Publications by authors named "Klaus Ballanyi"

58 Publications

Expiratory abdominal muscle nerve is active at flexor phase, while inspiratory phrenic nerve is not active during locomotion evoked by 5-HT and NMDA in the neonatal rat.

Neurosci Res 2021 Jul 27. Epub 2021 Jul 27.

Department of Physiology, Faculty of Medicine & Dentistry, 7-50 Medical Sciences Building, University of Alberta, Edmonton, T6G 2H7, Canada. Electronic address:

Abdominal muscles are involved in respiration and locomotion. In the isolated pons-spinal cord-rib attached preparation from neonatal rat, the phrenic nerve and abdominal muscles show inspiratory and expiratory activity, respectively. Using this preparation, we investigated whether the bath application of NMDA and 5-HT could evoke locomotor activities in the fourth cervical ventral root (C4VR), phrenic nerve, and abdominal muscle nerve (ilioinguinal nerve, IIG-n). We also observed rib and abdominal muscle movements visually. The phrenic nerve and C4VR showed inspiratory activity consistently under the control conditions, whereas IIG-n showed expiratory activity only at the beginning of the experiment. During the chemically-induced locomotion, both C4VR and IIG-n showed locomotor activity, and IIG-n in particular showed flexor activity. During the flexor activity, lateral bending of the rib cage to the recording site was observed. The phrenic nerve showed weak or no apparent locomotor activity. We concluded that the central pattern generator (CPG) for locomotion provides stronger excitatory synaptic inputs to C4 motoneurons innervating neck and shoulder muscles than the inputs to the phrenic motoneurons. Thus, the locomotor CPG provides a suitable amount of inputs to the functionally proper motoneurons. This preparation will be useful to explore how the respiratory and locomotor CPGs select proper motoneurons to give synaptic inputs and are coordinated with each other.
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http://dx.doi.org/10.1016/j.neures.2021.07.004DOI Listing
July 2021

Mapping the Dynamic Recruitment of Spinal Neurons during Fictive Locomotion.

J Neurosci 2020 12 13;40(50):9692-9700. Epub 2020 Nov 13.

Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2E1, Canada

The basic rhythmic activity that underlies stepping is generated by a neural network, situated in the spinal cord, known as the locomotor central pattern generator (CPG). While a series of lesion experiments have demonstrated that the mammalian locomotor CPG is distributed throughout the ventral portion of the caudal spinal cord, the specific transverse distribution of this neural network is unclear. Here we evoke fictive locomotor activity of various frequencies in upright spinal cords prepared from male and female neonatal mice. This preparation enables us to use an imaging approach to identify locomotor-related cells across the transverse plane of the spinal cord. Results indicate that there is a clear shift in the recruitment of cells toward the ventromedial, and away from the ventrolateral, spinal cord as the frequency of fictive locomotion increases. Surprisingly, the analysis of multiple frequencies of fictive locomotion in the same spinal cord indicates that few neurons are involved in locomotor outputs across multiple speeds. Collectively, these experiments allow us to map the transverse distribution of the locomotor CPG and highlight the pattern of dynamic recruitment that occurs within this neural circuit as the frequency is altered. Our findings are consistent with data indicating that there is a speed-dependent recruitment of interneuronal populations during locomotion and suggest that the locomotor CPG is not a static network, but rather the specific cells recruited vary extensively based on demand. In this article, we use an imaging approach to identify all those cells that are rhythmically active at the same frequency as fictive locomotion recorded from the ventral roots of the isolated spinal cord. These experiments allow us to map the distribution of locomotor-related cells across the transverse plane of the spinal cord and identify the recruitment pattern of these cells as the frequency of locomotor outputs is altered. Our results indicate that there are drastic changes in the specific neurons activated at different frequencies and provide support for the concept that the locomotor central pattern generator is a modular network with speed-dependent recruitment of interneuronal components.
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http://dx.doi.org/10.1523/JNEUROSCI.1885-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726538PMC
December 2020

Endoplasmic reticulum stress in the dorsal root ganglia regulates large-conductance potassium channels and contributes to pain in a model of multiple sclerosis.

FASEB J 2020 09 17;34(9):12577-12598. Epub 2020 Jul 17.

Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada.

Neuropathic pain is a common symptom of multiple sclerosis (MS) and current treatment options are ineffective. In this study, we investigated whether endoplasmic reticulum (ER) stress in dorsal root ganglia (DRG) contributes to pain hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory cells and increased levels of ER stress markers are evident in post-mortem DRGs from MS patients. Similarly, we observed ER stress in the DRG of mice with EAE and relieving ER stress with a chemical chaperone, 4-phenylbutyric acid (4-PBA), reduced pain hypersensitivity. In vitro, 4-PBA and the selective PERK inhibitor, AMG44, normalize cytosolic Ca transients in putative DRG nociceptors. We went on to assess disease-mediated changes in the functional properties of Ca -sensitive BK-type K channels in DRG neurons. We found that the conductance-voltage (GV) relationship of BK channels was shifted to a more positive voltage, together with a more depolarized resting membrane potential in EAE cells. Our results suggest that ER stress in sensory neurons of MS patients and mice with EAE is a source of pain and that ER stress modulators can effectively counteract this phenotype.
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http://dx.doi.org/10.1096/fj.202001163RDOI Listing
September 2020

The ER chaperone calnexin controls mitochondrial positioning and respiration.

Sci Signal 2020 06 30;13(638). Epub 2020 Jun 30.

Faculty of Medicine and Dentistry, Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

Chaperones in the endoplasmic reticulum (ER) control the flux of Ca ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis.
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http://dx.doi.org/10.1126/scisignal.aax6660DOI Listing
June 2020

Using an upright preparation to identify and characterize locomotor related neurons across the transverse plane of the neonatal mouse spinal cord.

