Andrew Moorhouse

Techniques: Applications of the nerve–bouton preparation in neuropharmacology

Publisher: Elsevier BV

Date: 2003

DOI: 10.1016/S0165-6147(02)00010-X

Abstract: Single mammalian neurons can be isolated with adherent functional synaptic terminals using an enzyme-free, mechanical dissociation procedure. This allows investigations of the effects of presynaptic modulators of synaptic transmission with unprecedented ease and accuracy. Furthermore, single presynaptic terminals and boutons can be visualized using fluorescent markers and can also be focally stimulated with electrical pulses. In this article, the isolated-nerve-adherent-synaptic-bouton preparation and some ex les of its general properties and uses are discussed.

Analyzing Ion Permeation in Channels and Pumps Using Patch‐Clamp Recording

Publisher: Wiley

Date: 24-09-2015

DOI: 10.1002/9781119085126.CH3

Overexpression of neuronal K+–Cl− co-transporter enhances dendritic spine plasticity and motor learning

Publisher: Springer Science and Business Media LLC

Date: 13-02-2019

DOI: 10.1007/S12576-018-00654-5

Abstract: The neuronal K

Differential modulation of evoked and spontaneous glycine release from rat spinal cord glycinergic terminals by the cyclic AMP/protein kinase A transduction cascade

Publisher: Wiley

Date: 17-09-2004

DOI: 10.1111/J.1471-4159.2004.02741.X

Abstract: The mechanisms underlying cyclic AMP modulation of action potential-dependent and -independent (spontaneous) release of glycine from terminals synapsing onto sacral dorsal commissural nucleus neurons of lamina X were studied in spinal cord slices using conventional patch-cl recordings. 3-Isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, and forskolin increased the litude of evoked inhibitory postsynaptic currents (eIPSCs) in a sensitive manner to protein kinase A (PKA) inhibition (with KT-5720). Direct activation (with adenosine 3',5'-cyclic-monophosphothioate, Sp-isomer) and inhibition (with adenosine 3',5'-cyclic-monophosphothioate, Rp-isomer) of PKA increased and decreased the eIPSC litude, respectively. Paired pulse experiments and direct injection of PKA inhibitor fragment 6-22 amide (PKI(6-22)) into the recording neuron revealed that these effects on eIPSC litude occurred presynaptically, indicating that evoked glycine release is regulated by presynaptic cAMP via changes in PKA activity. Increasing cAMP also increased spontaneous release of glycine, causing an increased frequency of miniature IPSCs (mIPSCs). In contrast to the effects on evoked release, this response was not solely mediated via PKA, as it was not occluded by PKA inhibition, and both direct inhibition and direct activation of PKA actually enhanced mIPSC frequency. Direct inhibition of cAMP (with SQ 22536) did, however, reduce mIPSC frequency. These results suggest cAMP modulation of evoked and spontaneous release involves different presynaptic mechanisms and proteins.

Early Changes in KCC2 Phosphorylation in Response to Neuronal Stress Result in Functional Downregulation

Publisher: Society for Neuroscience

Date: 14-02-2007

DOI: 10.1523/JNEUROSCI.3104-06.2007

Abstract: The K + Cl − cotransporter KCC2 plays an important role in chloride homeostasis and in neuronal responses mediated by ionotropic GABA and glycine receptors. The expression levels of KCC2 in neurons determine whether neurotransmitter responses are inhibitory or excitatory. KCC2 expression is decreased in developing neurons, as well as in response to various models of neuronal injury and epilepsy. We investigated whether there is also direct modulation of KCC2 activity by changes in phosphorylation during such neuronal stressors. We examined tyrosine phosphorylation of KCC2 in rat hippoc al neurons under different conditions of in vitro neuronal stress and the functional consequences of changes in tyrosine phosphorylation. Oxidative stress (H 2 O 2 ) and the induction of seizure activity (BDNF) and hyperexcitability (0 Mg 2+ ) resulted in a rapid dephosphorylation of KCC2 that preceded the decreases in KCC2 protein or mRNA expression. Dephosphorylation of KCC2 is correlated with a reduction of transport activity and a decrease in [Cl − ] i , as well as a reduction in KCC2 surface expression. Manipulation of KCC2 tyrosine phosphorylation resulted in altered neuronal viability in response to in vitro oxidative stress. During continued neuronal stress, a second phase of functional KCC2 downregulation occurs that corresponds to decreases in KCC2 protein expression levels. We propose that neuronal stress induces a rapid loss of tyrosine phosphorylation of KCC2 that results in translocation of the protein and functional loss of transport activity. Additional understanding of the mechanisms involved may provide means for manipulating the extent of irreversible injury resulting from different neuronal stressors.

Characterization of the Effects of Charged Residues in the Intracellular Loop on Ion Permeation in  1 Glycine Receptor Channels

Publisher: Elsevier BV

Date: 2008

DOI: 10.1074/JBC.M806618200

An optimised 3 M KCl salt-bridge technique used to measure and validate theoretical liquid junction potential values in patch-clamping and electrophysiology

Publisher: Springer Science and Business Media LLC

Date: 21-06-2013

DOI: 10.1007/S00249-013-0911-3

Abstract: Accurate potential measurements in electrophysiological experiments require correction for liquid junction potentials (LJPs), and, in patch-cl ing especially, these can often be ~5-10 mV or more. They can be either calculated, if ion mobilities are known, or measured directly. We describe an optimised system to directly measure LJPs with a patch-cl lifier, using as a reference electrode, a freshly-cut 3 M KCl-agar salt-bridge (in polyethylene tubing) with its tip cut off by at least 5 mm during solution changes to eliminate its solution-history-dependent effects. We quantify such history-dependent effects and complement this with a de-novo theoretical analysis of salt diffusion to and from the salt-bridge. Our analysis and experimental results validate the optimised methodology for measuring LJPs, and the use of the Henderson equation for accurately calculating them. The use of this equation is also assessed and generally validated in the light of rigorous Nernst-Planck-Poisson and other numerical simulations and analytical studies of LJPs over recent decades. Digitizing, recording and lifying the measured potentials increases their accuracy. The measured potentials still need correction for small, well-defined calculable, shifts in LJPs at the 3 M KCl-agar reference. Using this technique, we have measured changes in LJPs for diluted solutions of NaCl, LiCl, KCl, CsCl and NaF, obtaining excellent agreement within ±0.1 mV of predicted values, calculated using ion activities. Our de novo LJP measurements of biionic combinations of the above undiluted salts, and NaI and NaF (with halide anions I⁻ and F⁻), generally also gave excellent agreement with predicted values.

Microglial Contact Prevents Excess Depolarization and Rescues Neurons from Excitotoxicity

Publisher: Society for Neuroscience

Date: 05-2016

DOI: 10.1523/ENEURO.0004-16.2016

Abstract: Microglia survey and directly contact neurons in both healthy and damaged brain, but the mechanisms and functional consequences of these contacts are not yet fully elucidated. Combining two-photon imaging and patch cl ing, we have developed an acute experimental model for studying the role of microglia in CNS excitotoxicity induced by neuronal hyperactivity. Our model allows us to simultaneously examine the effects of repetitive supramaximal stimulation on axonal morphology, neuronal membrane potential, and microglial migration, using cortical brain slices from Iba-1 eGFP mice. We demonstrate that microglia exert an acute and highly localized neuroprotective action under conditions of neuronal hyperactivity. Evoking repetitive action potentials in in idual layer 2/3 pyramidal neurons elicited swelling of axons, but not dendrites, which was accompanied by a large, sustained depolarization of soma membrane potential. Microglial processes migrated to these swollen axons in a mechanism involving both ATP and glutamate release via volume-activated anion channels. This migration was followed by intensive microglial wrapping of affected axons and, in some cases, the removal of axonal debris that induced a rapid soma membrane repolarization back to resting potentials. When the microglial migration was pharmacologically blocked, the activity-induced depolarization continued until cell death ensued, demonstrating that the microglia–axon contact served to prevent pathological depolarization of the soma and maintain neuronal viability. This is a novel aspect of microglia surveillance: detecting, wrapping, and rescuing neuronal soma from damage due to excessive activity.

