Brain Topogr DOI 10.1007/s10548-015-0430-x

ORIGINAL PAPER

Effects of Hydrogen Sulfide on Modulation of Theta–Gamma Coupling in Hippocampus in Vascular Dementia Rats Xiaxia Xu1 • Chunhua Liu2 • Zhanyong Li1 • Tao Zhang1

Received: 12 July 2014 / Accepted: 4 March 2015  Springer Science+Business Media New York 2015

Abstract Our previous study showed that hydrogen sulfide (H2S) could alleviate the cognitive deficits in vascular dementia (VD) rats associated with the improvement of synaptic plasticity. Neural oscillations are reported to interact with each other through either identical-frequency or cross-frequency coupling. This study examined whether impaired neural couplings could be alleviated by H2S in the hippocampal CA3–CA1 of VD rats and explored its possible mechanism. A VD rat model was established by two-vessel occlusion. Sodium hydrosulfide (NaHS), a kind of H2S donor, was administered intraperitoneally (5.6 mg/kg/day) for 3 weeks. Local field potentials were simultaneously collected in the hippocampal CA3 and CA1. The effects of NaHS on the modulation of theta–gamma coupling were evaluated by using the measurements of both phase–phase coupling and phase–amplitude coupling, while several other approaches including behavior, electrophysiology, western blot, immunofluorescence staining were also employed. The results showed that NaHS significantly prevented spatial learning and memory impairments (p \ 0.01). NaHS considerably alleviated the impairment of neural coupling in VD rats in an identical-frequency rhythm and

Xiaxia Xu and Chunhua Liu have contributed equally to this work. & Tao Zhang [email protected] 1

College of Life Sciences and Key Laboratory of Bioactive Materials Ministry of Education, Nankai University, Tianjin 300071, People’s Republic of China

2

College of Medicine Science, Nankai University, Tianjin 300071, People’s Republic of China

between cross-frequency bands. Moreover, the expression of cystathionine-b-synthase (CBS) was markedly attenuated in VD rats. NaHS elevated the expression of CBS to maintain the intrinsic balance of H2S. Interestingly, it was observed that NaHS increased the protein expression of N-methyl-D-aspartic acid receptor 2A (NMDAR2A) in VD rats. In conclusion, the data suggest that NaHS played the neuroprotective role partly via modulating the expression of NMDAR2A in order to alleviate the impairments of neural couplings in VD rats. Keywords Theta  Gamma  Vascular dementia  Hydrogen sulfide  Identical frequency coupling  Cross frequency coupling Abbreviations CFC Cross frequency coupling CMI Conditional mutual information fEPSP Field excitatory postsynaptic potential H 2S Hydrogen sulfide NaHS Sodium hydrosulfide LFP Local field potential LTP Long term potentiation MWM Morris water maze MI Modulation index NMDAR N-methyl-D-aspartic acid receptor PAC Phase–amplitude coupling PAC–CMI Phase–amplitude coupling–conditional mutual information PAC–PLV Phase–amplitude coupling–phase locking value PLV Phase locking value VD Vascular dementia 2VO Two vessel occlusion PPC Phase–phase coupling

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Introduction Vascular dementia (VD) is commonly caused by cerebral ischemia and the second most common form of dementia after Alzheimer’s disease (AD) (Iadecola 2013). It is characterized by deficits of executive and memory functions (Gosselin et al. 2001; Billings et al. 2005; Xu et al. 2012) and there is no cure for this disease so far. The hippocampal formation is critical for spatial memory and the most sensitive brain area to the cerebral ischemia (Barth and Mody 2011). A previous study reported that the hippocampal formation played a key role in VD (Fein et al. 2000). In addition, VD not only presented cognitive dysfunction, but also showed some changes of spontaneous neural activity (Xu et al. 2012; Tuffrey-Wijne and Watchman 2014). Catching the alterations of neural activity might be useful for predicting or diagnosing VD (Goutagny and Krantic 2013; Tuffrey-Wijne and Watchman 2014). Further exploring the underlying mechanism of neural activity changes might help understand this disease (Goutagny and Krantic 2013; Tuffrey-Wijne and Watchman 2014) and contribute to develop new drug candidates. Moreover, it is well known that there is a close relationship between N-methyl-D-aspartic acid receptors (NMDARs) and neural activity rhythms (Hakami et al. 2009; Kittelberger et al. 2012; Kocsis 2012). Several studies report that NMDARs play a crucial role in VD disease (Tanovic and Alfaro 2006; Olivares et al. 2012). Considering the effect of NMDAR on neural activity and VD, some drug candidates targeting on NMDAR might be able to alleviate neural damage in VD rats. Hydrogen sulfide (H2S) has been recognized as the third gaseous signal molecule after NO and CO in the last two decades (Kimura 2002, Zhang and Bian 2014). It is synthesized by cystathionine-b-synthase (CBS) and shows a relatively high concentration in the brain (Abe and Kimura 1996; Dello Russo et al. 2000; Wang 2002). Studies showed that physiological concentrations of H2S selectively enhanced NMDAR-mediated responses and facilitated the induction of hippocampal long-term potentiation (LTP) (Abe and Kimura 1996). Accumulating evidences indicated that H2S showed potential therapeutic value in several CNS diseases, such as AD, ischemic stroke, and traumatic brain injury (Gupta et al. 2010; Li et al. 2011). In our lab, we found that H2S could improve the performance of VD rats in Morris water maze (MWM) test, which was in line with the increased LTP in the hippocampal CA3–CA1 pathway (Li et al. 2011). Accordingly, we wondered whether H2S could partly resume the altered pattern of neural activity induced by dementia, and if so, whether this effect was related to NMDAR. Spontaneous neural activity is classically identified as delta 1–3 Hz, theta 3–8 Hz, alpha 8–13 Hz, beta 13–30 Hz

