Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-013-1406-x
ION CHANNELS, RECEPTORS AND TRANSPORTERS
Kv7 potassium channel subunits and M currents in cultured hippocampal interneurons Alexej Grigorov & Anastasia Moskalyuk & Mykola Kravchenko & Nikolai Veselovsky & Alexei Verkhratsky & Svetlana Fedulova
Received: 4 November 2013 / Revised: 11 November 2013 / Accepted: 12 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Potassium channels of the Kv7 family that mediate the non-inactivating M current regulate the excitability of many types of neurons in the central nervous system, including some in the hippocampus. We report here that individual interneurons from newborn rat hippocampi in long-term culture strongly express messenger RNA specific for Kv7.2 and Kv7.3 and, to a lesser extent, Kv7.5 channel subunits but not for the Kv7.4 subunit. An M-like current was electrophysiologically identified in two subpopulations of interneurons distinct in their spiking behaviour (regular or fast spiking). The M-channel enhancer retigabine reduced interneuronal excitability by constraining the number of action potentials generated during imposed depolarisations; this effect was inhibited by specific the M-channel blocking drugs. In paired synaptically connected interneuron-target cell recordings, anatomically localised applications of retigabine indicated that M channels were present in both the interneuron soma and its GABA-ergic inhibitory axon. We conclude that M-channel subunits and functional M channels are broadly expressed in hippocampal interneurons and their axons and are potentially capable of strongly regulating their firing properties.
Keywords Paired neuron recording . Voltage clamp . Excitability . PCR . Retigabine A. Grigorov : A. Moskalyuk : N. Veselovsky : S. Fedulova (*) Bogomoletz Institute of Physiology NASU, Bogomoletz str., 4, 01024 Kiev, Ukraine e-mail: [email protected] M. Kravchenko Department of Pharmacology and Toxicology, University of Saarland, Homburg, Saarland, Germany A. Verkhratsky The University of Manchester, Manchester, UK
Introduction Interneurons play a fundamental role in information processing in the hippocampus and are equipped with a unique set of ion channels that govern their electrical behaviour [14, 19]. One particular ion channel that regulates the excitability of many neurones is the voltage-gated M-type potassium channel [2]. This K+ channel is composed of members of the Kv7 potassium channel family, most frequently (but not invariably) of Kv7.2/7.3 heteromers [7, 27, 34]. The Kv7.2 protein has been identified in some mouse hippocampal interneurons [3] and [12] showed the presence of Kv7.2 and Kv7.3 immunoreactivity in a subset of somatostatin-expressing mouse stratum oriens interneurons, plus a functional M current that affected the interspike interval and action potential frequency. In the present paper, we have explored the possible role of Kv7/M channels in rat hippocampal inhibitory interneurons in culture, in which they make synaptic connections with target cells. We show the presence of Kv7.2 and Kv7.3 transcripts in all individual inhibitory interneurons (and Kv7.5 transcripts in some). We also demonstrate that all such neurones possess potentially functional M channels, which, when activated, regulate neuronal firing activity and neuronal interactions with target neurones. Some of our preliminary observations have been published in Ukraininan [9].
