Ciliary neurotrophic factor (CNTF) activation of astrocytes decreases spreading depolarization susceptibility and increases potassium clearance

Abstract

Waves of spreading depolarization (SD) have been implicated in the progressive expansion of acute brain injuries. SD can persist over several days, coincident with the time course of astrocyte activation, but little is known about how astrocyte activation may influence SD susceptibility. We examined whether activation of astrocytes modified SD threshold in hippocampal slices. Injection of a lentiviral vector encoding Ciliary neurotrophic factor (CNTF) into the hippocampus in vivo, led to sustained astrocyte activation, verified by up-regulation of glial fibrillary acidic protein (GFAP) at the mRNA and protein levels, as compared to controls injected with vector encoding LacZ. In acute brain slices from LacZ controls, localized 1M KCl microinjections invariably generated SD in CA1 hippocampus, but SD was never induced with this stimulus in CNTF tissues. No significant change in intrinsic excitability was observed in CA1 neurons, but excitatory synaptic transmission was significantly reduced in CNTF samples. mRNA levels of the predominantly astrocytic Na(+) /K(+) -ATPase pump α2 subunit were higher in CNTF samples, and the kinetics of extracellular K(+) transients during matched synaptic activation were consistent with increased K(+) uptake in CNTF tissues. Supporting a role for the Na(+) /K(+) -ATPase pump in increased SD threshold, ouabain, an inhibitor of the pump, was able to generate SD in CNTF tissues. These data support the hypothesis that activated astrocytes can limit SD onset via increased K(+) clearance and suggest that therapeutic strategies targeting these glial cells could improve the outcome following acute brain injuries associated with SD.

Keywords: astrocyte activation; brain slice; ciliary neurotrophic factor (CNTF); hippocampus; spreading depolarization.

Figure 1. Astrocyte activation within murine hippocampus

A. Representative distribution of β-galactosidase immunohistochemistry demonstrates that intrahippocampal lentivirus injections lead to infection localized within the hippocampus, and high power confocal images of the CA1 and dentate gyrus showing localization to neuronal cell bodies (30 μm cryostat section, scale bar: 1 mm and 100 μm respectively). B. GFAP immunohistochemistry: Low power confocal projections (20 μm thick) were used to quantify changes in either total astrocyte number or simply increases in GFAP expression within resident astrocytes in hippocampal slice preparations (250 μm sections) from mice injected with lenti-CNTF versus lenti-LacZ as well as none injected controls (top panel, n= 8/8/7). Representative high power confocal images (20 μm thick) show increased GFAP within single astrocytes from CNTF hippocampal slices versus LacZ and controls (bottom panel). (GFAP: red, DAPI: blue, Scale bars: top panel 100 μm, bottom panel 50 μm). C. Quantification of area expressing GFAP and total astrocyte density. There was a significant increase in GFAP expression in CNTF preparations when compared with both LacZ and non-injected controls (F(2,22)=5.049, *p=.017, One-way ANOVA with Tukey's post-hoc test), but no significant difference in total number of astrocytes (F(2,22)=.968, p=.40, One-way ANOVA with Tukey's post-hoc test). D. RT-qPCR was used to measure GFAP mRNA levels in CNTF and LacZ hippocampal tissue. Cyclophilin A served as a housekeeping gene and was used to normalize the expression levels of the genes of interest. There was a significant increase in GFAP mRNA (t(10)=10.97, ***p=.0001, independent t-test) in CNTF tissue compared with LacZ controls.

Figure 2. Inhibition of SD in slices with CNTF-activated astrocytes

A. Representative DC recordings from LacZ preparations during 50 ms, 100 ms, and 200 ms KCl microinjections with 10 min intervals between stimuli. Recordings (R) were taken ~300 μm from the KCl injection site (S) as shown in bright field images in top corner of montages (B). B. Representative montage of 450 nm autofluorescence during the 100 ms KCl microinjection (shown electrically in A) from the LacZ preparation which resulted in the propagation of SD across the preparation beyond the field of view (~1300 μm). C. Representative DC recording from a CNTF preparation where KCl microinjections up to 200 ms in duration did not result in SD. D. Representative 450 nm autofluorescence recording during the 100 ms KCl microinjection from the CNTF preparation where the initial passive depolarization due to the exogenous KCl application occurred, but no propagating SD event. E. SD threshold determined with high K⁺ microinjections (1 M KCl). In LacZ slices SDs were induced in all preparations by the longest K⁺ microinjection (200 ms) (n=18/18). The same stimuli did not generate an SD in any CNTF preparations (n=0/18). F. Distance of the final tissue depolarization in CNTF preparations versus the initial passive depolarization due to the spread of exogenous K⁺. CNTF preparations showed no significant differences between the passive K⁺ depolarization (white triangles) and the final response (white boxes), providing further support for the lack of SD in CNTF preparations (n=12, dashed line represents the average distance of the view frame).

