Anoxia-induced hippocampal LTP is regeneratively produced by glutamate and nitric oxide from the neuro-glial-endothelial axis

Abstract

Transient anoxia causes amnesia and neuronal death. This is attributed to enhanced glutamate release and modeled as anoxia-induced long-term potentiation (aLTP). aLTP is mediated by glutamate receptors and nitric oxide (·NO) and occludes stimulation-induced LTP. We identified a signaling cascade downstream of ·NO leading to glutamate release and a glutamate-·NO loop regeneratively boosting aLTP. aLTP in entothelial ·NO synthase (eNOS)-knockout mice and blocking neuronal NOS (nNOS) activity suggested that both nNOS and eNOS contribute to aLTP. Immunostaining result showed that eNOS is predominantly expressed in vascular endothelia. Transient anoxia induced a long-lasting Ca²⁺ elevation in astrocytes that mirrored aLTP. Blocking astrocyte metabolism or depletion of the NMDA receptor ligand D-serine abolished eNOS-dependent aLTP, suggesting that astrocytic Ca²⁺ elevation stimulates D-serine release from endfeet to endothelia, thereby releasing ·NO synthesized by eNOS. Thus, the neuro-glial-endothelial axis is involved in long-term enhancement of glutamate release after transient anoxia.

Keywords: Biophysics; Molecular biology.

Graphical abstract

Figure 1

aLTP induced by transient anoxia is caused by increased transmitter release probability (A) Schematic illustration of hippocampal slice with recording and stimulation electrodes in CA1 region separated from CA3 by an incision. (B) Field EPSPs recorded from the CA1 stratum radiatum (SR) in response to Schaffer collateral (SC) stimulations with a pair of pulses (50 ms interval) repeated every 20 s (sample records). Time plots indicate the slope of fEPSPs and bar graphs show mean fEPSC and PPRs (with SEMs, n = 7 slices) at different epochs: (i) before and (ii) at the end of anoxic period (O₂ replaced by N₂ in aCSF for 10 min, red column), and (iii) 160 min after anoxia. A transient decrease of fEPSP slope during anoxia (18.7 ± 4%) was followed by a long-lasting increase (147.3 ± 12%, recorded up to 180 min after anoxia; n = 7 slices, p < 0.001 in one-way repeated-measures ANOVA). Scale bars: 0.2 mV. 20 ms. (C) EPSCs in whole-cell recording from individual CA1 pyramidal neurons. After anoxia, EPSC amplitude increased to 147.6 ± 9% (iii, n = 8 cells) accompanied by a decrease in PPR (bar graphs). Asterisks indicate ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 in paired-sample t test. Scale bars; 0.4 nA, 20 ms. (D) Variance-mean analysis of EPSCs before (sample EPSC traces in black) and 30 min after (red) the onset of anoxia at different Ca²⁺/Mg²⁺ concentrations (EPSC amplitudes shown in the upper panel). Variance-mean data plots were fitted to parabola. Bar graphs indicate quantal parameters before and after aLTP expression (from top to bottom; release probability, number of release sites and quantal size, respectively, n = 6 cells, ∗p < 0.05, N.S, not significant difference in paired-sample t test). aCSF contained the low-affinity AMPA receptor antagonist γ-D-glutamylglycine (γ-DGG, 1 mM) throughout to minimize saturation of AMPA receptors. Scale bars; 0.2 mV, 20 ms.