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and gamma 30–100 Hz (Buzsaki and Draguhn 2004), which are possibly associated with some special brain status. Among the five rhythms, theta and gamma rhythms are believed to be most related to cognitive function (Kahana et al. 2001; Behrendt 2010). A previous important review clearly indicated that the phase synchronization of oscillations between different brain regions supported memory and acted by facilitating neural communication and by promoting neural plasticity (Fell and Axmacher 2011). Here, phase synchronization refers to the phase coupling either within an identical frequency or between two different frequencies, i.e. cross-frequency phase coupling. To date, two vital types of cross-frequency phase coupling have been widely identified. One is phase–phase coupling (PPC), which reflects phase locking of n cycles of one oscillation to m cycles of another oscillation (Tass et al. 1998). Another is phase– amplitude coupling (PAC), which characterizes low frequency nesting high frequency (Canolty et al. 2006). Furthermore, supposing phase synchronization is associated with the neural communication between two brain regions, a time lag is expected, which corresponded to the conduction delay between these two regions (Fell and Axmacher 2011). Taken together, we certainly believe that the application of phase synchronization measurements, including both identical frequency and cross-frequency couplings (Ford et al. 2008), and directional couplings (Tropini et al. 2009; Taxidis et al. 2010) may help us to better understand how H2S retrieves the altered pattern of neural activity induced by dementia in network level. In this study, in order to examine the effect of H2S on spontaneous neural activity in VD rats, local field potentials (LFPs) were recorded at both the hippocampal CA3 and CA1 regions in the sham, VD and VD ? NaHS treated groups. Several analytic algorithms were applied to measure the phase synchronization and directional coupling respectively, including phase locking value (PLV), generalized partial directed coherence (gPDC), n:m phase locking value (n:m PLV), modulation index (MI), phase– amplitude coupling–phase locking value (PAC–PLV) and phase–amplitude coupling–conditional mutual information (PAC–CMI). In addition, the expressions of CBS and NMDAR2A were examined to explain the underlying molecular mechanisms of H2S on VD.

Materials and Methods Animals and Treatment The study was approved by the local Ethical Committee of Nankai University and carried out according to the guidelines of the Beijing Laboratory Animal Center. Twenty-two

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male Wistar rats (250–300 g) were provided by the Laboratory Animal Center, Academy of Military Medical Science of People’s Liberation Army and reared in the animal house of Medical School, Nankai University, under a constant temperature (24 ± 2 C) and a 12 h light/dark cycle (lights on at 7 a.m.) with freely available food and water. Rats were randomly divided into three groups: sham group (Sham, n = 8), VD group (VD, n = 8) and VD treated with NaHS group (VD ? NaHS, n = 6). The VD rat model was established by 2VO surgery described particularly in a previously paper (Li et al. 2011; Xu et al. 2012). Briefly, animals were fasted but freely accessed water during the night before 2VO surgery. The surgery was carried out under aseptic condition. Rats were anaesthetized with 10 % chloral hydrate intraperitoneally (i.p.) (3 ml/kg), and then they were sterilized and sheared in operation area. A ventral midline incision was made in the neck and a dissection was performed to expose the right and left common carotid arteries. A surgical line was thread beneath the common carotid arteries. After that, the vessels in the VD and VD ? NaHS groups were fully ligated before sutured the wound. The operative procedures were performed identically for the sham group, except for carotid arteries ligation. After the surgery, all the rats received reagents for 3 weeks, i.e. the sham group (saline, i.p.), the VD ? NaHS group (NaHS, 5.6 mg/kg/day, i.p.) and the VD group (saline, i.p.). Morris Water Maze Experiment The MWM test was used to examine the rats’ capacity of the spatial learning and memory after 3-week post-injury. The following sentences provide a brief description of the MWM process, but more detailed information can be found in our previous papers (Li et al. 2011; Zhang et al. 2011; Zheng et al. 2011). The MWM test consists of two phases, place navigation and spatial probe. The first phase covered 4 days with two sessions (8 hourly intervals) per day. In each section, a rat was placed into the water from four starting points spaced 90 apart around the tank’s perimeter, respectively. The rat swam freely until it found the platform within 60 s. If failed, it was placed on the platform for 10 s. In this process, the escape latency and the swimming speed were recorded. Spatial probe was performed on the 5th day. In the phase, the platform was removed and the rats were gently released into water and allowed them to swim for 60 s. In the process, the platform crossings and quadrant dwell time were collected. Electrophysiological Experiment Electrophysiological experiments were carried out after the MWM test. Rats were anaesthetized with 30 % urethane

with a dosage of 4 ml/kg, i.p. (Sigma-Aldrich, St. Louis, MO, USA), after that they were placed in a stereotaxic frame (Narishige, Japan). Two recording electrodes (stainless steel) were implanted into CA3 (4.2 mm posterior to the bregma, 3.5 mm lateral to midline, and 2.5 mm ventral below the dura) and CA1 (3.5 mm posterior to the bregma, 2.5 mm lateral to midline, and 2.0 mm ventral below the dura) regions. Both LFPs recordings were referenced to two ground reference electrodes placed symmetrically over the two hemispheres of the cerebellum. The signals were acquired simultaneously by the two recording electrodes at a sampling rate of 1000 Hz for 10 min. Neurodynamic Analysis LFP Preprocessing Before coupling estimation, all LFP recordings were preprocessed by locdetrend function in the Chronux 2.00 toolbox (http://www.chronux.org) (Mitra and Bokil 2008). We subtracted the linear regression line fit within a 20 s moving window by using locdetrend function. Consequently, direct current offsets and slowly changing components were effectively removed (van der Meer and Redish 2011). Multi-taper Spectral Estimation It is the most principled method to estimate the power spectrum of a signal (Thomson 1982), in which an orthogonal family of tapers called Slepian sequences were used. 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 2   K  X N K   1X 1X  1 MT k X~k ðf Þ2 S ðf Þ ¼ expð2pifnÞun Xn  ¼   K k¼1 K k¼1 N n¼1 where ukn ; n ¼ 1; 2. . .; N is the kth Slepian sequence, X~k ðf Þ is the tapered Fourier transform of Xk. In the study, a window length of 20,000 (20 s) with 50 % overlap was chosen to estimate the LFP power. Phase Locking Value (PLV) This was a conventional method to measure the strength of phase synchronization between two brain regions (Lachaux et al. 1999). The original LFPs were filtered into 3–8 Hz (theta band, bandwidths = 1 Hz, step = 1 Hz) and 30–50 Hz (gamma band, bandwidths = 1 Hz, step = 1 Hz). The process was realized by the mean of eegfilt.m from EEGLAB toolbox (Delorme and Makeig 2004).

