Morin improves learning and memory in healthy adult mice

Abstract Background Morin is a flavonoid found in many edible fruits. The hippocampus and entorhinal cortex play crucial roles in memory formation and consolidation. This study aimed to characterize the effect of morin on recognition and space memory in healthy C57BL/6 adult mice and explore the underlying molecular mechanism. Methods Morin was administered i.p. at 1, 2.5, and 5 mg/kg/24 h for 10 days. The Morris water maze (MWM), novel object recognition, novel context recognition, and tasks were conducted 1 day after the last administration. The mice's brains underwent histological characterization, and their protein expression was examined using immunohistochemistry and Western blot techniques. Results In the MWM and novel object recognition tests, mice treated with 1 mg/kg of morin exhibited a significant recognition index increase compared to the control group. Besides, they demonstrated faster memory acquisition during MWM training. Additionally, the expression of pro‐brain‐derived neurotrophic factor (BDNF), BDNF, and postsynaptic density protein 95 proteins in the hippocampus of treated mice showed a significant increase. In the entorhinal cortex, only the pro‐BDNF increased. Morin‐treated mice exhibited a significant increase in the hippocampus's number and length of dendrites. Conclusion This study shows that morin improves recognition memory and spatial memory in healthy adult mice.


BACKGROUND
Some cognitive abilities, such as acquisition, storage, manipulation, and retrieval of information, usually decline with age and in some neurodegenerative diseases (Murman, 2015).Epidemiological and clinical evidence suggests that the level of education, an active lifestyle, and diet can improve cognitive function in healthy people and are protective against the rate of memory decline.Other research has established that learning and memory, as well as mood, can be strongly influenced by diet during development and in adulthood (Stangl & Thuret, 2009).
Experimental evidence from in vitro and in vivo studies indicates that morin exerts neuroprotective actions against several neuropathologies that show cognitive decline.For instance, in Alzheimer´s disease, in vitro studies show that morin inhibits the formation of amyloid and disaggregates amyloid fibers (Noor et al., 2012), inhibits β-amyloid peptide (Aβ) fibrillogenesis (Kim et al., 2005), and its early stages of aggregation (Lemkul & Bevan, 2010).Moreover, morin blocks GSK3β-induced tau phosphorylation (Gong et al., 2011).In Alzheimer's disease transgenic mouse models, morin shows different protection mechanisms; in APPswe/PS1dE9 mice, it restores cognitive functions, reduces the activated glial cells and Aβ production, and increases synaptic expression markers (Du et al., 2016).Moreover, morin attenuates tau hyperphosphorylation in the transgenic 3xTg-AD mice model (Gong et al., 2011).In other neurodegenerative diseases, such as Parkinson's disease, in vitro studies showed that a decrease in the formation of reactive oxygen species and apoptosis in PC12 cells exposed to MPP+ (Zhang et al., 2010) as well as reduced lipid membrane damage by α-synuclein aggregates (Caruana et al., 2012), and significantly increased motor abilities (Zhang et al., 2010).Furthermore, morin protects neurons and oligodendrocytes from excitotoxic cell death (Gottlieb et al., 2006;Ibarretxe et al., 2006) and significantly improves spatial memory in an animal stroke model (Gottlieb et al., 2006).Recently, morin was described to produce an antipsychoticlike activity in mice, possibly mediated via mechanisms related to decreased inflammation and enhancement of GABAergic neurotransmission, brain-derived neurotrophic factor (BDNF), and suppression of NADPH-oxidase, which induce oxidative damage and neuroinflammation (Ben-Azu et al., 2019;Ben-Azu et al., 2018a, 2018b, 2018c, 2018d).
Given the association between psychosocial stress and neuropsychiatric disorders, morin also has been tested in animal models of social stress where it showed to reduce inflammatory mediators, protect hippocampus from damage, and improve motor and memory impairments induced by psychosocial stress (Ben-Azu et al., 2020;Elizabeth et al., 2020).Sleep deprivation is another stress factor that can cause several changes that induce learning and memory deficits (Chen et al., 2023), and morin treatment has shown significant reduction in memory impairment, anxiety-like behavior, and neuronal damage in a mouse model of sleep deprivation (Olonode et al., 2019).
BDNF plays a crucial role in processes that underlie learning and memory, such as excitatory synaptic transmission, regulation of the structure and functions of different neuronal circuits throughout life, and activity-dependent changes in synaptic strength and plasticity, among others, suggesting a pivotal role in these cognitive functions (Bekinschtein et al., 2008;Poo, 2001;Yamada & Nabeshima, 2004).
Recently, an essential role in Hebbian-type long-term potentiation, long-term depression (LTD), and homeostatic synaptic plasticity has been established for morin (Wang et al., 2012).In the adult brain, BDNF is synthesized and secreted by neurons and glial cells, such as microglia and astrocytes, and this neurotrophin regulates structural and physiological aspects of synapses ranging from short-term to longlasting in many brain regions, including the hippocampus (Lu et al., 2014).BDNF is required for learning and memory processes, and its increase is also correlated to changes in the number, size, and shape of dendrites, which are part of the structural synaptic plasticity (Zagrebelsky et al., 2020).On the other hand, reduced BDNF levels have been reported in disorders that produce cognitive impairments, including chronic stress, aging, and Alzheimer's diseases, principally in memoryrelated structures, like the hippocampus and entorhinal and frontal cortices (Brigadski & Leßmann, 2020;Miranda et al., 2019).Moreover, it is known that the downregulation of BDNF decreases learning and memory (Mizuno et al., 2003).
There has been a growing interest over the last decade in natural molecules with the potential to improve cognitive ability not only in sick people but also in healthy people, and several studies in humans and animals have shown that consuming flavonoids is associated with better cognitive function (Spencer, 2008).However, there is limited information regarding the effect of pure molecules as a cognitive enhancer, particularly in learning and memory.
Thus, the present study was designed to examine whether morin functions as a cognitive enhancer on the memory of healthy mice using

