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Several pathophysiological events occur after cis P-tau Background accumulation in the neurons, for example, microtubule Alzheimer’s disease (AD) is an age-related, progressive, networks and axonal mitochondrial transport disruption, and irretrievable neurodegenerative disorder. The clini - propagation of cis P-tau to other neurons , activation cal manifestation of AD is a progressive loss of cogni- of several kinases [12, 13], and finally, cell apoptosis. Cis tive ability and daily function activities [1, 2]. The most P-tau is aggregated more than trans P-tau because it is common pathophysiology of AD is an unusual extracel- resistant to dephosphorylation and degradation, cannot lular accumulation of amyloid-β peptide (Aβ) as amy- reinforce microtubule assembly, and is more prone to loid and senile plaques and hyperphosphorylated tau aggregation . Twenty hours after the onset of tauopa- protein aggregated as intracellular neurofibrillary tan - thy, cis P-tau was dramatically aggregated in the cerebral gles (NFTs) . Tau protein is known as a microtubule- cortex and remained high for up to 2 months, and was associated protein that is mainly expressed in the brain. propagated from the cerebral cortex into the hippocam- The main functions of tau are stabilizing and coordinat - pus 6 months later . Therefore, the accumulation of ing the molecule’s movement along the microtubule, cis P-tau and the possibility of dementia and AD is raised which is strongly regulated by phosphorylation. Phos- to 30 years later . phorylated tau has two isoforms: trans P-tau is physi- Pourhamzeh et al.  confirmed that bilateral intra- ological, promoting microtubule assembly, whereas the hippocampal injection of cis P-tau could produce Alz- cis form is pathogenic . heimer-like disease in rats. They reported that cis P-tau The mechanisms of phosphorylation and hyper - increased β-amyloid accumulation and tau protein aggre- phosphorylation of tau protein are unknown. Hyper- gation in the hippocampus and neocortex and impaired phosphorylated tau, especially cis P-tau, aggregates in learning and memory in the Morris water maze at 2, 4, some neurodegenerative diseases named tauopathies and 8 weeks after injection. In line with this data, another [4–8]. Tauopathies are progressive neurodegenerative study showed that P-tau accumulation following trau- disorders pathologically determined by tau deposits in matic brain injury results in Alzheimer-like changes and the brain, such as Alzheimer’s disease, frontotemporal impairment in spatial learning and memory in the Morris dementia, and chronic traumatic encephalopathy [9, water maze 6 weeks after P-tau accumulation. However, 10]. F atemeh et al. Behavioral and Brain Functions (2023) 19:9 Page 3 of 13 these changes disappeared at 7 months after cis-P tau increase in β-amyloid and cis P-tau accumulation up to injection . 2 months after intra-hippocampal cis P-tau injection In addition to memory impairment, pathological tau . Seven months after intra-hippocampal cis p-tau aggregation in the synapses can promote synaptic loss injection, no β-amyloid accumulation was observed and and synaptic plasticity impairment . The density of there was no statistical significant between cis P-tau and NFTs is significantly correlated with synaptic loss and control groups (P = 0.9118). However, there was a slight cognitive reduction, indicating that the pathological tau cis P-tau accumulation in the cis P-tau group, although may be a pathogenic factor in AD . Therefore, given it was not significant compared to the control group that pathologic tau protein disappears in the brain after (110.3 ± 1.613% vs. 105 ± 2.22% respectively; P = 0.0895). 7 months, in this study, we want to know whether dorsal hippocampal injection of cis P-tau causes impairment in Intra‑hippocampal cis‑P tau resulted in working memory learning and memory and disruption in synaptic plastic- impairment ity in dorsal and ventral hippocampus in 7 months after Working memory was evaluated by the Y maze test at the injection (Fig. 1). 