J Neurosci Methods 2019 07 25;323:90-97. Epub 2019 May 25.

Department of Physiology, University of Alberta, 3-020D Katz Building, Edmonton, Alberta, T6G 2E1, Canada; Neuroscience and Mental Health Institute, University of Alberta, 3-020D Katz Building, Edmonton, Alberta, T6G 2E1, Canada. Electronic address:

Background: The basic rhythmicity underlying stepping in mammals is generated by a neural network, situated in the spinal cord, known as the locomotor central pattern generator (CPG). While a molecular approach has provided information regarding neuronal populations that participate in locomotor activity and their specific function, the distributed nature of the locomotor CPG has made it difficult to identify and characterize the specific neurons belonging to each population that are rhythmically-active during stepping.

New Method: We describe a preparation in which we isolate the spinal cord from a neonatal mouse, section it at a lumbar segment, situate it in an upright orientation under the objective lens of a 2- photon microscope, and evoke fictive locomotion.

Results: This preparation allows us to image rhythmic Ca oscillations in spinal neurons, and visually identify those that are involved in fictive locomotor activity. We can then characterize unique features of these neurons.

Comparison With Existing Methods: This builds on existing fictive locomotor preparations and is the first which allows for the visual identification of locomotor related neurons spanning the transverse plane of the spinal cord, facilitating their electrophysiological and anatomical characterization CONCLUSIONS: This approach promises to provide new information regarding the distribution of the locomotor CPG in the transverse plane, the characteristics of its component interneurons, as well as the cellular mechanisms and network properties which underlie rhythm generation. By altering the location of Ca indicator application it can also be used to identify and characterize neurons involved in other facets of sensorimotor processing.
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http://dx.doi.org/10.1016/j.jneumeth.2019.05.010DOI Listing
July 2019

Receptor dependence of BDNF actions in superficial dorsal horn: relation to central sensitization and actions of macrophage colony stimulating factor 1.

J Neurophysiol 2019 06 17;121(6):2308-2322. Epub 2019 Apr 17.

Neuroscience and Mental Health Institute, University of Alberta , Edmonton , Canada.

Peripheral nerve injury elicits an enduring increase in the excitability of the spinal dorsal horn. This change, which contributes to the development of neuropathic pain, is a consequence of release and prolonged exposure of dorsal horn neurons to various neurotrophins and cytokines. We have shown in rats that nerve injury increases excitatory synaptic drive to excitatory neurons but decreases drive to inhibitory neurons. Both effects, which contribute to an increase in dorsal horn excitability, appear to be mediated by microglia-derived BDNF. We have used multiphoton Ca imaging and whole cell recording of spontaneous excitatory postsynaptic currents in defined-medium organotypic cultures of GAD67-GFP mice spinal cord to determine the receptor dependence of these opposing actions of BDNF. In mice, as in rats, BDNF enhances excitatory transmission onto excitatory neurons. This is mediated via presynaptic TrkB and p75 neurotrophin receptors and exclusively by postsynaptic TrkB. By contrast with findings from rats, in mice BDNF does not decrease excitation of inhibitory neurons. The cytokine macrophage colony-stimulating factor 1 (CSF-1) has also been implicated in the onset of neuropathic pain. Nerve injury provokes its de novo synthesis in primary afferents, its release in spinal cord, and activation of microglia. We now show that CSF-1 increases excitatory drive to excitatory neurons via a BDNF-dependent mechanism and decreases excitatory drive to inhibitory neurons via BDNF-independent processes. Our findings complete missing steps in the cascade of events whereby peripheral nerve injury instigates increased dorsal horn excitability in the context of central sensitization and the onset of neuropathic pain. Nerve injury provokes synthesis of macrophage colony-stimulating factor 1 (CSF-1) in primary afferents and its release in the dorsal horn. We show that CSF-1 increases excitatory drive to excitatory dorsal horn neurons via BDNF activation of postsynaptic TrkB and presynaptic TrkB and p75 neurotrophin receptors. CSF-1 decreases excitatory drive to inhibitory neurons via a BDNF-independent processes. This completes missing steps in understanding how peripheral injury instigates central sensitization and the onset of neuropathic pain.
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http://dx.doi.org/10.1152/jn.00839.2018DOI Listing
June 2019

Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration.

Commun Biol 2019 14;2:18. Epub 2019 Jan 14.

1Department of Urology, Boston Children's Hospital, Department of Microbiology and Immunobiology, Department of Surgery, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 USA.

Potassium ion (K) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are Förster resonance energy transfer (FRET)-based indicators with a bacterial K binding protein (Kbp) inserting between the fluorescent protein FRET pairs mCerulean3/cp173Venus and Clover/mRuby2, respectively. GINKO1 (green indicator of K for optical imaging) is a single fluorescent protein-based K indicator constructed by insertion of Kbp into enhanced green fluorescent protein (EGFP). These indicators are suitable for detecting K at physiologically relevant concentrations in vitro and in cells. KIRIN1 enabled imaging of cytosolic K depletion in live cells and K efflux and reuptake in cultured neurons. GINKO1, in conjunction with red fluorescent Ca indicator, enable dual-color imaging of K and Ca dynamics in neurons and glial cells. These results demonstrate that KIRIN1 and GINKO1 are useful tools for imaging intracellular K dynamics.
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http://dx.doi.org/10.1038/s42003-018-0269-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331434PMC
May 2020

TARP mediation of accelerated and more regular locus coeruleus network bursting in neonatal rat brain slices.

Neuropharmacology 2019 04 8;148:169-177. Epub 2019 Jan 8.