Reagents
Morin was obtained from Sigma-Aldrich Co.

Experimental design
Morin was dissolved in DMSO and diluted with saline solution.Mice were divided into four groups: vehicle (DMSO in saline solution 0.25 mL/L/24 h i.p. for 10 days) and morin (1, 2.5, and 5 mg/kg/24 h i.p. for 10 days).Ten mice per group were used for each test of learning and memory, whereas 10 additional mice per group were used for molecular studies.As it is known that DMSO can cause deleterious effects or epigenetic changes at concentrations higher than 1% (V/V) and 0.1% (V/V), respectively, in this work we used a concentration much lower than those reported to cause such effects (de Abreu Costa et al., 2017;Verheijen et al., 2019).

Object recognition and object recognition in a novel context
Morin and vehicle-treated mice were first tested on CNOR and Novel NOR, mainly hippocampal and cortex-dependent.The following groups were tested for the two experiments: DMSO, morin 1, 2.5, and 5 mg/kg.
The habituation for both tasks was performed on days 7-9 (of 10 days of treatment) by placing the mice in an empty arena for 5 min each day.
As previously described, 10 mice per group were trained in a NOR task (Martinez-Coria et al., 2010).The following four groups were tested: DMSO, morin 1, 2.5, and 5 mg/kg.The animals were trained by letting them explore two identical objects at opposite ends of the arena for 5 min.The next day, mice were tested for 3 min with one familiar object and one novel object of similar dimensions.The recognition index (RI) represents the percentage of time mice spent exploring the novel object.
In order to study the recognition by a different experimental approach, another four groups (DMSO, morin 1, 2.5, and 5 mg/kg) were trained in a novel context task as follows: The animals were trained by letting them explore a pair of identical objects in a first arena (context A) for 5 min.After 90 min, the mice were placed in a second arena (context B) with another pair of identical objects and allowed to explore for 5 min.Finally, 24 h later, mice were tested for 3 min with one familiar object in context A and one object out of context from the second arena (B).The RI represents the percentage of time mice spent exploring the object out of context.