7 month after bilateral intra-hippocampal injection of cis-P tau. Unpaired t-test showed an impaired percentage Results of spontaneous alternation in the cis-P tau group com- Cis P‑tau and β‑amyloid accumulation disappeared pared to the control group (57.13 ± 0.72 vs. 74.30 ± 1.73 at 7 months after intra‑hippocampal cis P‑tau injection respectively; P < 0.001; Fig. 2A). The percent time spent At the first step, β-amyloid and cis P-tau accumula - in center point decreased in cis P-tau compared to tion was assessed in the dorsal hippocampus by immu- control group (3.32 ± 0.27 vs. 5.4 ± 0.57 respectively, nostaining. Previous experiment showed a significant P < 0.001; Fig. 2B). The total distance did not affect by Fig. 1 Cis P‑tau and β‑amyloid accumulation decreased at 7 months after intra‑hippocampal cis Pinjection. A Cis P ‑tau and amyloid beta staining is shown in hippocampal area in control and cis P‑tau groups. Quantification of β‑amyloid B and cis P ‑tau C immunostaining showed that there was no hippocampal accumulation at 7 months after cis P‑tau injection. All data show mean ± SEM Fatemeh et al. Behavioral and Brain Functions (2023) 19:9 Page 4 of 13 Fig. 2 Impaired working memory in mice at 7 months after intra‑hippocampal cis P ‑tau injection. The percentage of spontaneous alternation (A) and the percentage of time spent in center point (B) significantly decreased at 7 months after intra‑hippocampal cis P ‑tau injection in cis P ‑tau compared to control group. There was no significant difference in total distance between cis P ‑tau and control groups (C) . Color maps depict the mean spontaneous alternation across all animals in each group, showing the percent time in each arm during the whole eight‑min test duration (D). All data represents mean ± SEM. **P < 0.01 and *** P < 0.001 compared to the control group intra-hippocampal cis P-tau injection (Fig. 2C). These increased in cis P-tau compared to the control group results indicated that cis- P tau administration might on day 2 (14.11 ± 0.96 vs. 10.69 ± 0.83; P < 0.05), day 3 cause working memory impairment at 7 month after (10.51 ± 0.74 vs. 6.2 ± 0.77; P < 0.01), and day 4 (8.48 ± 0.64 injection. vs. 5.15 ± 0.56; P < 0.05) (Fig. 3C). The total error was sig - nificantly higher in the cis P-tau group compared to the Cis P‑tau led to spatial learning and memory damage control group only on day 3 (10.58 ± 0.75 vs. 6.29 ± 0.77; Spatial learning and memory were assessed at 7 months P < 0.05) (Fig. 3D). There was no significant difference in after bilateral intra-hippocampal injection of cis-P tau in total distance and velocity in the cis P-tau compared to the Barnes maze test. A two-way ANOVA followed by the control group. These data showed that cis P-tau injec - Sidak’s multiple comparisons test showed a higher pri- tion did not interfere with the animal’s locomotors activ- mary latency to find the goal box in the cis P-tau com - ity (Fig. 3E, F). pared to the control group on day 2 (134.67 ± 2.59 vs. The strategy to find the goal box was also assessed in 76.64 ± 5.25; P < 0.01), day 3 (75.55 ± 4.12 vs. 36.42 ± 1.97; these two groups. The random strategy shows the defi - P < 0.05) and day 4 (56.796 ± 2.017 vs. 19.304 ± 2.021; ciency in animal’s learning and memory. Therefore, ani - P < 0.05) (Fig. 3A). Similarly, the total latency to find the mals search the entire maze environment to find the goal goal box increased in the cis P-tau group compared to box. In serial strategy, the animal’s learning increases, control group on day 2 (209.80 ± 12.27 vs. 130.66 ± 10.59; and the subject can find the goal box using its memory. P < 0.001), day 3 (99.80 ± 10.85 vs. 48.07 ± 4.01; P < 0.001) Finally, the animal detects the exact location of the goal and day 4 (77.83 ± 4.34 vs. 29.43 ± 4.63; P < 0.001) box using peripheral cues and memory in direct strategy. (Fig. 3B). All (100%) animals in both groups used a random strat- Primary and total errors were evaluated by two-way egy due to a lack of familiarity with the environment on repeated measures ANOVA and Sidak’s multiple compar- day 1. During the training days, subjects in the control isons post hoc test. The primary error was significantly group used serial and direct strategy on day 2 (23.33% F atemeh et al. Behavioral and Brain Functions (2023) 19:9 Page 5 of 13 Fig. 3 Impaired learning and spatial memory in mice 7 months after intra‑hippocampal cis P ‑tau injection. Graphs shows the effect of intra‑hippocampal cis P ‑tau injection on primary latency A total latency B primary errors C total errors D traveled distance E velocity F strategy G and hole exploration frequency in the goal sector (GS) I non‑ goal sector (NGS) J GS/NGS ratio K and target‑seeking activity L in the Barnes maze test at 7 months after cis P‑tau injection compared to the control group. All data represent mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001. Color maps M shows the percent time in each location of the maze on the probe day in control and cis P‑tau groups. A sample of exploration path of animals in control and cis P‑tau groups on the probe day is also showing N. The black circle marks the location of the