Impairment of Theta Oscillation in Hippocampus CA1 mediates age-dependent movements’ alternations in 5xFAD Mouse Model of Alzheimer's Disease

doi:10.21203/rs.3.rs-4531158/v1

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Impairment of Theta Oscillation in Hippocampus CA1 mediates age-dependent movements’ alternations in 5xFAD Mouse Model of Alzheimer's Disease

https://doi.org/10.21203/rs.3.rs-4531158/v1

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Clinical evidences indicate that multifaceted gait abnormalities may manifest in Alzheimer's disease (AD) patients, which associated with cognitive decline. Although the correlation between hippocampal theta power and locomotion has been known for a long time, the mechanisms by how hippocampus impairment participates in the altered gait seen in AD is not fully understood. To explore the manifestations of gait disorders in AD, we characterized gait performance in 3-, 6-, and 9-month-old male 5xFAD and control mice in the semi-automated, highly sensitive, Catwalk XT system. The 5xFAD mice displayed a decrease in kinetic parameters (average speed and cadence), and spatial parameters (paw area), while the temporal parameters (stance and swing time) were significantly increased. The parameters of interlimb coordination also displayed deficits. The majority of impairment variables related to the slow speed in 5xFAD mice at 9-month-old. We further explored the theta oscillations in the brain by tetrode in vivo recording of the hippocampus CA1. The results showed that the theta oscillations reduced in the hippocampus CA1 of 5xFAD mice, which related to the gait impairments. In conclusion, gait impairments started at 6 months of age, manifested at 9 months of age in 5xFAD mice. A reduction in theta oscillation power of the hippocampus CA1 might be responsible for the gait impairments.

Alzheimer's disease

5xFAD mouse

Gait

Hippocampus

Theta oscillation

Alzheimer's disease and other neurological disorders of the central nervous system (CNS), including Parkinson's disease, Huntington's disease, and stroke negatively impact coordination and motor functions, with one particular characteristic being abnormal gait patterns. Gait is a complex and learned biological activity that begins with voluntary action [1]. One leg bears the weight of the body while the other moves forward to begin the next support, requiring a repetitive, flowing, and naturally coordinated equilibrium at each step [2]. Such a process reveals different gait patterns that can subsequently be used to assess individual health, especially that of elderly people [3]. Gait is a complex activity for which proper cadence, speed adjustment, postural control, balance, and coordination of limb movement are required. In particular, speed regulation is an important aspect of locomotion. Since muscle activity is coordinated, changes in locomotor speed may also involve changes in coordination between limbs, leading to specific patterns of limb movement, or gaits. In this context, quadrupeds display various locomotor gaits which are speed-dependent [4].

As an age-related neurodegenerative disease, Alzheimer's disease (AD) is characterized by neurofibrillary tangles, Aβ plaques, and cognitive impairment [5]. In addition to the cognitive deficit that is the primary clinical hallmark of Alzheimer's disease, various studies have also reported a close relationship between a decline in gait function and AD [6–8]. In fact, it has been reported that the early stages of the disease can be characterized by impaired gaits and, as such, these can serve as biomarkers to predict cognitive impairment among the elderly [9–11]. It has even been shown that AD is more likely to develop in patients with mild cognitive impairment (MCI) that is accompanied by motor dysfunction compared with those with MCI only, with the motor dysfunction often related to the severity of cognitive dysfunction in patients with AD [12]. Compared to normal aging [13], AD was associated with gait characteristics characterized by a slower pace, impaired rhythm, and an increased variability. High gait variability was regarded as a sign for cognitive-cortical dysfunction, which may facilitate the diagnosis of Alzheimer's disease [14]. The study also demonstrated that patients with AD typically walk at a slower speed, and as the disease developed, they were affected more [15]. Furthermore, considering that reduced mobility has a negative impact on the quality of life while increased falls can lead to an increase in morbidity and mortality, gait disturbances are of particular concern [16]. It may be helpful to monitor the mobility performance of elderly persons continuously and regularly in order to diagnose and assess the degree of neurological impairment [17].

Due to similarities between rodent and human locomotion, motor deficits are often studied qualitatively in rodent models using the beam balance rod, inverted screen, grip strength and rotarod tests [18], as these help to quantify the efficacy of different therapeutic approaches. However, the main limitation of these motor tests is that they are primarily undertaken by a trained observer who is only able to detect major motor and coordination dysfunctions [19]. Recently, the application of technology as well as automated systems made it possible to quantitatively assess motor deficits, independently of the observer, through CatWalk gait analysis.

