Results
40 Hz light flicker entrained with hippocampal CA1 low gamma
To
demonstrate the effects of different frequencies of light flicker on
CA1 oscillations, LFPs were recorded in mice implanted with a
four-electrode array into the CA1 stratum pyramidale layer (Fig. ),
followed by treatment with 10 Hz, 30 Hz, 40 Hz, 50 Hz, 80 Hz,
arrhythmic frequency, or without light flicker (occluded LED) light
flicker (Fig. and Supplementary Fig.
1j–m). Because mouse movement speed affects the linearity of theta waves and potentially presents theta harmonics
20,21,
three similar low-speed movement states, i.e., approach/exploration of a
novel object and low-speed free walking, were selected for the LFP
study. The speeds/velocities were at 2~3 cm/s forward, as determined by
footfall patterns (Supplementary Fig.
1n)
22. Oscillation entrainment occurred in CA1 LFP in response to light flickers of 40 Hz (Fig. ), 10 Hz, 30 Hz, 50 Hz and 80 Hz (Supplementary Fig.
1j – m), but not the occluded LED or arrhythmic light (Fig. ).
The power spectral density (PSD) of CA1 low gamma, but not theta and
high gamma, oscillation increased significantly in response to 40 Hz
light flicker treatment compared with the controls (1ANOVA, F
(2, 15) = 4.512,
P = 0.0292; LSD’s post hoc test,
POccluded LED vs. 40 Hz LED = 0.0209,
P40 Hz LED vs. Random LED = 0.0192, Fig. ), confirming previous studies
10,12,23,24.
Recording of CA1 LFP in behaving mouse.
a
A multielectrode array was dorsally implanted into the CA1 7 d before
2VO experimentation. Recordings of LFP from the dorsal hippocampal CA1
region in vivo were performed on free-moving mice. b–d, LFP PSD of CA1 during light flicker. e–g Quantification of LFP PSD of the three groups of mice with the same legend shown in g (n = 6 sessions/5 mice each group). h
Example spectrograms for the four groups of mice recorded 10 s in CA1
during low-speed free walking. The excerpt of raw LFPs of theta, low
gamma, and high gamma oscillations are shown in the lower panel of h, and the PSD intensity is color-coded according to the color scale shown on the right. i–k
Quantification of the LFP powers of the four groups of mice in the
theta, low gamma, and high gamma frequencies. The same group legend is
shown in k (Sham: n = 5 sessions/3 mice; Sham+LED: n = 6 sessions/3 mice; 2VO: n = 7 sessions/3 mice; 2VO + LED; n = 8 sessions/3 mice). l,m PAC analysis of low gamma and high gamma, respectively, to the theta phase as indicated by the modulation index (n = 3 mice). n
Example of moving window spectrogram of hippocampal CA1 LFP time-locked
to the initiation of approach (<0 s) and explore (>0 s) a novel
object and the PSD intensity is color-coded according to the color scale
shown on the right. o–r The ratio of PSD between Exploration (PSDExp) and Approach (PSDAppr) of the four groups of mice at the indicated oscillation frequencies on the y-axis (n = 6 sessions/3 mice). Data represent the mean ± SEM. Error bars indicate SEM. *P < 0.05, ##P < 0.01, *** and ###P < 0.001; The test used in e–g 1ANOVA with LSD’s post hoc analysis; i–k 2ANOVA with LSD’s post hoc test; l, m 2ANOVA with Tukey's post hoc test; o–r RM-2ANOVA with Sidak’s post hoc test. Source data are provided as a Source Data file.
40 Hz light flicker restored CA1 low gamma reduction in 2VO
40 Hz
light flicker restores 2VO-affected CA1 LFP. Mice received light
flicker treatment 2 h after 2VO twice per day, 12 h apart, for up to 14
days (Supplementary Fig.
1a–d). 2VO group of mice CA1 LFP power (Fig. ) was significantly reduced in the 30–50 Hz low gamma range (2ANOVA, F
(1, 22) = 7.628,
P = 0.0114; LSD’s post hoc test,
PSham vs. 2VO = 0.0434, Fig. ), but not 4–12 Hz theta (2ANOVA, F
(1,22) = 0.04573,
P = 0.8326; LSD’s post hoc test,
PSham vs. 2VO = 0.8454, Fig. ) and 80–120 Hz high gamma (2ANOVA, F
(1,22) = 0.7381,
P = 0.3995; LSD’s post hoc test,
PSham vs. 2VO = 0.5716, Fig. ).
The 2VO mice also displayed decreased phase-amplitude coupling (PAC)
between theta phase and slow gamma amplitude compared with the Sham
group (2ANOVA, F
(1, 8) = 5.441,
P = 0.0480; Tukey’s post hoc test,
PSham vs. 2VO = 0.0006, Fig. ). Theta-high gamma PAC was not altered in 2VO mice (2ANOVA, F
(1,8) = 0.07855,
P = 0.7864; Tukey’s post hoc test,
PSham vs. 2VO = 0.9370, Fig. ).
Importantly, 40 Hz light flicker treatment significantly enhanced 2VO
group CA1 low gamma power (2ANOVA with LSD’s post hoc test,
P2VO vs. 2VO+LED = 0.0001, Fig. ), and restored theta-low gamma PAC in the 2VO + LED group (2ANOVA with Tukey’s post hoc test,
P2VO vs. 2VO+LED = 0.0021, Fig. ), indicating a specific reduction of hippocampal CA1 low gamma in cerebral ischemia.
Novel
object exploration elicits a distinct CA1 40 Hz low gamma oscillation,
which are related most specifically to object-location associative
memory encoding rather than overt behaviors
25,26. CA1 LFPs were therefore recorded in four groups of mice performing an approach/exploration of novel object task (Fig. ).
An example of a moving window spectrogram of a mouse time-locked as a
mouse initiated an approach (<0 s) and exploration (>0 s) of a
novel object was shown in Fig. .
A significant reduction in the 30–50 Hz low gamma range and the 40 Hz
specific gamma occurred in the PSD ratio between Exploration/Approach
compared between Sham vs. 2VO group (For low gamma: RM-2ANOVA, F
(1, 10) = 70.82,
P < 0.0001; Sidak’s post hoc test,
P3d < 0.0001,
P7d < 0.0001,
P14d = 0.0006, Fig. . For 40 Hz gamma: RM-2ANOVA, F
(1, 10) = 144.60,
P < 0.0001; Sidak’s post hoc test,
P3d < 0.0001,
P7d < 0.0001,
P14d < 0.0001, Fig. ). No change occurred in the 4–12 Hz theta (RM-2ANOVA, F
(1, 10) = 0.637,
P = 0.4433; Sidak’s post hoc test,
P3d = 0.7603,
P7d = 0.9061,
P14d = 0.9959, Fig. ) and the 80–120 Hz high gamma (RM-2ANOVA, F
(1, 10) = 0.4482,
P = 0.5184; Sidak’s post hoc test,
P3d = 0.7685,
P7d = 0.9998,
P14d = 0.9473, Fig. ).
The 2VO-induced low gamma reduction during exploration was
significantly rescued by the 40 Hz light flicker compared between
2VO + LED and 2VO (RM-2ANOVA with Sidak's post hoc test,
P3d < 0.0001,
P7d < 0.0001,
P14d < 0.0001, Fig. ). Therefore, 40 Hz light flicker rescued 2VO-induced impairment in CA1 low gamma and theta-low gamma PAC.
30–50 Hz light flicker conferred CA1 neuroprotection
To
demonstrate that
40 Hz light flicker confers neuroprotection, the 2VO
mouse model was empirically established and characterized to show
selective CA1 damage. A protocol of 5 min 2VO reduced hippocampal
regional blood flow (hrCBF) to 85%–95% (Supplementary Fig.
1a, b) with simultaneous hypotension at 35–40 mmHg during 2VO (Supplementary Fig.
1c). Strong Fluoro-Jade B (FJB) fluorescence occurred in the CA1 area at 3–14 d after 2VO (Fig. ). No FJB neurons were in the CA2, CA3, cortex, striatum, and thalamus nuclei (Supplementary Fig.
2a). The numbers of parvalbumin-positive (PV+) interneurons, as shown in Supplementary Fig.
2(b, c),
were also not significantly different compared amongst the four
experimental groups at 3 d, 7 d, and 14 d post 2VO, indicating selective
injury to CA1 excitatory neurons. 40 Hz light flicker treatment
significantly reduced CA1 FJB positive neurons in the 2VO + LED group
(2ANOVA: F
(1, 30) = 23.75,
P < 0.0001, with LSD’s post hoc test for 2VO vs. 2VO + LED:
P3day = 0.0014,
P7day = 0.0072,
P14day = 0.0495, Fig. ).
At 14 d post 2VO, FJB positive cells were not present in the CA1 region
of mice treated with 40 Hz LED light (2VO + LED group, Fig. ),
demonstrating remarkable neuroprotection to CA1 neurons by 40 Hz light flicker.
Protection to CA1 neurons conferred by 40 Hz light treatment.
a Coronal brain sections were stained with FJB. Hippocampal areas were shown in a highlighting 2VO-induced CA1 degenerating neurons (bright green colored cells). Scale bars = 50 µm. b
Enlarged CA1 area from 3 d, 7 d, and 14 d after 2VO with or without
40 Hz light treatment. Scale bars = 20 µm. The last column of b showed 40 Hz light treatment at 3 d after 2VO. c and d Quantification of CA1 FJB positive neurons within CA1 (n = 6 mice each group in c; LED after 2VO 2 h: n = 6 mice; LED after 2VO 3d: n = 5 mice). e
2VO mice were treated with different rhythmic frequencies, and
arrhythmic frequency of lights and FJB staining was performed 3 d after
2VO. n = 6 mice in 2VO and 40 Hz groups; n = 4 mice in Random group; n = 5 mice in other groups. Data represent the mean ± SEM. Error bars indicate SEM. ##
indicates a comparison between 2VO and 2VO + LED group. *indicates a
comparison between 2VO + LED group received 40 Hz light 2 h or 3 d after
2VO surgery, and ** in panel e indicates a comparison between the random group and 2VO mice received different frequencies of light treatment (*,#P < 0.05, and **P < 0.01). The test used in c: 2ANOVA with LSD’s post hoc analysis; d Two-tailed unpaired t-test; e 1ANOVA with Dunnett’s post hoc test. Source data are provided as a Source Data file.