Activation of presynaptic glycine receptors facilitates glycine release from presynaptic terminals synapsing onto rat spinal sacral dorsal commissural nucleus neurons

Publisher: Wiley

Date: 07-2003

DOI: 10.1113/JPHYSIOL.2003.041053

Very Negative Potential for Half-inactivation of, and Effects of Anions on, Voltage-dependent Sodium Currents in Acutely Isolated Rat Olfactory Receptor Neurons

Publisher: Springer Science and Business Media LLC

Date: 05-2000

DOI: 10.1007/S002320001061

Abstract: Previous measurements with CsF pipette solutions using whole-cell patch-cl techniques in dissociated rat olfactory receptor neurons (ORNs) indicated that the sodium currents had very negative inactivation characteristics with the implication that the cell resting potential must also normally have a very negative value. This study supports the conclusions that such an effect was real and not dependent on either the nature of the pipette anions or the recording situation previously used. For all pipette solutions, sodium currents showed a threshold activation approximately -80 mV and half-maximal activation voltages approximately -55 with half-inactivation potential < or =-100 mV, without being significantly affected by the replacement of F(-) by other pipette anions (H(2)PO(-)(4) and acetate(-)) or the addition of nucleotides and glutathione (which did cause a very slight positive shift). F(-), followed by H(2)PO(-)(4) and to a much lesser extent by acetate(-), was the most favorable pipette anion for obtaining good seals and whole-cell sodium currents in these extremely small ORNs. These results implied that resting potentials, for viable responsive cells, should be more negative than about -90 mV, as supported by the observation that action potentials could only be evoked from holding potentials more negative than -90 mV.

The sulphonylurea receptor confers diazoxide sensitivity on the inwardly rectifying K+ channel Kir6.1 expressed in human embryonic kidney cells.

Publisher: Wiley

Date: 08-1996

DOI: 10.1113/JPHYSIOL.1996.SP021526

Abstract: 1. We have examined the effects of diazoxide and intracellular ATP (ATPi) on whole-cell currents in HEK293 cells transfected transiently with the inwardly rectifying K+ channel Kir6.1 (uKATP1) or cotransfected with Kir6.1 and the sulphonylurea receptor (SUR1). 2. Kir6.1 currents were unaffected by the K+ channel opener diazoxide or by dialysis with 0.3 mM ATPi. 3. Kir6.1-SUR1 currents increased in litude when cells were dialysed with 0.3 mM ATP, but not with 5 mM ATP. This activation may be explained by the loss of endogenous ATP from the cell when the intracellular solution contains 0.3 mM ATP. Kir6.1-SUR1 currents were also activated by diazoxide this activation was greater with 0.3 mM ATP1 than with 5 mM ATP1. 4. We conclude that SUR1 is required to confer both diazoxide and ATP sensitivity on Kir6.1.

Ligand-gated ion channels: mechanisms underlying ion selectivity

Publisher: Elsevier BV

Date: 10-2004

DOI: 10.1016/J.PBIOMOLBIO.2003.09.002

External divalent cations increase anion–cation permeability ratio in glycine receptor channels

Publisher: Springer Science and Business Media LLC

Date: 03-03-2010

DOI: 10.1007/S00424-010-0792-6

Abstract: The functional role of ligand-gated ion channels in the central nervous system depends on their relative anion-cation permeability. Using standard whole-cell patch cl measurements and NaCl dilution potential measurements, we explored the effect of external alent ions on anion-cation selectivity in alpha1-homomeric wild-type glycine receptor channels. We show that increasing external Ca(2+) from 0 to 4 mM resulted in a sigmoidal increase in anion-cation permeability by 37%, reaching a maximum above about 2 mM. Our accurate quantification of this effect required rigorous correction for liquid junction potentials (LJPs) using ion activities, and allowing for an initial offset potential. Failure to do this results in a considerable overestimation of the Ca(2+)-induced increase in anion-cation permeability by almost three-fold at 4 mM external Ca(2+). Calculations of LJPs (using activities)_ were validated by precise agreement with direct experimental measurements. External SO (4) (2-) was found to decrease anion-cation permeability. Single-channel conductance measurements indicated that external Ca(2+) both decreased Na(+) permeability and increased Cl(-) permeability. There was no evidence of Ca(2+) changing channel pore diameter. Theoretical modeling indicates that the effect is not surface charge related. Rather, we propose that, under dilution conditions, the presence of an impermeant Ca(2+) ion in the channel pore region just external to the selectivity filter tends to electrostatically retard outward movement of Na(+) ions and to enhance movement of Cl(-) ions down their energy gradients.

IP3-Gated Channels and their Occurrence Relative to CNG Channels in the Soma and Dendritic Knob of Rat Olfactory Receptor Neurons

Publisher: Springer Science and Business Media LLC

Date: 05-2001

DOI: 10.1007/S00232-001-0013-5

Cellular Mechanisms of Neuronal Cl− Homeostasis and its Modulation by Neuronal Injury

Publisher: Springer New York

Date: 05-10-2010

DOI: 10.1007/978-1-4419-6978-1_9

A patch-clamp investigation of membrane currents in a novel mammalian retinal ganglion cell line

Publisher: Elsevier BV

Date: 04-2004

DOI: 10.1016/J.BRAINRES.2004.01.027

Anomalous mole-fraction effects in recombinant and native cyclic nucleotide-gated channels in rat olfactory receptor neurons

Publisher: The Royal Society

Date: 07-07-2001

DOI: 10.1098/RSPB.2001.1663

Physiological Implications of Microglia–Synapse Interactions

Publisher: Springer New York

Date: 2019

DOI: 10.1007/978-1-4939-9658-2_6

Abstract: Microglia are the sole immune responding cells in the central nervous system. Their role as neuroimmune cells in the pathogenesis of various neurodegenerative and infectious diseases of the brain have been extensively studied. Upon brain disease and infection, they adopt an activated phenotype associated with the release of cytokines and neurotrophic factors and resulting in neuroprotective or neurotoxic outcomes. However, microglia are resident also in the healthy or physiological brain, but much less is known about their role(s) in the healthy brain, partly due to technical limitations regarding investigation of these highly reactive cells in the intact brain. Recent developments in molecular probes and in vivo optical imaging techniques has now helped to characterize microglia in the physiological or healthy brain. In vivo two-photon imaging of fluorescently labeled microglia have revealed that they are highly motile cells in the healthy brain, extending and retracting their processes that extend from a largely stationary cell soma. In this chapter, we briefly summarize some of the physiological functions of microglia in the uninjured brain, with a focus on interactions they have with synapses.

Direct effects of tolbutamide on mitochondrial function, intracellular Ca 2+ and exocytosis in pancreatic β-cells

Publisher: Springer Science and Business Media LLC

Date: 23-02-1999

DOI: 10.1007/S004240050820

Abstract: Using the whole-cell voltage-cl method to measure ATP-sensitive K+(KATP) currents, changes in cell capacitance to measure secretion and microfluorimetry to monitor intracellular Ca2+ and mitochondrial function, we have investigated the direct effect of sulphonylureas on exocytosis in pancreatic beta-cells. Tolbutamide (100 microM) and 100 nM 4-beta-12-phorbolmyristate-13-acetate (PMA), which activates the protein kinase C (PKC) isoforms found in beta-cells, potentiated exocytosis in a non-additive manner. These effects were blocked by down-regulation of PKC. Our data support the idea that tolbutamide can potentiate secretion from beta-cells via a PKC-dependent pathway. Because PKC and sulphonylureas can modulate the activity of KATP channels, we explored whether the above effects are caused by inhibition of this channel. PMA increased whole-cell KATP currents but did not affect their sensitivity to tolbutamide. Down-regulation of PKC affected neither the magnitude nor the tolbutamide sensitivity of the KATP current. Both tolbutamide and the mitochondrial uncoupler FCCP (1 microM) mobilized intracellular Ca2+ and prolonged Ca2+ transients elicited by cholinergic mobilization of intracellular Ca2+ stores. Tolbutamide (0.1-0.5 mM), like FCCP, depolarized the mitochondrial membrane potential and activated KATP currents. We suggest that sulphonylureas can directly potentiate exocytosis by impairing mitochondrial function and Ca2+ handling, which ultimately leads to activation of Ca2+-dependent enzymes such as PKC.