Despite the fact that rodents use quadrupedal gait patterns that differ significantly from those of humans, yet the basis for analyzing gait patters is similar [20, 21]. Thus, advanced analysis of rodent gait can be essential for better modelling of human disorders. Given the increasing importance of the link between AD and human gait, several studies are now already focused on characterizing the change of gait in AD mouse models. For instance, Brown et al. found impaired locomotion activity in 11- to 13-month-old 5xFAD mice, possibly as a result of impaired gait [22, 23]. Similarly, in Tg2576, another type of AD mouse model, Seo et al. reported significant reductions in stride times as well as increases in cadence prior to plaque accumulation, regardless of body size or weight [24]. Gait abnormalities were also present in the 6-month-old 3xTg-AD mice for which strides were shorter compared with wild type ones [25]. However, the effects of AD on various gait parameters of 5xFAD mouse model of different ages are yet to be well characterized. Therefore, the current work compared gait function in freely moving 3-, 6- and 9-month-old male 5xFAD mice and age-matched wild type controls using Catwalk XT system to analyze gait parameters (e.g., stride time and length, cadence, swing speed and average speed). These parameters are thought to be sensitive indicators of human movement anomalies associated with Alzheimer's disease [14] to extend previous findings that AD mice exhibit gait abnormalities.

Furthermore, since variations in gait can be indicative of subtle changes that occur as a result of impaired cognition or aging, corresponding brain characteristics have also been widely studied. It is known that the hippocampus plays an important role in memory, navigation, and cognition [26]. Notably, the medial entorhinal cortex and the CA1 region of the hippocampus have been associated with linear speed modulation, either together with positional information or as speed cells [27, 28]. Additionally, theta power was found to be related to a locomotor activity which was measured at the hippocampal field potential, where speed was found to be influenced [29, 30]. The theta rhythms’ amplitude and frequency were found to increase with walking speed in the hippocampus CA1 [31], hence suggesting its involvement in coding speed. Gait speed is also directly encoded through changes in the firing rates of neurons within the entorhinal cortex and the hippocampus [27, 32–34]. Additionally, several entorhinal neurons demonstrate changes in firing rhythmicity with an increase in walking speed, most commonly increasing their frequency [35]. Over the past few decades, studies on patients and animal models of AD have reported reduced theta power in the CA1 region of the hippocampus [36–38]. For a long time, researchers have understood the correlation between the hippocampus and movement [39, 40], the mechanisms by how hippocampus impairment participates in the altered gait seen in AD are still not clear. Consequently, the role of the CA1 region of the hippocampus in modulating gait speed was additionally investigated using tetrode in-vivo recording during walking in an open field test (OFT) to determine whether it contributed to a gait deficit in 5xFAD mice.

Herein, we report that 5xFAD mice exhibit significant impairment in gait behavior, particularly at 9 months of age, possibly due to functional abnormalities such as reduced theta oscillation power and decreased speed correlated cell proportions within the hippocampus CA1.

2.1 Animal grouping

Male 5xFAD mice and age-matched C57BL/6 WT mice were obtained from the Model Animal Research Center of Nanjing University and bred in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. 3-, 6- and 9-m old mice (n = 10 in each group) were used in studies. All mice were housed in an environment with a temperature of 22 ± 2° C and humidity of 55 ± 10% under a 12-h light/dark cycle (light on 07:00–19:00) and had access to water and food ad libitum. Before experiment all mice were allowed to have seven days acclimation. All experimental protocols were in strict accordance with the regulations of the National Institutes of Health guide for the care and use of laboratory animals, formulated by the Ministry of Science and Technology of the People's Republic of China and were approved by the Animals Research Ethics Committee of Shanghai University of Chinese Medicine (No.PZSHUTCM200821019).

2.2 CatWalk gait analysis

To detect the gait differences between 5xFAD and WT mice at different ages, we tested 3-, 6- and 9-m male mice using a highly sensitive, automated computer-assisted method (Noldus Information Technology Inc, USA) as described [41]. Catwalk is a sophisticated apparatus that is capable of assessing footfall and motor performance quantitatively, as the animals freely walked on an illuminated glass platform (20cm wide × 130cm long), a high-speed high-resolution camera was used to record paw location and placement patterns, providing accurate and repeatable measurements of gait function as well as spatial and temporal aspects of interlimb coordination. We began by establishing the walkway area in accordance with the recommendation of the company. We did not include any light stimuli, food enticements or even the home cage at the end of the walkway as bait in our method in order to achieve unforced locomotion. The mice were placed in the testing room 30 minutes before the experiments to become accustomed to the conditions of the experiment. Every mouse had a 25-minute free walk along the corridor and walkway on training days. Every trial was followed by the cleaning of the glass plate. A minimum of three completed trials were collected during the testing days, during which the mice were allowed to freely move back and forth on the corridor for 20 minutes. For mice that were not able to complete the trial in 20 minutes, a second try was permitted the following day so that at least three compliant trials could be completed without stressing the mice. The high-speed camera was recorded at 100 frames per second (GP-2360C, GEViCAM) with lenses set at 1.5 apertures (DFGHA–1B Fujifilm). Trials that met compliance criteria were defined as those ranging in duration between 1 and 5 seconds (where the average speed should be between 8 and 40 cm/s), along with maximum speed variations of not more than 20%. Based on the manual and previous experience [42], the training and experimental protocols were conducted.