escape box in M and N and 14.17% respectively), day 3 (24.15% and 55.01% percentage of random and serial strategies throughout respectively), and day 4 (30.82% and 65.01% respectively). the training demonstrated that the cis P-tau group could (Fig. 3G, H). However, animals in the cis P-tau group did not learn the goal box’s location and could not memorize not learn to use direct strategy and mostly used random it. and serial strategies on days 2 (93.75% and 6.25% respec- Goal sector (GS) exploration significantly decreased in tively), day 3 (18.75% and 67.85% respectively), and day the cis P-tau group compared to control (3.33 ± 0.15 vs. 4 (37.5% and 43.75% respectively) (Fig. 3G, H). The high 5.17 ± 0.33; P < 0.001). In addition, non-goal sector (NGS) Fatemeh et al. Behavioral and Brain Functions (2023) 19:9 Page 6 of 13 exploration was significantly increased in the cis P-tau group compared to control (2.05 ± 0.08 vs. 1.20 ± 0.10; P < 0.05) during the probe test (Fig. 3I, J). GS/NGS ratio as a GS preference or a spatial memory index was signifi - cantly decreased in the cis P-tau group compared to the control (1.65 ± 0.11 vs. 3.13 ± 0.25; P < 0.001, Fig. 3K). Tar- get-seeking was also calculated in these two groups, and there was not any significant difference between cis P-tau and control groups (Fig. 3L). These data showed that animals in both groups explored all of the holes equally; however, the subjects in cis P tau group could not memo- rize the location of the target hole and goal box. Cis P‑tau decreased basic synaptic transmission on dorsal hippocampus We first compared the basal synaptic field potential responses in the dorsal and ventral CA1 stratum pyrami- dal in response to Schaffer collaterals stimulation in the control and cis P-tau groups. The fEPSP slope was meas - ured in response to different stimulation intensities, and the I-O curves were constructed. Obtained results showed that the dorsal and ventral hippocampus had no significant differences in the I-O curve in the control group (Fig. 4A). There was no significant difference in test pulse intensity between the dorsal and ventral hippocam- pus in the control group (64.40 ± 4.23 and 56.60 ± 2.93 respectively, Fig. 4B). Compared to the control group, Fig. 4 Basic synaptic transmission decreased in the dorsal the I-O curve shifted to the right in the dorsal but not the hippocampus at 7 months after intra‑hippocampal cis P ‑tau injection. ventral hippocampus in cis P-tau (Fig. 4C, E). Accord- Graphs show the basic synaptic transmission (I–O curve) A and test ingly, the test pulse intensity significantly decreased in pulse intensity B in the dorsal vs. ventral hippocampal slices in the the dorsal hippocampus of the cis P-tau group compared control group. Cis P‑tau injection shifted the I‑ O curve to the right C and decreased the test pulse intensity D in the dorsal hippocampus to the control (51.25 ± 2,39 µA vs. 64.4 ± 4.23 µA; P < 0.05, in the cis P‑tau compared to the control group. In the ventral Fig. 3D). No significant difference was observed in the hippocampal slices no significant difference observed in I‑ O curve E test pulse intensity in the ventral hippocampus between and test pulse intensity F between cis P‑tau compared to the control cis P-tau (51.00 ± 2.93 µA) and control (51.00 ± 2.52 µA) group. All data represent mean ± SEM. * P < 0.05 groups (Fig. 4F). Cis P‑tau disrupted LTP induction in the dorsal was no significant difference in LTP magnitude in the hippocampus ventral hippocampus between the two groups (Fig. 5E, In the next step, we assessed the generation of long-term F). potentiation (LTP) in fEPSP slope following primed burst stimulation (PBS) applying in the Schaffer collaterals in the stratum radiatum of the dorsal and ventral hip- Cis P‑tau decreased the number of survived cells pocampal CA1 area. There was not any significant dif - in the hippocampus ference between the dorsal (172.50 ± 11.66% of baseline) For assessing the changes in the number of survived and ventral (160.60 ± 2.43% of baseline) hippocampal cells in the dorsal and ventral hippocampus, Nissl stain- LTP magnitude in the control group (Fig. 5A, B). ing was performed in cis P-tau and control groups. As Then, we compared the LTP magnitude in the dor - Fig. 6 shows, the number of survived cells decreased sal and ventral hippocampus of the cis P-tau groups significantly (P < 0.001) in the dorsal hippocampus of with the control group. Data demonstrated that intra- the cis-P tau group (148 ± 10.8) compared to the con- hippocampal cis P-tau injection significantly (P < 0.01) trol (305 ± 15.2). Similarly, the number of