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Afterward, the Hilbert transform was used to determine the instantaneous phases of filtered LFPs signed as /CA3(f, t) and /CA1(f, t) in all the frequency bands. PLV was defined as,    1 X N   PLVðf Þ ¼  expði½/CA3 ðf ; jDtÞ  /CA1 ðf ; jDtÞÞ  N j¼1

locking between two oscillators (f1 and f2) occurs at an n:m ratio when there are m cycles of the f1 oscillators for every n f2 oscillators. The radial distance (r) values were calculated as,    1 X N  i½m/theta ðtÞn/gamma ðtÞ  rn:m ¼  e   N t¼1

N was the length of the signal and Dt1 was the sampling frequency. The PLV was in [0 1] where 1 and 0 standed for full synchronization and no synchronization at all, respectively.

At different n:m ratios, e.g., 1:1, 1:2,…,1:20, etc. The Rayleigh test was used for uniformity test with r = 0 for uniform and r = 1 for a perfect unimodal distribution. These distributions were regarded to show the coupling between these two oscillators and independent of the amplitude (Tass et al. 1998; Belluscio et al. 2012). For the analysis, the CA3 theta rhythm and CA1 gamma rhythm were previously band-filtered in 3–8 Hz range and in 30–50 Hz range using eegfilt.m in EEGLAB box, respectively. Subsequently, a time window of 0.5 s with an overlap of 25 % was adopted.

Generalized Partial Directed Coherence (gPDC) The gPDC was designed to identify the directional coupling between brain regions (Baccala et al. 2007) and was calculated based on vector autoregressive (VAR) modeling. Briefly, the bivariate VAR model of order p was described by the following:   X    X p  11 Xt Xtr e ar a12 r ¼ þ tY 21 22 Yt Y et a a tr r r r¼1 Fourier transformation of the VAR coefficients:  p  11 X ar a12 r Aðf Þ ¼  expði2pfrÞ a21 a22 r r r¼1

Modulation Index (MI) In the present study, the MI was used to quantify the cross frequency PAC between CA3 theta rhythm and CA1 gamma rhythm. The MI measure produced a complex valued composite signal Zfph;fam ðtÞ which was defined as a function of the instantaneous theta phase /fph and instantaneous gamma amplitude Afam(t)

 Þ stood for the difference between the identity Then Aðf matrix and A(f):   X  p  11 ar a12 r  Þ¼ 1 0  Aðf  expði2pfrÞ 0 1 a21 a22 r r r¼1   a ðf Þ a12 ðf Þ ¼ 11 a21 ðf Þ a22 ðf Þ

Zfph:fam ðtÞ ¼ Afam ðtÞ  expði  /fph ðtÞÞ

The gPDC, obtained from signals Y and X, could be written as follows:

MIraw ¼ absðmeanðZfph;fam ðtÞÞÞ

gPDCY!X ðf Þ ¼

a11 ðf Þ r11 a12 ðf Þ r11 þ a12 ðf Þ r12

,c2 ðf Þ

where r1 and r2 represented the standard deviation of VAR model in frequency domain. The gPDC from X to Y could be written analogously as c1(f). In this study, in order to assure a stationary and adequate fit of VAR model, a window length of 1 s with 50 % overlap was chosen. The model order p was determined by AIC (Akaike 1974). n:m Phase Locking Values It can be used to measure the cross frequency PPC between CA3 theta rhythm and CA1 gamma rhythm. The phase

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A joint probability density function on the complex plane was created whereas particular amplitude and phase value to co-occur could be identified. The MI value was calculated as the absolute value of average Zfph;fam ðtÞ

Surrogate data were generated by a time lag s between /fph(t) and Afam(t): Zsurr ðt; sÞ ¼ Afam ðt þ sÞ expði  /fph ðtÞÞ The normalized MI was defined as: MINorm ¼ ðMIraw  lÞ=r l was the surrogate mean vector length and r was the standard deviation. In the study, the convolution with complex Morlet wavelets of the depth 7 was used to generate analytic representations of CA3 low frequency bands (1–20 Hz, band = 1 Hz, step = 1 Hz) and CA1 gamma frequency bands (30–80 Hz, band = 1 Hz, step = 1 Hz). And CA3 /(t) and CA1 Agamma(t) were obtained by Hilbert transform, respectively. To calculate the MI values, a window length of 40 s with 50 % overlap and surrogating 100 times were used.

