Limits...
Astrocytes increase the activity of synaptic GluN2B NMDA receptors.

Hahn J, Wang X, Margeta M - Front Cell Neurosci (2015)

Bottom Line: Using primary hippocampal cultures with mature synapses, we found that the density of NMDA-evoked whole-cell currents was approximately twice as large in neurons cultured in the presence of glia compared to neurons cultured alone.Instead, we found that the peak amplitudes of total and NMDAR miniature excitatory postsynaptic currents (mEPSCs), but not AMPAR mEPSCs, were significantly larger in mixed than neuronal cultures, resulting in a decreased synaptic AMPAR/NMDAR ratio.Given that physiologic activation of synaptic NMDARs is neuroprotective and that an increase in the synaptic GluN2B current is associated with improved learning and memory, the astrocyte-induced potentiation of synaptic GluN2B receptor activity is likely to enhance cognitive function while simultaneously strengthening neuroprotective signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Francisco San Francisco, CA, USA.

ABSTRACT
Astrocytes regulate excitatory synapse formation and surface expression of glutamate AMPA receptors (AMPARs) during development. Less is known about glial modulation of glutamate NMDA receptors (NMDARs), which mediate synaptic plasticity and regulate neuronal survival in a subunit- and subcellular localization-dependent manner. Using primary hippocampal cultures with mature synapses, we found that the density of NMDA-evoked whole-cell currents was approximately twice as large in neurons cultured in the presence of glia compared to neurons cultured alone. The glial effect was mediated by (an) astrocyte-secreted soluble factor(s), was Mg(2+) and voltage independent, and could not be explained by a significant change in the synaptic density. Instead, we found that the peak amplitudes of total and NMDAR miniature excitatory postsynaptic currents (mEPSCs), but not AMPAR mEPSCs, were significantly larger in mixed than neuronal cultures, resulting in a decreased synaptic AMPAR/NMDAR ratio. Astrocytic modulation was restricted to synaptic NMDARs that contain the GluN2B subunit, did not involve an increase in the cell surface expression of NMDAR subunits, and was mediated by protein kinase C (PKC). Taken together, our findings indicate that astrocyte-secreted soluble factor(s) can fine-tune synaptic NMDAR activity through the PKC-mediated regulation of GluN2B NMDAR channels already localized at postsynaptic sites, presumably on a rapid time scale. Given that physiologic activation of synaptic NMDARs is neuroprotective and that an increase in the synaptic GluN2B current is associated with improved learning and memory, the astrocyte-induced potentiation of synaptic GluN2B receptor activity is likely to enhance cognitive function while simultaneously strengthening neuroprotective signaling pathways.

No MeSH data available.


The frequency and amplitude of directly recorded NMDAR mEPSCs are increased in the presence of glia. (A) Representative NMDAR mEPSC traces recorded at −60 mV from neuronal and mixed cultures in the presence of TTX, NBQX, bicuculline and strychnine; bottom traces show recordings on an expanded time scale. (B–E) Cumulative probability, mean frequency, and mean amplitude plots of NMDAR mEPSCs are shown; both frequency and amplitude of NMDAR mEPSCs were significantly larger in mixed cultures (*p < 0.05, t-test). Of note, the peak NMDAR mEPSC amplitudes obtained from this analysis were larger than the peak NMDAR mEPSC amplitudes obtained by subtracting the average AMPAR mEPSC traces from the average total mEPSC traces (Figure 4B); the most likely explanation for this discrepancy is our inability to resolve small and medium-sized (<7 pA) NMDAR mEPSC events from the background noise in direct recordings, biasing the overall analysis to the largest mEPSCs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400914&req=5

Figure 5: The frequency and amplitude of directly recorded NMDAR mEPSCs are increased in the presence of glia. (A) Representative NMDAR mEPSC traces recorded at −60 mV from neuronal and mixed cultures in the presence of TTX, NBQX, bicuculline and strychnine; bottom traces show recordings on an expanded time scale. (B–E) Cumulative probability, mean frequency, and mean amplitude plots of NMDAR mEPSCs are shown; both frequency and amplitude of NMDAR mEPSCs were significantly larger in mixed cultures (*p < 0.05, t-test). Of note, the peak NMDAR mEPSC amplitudes obtained from this analysis were larger than the peak NMDAR mEPSC amplitudes obtained by subtracting the average AMPAR mEPSC traces from the average total mEPSC traces (Figure 4B); the most likely explanation for this discrepancy is our inability to resolve small and medium-sized (<7 pA) NMDAR mEPSC events from the background noise in direct recordings, biasing the overall analysis to the largest mEPSCs.