survived cells reduced the LTP magnitude in the dorsal hippocampus decreased significantly (P < 0.001) in the ventral hip- (106.10 ± 5.52% of baseline; Fig. 5C, D). However, there pocampus of the cis-P tau group (284 ± 13.3) compared F atemeh et al. Behavioral and Brain Functions (2023) 19:9 Page 7 of 13 Therefore, it may be postulated that following microin - jection of cis P-tau into the dorsal hippocampus, an accu- mulation of cis-P tau in the ventral hippocampus and the prefrontal cortex probably caused neurodegeneration in both areas. In addition, animals in cis P-tau group spent less time in the center of Y-maze apparatus. It showed that when the subjects reached to the center point of the maze, they did not make a decision to go to next arm and moved without any goal. However, in the control group, after reaching the center point of the maze, the animals took more time to choose the correct arm. It has been reported that cis-P tau microinjection into the hip- pocampus may induce beta amyloid plaques and both cis P-tau and beta amyloid (Aβ1-42) induce each other and leads to similar and identical neurotoxicity . There - fore, the results of the present study may be contributed to a tau-induced Alzheimer’s-like disease model in mice. In this study, we used the Barnes maze, which is less stressful than the Morris water maze, especially for mice [21, 22]. In addition, many articles highlight the prior- ity of mice (especially C57BL/6 J) to rats in Barnes maze studies due to their innate curiosity and tendency to escape into the small holes [23, 24]. Animals in cis P-tau group showed spatial learning Fig. 5 Cis P‑tau injection disrupted LTP induction in the dorsal hippocampus at 7 months after injection. Graphs show the LTP and memory impairment in the Barnes maze test. The magnitude in dorsal vs. ventral hippocampal slices in the control increase in primary and total errors and latency to the group (A and B). Cis P‑tau injection decreased LTP magnitude in goal box and the decrease in using the direct strategy dorsal C and D but not in ventral E and F hippocampus in cis p‑tau showed a significant impairment in learning processes group compared to control group. All data represent mean ± SEM. ** in cis P-tau group. In addition, obtained data on probe P < 0.01 compared to the control group test day showed that subjects in the cis P-tau group spent less time in the goal sector than the control, indicating memory impairment in this group. Similar cognition to the control group (377.4 ± 9.9). In addition, the reduc- impairments were previously reported in Alzheimer’s like tion of survived cells in the dorsal hippocampus was disease models in laboratory mice [25–27]. In line with higher than in the ventral hippocampus. the present data, Ramsden et al. in  reported that spatial memory was dramatically impaired in tauopathic Discussion mice at 7 and 9.5 months of age in the Morris water maze While it has been reported that bilateral intra-hippocam- . Brunden et al.  also showed an increase in the pal injection of cis P-tau increases amyloid-beta accumu- number of errors in the Barnes maze test in a trans- lation and tau protein aggregation in the hippocampus at genic mice model of tauopathy . The spatial memory 2, 4, and 8 weeks after cis P-tau injection , results of impairment of transgenic mice in Barnes and Morris the current study demonstrated that there was no intra- water mazes may indicate hippocampal dysfunction. hippocampal β-amyloid and cis P-tau accumulation at The dorsal hippocampus has a crucial role in spa - 7 months after cis P-tau injection. However, a significant tial learning and memory [30–32] while the ventral deficit in learning and memory and synaptic plasticity hippocampus is mainly involved in emotional proce- dysfunction was observed at 7 months after intra-hip- dures [33, 34]. Therefore, our data confirmed a signifi - pocampal injection of cis P-tau. cant dysfunction in dorsal hippocampus of Alzheimer’s A significant decrease in spontaneous alternation in the like-disease animals. In consistent with our data, some Y-maze test was observed following cis P-tau injection, studies reported the neuropathological changes in the which showed a deficit in working memory in the cis hippocampus of transgenetic mice model of AD [35, 36]. P-tau group compared to the control group at 7 months In addition, O’Lear et al. reported that the APP/PS1 AD after injection. A normal working memory needs inter- transgenic model mice spent less time in the correct zone play between several areas of the brain, such as the than wild-type because of impaired spatial memory . ventral