Acidosis-Induced Dysfunction of Cortical GABAergic Neurons through Astrocyte-Related Excitotoxicity

Abstract

Background: Acidosis impairs cognitions and behaviors presumably by acidification-induced changes in neuronal metabolism. Cortical GABAergic neurons are vulnerable to pathological factors and their injury leads to brain dysfunction. How acidosis induces GABAergic neuron injury remains elusive. As the glia cells and neurons interact each other, we intend to examine the role of the astrocytes in acidosis-induced GABAergic neuron injury.

Results: Experiments were done at GABAergic cells and astrocytes in mouse cortical slices. To identify astrocytic involvement in acidosis-induced impairment, we induced the acidification in single GABAergic neuron by infusing proton intracellularly or in both neurons and astrocytes by using proton extracellularly. Compared the effects of intracellular acidification and extracellular acidification on GABAergic neurons, we found that their active intrinsic properties and synaptic outputs appeared more severely impaired in extracellular acidosis than intracellular acidosis. Meanwhile, extracellular acidosis deteriorated glutamate transporter currents on the astrocytes and upregulated excitatory synaptic transmission on the GABAergic neurons. Moreover, the antagonists of glutamate NMDA-/AMPA-receptors partially reverse extracellular acidosis-induced injury in the GABAergic neurons.

Conclusion: Our studies suggest that acidosis leads to the dysfunction of cortical GABAergic neurons by astrocyte-mediated excitotoxicity, in addition to their metabolic changes as indicated previously.

Fig 1. Extracellular and intracellular acidosis impairs the production of action potentials at the cortical GABAergic neurons.

Sequential spikes of GABAergic neurons in cortical slices were evoked by injecting depolarization pulse (200 ms) and recorded by using whole-cell current-clamp. Extracellular acidosis was made by perfusing the cortical slices with the acidic ACSF (pH 6.75) after the control ACSF (pH 7.35). Intracellular acidosis was made by using the recording pipettes whose tips were filled with control pipette solution (pH 7.35) and back-filled with acidification pipette solution (pH 6.75). A) shows the evoked spikes under the control (red trace) and subsequent intracellular acidification (blue trace), respectively. B) shows the values of spike frequencies under the conditions of control (pH 7.35, red bar) and intracellular acidification (pH 6.75; blue bar). Two asterisks show p<0.01 (n = 15, paired t-test). C) shows the spikes under the control (red trace) and extracellular acidification (dark blue), respectively. D) shows the values of spike frequencies under the conditions of control (pH 7.35, red bar) and extracellular acidification (pH 6.75; dark-blue). Two asterisks show p<0.01 (n = 15, paired t-test). Dash-lines in A) & C) show the levels of threshold potentials.

Fig 2. Extracellular and intracellular acidosis prolongs the refractory periods of action potentials at the cortical GABAergic neurons.

Refractory periods were measured by injecting paired-depolarization pulses and recorded under whole-cell current-clamp. A) shows the measurements of refractory periods under the control (red trace) and subsequent intracellular acidification (blue), respectively. B) shows the averaged values of spike refractory periods under the conditions of control (pH 7.35, red bar) and intracellular acidification (pH 6.75; blue). Two asterisks show p<0.01 (n = 15, paired t-test). C) shows the measurement refractory periods under the control (red trace) and extracellular acidification (dark blue), respectively. D) shows the values of refractory periods under the conditions of control (pH 7.35, red bar) and extracellular acidification (pH 6.75; dark-blue). Two asterisks show p<0.01 (n = 15, paired t-test).

Fig 3. Extracellular acidosis impairs GABAergic synaptic transmission at cortical pyramidal neurons dominantly.

Spontaneous IPSCs (sIPSC) were recorded by whole-cell voltage-clamp without stimulating presynaptic axons under the conditions of control and then extracellular acidification versus of control and intracellular acidification. A) shows the recorded sIPSCs under the control (top trace), intracellular acidification (middle trace) and extracellular acidification (bottom trace). B) illustrates the differences of sIPSC amplitudes between control and intracellular acidosis (∆IPSC amplitudes, red bar) as well as the ∆IPSC amplitudes between control and extracellular acidosis (blue bar; two asterisks, p<0.01, n = 15; one-way ANOVA). C) illustrates the differences of inter-sIPSC intervals between control and intracellular acidosis (∆inter-IPSC interval, red bar) as well as ∆inter-IPSC intervals between control and extracellular acidosis (blue bar; two asterisks, p<0.01, n = 15; one-way ANOVA).