Figure 2

Induction of aLTP is mediated by presynaptic ·NO signaling cascade Block of aLTP induction by (A) APV (100 μM, n = 7 slices), (B) PTIO (100 μM, n = 7), (C) KT-5823 (10 μM, n = 5), (D) Y-27632 (10 μM, n = 7), and (E) PAO (1 μM, n = 7). Sample records of fEPSPs are shown above each time plot of fEPSP slope. Bar graphs indicate normalized fEPSP slopes before (i) and after (ii) anoxic insult. (F) Rho activator II (1 μg/mL) potentiated fEPSP slope (ii, 139 ± 7%) and occluded aLTP (145.2 ± 8%, n = 5, one-way repeated-measures ANOVA: F_{(2, 12)} = 23.4). Bar graphs indicate PPR (ii, 80.3 ± 5%, iv, 82 ± 6%, n = 5, one-way repeated-measures ANOVA: F_{(2, 12)} = 20.16). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, N.S, no significance. Scale bars; 0.2 mV, 20ms. (G) ·NO released from the hippocampal CA1 area after 3 min anoxia (OGD) amperometrically monitored using a carbon fiber ·NO sensor probe. An averaged trace from 5 measurements is shown after subtracting the baseline. (H)·NO signaling cascade in presynaptic terminals causing enhancement of glutamate release probability.

Figure 3

Reversal of established aLTP by aLTP induction blockers Established aLTP was canceled by (A) APV (100 μM, n = 6), (B) PTIO (100 μM, n = 7), (C) KT-5823 (10 μM, n = 5), (D) Y-27632 (10 μM, n = 7), or (E) PAO (1 μM, n = 6). Bar graphs indicate normalized fEPSC slopes and PPRs before (i) and after (iii) aLTP induction and after application of aLTP induction blocker. Scale bars; 0.2 mV, 20 ms. (F) A positive feedback loop causing regenerative expression of aLTP. Glutamate released by transient anoxia induces ·NO production via activation of NMDA receptors. ·NO then activates presynaptic ·NO signaling cascade to enhance glutamate release. This positive loop can be disrupted by APV, PTIO, KT-5823, Y-27632 or PAO, all of which are aLTP-induction blockers.

Figure 4

Occlusion of sLTP by aLTP rescued by aLTP induction blockers (A) TBS-induced LTP of fEPSPs (n = 6) in hippocampal CA1 synapses (stimulation protocol shown in inset). PPR was unchanged during sLTP (lower panel). (B) PTIO (100 μM) had no effect on the TBS-induced LTP (n = 5). (C) aLTP (151 ± 9%) occluded TBS-induced LTP (n = 6), with no further potentiation induced by TBS (153 ± 9%, one-way repeated-measures ANOVA). (D) Block of established aLTP by PTIO rescued TBS-induced LTP (137 ± 8%, n = 6). Bar graphs indicate percentages of fEPSC slope at different epochs relative to controls (i). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, N.S, no significance. Scale bars; 0.2 mV, 20 ms.

Figure 5

·NO released after transient anoxia derived from nNOS and eNOS (A) aLTP of fEPSPs induced in hippocampal CA1 synapses from WT mice (147 ± 12%, black, n = 7 slices, duplicated from Figure 1B) and from eNOS-KO mice (125.4 ± 4%, purple, n = 6 slices, superimposed). Bar graphs show the fEPSP slope before (i, black) and 60 min after anoxic insult (iii). Scale bars; 0.4 mV, 20 ms. (B) The nNOS inhibitor NPA (1 μM) blocked aLTP induced in eNOS-KO mice (n = 5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, N.S, no significance. Scale bars; 0.4 mV, 20 ms. (C) Whole-cell washout of intracellular L-arginine in CA1 pyramidal neurons in slices from WT mice attenuated aLTP magnitude to 124 ± 3% (red, n = 8 cells) from control (147 ± 9% black, n = 9). Bath-application of fluoroacetate (FA, 10 mM) abolished the aLTP remaining after L-arginine washout (blue, n = 7). Bar graphs show normalized EPSC amplitude 35 min after anoxia onset (ii) relative to controls before anoxia (i). Scale bars; 0.2nA, 20 ms. (D) Whole-cell washout of intracellular L-arginine in CA1 pyramidal neurons in slices from eNOS-KO mice abolished aLTP (controls, purple, n = 7; L-Arg-free, red, n = 7). Scale bars; 0.2 nA, 20 ms. (E) Whole-cell loading of BAPTA (10 mM) in CA1 pyramidal neurons in slices from WT mice attenuated aLTP magnitude to 119 ± 5% (BAPTA, green, n = 7) compared to controls (147 ± 9% black, n = 9). Bath-application of FA (10 mM) abolished the aLTP remaining after BAPTA loading (orange, n = 8). Bar graphs show normalized EPSP amplitude 35 min after anoxic insult (ii) relative to controls before anoxia (i). Scale bar; 0.2 nA, 20 ms.