hippocampus and prefrontal cortex [19, 20]. Fatemeh et al. Behavioral and Brain Functions (2023) 19:9 Page 8 of 13 Fig. 6 Cis P‑tau decreased the hippocampal survived cells in the dorsal and ventral hippocampus at 7 months after injection. Representative hippocampal sections were stained with the Nissl method for evaluation of the survivor cells in control‑ dorsal A cis P‑tau‑ dorsal B control‑ ventral C and cis P‑tau‑ ventral D groups. Bar graph E shows the quantitative effect of cis P ‑tau injection on pyramidal survived cells in dorsal and ventral *** hippocampus compared to control group. All data represents mean ± SEM P < 0.001 In the present study, distance and velocity had no sig- models . According to the input–output curve, the nificant differences between cis P-tau and the control basal synaptic function had no significant difference groups, indicating no changes in motor activity between between the dorsal and ventral hippocampus of the con- the two groups. trol group. Consistent with our data, Tidball et al. , Since an AD brain dramatically loses synapses in the Milior et al. , and Schreurs et al.  showed that no temporal areas, the changes in synaptic strength are difference was detected in the relationship between stim - important signs to show the magnitude of Alzheimer’- ulus intensity and fEPSP slope in the dorsal versus the like disease models and tauopathogenesis in animal ventral hippocampus [39–41]. In contrast to the control F atemeh et al. Behavioral and Brain Functions (2023) 19:9 Page 9 of 13 group, in the cis P-tau group, excitability decreased in Conclusion the dorsal region but had no change in the ventral. In line The probable tau pathogenesis following intra-hip - with our study, basic synaptic transmission was reduced pocampal cis P-tau resulted in working and spatial in the hippocampus in a transgenic animal model of AD memory impairment and constructed a long-term (5xFAD mice) . In another study, Tulloch et al.  Alzheimer’s-like behavior for a long duration. In addi- compared basic synaptic transmission in several Alz- tion, basal synaptic transmission and synaptic plastic- heimer’s animal models and did not observe any signifi - ity were disrupted in the dorsal region of the cis P-tau cant difference between the control and the Alzheimer’s group. The number of neurons decreased in the hip - groups. In line with our study, the basic synaptic trans- pocampus, while this decrease was higher in the dorsal mission was slightly reduced (not significant) in mice hippocampus than in the ventral. Of course, because AD-transgenic models of APP/PS1 + Tau . cis P-tau was injected into the dorsal hippocampus LTP at Schaffer collateral synapses was not different and resulted in a higher neuronal loss (compared to the in the dorsal and ventral hippocampus of the control ventral area), it was logic to observe higher histological group. This data is contrary to the study performed by damage in the dorsal part, which was accompanied by Milior et al. . They investigated the electrophysiologi - impairment in synaptic potentiation and spatial learn- cal properties of CA1 pyramidal cells along the whole ing and memory impairment. More research needs to hippocampal dorsoventral axis in 2–3 months old mice. assay the accumulation of beta-amyloid, cis P-tau, and They found that LTP in Schaffer collateral synapses is apoptosis process in different hippocampal areas by lower in the ventral hippocampus than in the dorsal, a immunohistochemistry. phenomenon that is related to more excitability of ven- tral pyramidal neurons compared to the dorsal region of the hippocampus [43, 44] Papaleonidopoulos et al. Methods also reported that the dorsal hippocampus has a lower Cis‑P tau extraction threshold for LTP induction compared to the ventral Pathogenic P-tau formation was induced by the trau- region in 28–38 days and 2–3 months old rats. Because matic brain injury (TBI) model . In this model, TBI the main difference between the present study and the occurred by dropping a 450 g weight from 2 m height on previous report relates to the age of the subjects, we used skull of anesthetized adult male Wistar rats [47, 48]. It 11-month-old mice. Therefore, it may be suggested that was purified from the brain extract after cis pT231- tau the difference in excitability of neurons between dor - accumulation confirmation. The cortex tissues were sepa - sal and ventral areas of the hippocampus disappears by rated and lysed in an extraction buffer (2 ml/g tissue). The aging. The lack of difference between hippocampal dorsal contents of extraction solution include 20 mM PIPES pH and ventral areas also confirms this hypothesis. 