Intermittent fasting disrupts hippocampal-dependent memory and norepinephrine content in aged male and female mice

Kimberly Wiersielis; Ali Yasrebi; Thomas J Degroat; Nadja Knox; Catherine Rojas; Samantha Feltri; Troy A Roepke

doi:10.1016/j.physbeh.2023.114431

. Author manuscript; available in PMC: 2025 Jan 17.

Published in final edited form as: Physiol Behav. 2023 Dec 10;275:114431. doi: 10.1016/j.physbeh.2023.114431

Intermittent fasting disrupts hippocampal-dependent memory and norepinephrine content in aged male and female mice

Kimberly Wiersielis ^a,^b,^*, Ali Yasrebi ^a, Thomas J Degroat ^a,^c, Nadja Knox ^a,^c, Catherine Rojas ^a,^b, Samantha Feltri ^a, Troy A Roepke ^a,^b,^c,^d,^e

PMCID: PMC11740021 NIHMSID: NIHMS1958961 PMID: 38072036

Abstract

Intermittent fasting (IMF) is associated with many health benefits in animals and humans. Yet, little is known if an IMF diet affects mood and cognitive processing. We have previously identified that IMF in diet-induced obese males increases norepinephrine and dopamine content in the hypothalamus and increases arcuate neuropeptide Y (NPY) gene expression more than in ad libitum control males. This suggests that IMF may improve cognition through activation of the hindbrain norepinephrine neuronal network and reverse the age-dependent decline in NPY expression. Less is known about the association between anxiety and IMF. Although, in humans, IMF during Ramadan may alleviate anxiety. Here, we address the impact of IMF on anxiety-like behavior using the open field test, hippocampal-dependent memory using the Y-maze and spatial object recognition, and hippocampal-independent memory using novel object recognition in middle-aged male and female (12 mo) and aged male and female (18 mo) mice. Using ELISA, we determined norepinephrine (NE) content in the dorsal hippocampus (DH) and prefrontal cortex (PFC). We also investigated gene expression in the arcuate nucleus (ARC), the lateral hypothalamus (LH), and the locus coeruleus (LC). In IMF-treated females at both ages, we observed an improvement in spatial navigation although an impairment in spatial object orientation. IMF-treated females (12 mo) had a reduction and IMF-treated males (12 mo) displayed an improvement in novel object recognition memory. IMF-treated females (18 mo) exhibited anxiolytic-like behavior and increased locomotion. In the DH, IMF-treated males (12 mo) had a greater amount of NE content and IMF-treated males (18 mo) had a reduction. In the ARC, IMF-treated males (12 mo) exhibited an increase in Agrp and Npy and a decrease in Adr1a. In the ARC, IMF-treated males (18 mo) exhibited an increase in Npy and a decrease in Adr1a; females had a trending decrease in Cart. In the LH at 12 months, IMF-treated males had a decrease in Npy5r, Adr1a, and Adr1b; both males and females had a reduction in Npy1r. In the LH, IMF-treated females (18 mo) had a decrease in Hcrt. In the LC at both ages, mice largely exhibited sex effects. Our findings indicate that IMF produces alterations in mood, cognition, DH NE content, and ARC, LH, and LC gene expression depending on sex and age.

Keywords: Intermittent fasting, Cognition, Anxiety, Hippocampus, Prefrontal cortex, Norepinephrine

1. Introduction

Cognitive decline in aged adults is a growing public health issue [1]. Overall, the prevalence of cognitive decline is 11.1 %, or 1 in 9 adults [2]. Cognitive decline accelerates with age; between ages 45–64 the rate is 10.8 % and at 65 years and older the rate is 11.7 % [2]. A diagnosis of dementia is made when cognitive impairment in adults exhibits deficits in memory, language, and reasoning difficulties sufficiently severe to affect daily function [3]. Although the core symptoms of cognitive decline is memory impairment, it often shows comorbid symptoms with anxiety, often seen in the early phase of decline [4]. Core symptoms of anxiety are characterized by tenseness, irritability, and excessive worry [5]. Thus, it is imperative to elucidate the underlying cause of cognitive decline in order to improve the quality of life by reducing symptoms in those suffering from dementias.

Another public health concern that is related to deficits in cognition is obesity. The obesity epidemic in the United States poses a threat to life expectancy and contributes to the rising cost of the health care system [6,7]. Recent estimates show that in less than a decade, the prevalence of obesity in the United States has increased from 34.6 % in 2006 to 37.9 % in 2014 [8]. A relatively consistent finding associated with obesity is disruption in cognitive processing [9–15]. For example, a higher Body Mass Index (BMI) in adults is associated with a greater risk for cognitive decline [11] and poorer performance on cognitive tests [14,15]. Relatedly, diets high in fat are linked to cognitive decline, as an increased level of saturated fat is a risk factor for dementia [12,16].

One way to combat the symptoms of obesity-related cognitive decline is through dietary manipulations, such as intermittent fasting (IMF) [17]. IMF is a dietary pattern that cycles between periods of fasting and eating on a predetermined schedule [18,19]. IMF typically results in a healthy weight, improved health, and well-being [20]. Clinically, IMF has been shown to have neuroprotective properties in diseases related to anxiety [21–23] cognitive decline [24,25] and improve short-term memory [26]. For instance, during Ramadan, a period devoted to prayers in Islam [27], when pre-Ramadan anxiety scores were compared to post-Ramadan scores, anxiety was found to be lower at the end of Ramadan, when evaluated by the Depression Anxiety Stress Scale [21]. IMF-induced improvements in memory are thought to be through promoting associative plasticity in hippocampal pyramidal neurons [28]. Further research is needed into the mechanism of dietary manipulations on cognitive function in aged rodents and human populations, whether obese or not.

Aging is associated with deficits in memory, which may contribute to the impairments exhibited in cognitive processing and executive function [29–31]. Age-related changes in brain function have consistently been reported in a number of specialized brain regions involved in cognition. Studies in animal models show that adult hippocampal neurogenesis declines greatly with increasing age, particularly in the dentate gyrus (DG) [32]. In parahippocampal regions, such as the perirhinal cortex (PRC), cell loss occurs very early in the onset of age-related dementia [33,34]. Due to the prominent projections to the hippocampus, loss in medial septum (MS) cells during aging results in impaired hippocampal function [35–37]. In a region well known to be involved in cognition, the prefrontal cortex (PFC), pharmacological studies suggest that dopamine and norepinephrine (NE) modulate memory function through D1 and alpha-2 adrenergic receptor action, and their depletion in aged animals results in hippocampal-dependent memory deficits [38, 39]. The mechanisms of age-related cognitive decline are not fully characterized, yet the examination of areas that undergo age-related changes in brain function may uncover mechanism for cognition and memory deficits later in life.