Department of Physiology, Faculty of Medicine & Dentistry, 7-50 MSB, University of Alberta, Edmonton, T6G2H7, Alberta, Canada. Electronic address:

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARP) increase neuronal excitability. However, it is unknown how TARP affect rhythmic neural network activity. Here we studied TARP effects on local field potential (LFP) bursting, membrane potential and cytosolic Ca (Ca) in locus coeruleus neurons of newborn rat brain slices. LFP bursting was not affected by the unselective competitive ionotropic glutamate receptor antagonist kynurenic acid (2.5 mM). TARP-AMPAR complex activation with 25 μM CNQX accelerated LFP rhythm 2.2-fold and decreased its irregularity score from 63 to 9. Neuronal spiking was correspondingly 2.3-fold accelerated in association with a 2-5 mV depolarization and a modest Ca rise whereas Ca was unchanged in neighboring astrocytes. After blocking rhythmic activities with tetrodotoxin (1 μM), CNQX caused a 5-8 mV depolarization and also the Ca rise persisted. In tetrodotoxin, both responses were abolished by the non-competitive AMPAR antagonist GYKI 53655 (25 μM) which also reversed stimulatory CNQX effects in control solution. The CNQX-evoked Ca rise was blocked by the L-type voltage-activated Ca channel inhibitor nifedipine (100 μM). The findings show that ionotropic glutamate receptor-independent neonatal locus coeruleus network bursting is accelerated and becomes more regular by activating a TARP-AMPAR complex. The associated depolarization-evoked L-type Ca channel-mediated neuronal Ca rise may be pivotal to regulate locus coeruleus activity in cooperation with SK-type K channels. In summary, this is the first demonstration of TARP-mediated stimulation of neural network bursting. We hypothesize that TARP-AMPAR stimulation of rhythmic locus coeruleus output serves to fine-tune its control of multiple brain functions thus comprising a target for drug discovery.
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http://dx.doi.org/10.1016/j.neuropharm.2019.01.004DOI Listing
April 2019

Voluntary wheel running reveals sex-specific nociceptive factors in murine experimental autoimmune encephalomyelitis.

Pain 2019 Apr;160(4):870-881

Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada.

Multiple sclerosis (MS) is an inflammatory, neurodegenerative autoimmune disease associated with sensory and motor dysfunction. Although estimates vary, ∼50% of patients with MS experience pain during their disease. The mechanisms underlying the development of pain are not fully understood, and no effective treatment for MS-related pain is available. Previous work from our laboratory demonstrated that voluntary exercise (wheel running) can reduce nociceptive behaviours at the disease onset in female mice with experimental autoimmune encephalomyelitis (EAE), an animal model used to study the immunopathogenesis of MS. However, given the established sex differences in the underlying mechanisms of chronic pain and MS, we wanted to investigate whether wheel running would also be effective at preventing nociceptive behaviours in male mice with EAE. C57BL/6 mice of both sexes were given access to running wheels for 1 hour/day until the disease onset, when nociceptive behaviour was assessed using von Frey hairs. Daily running effectively reduced nociceptive behaviour in female mice, but not in male mice. We explored the potential biological mechanisms for these effects and found that the reduction in nociceptive behaviour in female mice was associated with reduced levels of inflammatory cytokines from myelin-reactive T cells as well as reduced dorsal root ganglia excitability as seen by decreased calcium responses. These changes were not seen in male mice. Instead, running increased the levels of inflammatory cytokines and potentiated Ca responses in dorsal root ganglia cells. Our results show that voluntary wheel running has sex-dependent effects on nociceptive behaviour and inflammatory responses in male and female mice with EAE.
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http://dx.doi.org/10.1097/j.pain.0000000000001465DOI Listing
April 2019

A Bioluminescent Ca Indicator Based on a Topological Variant of GCaMP6s.

Chembiochem 2019 02 31;20(4):516-520. Epub 2018 Jul 31.

University of Alberta, Department of Chemistry, 11227 Saskatchewan Drive, Edmonton, Alberta, T6G 2G2, Canada.

Fluorescent genetically encoded calcium ion indicators (GECIs) enable Ca dynamics to be monitored in a diverse array of cell types and tissues. One drawback of green fluorescent GECIs, such as the widely used GCaMP6, is that the blue wavelengths of light used to excite the GECI also activate optogenetic actuators such as channelrhodopsins. Accordingly, it is particularly challenging simultaneously to use both optogenetic actuators and GECIs to both control and image cell signaling. Bioluminescence is an alternative imaging modality that circumvents this problem by avoiding the need for illumination for fluorescence excitation. Here, we report the development of a bioluminescent GECI, designated LUCI-GECO1, based on efficient bioluminescent resonance energy transfer (BRET) between the NanoLuc luciferase and a topological variant of GCaMP6s. LUCI-GECO1 is a sensitive ratiometric GECI that retains the highly optimized properties of GCaMP6s, as we demonstrate by imaging of chemically and optogenetically induced Ca concentration changes in cultured cells and neurons.
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http://dx.doi.org/10.1002/cbic.201800255DOI Listing
February 2019

-Expressing Interneurons Regulate Left-Right Alternation during Mammalian Locomotor Activity.

J Neurosci 2018 06 22;38(25):5666-5676. Epub 2018 May 22.

