing.

Brain 2019 06;142(6):1535-1546

Department of Neuroscience, Carleton University, Ottawa, ON, Canada.

Dysregulated excitability within the spinal dorsal horn is a critical mediator of chronic pain. In the rodent nerve injury model of neuropathic pain, BDNF-mediated loss of inhibition (disinhibition) gates the potentiation of excitatory GluN2B N-methyl-d-aspartate receptor (NMDAR) responses at lamina I dorsal horn synapses. However, the centrality of this mechanism across pain states and species, as well as the molecular linker involved, remain unknown. Here, we show that KCC2-dependent disinhibition is coupled to increased GluN2B-mediated synaptic NMDAR responses in a rodent model of inflammatory pain, with an associated downregulation of the tyrosine phosphatase STEP61. The decreased activity of STEP61 is both necessary and sufficient to prime subsequent phosphorylation and potentiation of GluN2B NMDAR by BDNF at lamina I synapses. Blocking disinhibition reversed the downregulation of STEP61 as well as inflammation-mediated behavioural hypersensitivity. For the first time, we characterize GluN2B-mediated NMDAR responses at human lamina I synapses and show that a human ex vivo BDNF model of pathological pain processing downregulates KCC2 and STEP61 and upregulates phosphorylated GluN2B at dorsal horn synapses. Our results demonstrate that STEP61 is the molecular brake that is lost following KCC2-dependent disinhibition and that the decrease in STEP61 activity drives the potentiation of excitatory GluN2B NMDAR responses in rodent and human models of pathological pain. The ex vivo human BDNF model may thus form a translational bridge between rodents and humans for identification and validation of novel molecular pain targets.
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http://dx.doi.org/10.1093/brain/awz105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536915PMC
June 2019

Potentiation of Synaptic GluN2B NMDAR Currents by Fyn Kinase Is Gated through BDNF-Mediated Disinhibition in Spinal Pain Processing.

Cell Rep 2016 12;17(10):2753-2765

Program in Neurosciences & Mental Health, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Physiology, University of Toronto, Toronto, ON M5S 1A1, Canada. Electronic address:

In chronic pain states, the neurotrophin brain-derived neurotrophic factor (BDNF) transforms the output of lamina I spinal neurons by decreasing synaptic inhibition. Pain hypersensitivity also depends on N-methyl-D-aspartate receptors (NMDARs) and Src-family kinases, but the locus of NMDAR dysregulation remains unknown. Here, we show that NMDAR-mediated currents at lamina I synapses are potentiated in a peripheral nerve injury model of neuropathic pain. We find that BDNF mediates NMDAR potentiation through activation of TrkB and phosphorylation of the GluN2B subunit by the Src-family kinase Fyn. Surprisingly, we find that Cl-dependent disinhibition is necessary and sufficient to prime potentiation of synaptic NMDARs by BDNF. Thus, we propose that spinal pain amplification is mediated by a feedforward mechanism whereby loss of inhibition gates the increase in synaptic excitation within individual lamina I neurons. Given that neither disinhibition alone nor BDNF-TrkB signaling is sufficient to potentiate NMDARs, we have discovered a form of molecular coincidence detection in lamina I neurons.
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http://dx.doi.org/10.1016/j.celrep.2016.11.024DOI Listing
December 2016

The unusual suspects: Regulation of retinal calcium channels by somatostatin.

Channels (Austin) 2015 ;9(2):61-2

a Department of Neuroscience ; Carleton University ; Ottawa , ON Canada.

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http://dx.doi.org/10.1080/19336950.2015.1018000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594440PMC
September 2015

GluN2B and GluN2D NMDARs dominate synaptic responses in the adult spinal cord.

Sci Rep 2014 Feb 13;4:4094. Epub 2014 Feb 13.

1] Program in Neurosciences & Mental Health, Hospital for Sick Children, Toronto, ON, Canada [2] Department of Physiology, University of Toronto, Toronto, ON, Canada.

The composition of the postsynaptic ionotropic receptors that receive presynaptically released transmitter is critical not only for transducing and integrating electrical signals but also for coordinating downstream biochemical signaling pathways. At glutamatergic synapses in the adult CNS an overwhelming body of evidence indicates that the NMDA receptor (NMDAR) component of synaptic responses is dominated by NMDARs containing the GluN2A subunit, while NMDARs containing GluN2B, GluN2C, or GluN2D play minor roles in synaptic transmission. Here, we discovered NMDAR-mediated synaptic responses with characteristics not described elsewhere in the adult CNS. We found that GluN2A-containing receptors contribute little to synaptic NMDAR responses while GluN2B dominates at synapses of lamina I neurons in the adult spinal cord. In addition, we provide evidence for a GluN2D-mediated synaptic NMDAR component in adult lamina I neurons. Strikingly, the charge transfer mediated by GluN2D far exceeds that of GluN2A and is comparable to that of GluN2B. Lamina I forms a distinct output pathway from the spinal pain processing network to the pain networks in the brain. The GluN2D-mediated synaptic responses we have discovered in lamina I neurons provide the molecular underpinning for slow, prolonged and feedforward amplification that is a fundamental characteristic of pain.
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http://dx.doi.org/10.1038/srep04094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923208PMC
February 2014

Calcium-permeable ion channels in pain signaling.