Delayed
light treatment at 3 d after 2VO was not neuroprotective compared with
the early treatment group (2 h after 2VO) (Two-tailed unpaired t-test, t
(9) = 2.317, P = 0.0457, Fig. , the last column on the right and Fig. ).
Reparably, light flickers at 30 Hz, 40 Hz, and 50 Hz all showed
significant protection to CA1 pyramidal neurons compared with the
arrhythmic frequency (1ANOVA, F(3, 16) = 5.63, P = 0.0079, with Dunnett’s post hoc test at P30Hz vs. Random = 0.0106, P40Hz vs. Random = 0.0077, P50Hz vs. Random = 0.0076, Fig. ).
In contrast, the 10 Hz, 80 Hz, and random frequency light flickers were
not protective based on FJB staining of hippocampal brain sections
(1ANOVA, F(2, 11) = 0.02985, P = 0.9707, Dunnett’s post hoc test: P10Hz vs. Random = 0.9971, P80Hz vs. Random = 0.9617, Fig. ). These data suggested that light flicker protection was time- and frequency-dependent.
Several
factors were investigated to understand how light flickers protect
neurons. First, the cerebral blood flow (CBF) was not affected by light
flicker. The laser Doppler flowmetry of hrCFB showed no statistical
differences amongst the four experimental groups (Supplementary Fig.
1b). The CBF of the whole cortical area in general (Supplementary Fig.
1e, f), and the visual cortex area in particular (Supplementary Fig.
1e, g),
were also not significantly different at 3 d, 7 d, and 14 d between 2VO
and 2VO + LED groups, and between Sham and Sham+LED groups,
respectively, as measured using laser speckle contrast imaging.
FITC-lectin perfusion staining at the end of 14 d of light treatment
also showed that blood vessel densities in the hippocampal and visual
cortex areas were not significantly different between 2VO and 2VO + LED
groups (VC: 2ANVOA F
(1, 40) = 0.0559,
P = 0.8143; HPC: F
(1, 38) = 0.1657,
P = 0.6863, Supplementary Fig.
1h-i).
Second, 2VO activated microglia. However, the Sham and 2VO groups
showed no increase in microglia in response to light flicker
(Supplementary Fig.
2d–h),
indicating an inverse correlation with neuroprotection. The numbers of
GFAP positive astrocytes were similar compared amongst all four groups
of mice (Supplementary Fig.
3). These data indicated that light flicker might have a direct effect on neurons.
40 Hz light flicker improved cognitive functions of 2VO mice
A battery of cognitive-behavioral tests was performed (Fig. ).
The open-field test (OFT) showed that 2VO mice performed poorly
compared with the Sham group in terms of the total distance traveled
(2ANOVA, F(1, 28) = 8.338, P = 0.0074, Tukey’s post hoc test, PSham v.s. 2VO = 0.0308, Fig. ), the distance in the field center (F(1, 28) = 5.837, P = 0.0225; Tukey’s post hoc test, PSham vs. 2VO = 0.0477, Fig. ) and the percentage of time spent in the center (F(1, 28) = 12.05, P = 0.0017; Tukey’s post hoc test, PSham vs. 2VO = 0.0480, Fig. ). Light flicker treatment enhanced OFT performance of 2VO group (Tukey’s post hoc test for 2VO vs. 2VO + LED, P = 0.0458 in Fig. ; P = 0.0223 in Fig. ; P = 0.0015 in Fig. ), demonstrating that 40 Hz light flicker enhanced the locomotor ability, but did not produce anxiety-like behaviors.
Improvement of cognitive functions of 2VO mice after 40 Hz light visual stimulation.
a A schema showing experimental design and timeline. b–d OFT results of four groups of mice. Total distance traveled (b), distance in the center (c), and percentage of time spent in the center (d) were measured and compared amongst the four experimental groups (Sham: n = 7; Sham+LED: n = 8; 2VO: n = 9; 2VO + LED: n = 8 in OFT). e Water maze test (WMM) was also performed on the four experimental groups with days indicated. Escape latency to the platform (e) was measured during the acquisition test at 8, 9, 10, 11, and 12 d after 2VO surgery. Pathlength (f) and locomotion speed (g) of the four groups were also recorded and plotted (Sham: n = 8; Sham+LED: n = 8; 2VO: n = 9; 2VO + LED: n = 10 in the acquisition test of MWM). h The percentage of time spent in the target quadrant during the spatial exploring stage of the MWM probe test (Sham: n = 7; Sham+LED: n = 8; 2VO: n = 7; 2VO + LED: n = 8
in the probe test of MWM). Data were mean ± SEM. Error bars indicated
SEM. * indicates a statistical difference between Sham and 2VO group; # indicates a statistical difference between 2VO and 2VO + LED group. *, #P < 0.05, **, ##P < 0.01, ***, ###P < 0.001; Test used in b–d 2ANOVA with Tukey’s post hoc test; e–g RM-2ANOVA with LSD’s post hoc test; h 2ANOVA with LSD’s post hoc test. Source data are provided as a Source Data file.
The
water maze test showed that 40 Hz light flicker significantly
attenuated 2VO-induced impairments in spatial leaning ability and
reference memory (Fig. ). All four groups of mice showed no difference in swimming speed (Fig. ).
However, the escape latency and pathlength of 2VO mice were
significantly longer compared with the Sham group, respectively, during
the acquisition test (Escape latency: RM-2ANOVA, F(1, 15) = 14.52, P = 0.0017, LSD’s post hoc test, P8 = 0.579, P9 = 0.017, P10 = 0.016, P11 = 0.011, P12 = 0.001, Fig. ; Pathlength: F(1, 15) = 7.118, P = 0.0175, LSD’s post hoc test, P8 = 0.652, P9 = 0.162, P10 = 0.0505, P11 = 0.007, P12 = 0.015, Fig. ).
A lesser time spent in the target quadrant in the probe test was an
indication of poor spatial learning and memory abilities, which was
associated with the deficits of object-location associative memory
encoding caused by 2VO (2ANOVA with LSD’s post hoc test, PSham vs. 2VO = 0.0438, Fig. ).
40 Hz light flicker significantly enhanced the acquisition abilities of
spatial learning and memory compared with the 2VO group (RM-2ANOVA, F(1, 17) = 10.24, P = 0.0053, LSD’s post hoc test, P8 = 0.838, P9 = 0.179, P10 = 0.041, P11 = 0.034, P12 = 0.00017, Fig. ; 2ANOVA, F(1, 26) = 5.492, P = 0.0270, LSD’s post hoc test, P2VO vs. 2VO+LED = 0.0107, Fig. ). These data are strong indications that 40 Hz light flicker mitigated cognitive deficits caused by 2VO.
Enhancement of CA3-CA1 LTPs by 40 Hz light flicker
There is a strong correlation of theta-gamma PAC strength with LTP and learning memory
27,28.
Experiments were, therefore, designed to determine whether 40 Hz light
flicker enhances the fast excitatory synaptic LTPs of the CA3-CA1
network, including NMDA receptor-dependent LTP (LTP
NMDAR) and L-type voltage-gated calcium channel-dependent LTP (LTP
L-VGCC), both are important in cerebral ischemia-induced cognitive impairments
18,29. In the presence of NMDAR antagonist APV, LTP
L-VGCC can be induced with 200 Hz tetanic stimulation
30,
which served as a pharmacological approach to isolate the two LTPs. The
input/output (I/O) curves of field excitatory postsynaptic potentials
(fEPSP) amplitude and slope with a series of increasing stimulation
intensities were recorded and shown in Fig. ,
respectively. The basal synaptic transmission of 2VO mice was
significantly impaired compared with the Sham group in slope (Fig. , RM-2ANOVA, F
(1, 4) = 29.97,
P = 0.0054 with Sidak’s post hoc test at
P0.1 = 0.8291,
P0.2 = 0.0250,
P0.3 < 0.0001,
P0.4 < 0.0001,
P0.5 < 0.0001,
P0.6 < 0.0001), and in amplitude (Fig. , RM-2ANOVA, F
(1, 4) = 37.28,
P = 0.0036 with Sidak’s post hoc test at
P0.1 = 0.5231,
P0.2 < 0.0001,
P0.3 < 0.0001,
P0.4 < 0.0001,
P0.5 < 0.0001,
P0.6 < 0.0001). The other three groups of mice were not significantly different.
Increased LTPs in response to 40 Hz light treatment of 2VO mice.
The input/output curve (I/O) of fEPSP slope (a) and amplitudes (b) across stimulation intensities of all four groups of mice (n = 3). c The LTPNMDAR
was induced by 100 Hz frequency tetanic stimulation of the Schaffer
collaterals projected to the CA1 pyramidal neurons on acute hippocampal
slices 3 d after 2VO (d). e The first 30 min of evoked responses were normalized and used as the baseline responses of LTPNMDAR. The magnitude of LTPNMDAR was determined according to the responses between 0 and 60 min after the HFS (n = 3). f LTPL-VGCC
was induced by stimulation of the Schaffer collaterals with 200 Hz
tetanic stimulation + D-APV (50 μm) throughout the experiment. g The first 30 min of evoked responses were normalized and used as the baseline responses of LTPL-VGCC. LTPL-VGCC magnitude was determined according to the responses between 0 and 60 min after the HFS (Sham n = 3 mice, Sham+LED n = 4 mice, 2VO n = 3 mice, 2VO + LED n = 4 mice). Data represent the mean ± SEM. Error bars indicate SEM. * indicates a comparison between Sham and 2VO group; # indicates a comparison between 2VO and 2VO + LED group. *, #P < 0.05, **, ##P < 0.01, ***, ###P < 0.001. The test used in a, b, d, f RM-2ANOVA with Sidak’s post hoc test. e, g 2ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file.
