Effect of Alpha-7 Nicotinic Acetylcholine Receptor Activation on Beta-amyloid Induced Recognition Memory Impairment. Possible Role of Neurovascular Function

PURPOSE: To evaluate the effects of PHA-543613 (α7-nAChR agonist) and galantamine (acetylcholinesterase inhibitor (AChEI)) on recognition memory and neurovascular coupling (NVC) response in beta-amyloid (Aβ) 25–35-treated mice. METHODS: PHA-543613 (1 mg/kg, i.p.), and galantamine (3 mg/kg, s.c.), effects were tested in Aβ 25–35 mice model of AD. α7-nAChR antagonist, methyllycaconitine (MLA) (1 mg/kg, i.p.), was used for evaluation of receptor blockade effects. Recognition memory in animals was assessed by the novel object recognition (NOR) task. NVC response was analyzed by laser-doppler flow meter in barrel cortex by whisker stimulation method. RESULTS: Both, PHA-543613 and galantamine improve recognition memory in Aβ-treated animals. However, the advantageous effects of PHA-543613 were significantly higher than galantamine. Also, pretreatment with MLA reversed both galantamine and PHA-543613 effects on NOR. Impaired NVC response in AD animals was improved by PHA-543613 and galantamine. However, MLA pretreatment disrupts this function. CONCLUSION: Activation of α7-nAChR improved recognition memory possible through enhancement of neurovascular response in Alzheimer's disease in animals. Effect of alpha-7 nicotinic acetylcholine receptor activation on beta-amyloid induced recognition memory impairment. Possible role of neurovascular function


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder and most prevalent form of dementia which is characterized by amyloid beta (Aβ) accumulation in neuronal structures of the brain and cholinergic dysfunction 1 .In this way, the nicotinic acetylcholine receptor (nAChR) as a key component of cholinergic function plays axial role in disease progression 2 .Among nAChR subunits, homomeric α7 receptor seems to have an important role in cognitive-related processes 3 .It is the most abundant nAChR subtype in the hippocampus, cortex and hypothalamus of the mammalian brain 4 .Studies show that Aβ may cause cholinergic dysfunction through affecting α7-nAChR 5 .Moreover, α7-nAChR activation decreases Aβ-induced neurotoxicity 6 .Studies have shown that α7-nAChR activation may represent a therapeutic strategy for AD-related cognitive impairments 7 .
Also, the cholinergic-vascular hypothesis, suggests that the loss of cholinergic vascular innervation is responsible for the impairment of cerebral blood flow (CBF) in AD 8 .The tight coupling between neuronal activity and CBF is known as functional hyperemia or neurovascular coupling (NVC) which is altered both in AD patients and animal models 9 .Aβ is responsible for impairment of cerebrovascular auto-regulation and NVC response 10 .Also, Aβ could attenuate cerebral hemostasis by blocking α7-nAChRs and activation of this receptor could improve impaired cerebral hemodynamic and NVC response in AD 11 .
PHA-543613 is a potent and selective α7-nAChR agonist with ability of brain penetration.However, there are limited reports about its exact procognitive mechanisms.Hence, this study was conducted to assess the possible effect of this molecule on recognition memory in Aβ-treated animals and possible role of NVC response in this matter.

Experimental animals
The experimental procedures were approved by regional ethics committee of Tabriz University of Medical Sciences (TUOMS) (code number: 5.4.6251).BALB/c male mice weighing 25-30 grams were obtained (total number=105) from Razi Institute Laboratory Animal Care Center (Karaj, Iran).Before and during the study, animals were housed in standard cages (five animal per cage) under controlled conditions (12/12 h light/dark cycle starting at 7:00 AM and temperature of 25±2°C) with access to water and standard pellet food ad libitum.1).The dosages and paradigms of injections were chosen on the basis of previous studies [12][13][14] .

Intracerebroventricular (i.c.v.) injection of Aβ 25-35 peptides
Aβ administration and model preparation were done according to previously described method 15 .Briefly, Aβ 25-35 peptide was dissolved in phosphate buffer saline (PBS) at a concentration of 2 mg/ml and was incubated for peptide aging or aggregation at 37ºC for four days.
Administration of saline (i.c.v.) did not have significant effect on animals' survival or function in comparison to control (vehicle via i.p. route) group.Therefore, the results of sham group were not showed in the results section.