Figure 6

Absence of eNOS expression in astrocytes and astrocytic Ca²⁺ transients mirroring aLTP (A) Immunofluorescent staining of GFAP and eNOS in hippocampal tissue sections. In WT mice (upper panels), eNOS (green) is strongly expressed in blood vessels (BVs) in stratum lacunosum-moleculare (SLM) of CA1, with no overlap with the astrocyte marker GFAP (red), whereas in eNOS-KO mice (lower panels), BVs showed no significant signal besides non-specific background. Rightmost panels show higher magnification views of the boxed regions in merged pictures. GFAP-positive astrocytic endfeet surrounded distinct eNOS signals in vascular endothelia. In eNOS-KO mice, in the absence of eNOS signal, GFAP signals were seen around BVs. The graph in the right panel shows densitometric quantification of fluorescent signals in WT and eNOS-KO mice. Individual fluorescence signal intensities were normalized to that in WT, except for the astrocyte signal, which was normalized to the intensity of WT BVs. The eNOS fluorescence intensity in BVs in WT and eNOS-KO sections was significantly different (p < 0.0001, unpaired t-test), whereas the signal intensity in neurons and astrocytes was not different. (B) Two-photon Ca²⁺ imaging of GCaMP6f-expressing astrocytes (outlined in upper panels) from different periods (i-v) before (i), during (ii) and after (iii-v) 10-min anoxia (from time 0). Lower panels show 5 min samples of astrocytic Ca²⁺ signal intensity (DF/F) at different epochs. After astrocytic Ca²⁺ signal recovered from anoxia-induced decline (iii), it underwent a significant increase lasting >70 min (iv, Ca²⁺-aLTP). Bath-application of PTIO (100 μM) at 70 min, after establishment of Ca²⁺-aLTP, reversed it to the baseline level (v). Bar graphs indicate long-term potentiation of Ca²⁺ signal after anoxia (iv, 111.26 ± 3.1%) canceled by PTIO (v, 103.51 ± 3.4%; 12 slices, n = 25, one-way repeated-measures ANOVA: F_{(4, 120)} = 14.6).

Figure 7

Astrocyte-endothelium coupling mediated by D-serine (A) In WT mouse hippocampal CA1 neurons, the nNOS inhibitor NPA attenuated aLTP of fEPSP to 125.5 ± 7% (orange, n = 5). Co-treatment of NPA with DAAO abolished aLTP (105.1 ± 6%, green, n = 6; one-way ANOVA: F_(2,15) = 16.21). Bar graph shows the fEPSP slope 60 min after anoxic insult (ii) relative to baseline before anoxia (i). Scale bars; 0.4 mV, 20 ms. (B) In whole-cell recording with intracellular washout of L-arginine, aLTP was reduced (orange, data reproduced from Figure 5C) but was abolished by pre-treatment with DAAO (104 ± 6%, blue, n = 7; one-way ANOVA: F_(2,15) = 15.39). Bar graph shows normalized EPSC amplitude 35 min after anoxic insult relative to baseline before anoxia (i). ∗p < 0.05, ∗∗p < 0.01. Scale bars; 0.2 nA, 20 ms. (C) Neuro-glial-endothelial coupling for ·NO production by eNOS. Transient anoxia causes glutamate leakage, triggering the positive feedback loop (B). After anoxia, glutamate leakage ceases and the feedback loop is driven by elevated glutamate release from nerve terminals. High [Glu]_o in the synaptic cleft causes Ca²⁺ elevation throughout astrocytes by propagation from their processes at the synapses to their endfeet at the vascular endothelia and co-release glutamate and D-serine, thereby stimulating eNOS to produce ·NO, which together with that produced by neuronal nNOS contributes to the induction (Figure 2) and expression (Figure 3) of aLTP.