Neuroscience and Mental Health Institute and

The basic pattern of activity underlying stepping in mammals is generated by a neural network located in the caudal spinal cord. Within this network, the specific circuitry coordinating left-right alternation has been shown to involve several groups of molecularly defined interneurons. Here we characterize a population of spinal neurons that express the Wilms' tumor 1 () gene and investigate their role during locomotor activity in mice of both sexes. We demonstrate that -expressing cells are located in the ventromedial region of the spinal cord of mice and are also present in the human spinal cord. In the mouse, these cells are inhibitory, project axons to the contralateral spinal cord, terminate in close proximity to other commissural interneuron subtypes, and are essential for appropriate left-right alternation during locomotion. In addition to identifying -expressing interneurons as a key component of the locomotor circuitry, this study provides insight into the manner in which several populations of molecularly defined interneurons are interconnected to generate coordinated motor activity on either side of the body during stepping. In this study, we characterize -expressing spinal interneurons in mice and demonstrate that they are commissurally projecting and inhibitory. Silencing of this neuronal population during a locomotor task results in a complete breakdown of left-right alternation, whereas flexor-extensor alternation was not significantly affected. Axons of neurons are shown to terminate nearby commissural interneurons, which coordinate motoneuron activity during locomotion, and presumably regulate their activity. Finally, the gene is shown to be present in the spinal cord of humans, raising the possibility of functional homology between these species. This study not only identifies a key component of the locomotor circuitry but also begins to unravel the connectivity among the growing number of molecularly defined interneurons that comprise this neural network.
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http://dx.doi.org/10.1523/JNEUROSCI.0328-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6595980PMC
June 2018

Characterization of the Nile Grass Rat as a Unique Model for Type 2 Diabetic Polyneuropathy.

J Neuropathol Exp Neurol 2018 06;77(6):469-478

Department of Surgery, University of Alberta, Edmonton, Alberta, Canada.

Type 2 diabetes (T2D) has reached pandemic proportions worldwide. Almost half of T2D patients suffer from polyneuropathy that can present as paresthesia, hyperalgesia, allodynia, or hypoesthesia. Therapeutic treatment options are largely incomplete, suggesting new avenues of research are needed. Herein, we introduce the African Nile Grass rat (NGR), which develops T2D solely by diet manipulation, as a novel T2D polyneuropathy model. The purpose of this study was to first characterize T2D-induced polyneuropathy in the NGRs before highlighting their strength as a potential prediabetic model of T2D. NGRs with long-term T2D exhibit hallmark features of polyneuropathy such as decreased motor nerve conduction velocity, intraepidermal denervation, and hyposensitivity to noxious mechanical and thermal stimulation. At the dorsal root ganglia, T2D neurons have altered sodium channel expression, specifically increased Nav1.7 and Nav1.9, and their surrounding satellite glial cells express glial fibrillary acidic protein. Now that these T2D NGRs have been characterized and shown to have a similar presentation to human and other animal models of T2D, the strength of this diet-induced model can be exploited. The prediabetic changes can be observed over their long progression to develop T2D which may allow for a therapeutic window to prevent T2D before permanent damage occurs.
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http://dx.doi.org/10.1093/jnen/nly030DOI Listing
June 2018

Suction electrode recording in locus coeruleus of newborn rat brain slices reveals network bursting comprising summated non-synchronous spiking.

Neurosci Lett 2018 04 12;671:103-107. Epub 2018 Feb 12.

Department of Physiology, Faculty of Medicine & Dentistry, 750 MSB, University of Alberta, Edmonton, T6G2H7, Canada. Electronic address:

The brainstem locus coeruleus (LC) controling behaviors like arousal, sleep, breathing, pain or opioid withdrawal is an established model for spontaneous action potential synchronization. Such synchronous 'spiking' might produce an extracellular field potential (FP) which is a crucial tool for neural network analyses. We found using ≥10 μm tip diameter suction electrodes in newborn rat brainstem slices that the LC generates at ∼1 Hz a robust rhythmic FP (rFP). During distinct rFP phases, LC neurons discharge with a jitter of ±33 ms single spikes that summate to a ∼200 ms-lasting population burst. The rFP is abolished by blocking voltage-gated Na channels with tetrodotoxin (TTX, 50 nM) or gap junctions with mefloquine (100 μM) and activating μ-opioid receptors with [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin (DAMGO, 1 μM). Raising superfusate K from 3 to 7 mM either increases rFP rate or transforms its pattern to slower and longer multipeak bursts similar to those during early recovery from DAMGO. The results show that electrical coupling of neonatal LC neurons does not synchronize their spiking as previously proposed. They also indicate that both increased excitability (by elevated K) and recovery from inhibition (by opioids) can enhance spike desynchronization to transform the population burst pattern. Both observations show that this gap junction-coupled neural network has a more complex connectivity than currently assumed. These new findings along with the inhibitory drug effects that are in line with previous reports based on single neuron recording point out that field potential analysis is pivotal to further the understanding of this brain circuit.
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http://dx.doi.org/10.1016/j.neulet.2018.02.020DOI Listing
April 2018

A genetically encoded Ca indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

BMC Biol 2018 01 16;16(1). Epub 2018 Jan 16.

Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.

Background: Genetically encoded calcium ion (Ca) indicators (GECIs) are indispensable tools for measuring Ca dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores.

Results: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.

Conclusion: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.
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http://dx.doi.org/10.1186/s12915-018-0480-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771076PMC
January 2018

Genetically Encoded Glutamate Indicators with Altered Color and Topology.

ACS Chem Biol 2018 07 22;13(7):1832-1837. Epub 2018 Jan 22.

Department of Chemistry , University of Alberta , Edmonton , Alberta , Canada T6G 2G2.

Glutamate is one of the 20 common amino acids and of utmost importance for chemically mediated synaptic transmission in nervous systems. To expand the color palette of genetically encoded indicators for glutamate, we used protein engineering to develop a red intensity-based glutamate-sensing fluorescent reporter (R-iGluSnFR1). Manipulating the topology of R-iGluSnFR1, and a previously reported green fluorescent indicator, led to the development of noncircularly permutated (ncp) variants. R- and R-iGluSnFR1 display glutamate affinities of 11 μM and 0.9 μM, respectively. We demonstrate that these glutamate indicators are functional when targeted to the surface of HEK-293 cells. Furthermore, we show that G-iGluSnFR enabled reliable visualization of extrasynaptic glutamate in organotypic hippocampal slice cultures, while R-iGluSnFR can reliably resolve action potential-evoked glutamate transients by electrical field stimuli in cultures of dissociated hippocampal neurons.
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http://dx.doi.org/10.1021/acschembio.7b01085DOI Listing
July 2018

Characterization of Superficial Dorsal Horn Neurons from "Tamamaki" Mice and Stability of their GAD67-EGFP Phenotype in Defined-Medium Organotypic Culture.