The 2VO group showed a significant lack of LTP
NMDAR compared to the Sham group at 3 d (RM-2ANOVA, F
(1, 4) = 578.70,
P < 0.0001, Fig. ; 2ANOVA with Tukey’s post hoc test,
P < 0.0001, Fig. ), 7 d and 14 d (Supplementary Fig.
4a–c)
after 2VO. Light flicker treatment of 2VO mice with 40 Hz, but not
10 Hz, 80 Hz, and random frequency, significantly increased LTP
NMDAR in the 2VO + LED group after 3 d (RM-2ANOVA, F
(1, 4) = 25.26,
P = 0.0074, Fig. ; 2ANOVA with Tukey’s post hoc test,
P = 0.0006, Fig. and Supplementary Fig.
4e, f), and 7d and 14 d (Supplementary Fig.
4a–c), demonstrating enhanced CA3-CA1 excitatory synaptic strength after 40 Hz light stimulation.
LTP
L-VGCC was pharmacological isolated using D-APV (50 μM) or D-APV + nifedipine (10 μM) (Supplementary Fig.
4d, g). A near-complete loss of LTP
L-VGCC occurred in 3 d 2VO mice compared with the Sham group (RM-2ANOVA, F
(1, 4) = 8.917,
P = 0.0405, Fig. ; 2ANOVA with Tukey’s post hoc test,
P = 0.0059, Fig. ). In contrast, 40 Hz light flicker restored LTP
L-VGCC to the normal level at 3 d when compared with 2VO group (RM-2ANOVA, F
(1, 5) = 162.6,
P < 0.0001, Fig. ; 2ANOVA with Tukey’s post hoc test,
P = 0.0020, Fig. ). 2VO mice treated with 10 Hz, 80 Hz, or random frequency light flicker failed to restore 2VO-induced reduction in LTP
L-VGCC (Supplementary Fig.
4h, i). These data demonstrated that 40 Hz light flickers enhanced postsynaptic LTPs.
40 Hz light flicker enhanced CA3-CA1 presynaptic plasticity
To
further understand the effect of light flicker on CA3-CA1 synaptic
transmission, presynaptic short-term plasticity was measured using the
paired-pulse ratio (PPR), which is inversely related to the initial
presynaptic vesicle release probability
31,32.
Control experiments were performed to show that neither D-APV nor
nifedipine affected CA3-CA1 PPR, since both target postsynaptic
receptors (Supplementary Fig.
5a, b). The 2VO group significantly elevated PPR compared to the Sham group (2ANOVA, F
(1, 15) = 26.5,
P = 0.0001, Tukey’ post hoc test
PSham vs. 2VO = 0.0009, Fig. ),
indicating the reduced readily release probability of
neurotransmitters. 40 Hz light treatment restored presynaptic readily
release probability and enhanced presynaptic short-term plasticity as
evidenced by the reduction of 2VO group PPR (2ANOVA, F
(1, 15) = 37.01,
P < 0.0001, Tukey’ post hoc test
P2VO+LED vs. 2VO = 0.0003, Fig. ).
In contrast, stimulation with arrhythmic light, 10 Hz, and 80 Hz light
did not affect 2VO group PPR severing as negative controls
(Supplementary Fig.
4j, k).
40 Hz light stimulation enhanced CA3-CA1 presynaptic short-term plasticity.
a
Hippocampal slices from the Sham, Sham+LED, 2VO, and 2VO + LED groups
at 3 d after the 2VO surgery were subjected to PPR measurements. The
inter-stimulus intervals (ISI) were from 20–200 ms. The ratios of the
slope of the second response to that of the first at the 40 ms ISI for a were measured and plotted in b (Sham: n = 5 mice; Sham+LED: n = 5 mice; 2VO: n = 4 mice; 2VO + LED: n = 5 mice). c
The protocol for determining the kinetics of FM1-43 release from
presynaptic terminals. Presynaptic terminals were labeled by exposure to
5 µM FM1-43 (numbers indicate time in minutes and seconds; zero time
represents the beginning of the unloading stimulation; Scale
bar = 2 µm). The first image was taken before unloading stimulation. d
Normalized fluorescence intensity decay of puncta to the initial FM1-43
loading intensity were plotted. Each point represents the average of a
total of 10–15 boutons from different slices of mice. The rate of puncta
unloading in brain slices (1/t1/2) was plotted and shown in e (Sham: n = 3 mice; Sham+LED: n = 3 mice; 2VO: n = 3 mice; 2VO + LED: n = 4 mice). Data represent the mean ± SEM. Error bars indicate SEM. ***, ###P < 0.001. The test used in b, e 2ANOVA with Tukey’ post hoc test. Source data are provided as a Source Data file.
FM1-43,
a fluorescent marker of presynaptic activity, was used to directly
visualize the dynamic changes of presynaptic vesicle release
probabilities due to 40 Hz light treatment. Imaging of presynaptic
vesicle exocytosis at the presynaptic boutons of CA3-CA1 excitatory
synapses in acute hippocampal slices was performed using two-photon
excitation microscopy (Fig. ). The brief loading protocol used here selectively labeled presynaptic terminals (Fig. )
33. The puncta shown in Fig.
represent glutamatergic presynaptic terminals from CA3 pyramidal
neurons, which accounted for most of the terminals examined in this
region. During a train of 1.5 Hz stimuli, the intensity of puncta
staining decayed with an approximately exponential time course (Fig. ), reflecting the first-order kinetics of vesicle release.
The rate of FM1-43 unloading, shown as the inverse halftime of intensity decay (1/t
1/2), in 2VO CA3 glutamatergic presynaptic terminals was significantly decreased compared with the Sham group (2ANOVA, F
(1, 9) = 23.26,
P = 0.0009, Tukey’s post hoc test
PSham vs. 2VO = 0.0006, Fig. ).
The 2VO + LED group completely restored the unloading kinetics to the
levels of the Sham group (2ANOVA with Tukey’s post hoc test,
P2VO vs. 2VO+LED = 0.0006, Fig. ),
providing additional evidence to support the PPR findings. In contrast,
stimulation with arrhythmic light, 10 Hz and 80 Hz light flicker did
not affect 2VO group unloading kinetics, severing as negative controls
(Supplementary Fig.
4l, m).
40 Hz light flicker increased 2VO mice dendritic plasticity
The
dendritic spine formation and alterations were examined under
two-photon excitation microscopy using transgenic Thy1-YFP male mice.
Dendritic spines of YFP-expressing CA1, CA3 pyramidal neurons were
examined in hippocampal brain slices using the Sholl analysis
34 (Fig. ).
A Z-stack of over 500 μm optical scans of the CA3-CA1 area were
compiled, and individual pyramidal neurons of interests were highlighted
using camera lucida tracing (Fig. ). Three types of dendritic spines were identified based on their shapes, including stubby, mushroom, and thin (Fig. ). More than 95% of them were stubby or mushroom-shaped boutons (Fig. lower panel). At 3 d after 2VO, a significant reduction in total number of spines (2ANOVA, F
(1, 20) = 23.45,
P < 0.0001, Tukey’s post hot test,
PSham vs. 2VO < 0.0001, Fig. ) and stubby shaped spines (2ANOVA, F
(1, 20) = 45.59,
P < 0.0001, Tukey’s post hot test,
PSham vs. 2VO < 0.0001, Fig. )
occurred. These spines were on the primary and secondary branches of
the apical dendrites of CA1 pyramidal neurons. 2VO + LED mice showed
significantly increased spine numbers on CA1 apical dendrites (2ANOVA
with Tukey’s post hoc test,
P2VO vs. 2VO+LED < 0.0001, Fig. ), particularly the stubby-shaped spines (2ANOVA with Tukey’s post hoc test,
P2VO vs. 2VO+LED < 0.0001, Fig. ),
indicating enhanced CA3-CA1 synaptic plasticity. It is worth noting
that at 3 d post 2VO, CA3 neurons were intact with no significant loss
of dendritic spines (2ANOVA, Fig. , F
(1, 20) = 0.738,
P = 0.4005; Fig. , F
(1, 20) = 0.1321,
P = 0.7200; Fig. , F
(1, 20) = 1.479,
P = 0.2381; Fig. , F
(1, 20) = 0.2157,
P = 0.6473).
Structural plasticity of CA1 and CA3 neurons in response to 40 Hz light treatment.
a A diagram of CA1 and CA3 areas where two-photon images were taken and shown in panel c. b Camera lucida of a CA1 pyramidal neuron for Sholl analysis. Scale bar = 100 μm. c
Representative pyramidal neurons in CA1 of four groups (yellow colored
lines) were put together using a z-stack of over 500 optical sections (6
repeats from n = 3 mice in each group). Scale bars = 50 μm. d
Representative two-photon images of stubby, mushroom, and thin-formed
dendritic spines (top panels) of the apical dendrites of Thy1-YFP mice
of Sham, Sham+LED, 2VO, and 2VO + LED groups at 3 d post-surgery (6
repeats from n = 4 mice). Scale bars = 5 μm. The percentage of
stubby, mushroom and thin shaped boutons were averaged and shown in the
lower panel of d (n = 4 mice). e–l
Quantifications of the number of dendritic spines per unit length of
the CA1 and CA3 apical and basal dendrites, respectively, at 3 d
post-surgery (n = 6 slices/3 mice in each group). Data
represent the mean ± SEM. Error bars indicate SEM. *indicates
differences between the Sham group and 2VO group; # indicates differences between the 2VO group and 2VO + LED group. #P < 0.05, ##P < 0.01, ***, ###P < 0.001. The test used in e–l 2ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file.
To
demonstrate the long-term effects of 40 Hz light flicker on CA1
dendritic spines, CA1 and CA3 apical and basal dendritic spines were
examined at 3 d, 7 d, and 14 d post 2VO. 2VO caused a significant loss
of CA1 dendritic spines throughout the 14 d periods (Supplementary Fig.
6a, b), while 40 Hz light treatment prevented the loss of CA1 dendritic spines (Supplementary Fig.
6a, b). In contrast, the loss of CA3 dendritic spines only appeared at 7 d and 14 d post 2VO (Supplementary Fig.
6c, d), and such a loss was completely rescued in the 2VO + LED group (Supplementary Fig.
6c, d),
indicating that loss of connection with CA1 neurons may eventually lead
to CA3 dendritic spine loss in the absence of 40 Hz light treatment.