Materials and methods Hippocampal cell culture All experiments were carried in accordance with the European Communities Council Directive of 24 November 1986 (86/ 609/EEC) and approved by the Animal Use and Care Committee of the Bogomoletz Institute of physiology. Experiments were performed on neurons in 3–4-week-old primary culture
Pflugers Arch - Eur J Physiol
prepared from whole hippocampi of newborn Wistar rats. Pups were decapitated after cervical dislocation (in accordance with EU Directive and rules set by the local Ethics Committee), hippocampi isolated and treated with 0.025 % trypsin (type XII-S, Sigma) at room temperature (21–24 °С) for 5–6 min. After trituration, cells were plated on poly-L ornithine- and laminin-covered (Sigma) Petri dishes at a density of 30,000/cm2. Cells were cultured in Eagle’s minimum essential medium containing 10 % horse serum, 6 μg/ml insulin, 2.3 mg/ml NaHCO3 bicarbonate buffer, 25 U/ml benzylpenicillin sodium salt and 25 μg/ml streptomycin sulfate and maintained in a temperature-controlled (37 °С) incubator (Jouan) in an atmosphere of 5 % СО2. To inhibit glial cells proliferation, 5 μM of cytosine-β-D -arabinofuranoside (Sigma) was added into medium at the third day in vitro. After 24 h, the cells were transferred into normal medium. Isolated cultured rat sympathetic neurones Culture of neurons from rat superior cervical ganglia (SCG) was prepared according to the procedure described elsewhere [29]. Briefly, 17-day-old Wistar rats of either sex were anaesthetised with ether and killed by cervical dislocation. Superior cervical ganglia were dissected into small fragments and treated with 1.0 mg/ml collagenase (Sigma) for 20– 40 min at 37 °C. Cell suspension was placed on coverslips (Marienfeld, Germany) coated with poly-L -lysine, the cell density being 1×104/ml. Five to six drops of the culture medium were added to each coverslip 30–40 min later. SCG cells were cultured in a 4.5-g/l glucose-containing Dulbecco’s modified Eagle’s medium supplemented with 40 μg/ml gentamicin, 0.25 U/ml insulin, 10 % horse serum and 5 % fetal calf serum. A drop of cell suspension was placed on coverslips coated with poly-L -lysine, the cell density being 1×104/ml. Thirty- to 40-min later, five to six drops of the culture medium were added to each coverslip. The cells were incubated at 37 °C, 95 % humidity and 5 % CO2 for 2 days. Reverse transcriptase polymerase chain reaction Messenger RNA (mRNA) transcripts for Kv7.2, Kv7.3, Kv7.4 and Kv7.5 (products of KCNQ2, 3, 4 and 5 genes) were sought in whole isolated hippocampi from newborn and 21day-old rats and in single neurons cultured under the conditions that used for electrophysiological recording (see below), by reverse transcriptase polymerase chain reaction (RT-PCR), as described by [27]. DNase I treated RNA (0.2 mg) was reverse transcribed using M-MLV reverse transcriptase (Promega). The resulting complementary DNA (cDNA) template was subjected to PCR amplification using oligodeoxynucleotide primers based on rat KCNQ2 (GenBank accession number AF08453), KCNQ3 (AF091247), KCNQ4 (AF249748) and human KCNQ5
(AF202977) sequences. Primers (designed to be intron spanning) and cycling conditions were as described by [27]. Human and rat KCNQ5 base sequences covered by the primers are identical. Amplified products were analysed by electrophoresis through 2 % MetaPhor agarose (FMC BioProducts). Single-cell reverse transcriptase polymerase chain reaction Cytosol from single hippocampal interneurons that had been obtained from newborn rat pups and cultured for 20 days was collected into 7.5 ml of recording solution and eluted into a tube containing 2.5 ml first-strand buffer [2 mM each deoxynucleotide triphosphate, 20 mM oligo d(T)15, 40 mM dithiothreitol and 20 U RNase inhibitor (Roche)]. Reverse transcription of mRNA was initiated by addition of 100 U M-MLV reverse transcriptase RNase H (−) point mutant (Promega) followed by incubation at 37 °C for 1 h. A multiplex PCR protocol was then used to amplify simultaneously cDNAs for KCNQ2–5. Electrophysiological recordings