Mentions: Cells were lysed on ice with modified RIPA buffer (0.5% SOD, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 50 mM TrisCl, 1 mM EDTA) supplemented with a protease inhibitor cocktail (Roche). Crude lysates were cleared by centrifugation at 6000 rpm for 10 min at 4°C, solubilized with LDS sample buffer (Invitrogen) supplemented with TCEP reducing reagent (Pierce; final concentration 12.5 mM), and heated for 10 min at 70°C prior to loading; given different cellular compositions of the cultures, samples were loaded on an equal volume basis. Samples were electrophoretically resolved with 4–12% Bis-Tris NuPAGE gels (Invitrogen) and electroblotted to nitrocellulose membranes. Membranes were blocked for 1 h at RT in TBS (150 mM NaCl, 20 mM TrisCl; pH = 7.4) containing 3% nonfat dried milk, incubated with primary antibodies for 2–3 h at RT or overnight at 4°C, washed 4 times at RT, incubated with the corresponding secondary antibody for 1 h at RT, and washed for at least 30 min at RT. Following a final wash in TBS with 0.1% Tween for 10 min at RT, protein-antibody complexes were detected using an ECL chemiluminescent kit (Pierce Biotechnology) and CL-XPosure Film (Thermo Scientific) with a Konica SRX-101A film developer. In a subset of experiments (Figure 10), the Odyssey Fc infrared imaging system (LI-COR Biosciences) was used for detection of the protein-antibody complexes. In these experiments, electrophoresis was performed using 3–8% Tris-Acetate NuPAGE gels (Invitrogen). The membranes were dried at RT for at least 1 h and then blocked with Odyssey Blocking Buffer (LI-COR Biosciences) for 1 h at RT. Odyssey Blocking Buffer supplemented with 0.1% Tween 20 was used for dilution of primary and secondary antibodies, while PBS supplemented with 0.1% Tween 20 was used for washing. Membranes were incubated with primary antibodies for 2 h at RT or overnight at 4°C, washed 4 times (5 min/wash) at RT, incubated with secondary antibodies for 1 h at RT, washed 4 times (5 min/wash) at RT, and then imaged using Odyssey Fc (LI-COR Biosciences) at 700 nm and 800 nm; images were analyzed using Image Studio Imaging Software (LI-COR Biosciences). Primary antibodies included: mouse monoclonal anti-NR1 (BD Biosciences, 1:1000), rabbit monoclonal anti-GluN2A (Millipore, 1:1000), mouse monoclonal anti-GluN2B (BD Biosciences, 1:100 [film] or 1:500 [Odyssey Fc]), rabbit polyclonal anti-phospho-GluN2B (Ser1303) (Millipore, 1:1000), mouse monoclonal anti-GluR1-NT (Millipore, 1:500), mouse monoclonal anti-PSD-95 (NeuroMab, 1:1000), rabbit polyclonal anti-synaptophysin (Santa Cruz Biotechnology, 1:1000) and mouse monoclonal anti-actin (Sigma, 1:5000). Secondary antibodies included goat horseradish peroxidase (HRP)-conjugated anti-rabbit secondary H + L IgG antibody and goat HRP-conjugated anti-mouse secondary H + L IgG antibody (Jackson ImmunoResearch, 1:5000 for both) or goat IRDye® 800CW-conjugated anti-rabbit secondary H + L IgG antibody and goat IRDye® 680RD-conjugated anti-mouse secondary H + L IgG antibody (LI-COR Biosciences, 1:15000 for both).