The phase coupling parameter describes the temporal relationship between placement of two paws (anchor and target) within a step cycle. It can also be used to examine inter-paw coordination across six pairs of paws (diagonal pairs: RF-LH, LF-RH, ipsilateral pairs: RF-RH, LF-LH, and girdle pairs: LF-RF, LH-RH). A healthy young mouse with normal gait usually moves the diagonal paw pairs synchronously with the anchor, resulting in a 0% phase coupling values. A phase coupling of 50% is expected when ipsilateral and girdle pairs alternate. The phase coupling is expressed as a percentage of the step cycle time of the anchor paw by measuring the timing between initial contacts of paw pairs. In order to determine whether asynchronous stepping occurs, the absolute deviation from the expected coupling value should be calculated. Deviation from the expected value which represents a high level of gait coordination is shown as Δ Phase coupling [43, 44].

Subsequently, for the analysis, each foot print was manually checked and labelled as LF (Left Front), LH (Left Hind), RF (Right Front), and RH (Right Hind) paw to generate gait parameters. We categorized the gait parameters into four major categories to better present the data: (1) gait characteristics and kinetic parameters (average speed, cadence, number of steps, body speed and swing speed), (2) temporal parameters (stance and swing time, step cycle and maximum contact at [% of stance time]), (3) spatial parameters (print area and maximum intensity), (4) interlimb coordination parameters (base of support, print position stride length, support, phase coupling, number of patterns, regularity index and step sequences).

2.3 Open Field Test (OFT)

Implanted mice were allowed to freely move in the square arena (50×50×40 cm) with white walls and floor for 30 minutes. The speed and total distance travelled of the mice were recorded and analyzed by CinePlex video tracking system (Plexon, USA). The open field arena was fully cleaned with 75% ethanol after each trial.

2.4 Tetrode electrode implantation surgery and data collecting

We used the Self-made movable bundles of the 32-channel tetrode electrode [45] to target the hippocampus CA1 (AP -2 mm; ML ± 1.8 mm and -DV 1.1 mm) of the anesthetized mice on the stereotactic frame. The signals were amplified, band-pass filtered (0.5–1000 Hz for local field potentials [LFPs] and 0.6-6 kHz for spikes), and digitized by the Plexon OmniPle Neural Data Acquisition System (Plexon, USA). Neural signals were recorded by using the Plexon OmniPlex Neural Data Acquisition System (Dallas, TX, USA). Signals were digitized at a frequency of 40 kHz for spikes. When the recording is finished, open the data with Offline Sorter to open for offline processing. The processing includes: wave shape alignment, noise filtering, neuron classification, etc. Then we used custom-made Matlab and followed by manual adjustment using the software MClust 4.4 program (A. D. Redish, http://redishlab.neuroscience.umn.edu/MClust/MClust.html). Through the above steps single units were identified from multi-unit recordings. Last, we used Neuroexplorer 5.306 software (Nex Technologies, USA), Neuroexplorer software correlates neuronal signals with behavioral data.

2.5 Statistical analysis

Normality was checked by visual inspection of the data and a Shapiro-Wilks test and p value was more than 0.05. Unpaired Student t tests were performed to compare the data with 2 experimental groups (WT and 5xFAD) of the same age. Two-way ANOVA followed by multiple comparison for non-parametric tests was performed to investigate the effects of age at 3 m, 6 m, and 9 m 5xFAD mice. All correlations were assessed using a linear regression to find an r 2 value and the F-test to determine a p value of the linear fit. All data were presented as means ± SEM and analyzed by GraphPad 7.0 software (San Diego, CA). A p value of < 0.05 was considered as statistically significant.

3.1 Gait impairment in 5xFAD mice

Here, we found that compared to WT ones, 5xFAD mice exhibited significant functional impairment in gait that intensify with increasing age, manifesting by the following four aspects of gait parameters (1) decreased speed (including average speed and swing speed of all paws), stride length and cadence as well as increased number of steps; (2) spending more time (including stance time, swing time, step cycle time and time for all paws to make the maximum contact to the glass plate); (3) decreased print areas and maximum intensity of all paws; (4) increased base of support and phase coupling deviation of all paws, decreased print position, and regularity index as well as significantly altered step cycle and step patterns of all paws. All the gait parameters that we focused on in our study are shown for each paw, namely the left front (LF), left hind (LH), right front (RF), and right hind (RH) paw. For some data, we only presented single forelimb (RF) and hindlimb (RH) as the data of the other two limbs were similar. In detail, the following is described.

