Decrease in GABA_A immunoreactivity in APP mice.

<p>GABA_A immunoreactivity in the somatosensory cortex of a 4 month old wildtype littermate control mouse (<b>A</b>), and a 4 month old APP mouse (<b>B</b>). (<b>C</b>) Bar graph comparing intensity of GABA_A immunoreactivity between conditions as a percentage of wildtype level at 4 months (n = 3–4 mice/group). (<b>D</b>) Voltage-sensitive dye traces showing a decrease in power of slow oscillations 60 minutes after topical application of 50 μM picrotoxin to a 4 month old wildtype mouse brain. (<b>E</b>) Slow oscillation power (normalized to wildtype) and (<b>F</b>) mean slow oscillation frequency before (WT baseline) and after picrotoxin (PTX) application to brains of 2–4 month old wildtype mice (n = 4 mice). (<b>G</b>) Slow oscillation power (normalized to APP) and (<b>H</b>) mean slow oscillation frequency before (APP baseline) and after picrotoxin (PTX) application to brains of 2–4 month old APP mice (n = 4 mice). Scale bar, 50 μm. * p<0.05, *** p≤0.001.</p

Decrease in GABA_B expression in APP mice.

<p>GABA_B immunoreactivity in the somatosensory cortex of a 4 month old wildtype littermate control mouse (<b>A</b>), and a 4 month old APP mouse (<b>B</b>). B represents an extreme case. (<b>C</b>) Bar graph comparing GABA_B immunoreactivity between conditions as a percentage of wildtype level at 4 months (n = 3–4 mice/group, p≤0.001). (<b>D</b>) Slow oscillation power (normalized to wildtype) and (<b>E</b>) mean slow oscillation frequency before (baseline) and after 50 μM saclofen application to the brain of wildtype mice (n = 4 mice). (<b>F</b>) Slow oscillation power (normalized to wildtype) and (<b>G</b>) mean slow oscillation frequency before (baseline) and after 50 μM saclofen application to the brain of APP mice (n = 6 mice). Scale bar, 30 μm. * p<0.05, *** p≤0.001.</p

Optogenetic manipulation of slow oscillations.

<p>(<b>A</b>) Voltage-sensitive dye signal showing spatiotemporal resolution of neural circuit oscillations driven with light activation of ChR2 at 1.2 Hz in the somatosensory cortex of a Thy1-ChR2-YFP mouse. The images correspond to the first three oscillations in <b>B</b>. Scale bar, 20 μm. (<b>B</b>) WT: Trace of voltage-sensitive dye signal showing that slow oscillations can be driven at twice the normal rate with light activation of ChR2 at 1.2 Hz when light pulses are present (light pulses are depicted in blue) in the somatosensory cortex. The frequency of waves slows after cessation of light stimulation. APP: VSD trace depicting restoration of slow oscillations in APP mice expressing ChR2 virus under CamKIIα promoter when stimulated with light at 0.6 Hz. A decrease in power is evident after light stimulation is stopped. (<b>C</b>) Site of ChR2 viral injection under the CamKIIα promoter tagged with mCherry, or empty vector. (<b>D</b>) mCherry expression in the cortex in a coronal section of postmortem brain tissue. Scale bar, 100 μm. (<b>E</b>) Site of viral injection of YC3.6 in the contralateral hemisphere posterior to the site of ChR2 expression. (<b>F</b>) YFP expression in the cortex in a coronal section of postmortem brain tissue. Scale bar, 100 μm. (<b>G-H</b>) Neuronal activity driven with light activation of ChR2 in wildtype mice (n = 3–5 mice). Control corresponds to mCherry vector lacking ChR2. Light activation of ChR2 at 1.2 Hz increases the power and frequency of slow oscillations. Powers (<b>G</b>) and frequencies (<b>H</b>) of neuronal activity generated in the presence or absence of light with or without ChR2. (<b>I</b>) Slow oscillation power (normalized to APP without light activation) and (<b>J</b>) mean slow oscillation frequency before or after light activation of ChR2 in 4 month old APP mice expressing the virus (n = 6 mice). * p<0.05, ** p≤0.01.</p

Decrease in GABA in APP mice.