Astrocytes increase the activity of synaptic GluN2B NMDA receptors.

Hahn J, Wang X, Margeta M - Front Cell Neurosci (2015)

The frequency and amplitude of directly recorded NMDAR mEPSCs are increased in the presence of glia. (A) Representative NMDAR mEPSC traces recorded at −60 mV from neuronal and mixed cultures in the presence of TTX, NBQX, bicuculline and strychnine; bottom traces show recordings on an expanded time scale. (B–E) Cumulative probability, mean frequency, and mean amplitude plots of NMDAR mEPSCs are shown; both frequency and amplitude of NMDAR mEPSCs were significantly larger in mixed cultures (*p < 0.05, t-test). Of note, the peak NMDAR mEPSC amplitudes obtained from this analysis were larger than the peak NMDAR mEPSC amplitudes obtained by subtracting the average AMPAR mEPSC traces from the average total mEPSC traces (Figure 4B); the most likely explanation for this discrepancy is our inability to resolve small and medium-sized (<7 pA) NMDAR mEPSC events from the background noise in direct recordings, biasing the overall analysis to the largest mEPSCs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400914&req=5

Figure 5: The frequency and amplitude of directly recorded NMDAR mEPSCs are increased in the presence of glia. (A) Representative NMDAR mEPSC traces recorded at −60 mV from neuronal and mixed cultures in the presence of TTX, NBQX, bicuculline and strychnine; bottom traces show recordings on an expanded time scale. (B–E) Cumulative probability, mean frequency, and mean amplitude plots of NMDAR mEPSCs are shown; both frequency and amplitude of NMDAR mEPSCs were significantly larger in mixed cultures (*p < 0.05, t-test). Of note, the peak NMDAR mEPSC amplitudes obtained from this analysis were larger than the peak NMDAR mEPSC amplitudes obtained by subtracting the average AMPAR mEPSC traces from the average total mEPSC traces (Figure 4B); the most likely explanation for this discrepancy is our inability to resolve small and medium-sized (<7 pA) NMDAR mEPSC events from the background noise in direct recordings, biasing the overall analysis to the largest mEPSCs.
Mentions: Cells were lysed on ice with modified RIPA buffer (0.5% SOD, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 50 mM TrisCl, 1 mM EDTA) supplemented with a protease inhibitor cocktail (Roche). Crude lysates were cleared by centrifugation at 6000 rpm for 10 min at 4°C, solubilized with LDS sample buffer (Invitrogen) supplemented with TCEP reducing reagent (Pierce; final concentration 12.5 mM), and heated for 10 min at 70°C prior to loading; given different cellular compositions of the cultures, samples were loaded on an equal volume basis. Samples were electrophoretically resolved with 4–12% Bis-Tris NuPAGE gels (Invitrogen) and electroblotted to nitrocellulose membranes. Membranes were blocked for 1 h at RT in TBS (150 mM NaCl, 20 mM TrisCl; pH = 7.4) containing 3% nonfat dried milk, incubated with primary antibodies for 2–3 h at RT or overnight at 4°C, washed 4 times at RT, incubated with the corresponding secondary antibody for 1 h at RT, and washed for at least 30 min at RT. Following a final wash in TBS with 0.1% Tween for 10 min at RT, protein-antibody complexes were detected using an ECL chemiluminescent kit (Pierce Biotechnology) and CL-XPosure Film (Thermo Scientific) with a Konica SRX-101A film developer. In a subset of experiments (Figure 10), the Odyssey Fc infrared imaging system (LI-COR Biosciences) was used for detection of the protein-antibody complexes. In these experiments, electrophoresis was performed using 3–8% Tris-Acetate NuPAGE gels (Invitrogen). The membranes were dried at RT for at least 1 h and then blocked with Odyssey Blocking Buffer (LI-COR Biosciences) for 1 h at RT. Odyssey Blocking Buffer supplemented with 0.1% Tween 20 was used for dilution of primary and secondary antibodies, while PBS supplemented with 0.1% Tween 20 was used for washing. Membranes were incubated with primary antibodies for 2 h at RT or overnight at 4°C, washed 4 times (5 min/wash) at RT, incubated with secondary antibodies for 1 h at RT, washed 4 times (5 min/wash) at RT, and then imaged using Odyssey Fc (LI-COR Biosciences) at 700 nm and 800 nm; images were analyzed using Image Studio Imaging Software (LI-COR Biosciences). Primary antibodies included: mouse monoclonal anti-NR1 (BD Biosciences, 1:1000), rabbit monoclonal anti-GluN2A (Millipore, 1:1000), mouse monoclonal anti-GluN2B (BD Biosciences, 1:100 [film] or 1:500 [Odyssey Fc]), rabbit polyclonal anti-phospho-GluN2B (Ser1303) (Millipore, 1:1000), mouse monoclonal anti-GluR1-NT (Millipore, 1:500), mouse monoclonal anti-PSD-95 (NeuroMab, 1:1000), rabbit polyclonal anti-synaptophysin (Santa Cruz Biotechnology, 1:1000) and mouse monoclonal anti-actin (Sigma, 1:5000). Secondary antibodies included goat horseradish peroxidase (HRP)-conjugated anti-rabbit secondary H + L IgG antibody and goat HRP-conjugated anti-mouse secondary H + L IgG antibody (Jackson ImmunoResearch, 1:5000 for both) or goat IRDye® 800CW-conjugated anti-rabbit secondary H + L IgG antibody and goat IRDye® 680RD-conjugated anti-mouse secondary H + L IgG antibody (LI-COR Biosciences, 1:15000 for both).