The hypothalamic arcuate nucleus (ARC) is critical for food intake regulation and has been implicated in IMF. The ARC contains orexigenic neurons co-expressing agouti-related peptide (AgRP) and neuropeptide Y (NPY) and anorexigenic neurons expressing proopiomelanocortin (POMC). The neuropeptide orexin, (also called hypocretin) in the lateral hypothalamus (LH) has a critical role in arousal, reward, and mood [40, 41]. Although complex, orexin is involved in the modulation of metabolism, food intake, and wakefulness [40,42–44]. Orexin neurons are found in the LH and have widespread projections in the brain [45,46]. Two prominent orexin projections from the LH are to the dopaminergic ventral tegmental area (VTA), implicated in reward-based eating, and to the noradrenergic locus coeruleus (LC), critical for arousal [46–48]. Of particular focus is the LH-orexin projection to the LC brainstem nucleus, as alterations in the orexin system have been shown to modulate NE-induced arousal [49]. A vast majority of NE neurons are concentrated in the LC, and these NE-containing axons are distributed widely throughout the brain, particularly to brain areas associated with cognitive function and anxiety [48]. Because cognitive function relies on the activation of arousal pathways, our study centers on the LC projections to the hippocampus (HPC) and PFC, as both are involved in cognitive processing [50–53]. The influence of orexin on LC-mediated cognitive processing in males and females is not well understood; however, it is imperative to understand this interconnection through the impact of IMF and assessment of behaviors associated with cognition.

IMF has beneficial effects on anxiety and cognition. However, the underlying neural pathways are not fully understood. IMF may improve cognition through the modulation of a hypothalamic-to-hindbrain circuit leading to release of NE in brain regions involved in cognition and anxiety in mice. The current study seeks to elucidate the influence of IMF on cognition and anxiety. The goal of this study was to examine the relationship between IMF and cognitive decline on behavior and brain function. Here, we evaluate anxiety-like behavior with the open field test, hippocampal-dependent memory using the Y-maze and spatial object recognition, and hippocampal-independent memory using novel object recognition in middle-aged male and female (12 mo) and aged male and female (18 mo) mice. In addition, we used ELISA to determine NE content in the dorsal hippocampus (DH) and PFC and assessed gene expression in the ARC, the LH, and the LC. In the ARC, we investigated the melanocortin genes, norepinephrine receptors, and K⁺ channel subunits consisting of Agrp, Npy, Hcrtr1, Adr1a, Adr1b, Kcnq3, Kcnq5, Pomc, and Cart. In the LH, we assessed Hcrt (orexin), a regulator of arousal, the NPY receptors Npy1r and Npy5r, and α-adrenergic receptor 1a and 1b, Adr1a and Adr1b, respectively. In the LC, we examine Slc6a2, norepinephrine transporter gene, Dbh, dopamine β-hydroxylase, which catalyzes the conversion of dopamine to NE, and Hcrtr1, hypocretin receptor 1, which is involved in arousal. We determine how IMF improves cognition through the modulation of a hypothalamic to hindbrain circuit leading to release of NE in brain regions involved in cognition and memory. We hypothesised that enhanced NE signaling caused by IMF restores cognition and memory in aged male and female mice through the differential modulation of the NPY-orexin circuit.

2. Methods

2.1. Animals and housing conditions

Male and female C57BL/6 mice (6 months) were purchased from The Jackson Laboratory and housed in standard laboratory cages in our facility until 12 months or 18 months of age prior to dietary manipulatons. All were fed an ad libitum standard chow (13 % kCal fat, 3.48 kcal/g, Lab Diet 5V75; low phytoestrogen,<75 ppm). At the start of dietary manipulations, mice were separated into two groups: 1) Control ad libitum feeding and 2) IMF. As previously decribed [17], for the IMF group, food deprivation was for a 24-hour period beginning at 9:00 AM (fasting day), 2 h into the light cycle, on Monday, Wednesday and Fridays. On fasting days, all animals including controls were weighed, food intake was recorded, and cages were changed [17]. Food was returned to the IMF-treated mice at 9:00 AM the following day and fed ad libitum until the next fasting period. Mice were pair housed and maintained on a 12-hour light, 12-hour dark cycle; lights on from 7:00 AM to 7:00 PM. Mice were kept under controlled temperature (21–23 °C) and humidity (30–70 %) regulation. The estrous cycle was not evaluated for all females because the 18-month-old females had entered reproductive senescence. All procedures were approved by the Institutional Animal Care and Use Committee of Rutgers University.

2.2. Behavioral analysis

All testing was recorded (ANY-maze, Version 6, Stoelting, USA) by a camera suspended above the test arena. Mice underwent either ad libitum control or IMF for four weeks followed by a behavioral paradigm consisting of tests that evaluate cognition and anxiety-like behavior (Fig. 1). Experimental mice sustained the IMF paradigm during behavioral testing and were only evaluated on behavior on days in which they were food satiated. Mice were habituated to the experimental room 24 h prior to testing. Behavioral testing occurred between 0900 and 1200 h and was conducted during the light phase of the light/dark cycle. All behavior testing was conducted by an experimenter blind to experimental conditions. n’s per treatment group for behavior ranged from 10 to 16.

Fig. 1. — Experimental timeline. 12-month and 18-month-old male and female mice underwent either *ad libitum* control or an IMF paradigm (3 days/week) for four weeks prior to behavior testing. Mice were tested on the Y-maze, open field test (OFT), spatial object recognition (SOR), and novel object recognition (NOR). Experimental mice sustained the IMF paradigm during behavioral testing and were only assessed on days they were food satiated. Mice were fasted for five hours prior to euthanasia.

2.2.1. Y-maze

Mice were first tested on the Y-maze. The Y-maze is a white plexiglass apparatus consisting of three identical arms (8 cm wide, 15 cm high, and 30 cm long) connected to a triangular central platform. The three arms consisted of the start arm, the habituation arm, and the test arm. Directly before the habituation arm and the test arm, there was a white opaque removable door 1 cm from the triangular center platform. The maze was surrounded by outer walls with a distinct visual cue (white vertical striped cue) above the habituation and test arms that stayed stationary for the duration of the testing period. The test consisted of a 5 min habituation phase and 5 min test phase [54]. The habituation phase was started by placing the mouse in the distal end of the start arm and allowing the mouse to freely explore the start arm and one of the other arms (habituation arm) while the third arm (test arm) was blocked. The habituation arm and test arm were counterbalanced between mice (but not within mice) to reduce arm bias effects. At the end of the habituation phase the mouse was placed in a temporary holding cage away from the testing location for a 5 min intertrial delay. After the intertrial delay, the mouse was returned to the start arm of the Y-maze and allowed to freely explore all three arms for 5 min. All four paws needed to be in an arm to constitue an entry. Specific measures analyzed were the amount of time spent in the unknown arm (sec), the number of unknown arm entries, the percentage of unknown arm time (sec), and the percentage of unknown arm entries.

2.2.2. Open field test (OFT)

The next day of behavior testing was the first day of a four day object location paradigm which included an open field test (OFT). On the first day of this paradigm, mice underwent habituation to the spatial object recognition testing arena which served as the OFT. The OFT utilizes a square white opaque plexiglass arena (40 cm long × 40 cm wide × 40 cm high, open-top) with a 64-square grid floor (8 × 8 squares, 5 cm/side). At the start of the test, mice were placed in the same (10 cm long × 10 cm wide) corner square of the arena, they then were allowed to freely explore for 5 min. Automated ANY-maze computer-scored measures consisted of the amount of time spent in the center zone (sec) (4 × 4 center squares), the number of center entries (4 × 4 center squares), the percentage of time spent in the center zone (sec), the percentage of entries in the center zone, the distance traveled (m), and the mean speed (m/sec). All four paws of the animal’s body needed to be in a zone to be defined as an entry. After each animal trial, the surface of the arena was cleaned with a sterilizing solution.