Together, these results demonstrated that 40 Hz flicker light
strengthened CA3-CA1 synaptic connections.
40 Hz light flicker altered hippocampal protein expression
To
identify specific molecular pathways involved in CA3-CA1 network
responses to 2VO and 40 Hz light treatment, the whole hippocampi from
2VO, 2VO + LED, and the Sham group 3 d post-surgery were isolated and
subjected to proteomic analysis (Fig. ).
Using bioinformatic gene ontology analysis, 2VO altered the expression
of more than 60 proteins over 1.2-fold changes compared with the Sham
group, and 31 of those with altered expression in the 2VO + LED group
(Supplementary Fig
7).
The identified protein groups were related to synaptic functions (45%),
cell death (24%), and transcription and translation (14%) (Fig. ).
The remainders were related to cell differentiation (5%), epigenetic
regulations (5%), immune response (3%), oxidative stress (2%), and
mitochondrial functions (2%). We focused on genes potentially involved
in modulating synaptic neurotransmission, such as the regulator of
G-protein signaling 12 (RGS12), a regulator of the N-VGCC
35. The expression level of RGS12, shown by western blotting (Fig. ), was down-regulated by 20% and 30% in 2VO group at 3 d and 7 d, respectively, compared with Sham group (2ANOVA, F
(1, 8) = 5.3,
P = 0.0503, LSD’ post hot test,
PSham vs. 2VO = 0.0263, Fig. ), and up-regulated by 60% and 35% at 3 d and 7 d 2VO + LED group, respectively, compared with the 2VO group (2ANOVA, F
(1, 8) = 5.53,
P = 0.0466, LSD’ post hot test,
P = 0.0249, Fig. ).
Profiling hippocampal protein expression associated with 40 Hz light treatment.
a GO analysis according to the functions of 31 proteins, which significantly altered their expression in the 2VO + LED group. b Representative western blots are showing increased levels of RGS12 expression 3 d (4 repeats from n = 2 mice) and 7 d (6 repeats from n = 3 mice) post 2VO. c, d Quantifications of RGS12 expression using β-actin as an internal standard of 3 d (n = 2 mice in each group) and 7 d (n = 3
mice in each group) post 2VO groups, respectively. Data represent the
mean ± SEM. Error bars indicate SEM. * is for comparison between Sham
and 2VO groups; # is for comparison between 2VO and 2VO + LED groups. *,#P < 0.05. The test used in d 2ANOVA with LSD’s post hoc test. Source data are provided as a Source Data file.
Increased RGS12 modulation of N-VGCC short-term plasticity
To
demonstrate RGS12 involvement in presynaptic plasticity in response to
40 Hz light flicker, two experimental approaches were used: a decoy
peptide blocking the interaction of endogenous RGS12 with N-VGCC, and an
AAV-shRNA knocking down RGS12 expression in the 2VO brain (Fig. ).
The decoy RGS12 peptide GK23 has an identical peptide sequence to that
of the N-VGCC centered around Tyr-804. The GK23 sequence was completely
lacking in L-VGCC, confirming the specificity of GK23 (Supplementary
Fig.
8a). GK23 significantly increased PPR compared with the control peptide GK13 (Supplementary Fig.
5c, d),
demonstrating its effectiveness. RGS12 decoy peptide GK23 was delivered
to the left cerebroventricle using an Alzet® osmotic pump followed by
2VO surgery and 40 Hz light treatment. As shown in Fig. ,
GK23, and ω-conotoxin GVIA significantly enhanced PPR of the 3 d
2VO + LED group compared with the GK13-treated 2VO + LED group (1ANOVA, F
(2, 6) = 5.559,
P = 0.0431, Dunnett’s post hoc test,
PGK23 vs. GK13 = 0.0351,
PGK23 vs. ω-conotoxin GVIA = 0.7761, Fig.
left), confirming RGS12 regulation of 40 Hz light-induced presynaptic
plasticity. FM1-43 was also used to directly image presynaptic vesicle
exocytosis. Acute hippocampal slices from the above-treated 2VO + LED
mice were used. The halftime of puncta intensity decay (1/t
1/2) was significantly reduced in GK23, and ω-conotoxin GVIA treated 2VO + LED mice (1ANOVA, F
(2, 6) = 13.35,
P = 0.0062, Dunnett’s post hoc test,
PGK23 vs. GK13 = 0.0052,
PGK23 vs. ω-conotoxin GVIA = 0.6388, Fig. left). Furthermore, GK23 and ω-conotoxin GVIA completely inhibited the occurrence of postsynaptic LTP
NMDAR and LTP
L-VGCC in the 2VO + LED group (Fig. : RM-2ANOVA, GK13 vs. GK23: F
(1, 4) = 34.5,
P = 0.0042, GK23 vs. ω-conotoxin GVIA: F
(1, 4) = 0.001461,
P = 0.9713. Figure left: 1ANOVA with Dunnett’s post hoc test
PGK23 vs. GK13 = 0.0013,
PGK23 vs. ω-conotoxin GVIA = 0.9985. Figure : RM-2ANOVA, GK13 vs. GK23: F
(1, 5) = 9.099,
P = 0.0295, GK23 vs. ω-conotoxin GVIA: F
(1, 5) = 0.006603,
P = 0.9384. Figure left: 1ANOVA with Dunnett’s post hoc test
PGK23 vs. GK13 = 0.0170,
PGK23 vs. ω-conotoxin GVIA = 0.9945).
These data demonstrated that inhibition of presynaptic RGS12
interaction with N-VGCC suppressed light flicker restoration of
presynaptic release probability in 2VO mice.
RGS12 regulation of N-VGCC-dependent PPR, vesicle release, and LTPs.
a
Mouse was implanted with an osmotic pump delivering GK13, GK23, or
ω-conotoxin GVIA before the 2VO surgery and 40 Hz light treatment
throughout the 3 d periods. Hippocampal slices were subjected to PPR
measurements. b AAV-shRNA-Ctl and AAV-shRNA-RGS12 were
stereotaxic injected into the CA1 area for 3 weeks, followed by 2VO and
40 Hz light treatment (2VO + LED) for 3 d. Hippocampal slices were
measured for PPR. PPR at the 40 ms ISIs for both a and b were measured and plotted in c. a, b, and c share the same legends (n = 3 mice each group). The hippocampal brain slices of 2VO + LED mice treated either with GK13, GK23, and ω-conotoxin GVIA (d) or with AAV-shRNA-Ctl and AAV-shRNA-RGS12 (e)
were measured for presynaptic vesicle release probability based on the
kinetics of FM1-43 release from presynaptic terminals. Rate (1/t1/2) of puncta unloading in brain slices was plotted and shown in f. d, e, and f share the same legend (n = 3 mice each group): g–l Measurements of LTPs. The effect of osmotic pump application of RGS12 decoy peptide GK23 (g, i, j, l) and AAV-shRNA-RGS12 (h, i, k, l) on CA3-CA1 LTPs of 2VO mice 3 d after 40 Hz light treatment (2VO + LED) was determined using 100 Hz (g,h) or 200 Hz (j,k) tetanic stimulations (g–l
n = 3 mice in all groups except n = 4 mice in LTPL-VGCC GK 23 group). Data represent the mean ± SEM. Error bars indicate SEM. *P < 0.05, **P < 0.01. The test used on the left side of c, f, i, l 1ANOVA with Dunnett’s post hoc test; on the right side of c, f, i, l Two-tailed unpaired t-test; g, h, j, k RM-2ANOVA with Sidak’s post hoc test. Source data are provided as a Source Data file.
The
second approach was to use AAV-shRNA to knockdown CA1 RGS12 expression.
AAV-shRNA-RGS12 and control AAV-shRNA-CTL structures were shown in
Supplementary Fig.
8b. The hippocampal CA1 neuron expression of AAV-shRNA-RGS12 for 3 weeks (Supplementary Fig.
8c) successfully reduced the expression level of RGS12 in the hippocampus (Supplementary Fig.
8d).
Knocking down RGS12 expression level produced a similar effect to that
of the GK23 decoy peptide in terms of the increased CA3-CA1 PPR in
2VO + LED mice (Two-tailed unpaired t-test, t
(4) = 8.197,
P = 0.0012, Fig. , right), and reduced presynaptic vesicle release probability (Two-tailed unpaired t-test, t
(4) = 3.011,
P = 0.0395, Fig. right). AAV-shRNA-RGS12 also significantly reduced the levels of postsynaptic LTP
NMDAR and LTP
L-VGCC in the 2VO + LED group of mice compared with AAV-shRNA-CTL (Fig. : RM-2ANOVA, F
(1, 4) = 10.4,
P = 0.0321. Fig. right: Two-tailed unpaired t-test, t
(4) = 3.225,
P = 0.0321. Figure : RM-2ANOVA, F
(1, 4) = 63.5,
P = 0.0013. Figure right: Two-tailed unpaired t-test, t
(4) = 7.969,
P = 0.0013).
Importantly, knocking down RGS12 diminished the rescuing effect of
40 Hz light flicker on CA1 low gamma (Supplementary Fig.
8e, f).
In contrast, theta and high gamma oscillations PSD ratios were not
affected throughout the 14 d treatment (Fig. S8g, h), demonstrating the
important role of RSG12 in mediating 40 Hz light response in the CA1
neurons.
Collectively, these data
demonstrated that 40 Hz light flicker conferred neuroprotection through
increased RGS12-regulation of the N-VGCC-dependent CA3-CA1 synaptic
plasticity. Enhanced CA3-CA1 synaptic strength provided a survival
signal to CA1 neurons leading to neuroprotection during cerebral
ischemia.
Discussion
Data
presented here support a causal relationship between hippocampal low
gamma and cognitive impairment in behaving 2VO mice. Modulation of
hippocampal low gamma using 30–50 Hz light flicker (1) restored
hippocampal CA1 slow gamma and phase-amplitude coupling to theta
oscillations in free behaving 2VO mice; (2) protected CA1 neurons; (3)
improved locomotion, spatial learning, and memory; and (4) enhanced
CA3-CA1 synaptic strength through an RGS12 modulation of
N-VGCC-dependent pathway. Future investigations of the precise neural
circuitry modulated by the light flicker are warranted.