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Phase–Amplitude Coupling–Phase Locking Value (PAC–PLV) The PLV has been used to analyse the cross frequency PAC (Penny et al. 2008). In this study, we firstly obtained /theta (the phase of the wide-band filtered CA3 theta rhythm (3–8 Hz)) and ampc (the amplitude of the narrowband filtered CA1 gamma frequency bands (step = 1 Hz, from 30 to 50 Hz)) by Hilbert transform. And then the second Hilbert transform was applied to gain the phase of ampc, signed as /ampc . Finally, the PLV between /theta and /ampc was calculated. The process is named PAC_PLV to distinguish from the PLV method used for the identical frequency phase coupling. Phase–Amplitude Coupling–Conditional Mutual Information (PAC–CMI) In this study, conditional mutual information (CMI) (Palu et al. 2001; Palu and Stefanovska 2003; Xu et al. 2013a; Zheng and Zhang 2013) was used to investigate the directional coupling between the CA3 theta rhythm and CA1 gamma rhythm. This method was abbreviated to PAC– CMI. Simply, the conditional mutual information Ið/theta ; Ds /ampc j/ampc Þ, which estimated the information about the s-future of the process /ampc contained within the process /theta, was used to stand for /theta to /ampc directional coupling and was defined as: Ið/theta ; Ds /ampc j/ampc Þ ¼ Hð/theta j/ampc Þþ HðDs /ampc j/ampc Þ  Hð/theta ; Ds /ampc j /ampc Þ with the phase increments Ds /ampc ¼ /ampc ðt þ sÞ  /ampc ðtÞ . In the study, we applied the sliding window (length = 24 s) with 50 % overlap and s = 100 ms to calculate Ið/theta ; Ds /ampc j/ampc Þ.

Histological and Electrophysiological Localization of Recording Electrodes Sites In order to damage both the hippocampal CA1 and CA3 regions, a 2 mA direct current was performed through the two recording electrodes for 20 s after electrophysiological recordings. The brain samples were immersed in 4 % paraformaldehy at 4 C for 24 h, and then dehydrated in 30 % sucrose overnight at 4 C and embedded by OCT compound. The coronary slices were obtained for neutral red staining. It was performed in accordance with the standard procedure. Firstly, sections were washed in PBS for 5 min, stained by neutral red for 5 min and washed in PBS for 8 s. Afterward, sections were dehydrated as

follows, i.e. 50 % alcohol for 8 s, 75 % alcohol for 8 s, 95 % alcohol for 8 s, 100 % alcohol I and II for 8 s, respectively; and 100 % xylene I and II for 1 min respectively. Finally, sections were detected by light microscope. Immunofluorescence Staining According to the method described by Maerz et al. (2011), the immunofluorescence study was carried out as follows. Firstly, brain sections were washed in PBS for three times and blocked with 10 % goat serum for 1 h at room temperature. And then sections were incubated with NMDAR2A (1:1000, abcam, UK) for 18 h at 4 C. After three washes, they were incubated with Alexa 488-conjugated anti-rabbit IgG for 6 h at room temperature. Finally, they were incubated with DAPI (1:2000, Beyotime Biotechnology, Haimen, China) for 3 min and the fluorescent signals were detected by laser scanning confocal microscope. Western Blotting During the experiment, the western blotting was performed (Liu et al. 2014). Firstly, the hippocampus was separated from brain, homogenized in lysis buffer (Beyotime Biotechnology, Haimen, China), centrifugated them at 12,000 rpm for 15 min at 4 C, collected the supernatant and quantified the concentration of protein by the enhanced BCA Protein Assay Kit (Beyotime Biotechnology, Haimen, China).Secondly, 30 mg proteins were separated on SDS-PAGE gels. Thirdly, proteins were transferred onto 0.22-lm polyvinylidenedifluoride (PVDF) membranes (Milli pore Corporation). And then, PVDF membranes were blocked for 2 h at room temperature with Tris-buffered saline (TBS) including 5 % skim milk. Fourthly, the monoclonal antibody CBS (1:500) and bactin (1:1000) (Sata Cruz, USA) were diluted in Trisbuffered saline with Tween-20 (TBST), after that the PVDF membranes were incubated overnight at 4 C. Fifthly, the PVDF membranes were washed three times with TBST, and incubated the membranes with secondary antibody (1:3000; Cell Signaling Technology, Beverly, MA, USA) for 1 h at room temperature. Finally, the western blotting imaging system was employed to detect the protein band intensities. Data and Statistical Analysis All filters in the study were realized by means of eegfilt.m from EEGLAB toolbox (Delorme and Makeig 2004). In order to avoid edge effects caused by filtering, the first and last 10 s of data were deducted (Kramer et al. 2008). All

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data were presented as mean ± SEM. The differences of the MWM results were analyzed by Two-way repeated measures ANOVA. The comparisons of the differences of all the LFPs among the three groups were performed using one-way ANOVA. Post hoc analysis was made and significant differences were determined by least significant difference (LSD) tests with p \ 0.05. The differences of MI, PAC–PLV and PAC–CMI among the three groups were determined by Kruskal–Wallis test. Post hoc Nemenyi test was applied in determining the significant differences (p \ 0.05). All analyses were performed using SPSS 17.0 software.

Results NaHS Improved the Spatial Learning and Memory of VD Rats Table 1 showed the MWM test results. In the 4 days of place navigation phase, the escape latency was reduced gradually in all three groups. This indicated that after training, all rats had learned the position of underwater platform. However, on the 4th day, the VD rats spent more time to find the platform compared to that of the Sham rats (p \ 0.01). Importantly, it was found that NaHS could significantly alleviate the impairment of VD. In the spatial probe phase, it could be seen that there were remarkable reductions of platform crossings (1.25 ± 0.25) and quadrant dwell time (27.86 ± 1.09 %) in the VD group compared to that in the Sham group. Interestingly, both indexes above were significantly improved to 3.13 ± 0.40 and 36.85 ± 2.28 % respectively (p \ 0.01) after administrating with NaHS, compared with that of the VD group. NaHS Enhanced the Power of Theta and Gamma Rhythms in VD Rats The digitized LFPs signals were subjected to a fast Fourier transformation to generate a power spectrum. Figure 1 shows represented LFP traces and their power spectra, as

