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Neto2- mice have impaired GABAergic inhibition and are susceptible to seizures.

Mahadevan V, Dargaei Z, Ivakine EA, Hartmann AM, Ng D, Chevrier J, Ormond J, Nothwang HG, McInnes RR, Woodin MA - Front Cell Neurosci (2015)

Bottom Line: Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized.We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls.To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto Toronto, ON, Canada.

ABSTRACT
Neto2 is a transmembrane protein that interacts with the neuron-specific K(+)-Cl(-) cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl(-) gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2- mice would be prone to seizure activity. We found that Neto2- mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity.

No MeSH data available.


Related in: MedlinePlus

Hippocampal pyramidal neurons from adult Neto2- mice have depolarized EGABA. (Ai) Example of gramicidin perforated patch clamp recordings obtained from CA1 pyramidal neurons in acute hippocampal slices prepared from Neto2- mice (n = 15 neurons, gray) and wild-type mice (n = 21 neurons, black). The inhibitory postsynaptic potential (IPSP) amplitude was plotted against the holding potential of the postsynaptic membrane, and the intercept of this curve with the x-axis was taken as EGABA. Insets: traces of IPSPs for the examples shown. Scale bars: 5 mV, 10 ms. (Aii) Summary of all experiments similar to (Ai). (B) Tl+-uptake assay performed in HEK-293 cells (n = 4 sets), in the absence (white bars) and presence of 2 mM furosemide (black bars). ***p < 0.001.
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Figure 1: Hippocampal pyramidal neurons from adult Neto2- mice have depolarized EGABA. (Ai) Example of gramicidin perforated patch clamp recordings obtained from CA1 pyramidal neurons in acute hippocampal slices prepared from Neto2- mice (n = 15 neurons, gray) and wild-type mice (n = 21 neurons, black). The inhibitory postsynaptic potential (IPSP) amplitude was plotted against the holding potential of the postsynaptic membrane, and the intercept of this curve with the x-axis was taken as EGABA. Insets: traces of IPSPs for the examples shown. Scale bars: 5 mV, 10 ms. (Aii) Summary of all experiments similar to (Ai). (B) Tl+-uptake assay performed in HEK-293 cells (n = 4 sets), in the absence (white bars) and presence of 2 mM furosemide (black bars). ***p < 0.001.

Mentions: Transport activity was determined by measuring Cl−-dependent uptake of Tl+ in HEK-293 cells. Uptake measurements were done as described previously (Delpire et al., 2009; Hartmann et al., 2010; Weber et al., 2014). Cells were transiently transfected with empty plasmid or pcDNA3.1 KCC2 or pcDNA3.1 KCC2 alone + pcDNA3.1 Neto2 constructs. Briefly, 150 μl of Opti-MEM (Invitrogen), 6 μl of TurboFect (Fermentas, Karlsruhe, Germany), and appropriate cDNA were mixed and incubated for 20 min at room temperature prior to transfection (the total cDNA transfected were 750 ng, and the ratio of KCC2: Neto2 transfection was in 1:1 ratio). Twenty four hours after transfection, HEK-293 cells were plated in a black-walled 96-well culture dish (Greiner Bio-One, Frickenhausen, Germany) at a concentration of 100,000 cells/well. The HEK-293 cells were processed for flux measurements by replacing the medium with 80 μl of preincubation buffer (100 mM N-methyl-D-glucamine, 5 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, 5 mM HEPES, pH 7.4) with or without 2 μM FlouZin-2 AM dye (Invitrogen) plus 0.2% (w/v) Pluronic F-127 (Invitrogen). After incubation for 48 min at room temperature, cells were washed three times with 80 μl of preincubation buffer and incubated for 15 min with 80 μl of preincubation buffer plus 0.1 mM ouabain to block Na+/K+ ATPases. Thereafter, the culture dish was inserted into a fluorometer (Fluoroskan Accent, Thermo Scientific, Bremen, Germany), and the wells were injected with 40 μl of 5× thallium stimulation buffer (12 mM Tl2SO4, 100 mM N-methyl-D-glucamine, 5 mM HEPES, 2 mM CaSO4, 0.8 mM MgSO4, 5 mM glucose, pH 7.4). The fluorescence across the entire cell population in a single well was measured in a kinetic dependent manner (excitation 485 nm, emission 538 nm, 1 frame in 4 s in a 200-s time span). The activity was calculated with the initial values of the slope of Tl+-stimulated fluorescence increase by using linear regression. At least two independent DNA preparations were used per construct, giving similar results. At the end of each experiment, Tl+ flux was blocked in the presence of 2 mM furosemide, to demonstrate the specificity of the KCC2 transporter activity. Experiments in Figure 1B are representative results from four independent biological replicates.