The
current findings that cerebral ischemia affects low gamma in
free-behaving mouse and that the 30–50 Hz light flickers can restore low
gamma to confer neuroprotection through an RGS12-modulated enhancement
of CA3-CA1 synaptic plasticity are novel. Our data that 40 Hz flicker
light can modulate hippocampal low gamma are in agreement with published
studies
10,12,23,24. It is worth noting that our studies were performed when the mouse locomotion state was free from generating theta harmonics
20,21.
Flickering light did not cause the mouse to run faster since OFT data
shows that these mice had no anxiety behaviors during and after the
light treatments (Fig. ). The PAC strength depends on behavior and is one of the most predictive tools to demonstrate relations to learning and memory
36,37.
The present study revealed theta-low gamma PAC deficits after cerebral
ischemia and light flicker restore PAC in behaving mice engaged in an
object exploration behavior.
Neuroprotection conferred
by 40 Hz light flicker is striking. Strengthening of CA3-CA1 excitatory
synapses could send pro-survival signals to the CA1 neurons, making the
CA1 neurons more resistant to reperfusion-induced neuronal death. The
proteomic analysis identified groups of genes regulating synaptic
plasticity and cell death, which may contribute to CA1 neuronal
survival. The literature also supports that activation of synaptic NMDAR
promotes postsynaptic neuron survival, while extra-synaptic NMDAR
promotes neuronal death
38.
L-VGCC activity transmits calcium-regulated survival signaling to the
nucleus that supports learning and memory. Inhibiting L-VGCC and LTP
L-VGCC causes delayed death of CA1 neurons in 2VO
18,39. Thus, 40 Hz light drives low gamma to enhance CA3-CA1 synaptic strength to achieve neuroprotection.
There
are three lines of evidence supporting 40 Hz light flicker enhancement
of synaptic strength. First, the activity-triggered RGS12 modulation of
N-VGCCs-dependent presynaptic release probability, which serves as an
essential link between presynaptic membrane depolarization and
presynaptic vesicle exocytosis in tuning neurotransmission. PPR and
FM1-43 data are reliable readouts of presynaptic N-VGCC-initiated fast
release of neurotransmitters
40.
Preventing RGS12 binding to the tyrosine-phosphorylated N-VGCC
pore-forming α-subunit using RGS12 decoy peptide and rAAV-RGS12 lent
further support of RGS12’s novel role in enhancing synaptic strength to
40 Hz light flicker treatment.
Second, light flicker restored both LTP
NMDAR and LTP
L-VGCC
in the 2VO hippocampus. Enhancement of excitatory neurotransmitter
release by light flicker activated ionotropic NMDAR and could trigger
AMPA-type glutamate receptor trafficking to the dendritic surface in an
L-VGCC-dependent manner
41.
L-VGCC tunes the surface expression of key synaptic proteins to enhance
synaptic strength. Indeed, theta-burst stimulation induces LTP
L-VGCC42. Inactivation of the Ca
V1.2 gene causes deficiencies in NMDAR-independent LTP and impairment in spatial learning
18,32.
Third,
the formation of stable dendritic spines also represents signs of
enhancement of synaptic strength in response to 40 Hz light treatment.
The shapes of individual dendritic spines are proposed to correlate with
their capacity for structural changes. In particular, the stubby and
mushroom-shaped boutons located on CA1 apical dendrites were accounted
for 90% of the increased boutons. These two types of boutons are spines
that endure structural modifications resulting in enhanced LTPs. CA3
spine losses at 7 d and 14 d post-2VO were rescued by light flicker
treatment supporting the role of light flicker in the strengthening of
CA3-CA1 synaptic connections.
Microglia
activation in cerebral ischemia can be either beneficial or detrimental
to neurons depending on the timing. Although 40 Hz light activates
microglia in Alzheimer’s disease mouse brain to clear β-amyloids
10,11,
the current data failed to indicate microglia response to light flicker
in the ischemia brain. This is based on microglia immunostaining and
the lack of microglia-related gene expression in proteomic profiling.
The light treatment time may underlie the lack of response of microglia
in the current 2VO studies.
Methods
Key resources table
Which
contains information about the antibodies used, pharmacological agents,
peptides, and viral vectors. The table can be found in the online
Source Data file.
Animals
Adult
male C57Bl/6 mice (3-months-old) were purchased from the Guangdong
Medical Laboratory Animal Center. Adult male Thy1-YFP-H mice (3 months
old) were purchased from the Jackson Laboratory (USA). Mice were
maintained in a pathogen-free SPFII animal facility in a
condition-controlled room (23 ± 1°C, 50 ± 10% humidity). A 12 h
light/dark cycle was automatically imposed. Mice were housed in groups
of four to five per individually ventilated cages and given access to
food and water ad libitum unless otherwise stated in the methods below.
Experimenters were blinded to animals’ treatments and sample processing
throughout the subsequent experimentation and analyses.
Ethical approval and animal study design
All
animal experiments were conducted according to protocols approved by
the Animal Care Committee of the Southern University of Science and
Technology (Shenzhen, China). The ARRIVE guideline was followed when
designing and performing animal experimentation
43.
Efforts were made to minimize the number of mice used. The inclusion
criterion was based on the identical age and sex of the mice. The
exclusion criterion was when the mouse failed to survive 2VO surgery at
the end of the 14 d periods of the experimentation. To achieve
meaningful statistical differences, a minimal of five mice per group
were used in the LFP in vivo recordings, behavioral studies, RGS12 gene
expression modifications experiments as indicated in the figure legends.
For tissue section staining and western blotting experimentation, at
least three mice per group were used.
A
total of 112 mice with the same age (3-month-old) and sex (male) were
first randomly assigned to two groups (56 each) to receive Sham or 2VO
surgery, respectively. After the surgery, mice in each group were
randomly assigned to receive LED light or normal light treatment. In the
end, these mice became four groups to receive Sham, Sham+LED, 2VO,
2VO + LED treatments. Data derived from all qualified animals were
included in the analyses and presentation of the results.
Mouse two-vessel occlusion (2VO) ischemia model
Bilateral occlusion of the mouse common carotid arteries was performed
44.
Animals were fasted overnight, but free to drink, before surgery. Mice
were anesthetized with a mixture of 2.5% isoflurane in N
2:O
2
(70:30; flow rate 400 ml/min) in an induction chamber (Product #R540,
RWD Life Science, Shenzhen, China). When the animal showed no sign of
consciousness, it was moved to the anesthetic face mask supplied with
1.5% isoflurane in N
2:O
2 (70:30; flow rate
400 ml/min). The animal's body temperature was maintained at
37.0 ± 0.5 °C using a rectal temperature probe and a heating blanket
(Product # TCAT-2 Temperature Controller, Harvard Apparatus, USA). Furs
around the ventral neck region were shaved using a hair clipper to
expose the skin which was disinfected with iodine followed by 75%
ethanol. Under the operating microscope, an incision in the midline of
the ventral neck region was made with a scalpel. The superficial fascia
was dissected to expose the bilateral common carotid arteries.
Surgery for 2VO should be combined with systemic hypotension (35–40 mm Hg) to induce sufficient brain ischemia
19,44,45.
To do this, the isoflurane concentration was increased to 5% for 2 min,
and the carotid arteries were clamped with micro-vessel clamps for
5 min to induce global cerebral ischemia. The isoflurane level was
decreased to 0% 2 min before the release of the micro-vessel clamps and
resume of blood reperfusion. The neck incision was sutured with 5-0
sterile silk sutures. The animals were moved into a warm (37.0 ± 1 °C)
recovery chamber (Product #DW-1, Harvard Apparatus, USA) to prevent
post-ischemic hypothermia. For Sham operations, micro-vessel clamping
was performed on the bilateral common carotid arteries. The clamping was
immediately released to allow for instant reperfusion. The subsequent
operation is identical to that performed on animals undergoing cerebral
ischemia, including anesthesia processes.
Measurement of mean arterial blood pressure (MABP)
The
mean arterial blood pressure was measured using a non-invasive blood
pressure system (Product #CODA, Kent Scientific Corporation). Before
surgery, the occlusion tail-cuff was placed through the tail and towards
the base of the tail until encountering resistance. The volume pressure
recording (VPR) sensor cuff was then slid up the tail until reaching
the occlusion cuff. The MABP recording started 2 min before the onset of
administering of 5% isoflurane in the respiratory gases and continued
during the 5 min period of 2VO and into the first 2 min of reperfusion.
During the 2 min of administering of 5% isoflurane, the MABP dropped to
about 40 mmHg, at which time both common carotid arteries were occluded.
The MABP stabilized at 35 mmHg during the occlusion period without
further manipulation of isoflurane levels. MABP of every mouse
undergoing either sham-operation or 2VO surgery was subjected to
hypotension and measurement of MABP.
Measurement of regional cerebral blood flow (rCBF)
Mouse
hippocampal blood flow (hrCBF) was detected by using a laser Doppler
flowmetry (moorVMS-LDF2, Moor Instruments, Wilmington, DE) with a 0.5 mm
flexible fiber optics. The longitudinal hippocampal artery provides the
blood supply to the ventral and dorsal parts of the hippocampus. It
follows a path approximately parallel to the longitudinal axis of the
hippocampus and close to the hippocampal fissure. Localization of the
longitudinal hippocampal artery was determined using the
three-dimensional digital mouse brain atlases
9,46–48.
After shaving the head fur and disinfecting with iodine and 75%
ethanol, an incision was made on the head skin with a scalpel and the
tip of the probe was fixed on to the surface of the skull (bregma: −2.0
anterior/ posterior, 1.5 medial/lateral in mm). The intact skull was
moisturized by silicone oil to better visualize the vascular landmarks.
The hrCBF recording started 2 min before the onset of administering 5%
isoflurane in the respiratory gases. Recordings were made during the
entire 5 min of occlusion of the common carotid arteries and continued
for 2 min into reperfusion. Five mice per group were used to determine
2VO ischemia/reperfusion-induced changes in hrCBF.