3.1.1 Gait characteristics and kinetic parameters

Firstly, the body weights of mice were examined to determine suitable detection settings. The comparison between 5xFAD mice and WT ones of the same age (3-, 6- and 9-m) revealed no difference, while both of them showed significantly increased body weights with aging (Fig. 1A). The results of gait characteristics and kinetic parameters manifested that from 3- to 9-m old, 5xFAD mice showed a significant decreasing trend in the average speed (Fig. 1C) and cadence (Fig. 1D) and an increasing tendency in the number of steps with age (Fig. 1E) and differences were found in 3- and 9-m old groups. At the same time, we discovered that compared with 6-, and 9-m old WT mice, the age-matched 5×FAD ones showed decreased average speed and cadence and increased number of steps, but there was no difference between 3-m old groups (Fig. 1C, 1D, 1E). Further, the swing speed of 6- and 9-m old 5xFAD mice were decreased compared to WT ones (Fig. 1F). For stride length, all paws of 5×FAD mice showed significantly decreased with age, and there is significant difference between 3- and 9-m old groups. Compared to 3- and 6-m old WT mice, the four paws stride length of age-matched 5×FAD ones showed only a tendency towards decrease, while there was an obvious decrease in 9-m old group (Fig. 1G).These results indicated that 5xFAD mice showed decreased speed, cadence as well as stride length and increased number of steps, especially in 9-m old group.

3.1.2 Temporal parameters

The temporal parameters results illustrated that 5xFAD mice showed more stance time (Fig. 2B), swing time (Fig. 2C), step cycle time (Fig. 2D) and time for all paws to make the maximum contact to the glass (Fig. 2E) plate with age, and there were statistical differences between 3- and 9-m 5xFAD groups. Moreover, the 9-m old 5×FAD ones showed much more stance time, swing time and step cycle time of all paws (Fig. 2B, 2C, 2D). Relative to their stance time, the 9-m old 5×FAD ones also showed increased time for all paws to make the maximum contact at the glass plate relative to their stance time since the start of the trial (Fig. 2E). These results suggested that the 5xFAD mice showed significantly increased temporal parameters of all paws at 9-m old.

3.1.3 Spatial parameters

For spatial parameters results, 5×FAD mice revealed a decreasing trend of print areas (Fig. 3C) and maximum intensity (Fig. 3D) of all paws with age, and the statistic difference between 3- and 9-m old ones was significant. Meanwhile, 9-m old 5×FAD mice showed decreased print areas and maximum intensity of all paws (Fig. 3C, 3D). We also discovered the minimum intensity of all paws were highly increased at 9-m old in both WT and 5xFAD groups, but there was no statistic difference between WT and 5×FAD ones of all ages (Fig. 3E). These results revealed decreased print areas and maximum intensity of all paws in 5xFAD mice.

3.1.4 Interlimb coordination parameters

Comparing interlimb coordination parameters (Fig. 4A) between 5×FAD and WT mice, 5×FAD ones revealed a higher base of support of front and hind paws, and the statistic difference between 9-m old groups was significant. Due to the increasing tendency of 5xFAD ones with age, the difference in base of support of all paws between 3- and 9-m old groups was significant (Fig. 4B). 5xFAD mice also showed significantly reduced print position of both right and left paws with age, the difference between 3- and 9-m groups was significant. Meanwhile, 9-m old 5×FAD mice revealed less print position of both right and left paws in comparison with age-matched WT ones (Fig. 4C). It was found that WT mice were mainly supported by two paws in a diagonal shape during the trial (about 60%). In contrast, 5×FAD ones revealed a reduced tendency of diagonal shape with age, and there were statistic differences between 3- and 9-m old 5xFAD groups and also 9-m old WT and 5xFAD groups. However, 9-m 5×FAD mice revealed an increased percentage of other types of support patterns, such as girdle, lateral, and even three or four paws with an obvious difference (Fig. 4D).

Phase coupling measures inter-paw coordination across girdle, ipsilateral and diagonal paw pairs during spontaneous walk (Fig. 5A) [46]. Increased deviation from expected phase coupling values indicate worsening inter-paw coordination. In comparison with age-matched WT controls, 9-month-old 5xFAD mice displayed a significant increase in phase coupling deviation across most paw pairs (Fig. 5B, C, E, G). Nevertheless, no significant differences were observed between WT and 5xFAD mice at 3-months of age. In general, we observed an obvious decreasing of inter-paw coordination of 5xFAD mice of all paw pairs with aging.