Physiol Rev 2014 Jan;94(1):81-140

The detection and processing of painful stimuli in afferent sensory neurons is critically dependent on a wide range of different types of voltage- and ligand-gated ion channels, including sodium, calcium, and TRP channels, to name a few. The functions of these channels include the detection of mechanical and chemical insults, the generation of action potentials and regulation of neuronal firing patterns, the initiation of neurotransmitter release at dorsal horn synapses, and the ensuing activation of spinal cord neurons that project to pain centers in the brain. Long-term changes in ion channel expression and function are thought to contribute to chronic pain states. Many of the channels involved in the afferent pain pathway are permeable to calcium ions, suggesting a role in cell signaling beyond the mere generation of electrical activity. In this article, we provide a broad overview of different calcium-permeable ion channels in the afferent pain pathway and their role in pain pathophysiology.
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http://dx.doi.org/10.1152/physrev.00023.2013DOI Listing
January 2014

Identification of sodium channel isoforms that mediate action potential firing in lamina I/II spinal cord neurons.

Mol Pain 2011 Sep 12;7:67. Epub 2011 Sep 12.

Zalicus Pharmaceuticals Ltd, Vancouver, BC, Canada.

Background: Voltage-gated sodium channels play key roles in acute and chronic pain processing. The molecular, biophysical, and pharmacological properties of sodium channel currents have been extensively studied for peripheral nociceptors while the properties of sodium channel currents in dorsal horn spinal cord neurons remain incompletely understood. Thus far, investigations into the roles of sodium channel function in nociceptive signaling have primarily focused on recombinant channels or peripheral nociceptors. Here, we utilize recordings from lamina I/II neurons withdrawn from the surface of spinal cord slices to systematically determine the functional properties of sodium channels expressed within the superficial dorsal horn.

Results: Sodium channel currents within lamina I/II neurons exhibited relatively hyperpolarized voltage-dependent properties and fast kinetics of both inactivation and recovery from inactivation, enabling small changes in neuronal membrane potentials to have large effects on intrinsic excitability. By combining biophysical and pharmacological channel properties with quantitative real-time PCR results, we demonstrate that functional sodium channel currents within lamina I/II neurons are predominantly composed of the NaV1.2 and NaV1.3 isoforms.

Conclusions: Overall, lamina I/II neurons express a unique combination of functional sodium channels that are highly divergent from the sodium channel isoforms found within peripheral nociceptors, creating potentially complementary or distinct ion channel targets for future pain therapeutics.
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http://dx.doi.org/10.1186/1744-8069-7-67DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190347PMC
September 2011

A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain.

Pain 2011 Apr 23;152(4):833-843. Epub 2011 Feb 23.

Zalicus Pharmaceuticals, 301-2389 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3 Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada T2N 4N1 Department of Pharmacology and Anesthesiology, University of Arizona, Tucson, AZ 85724, USA Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada V6T 1Z4.

Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including Na(V)1.7 and Na(V)1.8 sodium channels and Ca(V)3.2 T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactivation of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics.
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http://dx.doi.org/10.1016/j.pain.2010.12.035DOI Listing
April 2011

Contributions of T-type voltage-gated calcium channels to postsynaptic calcium signaling within Purkinje neurons.

Cerebellum 2012 Sep;11(3):651-65

Institut des Neurosciences Cellulaires et Intégratives, CNRS-Université de Strasbourg, 5 rue Blaise Pascal, Strasbourg, France.