Dual microglia effects on blood brain barrier permeability induced by systemic inflammation

Publisher: Springer Science and Business Media LLC

Date: 20-12-2019

DOI: 10.1038/S41467-019-13812-Z

Abstract: Microglia survey brain parenchyma, responding to injury and infections. Microglia also respond to systemic disease, but the role of blood–brain barrier (BBB) integrity in this process remains unclear. Using simultaneous in vivo imaging, we demonstrated that systemic inflammation induces CCR5-dependent migration of brain resident microglia to the cerebral vasculature. Vessel-associated microglia initially maintain BBB integrity via expression of the tight-junction protein Claudin-5 and make physical contact with endothelial cells. During sustained inflammation, microglia phagocytose astrocytic end-feet and impair BBB function. Our results show microglia play a dual role in maintaining BBB integrity with implications for elucidating how systemic immune-activation impacts neural functions.

IP3-Gated Channels and their Occurrence Relative to CNG Channels in the Soma and Dendritic Knob of Rat Olfactory Receptor Neurons

Publisher: Springer Science and Business Media LLC

Date: 02-2001

DOI: 10.1007/S0023200100135

Microglia Enhance Synapse Activity to Promote Local Network Synchronization

Publisher: Society for Neuroscience

Date: 09-2018

DOI: 10.1523/ENEURO.0088-18.2018

The contribution of proline 250 (P-2′) to pore diameter and ion selectivity in the human glycine receptor channel

Publisher: Elsevier BV

Date: 11-2003

DOI: 10.1016/J.NEULET.2003.08.005

Abstract: The glycine receptor-channel (GlyR) mediates neuronal inhibition by selectively allowing the passage of Cl(-) ions through its channel. The pore region for ion selectivity is localised to the constricted internal end of the M2 transmembrane domain. This paper investigates the contribution of the P-2' residue in determining pore diameter and ion charge selectivity of the GlyR. The deletion of this proline has been shown to decrease the anion/cation permeability ratio, with P(Cl)/P(Na) decreasing from approximately 27 to approximately 4. We show that the P-2' deletion by itself produces a GlyR with a larger pore diameter ( approximately 0.69 nm) than the wild type value ( approximately 0.54 nm). This confirms that the P-2' residue reduces pore size, which suggests that, in addition to electrostatic effects, pore size also contributes to ion-charge selectivity.

The startle disease mutation Q266H, in the second transmembrane domain of the human glycine receptor, impairs channel gating

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 02-1999

DOI: 10.1124/MOL.55.2.386

Abstract: Hyperekplexia (startle disease) results from mutations in the glycine receptor chloride channel that disrupt inhibitory synaptic transmission. The Q266H missense mutation is the only hyperekplexia mutation located in the transmembrane domains of the receptor. Using recombinant expression and patch-cl ing techniques, we have investigated the functional properties of this mutation. The ability of glycine and taurine to open the channel was reduced in the mutated channel, as shown by a 6-fold shift in the concentration-response curve for both agonists. This was not accompanied by similar changes in agonist displacement of strychnine binding, suggesting that the mutation affects functions subsequent to ligand binding. Taurine was also converted to a weak partial agonist and antagonized the actions of glycine, consistent with changes in its channel gating efficacy. Because the Q266H mutation is within the pore-forming second transmembrane domain, we tested for a direct interaction with permeating ions. No change in either the cation/anion selectivity ratio or in single channel conductance levels was observed. No differential effects of Zn++, pH, and diethylpyrocarbonate were observed, implying that the histidine side chain is not exposed to the channel lumen. Single-channel recordings revealed a significant reduction in open times in the mutant receptors, at both high and low agonist concentrations, consistent with the open state of the channel being less stable. This study demonstrates that residues within the second transmembrane domain of ligand-gated ion channel receptors, even those whose side chains do not directly interact with permeating ions, can affect the kinetics of channel gating.

Enhanced GABAergic Activity in the Mouse Primary Somatosensory Cortex Is Insufficient to Alleviate Chronic Pain Behavior with Reduced Expression of Neuronal Potassium–Chloride Cotransporter

Publisher: Society for Neuroscience

Date: 21-11-2012

DOI: 10.1523/JNEUROSCI.2104-12.2012

Abstract: The correct balance between excitation and inhibition is crucial for brain function and disrupted in several pathological conditions. Excitatory neuronal circuits in the primary somatosensory cortex (S1) are modulated by local inhibitory neurons with the balance of this excitatory and inhibitory activity important for function. The activity of excitatory layer 2/3 neurons (L2/3) in the S1 cortex is increased in chronic pain, but it is not known how the local interneurons, nor the balance between excitation and inhibition, may change in chronic pain. Using in vivo two-photon calcium imaging and electrophysiology, we report here that the response of L2/3 local inhibitory neurons to both sensory stimulation and to layer 4 electrical stimulation increases in inflammatory chronic pain. Local application into L2/3 of a GABA A receptor blocker further enhanced the activity of S1 excitatory neurons and reduced pain thresholds, whereas local application of the GABA A receptor modulators (muscimol and diazepam) transiently alleviated the allodynia. This illustrates the importance of the local inhibitory pathways in chronic pain sensation. A reduction in the expression and function of the potassium–chloride cotransporter 2 occurred during chronic pain, which reduces the efficacy of the inhibitory inputs to L2/3 excitatory neurons. In summary, both excitatory and inhibitory neuronal activities in the S1 are enhanced in the chronic pain model, but the increased inhibition is insufficient to completely counterbalance the increased excitation and alleviate the symptoms of chronic pain.

Unpacking and validating the “cell membrane” core concept of physiology by an Australian team

Publisher: American Physiological Society

Date: 09-2023

DOI: 10.1152/ADVAN.00143.2022

Abstract: The “cell membrane” core concept was unpacked by a team of Australian physiology educators into a conceptual framework to provide guidance for students and educators. Key themes in the cell membrane core concept were cell membrane definition and structure, transport across cell membranes, and membrane potentials. Australian educators reviewing the framework identified cell membrane as an essential yet relatively simple core concept, suggesting that this is well-placed in foundational physiology courses across a erse range of degrees.

Functions of microglia in the central nervous system – beyond the immune response

Publisher: Cambridge University Press (CUP)

Date: 02-2011

DOI: 10.1017/S1740925X12000063

Abstract: Microglia cells are the immune cells of the central nervous system and consequently play important roles in brain infections and inflammation. Recent in vivo imaging studies have revealed that in the resting healthy brain, microglia are highly dynamic, moving constantly to actively survey the brain parenchyma. These active microglia can rapidly respond to pathological insults, becoming activated to induce a range of effects that may contribute to both pathogenesis, or to confer neuronal protection. However, interactions between microglia and neurons are being recognized as important in shaping neural circuit activity under more normal, physiological conditions. During development and neurogenesis, microglia interactions with neurons help to shape the final patterns of neural circuits important for behavior and with implications for diseases. In the mature brain, microglia can respond to changes in sensory activity and can influence neuronal activity acutely and over the long term. Microglia seem to be particularly involved in monitoring the integrity of synaptic function. In this review, we discuss some of these new insights into the involvement of microglia in neural circuits.

TRPC Channels Activated by G Protein-Coupled Receptors Drive Ca2+ Dysregulation Leading to Secondary Brain Injury in the Mouse Model

Publisher: Springer Science and Business Media LLC

Date: 18-07-2023

DOI: 10.1007/S12975-023-01173-1

Abstract: Canonical transient receptor potential (TRPC) non-selective cation channels, particularly those assembled with TRPC3, TRPC6, and TRPC7 subunits, are coupled to G αq -type G protein-coupled receptors for the major classes of excitatory neurotransmitters. Sustained activation of this TRPC channel-based pathophysiological signaling hub in neurons and glia likely contributes to prodigious excitotoxicity-driven secondary brain injury expansion. This was investigated in mouse models with selective Trpc gene knockout (KO). In adult cerebellar brain slices, application of glutamate and the class I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine to Purkinje neurons expressing the GCaMP5g Ca 2+ reporter demonstrated that the majority of the Ca 2+ loading in the molecular layer dendritic arbors was attributable to the TRPC3 effector channels ( Trpc3 KO compared with wildtype (WT)). This Ca 2+ dysregulation was associated with glutamate excitotoxicity causing progressive disruption of the Purkinje cell dendrites (significantly abated in a GAD67-GFP - Trpc3 KO reporter brain slice model). Contribution of the G αq -coupled TRPC channels to secondary brain injury was evaluated in a dual photothrombotic focal ischemic injury model targeting cerebellar and cerebral cortex regions, comparing day 4 post-injury in WT mice, Trpc3 KO , and Trpc1/3/6/7 quadruple knockout ( Trpc QKO ), with immediate 2-h (primary) brain injury. Neuroprotection to secondary brain injury was afforded in both brain regions by Trpc3 KO and Trpc QKO models, with the Trpc QKO showing greatest neuroprotection. These findings demonstrate the contribution of the G αq -coupled TRPC effector mechanism to excitotoxicity-based secondary brain injury expansion, which is a primary driver for mortality and morbidity in stroke, traumatic brain injury, and epilepsy.