For regularity index, we discovered that 5xFAD mice showed less and less during the trial with age, and there was obvious difference between 3- and 9-m old groups. Compared to age-matched WT mice, the regularity index of 6- and 9-m old 5xFAD ones were also much less (Fig. 6B). Even no statistic difference was found in the number of footfall patterns between 5xFAD and WT mice of all ages (Fig. 6C). But when it comes to gait-pattern frequency distribution, we found that start form 3-m old, compared with age-matched WT mice, 5xFAD ones exhibited a 15 to 20 percent increase in the frequency of the radial pattern AA (LH-RF-RH-LF). Meanwhile, the alternating contralateral footfall pattern CB (LH-RH-LF-RF) was used with increased frequency in 5xFAD mice of all ages. Thus, the compensatory adjustments in gait mechanics of 5xFAD mice, observed via gait pattern frequency distribution, revealed that since 3-m old, 5xFAD mice preferentially relied on the AA radial pattern and alternating CB pattern over the AB (LH-LF-RH-RF) and CA (LH-RF-LF-RH) gait patterns preferentially used by WT mice (Fig. 6D).

In the end, we use radar plot to illustrate the percentage changes for each gait parameter of 5xFAD mice at different ages in comparison with age-matched WT ones. Not much changes were found between these two groups at 3 months of age, but from 6 months of age, differences started to show in some gait parameters, and by 9 months of age, significant differences were found in all gait parameters apart from the minimum intensity and number of patterns between those two groups (Fig. 6E). These parameters exhibited a significant difference in average speed, cadence, number of steps, stance time, swing time, step cycle time, maximum contact time at the glass plate, print area, maximum intensity, base of support, stride length and regularity index of all paws in average in 5xFAD mice, especially at 9-month-old.

3.2 Correlation between speed and gait parameters of 5xFAD mice

Based on above results, we found that 5×FAD mice manifested significant functional impairment in gait, especially at the age of 9 months, hence, we focus on the time point of 9-m old for the following study. In addition, in light of findings by Batka et al. [47] and de Haas et al. [48] that CatWalk parameters vary with speed, we decreased this variation by selecting the trials with similar speed for the subsequent analysis of 9-m groups as shown in figure S1 (A-O). It was found that the difference in gait parameters between 5xFAD mice and WT mice disappeared when speeds were the same, suggesting that the difference in gait parameters was the result of a slower speed.

At the same time we evaluated a possible correlation of every gait parameter of 9-m old 5xFAD mice with speed by calculating Pearson’s correlation. As illustrated in Fig. 7 (all values represented by the average of four paws,) the cadence, stride length, print area, and maximum intensity showed a positive correlation to the speed of mice (Fig. S2A-D). In contrast, base of support, maximum contact time at the glass plate, stand time, number of steps and number of patterns showed a negative correlation to the speed of mice (Fig. S2E-I). While regularity index, step cycle time and swing time showed no significant correlation to the speed of mice (Fig. S2J-L). These results showed a strong correlation between speed and gait parameters, which also highlighted the speed of the mouse as a strong predictor of its gait performance.

3.3 Impairment of theta oscillation and speed correlated cells in hippocampus CA1 of 5xFAD mice

Then we carried out open field test (OFT) with local field potential (LFP) and single cell activity in vivo recording in the hippocampus CA1 of WT and 5xFAD mice by implanted 32-channel tetrodes. The results are presented below.

3.3.1 The change of theta oscillation in hippocampus CA1 of 5×FAD mice

Firstly, we detected the power spectra of theta rhythm (4–8Hz) [49, 50] at rest and no difference was found between WT and 5xFAD mice. After analyzing the relative power of theta oscillations during locomotor activity, we observed a significant decrease in the relative power of theta oscillation in 5xFAD mice compared with WT ones (Fig. 7A, D). Meanwhile, we found in WT mice the power of theta oscillation was positively correlated with speed, but it seemed no significant correlation between the power of theta oscillation and speed in 5xFAD ones (Fig. 7B, E). Consistent with the gait results, we also found the average speed of 5×FAD mice was significantly reduced compared with WT ones within one trial session of 1500 seconds (Fig. 7C). These results suggested that the moving speed and power of theta oscillation in the hippocampus CA1 were influenced synchronously in 5×FAD mice compared to WT ones and there might be a causal link between theta rhythm disruption and gait deficits..