Neto2- mice have impaired GABAergic inhibition and are susceptible to seizures.

Mahadevan V, Dargaei Z, Ivakine EA, Hartmann AM, Ng D, Chevrier J, Ormond J, Nothwang HG, McInnes RR, Woodin MA - Front Cell Neurosci (2015)

Hippocampal pyramidal neurons from adult Neto2- mice have depolarized EGABA. (Ai) Example of gramicidin perforated patch clamp recordings obtained from CA1 pyramidal neurons in acute hippocampal slices prepared from Neto2- mice (n = 15 neurons, gray) and wild-type mice (n = 21 neurons, black). The inhibitory postsynaptic potential (IPSP) amplitude was plotted against the holding potential of the postsynaptic membrane, and the intercept of this curve with the x-axis was taken as EGABA. Insets: traces of IPSPs for the examples shown. Scale bars: 5 mV, 10 ms. (Aii) Summary of all experiments similar to (Ai). (B) Tl+-uptake assay performed in HEK-293 cells (n = 4 sets), in the absence (white bars) and presence of 2 mM furosemide (black bars). ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585209&req=5

Figure 1: Hippocampal pyramidal neurons from adult Neto2- mice have depolarized EGABA. (Ai) Example of gramicidin perforated patch clamp recordings obtained from CA1 pyramidal neurons in acute hippocampal slices prepared from Neto2- mice (n = 15 neurons, gray) and wild-type mice (n = 21 neurons, black). The inhibitory postsynaptic potential (IPSP) amplitude was plotted against the holding potential of the postsynaptic membrane, and the intercept of this curve with the x-axis was taken as EGABA. Insets: traces of IPSPs for the examples shown. Scale bars: 5 mV, 10 ms. (Aii) Summary of all experiments similar to (Ai). (B) Tl+-uptake assay performed in HEK-293 cells (n = 4 sets), in the absence (white bars) and presence of 2 mM furosemide (black bars). ***p < 0.001.
Mentions: Transport activity was determined by measuring Cl−-dependent uptake of Tl+ in HEK-293 cells. Uptake measurements were done as described previously (Delpire et al., 2009; Hartmann et al., 2010; Weber et al., 2014). Cells were transiently transfected with empty plasmid or pcDNA3.1 KCC2 or pcDNA3.1 KCC2 alone + pcDNA3.1 Neto2 constructs. Briefly, 150 μl of Opti-MEM (Invitrogen), 6 μl of TurboFect (Fermentas, Karlsruhe, Germany), and appropriate cDNA were mixed and incubated for 20 min at room temperature prior to transfection (the total cDNA transfected were 750 ng, and the ratio of KCC2: Neto2 transfection was in 1:1 ratio). Twenty four hours after transfection, HEK-293 cells were plated in a black-walled 96-well culture dish (Greiner Bio-One, Frickenhausen, Germany) at a concentration of 100,000 cells/well. The HEK-293 cells were processed for flux measurements by replacing the medium with 80 μl of preincubation buffer (100 mM N-methyl-D-glucamine, 5 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, 5 mM HEPES, pH 7.4) with or without 2 μM FlouZin-2 AM dye (Invitrogen) plus 0.2% (w/v) Pluronic F-127 (Invitrogen). After incubation for 48 min at room temperature, cells were washed three times with 80 μl of preincubation buffer and incubated for 15 min with 80 μl of preincubation buffer plus 0.1 mM ouabain to block Na+/K+ ATPases. Thereafter, the culture dish was inserted into a fluorometer (Fluoroskan Accent, Thermo Scientific, Bremen, Germany), and the wells were injected with 40 μl of 5× thallium stimulation buffer (12 mM Tl2SO4, 100 mM N-methyl-D-glucamine, 5 mM HEPES, 2 mM CaSO4, 0.8 mM MgSO4, 5 mM glucose, pH 7.4). The fluorescence across the entire cell population in a single well was measured in a kinetic dependent manner (excitation 485 nm, emission 538 nm, 1 frame in 4 s in a 200-s time span). The activity was calculated with the initial values of the slope of Tl+-stimulated fluorescence increase by using linear regression. At least two independent DNA preparations were used per construct, giving similar results. At the end of each experiment, Tl+ flux was blocked in the presence of 2 mM furosemide, to demonstrate the specificity of the KCC2 transporter activity. Experiments in Figure 1B are representative results from four independent biological replicates.

Bottom Line: Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized.We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls.To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto Toronto, ON, Canada.

ABSTRACT
Neto2 is a transmembrane protein that interacts with the neuron-specific K(+)-Cl(-) cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl(-) gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2- mice would be prone to seizure activity. We found that Neto2- mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity.

No MeSH data available.


Related in: MedlinePlus