and solutions Experiments were performed on neurons cultured for 18– 28 days and maintained at room temperature (20–23 °С). By this time, cells had formed their dendritic arbours and synaptic connections, and their morphological characteristics could be clearly distinguished. Only bi- and multipolar cells with soma diameters 15–35 μm were chosen for the recordings. Triangular-shaped neurons and all cells bearing three processes were excluded since these might have been pyramidal neurones. Recording chambers were mounted on the stage of an inverted microscope (Axiovert 200, Carl Zeiss), and cells were voltage-clamped with electrodes filled with the following solution (in mM): К-gluconate, 100; KCl, 50; EGTA, 10; MgCl2, 5; and HEPES, 2 (resistances, 4–6 MΩ). The extracellular solution was composed of (in mM): NaCl, 140; KCl, 3; CaCl2, 2; MgCl2, 2; HEPES, 20; and glucose, 30. This was supplemented with ionotropic glutamate N-methylD -aspartate (NMDA)- and non-NMDA-receptor inhibitors: DL-2-amino-5-phosphonovaleric acid (DL-AP5) and 6,7dinitroquinoxaline-2,3-dione (DNQX, each at 20 μM). Experiments were restricted to cells with resting potentials more negative than −40 mV. The whole-cell patch-clamp technique [8] was used to measure transmembrane currents. Signals were recorded using an Axopatch-200B amplifier (Axon Instruments), digitised at 10 kHz, filtered using a low-pass Bessel filter with 5 kHz cut-off frequency and stored and analysed using an analog-digital converter LabMaster TL-1 and pClamp 6.0 software (Axon Instruments). Voltage-gated potassium currents and postsynaptic currents were recorded in voltageclamp mode at holding potentials varied between −110 and +60 mV. To isolate voltage-gated potassium currents, the
Pflugers Arch - Eur J Physiol
extracellular solution was further supplemented with 1 μM tetrodotoxin to block sodium channels and 0.1 mM CdCl2 to block calcium channels. Evoked electrical activity of the interneurons was recorded in current-clamp mode. For this, the resting potential was set at −70 mV, while series of action potentials were evoked by 500 ms depolarising current steps, with varying current amplitudes sufficient to evoke a maximal action potential frequency. For simultaneous recording of presynaptic action potentials and evoked postsynaptic currents, both current- and voltage-clamp modes were used. Depolarising current pulses were applied to the soma of the presynaptic cell to evoke action potential trains and postsynaptic currents recorded at a holding potential of −75 mV. Local electrical stimulation was performed by means of a stimulus isolator ISO-Flex (AMPI, Israel). The stimulating glass pipette was a patch pipette with a 2-μm outer tip diameter. Evoked postsynaptic currents were recorded in response to stimulating a single axon with a train of 1-ms negative voltage steps. The stimulus frequency was 40 Hz and train duration 500 ms, repeated at 0.2 Hz. The amplitude of voltage on the input terminals of the pipette varied from 20 to 60 V. Drugs were delivered by a fast local superfusion technique, which could be directed selectively to the soma or to the neurone processes [32].
fluorescein isothiocyanate coupled secondary antibodies (1:100/1:200; DAKO Corporation, CA, USA) were applied for 1 h at room temperature. After several more washes, cells were attached to microscope slides using a fluorescence mounting medium (DAKO Corporation) and visualised using an Axiovert 200 microscope. Specificity of the anti-Kv7.2 staining was determined by competing out the staining by pre-absorbing the antibody with the immunogenic peptide epitope (10:1 excess; [7]). Staining was visualised using an Axiovert 200 microscope. Stained neurons were identified by comparing their fluorescent images with bright field images taken during electrophysiological experiments.
Chemicals and drugs Unless mentioned otherwise, all chemicals and drugs were obtained from Sigma, USA. The M-channel blockers linopirdine and XE991 were obtained from Tocris (UK). Retigabine was a gift from NeuroSearch A/S (Ballerup, Denmark).