3.3.2 The proportion of speed correlated cells changes in hippocampus CA1 of 5×FAD mice

A speed correlated cell (Fig. S3A) was defined by the correlation between firing rates and locomotor speed (p < 0.05; linear regression F-test) as described before [51]. We recorded total 114 units in WT and 124 units in transgenic mice. Approximate 60.5% (69/114) units in WT mice (Fig. S3G) and 40.3% (50/124) units (Fig. S3I) in 5xFAD mice showed linearly correlated between single cell spike firing and speed (positive and negative) (Fig. S3B-E). The mean r² for a linear speed correlation of all speed cells was 0.31 ± 0.037 in WT mice (0.33 ± 0.033 in 5xFAD); cells with a significant correlation to speed had a mean r² of 0.62 ± 0.033 (0.63 ± 0.027 in 5xFAD); and non-significantly correlated cells had an r² mean of 0.08 ± 0.015 (0.22 ± 0.027 in 5xFAD) (Fig. S3F, H). These results revealed that there were speed cells in both WT and 5xFAD mice, but the proportion in WT mice was much higher than that in 5xFAD ones.

In patients with AD, high gait variability has been identified as a new marker of cognitive and cortical dysfunction [52]. Indeed, a close relationship has been reported between the severity of gait changes, such as increased support phase, reduced stride length and decreased speed, and that of cognitive decline in human patients. These changes include a decrease in speed, a decrease in stride length, an increase in support phase and so on [53]. The analysis of gait is an effective and complementary method for detecting motor abnormalities associated with neuropsychiatric disorders [54, 55]. Finally, as part of the overall morphological profile of rodent models of mental diseases, gait performance must be monitored because it is a sensitive indicator of rodent health and longevity and its effect on behavior [56].

It is well known that aging is accompanied by a slowing of the gait, with many factors such as age-related changes in neurological, cognitive and physiological functions, being responsible for the process [57]. As such, differentiating between impaired gaits that result from diseases or ageing can be challenging. This issue was addressed in the current study through the recruitment of older mice controls while controlling for age within the comparative analysis. The aim of this study was to evaluate the gait alternation of 5xFAD mice when compared with age-matched WT ones by CatWalk XT at three different ages (3-, 6-, and 9-m), and to explore the mechanisms by how hippocampus impairment participates in the altered gait. In the field of Alzheimer's disease research, the 5xFAD model is among the most well-studied and commonly employed mouse models. There are thymus antigen-1 (Thy-1) promoter–driven transgenes for mutated presenilin-1 (PS1) and amyloid precursor protein (APP) [58]. These mice are well adapted for the research of movement impairment in Alzheimer's disease due to the fast onset of AD-related pathology and cognitive failure they exhibit. In addition, consistent with the findings of others, at 6 months of age, these mice start to show motor dysfunction which progresses rapidly to profound motor dysfunction by 9 months [59].

There is evidence that walk, trot, and bound are mediated by a distinct group of neurons that are recruited in a speed-dependent manner [60]. However, in a laboratory, 5xFAD mice were fed ad libitum and kept in small cages in a confined environment where, except for the experimenter and occasionally, their littermates, there were no predators. Within such a controlled setting, in addition to the severe motor function deficits of the mice [22, 23], the animals had no reason to adopt various locomotor gaits, thus explaining the study of current gaits in this research. To the best of the authors’ knowledge, the present study objectively and reliably attempt to quantify large numbers of gait parameters of male 5xFAD mice of different ages. A computer-aided gait analysis then demonstrated that the gait differences between 5xFAD mice and WT ones were increasingly apparent with age, especially in terms of decreased average, body and swing speed, increased temporal parameters and reduced spatial ones. The freely moving 9-month-old 5xFAD mice displayed significant changes in their gait signatures, which highlight the aspects of gait changes observed in AD patients, including changes in speed, cadence, gait variability, footfall pattern distribution, amongst others, compared with age-matched controls [61]. Notably, the decreased print areas of all paws in aged 5xFAD mice could be a sign of incomplete stance of paws during locomotion, which may result in reduced efficiency of the whole process [62, 63], suggesting a less efficient walking style compared to normal mice. The lower maximum intensity of all paws in aged 5xFAD mice compared with WT ones indicated their decreased propulsion [64], which also showed the feebleness of 5xFAD mice. Additionally, the decreased average, body and swing speeds of 5xFAD mice could possibly negatively impact coordination.

Interlimb coordination is important for locomotion, and therefore, factors related to coordination tend to be particularly important. For the support percentage of normal control mice, two diagonal paws (60%) were the most frequently used simultaneous paw pair during floor contact. However, except for the reduced frequency of diagonal support in aged 5xFAD mice, all other types of paw support increased in comparison with WT mice, indicating that 5xFAD mice were severely affected in terms of their stability [65, 66]. Additionally, the degree of synchrony between diagonal, horizontal, and lateral limb couplets in each group was also analyzed, a procedure also known as phase coupling, to evaluate the deterioration of locomotor coordination in 5xFAD mice. Inter-limb coupling was assessed by computing the interval between the onset times of each paw in a pair contacting the ground. As a measure of asynchrony, the absolute difference between the actual and expected coupling values was examined [67]. The expected interlimb coupling value is zero, for instance, if the diagonal limbs are moving in synchronism. It is well-known that when strolling, horizontal pairs of extremities change more frequently than diagonal pairs. There is a 50% inter-limb coupling value when the two extremities swap exactly [42, 68]. In the current study, compared with WT ones, increased deviation of 9-month-old 5xFAD mice from expected phase coupling values suggest worsening inter-paw coordination which is also commonly shown in AD patients [48].