2.2.3. Spatial object recognition (SOR)

Mice underwent 3 days of habituation to the testing arena prior to the spatial object recognition test. Mice were habituated to a white plexiglass square arena (40 cm × 40 cm × 40 cm, open top) and a black and white vertical striped cue on the inside wall, for 5 mins/day for three consecutive days. On the test day, mice were exposed to a 5 min training phase to two identical plastic objects (50 ml conical tube placed upsidedown) at one end of the arena placed 7 cm from arena walls. Following training, mice were returned to a holding cage for a 5 min delay during which one of the objects was displaced to the opposite side of the arena. Starting location of object and object being displaced were counterbalanced. Mice were then returned to the arena for a 5 min test where they were allowed to freely explore each object. The paramaters measured were the amount of time spent with the displaced object (sec), the number of bouts with the displaced object, the percentage of time spent with the displaced object (sec), and the percentage of bouts with the displaced object.

2.2.4. Novel object recognition (NOR)

We ensured our NOR test did not require the hippocampus because we used a repeated habituation exposure training procedure rendering the NOR independent of the hippocampus [55,56]. Mice underwent the paradigm for novel object recognition following SOR. The day after completing SOR, mice were habituated to the same plexiglass arena for an additional 5 min/day for five consecutive days. The test day was similar to SOR in that it consisted of 5 min training, delay, and testing phases. However, in NOR, mice were exposed to two metal objects (eyeglass holder, 5 cm × 5 cm × 5 cm) during the training phase, and then one metal object was replaced with a glass object during the delay phase (125 mL flask, 11.5 cm × 6.5 cm × 6.5 cm), located in the same position, prior to the test phase. Then mice are returned to the arena and allowed to freely explore the objects. Objects and their replacement location were counterbalanced. Behavior was automatically scored with ANY-maze software. Object exploration was defined as being within 2 cm of the object and actively sniffing the object. Mice that failed to explore both objects from SOR and NOR were dropped from analysis. For both the SOR and the NOR test, preference scores were calculated by determining the ratio of time spent with the displaced or novel object, respectively, divided by the time spent with the familiar object. The dependent variables for NOR were the amount of time spent with the novel object (sec), the number of bouts with the novel object, the percentage of time spent with the novel object (sec), and the percentage of bouts with the novel object.

2.3. Norepinephrine ELISA

2.3.1. Tissue processing

The PFC and HPC (dorsal section) were dissected for NE ELISA. The PFC and DH were cut into 1 mm coronal slices using a brain matrix (Ted Pella, Redding, CA, USA), (PFC bregma 1.34 to 2.68 mm; DH bregma −1.06 to −2.46 mm). The fresh slices were immediately microdissected using a dissecting microscope (Motic Microscopes) and flash frozen. For tissue collection, mice (n = 85) were dissected to collect 85 PFC and 73 DH tissue samples, with these samples being stored in a freezer at −80 °C to ensure that the samples are properly preserved overtime. Throughout the experiment, the samples were stored on ice as frequently as possible for conservation purposes, ensuring that none of the collected tissue sample was lost. Preparation for supernatant collection began by homogenizing the tissue samples using 40X buffer. As the samples contained different amounts of tissue in each tube attributed to human tissue collection error, this was accounted for by adding different amounts of the 40X buffer to each tube. The volume of buffer added was determined by accounting for the tissue mass inside the tube and adding the respective amount necessary for proper homogenization. The samples were placed in a centrifuge at 1300 rpm for 10 min at 4 °C. Centrifuging the samples produced a supernatant, where each supernatant sample was pipetted into a fresh 1.7 microcentrifuge tube, and stored at −80 °C.

2.3.2. ELISA procedure

To determine norepinephrine concentrations, the Noradrenaline Research ELISA test kit (Rocky Mountain Diagnostics BA E-5200R) was used for the enzyme immunoassay according to manufacturers instructions allowing for the quantitative data analysis of each sample’s norepinephrine concentration. Briefly, four ELISA kits were used (each kit containing two plates) for the samples (n = 85). The extraction wells, including the blanks, controls, standards, and samples, were equalized to a final volume of 100 μl using deionized water. Then, 25 μl of TE buffer was pipetted into each well. The plates were shaken at 600 rpm for 60 min at room temperature (RT=20–25 °C). After that, 150 μl of Acylation Reagent and 25 μl of Acylation Buffer were shaken at RT (20 min). Then, 100 μl of hydrochloric acid was shaken at RT (10 min). 90 μl of the solution with hydrochloric acid was transferred to the ELISA microtiter plate, where 25 μl of enzyme solution was pipetted into each well. This solution was shaken at RT (1 min), with a 2 hour incubation period at 37 °C. After the incubation period, 100 μl of the solution was pipetted into the ELISA pre-coated Noradrenaline Microtiter Strips. 50 μl of Noradrenaline Antiserum was then shaken at RT (1 min). The Noradrenaline Microtiter Strips were incubated for 15–20 h overnight at 4 °C. Once incubated, 100 μl of enzyme conjugate was shaken at RT (30 min). The plates were washed again, then 100 μl of substrate was shaken at RT (30 min). Lastly, 100 μl of stop solution was used and within 10 min, the absorbance was read using a microplate reader set to 450, 620, and 650 nm in wavelength. n’s per treatment group for ELISA ranged from 6 to 13.

2.4. RNA extraction and quantitative real-time PCR

The ARC, LH, and LC were microdissected for RNA extraction and gene expression analysis. The regions were cut into 1-mm coronal slices using a brain matrix (Ted Pella, Redding, CA, USA), (ARC bregma −1.34 to −2.70 mm; LH bregma −2.18 to −2.54 mm; LC bregma −5.34 to −5.80 mm). The brain blocks were transferred to RNAlater (Life Technologies, Inc) and stored overnight at 4 °C. Samples were dissected from slices using a dissecting microscope (Motic Microscopes). The dissected tissue was stored at −80 °C.

Complementary DNA (cDNA) was synthesized from brain tissue using Superscript III reverse transcription, 100 ng random hexamer primers, 4 μL 5 × buffer, superscript, 25 mM MgCl2,10 mM dNTP, 40 U/μL RNAsin, and 100 mM dithiothreitol in diethylpyrocarbonate-treated water (DTT) for a total volume of 20 μl. Reverse transcription was conducted using the following reverse transcription protocol: 5 min at 25 °C, 60 min at 50 °C, and 15 min at 70 °C. The cDNA samples were diluted to 1:20 with nuclease-free water (Bioexpress) stored at −20 °C. For qPCR, 4 μL of cDNA template was amplified using either PowerSYBR Green (Life Technologies) or Sso Advanced SYBR Green (BioRad, Hercules, CA) on CFX-Connect or Opus-96 Real-time PCR instrument (BioRad). The amplification protocol for all the genes was as follows: initial denaturing at 95 °C for 10 min (PowerSYBR) or 3 min (SsoAdvanced) followed by 40 cycles of amplification at 94 °C for 10 s (denaturing), 60 °C for 45 s (annealing), and completed with a dissociation step for melting point analysis with 60 cycles of 95 °C for 10 s, 65 °C to 95 °C (in increments of 0.5 °C) for 5 s, and 95 °C for 5 s. Negative controls and water blank were included in the qPCR plate design. n’s per treatment group for gene expression ranged from 5 to 8.

