Intermittent Hypoxia Disrupts Adult Neurogenesis and Synaptic Plasticity in the Dentate Gyrus

Abstract

Individuals with sleep apnea often exhibit changes in cognitive behaviors consistent with alterations in the hippocampus. It is hypothesized that adult neurogenesis in the dentate gyrus is an ongoing process that maintains normal hippocampal function in many mammalian species, including humans. However, the impact of chronic intermittent hypoxia (IH), a principal consequence of sleep apnea, on hippocampal adult neurogenesis remains unclear. Using a murine model, we examined the impact of 30 d of IH (IH₃₀) on adult neurogenesis and synaptic plasticity in the dentate gyrus. Although IH₃₀ did not affect paired-pulse facilitation, IH₃₀ suppressed long-term potentiation (LTP). Immunohistochemical experiments also indicate that IH perturbs multiple aspects of adult neurogenesis. IH₃₀ increased the number of proliferating Sox2⁺ neural progenitor cells in the subgranular zone yet reduced the number of doublecortin-positive neurons. Consistent with these findings, cell lineage tracing revealed that IH₃₀ increased the proportion of radial glial cells in the subgranular zone, yet decreased the proportion of adult-born neurons in the dentate gyrus. While administration of a superoxide anion scavenger during IH did not prevent neural progenitor cell proliferation, it mitigated the IH-dependent suppression of LTP and prevented adult-born neuron loss. These data demonstrate that IH causes both reactive oxygen species-dependent and reactive oxygen species-independent effects on adult neurogenesis and synaptic plasticity in the dentate gyrus. Our findings identify cellular and neurophysiological changes in the hippocampus that may contribute to cognitive and behavioral deficits occurring in sleep apnea.SIGNIFICANCE STATEMENT Individuals with sleep apnea experience periods of intermittent hypoxia (IH) that can negatively impact many aspects of brain function. Neurons are continually generated throughout adulthood to support hippocampal physiology and behavior. This study demonstrates that IH exposure attenuates hippocampal long-term potentiation and reduces adult neurogenesis. Antioxidant treatment mitigates these effects indicating that oxidative signaling caused by IH is a significant factor that impairs synaptic plasticity and reduces adult neurogenesis in the hippocampus.

Keywords: adult neurogenesis; hypoxia.

Figure 1.

Prolonged IH exposure attenuates LTP within the dentate gyrus. A, LTP of the fEPSP following HFS in control (blue circles; n = 9 slices, 7 animals), IH₁₀ (yellow triangles; n = 4 slices, 2 animals), IH₂₀ (magenta diamonds; n = 8 slices, 3 animals), and IH₃₀ (red squares; n = 10 slices, 7 animals) illustrate differences in potentiation following HFS. Representative traces of evoked fEPSPs are shown above the graph with baseline (black trace) and post-HFS induction indicated (color traces: control, blue; IH₁₀, yellow; IH₂₀, magenta; IH₃₀, red). Arrows at the bottom indicate the time sampled for B and C. Calibration: 0.2 mV, 10 ms. B, Immediately following HFS, a difference among groups was observed (F_(3,27) = 6.667, p = 0.0016). A post hoc Dunnett's test revealed no difference immediately following HFS between control and IH₁₀ groups, yet did in both IH₂₀ and IH₃₀ groups in the fEPSP slope when compared with control. C, Sixty minutes post-HFS, a difference among groups was detected (F_(3,27) = 9.529, p = 0.0002). Post hoc Dunnett's test revealed that, while no difference was present between the control and IH₁₀ groups, the fEPSP in both the IH₂₀ and IH₃₀ groups was reduced compared with control group. In a subset of experiments (n = 4 slices, 3 animals), applying to a larger stimulation current during HFS did not to evoke LTP in the IH₃₀ group (B and C, white triangles). *p < 0.05.

Figure 2.