Laser
speckle flowmetry: Total cortical blood flow was also measured using a
laser speckle contrast imager (MOORFLPI-2, Moor Instruments, Wilmington,
DE). Four groups of mice were measured after 3 d, 7 d, and 14 d of 2VO.
To reduce movement-induced blood flow artifacts, mice were anesthetized
during the measurement following the instrument manufacturer’s
instructions. The skin of the scalp was cut along the sagittal plane to
expose the braincase for imaging. A bolus of saline was applied to the
skullcap to normalize the effect of light refraction between animals.
Blood flow of the region of interest, such as the visual cortex, was
selected and measured using the onboard software (mFLPI2MeasV2.0 for
recording and moorFLPIReviewV50 for analysis).
Visual stimulation protocol
The visual stimulation equipment, TangGuangTM,
consisted of seven modules, including a tunable frequency signal
generator and six LED lamps (36 V). The six LED lamps were connected by
parallel circuits and equally distributed around the two transparent
cages, which separately housing the control and experimental groups,
respectively, at the same time in order for the animals to
simultaneously receive light stimulation. The first session (1 h) of LED
light flicker visual stimulation was performed 2 h after surgery. The
stimulation was conducted twice per day with the first session between
8–9 a.m. and the second session between 8–9 p.m. throughout the whole
experimental duration (up to 14 d). Delayed light treatment was also
performed 3 d after the 2VO and lasted up to 7 d post-ischemia. In
total, 10 Hz, 30 Hz, 50 Hz, 80 Hz, and random frequency LED light
flicker visual stimulation were conducted on mice using the same
protocol. The animal's body temperature was monitored and maintained at
37.0 ± 0.5 °C using a rectal temperature probe during the light
treatment.
Intracranial electrode implantation surgery
Seven
days before 2VO or Sham surgery, mice were prepared for implantation of
electrodes to record local field potentials (LFPs). Animals were fasted
overnight, but freed to drink, before surgery. Mice were anesthetized
with a mixture of 2.5% isoflurane in N2:O2 (70:30; flow rate 400 ml/min) in an induction chamber. Anesthesia was maintained with 1.5% isoflurane in N2:O2
mixture during surgery using an isoflurane vaporizer (Product #R540,
RWD Life Science, Shenzhen, China). The animal's body temperature was
maintained at 37.0 ± 0.5 °C using a rectal temperature probe and a
heating pad (Product #TCAT-2DF, Harvard Apparatus, USA).
A
cranial window (1.2 mm in diameter, anteroposterior, A/P: 2.0 mm;
mediolateral, M/L: 1.5 mm) over the left hippocampus was created using a
dental drill (Product #78001, RWD Life Science, Shenzhen, China) guided
by stereotaxis (Product # 68861 N, RWD Life Science, Shenzhen, China). A
4-channels microwire array electrode (35 μm, Stablohm 650, California
Fine Wire Co., USA) was inserted into the hippocampal stratum radiatum
in area CA1 and immobilized using kwik-sil (Item #KWIK-SIL, Microprobes
for Life Science, Gaithersburg, MD, USA). The electrode was anchored to
the skull bone using 4 skull screws (1.0 mm diameter) and embedded in
dental cement. Mice were then moved into a warm (37.0 ± 1 °C) recovery
chamber (Product #DW-1, Harvard) for 1 h.
LFP recording and analysis
After
4 d recovery from electrode implantation surgery, mice were adapted to
recording position by placing them in a box (50 depth X 50 width X 50
length in cm) 10 min per day until the first recording session. Before
each recording session, the box was cleaned by 75% ethanol. During
recording, the implant was gently held by a helium balloon allowing the
mouse to move freely in the box. To avoid the impact of movement speed
on the linearity of theta waves
22,
three low-speed movement states were used for the study, i.e.,
approach, exploration, and low-speed free walking. These low-speed
states are relevant to 2VO mice since the mouse does not normally engage
in fast running after the surgery. The speed/velocity of movement for
the three states was at 2~3 cm/s forward, as determined by footfall
patterns
22.
Head-stage
was connected to the OmniPlex Neural Recording Data Acquisition System
(Plexon Inc, Dallas, TX). A camera was set in the front of the box to
record the behavioral states of the mouse. An LED lamp was put in front
of the box to generate different frequency lights. LFPs in the CA1
region were recorded for at least 20 min for each mouse. Each mouse
received the same pattern of frequencies of light stimulation with a 2 h
break in between. These light flickers were at 10 Hz, 30 Hz, 40 Hz,
50 Hz, 80 Hz and an arrhythmic frequency. A total of five mice in each
group were used for statistical analysis. The LFP signals were sampled
at 1000 Hz with a band-pass filter set at 0.5−300 Hz. Raw data were
stored for later off-line analysis.
For LFP data
analyses, a multitaper fast Fourier transform method was implemented for
power spectral analyses using the NeuroExplorer software (Version 5,
Nex Technologies, Colorado Springs, CO, USA). Data were filtered from 0
to 120 Hz using the Digital Filter of Continuous Variables function
within the NeuroExplorer software. Frequency-Power curve was generated
using NeuroExplorer software Power Spectra for Continuous function.
Fig.
low-speed walking spectrogram was generated using the NeuroExplorer
software Spectrograms function. Time-frequency spectrograms were
generated using the NeuroExplorer software
Perievent Spectrograms function. The power spectrum in Fig. was given by a multi-taper estimation method using MATLAB using Eq. (
1). Given a time series
Xn,
n = 1, 2, …,
N, the number of the Slepian sequences is
K = 2
NW−1. The simplest multi-taper estimate of the spectrum is given by
Where
ukn,n=1,2,…,N is the
kth Slepian sequence.
Phase-amplitude
coupling analyses of coupled oscillations were obtained by bandpass
filtering signals between 4 and 12 Hz with a Hilbert transform
36. The MATLAB code used can be referenced in previous published works
49. The phase-amplitude comodulogram scans multiple frequency pairs searching for cross-frequency coupling.
Two
marks were manually added to the signal traces to indicate the times of
recording during approach and exploration, and a stable free walking
period. Only signal segments between these marks were analyzed. Among
the overall recording during these periods, 2 s × 10 representative
epochs free of major artifacts were randomly selected for calculating
LFP power and coherence. Sections of LFP data from at least 2 s of rest
period were selected for the performance of event-aligned Time-frequency
analysis using Perievent Spectrograms function [Frequency
Range: 0–120 Hz or 30–50 Hz; Shift Type: Absolute (s); Number of Shifts :
20; Shift (s): 0.1; Tapers : Single; Windowing Function: Hann]. We
calculated the ratio of power spectral density between PSDexploration and PSDapproach in theta, low gamma, 40 Hz region, and high gamma.
Mouse behavioral tests
All
behavioral tests were performed between 2 and 6 p.m. The apparatus was
carefully cleaned by wiping with 75% ethanol after each experiment to
remove the olfactory distraction. The person performing the test was
blind to the animal groups. All mice were familiarized with the
researchers
50.
Open field test
Mouse
spontaneous activity in the open field was tested using a clear plastic
cube box (41× 41× 38 cm) under a camera. The center region of the box
field was defined as a 20 cm × 20 cm virtual area. Each mouse was given
10 min to move freely in the box. All traveled distances and the
distance trace center were recorded and analyzed using Panlab software
Smart (V3.0, Harvard Apparatus, Holliston, MA). The rearing numbers,
representing the numbers of times mouse lifting the forepaws to explore
upwards, were recorded by the experimenter.
Morris water maze (MWM)
Morris
water maze was performed to determine the spatial learning ability and
reference memory. MWM was performed in a blue water pool (120 cm in
diameter with 2/3 of transparent water) containing a circular bright
blue platform (14 cm in diameter and submerged 1.5 cm beneath the water
surface). Various patterns were equally distributed around the wall as
visual cues for mice during the experiment.
For spatial
acquisition test, the spatial acquisition learning ability training
consisted of five consecutive days with every training day comprising of
four trials with 15 min inter-trial interval. The entry points were
randomly selected each time from the different designated locations.
Once the mouse successfully found the platform within 60 s, it was
placed into a cage under a warming lamp as a reward. Otherwise, the
mouse was gently and manually guided to the platform and allowed to
remain there for at least 20 s. The escape latency and pathlengh to the
platform was recorded each day to assess the spatial learning ability.
On
day 6, a probe trial test was performed. The hidden platform was
removed from the pool, and the mouse was placed in the quadrant
diagonally opposite the target quadrant and allowed to swim for 60 s.
The percentage of time spent in the target quadrant was recorded and
analyzed as a measure of spatial memory retention (or the reference
memory). Mice were monitored by a camera, and its trajectory was
analyzed using the Smart (V3.0, Panlab Harvard Apparatus).
Y-maze spontaneous alternations
To
assess the spatial working memory of the mice, spontaneous alternation
behavior was assessed in a Y maze after the MWM. The Y maze used in this
test was composed of three identical acrylic light blue arms (31 cm
long, 17 cm high, and 9.5 cm wide). During each trial, the mouse was
placed at the maze center and allowed to explore freely through the maze
for 5 min. The sequence of arm entries was recorded by a monitor right
above the apparatus and analyzed using the video tracking Panlab
software (Smart V3.0, Harvard Apparatus). Alternation percentage (%) was
calculated as a proportion of arm choices differing from the previous
two choices. To minimize the difference made by experimenter handling,
mice were handled by the same one experimenter throughout all behavioral
experiments.
Acute hippocampal slices
Mice
(either C57BL/6 or Thy1-YFP-H strains) were euthanized by cervical
dislocation. Brains were immediately harvested and placed into ice-water
cutting solution composed of (in mM): 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 2 MgSO4, 15 glucose, and 220 sucrose, saturated with 95% O2/5% CO2
(v/v). Coronal slices (300 μm thickness for electrophysiology recording
and 400 μm thickness for two-photon imaging) containing the hippocampus
were cut using a vibratome (VT1120S, Leica Systems). Hippocampal slices
were rinsed twice in artificial cerebrospinal fluid (aCSF), which
composed of (in mM) 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 2 MgSO4, and 15 glucose. Slices were gently moved to a brain slice keeper containing aCSF saturated with 95% O2/5% CO2
(v/v). Incubation of slices at temperature 37 °C for 30 min before
transferring to room temperature for at least 1 h prior to recording or
imaging. Slices were then individually transferred to a recording
chamber fixed to the stage of an upright microscope. During recording or
imaging, slices were continuously perfused with aCSF saturated with 95%
O2/5% CO2 (v/v) at a rate of 3 ml /min.