6.9, 1 mM MgSO4, 1 mM EGTA in the presence of 2 mM The present results also showed that hippocampal cis DTT, 1 mM PMSF, 1 mM EDTA, and 1 M NaCl. Then P-tau injection disrupted LTP induction in the dorsal we pellet the homogenate by centrifugation at 6000 × g hippocampus but did not significantly affect the ventral for 20 min at 4 ℃, and we sonicated the supernatant on region. Similar to our data, Crouzin et al. reported that ice four times (15 s on, the 30 s off ) and boiled in 5 M LTP was not induced in a mice model of Alzheimer’s- NaCl for 10 min. We chilled the extract on ice and then like disease . They concluded a reverse correlation ultra-centrifuged (Beckman coulter optima L-100XP) at between LTP incidence and pathophysiological changes 100,000 × g for 60 min at 4 ℃. The supernatant was dia - in the hippocampus. Thus, the severity of Alzheimer’s lyzed against PEM buffer (3 × 1). Finally, we purified tau pathological changes is matched with the extent of the protein by ion-exchange chromatography, as explained synaptic plasticity losses and, eventually, nerve damage , and stored it at—80 ℃ until use. occurrence. The decrease in the number of pyramidal cells and thickness of CA1 and CA3 regions have also been reported in Alzheimer’s-like disease induced by sco- Animals polamine  and in APP/PS1 transgenic mouse mod- Thirty adult male C57BL/6 mice (4 months old, weighing els of Alzheimer’s disease . In line with the previous 25–27 g) were obtained from Tarbiat Modares University studies, the present data also showed that the number of (Tehran, Iran) and housed in 21 ± 2 ℃, 12 h light–dark hippocampal neurons decreased after cis P-tau injection cycle, with free access to food and water. Animals were in dorsal and ventral areas. However, the percentage of divided into control and cis-P tau groups. Cis-P tau or decrease in the dorsal was higher than ventral area. This its solvent (saline) were injected intra-hippocampally difference may be considered as a reason for LTP disrup - in cis-P tau and control groups respectively. All experi- tion in the dorsal, but not in the ventral hippocampus. ments were run 7 months after chemical injection. Fatemeh et al. Behavioral and Brain Functions (2023) 19:9 Page 10 of 13 Stereotaxic surgery trial, the mouse was put in the start box, and 10 s later, Mice were anesthetized with an intraperitoneal injec- a light was turned on, the box was raised, and the mouse tion of ketamine/xylazine (100 mg/kg to 10 mg/kg) and explored the maze freely. The trial finished when the immobilized in a stereotaxic frame (Stoelting, USA). AD mouse entered the goal box or after 5 min had elapsed. model was induced by cis-P tau injection (1 μg/1 μl) into After entering the mouse into the goal box, the light was the dorsal hippocampus (stereotaxic coordination: 2 mm turned off, and the mouse remained in the goal box for posterior and 1.7 mm bilaterally to the right and left of 1 min. bregma and—1.6 mm below dura ) in a volume of 2 μl Mice trained for four trials (at 15 min intervals) per day over 6 min via a 10 μl Hamilton syringe using a microsy- for 4 days. After each trial, the maze was cleaned with ringe pump (WPI, UK). All microinjections were done at 70% ethylic alcohol solution. A probe trial was done on a speed of 0.5 μl/min, and the injection needle was left in the 5 day when the goal box was closed to assess maze place for an additional 10 min to allow the solution to dif- learning and memory retention. The probe experiment fuse from the tip entirely. allows determining whether trained animals use envi- ronmental cues to create a spatial map of their environ- Y‑ maze test ment and find a hole that was previously a goal box. The We selected a Y-shaped gray Plexiglas maze with 30 cm delay and time spent to find the last correct hole was length, 10 cm width, and 15 cm height for the working measured. Total trials were recorded by using a ceiling- memory task. The animal was put on the end of one arm mounted video camera. to explore for 8 min session freely. An entry happens The measured behavioral parameters included: 1— when all four mouse limbs are inside an arm. An alter- Primary and total latency evaluated as the time spent nation is determined as successive entries into all three by the mouse to find the goal box for the first time (pri - arms. Next, after recording the number of arm entries mary latency) and entering (total latency) during a learn- and alternations, the percentage of the alternation behav- ing trial; 2—Errors measured as the number of incorrect ior was calculated by the below formula: holes