Results Data analysis Identification of interneurons Parameters of whole-cell currents (amplitude and time constants) and parameters of action potentials (frequency, threshold and duration) were determined using Clampfit 9.0 software (Axon Instruments). Further data processing, including statistical analysis, was performed in Microsoft Excel. All data are presented as mean±standard error of the mean (SEM). Student’s t test was used to determine statistical significance of the difference between the mean values of two data sets. Significance levels are depicted in figures as follows: *P < 0.05, **P
ION CHANNELS, RECEPTORS AND TRANSPORTERS
Kv7 potassium channel subunits and M currents in cultured hippocampal interneurons Alexej Grigorov & Anastasia Moskalyuk & Mykola Kravchenko & Nikolai Veselovsky & Alexei Verkhratsky & Svetlana Fedulova
Received: 4 November 2013 / Revised: 11 November 2013 / Accepted: 12 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Potassium channels of the Kv7 family that mediate the non-inactivating M current regulate the excitability of many types of neurons in the central nervous system, including some in the hippocampus. We report here that individual interneurons from newborn rat hippocampi in long-term culture strongly express messenger RNA specific for Kv7.2 and Kv7.3 and, to a lesser extent, Kv7.5 channel subunits but not for the Kv7.4 subunit. An M-like current was electrophysiologically identified in two subpopulations of interneurons distinct in their spiking behaviour (regular or fast spiking). The M-channel enhancer retigabine reduced interneuronal excitability by constraining the number of action potentials generated during imposed depolarisations; this effect was inhibited by specific the M-channel blocking drugs. In paired synaptically connected interneuron-target cell recordings, anatomically localised applications of retigabine indicated that M channels were present in both the interneuron soma and its GABA-ergic inhibitory axon. We conclude that M-channel subunits and functional M channels are broadly expressed in hippocampal interneurons and their axons and are potentially capable of strongly regulating their firing properties.
Keywords Paired neuron recording . Voltage clamp . Excitability . PCR . Retigabine A. Grigorov : A. Moskalyuk : N. Veselovsky : S. Fedulova (*) Bogomoletz Institute of Physiology NASU, Bogomoletz str., 4, 01024 Kiev, Ukraine e-mail: [email protected] M. Kravchenko Department of Pharmacology and Toxicology, University of Saarland, Homburg, Saarland, Germany A. Verkhratsky The University of Manchester, Manchester, UK
Introduction Interneurons play a fundamental role in information processing in the hippocampus and are equipped with a unique set of ion channels that govern their electrical behaviour [14, 19]. One particular ion channel that regulates the excitability of many neurones is the voltage-gated M-type potassium channel [2]. This K+ channel is composed of members of the Kv7 potassium channel family, most frequently (but not invariably) of Kv7.2/7.3 heteromers [7, 27, 34]. The Kv7.2 protein has been identified in some mouse hippocampal interneurons [3] and [12] showed the presence of Kv7.2 and Kv7.3 immunoreactivity in a subset of somatostatin-expressing mouse stratum oriens interneurons, plus a functional M current that affected the interspike interval and action potential frequency. In the present paper, we have explored the possible role of Kv7/M channels in rat hippocampal inhibitory interneurons in culture, in which they make synaptic connections with target cells. We show the presence of Kv7.2 and Kv7.3 transcripts in all individual inhibitory interneurons (and Kv7.5 transcripts in some). We also demonstrate that all such neurones possess potentially functional M channels, which, when activated, regulate neuronal firing activity and neuronal interactions with target neurones. Some of our preliminary observations have been published in Ukraininan [9].