Low threshold voltage-gated T-type calcium channels have long been implicated in the electrical excitability and calcium signaling of cerebellar Purkinje neurons although the molecular composition, localization, and modulation of T-type channels within Purkinje cells have only recently been addressed. The specific functional roles that T-type channels play in local synaptic integration within Purkinje spines are also currently being unraveled. Overall, Purkinje neurons represent a powerful model system to explore the potential roles of postsynaptic T-type channels throughout the nervous system. In this review, we present an overview of T-type calcium channel biophysical, pharmacological, and physiological characteristics that provides a foundation for understanding T-type channels within Purkinje neurons. We also describe the biophysical properties of T-type channels in context of other voltage-gated calcium channel currents found within Purkinje cells. The data thus far suggest that one specific T-type isoform, Ca(v)3.1, is highly expressed within Purkinje spines and both physically and functionally couples to mGluR1 and other effectors within putative signaling microdomains. Finally, we discuss how the selective potentiation of Ca(v)3.1 channels via activation of mGluR1 by parallel fiber inputs affects local synaptic integration and how this interaction may relate to the overall excitability of Purkinje neuron dendrites.
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http://dx.doi.org/10.1007/s12311-010-0195-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411289PMC
September 2012

Functional coupling between mGluR1 and Cav3.1 T-type calcium channels contributes to parallel fiber-induced fast calcium signaling within Purkinje cell dendritic spines.

J Neurosci 2009 Aug;29(31):9668-82

Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.

T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Ca(v)3.1 T-type currents via a G-protein- and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Ca(v)3.1 gene knock-out animals show that Ca(v)3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Ca(v)3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s.
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http://dx.doi.org/10.1523/JNEUROSCI.0362-09.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6666586PMC
August 2009

Activation of corticotropin-releasing factor receptor 1 selectively inhibits CaV3.2 T-type calcium channels.

Mol Pharmacol 2008 Jun 21;73(6):1596-609. Epub 2008 Feb 21.

Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597.

The corticotropin-releasing factor (CRF) peptides CRF and uro-cortins 1 to 3 are crucial regulators of mammalian stress and inflammatory responses, and they are also implicated in disorders such as anxiety, depression, and drug addiction. There is considerable interest in the physiological mechanisms by which CRF receptors mediate their widespread effects, and here we report that the native CRF receptor 1 (CRFR1) endogenous to the human embryonic kidney 293 cells can functionally couple to mammalian Ca(V)3.2 T-type calcium channels. Activation of CRFR1 by either CRF or urocortin (UCN) 1 reversibly inhibits Ca(V)3.2 currents (IC(50) of approximately 30 nM), but it does not affect Ca(V)3.1 or Ca(V)3.3 channels. Blockade of CRFR1 by the antagonist astressin abolished the inhibition of Ca(V)3.2 channels. The CRFR1-dependent inhibition of Ca(V)3.2 channels was independent of the activities of phospholipase C, tyrosine kinases, Ca(2+)/calmodulin-dependent protein kinase II, protein kinase C, and other kinase pathways, but it was dependent upon a cholera toxin-sensitive G protein-mediated mechanism relying upon G protein betagamma subunits (Gbetagamma). The inhibition of Ca(V)3.2 channels via the activation of CRFR1 was due to a hyperpolarized shift in their steady-state inactivation, and it was reversible upon washout of the agonists. Given that UCN affect multiple aspects of cardiac and neuronal physiology and that Ca(V)3.2 channels are widespread throughout the cardiovascular and nervous systems, the results point to a novel and functionally relevant CRFR1-Ca(V)3.2 T-type calcium channel signaling pathway.
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http://dx.doi.org/10.1124/mol.107.043612DOI Listing
June 2008

Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11-coupled muscarinic acetylcholine receptors.

J Biol Chem 2007 Jul 29;282(29):21043-55. Epub 2007 May 29.

Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4.

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca(2+) channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca(2+) channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (alpha(1G)), Cav3.2 (alpha(1H)), and Cav3.3 (alpha) T-type Ca(2+)(1I)channels were co-expressed with the M1 Galpha(q/11)-coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca(2+) currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca(2+) channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of Galpha and Gbetagamma proteins and gain-of-function experiments with genetically encoded Galpha subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through Galpha(q/11). This is supported by experiments showing that activation of the M3 and M5 Galpha(q/11)-coupled mAChRs also causes inhibition of Cav3.3 currents, although Galpha(i)-coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation.
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http://dx.doi.org/10.1074/jbc.M611809200DOI Listing
July 2007

Inhibition of high voltage-activated calcium channels by spider toxin PnTx3-6.

J Pharmacol Exp Ther 2005 Sep 2;314(3):1370-7. Epub 2005 Jun 2.

Biotechnology Laboratory, University of British Columbia, Vancouver, Canada.