Pain induces stable, active microcircuits in the somatosensory cortex that provide a therapeutic target

Publisher: American Association for the Advancement of Science (AAAS)

Date: 25-09-2020

DOI: 10.1126/SCIADV.ABD8261

Abstract: Upregulation of N-type Ca 2+ channel dependent subunits increases functional connections and synchronization for pain formation.

Microglia: actively surveying and shaping neuronal circuit structure and function

Publisher: Elsevier BV

Date: 04-2013

DOI: 10.1016/J.TINS.2012.11.007

Abstract: The traditional role of microglia has been in brain infection and disease, phagocytosing debris and secreting factors to modify disease progression. Recent evidence extends their role to healthy brain homeostasis, including the regulation of cell death, synapse elimination, neurogenesis, and neuronal surveillance. These actions contribute to the maturation and plasticity of neural circuits that ultimately shape behavior. Here we review microglial contributions to the development, plasticity, and maintenance of neural circuits with a focus on interactions with synapses. We introduce this topic by reviewing recent studies on the migration and proliferation of microglia within the brain, and conclude with the proposal that microglia dysfunction may adversely affect brain function, and thereby contribute to the development of psychiatric and neurological disorders.

Depolarizing shift in the GABA-induced current reversal potential by lidocaine hydrochloride

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.BRAINRES.2010.05.052

Abstract: Lidocaine hydrochloride (LC-HCl) is widely used as a local anesthetic, while various adverse effects of LC-HCl, such as seizures have also been reported. Lidocaine is reported to inhibit various channels and receptors including GABA(A) receptors. Although the GABA(A) receptor-mediated response depends on Cl(-) equilibrium potential (E(Cl)), little is known about the effect of LC-HCl on E(Cl). In the present study, we investigated the effect of LC-HCl on GABA-induced currents in cultured rat hippoc al neurons with gramicidin-perforated patch-cl recording which is known to keep the intracellular Cl(-) concentration intact. LC-HCl inhibited outward GABA-induced currents with depolarizing shift of the GABA reversal potential (E(GABA)). The LC-HCl-induced positive E(GABA) shift was not observed with conventional whole-cell patch-cl method which cannot retain intact intracellular Cl(-) concentration. The LC-HCl action on E(GABA) was inhibited by either furosemide, a blocker of both Na(+)-K(+)-Cl(-) cotransporter (NKCC) and K(+)-Cl(-) cotransporter (KCC), or an increase in extracellular K(+) concentrations. Neither bumetanide, a specific inhibitor of NKCC, nor Na(+)-free external solution had any effect on the LC-HCl-induced E(GABA) shift. QX-314, a membrane impermeable lidocaine derivative, failed to shift E(GABA) to positive potential. Furthermore, LC-HCl caused a depolarizing shift of E(GABA) in cultured GT1-7 cells expressing KCC2 but failed to change E(GABA) in GT1-7 cells without expression of KCC2. These results suggest that the LC-HCl-induced positive E(GABA) shift is due to a blockade of KCC2. Together with the direct LC-HCl action to GABA(A) receptors, the positive E(GABA) shift induced by LC-HCl reduces the GABAergic inhibition in the central nervous system.

Establishing consensus for the core concepts of physiology in the Australian higher education context using the Delphi method

Publisher: American Physiological Society

Date: 09-2023

DOI: 10.1152/ADVAN.00140.2022

Abstract: This is the first time Australia-wide agreement has been reached on the core concepts of physiology with the Delphi method. Embedding of the core concepts will result in consistency in physiology curricula, improvements to teaching and learning, and benchmarking across Australian universities.

Zinc modulation of glycine receptors in acutely isolated rat CA3 neurons

Publisher: Elsevier BV

Date: 08-2008

DOI: 10.1016/J.LFS.2008.04.024

Abstract: Glycine and GABA are the primary inhibitory neurotransmitters in the spinal cord and brain stem, with glycine exerting its physiological roles by activating strychnine-sensitive ionotropic receptors. Glycine receptors are also expressed in the brain, including the cortex and hippoc us, but their physiological roles and pharmacological properties are largely unknown. Here, we report the pharmacological properties of functional glycine receptors in acutely isolated rat CA3 neurons using conventional whole-cell patch cl techniques. Both glycine and taurine, which are endogenous agonists of glycine receptors, elicited Cl(-) currents in a concentration-dependent manner. The glycine-induced current (I(Gly)) was inhibited by strychnine, picrotoxin or cyclothiazide in a concentration-dependent manner. At lower concentrations (0.01-1 microM), ICS-205,930 potentiated I(Gly), but at higher concentrations (>10 microM) it inhibited I(Gly). These pharmacological properties strongly suggest that CA3 neurons express functional strychnine-sensitive glycine receptors containing alpha2 subunits. Furthermore, at lower concentrations (1-30 microM), Zn(2+) potentiated I(Gly), but at higher concentrations (>100 microM) it inhibited I(Gly). Considering that Zn(2+) is synaptically co-released with glutamate from mossy fiber terminals that make excitatory synapses onto CA3 neurons, these results suggest that endogenous Zn(2+) modulation of these glycine receptors may have an important role in the excitability of CA3 neurons.

Alternative splicing of the TRPC3 ion channel calmodulin/ IP 3 receptor-binding domain in the hindbrain Enhances cation flux

Publisher: Society for Neuroscience

Date: 15-08-2012

DOI: 10.1523/JNEUROSCI.6446-11.2012

Abstract: Canonical transient receptor potential (TRPC3) nonselective cation channels are effectors of G-protein-coupled receptors (GPCRs), activated via phospholipase C–diacylglycerol signaling. In cerebellar Purkinje cells, TRPC3 channels cause the metabotropic glutamate receptor (mGluR)-mediated slow EPSC (sEPSC). TRPC3 channels also provide negative feedback regulation of cytosolic Ca 2+ , mediated by a C terminus “calmodulin and inositol trisphosphate receptor binding” (CIRB) domain. Here we report the alternative splicing of the TRPC3 mRNA transcript (designated TRPC3c), resulting in omission of exon 9 (approximately half of the CIRB domain) in mice, rats, and guinea pigs. TRPC3c expression is brain region specific, with prevalence in the cerebellum and brainstem. The TRPC3c channels expressed in HEK293 cells exhibit increased basal and GPCR-activated channel currents, and increased Ca 2+ fluorescence responses, compared with the previously characterized (TRPC3b) isoform when activated via either the endogenous M3 muscarinic acetylcholine receptor, or via coexpressed mGluR1. GPCR-induced TRPC3c channel opening rate (cell-attached patch) matched the maximum activation achieved with inside-out patches with zero cytosolic Ca 2+ , whereas the GPCR-induced TRPC3b activation frequency was significantly less. Both TRPC3 channel isoforms were blocked with 2 m m Ca 2+ , attributable to CIRB domain regulation. In addition, genistein blocked Purkinje cell ( S )-2-amino-2-(3,5-dihydroxyphenyl) acetic acid (mGluR1)-activated TPRC3 current as for recombinant TRPC3c current. This novel TRPC3c ion channel therefore has enhanced efficacy as a neuronal GPCR-Ca 2+ signaling effector, and is associated with sensorimotor coordination, neuronal development, and brain injury.