Bottom Line: Using primary hippocampal cultures with mature synapses, we found that the density of NMDA-evoked whole-cell currents was approximately twice as large in neurons cultured in the presence of glia compared to neurons cultured alone.Instead, we found that the peak amplitudes of total and NMDAR miniature excitatory postsynaptic currents (mEPSCs), but not AMPAR mEPSCs, were significantly larger in mixed than neuronal cultures, resulting in a decreased synaptic AMPAR/NMDAR ratio.Given that physiologic activation of synaptic NMDARs is neuroprotective and that an increase in the synaptic GluN2B current is associated with improved learning and memory, the astrocyte-induced potentiation of synaptic GluN2B receptor activity is likely to enhance cognitive function while simultaneously strengthening neuroprotective signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Francisco San Francisco, CA, USA.

ABSTRACT
Astrocytes regulate excitatory synapse formation and surface expression of glutamate AMPA receptors (AMPARs) during development. Less is known about glial modulation of glutamate NMDA receptors (NMDARs), which mediate synaptic plasticity and regulate neuronal survival in a subunit- and subcellular localization-dependent manner. Using primary hippocampal cultures with mature synapses, we found that the density of NMDA-evoked whole-cell currents was approximately twice as large in neurons cultured in the presence of glia compared to neurons cultured alone. The glial effect was mediated by (an) astrocyte-secreted soluble factor(s), was Mg(2+) and voltage independent, and could not be explained by a significant change in the synaptic density. Instead, we found that the peak amplitudes of total and NMDAR miniature excitatory postsynaptic currents (mEPSCs), but not AMPAR mEPSCs, were significantly larger in mixed than neuronal cultures, resulting in a decreased synaptic AMPAR/NMDAR ratio. Astrocytic modulation was restricted to synaptic NMDARs that contain the GluN2B subunit, did not involve an increase in the cell surface expression of NMDAR subunits, and was mediated by protein kinase C (PKC). Taken together, our findings indicate that astrocyte-secreted soluble factor(s) can fine-tune synaptic NMDAR activity through the PKC-mediated regulation of GluN2B NMDAR channels already localized at postsynaptic sites, presumably on a rapid time scale. Given that physiologic activation of synaptic NMDARs is neuroprotective and that an increase in the synaptic GluN2B current is associated with improved learning and memory, the astrocyte-induced potentiation of synaptic GluN2B receptor activity is likely to enhance cognitive function while simultaneously strengthening neuroprotective signaling pathways.

No MeSH data available.