Materials and methods Hippocampal cell culture All experiments were carried in accordance with the European Communities Council Directive of 24 November 1986 (86/ 609/EEC) and approved by the Animal Use and Care Committee of the Bogomoletz Institute of physiology. Experiments were performed on neurons in 3–4-week-old primary culture
Pflugers Arch - Eur J Physiol
prepared from whole hippocampi of newborn Wistar rats. Pups were decapitated after cervical dislocation (in accordance with EU Directive and rules set by the local Ethics Committee), hippocampi isolated and treated with 0.025 % trypsin (type XII-S, Sigma) at room temperature (21–24 °С) for 5–6 min. After trituration, cells were plated on poly-L ornithine- and laminin-covered (Sigma) Petri dishes at a density of 30,000/cm2. Cells were cultured in Eagle’s minimum essential medium containing 10 % horse serum, 6 μg/ml insulin, 2.3 mg/ml NaHCO3 bicarbonate buffer, 25 U/ml benzylpenicillin sodium salt and 25 μg/ml streptomycin sulfate and maintained in a temperature-controlled (37 °С) incubator (Jouan) in an atmosphere of 5 % СО2. To inhibit glial cells proliferation, 5 μM of cytosine-β-D -arabinofuranoside (Sigma) was added into medium at the third day in vitro. After 24 h, the cells were transferred into normal medium. Isolated cultured rat sympathetic neurones Culture of neurons from rat superior cervical ganglia (SCG) was prepared according to the procedure described elsewhere [29]. Briefly, 17-day-old Wistar rats of either sex were anaesthetised with ether and killed by cervical dislocation. Superior cervical ganglia were dissected into small fragments and treated with 1.0 mg/ml collagenase (Sigma) for 20– 40 min at 37 °C. Cell suspension was placed on coverslips (Marienfeld, Germany) coated with poly-L -lysine, the cell density being 1×104/ml. Five to six drops of the culture medium were added to each coverslip 30–40 min later. SCG cells were cultured in a 4.5-g/l glucose-containing Dulbecco’s modified Eagle’s medium supplemented with 40 μg/ml gentamicin, 0.25 U/ml insulin, 10 % horse serum and 5 % fetal calf serum. A drop of cell suspension was placed on coverslips coated with poly-L -lysine, the cell density being 1×104/ml. Thirty- to 40-min later, five to six drops of the culture medium were added to each coverslip. The cells were incubated at 37 °C, 95 % humidity and 5 % CO2 for 2 days. Reverse transcriptase polymerase chain reaction Messenger RNA (mRNA) transcripts for Kv7.2, Kv7.3, Kv7.4 and Kv7.5 (products of KCNQ2, 3, 4 and 5 genes) were sought in whole isolated hippocampi from newborn and 21day-old rats and in single neurons cultured under the conditions that used for electrophysiological recording (see below), by reverse transcriptase polymerase chain reaction (RT-PCR), as described by [27]. DNase I treated RNA (0.2 mg) was reverse transcribed using M-MLV reverse transcriptase (Promega). The resulting complementary DNA (cDNA) template was subjected to PCR amplification using oligodeoxynucleotide primers based on rat KCNQ2 (GenBank accession number AF08453), KCNQ3 (AF091247), KCNQ4 (AF249748) and human KCNQ5
(AF202977) sequences. Primers (designed to be intron spanning) and cycling conditions were as described by [27]. Human and rat KCNQ5 base sequences covered by the primers are identical. Amplified products were analysed by electrophoresis through 2 % MetaPhor agarose (FMC BioProducts). Single-cell reverse transcriptase polymerase chain reaction Cytosol from single hippocampal interneurons that had been obtained from newborn rat pups and cultured for 20 days was collected into 7.5 ml of recording solution and eluted into a tube containing 2.5 ml first-strand buffer [2 mM each deoxynucleotide triphosphate, 20 mM oligo d(T)15, 40 mM dithiothreitol and 20 U RNase inhibitor (Roche)]. Reverse transcription of mRNA was initiated by addition of 100 U M-MLV reverse transcriptase RNase H (−) point mutant (Promega) followed by incubation at 37 °C for 1 h. A multiplex PCR protocol was then used to amplify simultaneously cDNAs for KCNQ2–5. Electrophysiological recordings and solutions Experiments were performed on neurons cultured for 18– 28 days and maintained at room temperature (20–23 °С). By this time, cells had formed their dendritic arbours and synaptic connections, and their morphological characteristics could be clearly distinguished. Only bi- and multipolar