Heteromeric channel formation and Ca²⁺‐free media reduce the toxic effect of the weaver K_ir 3.2 allele

Publisher: Wiley

Date: 29-07-1996

DOI: 10.1016/0014-5793(96)00635-7

Abstract: Weaver mice have a severe hypoplasia of the cerebellum with an almost complete loss of the midline granule cells. Recent genetic studies of weaver mice have identified a mutation resulting in an amino acid substitution (G156S) in the pore of the inwardly rectifying potassium channel subunit Kir 3.2. When expressed in Xenopus oocytes the weaver mutation alters channel selectivity from a potassium-selective to a nonspecific cation-selective pore. In this study we confirm by cell-attached patch-cl recording that the mutation produces a non-selective cation channel. We also demonstrate that the cell death induced by weaver expression may be prevented by elimination of calcium from the extracellular solution as well as by coexpression with the wild-type Kir 3.2 allele, or other members of the Kir 3.0 subfamily. These results suggest that the weaver defect in Kir 3.2 may cause cerebellar cell death by cell swelling and calcium overload. Cells which express the weaver subunit, but which normally survive, may do so because of heteromeric subunit assembly with wild-type subunits of the Kir 3.0 subfamily.

Promiscuous coupling between the sulphonylurea receptor and inwardly rectifying potassium channels

Publisher: Springer Science and Business Media LLC

Date: 02-1996

DOI: 10.1038/379545A0

Abstract: Sulphonylureas are a class of drugs widely used to treat non-insulin-dependent diabetes mellitus. These drugs act by binding to a sulphonylurea receptor (SUR) in the pancreatic beta-cell membrane which inhibits an ATP-sensitive potassium (K-ATP) channel and thereby stimulates insulin secretion. There has been much debate as to whether SUR and the K-ATP channel are the same or separate proteins, whether SUR confers ATP-sensitivity on an ATP-insensitive pore-forming subunit, and whether sulphonylureas can also modulate other types of K-channel. We show here that SUR itself does not possess intrinsic channel activity but that it endows sulphonylurea sensitivity on several types of inwardly-rectifying K-channels. It does not necessarily confer ATP-sensitivity on these channels.

Maternal immune activation induces sustained changes in fetal microglia motility

Publisher: Springer Science and Business Media LLC

Date: 07-12-2020

DOI: 10.1038/S41598-020-78294-2

Abstract: Maternal infection or inflammation causes abnormalities in brain development associated with subsequent cognitive impairment and in an increased susceptibility to schizophrenia and autism spectrum disorders. Maternal immune activation (MIA) and increases in serum cytokine levels mediates this association via effects on the fetal brain, and microglia can respond to maternal immune status, but consensus on how microglia may respond is lacking and no-one has yet examined if microglial process motility is impaired. In this study we investigated how MIA induced at two different gestational ages affected microglial properties at different developmental stages. Immune activation in mid-pregnancy increased IL-6 expression in embryonic microglia, but failed to cause any marked changes in morphology either at E18 or postnatally. In contrast MIA, particularly when induced earlier (at E12), caused sustained alterations in the patterns of microglial process motility and behavioral deficits. Our research has identified an important microglial property that is altered by MIA and which may contribute to the underlying pathophysiological mechanisms linking maternal immune status to subsequent risks for cognitive disease.

Biophysics of CNG Ion Channels

Publisher: Springer Berlin Heidelberg

Date: 2008

DOI: 10.1007/978-3-540-72739-2_9

M2 pore mutations convert the glycine receptor channel from being anion- to cation-selective

Publisher: Elsevier BV

Date: 07-2000

DOI: 10.1016/S0006-3495(00)76287-4

Membrane Potential: Concepts

Publisher: Elsevier

Date: 2016

DOI: 10.1016/B978-0-12-394447-4.10027-6

Global transgenic upregulation of KCC2 confers enhanced diazepam efficacy in treating sustained seizures

Publisher: Wiley

Date: 17-11-2022

DOI: 10.1111/EPI.17097

Abstract: Reduced anticonvulsant efficacy of benzodiazepines is a problem in the treatment of status epilepticus, with up to 50% of patients failing to respond to their first dose. KCC2 is a neuronal K + ‐Cl − co‐transporter that helps set and maintain intracellular Cl − concentrations. KCC2 functional downregulation is a potential contributor to benzodiazepine resistance. We tested this idea using male and female doxycycline‐inducible, conditional transgenic mice to increase the functional expression of KCC2 in pyramidal neurons. We administered mice with two doses of the chemoconvulsant kainic acid (5 mg/kg, i.p.) 60 min apart and quantified the resultant seizures with electroencephalography (EEG) recordings. Overexpression of KCC2 prior to the chemoconvulsant challenge did not affect seizure latency or other measures of seizure severity, but it did increase diazepam's efficacy in stopping EEG seizures. Spike rate, time in seizure, and EEG spectral power following diazepam (5 mg/kg, i.p) were all significantly lower in KCC2 overexpression mice as compared to control mice. Our results indicate that, in the context of benzodiazepine resistance during sustained seizures, addressing impaired Cl − homeostasis alone appreciably improves the efficacy of γ‐aminobutyric acid (GABA)ergic inhibition. We therefore suggest the simultaneous targeting of KCC2 and GABA A receptors as a pathway for improving current anticonvulsant therapeutic strategies.

Charged residues at the 2 ' position of human GABA(c) rho 1 receptors invert ion selectivity and influence open state probability

Publisher: Elsevier BV

Date: 12-2004

DOI: 10.1074/JBC.M410625200

Cation-selective Mutations in the M2 Domain of the Inhibitory Glycine Receptor Channel Reveal Determinants of Ion-Charge Selectivity

Publisher: Rockefeller University Press

Date: 15-04-2002

DOI: 10.1085/JGP.20028552

Abstract: Ligand-gated ion channel receptors mediate neuronal inhibition or excitation depending on their ion charge selectivity. An investigation into the determinants of ion charge selectivity of the anion-selective α1 homomeric glycine receptor (α1 glycine receptor [GlyR]) was undertaken using point mutations to residues lining the extra- and intracellular ends of the ion channel. Five mutant GlyRs were studied. A single substitution at the intracellular mouth of the channel (A-1′E GlyR) was sufficient to convert the channels to select cations over anions with PCl/PNa = 0.34. This result delimits the selectivity filter and provides evidence that electrostatic interactions between permeating ions and pore residues are a critical factor in ion charge selectivity. The P-2′Δ mutant GlyR retained its anion selectivity (PCl/PNa = 3.81), but it was much reduced compared with the wild-type (WT) GlyR (PCl/PNa = 27.9). When the A-1′E and the P-2′Δ mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced (PCl/PNa = 0.13). The SDM GlyR was also Ca2+ permeable (PCa/PNa = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19′A GlyR) produced a more Ca2+-permeable channel (PCa/PNa = 0.73), without drastically altering monovalent charge selectivity (PCl/PNa = 0.23). The SDM+R19′E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced Ca2+ permeability (PCa/PNa = 0.92), with little effect on monovalent selectivity (PCl/PNa = 0.19). Estimates of the minimum pore diameter of the A-1′E, SDM, SDM+R19′A, and SDM+R19′E GlyRs revealed that these pores are larger than the α1 GlyR, with the SDM-based GlyRs being comparable in diameter to the cation-selective nicotinic acetylcholine receptors. This result provides evidence that the diameter of the ion channel is also an important factor in ion charge selectivity.