Neuroscience 2018 02 30;372:126-140. Epub 2017 Dec 30.

Neuroscience and Mental Health Institute, University of Alberta, Edmonton T6G 2H7, AB, Canada; Department of Pharmacology, University of Alberta, Edmonton T6G 2H7, AB, Canada. Electronic address:

Defined medium organotypic cultures (DMOTC) containing spinal dorsal horn neurons are especially useful in studying the etiology and pharmacology of chronic pain. We made whole-cell recordings from neurons in acutely isolated mouse spinal cord slices or from those maintained in DMOTC for up to 6 weeks. In acute slices, neurons in the substantia gelatinosa exhibited 7 different firing patterns in response to 800-ms depolarizing current commands; delay (irregular), delay (tonic), tonic, regular firing, phasic, initial bursting and single spiking. Initial bursting and regular firing neurons are not found in rat substantia gelatinosa. In acute slices from "Tamamaki" mice that express enhanced green fluorescent protein (EGFP) under the control of the glutamic acid decarboxylase 67 (GAD67) promotor, tonic, phasic and regular firing neurons exhibited the strongest GABAergic (GAD67-EGFP+) phenotype. Delay (tonic) and delay (irregular) neurons almost never expressed GAD67 (GAD67-EGFP-) and are likely glutamatergic. All seven phenotypes were preserved in mouse spinal cord neurons in DMOTC prepared from e12 embryos and the GAD67-EGFP+ phenotype continued to associate with phasic and regular firing neurons. Only 3 out of 51 GAD67-EGFP+ neurons exhibited a delay (tonic) firing pattern. Modifications to the mouse genome thus continue to be expressed when embryonic neurons develop in vitro in DMOTC. However, analysis of the amplitude and interevent interval of spontaneous EPSCs (sEPSCs) indicated substantial re-arrangement of synaptic connections within the cultures. Despite this, the characteristics and age-dependence of asynchronous oscillatory activity, as monitored by multiphoton Ca imaging, were similar in acute slices and in DMOTC.
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http://dx.doi.org/10.1016/j.neuroscience.2017.12.047DOI Listing
February 2018

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca -dependent P2Y receptor mechanism.

J Physiol 2018 08 27;596(15):3245-3269. Epub 2017 Jul 27.

Department of Physiology, Neuroscience and Mental Health Institute (NMHI), Women and Children's Health Research Institute (WCHRI), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.

Key Points: The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y receptor-mediated release of Ca from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure.

Abstract: The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Bötzinger Complex (preBötC) inspiratory rhythm-generating network acts via P2Y receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y receptor-mediated increase in fictive inspiratory frequency involves Ca recruitment from intracellular stores leading to increases in intracellular Ca ([Ca ] ) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y receptors on inspiratory neurons (and/or glia) to evoke Ca release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.
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http://dx.doi.org/10.1113/JP274727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068109PMC
August 2018

Acute anti-allodynic action of gabapentin in dorsal horn and primary somatosensory cortex: Correlation of behavioural and physiological data.

Neuropharmacology 2017 02 14;113(Pt A):576-590. Epub 2016 Nov 14.

Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, T6G 2H7, Canada; Department of Pharmacology, University of Alberta, Edmonton, AB, T6G 2H7, Canada. Electronic address:

Neuropathic pain is a debilitating consequence of neuronal injury or disease. Although first line treatments include the alpha-2-delta (α2δ)-ligands, pregabalin and gabapentin (GBP), the mechanism of their anti-allodynic action is poorly understood. One specific paradox is that GBP relieves signs of neuropathic pain in animal models within 30min of an intraperitoneal (IP) injection yet its actions in vitro on spinal dorsal horn or primary afferent neurons take hours to develop. We found, using confocal Ca imaging, that substantia gelatinosa neurons obtained ex vivo from rats subjected to sciatic chronic constriction injury (CCI) were more excitable than controls. We confirmed that GBP (100 mg/kg) attenuated mechanical allodynia in animals subject to CCI within 30min of IP injection.Substantia gelatinosa neurons obtained ex vivo from these animals no longer displayed CCI-induced increased excitability. Electrophysiological analysis of substantia gelatinosa neurons ex vivo suggest that rapidly developing in vivo anti-allodynic effects of GBP i) are mediated intracellularly, ii) involve actions on the neurotransmitter release machinery and iii) depend on decreased excitatory synaptic drive to excitatory neurons without major actions on inhibitory neurons or on intrinsic neuronal excitability. Experiments using in vivo Ca imaging showed that 100 mg/kg GBP also suppressed the response of the S1 somatosensory cortex of CCI rats, but not that of control rats, to vibrotactile stimulation. Since the level of α2δ1 protein is increased in primary afferent fibres after sciatic CCI, we suggest this dictates the rate of GBP action; rapidly developing actions can only be seen when α2δ1 levels are elevated.
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http://dx.doi.org/10.1016/j.neuropharm.2016.11.011DOI Listing
February 2017

TMX1 determines cancer cell metabolism as a thiol-based modulator of ER-mitochondria Ca2+ flux.

J Cell Biol 2016 08 8;214(4):433-44. Epub 2016 Aug 8.

Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G2H7, Canada

The flux of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria regulates mitochondria metabolism. Within tumor tissue, mitochondria metabolism is frequently repressed, leading to chemotherapy resistance and increased growth of the tumor mass. Therefore, altered ER-mitochondria Ca(2+) flux could be a cancer hallmark, but only a few regulatory proteins of this mechanism are currently known. One candidate is the redox-sensitive oxidoreductase TMX1 that is enriched on the mitochondria-associated membrane (MAM), the site of ER-mitochondria Ca(2+) flux. Our findings demonstrate that cancer cells with low TMX1 exhibit increased ER Ca(2+), accelerated cytosolic Ca(2+) clearance, and reduced Ca(2+) transfer to mitochondria. Thus, low levels of TMX1 reduce ER-mitochondria contacts, shift bioenergetics away from mitochondria, and accelerate tumor growth. For its role in intracellular ER-mitochondria Ca(2+) flux, TMX1 requires its thioredoxin motif and palmitoylation to target to the MAM. As a thiol-based tumor suppressor, TMX1 increases mitochondrial ATP production and apoptosis progression.
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http://dx.doi.org/10.1083/jcb.201512077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987292PMC
August 2016

A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices.

J Neurosci 2016 Feb;36(8):2458-72

Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada,

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation.
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http://dx.doi.org/10.1523/JNEUROSCI.3484-15.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764664PMC
February 2016

Progressive postnatal decline in leptin sensitivity of arcuate hypothalamic neurons in the Magel2-null mouse model of Prader-Willi syndrome.

Hum Mol Genet 2015 Aug 29;24(15):4276-83. Epub 2015 Apr 29.

Department of Medical Genetics, Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada

Prader-Willi syndrome (PWS) is a multigene disorder associated with neonatal failure to thrive, developmental delay and endocrine abnormalities suggestive of hypothalamic dysfunction. Children with PWS typically develop overt hyperphagia and obesity ∼8 years of age, later than children with other genetic forms of obesity. This suggests a postnatal developmental or degenerative component to PWS-associated obesity. De novo inactivating mutations in one PWS candidate gene, MAGEL2, have been identified in children with features of PWS. Adult mice lacking Magel2 are insensitive to the anorexic effect of leptin treatment, and their hypothalamic pro-opiomelanocortin (POMC) neurons fail to depolarize in response to leptin. However, it is unclear whether this leptin insensitivity is congenital, or whether normal leptin sensitivity in neonatal Magel2-null mice is lost postnatally. We used in vitro cytosolic calcium imaging to follow the postnatal development of leptin responses in POMC neurons in these mice. Leptin caused an activation of POMC neurons in wild-type acute hypothalamic slice preparations at all ages, reflecting their normal leptin-invoked depolarization. Normal leptin responses were found in Magel2-null mice up to 4 weeks of age, but the proportion of leptin-responsive POMC neurons was reduced in 6-week-old Magel2-null mice. The number of α-melanocyte-stimulating hormone immunoreactive fibers in the paraventricular hypothalamic nucleus was also reduced in mutant mice at 6 weeks of age. A similar progressive loss of leptin sensitivity caused by loss of MAGEL2 in children with PWS could explain the delayed onset of increased appetite and weight gain in this complex disorder.
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http://dx.doi.org/10.1093/hmg/ddv159DOI Listing
August 2015

A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging.

Nat Commun 2014 Oct 31;5:5262. Epub 2014 Oct 31.

Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.

The introduction of calcium ion (Ca(2+)) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca(2+) dynamics. However, one drawback of these indicators is that they have optimal two-photon excitation outside the near-infrared window (650-1,000 nm) where tissue is most transparent to light. To address this shortcoming, we developed a long Stokes shift RFP-based Ca(2+) indicator, REX-GECO1, with optimal two-photon excitation at <1,000 nm. REX-GECO1 fluoresces at 585 nm when excited at 480 nm or 910 nm by a one- or two-photon process, respectively. We demonstrate that REX-GECO1 can be used as either a ratiometric or intensiometric Ca(2+) indicator in organotypic hippocampal slice cultures (one- and two-photon) and the visual system of albino tadpoles (two-photon). Furthermore, we demonstrate single excitation wavelength two-colour Ca(2+) and glutamate imaging in organotypic cultures.
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http://dx.doi.org/10.1038/ncomms6262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920544PMC
October 2014

Identification of the pre-Bötzinger complex inspiratory center in calibrated "sandwich" slices from newborn mice with fluorescent Dbx1 interneurons.

Physiol Rep 2014 Aug 19;2(8). Epub 2014 Aug 19.

Department of Applied Science, The College of William & Mary, Williamsburg, Virginia.

Inspiratory active pre-Bötzinger complex (preBötC) networks produce the neural rhythm that initiates and controls breathing movements. We previously identified the preBötC in the newborn rat brainstem and established anatomically defined transverse slices in which the preBötC remains active when exposed at one surface. This follow-up study uses a neonatal mouse model in which the preBötC as well as a genetically defined class of respiratory interneurons can be identified and selectively targeted for physiological recordings. The population of glutamatergic interneurons whose precursors express the transcription factor Dbx1 putatively comprises the core respiratory rhythmogenic circuit. Here, we used intersectional mouse genetics to identify the brainstem distribution of Dbx1-derived neurons in the context of observable respiratory marker structures. This reference brainstem atlas enabled online histology for generating calibrated sandwich slices to identify the preBötC location, which was heretofore unspecified for perinatal mice. Sensitivity to opioids ensured that slice rhythms originated from preBötC neurons and not parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) cells because opioids depress preBötC, but not pFRG/RTN rhythms. We found that the preBötC is centered ~0.4 mm caudal to the facial motor nucleus in this Cre/lox reporter mouse during postnatal days 0-4. Our findings provide the essential basis for future optically guided electrophysiological and fluorescence imaging-based studies, as well as the application of other Cre-dependent tools to record or manipulate respiratory rhythmogenic neurons. These resources will ultimately help elucidate the mechanisms that promote respiratory-related oscillations of preBötC Dbx1-derived neurons and thus breathing.
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http://dx.doi.org/10.14814/phy2.12111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246597PMC
August 2014

Analysis of the long-term actions of gabapentin and pregabalin in dorsal root ganglia and substantia gelatinosa.