Prolonged IH exposure does not influence paired-pulse facilitation (PPF). A, Representative traces of evoked fEPSPs during PPF are shown. Calibration: 0.2 mV, 10 ms. B, PPF of the fEPSP was similar between control (blue circles, n = 6 slices; 4 animals) and IH₃₀ (red squares, n = 6 slices, 5 animals) at all six interpulse intervals (IPIs) tested: 40 ms IPI (IPI 40), t_(9.990) = 0.904, p = 0.386; IPI 80, t_(9.890) = 0.813, p = 0.4352; IPI 200, t_(9.963) = 1.169, p = 0.269; IPI 300, t_(9.79) = 0.406, p = 0.693; IPI 400, t_(8.512) = 0.515, p = 0.619; IPI 500, t_(9.997) = 0.556, p = 0.591. C, PPF at IPI 50 was similar before and following high-frequency stimulation for control (blue, n = 6 slices, 3 animals; t₍₅₎ = 0.8110, p = 0.4542) and IH₃₀ (red, n = 8 slices, 3 animals; t₍₇₎ = 1.777, p = 0.1188). Black-filled symbols represent mean for each group.

Figure 3.

IH₃₀ increases the number of neural progenitor cells. A, Representative section of dentate gyrus stained for Sox2 (green) and DAPI (blue). SGZ is shown outlined by yellow dotted lines. Scale bar, 100 μm. B, Representative images of Sox2⁺ labeling in control (top) and IH₃₀ (bottom) animals. SGZ is outlined in yellow. Blue channel depicts DAPI-labeled nuclei on left. Green channel depicts Sox2⁺ labeling in middle, and a merge is on the right. Scale bars, 100 μm. C, The number of Sox2⁺ cells in the SGZ increases following IH₃₀ (control, n = 10; IH₃₀, n = 7; t_(14.24) = 2.327, p = 0.035). *p < 0.05.

Figure 4.

IH₃₀ stimulates region-specific SOX2⁺ cell proliferation in the dentate gyrus. A, Representative section of dentate gyrus stained for Sox2 (green), Ki67 (red), and DAPI (blue). SGZ is outlined by yellow dotted lines. Counts were performed in the ML, GCL, SGZ, and hilus. The yellow arrow indicates a Ki67⁺/Sox2⁺ double-positive cell residing within the SGZ. Scale bar, 100 μm. B, Representative images of Ki67 and Sox2⁺ labeling in control (top) and IH₃₀ (bottom) animals. SGZ is outlined in yellow. Blue channel depicts DAPI-labeled nuclei on left, red channel depicts Ki67⁺ labeling second from left, green channel depicts Sox2⁺ labeling second from right, and a merge is on the right. Scale bars, 100 μm. C–F, Quantified proportions of double-labeled Sox2⁺/Ki67⁺ cells (control, n = 4; IH₃₀, n = 4) in the SGZ (t_(5.993) = 2.747, p = 0.034), hilus (t_(4.415) = 4.775, p = 0.0069), GCL (t_(3.580) = 2.414, p = 0.0808), and ML (t_(5.89) = 0.4592, p = 0.6625). *p < 0.05.

Figure 5.

IH₃₀ decreases the number of newly born neurons. A, Representative image of DCX⁺-labeled cells (gray) at low and high magnification. The yellow arrow shows a DCX⁺ immature neuron that was included in the analysis based on morphology. The red arrowhead points to a DCX⁺-labeled cell without a process extending into the GCL. Scale bars, 100 μm. B, Representative images of DCX⁺ labeling in control (top) and IH₃₀ (bottom) animals. Blue channel depicts DAPI-labeled nuclei on left, gray channel depicts DCX⁺ labeling in middle, and a merge is on the right. Scale bars, 100 μm. C, IH₃₀ reduced the number of DCX⁺ cells with neuronal morphology exhibiting clear dendritic projections from the dentate gyrus to the molecular layer (control, n = 5; IH₃₀, n = 5; t_(7.744) = 2.368, p = 0.046). *p < 0.05.

Figure 6.