Electrophysiology of LTP and paired-pulse ratio (PPR)
Coronal
hippocampal slices (300 μm) were prepared using a Vibratome slicer
(VT1120S; Leica) in ice-cold aCSF. All recordings were conducted in a
submerged recording chamber with aCSF (32 ± 0.5 °C, 3 ml/min), saturated
with 95% O
2/5% CO
2 (v/v)
50,51.
Injection of aCSF into the glass pipettes (1–5 MΩ) allowed the
recording of the field excitatory postsynaptic potentials (fEPSPs)
evoked in stratum radiatum of CA1 region by stimulating Schaffer
collateral/commissural (Sch/com) through a concentric bipolar
stimulating electrode. After slice recovery in the recording chamber
(10 min), input-output curves relating stimulus current intensity to
fEPSP slope and amplitude were generated. The stimulus intensity
required for the half-maximal fEPSP slope was selected.
PPR
experiments were performed using inter-stimulus intervals (ISIs) of 20,
40, 60, 80, 100, and 200 ms. PPF (%) was estimated by the ratio of the
second pulse-induced fEPSP slope to the first one. Baseline responses
were recorded per 30 s for 30 min before tetanus stimulation and
normalization.
NMDA receptor-dependent LTP (LTPNMDAR) was induced by applying a high-frequency stimulation (100 Hz, 1 s). L-type voltage-gated calcium channel-dependent LTP (LTPL-VGCC)
was induced with four stimulus trains (200 Hz, 0.5 s) delivered at a
rate of 1 train per 5 s in the presence of an NMDA receptor antagonist,
D-APV (50 μM, SIGMA). An L-VGCC blocker, Nifedipine (10 μM, SIGMA), was
applied to reduce the evoked responses induced in the presence of D-APV.
For PPF experiments, brain slices were preincubated in D-APV or
Nifedipine for 20 min before recording. For LTP recording, D-APV and
Nifedipine were delivered after 10 min onset of baseline response
recordings. ACSF containing D-APV or Nifedipine or both were used to
perfused brain slices during recording. Individual brain slices were
used to PPF, LTPNMDAR, and LTPL-VGCC recoding, respectively.
Both LTPNMDAR and LTPL-VGCC
were determined according to responses between 45 and 60 min after
conditioning stimulation. The evoked responses were recorded in
current-clamp mode using Axon MultiClamp 700B (Molecular Devices, USA)
amplifier.
Data were acquired and
analyzed with pClamp 9 (Molecular Devices, Sunnyvale, CA) and GraphPad
Prizm7.0 (San Diego, CA). Slices from each animal were averaged together
to give an n value of one. Statistical analyses of pooled data were
performed by Students t-test, one sample t-test vs. a theoretical mean
of zero or 100%, or repeated measure two-way ANOVA. P < 0.05 was considered to be statistically significant. Data are presented as mean ± SEM with the number of determinations (n) representing the number of recordings.
Two-photon excitation microscopy
The
Thy1-YFP-H transgenic mice selectively express yellow fluorescent
protein at high levels in hippocampal pyramidal neurons as well as
subsets of central neurons. Axons are brightly fluorescent all the way
to the terminals making this type of mice widely used for in vivo
imaging studies. An upright FVMPE-RS multiphoton microscopy system
(Olympus, Japan) based on an Olympus upright microscope equipped with a
water objective (25X/1.05, W.D. = 2 mm) was used. A femtosecond-pulsed
Ti:Sa laser (Mai Tai DeepSee, Spectra-physics, USA) was applied to
produce 960 nm laser for excitation of YFP protein expressed in
hippocampal pyramidal neurons.
Laser power was 120 mW
under the objective. Hippocampal slices were continuously perfused at
3 ml / min with aCSF, saturated with 95% O
2/5% CO
2
(v/v) for imaging. Images of CA1 and CA3 regions were taken with 512 x
512pixel resolutions and 0.99 μm per pixel size. Both apical dendrite
and basal dendrite were imaged under the same resolution with 0.1 μm per
pixel size. To quantify apical dendritic spines of CA1 and CA3 neurons,
Scholl analysis
34
was performed on the second and third-level branches within the region
of interest (ROI). At least 6 dendrites in CA3 and CA1 region for both
apical and basal dendrite on each hippocampal slice were imaged.
All
images were analyzed using ImageJ (NIH) and Imaris (Version 9.2, Oxford
Instruments) software. Dendritic spines were classified into a stubby,
mushroom, and thin shapes based on their morphology, and each class was
counted separately on a given length of the dendrite. Spine density was
averaged per length of dendrite and plotted.
Visualization of presynaptic vesical release: loading
Presynaptic boutons were loaded by bath-applying 5 μM FM1-43 (Molecular Probes, Eugene, OR) in 45 mM K
+
aCSF for 15 min with D-APV (50 μM) added to the external solution to
prevent synaptic driven action potentials from accelerating dye release
33,52.
After dye-loading, slices were transferred to aCSF containing ADVASEP-7
(0.1–0.15 mM) for 30–35 min to remove any dye bound to extracellular
tissue. The stimulus-induced distaining was evoked using 1.5 Hz
stimulation, as the bipolar tungsten electrode placed into the CA1
stratum radiatum region at a depth of approximately 50–100 µm. During
this process, the D-APV was always in the external solution to prevent
excitotoxic damage and the induction of plasticity caused by synaptic
stimulation.
Visualization of presynaptic vesical release: imaging
A
Two-photon patch-clamp system (Scientifica, Hyperscope, MMTP-3000, UK;
HEKA, EPC-10 USB Double, GER) equipped with a Ti:sapphire laser (900 nm;
Coherent, Chameleon Ultra II) was used for two-photon excitation
microscopy. Images were acquired using a 16 X 0.9 NA water-immersion
ultraviolet objective (CFI75 LWD) with Lasersharp software provided by
BioRad. A series of 6 images (512 × 512 pixels, 0.077 µm/pixel in the x–y axes) at different focal planes were acquired every 26–27 s with a 1-µm step in the z-direction. Then a subset of 3–6 contiguous in the z-axis
series was selected and aligned with the first z-series by shifting
each image in 3 dimensions based on the location of the peak of their
cross correlograms with the first z-series. The puncta (4–5 in each
slice), which was within 50–60 µm of the stimulating electrode and
stimulus-dependent unloading, were selected according to the three
criteria: first, a fluorescence intensity more than two standard
deviations above the mean background; second, a diameter between 0.3 to
1.8 µm; and third, a roughly circular shape. Fluorescence measurements
were analyzed using Image J by spatially averaging signals over a region
centered over each of the puncta for each time point during the
unloading protocol. The activity-dependent destaining time courses were
generated by each punctum subtracting its residual fluorescence
intensity (<10% of initial intensity), then normalized to the maximal
fluorescence intensity of punctum that was in the unloading procedure.
The inverse halftime of decay of intensity during unloading (1/t1/2) was then calculated.
Immunofluorescence staining
Mice were perfused with 0.01 M PBS, and 4% freshly made paraformaldehyde under deep anesthesia
53.
Coronal brain sections were cut at 10 μm thickness using a cryostat
microtome (CM1950, Leica). After post-fixation in 4% paraformaldehyde
for 15 min, sections were washed with PBS for 5 min, permeabilized in
TBS containing 0.3% Triton X-100 for 20 min, and blocked in TBST (0.1%
Tween) containing 5% BSA, 0.3% Triton X-100 for 1 h at room temperature.
Sections were incubated with the primary antibody overnight at 4 °C in
TBST with 1% BSA, 0.3%Triton X-100. Primary antibodies (anti-Iba1:
1:250, ab178847; anti-GFAP: 1:500, ab7260; anti- Parvalbumin: 1:500,
ab11427) were visualized with an appropriate secondary antibody (goat
Anti-Rabbit IgG H&L (Alexa Fluor® 488), 1:1000~1500, ab150077)
conjugated with Alexa-Fluor 488 (Jackson Immunoresearch) after washing 3
times with TBST. Slides were covered using a coverslip under the
Fluoroshield Mounting Medium spiked with DAPI (Abcam; ab104139). Images
were acquired using a fluorescence microscope (M2, Zeiss), a confocal
microscope (LSM 710; Zeiss), and quantified using Image J.
Fluoro-Jade B Staining
Fluoro-Jade
B staining was performed to visualize degenerating neurons. Brain
tissue sections were cut on a cryostat microtome (CM1950, Leica) at a
thickness of 25 μm. Sections were collected in 0.1 M PBS and mounted on a
2% gelatin-treated microscope glass slides. All tissue slices were
dried at 50 °C for at least 0.5 h. Tissue slices were first immersed
into a solution of 100% EtOH for 5 min, followed by rinsing in 70%
ethanol and distilled water for 2 min each session. After incubating in
0.06% potassium permanganate solution for 15 min, slides were washed
with distilled water and transferred to a 0.001% solution of Fluoro-Jade
B (dissolved in 0.1% acetic acid, Millipore, USA) for 30 min. Finally,
the stained slides were washed in distilled water and dehydrated
thoroughly in a heated incubator at 50 °C for 5 min. Slides were then
cleared in xylene for 2 min and mounted using a coverslip under DPX
mounting medium (Thermo Scientific). FJB positive cells were reported as
the number of FJB-positive cells per CA1 region.
Blood vessel quantification
Mice
were anesthetized via i.p. injection of sodium pentobarbital (50 mg/kg)
in a vehicle containing 0.9% sodium chloride. Upon sedation, mice were
transcardially perfused with 100 µg of FITC-tagged tomato lectin
(FL-1171, Vector Laboratories, Burlingame, CA) in 100 µl volume injected
directly into the left ventricle of the heart, over a period of
approximately 30 s. The heart continued to beat for approximately 1 min
following the injection. The animal was then perfused with 20 ml PBS
followed by 30 ml 4 % paraformaldehyde in PBS. The brain was dissected,
embedded in O.C.T. compound. Brain sections at 20 µm were cut on a
cryostat directly onto Superfrost Plus slides. The FITC–lectin signal in
the hippocampus and visual cortex was determined using a fluorescent
microscope, and the blood vessel density was measured using Image J.