Fig 4. Extracellular acidosis impairs the active intrinsic properties of the cortical GABAergic neurons dominantly.

A) shows the differences of spike frequencies between control and intracellular acidosis (red bar) as well as between control and extracellular acidosis (blue bar; asterisk, p<0.05; one-way ANOVA). B) shows the differences of spike refractory periods between control and intracellular acidosis (red bar) as well as between control and extracellular acidosis (blue bar; one asterisk, p<0.05; one-way ANOVA). C) shows the differences of spike threshold potentials between control and intracellular acidosis (red bar) as well as between control and extracellular acidosis (blue bar; one asterisk, p<0.05; one-way ANOVA).

Fig 5. Extracellular acidosis impairs the glutamate transporter current (GTC) on the cortical astrocytes.

The GTCs were recorded on cortical astrocytes and evoked by stimulating presynaptic axons. A) shows the superimposed waveforms of GTCs before (red trace) and after extracellular acidification (blue trace). B) shows the averaged values of GTCs before (red bar) and after extracellular acidification (blue bar; two asterisks, p<0.01, n = 16; paired t-test).

Fig 6. Extracellular acidosis upregulates glutamatergic synaptic transmission at cortical GABAergic neurons dominantly.

Spontaneous EPSCs were recorded on GABAergic neurons by whole-cell voltage-clamp without stimulating presynaptic axons. A) shows the recorded sEPSCs under the control (top trace), intracellular acidification (middle trace) and extracellular acidification (bottom trace). B) illustrates the differences of sEPSC amplitudes between intracellular acidosis and control (∆EPSC amplitudes, red bar) as well as the differences between extracellular acidosis and control (∆EPSC amplitudes, blue bar; p<0.01, n = 15; one-way ANOVA). C) shows the differences of inter-sEPSC interval between intracellular acidosis and control (∆inter-IPSC interval, red bar) as well as the differences between extracellular acidosis and control (blue bar; p<0.01, n = 15; one-way ANOVA).

Fig 7. The inhibition of glutamate receptors partially reverses the impairment of GABAergic synaptic outputs to cortical pyramidal neurons induced by extracellular acidosis.

sIPSCs were recorded by whole-cell voltage-clamp under the conditions of sequential manipulations, i.e., control, extracellular acidosis and extracellular acidosis plus 10 μM CNQX and 40 μM D-AP5. A) shows the recorded sIPSCs under the control (top trace), extracellular acidification (middle trace) and extracellular acidification plus glutamate receptor blockers (bottom trace). B) shows the averaged values of sIPSC amplitudes under the conditions of control (red bar), extracellular acidosis (blue bar) and extracellular acidosis plus glutamate receptor blockers (green bar; two asterisks, p<0.01; ##, p<0.01; n = 15; one-way ANOVA). C) shows the averaged values of inter-sIPSC intervals under the conditions of control (red bar), extracellular acidosis (blue bar) and extracellular acidosis plus glutamate receptor blockers (green bar; two asterisks, p<0.01; ##, p<0.01; n = 15; one-way ANOVA).

Fig 8. The inhibition of glutamate receptors partially reverses the impairment of intrinsic properties at cortical GABAergic neurons induced by extracellular acidosis.

Sequential spikes, threshold potentials and refractory period at GABAergic neurons were recorded by whole-cell voltage-clamp under the conditions of sequential manipulations, i.e., control, extracellular acidosis and extracellular acidosis plus 40 μM D-AP5 and 10 μM CNQX. A) shows the averaged values of spike frequency under the conditions of control (red bar), extracellular acidosis (blue bar) and extracellular acidosis plus glutamate receptor blockers (green bar). B) shows the averaged values of spike refractory periods under the conditions of control (red bar), extracellular acidosis (blue bar) and extracellular acidosis plus glutamate receptor blockers (green bar). C) shows the averaged values of spike threshold potentials under the conditions of control (red bar), extracellular acidosis (blue bar) and extracellular acidosis plus glutamate receptor blockers (green bar). Two asterisks present p<0.01, such as blue and green bars versus red bar. An asterisk presents p<0.05, such as green bar versus red bar. # presents p<0.05, such as green bar versus blue bar (n = 15; one-way ANOVA).

Fig 9. The dysfunction of glutamate transporter in the astrocyte leads to the impairment of GABAergic neuron during acidosis.

Extracellular acidification impairs the function of astrocytic glutamate transporter (Glu-T), and the subsequent glutamate accumulation deteriorates GABAergic neurons through activating ionotropic glutamate receptors, such as NMDAR and AMPAR.