explore before finding (primary errors) and entering (total errors) the goal box. Errors are explained as explor- Number of alternation ing any hole that does not contain the goal box; 3—Total Alternation precentage = × 100 Total number of arm entry distance and velocity for each trial are also calculated by using EthoVision XT; 4—For each trial, the search strat- A spontaneous alternation happens when a mouse egy (exploration patterns) is classified as direct (mov - enters a different arm of the maze in each of 3 consecu - ing directly to the target hole), serial (systematic search tive arm entries (i.e., visit from A to B or C, which are of sequential holes in a clockwise or counterclockwise designated to the other arms, respectively). In addition, direction), and random (unordered and random explora- incorrect trials are considered to travel back to a previ- tion of the maze); 5—The frequency of target hole explo - ously experienced arm, such as CBC moving. All move- ration was assessed by the goal sector (GS) parameter, ments were recorded using a computer-linked video and it is the sum target and a neighbor right or left holes camera mounted above the platform , and data were explorations divided by 3; 6—The frequency of non-tar - analyzed by Ethovision software 11 (Noldus Information get hole exploration was assessed by the non-goal sector Technology, Wageningen, The Netherlands). (NGS): the sum of explorations of the 17 non-goal holes divided by 17; 7—Goal sector preference: the ratio of GS Barnes maze test to NGS explorations; 8—Target-seeking activity: the total We assessed spatial hippocampal-dependent learn- explorations for whole, divided by 20 . ing and memory using by Barnes maze test. The maze includes a circular platform (92 cm in diameter) with 20 holes (hole diameter: 5 cm) along with the surround- Field potential recording in the hippocampal slices ings. During the experiment, the mouse learned the spa- The mice were anesthetized with carbon dioxide (CO ) tial position of the goal box (17.5 cm in length, 7.5 cm and decapitated at 7 months after bilateral hippocampal in width, and 8 cm in height). Three-maze cues were cis-P tau injection. We removed the mouse brain rapidly, placed all around the room to show the location of the and then the fresh brain was transferred into a chilled goal box hole. In the pre-training trial, the mouse was put artificial cerebrospinal fluid (aCSF) chamber. This cham - in the maze’s center in a white-colored cubed start box ber bubbled with carbogen (95% O and 5% CO ). The 2 2 (12.5 cm × 8 cm). After 10 s, the start box was raised, and aCSF contained (in mµ): NaCl 124, N aHCO 26, K H PO 3 2 4 the mouse learned to enter the goal box by guiding it to 1.25, KCl 5, C aCl 2, MgCl 2.06, and d-glucose 10 and its 2 2 the goal box and staying there for 2 min. After the pre- pH was 7.3–7.4. Next, we prepared coronal 400 µm thick training trial, the first trial began. At the onset of each slices containing the hippocampus using a vibratome F atemeh et al. Behavioral and Brain Functions (2023) 19:9 Page 11 of 13 (model VT 1200, Leica, Germany). After slice prepara- and six squares were counted to measure survived cells, tion, we put the slices in a recovery chamber for at least which were calculated as the number of cells/mm . 60 min at room temperature. Then we transferred slices (one by one) to an interface-type recording chamber con- Immunofluorescence taining 32 ℃ aCSF solution in a warm, humid oxygenated After washing with PBS-Tween, brain sections were per- environment. meabilized for 10 min with 0.2% (v/v) Triton X-100 and We recorded field potentials from the stratum radia - blocked for 1 h with NGS 10%. Afterward, the samples tum in the dorsal and ventral hippocampus. We used a were incubated with the following primary antibod- stimulating electrode (stainless steel, Teflon coated, A–M ies: cis pT231-tau mAb (1:500, gift from KP. Lu), and Aβ Systems, USA) that was placed on the Schaffer collat - oligomers (1:500, Abcam) at 4 ℃ in a moist and humid eral path and a recording glass electrode (borosilicate, chamber overnight. A secondary antibody anti-rabbit or O.D.: 1.5 mm, I.D.: 0.86 mm, Sutter instrument, USA). anti-mouse was added after washing the samples at 37 ℃ The recording electrode (2–5 MΩ) was filled with aCSF for one hour (Alexa Fluor 488, Thermo Fisher Scientific, and was placed on the stratum radiatum of hippocampal Rockford, USA). DAPI was used for staining the nuclei. CA1. A reference electrode was also put in the recording The samples were visualized by a fluorescent microscope chamber. The recording electrode