well as the associated group data in these three groups. Figure 1a exhibits representative examples of the LFP traces and their associated power spectra in three groups in the CA3 region, which is a rat in the Sham group (left column), a rat in the VD group (middle column) and another in the VD ? NaHS group (right column). Figure 1b was the same as above in the CA1 regions. There were theta and gamma rhythms, which are two prominent oscillatory neuronal activities, in both CA3 and CA1 regions in the Sham group. However, the above two major components were almost disappeared in the VD group. Interestingly, both of them were returned after administrating with NaHS (Fig. 1a, b). The statistical measurements of the relative theta powers (the ratio of 3–8 Hz power/1–50 Hz power) and gamma (the ratio of 30–50 Hz power/1–50 Hz power) are shown in Fig. 1c and d. One way ANOVA test showed that there were significant differences of the relative theta power among the three groups in CA3 region (Fig. 1c, F(2,19) = 4.065; p = 0.034). In addition, Post hoc test was performed. There was a significant decrease of power in the VD group compared to that in the Sham group (p = 0.018). After NaHS treatment, the theta power was apparently enhanced and there was a statistical difference of power between the VD group and the VD ? NaHS group (p \ 0.036). On the other side, one way ANOVA test showed that there were significant differences of the relative theta power among the three groups in CA3 region (Fig. 1d, F(2,19) = 8.480; p = 0.002). Post hoc test indicated that there was a significant decrease of the power in the VD group compared to that in the Sham group (p = 0.011). After NaHS treatment, the theta power was significantly enhanced and there was a statistical difference of power between the VD group and the VD ? NaHS group (p \ 0.036). NaHS Alleviated the Impairment of Synchronization in VD Rats The results, obtained from the PLV measurement of both theta and gamma frequency bands, were shown in Fig. 2a.

Table 1 The results of the MWM test Group

Escape latency (s) Day 1

Day 2

Platform crossings Day 3

Quadrant dwell time (%)

Day 4

Sham

39.60 ± 3.85

18.11 ± 2.33

11.75 ± 1.39

5.38 ± 0.62

53.21 ± 1.87

VD

50.61 ± 1.16

37.09 ± 2.75*

21.80 ± 1.50**

21.49 ± 1.64**

6.58 ± 0.79

1.25 ± 0.25**

27.86 ± 1.09**

VD ? NaHS

51.69 ± 2.49

17.58 ± 2.23#

14.52 ± 1.72##

10.75 ± 1.57##

3.13 ± 0.40##

36.85 ± 2.28##

The Escape latency, platform crossings and quadrant dwell time were compared among the Sham, VD and VD ? NaHS groups, respectively * p \ 0.05 and ** p \ 0.001 for comparison between the Sham group and the VD group; # p \ 0.05 and ## p \ 0.001 for comparison between the VD group and the VD ? NaHS group; No significant differences between the Sham group and the VD ? NaHS group

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Fig. 1 The power within theta and gamma frequency bands was significantly increased by sodium hydrosulfide in the VD ? NaHS group. a Representative traces of CA3 LFPs and their associated power spectra in the Sham (left), the VD (middle) and the VD ? NaHs (right) groups. b As same as an except for the region, which is the hippocampal CA1 area. c The group data of the power of

theta and d the power of gamma in both CA3 and CA1 regions in the three groups. *p \ 0.05 and **p \ 0.01 represent significant differences of power between the Sham group and the VD group. #p \ 0.05 represents significant differences of power between the VD group and the VD ? NaHS group. There are no significant differences of power between the Sham group and the VD ? NaHS group

It can be seen that all of the PLV values in these three groups are bigger than 0.5, indicating there is a relatively strong synchronization between CA3 and CA1. Furthermore, there were significant differences of the PLVs among the three groups by one-way ANOVA (theta: F(2,19) = 5.889, p = 0.010; gamma: F(2,19) = 4.366,

p = 0.028). Post hoc turkey test showed that there were remarkably decreased PLVs in the VD group compared to that in the Sham group (theta: p = 0.006; gamma: p = 0.010), while NaHS significantly alleviated the impairment of PLVs in the VD ? NaHS group in theta band (theta: p = 0.013; gamma: p = 0.066). There were no

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Fig. 2 NaHS alleviated the impairment of phase synchronization between hippocampal CA3 and CA1 regions in either theta or gamma rhythms in the VD ? NaHS group. a The phase locking values (PLVs) between CA3 and CA1 in the three groups. b The values of directional phase coupling gPDCCA3?CA1 between CA3 and CA1 in the three groups. The gray lines indicate the directional coupling from

CA1 to CA3 which were no remarkable differences among the three groups. *p \ 0.05, **p \ 0.01 and ***p \ 0.001 represent significant differences between the Sham group and the VD group. # p \ 0.05 and ##p \ 0.01 represent significant differences between the VD group and the VD ? NaHS group

significant differences of PLV values between the Sham and the VD ? NaHs groups (theta: p = 0.893; gamma: p = 0.491). In order to examine the enhanced CA3–CA1 phase synchronization at both theta and gamma rhythms in VD rats treated by NaHS, gPDC approach was applied to measure the directional coupling in the hippocampal CA3–CA1 pathway. The result was displayed in Fig. 2b. One way ANOVA showed that there were significant differences of driving strength gPDCCA3?CA1 in these three groups in either theta (F(2,19) = 6.554, p = 0.007) or gamma (F(2,19) = 10.856, p = 0.001) rhythms. Furthermore, post hoc turkey test showed that there was a statistical decrease of gPDCCA3?CA1 in the VD group compared to that in the Sham group in either theta rhythms or gamma rhythms (theta: p = 0.009; gamma: p = 0.0003). Interestingly, after NaHS treatment, the value of gPDCCA3?CA1 was considerably increased in the VD ? NaHS group compared to that in the VD group (theta: p = 0.004; gamma: p = 0.004), which was more or less back to the normal level in the Sham group (comparison between the Sham and the VD ? NaHS groups: theta, p = 0.573 and gamma, p = 0.397).

the third panel of Fig. 3a were detected by Rayleigh test (p \ 0.05). Furthermore, Fig. 3b displayed the statistical results of phase synchronization strength at different n:m ratios in the three groups. One way ANOVA showed that there were significant differences at 1:9 ratio among the three groups (F(2,19) = 14.865, p = 0.0002). In addition, post hoc turkey test exhibited that there was a considerable decrease of the phase synchronization strength at 1:9 ratio in the VD group compared to that in the Sham group (p = 0.0001). However, NaHS significantly increased the phase synchronization strength at 1:9 ratio (p = 0.006).