2.5. Statistical analysis

All 12 month and 18 month data were analyzed with a 2 × 2 ANOVA with sex (male × female), and treatement (IMF × control) as factors on Prism 6 (GraphPad Software, La Jolla, CA, USA version 9). Outliers that exceeded 2 standard deviations (SD) above or below the group mean were excluded using the Grubbs test (α < 0.05). Our results are designated as mean (± SEM) and were considered statistically significant at p < .05. Holm-Sidak’s multiple comparison tests were performed.

2.5.1. Outliers

2.5.1.1. Behavior.

Statistical outliers for the Y-maze: unknown arm time (sec), n = 1, 12 month:female:control; the number of unknown arm entries, n = 0; the percentage of unknown arm time (sec), n = 0; the percentage of unknown arm entries, n = 0.

Statistical outliers for the OFT: the amount of time spent in the center zone (sec), n = 4, 12 month:male:control, 12 month:male:IMF, 12 month:female:IMF, 18 month:female:control; the number of center entries, n = 2, 12 month:male:IMF, 18 month:male:IMF; the percentage of time spent in the center zone (sec), n = 4, 12 month:male:control, 12 month:male:IMF, 12 month:female:IMF, 18 month:female:control; the percentage of entries in the center zone, n = 1, 12 month:male:IMF; the distance traveled (m), n = 3, 18 month:male:control, 12 month:female: IMF, 18 month:female:IMF; the mean speed (m/sec), n = 3, 18 month: male:control, 12 month:female:control, 12 month:female:IMF.

Statistical outliers for SOR: the amount of time spent with the displaced object (sec), n = 2, 18 month:male:IMF, 12month:female:IMF; the number of bouts with the displaced object, n = 3, 18 month:male:control, 18 month:female:control, 18 month:female:IMF; the percentage of time spent with the displaced object (sec), n = 1, 12 month:male:IMF; the percentage of bouts with the displaced object, n = 0.

Statistical outliers for NOR: the amount of time spent with the novel object (sec), n = 3, 12 month:male:IMF, 18 month:female:control, 12 month:female:IMF; the number of bouts with the novel object, n = 2, 12 month:male:IMF, 18 month:female:control; the percentage of time spent with the novel object (sec), n = 0; the percentage of bouts with the novel object, n = 0.

2.5.1.2. ELISA.

Statistical outliers for ELISA: dorsal hippocampus, n = 2, 12 month:female:IMF, 18 month:male:IMF; prefrontal cortex, n = 2, 18 month:male:IMF, 18 month:male:control.

2.5.1.3. qPCR.

Statistical outliers for ARC: Agrp, n = 1, 12 month:female:control, 18 month:female:control; Npy, n = 2, 18 month:female: IMF (2); Hcrtr1, n = 0; Adr1a, n = 1, 18 month:male:IMF, Adr1b, 18 month:male:control, 18 month:female:IMF; Kcnq2, n = 0, Kcnq3, n = 1, 12 month:female:IMF, Kcnq5, n = 0; Pomc, n = 0; Cart, n = 0.

Statistical outliers for LH: Hcrt (orexin), n = 2, 18 month:male: control, 18 month:female:IMF; Npy1r, n = 0; Npy5r, n = 2, 18 month: male:control, 18 month:female:IMF; Adr1a, n = 0; Adr1b, n = 1, 12 month:female:control.

Statistical outliers for the LC: Slc6a2, n = 0; Dbh, n = 0; Hcrtr1, n = 0.

3. Results

3.1. Y-Maze

3.1.1. 12 months

On time spent in the unknown arm (Fig. 2A), we did not observe an effect of sex or treatment; however, we did observe an interaction effect (F(1,52) = 7.56, p = .0082). IMF-treated females exhibited an increase in time spent in the unknown arm in contrast to control females (p = .0169); however, there was no observable effect in IMF-treated males on this measure. Next, we evaluated the number of entries in the unknown arm (Fig. 2B). Although no main effect of treatment or an interaction was observed, an effect of sex was found (F(1,53) = 10.72, p = .0019). The percentage of time spent in the unknown arm (Fig. 2C) indicated no effect of sex or treatment, but an interaction of sex and treatment was identified (F(1,53) = 4.63, p = .0358), with no significant pairwise differences. The percentage of entries in the unknown arm (Fig. 2D) resulted in no differences in sex, treatment, or an interaction between sex and treatment.

3.1.2. 18 months

The assessment of time spent in the unknown arm (Fig. 3A) did not result in an effect of sex, treatment, or interaction between sex and treatment. No effect of treatment was observed on the number of unknown arm entries, but we did observe an effect of sex (F(1,41) = 22.56, p < .0001) and an interaction effect (F(1,41) = 4.810, p = .0340) (Fig. 3B). IMF-treated females exhibited a greater number of unknown arm entries than control females (p = .0166). No difference was observed between control and IMF-treated males. A difference was also observed between male and female IMF-treated mice (p < .0001), but not between control male and females. Both the percentage of time spent in the unknown arm (Fig. 3C) and the percentage of entries in the unknown arm (Fig. 3D) did not uncover an effect of sex, treatment, or interaction between sex and treatment.

3.2. Open field test

3.2.1. 12 months

On the amount of time spent in the center zone (Fig. 4A), we did not find an effect of treatment or an interaction between sex and treatment, but we did find a trending effect of sex (F(1,50) = 3.656, p = .0616). IMF-treated females had a reduction in center time in contrast to IMF-treated males (p = .0440). No differences were observed between control male and females. Next, the number of entries into the center zone was evaluated (Fig. 4B). There was no effect of sex, treatment, or interaction between sex and treatment on this measure. For the percentage of time spent in the center zone (Fig. 4C), no effect of sex, treatment, or interaction between sex and treatment was observed, although we observed at trending difference such that IMF-treated females had a decreased percentage of time spent in the center zone than IMF-treated males (p = .0551). No differences were observed between control males and females on this assessment. Considering percentage of entries in the center zone (Fig. 4D), we found an effect of sex (F(1,52) = 5.994, p = .0178), with no differences in treatment or an interaction. IMF-treated females had a reduction in the percentage of entries in the center zone in contrast to IMF-treated males (p = .0330). No differences were detected between control males and females. We next evaluated the distance traveled (Fig. 4E) and did not detect an effect of treatment or an interaction between sex and treatment; however, we did uncover an effect of sex (F(1,52) = 17.63, p = .0001). IMF-treated females had a decreased distance traveled in contrast to IMF-treated males (p = .0006). Further, control females had a reduced distance traveled in contrast to control males (p = .0405). We evaluated mean speed (Fig. 4F) and did not observe an effect of treatment or an interaction between sex and treatment, but did find an effect of sex (F(1,52) = 17.43, p = .0001). IMF-treated females had a decreased mean speed in contrast to IMF-treated males (p = .0006), and control females had a reduced mean speed in contrast to control males (p = .0418).