Morris water maze
Hippocampal-dependent learning and memory were examined using the MWM following standard protocols (Martinez-Coria et al., 2010).
After 6 days of treatment, DMSO and morin (1 mg/kg) mice groups were trained in a 1.2-meter diameter circular pool filled with opaque water at 21 • C.During the 4 days of training, mice were placed into the pool and allowed to find a submerged escape platform (4 trials/day).On the 5th day, the platform was removed to assess memory retention of the former platform location.Latency to cross the area and the number of crosses were measured.

Tissue processing
Tissue processing for immunohistochemical detection of proteins and quantification was performed following previously reported methods (Serrano-García et al., 2018).

Western blot
Equal amounts of protein (20-50 μg, depending on the protein of interest) were separated on 10% or 4% to 12% Bis/Tris gels (Bio-Rad) and transferred to a nitrocellulose membrane.Membranes were blocked for 1 h in 5% (v/v) suspension of nonfat milk Tris-buffered saline (pH 7.5) and incubated overnight with primary antibody at 4 • C. The primary antibodies used for Western blots are summarized above.After washing, the membranes were incubated with adjusted secondary anti-bodies coupled to horseradish peroxidase.Quantitative densiometric analyses were performed using ImageJ 1.4 software (NIH).

Golgi-Cox staining of neural dendrites
Coronal sections (Bregma 2.40 mm) of mouse brain (14 weeks old), control and treated with 1 mg/kg morin, were stained using the Golgi-Cox staining of neural dendritic silver impregnation technique.We used an FD Rapid GolgiStain Kit, FD NeuroTechnologies #PK401.The photographs were taken under a NIKON KCC-REM-NCY-E200 DMV model Eclipse E200LED MV R microscope.Fifteen neurons per individual were chosen, and those neurons in which their individuality was evident were selected, and the dendrites were drawn in Paint 3D to facilitate visualization.The dendrites of each selected neuron were counted for their subsequent statistical analysis with a two-way t-test, considering n = 45 for each treatment.Finally, the dendritic length was evaluated with the ImageJ 1.4 software, and the images were converted to an 8-bit format to transfer them to the NeuronStudio program and perform the Sholl analysis for each neuron.Subsequently, all the values of the dendrite length column were then added (values reflected in the results of the Sholl analysis).Figure 1 summarizes the experimental strategy used in this work.

Statistical analysis
Animals were randomly assigned to treatment versus control groups in this double-blind study.For immunohistochemistry and western blot-

Behavior: morin improves learning and memory
Morin-treated mice at 1 mg/kg showed a significant increase in the RI in both the NOR and CNOR tests (Figure 2a,b, respectively), suggesting a memory improvement.Moreover, they exhibited significantly improved memory acquisition in the MWM training, as revealed by the faster time to find the hidden platform on the 2nd and 3rd days of training (Figure 2c).Nevertheless, during the memory test 24 h after the last training day, the experimental group did not show any differences in the time spent in the target zone or the number of crosses (Figure 2d).

Morin treatment increased pro-BDNF, BDNF, and PSD-95 in the hippocampus and cortex differentially
To determine the possible molecular mechanism involved in memory improvement in mice treated with morin, we evaluated the expression of PSD-95, an abundant postsynaptic protein of excitatory synapsis that plays a key role in regulating synaptic strength (Keith & El-Husseini, 2008).In addition, we analyzed pro-BDNF and BDNF, a neuronal growth factor well known for its involvement in memory processes.
These molecular targets were analyzed after the administration of morin at 1 mg/kg in accordance with the memory tests.The results showed that morin significantly increased PSD95 levels in the mouse hippocampus but not in the cortex (Figure 3a,c).Regarding the precursors of BDNF, the pro-BDNF and the BDNF, morin significantly increased pro-BDNF in both structures, cortex and hippocampus, but BDNF expression only considerably increased in the hippocampus (Figure 3a,b).