Mutation of the pore glutamate affects both cytoplasmic and external dequalinium block in the rat olfactory CNGA2 channel

Publisher: Springer Science and Business Media LLC

Date: 06-2005

DOI: 10.1007/S00249-005-0479-7

Abstract: Dequalinium has recently been reported to block CNGA1 and CNGA2 channels expressed in Xenopus laevis. Using the inside-out configuration of the patch-cl technique, we examined the effects of dequalinium on rat olfactory CNGA2 channels expressed in human embryonic kidney (HEK293) cells and studied aspects of its molecular mechanism of action. We found that cytoplasmic dequalinium blocked wild-type (WT) CNGA2 channels in a voltage-dependent manner with an IC(50) of approximately 1.3 muM at a V(m) of + 60 mV, and an effective fractional charge, zdelta, of +0.8 (z=2, delta=+0.4), suggesting that cytoplasmic dequalinium interacts with a binding site that is about two fifths of the way along the membrane electric field (from the intracellular side). Neutralizing the negatively charged pore lining glutamate acid residue (E342Q) still allows effective channel block by cytoplasmic dequalinium with an IC(50) of approximately 2.2 muM at a V(m) of +60 mV but now having a zdelta of +0.1 (delta=+0.05), indicating a profoundly decreased level of voltage-dependence. In addition, by comparing the extent of block under different levels of channel activation, we show that the block by cytoplasmic dequalinium displayed clear state-dependence in WT channels by interacting predominantly with the closed channel, whereas the block in E342Q channels was state-independent. Application of dequalinium to the external membrane surface also blocked currents through WT channels and the E342Q mutation significantly increased the IC(50) for external block approximately fivefold. These results confirm dequalinium as a potent, voltage-dependent and state-dependent blocker of cyclic-nucleotide-gated channels, and show that neutralization of the E342 residue profoundly affects the block by both cytoplasmic and external application of dequalinium.

GABAB receptor transduction mechanisms, and cross-talk between protein kinases A and C, in GABAergic terminals synapsing onto neurons of the rat nucleus basalis of Meynert

Publisher: Wiley

Date: 14-08-2003

DOI: 10.1113/JPHYSIOL.2003.046524

Single Channel Analysis of Conductance and Rectification in Cation-selective, Mutant Glycine Receptor Channels

Publisher: Rockefeller University Press

Date: 15-04-2002

DOI: 10.1085/JGP.20028553

Abstract: Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of α1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2'Δ) GlyR, had mean inward chord conductances (at −60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively. This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at the extracellular mouth of the channel (SDM+R19'A GlyR) made the conductance–voltage relationship linear (14 pS at both 60 and −60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external alent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal alent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity mutations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels.

Activation-Dependent Rapid Postsynaptic Clustering of Glycine Receptors in Mature Spinal Cord Neurons

Publisher: Society for Neuroscience

Date: 2017

DOI: 10.1523/ENEURO.0194-16.2017

Abstract: Inhibitory synapses are established during development but continue to be generated and modulated in strength in the mature nervous system. In the spinal cord and brainstem, presynaptically released inhibitory neurotransmitter dominantly switches from GABA to glycine during normal development in vivo . While presynaptic mechanisms of the shift of inhibitory neurotransmission are well investigated, the contribution of postsynaptic neurotransmitter receptors to this shift is not fully elucidated. Synaptic clustering of glycine receptors (GlyRs) is regulated by activation-dependent depolarization in early development. However, GlyR activation induces hyperpolarization after the first postnatal week, and little is known whether and how presynaptically released glycine regulates postsynaptic receptors in a depolarization-independent manner in mature developmental stage. Here we developed spinal cord neuronal culture of rodents using chronic strychnine application to investigate whether initial activation of GlyRs in mature stage could change postsynaptic localization of GlyRs. Immunocytochemical analyses demonstrate that chronic blockade of GlyR activation until mature developmental stage resulted in smaller clusters of postsynaptic GlyRs that could be enlarged upon receptor activation for 1 h in the mature stage. Furthermore, live cell-imaging techniques show that GlyR activation decreases its lateral diffusion at synapses, and this phenomenon is dependent on PKC, but neither Ca 2+ nor CaMKII activity. These results suggest that the GlyR activation can regulate receptor diffusion and cluster size at inhibitory synapses in mature stage, providing not only new insights into the postsynaptic mechanism of shifting inhibitory neurotransmission but also the inhibitory synaptic plasticity in mature nervous system.

Controlled activation of cortical astrocytes modulates neuropathic pain-like behaviour

Publisher: Springer Science and Business Media LLC

Date: 14-07-2022

DOI: 10.1038/S41467-022-31773-8

Abstract: Chronic pain is a major public health problem that currently lacks effective treatment options. Here, a method that can modulate chronic pain-like behaviour induced by nerve injury in mice is described. By combining a transient nerve block to inhibit noxious afferent input from injured peripheral nerves, with concurrent activation of astrocytes in the somatosensory cortex (S1) by either low intensity transcranial direct current stimulation (tDCS) or via the chemogenetic DREADD system, we could reverse allodynia-like behaviour previously established by partial sciatic nerve ligation (PSL). Such activation of astrocytes initiated spine plasticity to reduce those synapses formed shortly after PSL. This reversal from allodynia-like behaviour persisted well beyond the active treatment period. Thus, our study demonstrates a robust and potentially translational approach for modulating pain, that capitalizes on the interplay between noxious afferents, sensitized central neuronal circuits, and astrocyte-activation induced synaptic plasticity.

Microglia contact induces synapse formation in developing somatosensory cortex

Publisher: Springer Science and Business Media LLC

Date: 25-08-2016

DOI: 10.1038/NCOMMS12540

Abstract: Microglia are the immune cells of the central nervous system that play important roles in brain pathologies. Microglia also help shape neuronal circuits during development, via phagocytosing weak synapses and regulating neurogenesis. Using in vivo multiphoton imaging of layer 2/3 pyramidal neurons in the developing somatosensory cortex, we demonstrate here that microglial contact with dendrites directly induces filopodia formation. This filopodia formation occurs only around postnatal day 8–10, a period of intense synaptogenesis and when microglia have an activated phenotype. Filopodia formation is preceded by contact-induced Ca 2+ transients and actin accumulation. Inhibition of microglia by genetic ablation decreases subsequent spine density, functional excitatory synapses and reduces the relative connectivity from layer 4 neurons. Our data provide the direct demonstration of microglial-induced spine formation and provide further insights into immune system regulation of neuronal circuit development, with potential implications for developmental disorders of immune and brain dysfunction.

Making light work of fine-tuning channelrhodopsins

Publisher: Elsevier BV

Date: 03-2019

DOI: 10.1074/JBC.H119.007749

In Vivo Two-Photon Imaging of Microglial Synapse Contacts

Publisher: Springer New York

Date: 2019

DOI: 10.1007/978-1-4939-9658-2_20

Abstract: Microglia are traditionally known as immune sentinels of the brain and as key player in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson disease, or amyotrophic lateral sclerosis. Recently, they were also identified as synaptic organizer, promoting formation and maturation of synapses as well as modifying synaptic activity. Interestingly, microglia-mediated synaptic pruning and microglia-mediated changes in synaptic plasticity were observed both during brain development and in neurodegenerative diseases, stressing the key role of microglia-synapse interaction in these processes. Here we descried a technique for noninvasive in vivo monitoring of microglia-synapse interactions by means of two-photon microscopy.

Focal Ischaemic Infarcts Expand Faster in Cerebellar Cortex than Cerebral Cortex in a Mouse Photothrombotic Stroke Model.

Publisher: Springer Science and Business Media LLC

Date: 17-02-2018

DOI: 10.1007/S12975-018-0615-1

Abstract: It is generally accepted that the cerebellum is particularly vulnerable to ischaemic injury, and this may contribute to the high mortality arising from posterior circulation strokes. However, this has not been systematically examined in an animal model. This study compared the development and resolution of matched photothrombotic microvascular infarcts in the cerebellar and cerebral cortices in adult 129/SvEv mice of both sexes. The photothrombotic lesions were made using tail vein injection of Rose Bengal with a 532 nm laser projected onto a 2 mm diameter aperture over the target region of the brain (with skull thinning). Infarct size was then imaged histologically following 2 h to 30-day survival using serial reconstruction of haematoxylin and eosin stained cryosections. This was complemented with immunohistochemistry for neuron and glial markers. At 2 h post-injury, the cerebellar infarct volume averaged ~ 2.7 times that of the cerebral cortex infarcts. Infarct volume reached maximum in the cerebellum in a quarter of the time (24 h) taken in the cerebral cortex (4 days). Remodelling resolved the infarcts within a month, leaving significantly larger residual injury volume in the cerebellum. The death of neurons in the core lesion at 2 h was confirmed by NeuN and Calbindin immunofluorescence, alongside activation of astrocytes and microglia. The latter persisted in the region within and surrounding the residual infarct at 30 days. This comparison of acute focal ischaemic injuries in cerebellar and cerebral cortices provides direct confirmation of exacerbation of neuropathology and faster kinetics in the cerebellum.