3.2.2. 18 months

Considering the amount of time spent in the center zone (Fig. 5A), we found an effect of sex (F(1,37) = 22.53, p < .0001), treatment (F(1,37) = 4.879, p = .0335), and interaction between sex and treatment (F(1,37) = 7.472, p = .0096). IMF-treated females exhibited an increase in center time in contrast to control females (p = .0017). No differences were observed between control and IMF-treated males. In addition, we detected a difference in IMF-treated mice such that, IMF-treated females had spent a greater amount of time in the center zone in contrast to IMF-treated males (p < .0001). No differences between control male and females was detected. We then evaluated the number of entries into the center zone (Fig. 5B). There was no effect of treatment or interaction between sex and treatment on this measure, but we did uncover an effect of sex (F(1,37) = 85.64, p < .0001). IMF-treated females had an increased number of entries into the center zone in contrast to IMF-treated males (p < .0001). Moreover, we detected a difference in control male and female mice as control females exhibited a greater number of entries into the center zone in contrast to control males (p < .0001). On the percentage of time spent in the center zone (Fig. 5C), we demonstrated an effect of sex (F(1,37) = 21.07, p < .0001), treatment (F (1,37) = 4.980, p = .0318), and interaction between sex and treatment (F(1,37) = 7.055, p = .0116). IMF-treated females exhibited an elevated percentage of time spent in the center zone than control females (p = .0019). No differences were observed between control and IMF-treated males. We also detected that IMF-treated females had a greater percentage of time spent in the center zone in contrast to IMF-treated males (p < .0001). No difference was uncovered between control males and females on this measure. The analysis of percentage of entries in the center zone (Fig. 5D) yielded no differences in treatment or an interaction between sex and treatment, but we did demonstrate an effect of sex (F(1,38) = 30.19, p < .0001). IMF-treated females had an increased percentage of entries in the center zone in contrast to IMF-treated males (p = .0002). In addition, control females had an increased percentage of entries in the center zone in contrast to control males (p = .0019). On the evaluation of distance traveled (Fig. 5E), we did not detect an effect of treatment or an interaction between sex and treatment, however we did illustrate an effect of sex (F(1,36) = 113.9, p < .0001). IMF-treated females had a greater distance traveled in contrast to control females (p = .0444). We did not uncover a difference in males on this measure. Further, IMF-treated females had an increased distance traveled in contrast to IMF-treated males (p < .0001) and control females had a greater distance traveled in contrast to control males (p < .0001). On the evaluation of mean speed (Fig. 5F), we did not observe an effect of treatment or an interaction between sex and treatment, but did demonstrate an effect of sex (F(1,36) = 112.9, p < .0001). IMF-treated females exhibited a greater mean speed in contrast to control females (p = .0476). No differences were observed between males on this measure. Moreover, IMF-treated females showed a greater mean speed in contrast to IMF-treated males (p < .0001), and control females had an increased mean speed in contrast to control males (p < .0001).

3.2.3. Comparison of the tested and calculated results with non-penetrating impact effect

Based on the drop hammer impact rig established in Section 3.2.1, the non-penetrating impact tests are conducted on specimens I to III using the impact velocities of 0.32, 0.42, and 0.51 m/s, respectively, which can be indirectly obtained via the height of the impactor with its vertical position being precisely adjusted by the control box. The time-domain ICF curves and load-displacement curves can be obtained through the force and laser displacement sensors. Also, considering the same excitation parameters such as impact energy and position of the drop hammer, the theoretical model is utilized to predict the ICF and load-displacement curve when the non-penetrating impact effect is considered. The comparison of the tested and calculated results with the impact velocity of 0.32, 0.42, and 0.51 m/s are shown in Figs. 8, 9 and 10, respectively, in which the related maximum calculation errors are also provided.

Fig. 8. — IMF-treated 12-month-old females had a reduction and IMF-treated 12-month-old males had an improvement in novel object recognition memory. A) IMF-treated females had a reduction in time spent with the novel object (*sec*) in contrast to control females. B) IMF-treated females displayed a reduction in the number of novel object bouts in contrast to control counterparts. C) For the percentage of time spent with the novel object (*sec*), IMF-treated males spent greater amounts of time in contrast to control counterparts. D) The percentage of novel object bouts did not demonstrate any pairwise differences. Data are represented as mean ± SEM. * = p < .05.

Fig. 9. — IMF-treated 18-month-old males and females exhibited no pairwise differences in novel object recognition memory. A) No pairwise differences observed in time spent with the novel object (*sec*). B) For the number of novel object bouts no pairwise differences were detected. C) For the percentage of time spent with the novel object (*sec*), no pairwise differences were observed. D) The percentage of novel object bouts did not demonstrate any pairwise differences. Data are represented as mean ± SEM.

Fig. 10. — 12-month-old males had an increase and 18-month-old females had a decrease in dorsal hippocampal NE content. A) On the examination of NE content in the dorsal hippocampus of 12-month-old mice, IMF-treated males had an increased amount of NE content in contrast to control males. Both control and IMF-treated males had a greater amount of NE content than control and IMF-treated females. B) For NE content in the prefrontal cortex of 12-month-old mice, both control and IMF-treated males had a greater amount of NE content than control and IMF-treated females. C) On the examination of NE content in the dorsal hippocampus of 18-month-old mice, IMF-treated females had a decreased amount of NE content in contrast to control females. Control females had an increased amount of NE content in contrast to control males. D) For NE content in the prefrontal cortex of 18-month-old mice, we did not detect any pairwise differences. Data are represented as mean ± SEM. * = p < .05; ** = p < .01; **** = p < .0001.

3.3. Spatial object recognition

3.3.1. 12 months

The analysis of time spent with the displaced object (Fig. 6A) revealed no effect of sex or treatment but did yield a trending interaction between sex and treatment (F(1,45) = 3.333, p = .0746). IMF-treated females had a reduction in time spent with the displaced object in contrast to control females (p = .0400). There were no differences seen in males on this assessment. The number of displaced object bouts (Fig. 6B), the percentage of time spent with the displaced object (Fig. 6C), and the percentage of displaced object bouts (Fig. 6D) did not demonstrate a difference in sex, treatment, or an interaction between sex and treatment.