IL-4 expression does not increase in morin treatment mice
One of the molecular mechanisms involved in the increase of BDNF in astrocytes is an extracellular increment of anti-inflammatory cytokines such as IL-4 (Gadani et al., 2012); however, we did not find significant changes in the expression of IL-4 in the hippocampus of mice treated with morin compared to controls (Figure 4a).

GFAP and pro-BDNF colocalize in the hippocampus in mice treated with morin
Following the same line of research above, we decided to analyze the expression of GFAP in astrocytes.A recent report showed that BDNF induced reactive astrogliosis in the CNS (Ding et al., 2020).Based on this background, we used western blot to analyze the GFAP protein, and we observed no alterations in the morin-administrated group (Figure 4b).When we analyzed the different hippocampus layers, no F I G U R E 4 Morin effect on astrocytes.Brain tissue was isolated from C57BL/6 mice treated with 1 mg/kg morin for 10 days.(a) IL-4 level was determined by immunohistochemistry. GFAP level was also determined by (b) Western blot and (c) immunohistochemistry in the hippocampus.Bars represent the mean ± standard error of the mean (SEM) of three animals/group and compared morin-treated mice versus vehicle, using t-student test or by one-way ANOVA.No significant difference was found.Finally, (d) GFAP colocalization with the pro-brain-derived neurotrophic factor (BDNF) was performed with immunofluorescence.significant differences were observed in the number of GFAP-positive cells compared to the control, despite a decrease in some layers (Figure 4c).Thus, our results show that BDNF does not increase GFAP expression in morin treatment mice.However, another parameter we analyzed to test if astrocytes were involved in the BDNF increase medi-ated by morin in the hippocampus was the colocalization of GFAP with the BDNF precursor, the pro-BDNF.Figure 4d shows that GFAP and pro-BDNF colocalize in the hippocampus in the animals treated with 1 mg/kg of morin, suggesting that the astrocytes could be the source of BDNF in mice stimulated by morin.

Morin treatment increases dendritic complexity
The rise in BDNF is related to the increase in the number and extension of dendrites, which in turn is a reflection of synaptic plasticity and is related to the acquisition of new information (Holtmaat & Caroni, 2016;Zagrebelsky et al., 2020).Our results show that 1 mg/kg morin increases the number and length of neuronal dendrites compared to the control.In addition, morin increases the extension of dendrites (Figure 5a,b).All the results shown here explain in part how morin improves mouse memory.