Animal peptide toxins have become powerful tools to study structure-function relationships and physiological roles of voltage-activated Ca(2+) channels. In the present study, we investigated the effects of PnTx3-6, a neurotoxin purified from the venom of the spider Phoneutria nigriventer on cloned mammalian Ca(2+) channels expressed in human embryonic kidney 293 cells and endogenous Ca(2+) channels in N18 neuroblastoma cells. Whole-cell patch-clamp measurements indicate that PnTx3-6 reversibly inhibited L-(alpha(1C)/Ca(v)1.2), N-(alpha(1B)/Ca(v)2.2), P/Q-(alpha(1A)/Ca(v)2.1), and R-(alpha(1E)/Ca(v)2.3) type channels with varying potency (alpha(1B) > alpha(1E) > alpha(1A) > alpha(1C)) and IC(50) values of 122, 136, 263, and 607 nM, respectively. Inhibition occurred without alteration of the kinetics or the voltage dependence of the exogenously expressed Ca(2+) channels. In N18 cells, PnTx3-6 exhibited highest potency against N-type (conotoxin-GVIA-sensitive) current. In contrast to its effects on high voltage-activated Ca(2+) channels subtypes, application of 1 microM PnTx3-6 did not affect alpha(1G)/Ca(v)3.1 T-type Ca(2+) channels. Based on our study, we suggest that PnTx3-6 acts as a omega-toxin that targets high voltage-activated Ca(2+) channels, with a preference for the Ca(v)2 subfamily (N-, P/Q-, and R-types).
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http://dx.doi.org/10.1124/jpet.105.087023DOI Listing
September 2005

Functional implications of a novel EA2 mutation in the P/Q-type calcium channel.

Ann Neurol 2004 Aug;56(2):213-20

Division of Neurology, University of British Columbia, Vancouver, British Columbia, Canada.

Episodic ataxia type 2 (EA2) is an autosomal dominant condition characterized by paroxysmal attacks of ataxia, vertigo, and nausea, typically lasting minutes to days in duration. These symptoms can be prevented or significantly attenuated by the oral administration of acetazolamide; however, the mechanism by which acetazolamide ameliorates EA2 symptoms is unknown. EA2 typically results from nonsense mutations in the CACNA1A gene that encodes the alpha1A (Cav2.1) subunit of the P/Q-type calcium (Ca2+) channel. We have identified a novel H1736L missense mutation in the CACNA1A gene associated with the EA2 phenotype. This mutation is localized near the pore-forming region of the P/Q-type Ca2+ channel. Functional analysis of P/Q-type channels containing the mutation show that the H1736L alteration affects several channel properties, including reduced current density, increased rate of inactivation, and a shift in the voltage dependence of activation to more positive values. Although these findings are consistent with an overall loss of P/Q-type channel function, the mutation also caused some biophysical changes consistent with a gain of function. We also tested the direct effect of acetazolamide on both wild-type and H1736L mutated P/Q-type channels and did not observe any direct action on channel properties of this pharmacological agent used to treat EA2 patients.
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http://dx.doi.org/10.1002/ana.20169DOI Listing
August 2004

Mammalian voltage-gated calcium channels are potently blocked by the pyrethroid insecticide allethrin.

J Pharmacol Exp Ther 2004 Mar 21;308(3):805-13. Epub 2003 Nov 21.

Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, Canada.

Pyrethroids are commonly used insecticides for both household and agricultural applications. It is generally reported that voltage-gated sodium channels are the primary target for toxicity of these chemicals to humans. The phylogenetic and structural relatedness between sodium channels and voltage-gated calcium (Ca) channels prompted us to examine the effects of the type 1 pyrethroid allethrin on the three major classes of mammalian calcium channels exogenously expressed in human embryonic kidney 293 cells. We report that all classes of mammalian calcium channels are targets for allethrin at concentrations very similar to those reported for interaction with sodium channels. Allethrin caused blockade with IC(50) values of 7.0 microM for T-type alpha(1G) (Ca(v)3.1), 6.8 microM for L-type alpha(1C) (Ca(v)1.2), and 6.7 microM for P/Q-type alpha(1A) (Ca(v)2.1) channels. Mechanistically, the blockade of calcium channels was found to be significantly different than the prolonged opening of mammalian sodium channels caused by pyrethroids. In all calcium channel subtypes tested, allethrin caused a significant acceleration of the inactivation kinetics and a hyperpolarizing shift in the voltage dependence of inactivation. The high-voltage-activated P/Q- and L-type channels showed a frequency of stimulation-dependent increase in block by allethrin, whereas the low-voltage-activated alpha(1G) subtype did not. Allethrin did not significantly modify the deactivation kinetics or current-voltage relationships of any of the calcium channel types. Our study indicates that calcium channels are another primary target for allethrin and suggests that blockade of different types of calcium channels may underlie some of the chronic effects of low-level pyrethroid poisoning.
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http://dx.doi.org/10.1124/jpet.103.058792DOI Listing
March 2004
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