<p>GABA immunoreactivity in the cortex of a 4 month old wildtype littermate control (<b>A</b>), and a 4 month old APP mouse (<b>B</b>). An example in B shows an extreme case. (<b>C</b>) Bar graph comparing cortical GABA levels measured with HPLC in mice older than 4 months (n = 5–6 mice/group). (<b>D</b>) Slow oscillation traces before and after topical application of 0.5 mM GABA to somatosensory cortices of WT and APP mouse brains. (<b>E</b>) Cortical GABA levels measured with HPLC in 2 month old WT and APP mice. (<b>F</b>) Bar graph showing a dose response to a topical application of 0 (PBS+/+), 0.05, 0.5 and 5mM GABA to the brains of APP mice; 0 and 5mM GABA to brains of WT mice (n = 3–4 mice/group). (<b>G</b>) Bar graph showing a dose response to a topical application of 0 (PBS+/+), 0.05, 0.5 and 5mM GABA to the brains of APP mice, normalized to the power after PBS+/+ application (n = 3–4 mice/group). (<b>H</b>) Slow oscillation power (normalized to 0 mM GABA or PBS+/+) before (0 mM GABA or PBS+/+) and after 5mM GABA application to wildtype mice (n = 3 mice/group). (<b>I</b>) Slow oscillation power (normalized to baseline) before (baseline) and after PBS application to APP mice (n = 7 mice/group). (<b>J</b>) Mean slow oscillation frequency in response to various GABA applications to APP mice (n = 3–4 mice/group). (<b>K</b>). Mean slow oscillation frequency before (0mM GABA or PBS+/+) and after 5mM GABA application to wildtype mice (n = 3 mice). (<b>L</b>) Slow oscillation power (normalized to baseline) before (baseline) and after PBS application to wildtype mice (n = 3 mice/group). Scale bar, 50 μm. * p<0.05.</p

Driving slow oscillations with light restores GABA receptor levels.

<p>(<b>A-C</b>). GABA immunoreactivity in the cortex of a 7 month old wildtype littermate control mouse (<b>A</b>), an APP transgenic (<b>B</b>) and an APP mouse whose slow oscillations were recovered with light (<b>C</b>). (<b>D-F</b>) GABA_A immunoreactivity in the cortices of a 7 month old wildtype littermate control mouse (<b>D</b>), an APP mouse (<b>E</b>), and an APP mouse whose slow oscillations were restored with light (<b>F</b>). (<b>G-I</b>) GABA_B immunoreactivity in the cortex of a 7 month old wildtype littermate control mouse (<b>G</b>), an APP mouse (<b>H</b>), and an APP mouse whose slow oscillations were restored with light activation of ChR2 (<b>I</b>). (<b>J</b>) Bar graph comparing cortical GABA levels measured with HPLC (n = 4 mice/group). (<b>K</b>). A bar graph comparing GABA_A immunoreactivity between conditions as a percentage of wildtype level at 7 months (n = 3–4 mice/group). (<b>L</b>) A bar graph comparing GABA_B immunoreactivity between conditions as a percentage of wildtype level at 7 months (n = 3–4 mice/group). ** p≤0.01, *** p≤0.001.</p

Calcium overload is prevented in APP mice whose slow oscillations were driven with light activation of ChR2.

<p>(<b>A,B</b>) In vivo multiphoton images of cortical neurites, pseudocolored according to [Ca2+]i, show the presence of elevated levels of calcium (yellow-red neurites) in a 6 months old APP mouse (<b>A,</b> arrows) in addition to neurites displaying normal calcium levels (for instance, blue neurites). Restoring slow oscillations with light activation of ChR2 prevented elevations of calcium (calcium overload) in cortical neurites (<b>B</b>). (<b>C</b>) Histograms showing distribution of YFP/CFP ratios in neurites with YC3.6 in APP mice at 5 and 6 months of age (n = 746 neurites in 7 mice). Calcium overload was defined as ratios greater than 2 standard deviations above the mean in wildtype mice (1.79) (n = 321 neurites in 5 mice). (<b>D</b>) Distribution of neurite YFP/CFP ratios in 5, 6, and 7 month old APP mice whose slow oscillations were restored with light activation of ChR2 (n = 369 neurites in 6 mice). (<b>E</b>) A bar graph showing the percentage of neurites exhibiting calcium overload across conditions at 6 months. (<b>F,G</b>) In vivo multiphoton images of cortical neurites, pseudocolored according to [Ca2+]i, show limited calcium overload within neurites in a 6 months old WT mouse (<b>F</b>). Driving slow oscillations with light activation of ChR2 failed to significantly alter calcium overload in cortical neurites in wildtype mice (<b>G</b>). (<b>H</b>) Histograms showing distribution of YFP/CFP ratios in neurites with YC3.6 in WT mice at 5 and 6 months of age (n = 321 neurites in 5 mice). (<b>I</b>) Distribution of neurite YFP/CFP ratios in 5 and 6 month old WT mice whose slow oscillations were driven with light activation of ChR2 at normal frequency (n = 326 neurites in 5 mice). (<b>J</b>) A bar graph showing the percentage of neurites exhibiting calcium overload across conditions in wildtype mice at 6 months. (<b>K</b>) Percentage of total time spent mobile during the day and night by APP and wildtype littermates whether treated with light or not. (Scale bar, 100 μm) ** p≤0.01.</p