DISCUSSION
Strong evidence suggests that dietary flavonoid intake can improve cognitive function in healthy people (Devore et al., 2012;Godos et al., 2020;Travica et al., 2020), delaying the impairment of age-related cognitive function.Animal studies using diets containing flavonoids have shown that these compounds can improve learning and memory function in healthy animals (Joseph et al., 1998;Shif et al., 2006;Vauzour et al., 2021;Winter, 1998).The beneficial effects of morin have been demonstrated in numerous in vitro and in vivo studies and include its anti-oxidant, anti-inflammatory, antiapoptotic, anticancer, neuroprotective, and neuroplastic properties in different human disease models (Rajput et al., 2021); however, less is known about the effects of pure flavonoids on cognitive functions, providing a causation to these improvements.In the present study, we have shown, for the first time, that morin treatment improves recognition and spatial memory function in healthy animals and increases the levels of pro-BDNF, BDNF, and PSD95 in the hippocampus cortex and pro-BDNF in the cortex.Recognition and spatial memory probably involve cortical and subcortical structures typically associated with the perirhinal cortex and hippocampal formation.Solid evidence is that increased BDNF in those structures is associated with memory performance (Falkenberg et al., 1992;Hall et al., 2000;Hopkins et al., 2011;Schaaf et al., 2001).
BDNF is a ligand for the TrkB receptor, and their interaction results in the PSD95-TrkB complex, which induces the transport of PSD95 to dendrites and synapses (Ji et al., 2005;Yoshii & Constantine-Paton, 2007).Furthermore, changes in the number and morphology of dendrites observed in the hippocampus of treated animals are consistent with previous work in which similar results are observed in the neuronal ear (He et al., 2017), and also these changes support memory improvement, as these kinds of modifications are associated with cognitive processes (Zagrebelsky et al., 2020).Moreover, increases in postsynaptic protein PSD95 are related to improvements in different forms of memories, including spatial memory (Fitzgerald et al., 2015).The results also showed an increase in pro-BDNF in both structures.This molecule is not only the precursor form of BDNF but also participates in another relevant process of plasticity as crucial as LTD, through which there is a refinement of connections by eliminating those abnormalities that are ineffective for the formation of synaptic plasticity, memory, and cognition (Borodinova & Salozhin, 2017;Kowiański et al., 2018).Our study presents evidence that morin can induce improvements in memory but some relevant aspects must be resolved.For example, there is little information available about the pharmacokinetics of morin, and the data that is available suggest that this flavonol has poor bioavailability after oral ingestion (Li et al., 2019;Zhang et al., 2011).In this study, we administrate morin intraperitoneally, which generally shows higher bioavailability than oral administration for small molecules (MW < 5000) like morin (Al Shoyaib et al., 2019); moreover, many studies have consistently demonstrated that morin is able to modify various pathological cascades in different neuropathologies such as psychosocial stress, sleep deprivation, schizophrenia, and Alzheimer's disease resulting in cognitive enhancement including memory and learning, (Ben-Azu et al., 2019;Ben-Azu et al., 2018;Ben-Azu et al., 2020;Du et al., 2016;Elizabeth et al., 2020;Olonode et al., 2019) suggesting that morin and/or its metabolites or conjugated forms can not only cross the blood-brain barrier but also interact with different types of neurotransmitter receptors, in particular serotonin and acetylcholine, which are related to memory-enhancing effects (Ben-Azu et al., 2021;Thilakarathna & Rupasinghe, 2013).However, it is necessary to perform more research to confirm this pharmacologic characteristic.This work shows that GFAP colocalizes with pro-BDNF, suggesting that at least astrocytes are involved in the BDNF secreted stimulated by morin.Still, this fact does not discard that morin could stimulate BDNF synthesis and its secretion from another cell type, such as neurons.Lastly, one gap in this study is that only males were included, due mainly to the idea that behavioral responses may be highly variable due to hormonal changes that occur in the estrous cycle; however, a recent study in healthy mice of both sexes shows that there is no greater variability in females than in males in any parameter evaluated and therefore there is no significant difference in the tests of spatial memory, recognition, or BDNF production (Melgar-Locatelli et al., 2024).This new information encourages future studies to include both sexes.

CONCLUSIONS
This study shows that morin improves recognition and spatial memory in healthy adult mice.We suggest that the molecular mechanism for these effects involves the increased production and secretion of BDNF by astrocytes.