Test apparatus and condition
The test apparatus was a plexiglas open-field box (33×33×20 cm).The box was placed in an acoustically-isolated room and light was provided by overhead lighting (15 lux).We used common objects that were different in shape and texture, however, the objects were counterbalanced in complexity.Direction of animal nose to the object (distance of ≤ 2 cm) and rearing up against the object to investigation were considered as exploration.After each trial, the arena and objects were cleaned with a 70% ethanol solution to eliminate the presence of any olfactory cues.All sessions were carried out during the morning (from 09:00).Data were acquired through a video camera that was fixed above the center of the task apparatus.All observations were done by an experimenter that was unaware of the entity of experimentations.

Habituation session
The habituation session of the NOR started 1 day prior to the training step.Before testing, animals were transferred to the test room and were placed there for 15 minutes for acclimation.Then, each mouse was placed in the box for habituation in the absence of objects for 10 min.Moreover, locomotor activity was recorded during the habituation session.
Training session One day after the habituation session, the animals were subjected to a training session.During the training, two identical objects (A and A') were placed in the box, 12 cm from lateral walls.Then, each animal was placed in the middle of the box and the total time spent to explore both objects was recorded over 10 minutes.

Retention session
For recognition memory evaluation, the animals were subjected to a single retention session the next day after the training trial.For this purpose, the animals were returned into the same task, but one of the familiar objects applied during the training session was replaced by a novel object B. Cognitive function was measured by exploratory preference, which was calculated by percent of time spent exploring each of the same objects or the novel object in the training and retention sessions, over the total time spent exploring both objects.

Measurement of NVC response
Neuronal activity-evoked CBF response provides an index for NVC.For evaluation of this response, on the day after behavioral testing, the rCBF on the surface of the right barrel cortex was measured using 0.6 mm diameter probe of Laser Doppler Flow meter (LDF) (Laserflo, Vasamedics, USA).In brief, animals were anaesthetized with 2% isoflurane, which was administered in a mixture of 70% N 2 and 30% O 2 .Mice were mounted in a stereotaxic frame.Because CBF measurements by LDF can vary depending on the monitoring site, the probe was placed at the same stereotaxic coordinates based on the mouse brain in stereotaxic coordinates AP = -1.8mm and ML = +2.5 mm 16 .The skull was thinned until being translucent using a dental drill.The tip of LDF was positioned on the thinned skull perpendicular to the surface of the brain.While we avoided large blood vessel areas we waited until the stable baseline was recorded for 10 minutes.Contralateral vibrissae were cut to an equal length of 0.5 cm and stimulated using a cotton swab.Stimulations (3-6 stimulations, 20 seconds at 8-10 Hz) were recorded, with four to six recordings that were acquired every 30-40 seconds and were averaged for each mouse.CBF change was defined as percentage increase relative to the baseline.The rectal temperature was maintained at 36.5 ± 0.5°C by placing the animals on a heating pad.

Statistical analysis
Descriptive data were defined as means ± SEM.

Locomotor activity
The

Effect of treatments on recognition memory
Treatments did not have any effect on exploratory preference for the objects in the training session [F (4, 70) = 0.07, p>0.05].However, in the retention session, there was a statistically significant difference between groups as determined by one-way ANOVA (F (4, 70) =25.89, p<0.001).
Post hoc analysis of retention session data revealed that, Aβ significantly reduced the exploratory preference for a novel object in comparison to control group (p<0.001) and treatment with PHA-543613 and galantamine significantly improved Aβ-induced recognition memory impairment (p<0.001).Also, in comparison to the control group, PHA-543613-treated AD animals did not have significant difference (p>0.05) in exploratory preference, but in the galantamine group it was significantly lower (p<0.05)(Figure 2).Pretreatment with MLA significantly prevented the improving effects of PHA-543613 on the impairment of recognition memory in Aβ-treated mice (p<0.001).Furthermore, galantamine effect on NOR performances of AD-animals could be abolished by the pretreatment with MLA (p<0.001).Pretreatment with normal saline, had no effect on the treatments results.α7-nAChR blockage in galantamine group had a lower effect on recognition memory in comparison to blockage of that receptor in the PHA-543613 group (p<0.05)(Figure 3).

Effect of treatments on NVC response
There was a significant difference in NVC response between groups as determined by one-way ANOVA [F (3, 28) =56.6, p<0.001].Post hoc analysis revealed that NVC response in AD animals was significantly lower as compared with control group (p<0.001).PHA-543613 and galantamine treatment significantly increased NVC response in Aβ-treated mice (p<0.001)(Figure 4).
MLA significantly prevented the improving effects of PHA-543613 on NVC response in AD animals (p<0.001).Moreover, the galantamine effect was abolished by the pretreatment with MLA, too (p<0.05)(Figure 5).