Four common step-sequence patterns (AB, AA, CB, CA) have been described in rodents, and these are known to be different as a result of stress or motor diseases [69, 70]. This study revealed that the main step sequence pattern (AB) was the same for WT mice of all ages being investigated, and this was in accordance with a previous report whereby 80–95% of the time, intact rodents showed preference for that regular step pattern [71]. However, in this study, for 5xFAD mice of as early as three months, there was a decrease in the AB pattern, although a corresponding increase of 15 to 20 percent in the frequency of the radial pattern AA, known as the "giraffe walk" [72]. This gait pattern was found to be elevated in aged people [73] and mouse models with increased gait instability [74]. These results highlight the significant deficiency of 5xFAD mice in interlimb coordination which is consistent with AD patients [75, 76].

Due to the fact that locomotion speed affects four different movement metrics, the statistical analysis reveals both intra- and inter-individual variation [42, 47, 77], the reduced speed of 5xFAD mice can be a critical reason for the impairment of gait behavior at 9-month-old compared to WT ones. Indeed, approximately 75% of the 12 locomotor characteristics studied in 9-month-old 5xFAD mice were contingent on the movement pace of the mouse, as verified by a correlation analysis. The results of research conducted on male C57BL/6 mice indicated that a positive relationship existed between kinetic parameters like movement and body speed and certain correlations to the spatial and interlimb coordination groups, while a negative relationship existed between speed and time parameters [42]. In a different extensive study of 16 wild-type mice conducted to examine the relation between speed and 162 gait parameters reported by the CatWalk software, revealing that over 90% of these parameters were speed-dependent [78].

Major parts of the central nervous system, namely the cardiorespiratory system, joints, muscles and the peripheral nervous system, interact to produce a normal gait, and therefore this can be used as an indicator of cognitive function, as intact cognition and attention processes are necessary to ensure proper gait [79]. For older adults and aged mice, it is likely that multiple factors could be involved in the mechanisms underlying AD-related changes in gait function [2]. In some research, motor defect in 5xFAD mice had been attributed to AD-related pathology in the pyramidal and extrapyramidal motor systems of the brain and spinal cord [59]. An altered gait may result from pathological processes affecting central nervous system networks, including motor, sensory, and cognitive functions, which is a complex interplay [80]. The accumulation of neuroinflammation, neurofibrillary tangles, amyloid-β plaques and other AD-related pathologies can negatively impact the neuronal circuits that regulate gait, resulting in gait impairments in AD patients [81]. Furthermore, it has been reported that, in AD patients, cerebral microhemorrhages (CMHs) can progressively contribute to impaired gate, with this factor now considered to be of emerging importance [82, 83]. In addition, results of isometric tension recordings of the soleus muscle suggest that the ability of 5xFAD mice’s motor nerves to generate output are not impaired, and therefore, impaired motor performance of the animals may not be attributed to denervation or impaired signaling at the neuromuscular junction [22]. Consequently, further research is warranted to examine the contribution of AD-related pathology within different regions of the brain and spinal cord to the motor dysfunction observed in 5xFAD mice.