C57BL/ 6 (
RRID:MGI:2159769) male adult mice (3-4 months old, weighing between 25 and 35 g) were obtained from the vivarium at the Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez.The mice were kept in standard conditions (12/12 h light/dark cycle, 21 ± 2 • C, and 40% relative humidity) with access to food and water ad libitum.The experiments were performed according to the Official Mexican Standard on Technical Specifications for the Production, Care and Use of Laboratory Animals (NOM-062-ZOO-1999).The institutional ethical committee for the Care and Use of Laboratory Animals (CICUAL-INNNMVS) evaluated and approved the protocol on March 29, 2021, approval number INNN-138/19.
The mice were sacrificed by pentobarbital overdose and cardiac perfusion with 0.01 M phosphate-buffered saline.Their brains were removed and cut along the sagittal midline.The half brain wasF I G U R E 1The figure shows the timeline of the experimental procedures.The experimental approach is represented on a timeline by a sequence of colored boxes paired with a colored line, which highlights pertinent details about the method and the procedure's time frame.micro-dissected into the hippocampus and entorhinal cortex and frozen on dry ice for subsequent biochemical analysis, whereas the other half was fixed in 4% paraformaldehyde (pH-7.4,48 h).Fixed brains were embedded in paraffin and serially sectioned to obtain sagittal cuts of 7 μm to analyze the hippocampus.Then, GFAP and IL-4 were determined by immunostaining.The stained slices were examined at 10× magnification; micrographs were used to determine the number of positive cells per slice for GFAP and integrated optical density for IL-4.The quantitative analysis of the GFAP-positive cells was carried out using ImageJ version 1.45.The GFAP-immunoreactive cells were manually counted at 10× in CA1, CA2, CA3, CA4, molecular layer dorsal (ML dorsal), molecular layer ventral (ML ventral), granular layer dorsal (GL dorsal), granular layer ventral (GL ventral), mossy fibers, radiate layer, and oriens layer.Measurements were taken of four to six tissue sections from each animal in the entire layer of the hippocampus, and three animals were analyzed for each condition.Results were expressed as numbers of GFAP-positive cells.

F
Morin effect on learning and memory.C57BL/6 mice were treated with increasing morin doses (0-5 mg/kg of body weight) for 10 days.Then, (a) novel object recognition test and (b) context novel object recognition tests were performed.After this, the responsive morin concentration of 1 mg/kg was tested for memory tests.(c) Acquisition training started after 6 days of treatment, and (d) retention test was 1 day after treatment with morin.Bars represent the mean ± standard error of the mean (SEM) of 10 animals/group.*p < .05compared 1 mg/kg of morin-treated mice versus vehicle-treated mice, using one-way ANOVA, repeated measures ANOVA followed by the Tukey post hoc test (Parts A and B).Parts C, D, and E were analyzed with t-student test.ting,images were processed using ImageJ automated software and the values obtained were analyzed with Student's t-test.The RI values between familiar and novel objects obtained during NOR and CNOR tests were compared with one-way ANOVA (between groups), followed by multiple comparisons tests with statistical significance determined using Tukey's method.The MWM test was analyzed by repeated-measures ANOVA and Turkey post hoc for between-group interaction and one-tailed unpaired t-student for both trials of the spatial training retention probe.To analyze differences in dendrites, the Mann-Whitney U test was used.All values were analyzed with Graph-Pad Prism 9.0.0 and all graphs were shown as the mean ± standard error of the mean and a p value of < 0.05 was considered statistically significant.

F
Morin effect on neuronal growth factors and synaptic proteins.Fresh brain tissue was extracted from C57BL/6 mice treated with 1 mg/kg morin for 10 days.(a) Representative immunoblotting and densitometric analysis of the levels of (b) pro-brain-derived neurotrophic factor (BDNF) and BDNF and (c) postsynaptic density protein (PSD95) in the hippocampus and cortex are shown.Bars represent the mean ± standard error of the mean (SEM) of three animals/group.*p < .05compared morin-treated mice versus vehicle, using t-student test.

F
I G U R E 5 Morin improves synaptic plasticity.Coronal sections of mouse brain from 14-week old control and treated with 1 mg/kg morin were stained using the Golgi-Cox staining of neural dendritic silver impregnation technique: (a) representative photographs; (b) representative images of Golgi-positive cells used form the dendritic length and Sholl analysis; (c) graph values of the quantification and extension of the dendrites.Bars represent the mean ± standard error of the mean (SEM) of three animals/group.*p < .05,compare morin-treated mice versus vehicle, using two-way student t.**p < .001compared morin-treated mice versus vehicle, using Mann-Whitney U test.