NaHS Mitigated the Impairment of Phase–Phase (n:m) Coupling in VD Rats Figure 3 displayed the PPC results between CA3 theta and CA1 gamma in the three groups. An example of phase– phase (n:m) coupling in a normal animal is shown in Fig. 3a, in which there are 9, 10 and 8 CA1 gamma oscillations in one CA1 theta circle, respectively. The corresponding radial distance values (r) of phase difference between CA3 theta and CA1 gamma rhythms were plotted and the ratios of n:m ranged from 1:1 to 1:20. The peaks in

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NaHS Ameliorated the Cross Frequency Phase–Amplitude Coupling in VD Rats Since it was found that the PAC between hippocampal CA3 and CA1 regions was significantly reduced in the VD rats (Xu et al. 2013b), a further study was performed on investigating the effect of NaHS on theta–gamma PAC by using three different algorithms, which were MI, PAC– PLV and PAC–CMI. MI Results The PAC between the CA3 low frequency bands (1–20 Hz) and the CA1 high frequency bands (30–100 Hz) in the three groups was measured and the results were presented in Fig. 4. Representative examples of MI results was displayed in Fig. 4a (one Sham rat), Fig. 4b (one VD rat) and Fig. 4c (one VD ? NaHS rat), respectively. It can be seen that there is a strong theta–gamma PAC in the Sham group and a distinct coupling occurred at low gamma (30–50 Hz) bins (Fig. 4a), while the coupling was almost disappeared

Brain Topogr Fig. 3 NaHS mitigated the impairment of phase–phase (n:m) coupling. a An example of CA3 theta-CA1 gamma PPC in a Sham rat. Top the trace of the filtered CA3 theta (3–8 Hz) rhythm; Middle the phase of the filtered CA1 gamma (30–48 Hz) rhythm. The interval between adjacent dotted lines represents one theta circle; Bottom mean radial distance values (r values) for different n:m ratios show large peaks at 1:9,1:10 and 1:8 in the above three theta circles, respectively. b The distribution of the peaks in the three groups. The arrows indicate n:m ratios at 9:1 and 10:1 where exist clear differences among the three groups. c The mean r values for all the n:m ratios of all calculated windows in the three groups. Large values at 9:1 appear in the Sham and the VD ? NaHS groups and disappear in the VD group. ***p \ 0.001 represents significant differences between the Sham group and the VD group. ##p \ 0.01 represents significant differences between the VD group and the VD ? NaHS group. No significant differences are found between the Sham group and the VD ? NaHS group

in the VD group (Fig. 4b). Interestingly, there is an observable theta-gamma PAC in the VD ? NaHS group (Fig. 4c), indicating that NaHS can attenuate the impairment of cross frequency PAC induced by VD to some extent. Kruskal–Wallis Test showed that there were significant differences of the index among the three groups (Fig. 4d, v2 = 8.648, p = 0.005). Post hoc Nemenyi test further showed that there was a remarkable decrease of MI between the Sham group and the VD group (p = 0.0011). However, NaHS significantly increased MI value in the NaHS group compared to that in the VD group Nemenyi test, p = 0.046). PAC–PLV and PAC–CMI Results Figure 5 showed the theta–gamma PAC measurements using both PAC–PLV and PAC–CMI algorithms in the

three groups. There are three typical examples of the PAC between CA3 theta and CA1 low gamma amplitude (Fig. 5a: a Sham rat; Fig. 5b: a VD rat; Fig. 5c: a VD ? NaHS rat). It can be seen that there is visible phase– amplitude synchronization between CA3 theta and CA1 gamma amplitude (indicated by dotted lines) in normal state, while reduced in VD condition and interestingly recovered after NaHS treatment. The group data of the measurements of PAC–PLV and PAC–CMI were showed in Fig. 5d and e, respectively. Kruskal–Wallis Test showed that there were significant differences of PAC among the three groups (PAC–PLV: v2 = 12.790, p = 0.002; PAC– CMI: v2 = 12.398, p = 0.002). Moreover, post hoc Nemenyi test showed that the phase-amplitude coupling was significantly reduced in the VD group compared to that in the Sham group (PAC–PLV: p = 0.0002; PAC–CMI: p = 0.0003). However, NaHS considerably enhanced the

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Fig. 4 Phase–amplitude coupling between hippocampal CA3 low frequency rhythm (1–20 Hz) and CA1 gamma rhythm (30–100 Hz) measured by MI. a–c Representative PAC between CA3 low frequency phase (x-axis 1–20 Hz, step = 1 Hz) and CA1 gamma amplitude (y-axis 30–100 Hz, step = 1 Hz) in one Sham (a), one VD (b) and one VD ? NaHS rat (c). Larger value indicates stronger

coupling. d The statistical MI results in the three groups. **p \ 0.01 represents significant differences between the Sham group and the VD group. #p \ 0.05 represents significant differences between the VD group and the VD ? NaHS group. $p \ 0.05 represents significant differences between the VD ? NaHS group and the Sham group

PAC in the VD ? NaHS group (Nemenyi test, PAC–PLV: p = 0.045, PAC–CMI: p = 0.008).

expressions of CBS and NMDAR2A were observed by either the western blotting or immunofluorescence staining. As shown in Fig. 7, there was a significant reduction of CBS expression (0.60-fold, p \ 0.01) in the hippocampus in the VD group compared to that in the Sham group, whereas NaHS significantly increased the expression of CBS in order to improve the level of H2S in the hippocampus (p \ 0.05). Figure 8 showed that the expression of NMDAR2A was obviously decreased in the VD group (p \ 0.01), however, the expression of NMDAR2A was considerably enhanced by NaHS (p \ 0.05).