J Neurophysiol 2014 Nov 13;112(10):2398-412. Epub 2014 Aug 13.

Neuroscience and Mental Health Institute, University of Alberta, Edmonton, Canada; Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada; and

The α2δ-ligands pregabalin (PGB) and gabapentin (GBP) are used to treat neuropathic pain. We used whole cell recording to study their long-term effects on substantia gelatinosa and dorsal root ganglion (DRG) neurons. Spinal cord slices were prepared from embryonic day 13 rat embryos and maintained in organotypic culture for >5 wk (neuronal age equivalent to young adult rats). Exposure of similarly aged DRG neurons (dissociated and cultured from postnatal day 19 rats) to GBP or PGB for 5-6 days attenuated high-voltage-activated calcium channel currents (HVA ICa). Strong effects were seen in medium-sized and in small isolectin B4-negative (IB4-) DRG neurons, whereas large neurons and small neurons that bound isolectin B4 (IB4+) were hardly affected. GBP (100 μM) or PGB (10 μM) were less effective than 20 μM Mn(2+) in suppression of HVA ICa in small DRG neurons. By contrast, 5-6 days of exposure to these α2δ-ligands was more effective than 20 μM Mn(2+) in reducing spontaneous excitatory postsynaptic currents at synapses in substantia gelatinosa. Spinal actions of gabapentinoids cannot therefore be ascribed to decreased expression of HVA Ca(2+) channels in primary afferent nerve terminals. In substantia gelatinosa, 5-6 days of exposure to PGB was more effective in inhibiting excitatory synaptic drive to putative excitatory neurons than to putative inhibitory neurons. Although spontaneous inhibitory postsynaptic currents were also attenuated, the overall long-term effect of α2δ-ligands was to decrease network excitability as monitored by confocal Ca(2+) imaging. We suggest that selective actions of α2δ-ligands on populations of DRG neurons may predict their selective attenuation of excitatory transmission onto excitatory vs. inhibitory neurons in substantia gelatinosa.
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http://dx.doi.org/10.1152/jn.00168.2014DOI Listing
November 2014

Suppression of network activity in dorsal horn by gabapentin permeation of TRPV1 channels: implications for drug access to cytoplasmic targets.

Neurosci Lett 2015 Jan 29;584:397-402. Epub 2014 Jul 29.

Neurosciences and Mental Health Institute, University of Alberta, Edmonton, Alberta, Canada T6G 2H7; Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. Electronic address:

The effectiveness of gabapentin (GBP) in the treatment of neuropathic pain depends on access to the α2δ-1 accessory subunit of voltage-gated Ca(2+) channels. Access may be limited by its rate of entry via the neuronal system L-neutral amino acid transporter. The open pore of capsaicin-activated TRPV1 channel admits organic molecules such as local anesthetics and we calculated that GBP entry via this route would be 500× more rapid than via the transporter. Capsaicin should therefore increase GBP effectiveness. We used a quaternary GBP derivative (Q-GBP) as sole charge carrier in whole-cell recording experiments on rat dorsal root ganglion (DRG) neurons. Under these conditions, capsaicin produced a capsazepine-sensitive inward current thereby confirming Q-GBP permeation of TRPV1 channels. We have previously established that 5-6 days exposure to 100 μM GBP decreases excitability of dorsal horn neurons whereas 10 μM is ineffective. Excitability was monitored using confocal Ca(2+) imaging of rat spinal cord slices in organotypic culture. GBP effectiveness was augmented by transient exposures of cultures to capsaicin and robust suppression of excitability was seen with 10 μM GBP. Experiments with an inhibitor of the neutral amino acid transporter, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH 300 μM), showed the actions of GBP seen in the presence of capsaicin were independent of its entry by this route. Capsaicin potentiation of GBP depression of dorsal horn activity may therefore reflect drug permeation of TRPV1 channels. Agonist activation of TRP channels may provide a means for improving drug access to cytoplasmic targets in selective neuronal populations defined on the basis of type of TRP channel expressed.
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http://dx.doi.org/10.1016/j.neulet.2014.07.033DOI Listing
January 2015

Microfluidic cell sorter-aided directed evolution of a protein-based calcium ion indicator with an inverted fluorescent response.

Integr Biol (Camb) 2014 Jul 19;6(7):714-25. Epub 2014 May 19.

Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, Alberta T6G 2G2, Canada.

We demonstrate a simple, low cost and disposable microfluidic fluorescence activated cell sorting system (μFACS) for directed evolution of fluorescent proteins (FP) and FP-based calcium ion (Ca(2+)) indicators. The system was employed to pre-screen libraries of up to 10(6) variants of a yellow FP-based Ca(2+) indicator (Y-GECO) with throughput up to 300 cells per s. Compared to traditional manual screening of FP libraries, this system accelerated the discovery of improved variants and saved considerable time and effort during the directed evolution of Y-GECO. Y-GECO1, the final product of the μFACS-aided directed evolution, has a unique fluorescence hue that places it in the middle of the spectral gap that separates the currently available green and orange FP-based Ca(2+) indicators, exhibits bright fluorescence in the resting (Ca(2+) free) state, and gives a large response to intracellular Ca(2+) fluctuations in live cells.
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http://dx.doi.org/10.1039/c4ib00039kDOI Listing
July 2014

Anoxia response in physiological potassium of the isolated inspiratory center in calibrated newborn rat brainstem slices.