transferred signals to (Olympus, BX51 with Olympus DP72 digital camera), an amplifier (ME208300, Nihon-kohden, Japan), and the and the images were analyzed by using ImageJ software signals were visualized by custom-made software (Poten- v1.43 (NIH, Bethesda, MD, USA). tialize; ScienceBeamCo., Iran). We plotted the Input/ output curve to calculate test pulse intensity. The evoked Statistical analysis field potential was recorded from the CA1 area at the Spontaneous alternation, percent time in center point, test pulse intensity (50% of an intensity producing maxi- total distance, test pulse, and percentage of potentia- mum response) for 20 min. Then, primed-burst stimula - tion were analyzed by unpaired t-test. Primary and total tion (PBS; a single pulse followed 170 µs later by a burst errors and latency, as well as field potentials (before of 10 pulses at 200 Hz, and the entire train was repeated and after LTP induction), were analyzed using two-way ten times) was applied, and post-PBS responses were ANOVA followed by Sidak’s multiple comparisons test. recorded for 60 min. The correlation between the direct strategies in trial days was analyzed using the correlation test and Pear- son correlation coefficient. The values were expressed as Tissue processing and sectioning means ± standard error of the mean (SEM). All statistical The mice were deeply anesthetized with ketamine/xyla - analyses were conducted using Graphpad Prism (version zine (100 mg/kg to 10 mg/kg) 7 months after cis P-tau 6.0). The probability level is interpreted as statistically injection. Then transcardial perfusion was performed significant when P < 0.05. with phosphate-buffered saline (20 mL) followed by 4% phosphate-buffered formalin (15–20 mL). Brain tissues Acknowledgements We thank our colleagues Dr. Nahid Roohi, Mr. Meysam Zare and Mr. Mahmoud were fixed overnight in the solution (4% paraformalde - Rezaei for thir helps in running the experiments. hyde in phosphate-buffered saline), next embedded in OCT compound (Sakura; Finetek; Torrance, CA), and Author contributions FB carried out the experiment, and analysed the data and wrote the first draft cut into 8 μm thick serial sections. of the manuscript; KS, AS and YF participated in the design of the study and in the results interpretation; JM‑Z contributed in the design of the experiments, data analysis, writing the manuscript and preparing the funds. All authors read Nissl staining and approved the final version of the manuscript. The number of survived cells was determined using Funding the Nissl staining method. In brief, the sections were This work was supported by a frant from Iran National Science Foundation rehydrated with graded series alcohols (96%, 80%, and (INSF) (grant number 4014516) and Tarbiat Modares University (Grant Number 70%) and stained with 0.1% Cresyl Fast Violet (Merck, IG‑39709). The funders had no role in the study. Germany) at room temperature for 2 min. After wash- Availability of data and materials ing, the sections were dehydrated by a graded series The data that support the findings of this study are available on request from of alcohols (70%, 80%, 96%, and 100%). Then they were the corresponding author. cleaned in xylene, cover slipped with Entellan (Merck, Chemical, Germany), and photographed. An Olym- Declarations pus BX-51 microscope and DP72 camera captured con- Ethics approval consent to participate secutive images at 400 × magnification. Using a grid All experiments and procedures were performed in accordance with the Tar‑ (200 μm × 200 μm), the images were randomly assigned, biat Modares University guidelines for animal care and approved by the Ethics Fatemeh et al. Behavioral and Brain Functions (2023) 19:9 Page 12 of 13 Committee of the Faculty of Medical Sciences, Tarbiat Modares University (IR. 18. Nelson PT, Alafuzoff I, Bigio EH, Bouras C, Braak H, Cairns NJ, et al. Correla‑ MODARES.REC.1397.020). tion of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. J Neuropathol Exp Neurol. 2012;71(5):362–81. Consent for publication 19. Swonger AK, Rech RH. Serotonergic and cholinergic involvement in Not applicable. habituation of activity and spontaneous alternation of rats in a Y maze. J Comp Physiol Psychol. 1972;81(3):509–22. Competing interests 20. Sarnyai Z, Sibille EL, Pavlides C, Fenster RJ, McEwen BS, Toth M. 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Behavioral and Brain Functions – Springer Journals
Published: May 25, 2023
Keywords: Alzheimer’s like disease; Cis P-tau; Dorsal and ventral hippocampus; Learning and memory and synaptic plasticity
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