A Single P-loop Glutamate Point Mutation to either Lysine or Arginine Switches the Cation–Anion Selectivity of the CNGA2 Channel

Publisher: Rockefeller University Press

Date: 13-03-2006

DOI: 10.1085/JGP.200509378

Abstract: Cyclic nucleotide-gated (CNG) channels play a critical role in olfactory and visual transduction. Site-directed mutagenesis and inside-out patch-cl recordings were used to investigate ion permeation and selectivity in two mutant homomeric rat olfactory CNGA2 channels expressed in HEK293 cells. A single point mutation of the negatively charged pore loop (P-loop) glutamate (E342) to either a positively charged lysine or arginine resulted in functional channels, which consistently responded to cGMP, although the currents were generally extremely small. The concentration–response curve of the lysine mutant channel was very similar to that of wild-type (WT) channels, suggesting no major structural alteration to the mutant channels. Reversal potential measurements, during cytoplasmic NaCl dilutions, showed that the lysine and the arginine mutations switched the selectivity of the channel from cations (PCl/PNa = 0.07 [WT]) to anions (PCl/PNa = 14 [Lys] or 10 [Arg]). Relative anion permeability sequences for the two mutant channels, measured with bi-ionic substitutions, were NO3− & I− & Br− & Cl− & F− & acetate−, the same as those obtained for anion-selective GABA and glycine channels. The mutant channels also seem to have an extremely small single-channel conductance, measured using noise analysis of about 1–2 pS, compared to a WT value of about 29 pS. The results showed that it is predominantly the charge of the E342 residue in the P-loop, rather than the pore helix dipoles, which controls the cation–anion selectivity of this channel. However, the outward rectification displayed by both mutant channels in symmetrical NaCl solutions suggests that the negative ends of the pore helix dipoles may play a role in reducing the outward movement of Cl− ions through these anion-selective channels. These results have potential implications for the determinants of anion–cation selectivity in the large family of P-loop–containing channels.

Regulation of lipid synthesis in myelin modulates neural activity and is required for motor learning

Publisher: Wiley

Date: 20-07-2023

DOI: 10.1002/GLIA.24441

Abstract: Brain function relies on both rapid electrical communication in neural circuitry and appropriate patterns or synchrony of neural activity. Rapid communication between neurons is facilitated by wrapping nerve axons with insulation by a myelin sheath composed largely of different lipids. Recent evidence has indicated that the extent of myelination of nerve axons can adapt based on neural activity levels and this adaptive myelination is associated with improved learning of motor tasks, suggesting such plasticity may enhance effective learning. In this study, we examined whether another aspect of myelin plasticity—changes in myelin lipid synthesis and composition—may also be associated with motor learning. We combined a motor learning task in mice with in vivo two‐photon imaging of neural activity in the primary motor cortex (M1) to distinguish early and late stages of learning and then probed levels of some key myelin lipids using mass spectrometry analysis. Sphingomyelin levels were elevated in the early stage of motor learning while galactosylceramide levels were elevated in the middle and late stages of motor learning, and these changes were correlated across in idual mice with both learning performance and neural activity changes. Targeted inhibition of oligodendrocyte‐specific galactosyltransferase expression, the enzyme that synthesizes myelin galactosylceramide, impaired motor learning. Our results suggest regulation of myelin lipid composition could be a novel facet of myelin adaptations associated with learning.

Activity-Dependent Global Downscaling of Evoked Neurotransmitter Release across Glutamatergic Inputs inDrosophila

Publisher: Society for Neuroscience

Date: 14-09-2020

DOI: 10.1523/JNEUROSCI.0349-20.2020

Abstract: Within mammalian brain circuits, activity-dependent synaptic adaptations, such as synaptic scaling, stabilize neuronal activity in the face of perturbations. Stability afforded through synaptic scaling involves uniform scaling of quantal litudes across all synaptic inputs formed on neurons, as well as on the postsynaptic side. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. We tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. We used motoneuron-specific genetic manipulations to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. We discovered an adaptation which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This “presynaptic downscaling” maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. Our results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side. SIGNIFICANCE STATEMENT To date, compensatory adaptations which stabilise target cell activity through activity-dependent global scaling have been observed only within central circuits, and on the postsynaptic side. Considering that maintenance of stable activity is imperative for the robust function of the nervous system as a whole, we tested whether activity-dependent global scaling could also manifest within peripheral circuits. We uncovered a compensatory adaptation which causes global scaling within a peripheral circuit and on the presynaptic side through uniform downscaling of evoked neurotransmitter release. Unlike in central circuits, uniform scaling maintains functionality over a wide, rather than a narrow, operational range, affording robust and stable activity. Activity-dependent global scaling therefore operates on both the presynaptic and postsynaptic sides to maintain target cell activity.

Motor learning requires myelination to reduce asynchrony and spontaneity in neural activity

Publisher: Wiley

Date: 29-08-2019

DOI: 10.1002/GLIA.23713

Cortical astrocytes prime the induction of spine plasticity and mirror image pain

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 16-04-2018

DOI: 10.1097/J.PAIN.0000000000001248

Abstract: Peripheral nerve injury causes maladaptive plasticity in the central nervous system and induces chronic pain. In addition to the injured limb, abnormal pain sensation can appear in the limb contralateral to the injury, called mirror image pain. Because synaptic remodeling in the primary somatosensory cortex (S1) has critical roles in the induction of chronic pain, cortical reorganization in the S1 ipsilateral to the injured limb may also accompany mirror image pain. To elucidate this, we conducted in vivo 2-photon calcium imaging of neuron and astrocyte activity in the ipsilateral S1 after a peripheral nerve injury. We found that cross-callosal inputs enhanced the activity of both S1 astrocytes and inhibitory neurons, whereas activity of excitatory neurons decreased. When local inhibitory circuits were blocked, astrocyte-dependent spine plasticity and allodynia were revealed. Thus, we propose that cortical astrocytes prime the induction of spine plasticity and mirror image pain after peripheral nerve injury. Moreover, this result suggests that cortical synaptic rewiring could be sufficient to cause allodynia on the uninjured periphery.

Clustering of Neuronal K+-Cl− Cotransporters in Lipid Rafts by Tyrosine Phosphorylation

Publisher: Elsevier BV

Date: 10-2009

DOI: 10.1074/JBC.M109.043620

Ion Permeation and Selectivity of Wild-Type Recombinant Rat CNG (rOCNC1) Channels Expressed in HEK293 Cells

Publisher: Springer Science and Business Media LLC

Date: 15-11-2000

DOI: 10.1007/S002320010021

Abstract: The permeation properties of adenosine 3', 5'-cyclic monophosphate (cAMP)-activated recombinant rat olfactory cyclic nucleotide-gated channels (rOCNC1) in human embryonic kidney (HEK 293) cells were investigated using inside-out excised membrane patches. The relative permeability of these rOCNC1 channels to monovalent alkali cations and organic cations was determined from measurements of the changes in reversal potential upon replacing sodium in the bathing solution with different test cations. The permeability ratio of Cl(-) relative to Na(+) (P(Cl)/P(Na)) was about 0.14, confirming that these channels are mainly permeable to cations. The sequence of relative permeabilities of monovalent alkali metal ions in these channels was P(Na) > or = P(K) > P(Li) > P(Cs) > or = P(Rb), which closely corresponds to a high-strength field sequence as previously determined for native rat olfactory receptor neurons (ORNs). The permeability sequence for organic cations relative to sodium was P(NH3OH) > P(NH4) > P(Na) > P(Tris) > P(Choline) > P(TEA), again in good agreement with previous permeability ratios obtained in native rat ORNs. Single-channel conductance sequences agreed surprisingly well with permeability sequences. These conductance measurements also indicated that, even in asymmetric bi-ionic cation solutions, the conductance was somewhat independent of current direction and dependent on the composition of both solutions. These results indicate that the permeability properties of rOCNC1 channels are similar to those of native rat CNG channels, and provide a suitable reference point for exploring the molecular basis of ion selectivity in recombinant rOCNC1 channels using site-directed mutagenesis.