Figure 3. CNTF-activated astrocytes display unchanged gap junctional coupling, but increased mRNA for the α2 subunit of the Na⁺/K⁺-ATPase

A. Representative low-power images from slice preparations where single astrocytes were loaded with biocytin to assess astrocytic coupling (scale bar: 150 μm). B. No significant difference in astrocyte coupling between LacZ and CNTF preparations, as assessed by the number of biocytin positive astrocytes (t(15)=.335, p=.74, independent t-test). C. RT-qPCR was used to measure mRNA levels in CNTF and LacZ hippocampal tissue. Cyclophilin A served as a housekeeping gene and was used to normalize the expression levels of the genes of interest. There was a significant increase in ATP-2alpha1 (t(10)=2.496, *p=.032, independent t-test), but no significant difference in K_ir4.1 (t(10)= 1.922, p=.0835, independent t-test) and NKCC1 (t(10)= 1.571, p=.15) expression in CNTF tissue compared with LacZ controls.

Figure 4. CNTF astrocytes activation results in reduced glutamatergic synaptic efficacy and increased K⁺ homeostasis

A. Input-output curves were generated from the slope of fEPSP at increasing currents (0.0-0.3 mA, 0.02 mA interval). There was a significant decrease in the slope of the fEPSPs in CNTF hippocampal slices when compared to LacZs (n=6 and n=7 respectively, F(2,10)=4.32, * p<.05, One-way ANOVA with Tukey's post-hoc test). Representative fEPSP traces from LacZ (black) and CNTF (grey) preparations are from a stimulus of 0.2 mA. B. Intrinsic membrane properties of neurons were also not significantly different between CNTF and LacZ controls. C. Extracellular K⁺ transients were evoked using electrical stimulation of Schaffer collateral inputs (20 Hz, 10 s), and recorded with double barreled K⁺ sensitive microelectrodes placed in stratum radiatum of area CA1. K⁺ accumulation and clearance rates were evaluated using two different stimulation intensities; 1) 70% stimulation intensity required to generate just maximal amplitude fEPSPs, and 2) stimulation intensity adjusted to generate matched 1 mV amplitude fEPSP. D. Representative individual responses to matched (1 mV fEPSP) stimuli in LacZ (black) and CNTF (red) preparations. Responses are normalized to peak amplitudes. E. Decay phase of evoked K⁺ transients from populations of experiments illustrated in panel D. (mean ± SEM shown, n=12 preparations for each group). F. Significantly shorter decay time constants in CNTF preparations. Data were derived from same preparations illustrated in panel E, with stimuli matched to generate 1 mV fEPSPs (n=12 for each group, *p=.026, independent t-test).

Figure 5. Contribution of Na⁺/K⁺-ATPase pump in the reduced susceptibility to SD of CNTF tissues

During 100 μM ouabain exposures, both electrical recordings and intrinsic optical signals were used to measure SD in the sr of the CA1. A. Although SD could be induced in all CNTF preparations (n=12), the latency to SD was significantly longer when compared to LacZ controls (n=12) (t(22)=3.12, **p=.005, independent t-test). B. The rate of propagation was also significantly slower in CNTF preparations when compared to LacZ slices (n=12, t(22)=6.53, ***p<.0001, independent t-test). C. Examples of K⁺ responses (in mM) from a LacZ preparation (black) and a CNTF preparation (red) during SD induced by 100 μM ouabain. Black arrows indicate SD onset. D. Extracellular changes in K⁺ were measured during ouabain-induced SD. While not significant, there was a trend towards a decrease in the peak concentration of extracellular K⁺ in CNTF preparations compared with LacZ controls (n=12, (t(22)=1.887, p=.0724, independent t-test)).