Discussion
The main findings of this study showed that activation of α7-nAChRs is able to improve Aβ 25-35 -induced recognition memory dysfunction possibly through modulation of NVC response.Aspects of AD can be mimicked by i.c.v.administration of Aβ peptides in the rodent brain 1 .
In this study, we observed that single i.c.v.administration of 10 nmol Aβ 25-35 could impair recognition memory in mice.This result is consistent with the findings of other studies 1,15,17 .The NOR task was selected for recognition memory evaluations in this study.The NOR test evaluates recognition memory in rodents 18 .Recognition memory is strongly correlated with hippocampal and cortical functional integrity in both rodents' and primates' brains 19 .Moreover, both cortical and hippocampal dysfunctions affect NOR paradigm results 18 .These compartments of the brain are well-documented to be adversely affected in aging and AD 20 .Also, the NOR task is a simple and non-stressful method in which external motivations are not necessary, so it is a suitable method for evaluation of recognition memory in aged or AD animals 18 .
According to the results, significant enhancement was seen in the exploratory preference of novel object in PHA-543613 and galantamine-received AD animals.This showed that both PHA-543613 and galantamine could improve recognition memory in Aβ-treated mice.However, in NOR task performance, the effect of galantamine (as an AChEI) was lower than PHA-543613 (as a selective agonist of receptor) which showed the importance of α7-nAChR selective activation versus general acetylcholine increasing in improvement of recognition memory in AD-animals.
Interestingly, PHA-543613 or galantamine-induced enhancement in recognition memory is completely or partially blocked by MLA pretreatment.It showed that α7-nAChR plays a cardinal role not only in PHA-543613, but also in galantamine procognitive effects.
There is very limited information about procognitive effects of PHA-543613.Wishka et al. first synthesized the PHA-543613 and evaluated its cognitive effects on normal rats 12 .Our recently-published paper showed its ameliorative effects on spatial memory in Aβ-treated animals 15 .Bali et al. described PHA-543613's procognitive effects on T-maze in the scopolamineinduced memory impairment model 7 .
Activation of α7-nAChR by various agonists showed improvement in recognition memory in different studies.In the Roncarati et al. study, SEN12333 (α7-nAChR agonist) prevented scopolamine-induced deficit in a recognition test 21 .In another study, Boess et al. 22 , showed that the α7-nAChR agonist ABBF improves social recognition memory in rats.Also, a study by McLean et al. showed that, recognition deficits in NOR could be attenuated by PNU-282987, a full agonist of α7-nAChR 23 .
According to a study by Tinsley et al. 24 , blockade of α7-nAChR by MLA could significantly impair object recognition memory.In a study by Pichat et al., acute treatment with SSR180711 improved recognition memory in object recognition tasks in rats and it was reversed by the α7-nAChR antagonist MLA 14 .Callahan et al. 25 , assessed recognition memory using the NOR in aged rats.They showed pretreatment with MLA reversed the recognition memory enhancement produced by the donepezil.
Evidence showed that Aβ could impair NVC response in AD patients and experimental models 26 .In our study, Aβ-treated mice showed impaired NVC response during whisker stimulation.Also, in Badhwar et al study impaired NVC response was seen in APP mice in the same situation 27 .Moreover, it has been shown that topical superfusion of soluble Aβ on the mouse neocortex attenuated CBF increase evoked by whisker stimulation 17 .In this study, PHA-543613 and galantamine treatment significantly increased NVC response in Aβ-treated mice and MLA pretreatment completely or partially prevented their improving effects.Si et al. 28 , hypothesized that activation of α7-nAChRs on cerebral perivascular sympathetic nerves leads to vasodilation and blockade of this receptor which reverses the effect.Rosengarten et al. 29 found that impaired NVC response was recovered under AChEI treatment in AD patients.Moreover, according to Bar et al. 30 , galantamine treatment improved vascular reactivity in AD.Overall, the cholinergic system, especially α7-nAChRs, plays a curtail role in neurovascular function in AD 8,26 .
locomotor activity was recorded during the NOR habituation session.Treatments in used doses had no significant effect on locomotor activity in different groups [F (4, 70) = 0.05, p>0.05].
Effect of alpha-7 nicotinic acetylcholine receptor activation on beta-amyloid induced recognition memory impairment.Possible role of neurovascular function Tukey test.T-test analysis was used to two-group comparison.All analyses were performed using IBM SPSS Statistics software (version 22 for Windows; SPSS Inc., Chicago, IL, USA).In all comparisons, p<0.05 was considered significant.