Measurement of the limiting equivalent conductivities and mobilities of the most prevalent ionic species of EGTA (EGTA(2-) and EGTA(3-)) for use in electrophysiological experiments

Publisher: Elsevier BV

Date: 07-1999

DOI: 10.1016/S0165-0270(99)00036-9

Abstract: In many experimental biological situations, chelating agents like EGTA (ethylene glycol-bis-(beta-amino-ethyl ether) N,N,N',N'-tetra-acetic acid) are commonly used to control or suppress the concentration of alent ions like Ca2+. The evaluation of liquid junction potentials in electrophysiological measurements, and particularly in patch-cl situations, requires information about the ions within the solution. Where there is a significant concentration of EGTA present, it is necessary to know the values of the relative mobility of at least the most predominant ionic species of EGTA in order to complete these calculations. EGTA, with four negative charges with different pKas, can therefore exist as four differently charged ions in solution (EGTA-, EGTA2-, EGTA3- and EGTA4-) or as uncharged, although between pH 5.5 and 8 it is almost exclusively EGTA2-. We have measured limiting equivalent conductivities of the most common ionic forms of EGTA (EGTA2- and EGTA3-) encountered at physiological pHs. These were 35.9 +/- 0.7 and 56 +/- 2.5 S cm2 equiv(-1) respectively. Their mobilities relative to K+ were 0.24 +/- 0.01 for EGTA2- and 0.25 +/- 0.01 for EGTA3-. Thus for typical electrophysiological solutions, the contribution of EGTA to the liquid junction potential should be small (e.g. approximately 0.4 mV).

GLYCINE RECEPTORS: WHAT GETS IN AND WHY?

Publisher: Wiley

Date: 04-11-1999

DOI: 10.1046/J.1440-1681.1999.03149.X

Abstract: 1. The glycine receptor channel (GlyR), a member of the ligand-gated ion channel superfamily, shares many similar permeation properties with the GABAA receptor channel. 2. The GlyR is anion permeable, with PK/PCl < 0.05, has a 5-6 A minimum pore diameter and a permeation selectivity sequence dominated by hydration energies. 3. The channels, which display multiple subconductance states, can be multiply occupied. 4. Two positive arginine rings at the ends of the pore region may contribute to the anion selectivity of the GlyR. 5. Mutation of the extracellular charged arginine ring can impair channel function by decreasing the sensitivity of glycine activation, reducing channel conductance, shifting the normal multi-subconductance states to lower values and by decoupling the link between ligand binding and channel gating. 6. These and other site-directed mutagenesis studies of recombinant GlyR, together with studies of native GlyR, are providing further insights into what controls gating and ion permeation and selectivity through this channel.

Microglia and synapse interactions: fine tuning neural circuits and candidate molecules

Publisher: Frontiers Media SA

Date: 2013

DOI: 10.3389/FNCEL.2013.00070

Further analysis of counterion permeation through anion-selective glycine receptor channels

Publisher: Informa UK Limited

Date: 05-2010

DOI: 10.4161/CHAN.4.3.11020

Abstract: The functional role of ion channels, which allow counterion permeation, depends critically on their relative anion-cation relative selectivity. From whole-cell patch cl reversal potential measurements under dilution potential conditions, we have already shown that anion-cation permeabilities of anion-selective wild-type (WT) and mutant (with larger pore diameter) glycine receptor (GlyR) channels in the presence of Li(+), Na(+) and Cs(+) counterions, were inversely correlated with the equivalent hydration diameter of the counterion, with chloride-cation permeability increasing as counterion equivalent hydration diameter increased with respect to the channel minimum pore diameter. Corrected for liquid junction potentials (LJPs using ion activities), the previous chloride-cation permeabilities for the alkali cations were 23.4 (Li(+)), 10.9 (Na(+)) and 5.0 (Cs(+)) for the smaller WT channel. Further analysis to incorporate an initial offset potential correction, to fully allow for slight differences between internal cell composition and external control salt solution, changed the above permeability ratios to 30.6 (Li(+)), 11.8 (Na(+)) and 5.0 (Cs(+)), adding enhanced support for the inverse correlation between anion-to-counterion permeability ratio and equivalent hydrated counterion diameter relative to channel pore diameter (erroneously ignoring LJPs reduces each permeability ratio to about 4). Also, new direct measurements of LJPs (for NaCl and LiCl salt dilutions) using a 3M KCl-agar reference salt bridge (with freshly-cut end for each solution composition change) have shown excellent agreement with calculated LJPs (using ion activities), validating calculated LJP values. We continue to suggest that counterion cations permeate with chloride ions as neutral pairs.

Controlled activation of cortical astrocytes can reverse neuropathic chronic pain

Publisher: Research Square Platform LLC

Date: 08-02-2021

DOI: 10.21203/RS.3.RS-150434/V1

Abstract: Chronic pain is a major public health problem that currently lacks effective treatment options. Here, we report a novel combination therapy that can effectively reverse chronic pain induced by nerve injury in mice. By combing transient nerve block to inhibit noxious afferent input from injured peripheral nerves, with transient concurrent activation of astrocytes in the somatosensory cortex (S1) by either transcranial direct current stimulation (tDCS) or via the chemogenetic DREADD system, we could reverse allodynia previously established by partial sciatic nerve ligation (PSL). Activation of astrocytes initiated spine plasticity to reduce synapses formed shortly after PSL. The cure from allodynia persisted long after ceasing active treatment. Thus, our study represents the first report of a robust, readily translatable approach for treating chronic pain that capitalizes on the causative interplay between noxious afferents, sensitized central neuronal circuits and astrocytic-activation induced plasticity.

Perforated Whole-Cell Patch-Clamp Technique: A User’s Guide

Publisher: Springer Japan

Date: 2012

DOI: 10.1007/978-4-431-53993-3_4

Anion-Cation Permeability Correlates with Hydrated Counterion Size in Glycine Receptor Channels

Publisher: Elsevier BV

Date: 11-2008

DOI: 10.1529/BIOPHYSJ.107.125690

Resting Microglia Directly Monitor the Functional State of SynapsesIn Vivoand Determine the Fate of Ischemic Terminals

Publisher: Society for Neuroscience

Date: 04-2009

DOI: 10.1523/JNEUROSCI.4363-08.2009

Abstract: Recent studies have identified the important contribution of glial cells to the plasticity of neuronal circuits. Resting microglia, the primary immune effector cells in the brain, dynamically extend and retract their processes as if actively surveying the microenvironment. However, just what is being s led by these resting microglial processes has not been demonstrated in vivo , and the nature and function of any interactions between microglia and neuronal circuits is incompletely understood. Using in vivo two-photon imaging of fluorescent-labeled neurons and microglia, we demonstrate that the resting microglial processes make brief (∼5 min) and direct contacts with neuronal synapses at a frequency of about once per hour. These contacts are activity-dependent, being reduced in frequency by reductions in neuronal activity. After transient cerebral ischemia, the duration of these microglia–synapse contacts are markedly prolonged (∼1 h) and are frequently followed by the disappearance of the presynaptic bouton. Our results demonstrate that at least part of the dynamic motility of resting microglial processes in vivo is directed toward synapses and propose that microglia vigilantly monitor and respond to the functional status of synapses. Furthermore, the striking finding that some synapses in the ischemic areas disappear after prolonged microglial contact suggests microglia contribute to the subsequent increased turnover of synaptic connections. Further understanding of the mechanisms involved in the microglial detection of the functional state of synapses, and of their role in remodeling neuronal circuits disrupted by ischemia, may lead to novel therapies for treating brain injury that target microglia.

UNSW

Location: Australia

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National Institute For Physiological Sciences

Location: Japan

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University Of Sydney

Location: Australia

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UNSW Sydney

Location: Australia

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University Of Oxford

Location: United Kingdom of Great Britain and Northern Ireland

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How Molecular Structure Determines How Ions Are Selected And Pass Through Neurotransmitter Channels

Start Date: 2003

End Date: 2005

Funder: National Health and Medical Research Council

Discovery Projects - Grant ID: DP0557657

Start Date: 2005

End Date: 12-2008

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE180100066

Start Date: 2018

End Date: 12-2018

Amount: $443,311.00

Funder: Australian Research Council