3.3.2. 18 months

The amount of time spent with the displaced object (Fig. 7A) did not illustrate an effect of sex or treatment, but did illustrate an interaction between sex and treatment (F(1,39) = 4.829, p = .0340). IMF-treated females have a reduction in time spent with the displaced object in contrast to control females (p = .0078). No effect was seen in males on this analysis. In addition, a difference was observed between control males and females (p = .0119); no effect was detected between IMF-treated males and females. On the number of displaced object bouts (Fig. 7B), no effects were demonstrated for sex and treatment although a trend for an interaction between sex and treatment was observed (F (1,37) = 3.444, p = .0715). For the analysis of percentage of time spent with the displaced object (Fig. 7C), there was no effect of sex nor an interaction between sex and treatment, but we did detect an effect of treatment (F(1,40) = 5.428, p = .0249). Although no interaction effect was observed, we did observe a trending difference between control and IMF-treated females such that IMF-treated females had a reduction in the percentage of time spent with the displaced object (p = .0801). No differences among males were detected. Furthermore, percentage of displaced object bouts (Fig. 7D) identified a trend for treatment (F(1,40) = 3.754, p = .0598), yet no effect of sex or interaction between sex and treatment.

3.4. Novel object recognition

3.4.1. 12 months

On the analysis of time spent with the novel object (Fig. 8A), we did not detect an effect of sex or treatment, but we did detect an interaction between sex and treatment (F(1,40) = 5.149, p = .0287). IMF-treated females spent a decreased amount of time with the novel object in contrast to control females (p = .0469); no differences were observed between males. The number of novel object bouts (Fig. 8B) demonstrated no effect of sex or treatment, but a trend for an interaction between sex and treatment (F(1,41) = 3.915, p = .0546). Subsequent examination detected a difference between IMF-treated and control females such that IMF-treated females had a reduction in novel object bouts in contrast to control females (p = .0370). There were no differences between males on this measure. Next, on the percentage of time spent with the novel object (Fig. 8C), we did not demonstrate an effect of sex or treatment, but did demonstrate a trending interaction between sex and treatment (F(1,42) = 3.339, p = .0748). IMF-treated males had an increased percentage of time spent with the novel object in contrast to control males (p = .0346). No effects were demonstrated between females on this evaluation. On the percentage of novel object bouts (Fig. 8D), we did not illustrate an effect of sex or treatment, but we did illustrate a trending interaction effect (F(1,42) = 4.057, p = .0504).

3.4.2. 18 months

On the analysis of time spent with the novel object (Fig. 9A), the number of novel object bouts (Fig. 9B), the percentage of time spent with the novel object (Fig. 9C), and the percentage of novel object bouts (Fig. 9D), we did not detect an effect of sex, treatment, or an interaction effect between sex and treatment.

3.5. Norepinephrine assay

3.5.1. 12 months

Considering the NE content in the dorsal hippocampus (Fig. 10A), we detected an effect of sex (F(1,34) = 33.02, p < .0001) and an interaction between sex and treatment (F(1,34) = 4.394, p = .0436). We also observed a trending effect of treatment (F(1,34) = 3.164, p = .0842). IMF-treated males had an increased amount of NE content in contrast to control males (p = .0083). No differences were observed in females on this measure. In addition, both control and IMF-treated males had a greater amount of NE content than control and IMF-treated females (p = .0149) and (p < .0001), respectively. Next, examining NE content in the prefrontal cortex (Fig. 10B), we did not find an effect of treatment or an interaction between sex and treatment, but we did find an effect of sex (F (1,35) = 64.95, p < .0001). Both control and IMF-treated males had a greater amount of NE content than control and IMF-treated females (p < .0001) and (p < .0001), respectively.

3.5.2. 18 months

On the examination of NE content in the dorsal hippocampus (Fig. 10C), we demonstrated an effect of sex (F(1,29) = 4.431, p = .0441), treatment (F(1,29) = 5.173, p = .0305), and an interaction between sex and treatment (F(1,29) = 6.928, p = .0135). IMF-treated females had a decreased amount of NE content in contrast to control females (p = .0091). No differences were observed in males on this measure. Moreover, control females had an increased amount of NE content in contrast to control males (p = .0042). No difference was detected between IMF-treated males and females. On the analysis of NE content in the prefrontal cortex (Fig. 10D), we did not detect an effect of sex or an interaction between sex and treatment; however, we did observe a trending effect of treatment (F(1,40) = 3.269, p = .0781).

3.6. qPCR

3.6.1. ARC

3.6.1.1. 12 months.

As we hypothesized that the influences of IMF on cognition may originate, in part, from the hypothalamus, we next investigated gene expression in the arcuate nucleus, the lateral hypothalamus, and the locus coeruleus to which the hypothalamus sends projections. In the ARC, we examined the melanocortin genes, norepinephrine receptors, and K⁺ channel subunits in all mice. In the ARC of 12-month-old mice, we found an effect of sex (F(1,20) = 5.912, p = .0246), and a trending effect of treatment (F(1,20) = 3.886, p = .0627), but no interaction between sex and treatment in Agrp expression (Fig. 11A). IMF-treated males had a greater fold change in contrast to control males (p = .0245), although no differences were detected between IMF-treated and control females. In addition, there was a trend for IMF-treated males to have an increased fold change compared to IMF-treated females (p = .0852). No differences were observed in control mice on this assessment. Next, on the examination of Npy (Fig. 11B), we found an interaction effect between sex and treatment (F(1,21) = 5.519, p = .0287) and a trending effect of treatment (F(1,21) = 3.068, p = .0944). IMF-treated males had a greater fold change in contrast to control males (p = .0149); no differences were exhibited between IMF-treated and control females. Moreover, IMF-treated males had an increased fold change compared to IMF-treated females (p = .0156). No differences were observed between control males and females. For Hcrtr1 (Fig. 11C), we observed an effect of sex (F(1,21) = 10.20, p = .0044) with control males exhibiting elevated fold change compared to control females (p = .0135); no differences were detected between IMF-treated males and females. On the assessment of Adr1a (Fig. 11D), we did not find an effect of sex or treatment, but we did observe an interaction between sex and treatment (F(1,21) = 8.912, p = .0071). IMF-treated males had a decreased fold change compared to control males (p = .0294). No differences were detected in females on this measure. Also, control males had an increased fold change in contrast to control females (p = .0130); no differences were exhibited between IMF-treated males and females. On the measure of Adr1b (Fig. 11E), we did not observe an interaction effect between sex and treatment, but we did observe a trend for sex and treatment (F(1,21) = 3.217, p = .0873) and (F(1,21) = 3.288, p = .0841), respectively. For Kcnq2 (Fig. 11F), we demonstrated a trending effect of treatment (F(1,21) = 4.059, p = .0569), but no effects of sex or an interaction between sex and treatment. Next, on the examination of Kcnq3 (Fig. 11G), we exhibited an effect of sex (F(1,20) = 7.737, p = .0115); however, no effect of treatment or interaction between sex and treatment. Control males had a trending greater fold change in contrast to control females (p = .0511); no differences were exhibited between IMF-treated males and females. On the investigation of Kcnq5 (Fig. 11H), we exhibited an effect of sex (F (1,21) = 7.373, p = .0130), but no effect of treatment or interaction between sex and treatment. Control males had a trending greater fold change in contrast to control females (p = .0550); no differences were exhibited between IMF-treated males and females. For both Pomc (Fig. 11I) and Cart (Fig. 11J), we did not find an effect of sex, treatment, or interaction between sex and treatment.

