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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 neurons from Neto2- mice have decreased surface KCC2 levels. (A) Representative immunoblots of KCC2 levels from the surface and total fractions, from wild-type and Neto2- neurons. The first two lanes in (A) correspond to biotinylated surface proteins (50 μg) recovered from the neutravidin beads. The last two lanes correspond to total proteins (5 μg). (B) Summary figures showing levels of surface and total KCC2 levels, normalized to β III Tuj1 in Neto2  neurons, relative to that of wild type (n = 3). Summary figures represent mean ± sem. *p < 0.05, **p < 0.01.
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Figure 2: Hippocampal neurons from Neto2- mice have decreased surface KCC2 levels. (A) Representative immunoblots of KCC2 levels from the surface and total fractions, from wild-type and Neto2- neurons. The first two lanes in (A) correspond to biotinylated surface proteins (50 μg) recovered from the neutravidin beads. The last two lanes correspond to total proteins (5 μg). (B) Summary figures showing levels of surface and total KCC2 levels, normalized to β III Tuj1 in Neto2 neurons, relative to that of wild type (n = 3). Summary figures represent mean ± sem. *p < 0.05, **p < 0.01.

Mentions: Biotinylation studies were performed as previously described (Mahadevan et al., 2014) with modifications. Briefly, DIC10–11 hippocampal neurons from wild type and Neto2- mice were washed three times with ice cold 1 × PBS then incubated for 30 min on ice in 1 × PBS containing 1 mg/ml biotin (Pierce Protein Research Products, Thermo Scientific). Cultures were then incubated in 10 mM Tris/HCl [pH 7.4], washed three times with 1 × PBS, homogenized with 0.5 mL of RIPA buffer [50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 0.5% DOC, protease inhibitors and phosphatase inhibitor mixture (Roche)], and incubated on ice for 30 min. The homogenate was centrifuged, supernatant was collected and quantified using the BioRad DC protein quantification kit. 50 μg of total protein in a total volume of 400 μL was mixed with 100 μL of a 50% slurry of Neutravidin beads (Pierce Protein Research Products, Thermo Scientific) and rotated for 1 h at 4C. The beads (first bound fraction) were harvested by centrifugation and washed three times in RIPA buffer. After the last wash all buffer was thoroughly removed from beads, and the biotin-bound and total input fractions were denatured in 6 × SDS sample buffer containing DTT at 37°C for 1 h before resolving them on onto 6% SDS-PAGE. Subsequent immunoblot analysis was performed as described elsewhere (Mahadevan et al., 2014). Experiments in Figure 2 are representative results from three 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 neurons from Neto2- mice have decreased surface KCC2 levels. (A) Representative immunoblots of KCC2 levels from the surface and total fractions, from wild-type and Neto2- neurons. The first two lanes in (A) correspond to biotinylated surface proteins (50 μg) recovered from the neutravidin beads. The last two lanes correspond to total proteins (5 μg). (B) Summary figures showing levels of surface and total KCC2 levels, normalized to β III Tuj1 in Neto2  neurons, relative to that of wild type (n = 3). Summary figures represent mean ± sem. *p < 0.05, **p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Hippocampal neurons from Neto2- mice have decreased surface KCC2 levels. (A) Representative immunoblots of KCC2 levels from the surface and total fractions, from wild-type and Neto2- neurons. The first two lanes in (A) correspond to biotinylated surface proteins (50 μg) recovered from the neutravidin beads. The last two lanes correspond to total proteins (5 μg). (B) Summary figures showing levels of surface and total KCC2 levels, normalized to β III Tuj1 in Neto2 neurons, relative to that of wild type (n = 3). Summary figures represent mean ± sem. *p < 0.05, **p < 0.01.
Mentions: Biotinylation studies were performed as previously described (Mahadevan et al., 2014) with modifications. Briefly, DIC10–11 hippocampal neurons from wild type and Neto2- mice were washed three times with ice cold 1 × PBS then incubated for 30 min on ice in 1 × PBS containing 1 mg/ml biotin (Pierce Protein Research Products, Thermo Scientific). Cultures were then incubated in 10 mM Tris/HCl [pH 7.4], washed three times with 1 × PBS, homogenized with 0.5 mL of RIPA buffer [50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 0.5% DOC, protease inhibitors and phosphatase inhibitor mixture (Roche)], and incubated on ice for 30 min. The homogenate was centrifuged, supernatant was collected and quantified using the BioRad DC protein quantification kit. 50 μg of total protein in a total volume of 400 μL was mixed with 100 μL of a 50% slurry of Neutravidin beads (Pierce Protein Research Products, Thermo Scientific) and rotated for 1 h at 4C. The beads (first bound fraction) were harvested by centrifugation and washed three times in RIPA buffer. After the last wash all buffer was thoroughly removed from beads, and the biotin-bound and total input fractions were denatured in 6 × SDS sample buffer containing DTT at 37°C for 1 h before resolving them on onto 6% SDS-PAGE. Subsequent immunoblot analysis was performed as described elsewhere (Mahadevan et al., 2014). Experiments in Figure 2 are representative results from three 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