cells with soma diameters 15–35 μm were chosen for the recordings. Triangular-shaped neurons and all cells bearing three processes were excluded since these might have been pyramidal neurones. Recording chambers were mounted on the stage of an inverted microscope (Axiovert 200, Carl Zeiss), and cells were voltage-clamped with electrodes filled with the following solution (in mM): К-gluconate, 100; KCl, 50; EGTA, 10; MgCl2, 5; and HEPES, 2 (resistances, 4–6 MΩ). The extracellular solution was composed of (in mM): NaCl, 140; KCl, 3; CaCl2, 2; MgCl2, 2; HEPES, 20; and glucose, 30. This was supplemented with ionotropic glutamate N-methylD -aspartate (NMDA)- and non-NMDA-receptor inhibitors: DL-2-amino-5-phosphonovaleric acid (DL-AP5) and 6,7dinitroquinoxaline-2,3-dione (DNQX, each at 20 μM). Experiments were restricted to cells with resting potentials more negative than −40 mV. The whole-cell patch-clamp technique [8] was used to measure transmembrane currents. Signals were recorded using an Axopatch-200B amplifier (Axon Instruments), digitised at 10 kHz, filtered using a low-pass Bessel filter with 5 kHz cut-off frequency and stored and analysed using an analog-digital converter LabMaster TL-1 and pClamp 6.0 software (Axon Instruments). Voltage-gated potassium currents and postsynaptic currents were recorded in voltageclamp mode at holding potentials varied between −110 and +60 mV. To isolate voltage-gated potassium currents, the
Pflugers Arch - Eur J Physiol
extracellular solution was further supplemented with 1 μM tetrodotoxin to block sodium channels and 0.1 mM CdCl2 to block calcium channels. Evoked electrical activity of the interneurons was recorded in current-clamp mode. For this, the resting potential was set at −70 mV, while series of action potentials were evoked by 500 ms depolarising current steps, with varying current amplitudes sufficient to evoke a maximal action potential frequency. For simultaneous recording of presynaptic action potentials and evoked postsynaptic currents, both current- and voltage-clamp modes were used. Depolarising current pulses were applied to the soma of the presynaptic cell to evoke action potential trains and postsynaptic currents recorded at a holding potential of −75 mV. Local electrical stimulation was performed by means of a stimulus isolator ISO-Flex (AMPI, Israel). The stimulating glass pipette was a patch pipette with a 2-μm outer tip diameter. Evoked postsynaptic currents were recorded in response to stimulating a single axon with a train of 1-ms negative voltage steps. The stimulus frequency was 40 Hz and train duration 500 ms, repeated at 0.2 Hz. The amplitude of voltage on the input terminals of the pipette varied from 20 to 60 V. Drugs were delivered by a fast local superfusion technique, which could be directed selectively to the soma or to the neurone processes [32].
fluorescein isothiocyanate coupled secondary antibodies (1:100/1:200; DAKO Corporation, CA, USA) were applied for 1 h at room temperature. After several more washes, cells were attached to microscope slides using a fluorescence mounting medium (DAKO Corporation) and visualised using an Axiovert 200 microscope. Specificity of the anti-Kv7.2 staining was determined by competing out the staining by pre-absorbing the antibody with the immunogenic peptide epitope (10:1 excess; [7]). Staining was visualised using an Axiovert 200 microscope. Stained neurons were identified by comparing their fluorescent images with bright field images taken during electrophysiological experiments.
Chemicals and drugs Unless mentioned otherwise, all chemicals and drugs were obtained from Sigma, USA. The M-channel blockers linopirdine and XE991 were obtained from Tocris (UK). Retigabine was a gift from NeuroSearch A/S (Ballerup, Denmark).
Results Data analysis Identification of interneurons Parameters of whole-cell currents (amplitude and time constants) and parameters of action potentials (frequency, threshold and duration) were determined using Clampfit 9.0 software (Axon Instruments). Further data processing, including statistical analysis, was performed in Microsoft Excel. All data are presented as mean±standard error of the mean (SEM). Student’s t test was used to determine statistical significance of the difference between the mean values of two data sets. Significance levels are depicted in figures as follows: *P < 0.05, **P