Fig 11. — In the ARC, IMF-treated 12-month-old males had a fold change increase in *Agrp* and *Npy* and a decrease in *Adr1a*. A) IMF-treated males had a greater fold change in *Agrp* in contrast to control males. There was a trend for IMF-treated males to have an increased fold change in *Agrp* in contrast to IMF-treated females. B) IMF-treated males had a greater fold change in *Npy* in contrast to control males. IMF-treated males had an increased fold change in *Npy* in contrast to IMF-treated females. C) For *Hcrtr1*, control males exhibited an elevated fold change in contrast to control females. D) On the assessment of *Adr1a*, IMF-treated males had a decreased fold change compared to control males. Control males had an increased fold change in contrast to control females. E) For *Adr1b*, no pairwise differences were observed. F) On the analysis of *Kcnq2*, no pairwise differences were detected. G) On the examination of *Kcnq3*, control males had a trending greater fold change in contrast to control females. H) On the investigation of *Kcnq5*, control males had a trending greater fold change in contrast to control females I) For *Pomc* no pairwise differences were demonstrated. J) *Cart* analysis resulted in no pairwise differences. Data are represented as mean ± SEM. * = p < .05.

3.6.1.2. 18 months.

In 18-month-old mice, Agrp in the ARC (Fig. 12A) was not altered by sex, treatment, or an interaction between sex and treatment. Next, on the examination of Npy (Fig. 12B), we exhibited an effect of sex (F(1,23) = 20.55, p = .0001), treatment (F(1,23) = 19.71, p = .0002), and a trending interaction effect between sex and treatment (F (1,23) = 3.738, p = .0656). IMF-treated males had a greater fold change in contrast to IMF-treated females (p = .0003). Control males showed a trending greater fold change in contrast to control females (p = .0748). Moreover, IMF-treated males had an increased fold change compared to control males (p = .0002). No differences were observed between control and IMF-treated females. For Hcrtr1 (Fig. 12C), we did not find an effect of sex, treatment or interaction between sex and treatment. On the assessment of Adr1a (Fig. 12D), we found an effect of sex (F(1,25) = 311.07, p < .0001) and treatment (F(1,25) = 4.488, p = .0442), but no interaction between sex and treatment. Control males had an increased fold change in contrast to control females (p < .0001), and IMF-treated males had a greater fold change in contrast to IMF-treated females (p < .0001). In addition, IMF-treated males had a decreased fold change in contrast to their control counterparts (p = .0287). No differences were observed in females on this measure. On the examination of Adr1b (Fig. 12E), we did not observe an effect of sex or treatment, but we did observe an interaction between sex and treatment (F(1,24) = 6.146, p = .0206). Control females trended toward an increased fold change in contrast to control males (p = .0982); no differences were detected in IMF-treated males and females. For Kcnq2 (Fig. 12F), we demonstrated an effect of sex (F(1,26) = 17.45, p = .0003), but no effect of treatment or an interaction between sex and treatment. control males had an increased fold change in contrast to control females (p = .0362), and IMF-treated males had a greater fold change in contrast to IMF-treated females (p = .0020). Next, on the assessment of Kcnq3 (Fig. 12G), we found an effect of sex (F(1,26) = 64.21, p < .0001); however, no effect of treatment or interaction between sex and treatment. Control males had an increased fold change in contrast to control females (p < .0001), as well as IMF-treated males had a greater fold change in contrast to IMF-treated females (p < .0001). On the analysis of Kcnq5 (Fig. 12H), we found an effect of sex (F(1,26) = 33.29, p < .0001), but no effect of treatment or interaction between sex and treatment. Control males had an increased fold change in contrast to control females (p = .0004), and IMF-treated males had a greater fold change in contrast to IMF-treated females (p = .0008). On the analysis of Pomc (Fig. 12I), we exhibited a trending interaction effect between sex and treatment (F(1,26) = 2.916, p = .0996), but no effect of sex or treatment. For Cart (Fig. 12J), we exhibited an effect of sex (F(1,26) = 26.96, p < .0001) and treatment (F(1,26) = 5.432, p = .0278); however, no interaction effect between sex and treatment. Control males had an increased fold change in contrast to control females (p = .0046), as well as IMF-treated males had a greater fold change in contrast to IMF-treated females (p = .0005). Moreover, we found that in IMF-treated females, Cart expression had a trending fold change decrease in contrast to their control counterparts (p = .0805). No differences were illustrated between males on this assessment.

3.6.2. LH

3.6.2.1. 12 months.

In the LH, we examined Hcrt (orexin), a regulator of arousal, the NPY receptors Npy1r and Npy5r, and α-adrenergic receptor 1a and 1b, Adr1a and Adr1b, respectively. In 12-month-old mice, for Hcrt (Fig. 13A), we demonstrated an effect of sex (F(1,22) = 8.126, p = .0093), but no effect of treatment or an interaction between sex and treatment. IMF-treated females had an increased fold change in contrast to IMF-treated males (p = .0177). No differences were detected between control mice. Next, for Npy1r (Fig. 13B), we found an effect of treatment (F(1,22) = 28.01, p < .0001), and an interaction between sex and treatment (F(1,22) = 4.538, p = .0446); however, no effect of sex. Control males had an increased fold change in contrast to control females (p = .0466), no differences were observed between IMF-treated males and females. Moreover, both IMF-treated males and females had a decreased fold change in contrast their control counterparts, (p < .0001) and (p = .0496), respectively. On the examination of Npy5r (Fig. 13C), we did not observe an effect of sex or treatment, but we did find an interaction between sex and treatment (F(1,22) = 5.317, p = .0309). IMF-treated males had a decreased fold change in contrast to control males (p = .0343); no differences were detected between IMF-treated and control females. Next, on the assessment of Adr1a (Fig. 13D), we found an effect of treatment (F(1,22) = 7.270, p = .0132), and an interaction effect between sex and treatment (F(1,22) = 5.695, p = .0260). IMF-treated males had a reduced fold change in contrast to control males (p = .0015); no differences were exhibited between IMF-treated and control females. For Adr1b (Fig. 13E), we illustrated an effect of treatment (F(1,23) = 6.259, p = .0199), and an interaction effect between sex and treatment (F(1,23) = 6.067, p = .0217). Control males had a greater fold change in contrast to control females (p = .0271); no differences were demonstrated between IMF-treated males and females. In addition, IMF-treated males had a decreased fold change in contrast to control males (p = .0014); no differences were demonstrated between control and IMF-treated females.

Fig. 13. — In the LH, 12-month-old IMF-treated males had a decrease fold change in *Npy5r, Adr1a*, and *Adr1b*; both males and females had a reduction in *Npy1r*. A) For *Hcrt*, IMF-treated females had an increased fold change in contrast to IMF-treated males. B) On the examination of *Npy1r*, control males had an increased fold change in contrast to control females. Both IMF-treated males and females had a decreased fold change in contrast their control counterparts. C) For *Npy5r*, IMF-treated males had a decreased fold change in contrast to control males. D) On the assessment of *Adr1a*, IMF-treated males had a reduced fold change in contrast to control males. E) For *Adr1b*, control males had a greater fold change in contrast to control females. IMF-treated males had a decreased fold change in contrast to control males. Data are represented as mean ± SEM. * = p < .05; ** = p < .01; **** = p < .0001.