Histological and Electrophysiological Localization of Recording Electrodes Sites In order to verify the location of the LFPs recording sites, neutral red staining was performed. According to the rat brain in stereotaxic coordinates (Paxinos and Watson 2007), the CA1 region (red arrow indicated in Fig. 6a) and CA3 region of hippocampus (yellow arrow indicated in Fig. 6b) are confirmed and the location is consistent with our experiments.

Discussion NaHS Modulated Protein Expressions of CBS and NMADR2A With the purpose of investigating the underlying mechanisms of NaHS in cognitive dysfunction induced by VD, the

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In one of our previous studies, we found that H2S could effectively improve the cognitive performance of VD rats and its effect was through the recovered synaptic plasticity in the hippocampal CA3–CA1 pathway (Li et al. 2011). In

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Fig. 5 Phase–amplitude coupling between hippocampal CA3 theta rhythm (3–8 Hz) and CA1 low gamma rhythm (30–60 Hz) measured by the PAC_PLV and PAC_CMI. a Representative example of PAC between CA3 theta and CA1 low gamma in one Sham rat. The traces represent the filtered CA3 theta (3–8 Hz) and CA1 gamma (30–60 Hz) frequency bands, respectively. The light gray curve stands for the amplitude of the filtered gamma rhythm. The dotted curve shows the synchronization between the CA3 theta phase and

CA1 gamma amplitude. b, c Same display as in A in one VD rat (b) and one VD ? NaHS rat (c). d, e The statistical PAC_PLV (d) and PAC_CMI (e) results in the three groups. ***p \ 0.001 represent significant differences between the Sham group and the VD group. #p \ 0.05 and ##p \ 0.01 represent significant differences between the VD group and the VD ? NaHS group. $p \ 0.05 represents significant differences between the VD ? NaHS group and the Sham group

this investigation, it was focused on exploring the effect of H2S on the neural oscillatory coupling in VD rats. Furthermore, both the western blotting and immunofluorescence staining were used to explore the underlying mechanism based on neural network.

The MWM is a valuable approach to assess the ability of spatial learning and memory in animal models. It was found that the rats performed a significant prolongation of escape latency in the VD group compared to that in the Sham group in the place navigation phase, suggesting that

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Fig. 6 The location of the LFP recording sites was proved by neutral red staining (940). The red arrow indicates CA1 region of the hippocampus (a) and the yellow arrow indicates CA3 region of the hippocampus in (b) (Color figure online)

the learning ability was significantly damaged in rats with VD. The data were in line with our previous report (Li et al. 2011). However, NaHS significantly improved the ability of spatial learning of VD rats (in days 3 & 4: p \ 0.01, Table 1). Furthermore, the time spent in the target quadrant and the number of crossing target quadrant in the spatial probe period were noticeably reduced in the VD group compared to that in the Sham group (p \ 0.01, Table 1), indicating that the ability of memory was much undermined in the VD animals. Nevertheless, the impairment of memory was noticeably alleviated in the VD ? NaHS group (p \ 0.01, Table 1). In addition, the experiment also included the Sham rats treated with NaHS. It showed that there were no significant differences of either the MWM test or LTP results between the Sham rats and the Sham ? NaHS rats (data not presented). It suggests that the Sham rats are not significantly affected by NaHS. Consequently, we did not present the detailed information about the data obtained from the Sham ? NaHS rats.

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It was well known that the phase synchronization in an identical frequency band between different brain areas had physiological significances. For example, theta and gamma phase synchronization was suggested to be important mechanisms of regulating inter-area (von Stein and Sarnthein 2000; Buzsaki and Draguhn 2004) and intra-area (Fell et al. 2001) communication, respectively. These functions were supposed to link single-neuron activity to behavior and mental disorders (Gallinat et al. 2006). In the present study, a visible decrease of either theta or gamma rhythm synchronization in the VD group was detected, which was in good agreement with the reduced synchronization in the EEG of Alzheimer subjects (Yener et al. 2007; Ford et al. 2008). Interestingly, our data showed that NaHS treatment could significantly alleviated the impairment of phase synchronization, induced by VD, at either theta or gamma frequency band (Fig. 2). These observations, obtained from phase synchronization analysis, suggested that the connection strength between neurons in the hippocampal CA3 and CA1 was greatly weakened, while

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Fig. 7 The expression of selected proteins detected by the western blotting assay in the hippocampus of the Sham, VD and VD ? NaHS groups. Pictures demonstrated the expression of CBS and b-actin. Quantitative analysis of protein expressions of CBS (n = 4). Data are expressed as mean ± SEM. ##p \ 0.01 represent significant differences between the Sham group and the VD group. *p \ 0.05 represent significant differences between the VD group and the VD ? NaHS group

NaHS significantly mitigated the impairment of phase coupling. To further elucidate how the phase synchronization alterations happened, we applied the gPDC approach to examine the changes of the directional coupling from CA3 to CA1. It was found that a more predominant driving effect occurred from CA3 to CA1 in these three groups (Fig. 2b), which was in line with the anatomy synaptic projections from CA3 to CA1. Additionally, a more predominant driving effect took place from CA3 to CA1 in the three groups (Fig. 2b), which was in line with the anatomy synaptic projections from CA3 to CA1. The cross-frequency PPC between different brain regions measured by the n:m phase locking was speculated to provide multiple time scales for the coordination of neuronal spikes, which facilitated information transferring and spike timing-dependent plasticity (Gerstner et al. 1996; Markram et al. 1997; Fell and Axmacher 2011). The analysis of PPC between CA3 theta and CA1 gamma showed that the n:m (1:9) theta–gamma rhythm coding was involved in cognitive function (Fig. 3). It could be seen that the PPC was diminished in the VD group, but significantly regained after NaHS treatment. The results suggested that the phase–phase synchronization of both theta and gamma rhythms in CA3–CA1 pathway was implicated in the information coding associated with learning and memory. Since the decreases of phase coupling on both theta and gamma oscillations between the hippocampal CA3 and CA1 were observed in the VD rats (Xu et al. 2013b), it was logically to make an assessment of NaHS effect on theta–