IH₃₀ exposure alters neural progenitor cell fate within the dentate gyrus. A, Representative images of tissue stained for birth-labeled RFP⁺ cells (red), GFAP (green), DCX (gray), and DAPI (blue) from control (left) and IH₃₀-exposed (right) mice. Scale bars, 100 μm. B–F, The proportion of: RFP⁺/GFAP⁺-colabeled cells with radial glial morphology were significantly different between groups (control, n = 9; IH₃₀, n = 11; t_(15.16) = 2.635, p = 0.0186; B); RFP⁺ neural progenitor cells in the SGZ were unchanged between groups (control, n = 8; IH₃₀, n = 11; t_(12.60) = 0.4915, p = 0.6315; C); RFP⁺/DCX⁺-colabeled progenitor cells were unchanged between groups (control, n = 8; IH₃₀, n = 11; t_(17.13) = 0.7422, p = 0.4680; D); RFP⁺/GFAP⁺-colabeled cells with astrocytic morphology were not significantly different between the two groups (control, n = 8; IH₃₀, n = 11; t_(14.68) = 1.267, p = 0.2250; E); RFP⁺ cells that exhibit neuronal morphology were reduced in IH₃₀ mice (control, n = 8; IH₃₀, n = 11; t_(13.97) = 2.730, p = 0.0163; F). Scale bars: B–F, 50 μm. G, Sholl analysis revealed that there were no significant changes in morphology as characterized by the number of intersections in dendritic arborization (control, n = 7; IH₃₀, n = 10; F_(30,320) = 0.750, p = 0.828). Representative images of neurons from control (top) and IH₃₀ (bottom) used for analysis on left. Scale bars: 50 μm. *p < 0.05.

Figure 7.

MnTMPyP administration reveals that neural progenitor cell proliferation is ROS independent. A, Representative images of Ki67 and Sox2⁺ labeling in control_MnTMPyP (top) and IH_MnTMPyP (bottom) animals are shown. Blue channel depicts DAPI-labeled nuclei on left, red channel depicts Ki67⁺ labeling second from left, green channel depicts Sox2⁺ labeling second from right, and a merge is on the right. Scale bars, 100 μm. B–E, The proportion of Ki67⁺/Sox2⁺-colabeled cells (control_MnTMPyP, n = 5; IH_MnTMPyP, n = 6) was increased following IH_MnTMPyP in the SGZ (t_(6.773) = 3.390, p = 0.0122; B), yet no differences were observed in the hilus (t_(8.869) = 1.293, p = 0.2287; C), GCL (t_(5.953) = 0.0863, p = 0.9340; D), and ML (t_(8.532) = 0.6589, p = 0.5273; E). *p < 0.05.

Figure 8.

MnTMPyP administration reveals that neuron development is ROS dependent. A, Representative images of DCX⁺ labeling in control_MnTMPyP (top) and IH_MnTMPyP (bottom) animals are shown. Blue channel depicts DAPI-labeled nuclei on left, gray channel depicts DCX⁺ labeling in middle, and a merge is on the right. Scale bars, 100 μm. B, Immature granule neurons labeled with DCX⁺ showed no significant difference between IH_MnTMPyP and control_MnTMPyP groups (control_MnTMPyP, n = 4; IH_MnTMPyP, n = 6; t_(7.708) = 2.144, p = 0.066). C, Representative images of RFP⁺ labeling in control_MnTMPyP (top) and IH_MnTMPyP (bottom) animals. Blue channel depicts DAPI-labeled nuclei on left, red channel depicts RFP⁺ labeling in middle, and a merge is on the right. Scale bars, 100 μm. D, The percentages of RFP⁺ neurons were not different between IH_MnTMPyP and control_MnTMPyP groups (control_MnTMPyP, n = 6; IH_MnTMPyP, n = 6; t_(7.361) = 0.402, p = 0.699).

Figure 9.

MnTMPyP administration reveals that LTP is a ROS-dependent process. A, LTP of the fEPSP following HFS showed that synaptic plasticity was maintained in IH_MnTMPyP-treated animals. The dashed blue line represents the mean from the control LTP experiments. Representative evoked EPSP traces illustrate pre-HFS (black trace) and post-HFS (green trace) induction. Calibration: 0.2 mV, 10 ms (inset). B, At 10 and 60 min post-HFS, there is a significant increase in EPSP slope when compared with pre-HFS baseline (F_(2,10) = 7.627, p = 0.009). *p < 0.05.