Proteomics analysis of ischemic mouse hippocampus
C57BL/6
mice were euthanized by cervical dislocation. The hippocampus was
quickly harvested on ice and ground to powder in liquid nitrogen. Four
volumes of lysis buffer (8 M urea, 1%. protease inhibitor cocktail) were
added to the cell powder, followed by sonication three times on ice
using a high-intensity ultrasonic processor (Ningbo Scientz
Biotechnology Co., Ltd. China). The remaining non-soluble components
were removed by centrifugation at 12,000 × g at 4 °C for
10 min. The supernatant was collected, and the protein concentration was
determined with the BCA kit according to the manufacturer’s
instructions.
To digest proteins, the protein solution
was reduced with 5 mM dithiothreitol for 30 min at 56 °C and alkylated
with 11 mM iodoacetamide for 15 min at room temperature in darkness. The
protein sample was then diluted by adding 100 mM Triethylammonium
bicarbonate (TEAB) to reduce urea concentration to less than 2 M.
Trypsin was added at 1:50 trypsin-to-protein mass ratio for the first
digestion overnight and 1:100 trypsin-to-protein mass ratio for a second
time for 4 h of digestion.
To conduct LC-MS/MS
analysis, the tryptic peptides were dissolved in 0.1% formic acid
(solvent A), and directly loaded onto a home-made reversed-phase
analytical column (15 cm length, 75 μm i.d.). The column gradient
comprised of an increase from 6% to 23% of solvent B (0.1% formic acid
in 98% acetonitrile) over 26 min, 23% to 35% in 8 min and to 80% in
3 min then holding at 80% for the last 3 min, all at a constant flow
rate of 400 nL/min on an EASY-nLC 1000 UPLC system.
The
peptides were subjected to NSI source followed by tandem mass
spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the
UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range
was 350–1800 for a full scan, and intact peptides were detected in the
Orbitrap at a resolution of 70,000. Peptides were then selected for
MS/MS using the NCE setting as 28, and the fragments were detected in
the Orbitrap at a resolution of 17,500. A data-dependent procedure that
alternated between one MS scan followed by 20 MS/MS scans with 15.0 s
dynamic exclusion was performed. Automatic gain control (AGC) was set at
5E4. The fixed first mass was set as 100 m/z.
To
identify proteins, the resulting MS/MS data were processed using the
Maxquant search engine (Version 1.5.2.8, Max Planck Institute of
Biochemistry). Tandem mass spectra were searched against the E. coli
SwissProt database concatenated with reverse decoy database. Trypsin/P
was specified as a cleavage enzyme allowing up to 2 missing cleavages.
The mass tolerance for precursor ions was set as 20 ppm in the First
search and 5 ppm in the Main search, and the mass tolerance for fragment
ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as a
fixed modification, and oxidation on Met was specified as variable
modifications. False discovery rate was adjusted to < 1%, and the
minimum score for peptides was set > 40.
Proteins
with >1.2-fold of change were selected based on the result of
LC-MS/MS analysis for clustering analysis and to generate a heat map.
The content of the filtered proteins matrix was transformed by the
function
x = −log10 (content). The
x values were
z-transformed. One-way hierarchical clustering (Euclidean distance,
average linkage clustering) was employed to cluster z-scores in genesis.
Cluster membership was visualized by MeV software (Version 4.90,
http://mev.tm4.org/).
Gene Ontology (GO) annotation of proteome was derived from the UniProt-GOA database (
http://www.ebi.ac.uk/GOA/).
Identified protein IDs were changed to UniProt IDs and matched to GO
IDs by protein IDs. Based on GO IDs, retrieved the corresponding
information from the UniProt-GOA database. The InterProScan software
(Version 5.14–53.0, EMBL-EBI) was used to annotate protein’s GO function
according to the protein sequence alignment method. If the identified
proteins were absent in the UniProt-GOA database, then the proteins were
classified by Gene Ontology annotation based on cellular function.
RGS12 decoy peptides
The
phosphotyrosine binding (PTB) domain at the N-terminus of RGS12
interacts with the SNARE-binding or “synprint” region of N-VGCC in a
phosphotyrosine-dependent manner
54.
GK23 amino acid sequence consisting of “RASCEALY(P)NE” was linked with a
TAT leader sequence (GRKKRRQRRRPQ) at the N-terminus and a FITC tag
added to the C-terminus for easy visualization. The chimeric polypeptide
was chemically synthesized commercially by GL Biochem Ltd. (Shanghai).
The sequence is
TAT-RGS12-PTB(GK23) = GRKKRRQRRRPQ-Arg-Ala-Ser-Cys-Glu-Ala-Leu-Tyr(P)-Asn-
Glu-Lys-FITC; The GK13 control peptide sequence is
TAT-FITC(GK13) = GRKKRRQRRRPQ -Lys-FITC. Hippocampal slices were
pre-incubated in aCSF containing 250 nM TAT peptides for 1 h at room
temperature. Slice was then continuously perfused at 3 ml /min with aCSF
saturated with 95% O
2/5% CO
2 (v/v) in the absence
of TAT peptide during electrophysiological recording. The penetration
of the TAT peptide into the hippocampal tissue was confirmed by
examining the slices under a fluorescent miscopy to detect
peptide-linked FITC. The FITC fluorescence remained in the slices after
1.5 h of electrophysiological recording.
Intracerebroventricular administration of peptides
GK23
(2 mg/kg/day), GK13 (2 mg/ kg / day), ω-Conotoxin GVIA (2.28 ng/kg/day)
were dissolved in aCSF and infused into osmotic pumps (#1003D, Alzet,
CA), respectively. The pumps were filled, connected to a cannula (Brain
infusion kit 3, Alzet, CA), and incubated in sterile saline (0.9 %) at
37 °C overnight.
Mice were fasted
overnight but freed to drink before the surgery. The mouse was fixed to a
stereotaxic apparatus (Product # 68861 N, RWD Life Science, Shenzhen,
China), and the midsagittal incision was made by a scalpel. After
cleaning the periosteum, an aperture (0.5 mm in diameter) was drilled by
a dental drill (Product #78001, RWD Life Science, Shenzhen, China)
following the coordinates from bregma: anterior-posterior = −0.5 mm;
lateral = 1.0 mm. The cannula was inserted into the hole (depth = 2.5 mm
from flat skull surface) by a cannula holder and anchored with dental
cement. The osmotic pump was placed into the subcutaneous pocket on the
back of the mouse and closed the incision with surgical sutures. After
osmotic pump implantation, the 2VO surgery was performed. Mice were then
moved into a warm (37 ± 1 °C) recovery chamber (Product #DW-1, Harvard)
for 1 h. Visual stimulation was performed 2 h after the 2VO surgery.
Peptide infusion and light stimulation lasted for 3 days. Mice were then
sacrificed for brain slicing and electrophysiology recordings.
Stereotaxic injection
Recombinant Adeno-Associated Viral Vectors (rAAVs, 1.0 × 1012
viral particles/ml) were custom made by Hanbio Biotechnology Co. Ltd
(Shanghai, China). rAAVs (2 ul). rAAVs were bilaterally injected into
the dorsal CA1 area (anteroposterior, A/P: 2.0 mm; mediolateral, M/L:
1.50 mm; dorsoventral, D/V: 1.60 mm) at a rate of 0.2 μl/min using a
pump (Legato185, KD Scientific). Mice were allowed to recover for 3
weeks before electrophysiological recordings and visualization of
presynaptic vesicle release.
Western blotting
The
hippocampus was removed and lysed in 200 ml lysis buffer (Product
#C500008, Sangon Biotech, China) containing proteinase inhibitor and
phosphatase inhibitor. The lysates were centrifuged at 13,680 g for
10 min at 4 °C, and the supernatant was collected and boiled in boiling
water for 10 min with loading buffer (4:1 ratio). Whole proteins were
electrophoresed in a 10% SDS-PAGE gel and then transferred to
polyvinylidene fluoride (PVDF) membranes (0.45 μm) using iBlot 2 dry
blotting system (Thermo Fisher Scientific Systems, USA). After blocking
in 5% skim milk, the PVDF membranes were incubated with the primary
antibody (anti-RGS12: 1:500, sc-514173) overnight at 4 °C. The PVDF
membranes were then incubated with a secondary antibody after washing
three times with TBST. Protein band intensities were detected with Tanon
(Shanghai, China). Equal protein loading was ensured using an internal
control β-actin detected by a mouse monoclonal antibody at 1:2000
(#ab6276, Abcam) dilution. Every brain sample was repeated twice
independently.
Statistical analysis
All
analyses were performed using GraphPad Prizm (Version 7.0, San Diego,
CA) or SPSS software (Version 22.0, IBM, USA). All data were presented
as mean ± standard error of the mean (SEM). The 2VO surgery and LED
treatment were used as two main factors for the 2ANOVA analysis.
Multiple group comparisons using a 1ANOVA and 2ANOVA were followed by a
post hoc Fisher’s LSD, Tukey’s, Sidak’s or Dunnett’s test to identify
significant groups as indicated in the figure legends. A 2ANOVA with
repeated measures over time was applied to the analysis of LFPs, LTP
recordings, distance traveled to the platform, and escape latency in the
spatial acquisition test of MWM to detect significant differences
between groups on different days or treatments. Statistical details for
specific experiments - including exact n values and what n represents,
precision measures, statistical tests used, and definitions of
significance - can be found in figure legends. Where representative
images were shown, each experiment was repeated independently at least
three times with similar results. A p value < 0.05 was taken
to indicate statistical significance. 1ANOVA: one-way ANOVA; 2ANOVA:
two-way ANOVA; RM-2ANOVA: repeated measure two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.
Hongjia Xu
Yiming Ding2,3†
Dewei Wang
Shunliang Xu
1