gamma PAC. As we all known that the PAC was functional connected with cognitive task demands, and it might be benefit for spatio-temporal organization of cell assemblies (Lisman and Idiart 1995, Varela et al. 2001, Buzsaki 2010) and neural coding illustrated by computational models (Lisman and Idiart 1995; Varela et al. 2001; Fell and Axmacher 2011). In the study, the three algorithms, including MI, PAC–PLV and PAC–CMI, were applied in measuring the theta–gamma PAC between hippocampal CA3 and CA1 regions. Firstly, MI measurement was applied to compute the cross frequency coupling strength, which showed a visible theta-gamma PAC in the Sham group and a distinct coupling pattern on lower gamma frequency band at about 35–45 Hz (Fig. 4a). However, the theta–gamma PAC was noticeably diminished in the VD group (Fig. 4b), while it was visibly regained by NaHS (Fig. 4c). The group data showed that NaHS significantly mitigated the impairment of PAC induced by VD (Fig. 4d, p \ 0.05). Secondly, the PAC–PLV method was employed in order to remove the power effect of gamma oscillations in the MI computation. Interestingly, the results from PAC–PLV further verified that the averaged phase coupling between theta phase and gamma amplitude was obviously weakened in the VD group (post hoc Nemenyi test: p = 0.0002, Fig. 5d), however, it was significantly regained by NaHS (post hoc Nemenyi test: p = 0.045, Fig. 5d). The CMI has been a widely used directional algorithm (Palu et al. 2001; Palu and Stefanovska 2003; Zheng and Zhang 2013) and the PAC–CMI was developed based on the CMI algorithm. Thirdly, it was consequently applied to examine if there was a directional PAC between CA3 theta and CA1 gamma. It can be seen that the directional PAC strength is extensively reduced in the VD group (post hoc Nemenyi test: p = 0.0003, Fig. 5e), but significantly recovered by NaHS (post hoc Nemenyi test: p = 0.008, Fig. 5e). The above data suggested that the impairment of PAC brought about the cognitive dysfunction, while the improvement of cognitive deficits, induced by NaHS, was associated with enhancing the strength of PAC. H2S is considered as neuromodulator and synthesized by CBS in the central nervous system. Several studies showed that physiological concentrations of H2S selectively enhanced NMDAR-mediated responses and facilitated the induction of the hippocampal LTP, which was possibly mediated by the cAMP/PKA pathway (Abe and Kimura 1996; Kimura 2000). Moreover, it was reported that the NMDAR in the hippocampus was a slow type of NDMA channel (Debanne et al. 1995), which had two important kinetics features. One was its slow open process, which corresponded to one gamma cycle (Jensen and Lisman 1996). Another was its deactivation time, which corresponded to one theta cycle (Jensen and Lisman 1996).

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Brain Topogr Fig. 8 The expression of NMDAR2A detected by immunofluorescence staining (9400) in the hippocampus of the Sham, VD and VD ? NaHS groups. a Pictures demonstrated the expression of NMDAR2A. b Quantitative analysis of NMDAR2A intensity (n = 6). Data are expressed as mean ± SEM. ##p \ 0.01 represent significant differences between the Sham group and the VD group. **p \ 0.01 represent significant differences between the VD group and the VD ? NaHS group

Certainly, a number of investigations showed that there was a close relationship between NMDAR and neural activity rhythms (Hakami et al. 2009; Kittelberger et al. 2012; Kocsis 2012). For example, it was reported that blockade of NMDAR by ketamine, as a non-selective NMDAR antagonist, reduced the PAC between theta and gamma rhythms in the hippocampus (Caixeta et al. 2013). In addition, the blockade of NMDAR by selective NMDA2A antagonist (PEAQX and NVP-AAM077) diminished the

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theta modulation of gamma (Kocsis 2012). Liu et al. showed that preferential inhibition of NR2A prevented the induction of LTP (Li et al. 2011). Moreover, it reported that the activation of NR2A promoted neuronal survival and presented a neuroprotective action against both NMDA receptor-mediated and non-NMDA receptor-mediated neuronal damage (Hardingham et al. 2002). In our previous study, it was found that H2S significantly improved spatial learning and memory deficits induced (P \ 0.01), inhibited

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the neuronal death induced by VD and remarkably enhanced LTP in the hippocampus of VD rats and (Li et al. 2011). Our data showed that NaHS considerably alleviated the impairment of neural coupling in the VD rats’ either in an identical-frequency band (Fig. 2) or between cross-frequency bands (Figs. 3, 4, 5). Most importantly, the present investigation showed that the expression of NMDAR2A was significantly decreased in the hippocampus of VD rats but visibly recovered by NaHS (Fig. 8), which was associated with the alterations of neural oscillatory patterns (Figs. 2, 3, 4, 5). In summary, our results elucidated the relationship between the modulation of oscillatory coupling in the hippocampus and the effects of H2S from a novel angle. The data suggested that H2S played an important role on regulating the phase coupling either in an identical frequency band or between cross frequency bands (PPC and PAC) in the hippocampal CA3–CA1 pathway, which was associated with the alterations of cognitive functions. Moreover, the effects of H2S on VD might be linked to the modulation of NMDAR2A expression in the hippocampus, which could be a potential molecular mechanism of H2S neuroprotection. Acknowledgments This work was supported by Grants from the National Natural Science Foundation of China (31171053, 11232005 to TZ),Tianjin Research Program of Application Foundation and Advanced Technology (12JCZDJC22300 to TZ), Tianjin Applied Basic Research Programs of Science and Technology Commission Foundation (14JCQNJC11800 to CHL) and 111 Project (B08011 to TZ).

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