There is evidence of a close link between gait variability in AD patients and structural and functional differences of the hippocampus [76, 84, 85]. Hence, in addition to learning and memory, abnormal hippocampal function may also significantly affect the gait. One of the most prominent topics to study in relation to hippocampus dysfunction is theta oscillation, which shows intriguing correlations with different behaviors that include both aspects of movement and memory function [39]. Theta rhythm is related to different types of movement behaviors, including running on a treadmill [86], track [87], and running wheel [88]. Similarly, theta rhythm is also correlated with learning and memory functions [89]. Consequently, a reduction in theta rhythm could be linked to memory impairment, as it is the case for AD [50, 90]. Less studied than movement and memory correlates, theta rhythm has also been linked to arousal [91], attention [31], and sensorimotor integration [92]. Moreover, speed has been reported to be affected by the oscillatory activity recorded in the hippocampal field potential, where theta power appears to be correlated with locomotor activity [93–95]. Hence, it seems that hippocampal theta band oscillations are prominent during locomotion [96]and suggest that they differ from passive and active waking [97]. Shin proposed that these oscillations might encode motor behavior information [98]. Meanwhile, multiple studies have shown that the frequency and amplitude of theta rhythm in the field potential of hippocampal CA1 are positively correlated with locomotor speed [99–102]which can be observed across three simultaneous recording sites in CA1 [103], and optogenetically increasing the amplitude of theta oscillations in hippocampus CA1 resulted in locomotion speed enhancement [104], suggesting crucial roles for the hippocampal theta rhythm in coding of locomotion velocity. The adjacent structures of the hippocampal formation and the medial septum are likely to convey information about head direction and locomotion speed to the hippocampus [105, 106]. However, in animal models and humans, theta band activity appears to modulate episodic memory and to be associated with AD pathology. Researchers have in fact reported varying degrees of reduced theta power and synchrony in patients with AD over the past few decades [107, 108]. Additionally, it has been reported that hippocampal theta oscillation declines with age, with a strong correlation between it and an accumulation of hippocampal Aβ in APP/PS1 mice [109]. In fact, impaired theta oscillation in the hippocampal CA1 region was considered one of the causes of impaired hippocampal-dependent cognitive function in AD rats [110] and 5xFAD mice [111]. Here, based on the findings, theta oscillations in the hippocampus CA1 were significantly reduced in comparison with the WT controls during locomotor activity, thus explaining the slower speed seen in 5×FAD mice and the subsequently gait impairment.

Furthermore, it has been demonstrated that the spiking activity of the hippocampal neurons oscillates at theta frequency, which is modulated by the LFP theta phase [112, 113] Consequently, changes in the firing rate of CA1 speed cells could be correlated with changes in theta frequency with speed, such that a higher frequency of theta cycles would result in a higher number of spikes per unit of time, with such an effect known as the “oscillatory coding” [114]. At the same time, the findings suggest that, neurons in the hippocampal formation and entorhinal cortex directly code locomotor speed with changes in firing rate [114, 115]. There are a number of neurons that can code locomotor speed, including grid cells and head direction cells in hippocampus CA1 [116]. With increasing speed, these speed cells commonly increase their frequency [35, 117]. In this study, a population of speed cells was identified in the hippocampus CA1 which, in a similar way to studies here, have a linear spiking correlation with locomotor speed in both WT and 5xFAD groups. We additionally described a partially reduced proportion of speed cells over normal control group in hippocampus CA1 of 5xFAD mice, which might also contribute to the reduced speed.

In conclusion, through this study, age-related changes in the voluntarily walking gait performance of male 5xFAD mice was characterized. Meanwhile, at the age of 9 months, 5xFAD mice exhibited quantifiable, clinically relevant alterations in gait function compared with age-matched WT ones which highlight the findings in human AD patients. Furthermore, it is possible that the abnormal operation of the hippocampus CA1, including reduced theta oscillation power and decreased proportion of speed cells could have been responsible for all the impaired gaits.

Ethical Approval

All animal experiments shall comply with the experimental animal management and ethics regulations of Shanghai University of Traditional Chinese Medicine, the ethics number of which is PZSHUTCM2212260003, and minimize animal suffering and unnecessary death during experimental operations.

Funding

This work was supported by the Chinese National Natural Science Foundation of China (Grant No. 62127810)

Authors’ contributions

Hong Ni and Zhongzhao Guo wrote the manuscript and designed the study; Jie Wang designed the study study; and Zilu Zhu analyzed the data; Chenyi Xia and Ming Xu performed the study; Guohui Zhang and Deheng Wang performed critical comments on the whole process of this study.

Consent for publication

All authors agree to the publication of this manuscript.

Competing interests

The authors declare no financial conflict of interest that might be construed to influence the results or interpretation of the manuscript.

Data availability statement

All data analyzed and presented in this study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Impairment of Theta Oscillation in Hippocampus CA1 mediates age-dependent movements’ alternations in 5xFAD Mouse Model of Alzheimer's Disease

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Abstract

Figures

1. Introduction

2. Methods

2.1 Animal grouping

2.2 CatWalk gait analysis

2.3 Open Field Test (OFT)

2.4 Tetrode electrode implantation surgery and data collecting

2.5 Statistical analysis

3. Results

3.1 Gait impairment in 5xFAD mice

3.1.1 Gait characteristics and kinetic parameters

3.1.2 Temporal parameters

3.1.3 Spatial parameters

3.1.4 Interlimb coordination parameters

3.2 Correlation between speed and gait parameters of 5xFAD mice

3.3 Impairment of theta oscillation and speed correlated cells in hippocampus CA1 of 5xFAD mice

3.3.1 The change of theta oscillation in hippocampus CA1 of 5×FAD mice

3.3.2 The proportion of speed correlated cells changes in hippocampus CA1 of 5×FAD mice

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

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