Adv Exp Med Biol 2012 ;758:91-8

Department of Physiology, University of Alberta, Edmonton, Canada.

Using newborn rat brainstem-spinal cords, we were the first to show that medullary inspiratory networks can generate the neonatal biphasic (initial acceleration-secondary slowing) respiratory response to severe hypoxia causing tissue anoxia. Our findings also indicated that medullary inspiratory interneurons remain functional during sustained anoxia due to effective utilization of anaerobic metabolism. In that previous work by us and related studies by others on respiratory anoxia responses in the above en bloc model or brainstem slices, presumptive recording sites within the pre-Bötzinger complex (preBötC) inspiratory center were not histologically verified. Moreover, preBötC slices were studied in 7-9 mM K(+) to stabilize rhythm which can, however, affect respiratory neuromodulation. Here, we summarize our previous findings on respiratory anoxia responses in the en bloc model in physiological (3 mM) K(+). Using our recently developed 'calibrated' slices, we also exemplify anoxia effects in anatomically identified preBötC cells in physiological K(+) based on recording electrophysiological population activity in conjunction with either membrane potential or cytosolic Ca(2+).
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http://dx.doi.org/10.1007/978-94-007-4584-1_12DOI Listing
April 2013

Amyloid β (Aβ) peptide directly activates amylin-3 receptor subtype by triggering multiple intracellular signaling pathways.

J Biol Chem 2012 May 12;287(22):18820-30. Epub 2012 Apr 12.

Division of Neurology, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.

The two age-prevalent diseases Alzheimer disease and type 2 diabetes mellitus share many common features including the deposition of amyloidogenic proteins, amyloid β protein (Aβ) and amylin (islet amyloid polypeptide), respectively. Recent evidence suggests that both Aβ and amylin may express their effects through the amylin receptor, although the precise mechanisms for this interaction at a cellular level are unknown. Here, we studied this by generating HEK293 cells with stable expression of an isoform of the amylin receptor family, amylin receptor-3 (AMY3). Aβ1-42 and human amylin (hAmylin) increase cytosolic cAMP and Ca(2+), trigger multiple pathways involving the signal transduction mediators protein kinase A, MAPK, Akt, and cFos. Aβ1-42 and hAmylin also induce cell death during exposure for 24-48 h at low micromolar concentrations. In the presence of hAmylin, Aβ1-42 effects on HEK293-AMY3-expressing cells are occluded, suggesting a shared mechanism of action between the two peptides. Amylin receptor antagonist AC253 blocks increases in intracellular Ca(2+), activation of protein kinase A, MAPK, Akt, cFos, and cell death, which occur upon AMY3 activation with hAmylin, Aβ1-42, or their co-application. Our data suggest that AMY3 plays an important role by serving as a receptor target for actions Aβ and thus may represent a novel therapeutic target for development of compounds to treat neurodegenerative conditions such as Alzheimer disease.
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http://dx.doi.org/10.1074/jbc.M111.331181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365692PMC
May 2012

Proteinase-activated receptor-1 mediates dorsal root ganglion neuronal degeneration in HIV/AIDS.

Brain 2011 Nov 21;134(Pt 11):3209-21. Epub 2011 Oct 21.

Department of Medicine, University of Alberta, Edmonton, AB T6G 2S2, Canada.

Distal sensory polyneuropathy is a frequent complication of lentivirus infections of the peripheral nervous system including both human immunodeficiency virus and feline immunodeficiency virus. Proteinase-activated receptors are G protein-coupled receptors implicated in the pathogenesis of neuroinflammation and neurodegeneration. Proteinase-activated receptor-1 is expressed on different cell types within the nervous system including neurons and glia, but little is known about its role in the pathogenesis of inflammatory peripheral nerve diseases, particularly lentivirus-related distal sensory polyneuropathy. Herein, the expression and functions of proteinase-activated receptor-1 in the peripheral nervous system during human immunodeficiency virus and feline immunodeficiency virus infections were investigated. Proteinase-activated receptor-1 expression was most evident in autopsied dorsal root ganglion neurons from subjects infected with human immunodeficiency virus, compared with the dorsal root ganglia of uninfected subjects. Human immunodeficiency virus or feline immunodeficiency virus infection of cultured human or feline dorsal root ganglia caused upregulation of interleukin-1β and proteinase-activated receptor-1 expression. In the human immunodeficiency virus- or feline immunodeficiency virus-infected dorsal root ganglia, interleukin-1β activation was principally detected in macrophages, while neurons showed induction of proteinase-activated receptor-1. Binding of proteinase-activated receptor-1 by the selective proteinase-activated receptor-1-activating peptide resulted in neurite retraction and soma atrophy in conjunction with cytosolic calcium activation in human dorsal root ganglion neurons. Interleukin-1β exposure to feline or human dorsal root ganglia caused upregulation of proteinase-activated receptor-1 in neurons. Exposure of feline immunodeficiency virus-infected dorsal root ganglia to the interleukin-1 receptor antagonist prevented proteinase-activated receptor-1 induction and neurite retraction. In vivo feline immunodeficiency virus infection was associated with increased proteinase-activated receptor-1 expression on neurons and interleukin-1β induction in macrophages. Moreover, feline immunodeficiency virus infection caused hyposensitivity to mechanical stimulation. These data indicated that activation and upregulation of proteinase-activated receptor-1 by interleukin-1β contributed to dorsal root ganglion neuronal damage during lentivirus infections leading to the development of distal sensory polyneuropathy and might also provide new targets for future therapeutic interventions.
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http://dx.doi.org/10.1093/brain/awr242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3212716PMC
November 2011
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