3.6.2.2. 18 months.

In 18-month-old mice, on the assessment of Hcrt (Fig. 14A), we demonstrated an effect of treatment (F(1,24) = 4.474, p = .0450), and a trending interaction effect between sex and treatment (F (1,24) = 3.428, p = .0764). IMF-treated females had a reduced fold change in contrast to control females (p = .0244). No differences were identified between IMF and control males. For Npy1r (Fig. 14B), we observed an effect of sex (F(1,26) = 9.987, p = .0040), but no effect of treatment or interaction between sex and treatment. Control males had a trending greater fold change in contrast to control females (p = .0140), no differences were observed between IMF-treated males and females. Next, for Npy5r (Fig. 14C), we did not find an effect of treatment or an interaction between sex and treatment, but we did find a trending effect of sex (F(1,24) = 4.244, p = .0504). IMF-treated males had an increased fold change in contrast to IMF-treated females (p = .0340); no differences were detected between control males and females. On the examination of Adr1a (Fig. 14D), we observed an effect of sex (F(1,26) = 22.73, p < .0001); however, no effect of treatment or interaction between sex and treatment. Both control and IMF-treated females had a decreased fold change in contrast to control and IMF-treated males (p = .0027) and (p = .0040), respectively. For Adr1b (Fig. 14E), we observed a trending effect of treatment (F(1,26) = 3.671, p = .0664), but no effect of sex or interaction between sex and treatment.

3.6.3. LC

3.6.3.1. 12 months.

In the LC, we investigated Slc6a2, norepinephrine transporter gene, Dbh, dopamine β-hydroxylase, which catalyzes the conversion of dopamine to norepinephrine, and Hcrtr1, hypocretin receptor 1, which is involved in arousal. In 12-month-old mice, for Slc6a2 (Fig. 15A), we detected an effect of sex (F(1,22) = 17.88, p = .0003), but no effect of treatment or an interaction between sex and treatment. Both control and IMF-treated females had an increased fold change in contrast to control and IMF-treated males (p = .0083) and (p = .0112), respectively. For Dbh (Fig. 15B), we found an effect of sex (F(1,22) = 9.317, p = .0058), but no effects of treatment or an interaction between sex and treatment. Control females had a greater fold change in contrast to control males (p = .0296); no differences were observed between IMF-treated males and females. For the examination of Hcrtr1 (Fig. 15C), we observed a trending effect of sex (F(1,22) = 3.668, p = .0686), but no effect of treatment or an interaction between sex and treatment.

Fig. 15. — In the LC, 12-month-old mice exhibited sex effects for *Slc6a2* and *Dbh*. A) For *Slc6a2*, both control and IMF-treated females had an increased fold change in contrast to control and IMF-treated males B) For the examination of *Dbh*, control females had a greater fold change in contrast to control males. C) For *Hcrtr1*, no pairwise differences were observed.

3.6.3.2. 18 months.

In 18-month-old mice, for Slc6a2 (Fig. 16A), we did not observe an effect of treatment, sex, or an interaction between sex and treatment. For Dbh (Fig. 16B), we detected an effect of sex (F(1,27) = 14.31, p = .0008), but no effect of treatment or an interaction between sex and treatment. Both control and IMF-treated females had a greater fold change in contrast to control and IMF-treated males (p = .0105) and (p = .0294), respectively. For the investigation of Hcrtr1 (Fig. 16C), we found an effect of sex (F(1,27) = 25.95, p < .0001), but no effect of treatment or an interaction between sex and treatment. Both control and IMF-treated females had an increased fold change in contrast to control and IMF-treated males (p = .0007) and (p = .0048), respectively.

4. Discussion

Little is known about the effects of IMF on cognition and anxiety and their associated neurological underpinnings. Here, we addressed the influence of IMF in middle-aged male and female (12 mo) and aged male and female (18 mo) mice using behavior, NE ELISA in the DH and PFC, and gene expression in the ARC, LH, and LC. For behavior, we found that IMF-treated females at 12 and 18 months, showed an improvement in hippocampal-dependent spatial navigation; however, a deficit in hippocampal-dependent spatial object orientation. At 12 months, IMF-treated females exhibited a decrease and IMF-treated males displayed an enhancement in novel object recognition memory. In the OFT, 18-month-old IMF-treated females exhibited anxiolytic-like behavior and increased locomotion. Behavior data has been summarized in Table 1. For the NE ELISA, we found no differences in the PFC; however, in the DH 12-month-old IMF-treated males had an increased amount of NE content and 18-month-old IMF-treated males had a reduction. For gene expression, in the ARC, IMF-treated 12-month-old males had a fold change increase in Agrp and Npy, but a decline in Adr1a. Also in the ARC, IMF-treated 18-month-old males had an increase in Npy and a decrease in Adr1a; females had a trending decrease in Cart. Gene expression in the LH at 12 months revealed IMF-treated males had a decrease in Npy5r, Adr1a, and Adr1b; both males and females had a reduction in Npy1r. In the LH at 18 months, IMF-treated females had a decrease in Hcrt. Gene expression in the LC at both ages largely illustrated sex effects. Gene expression data has been summarized in Table 2.

Table 1.

Summary of IMF treatment differences in behavior between male and female 12-month-old and 18-month-old mice.

Behavior Test	Measure	12-month-old		18-month-old
Behavior Test	Measure	Male	Female	Male	Female
Y-maze	Unknown Arm Time (sec)	n.s.	↑	n.s.	n.s.
	Unknown Arm Entries	n.s.	n.s.	n.s.	↑
	% Time in Unknown Arm	n.s.	n.s.	n.s.	n.s.
	% Entries in Unknown Arm	n.s.	n.s.	n.s.	n.s.
OFT	Time in Center Zone (sec)	n.s.	n.s.	n.s.	↑
	Entries in Center Zone	n.s.	n.s.	n.s.	n.s.
	% Time in Center Zone (sec)	n.s.	n.s.	n.s.	↑
	Entries in Center Zone	n.s.	n.s.	n.s.	n.s.
	Distance (m)	n.s.	n.s.	n.s.	↑
	Mean Speed (m/sec)	n.s.	n.s.	n.s.	↑
SOR	Time with Displaced Object (sec)	n.s.	↓	n.s.	↓
	Number of Displaced Object Bouts	n.s.	n.s.	n.s.	n.s.
	% Time with Displaced Object (sec)	n.s.	n.s.	n.s.	↓ trend
	% Number of Displaced Object Bouts	n.s.	n.s.	n.s.	n.s.
NOR	Time with Novel Object (sec)	n.s.	↓	n.s.	n.s.
	Number of Novel Object Bouts	n.s.	↓	n.s.	n.s.
	% Time with Novel Object (sec)	↑	n.s.	n.s.	n.s.
	% Number of Novel Object Bouts	n.s.	n.s.	n.s.	n.s.

Open in a new tab

NOR, novel object recognition; OFT, open field test; SOR, spatial object recognition. ↑ = increase in behavioral measure in IMF-treated mice in contrast to controls; ↓ = decrease in behavioral measure in IMF-treated mice in contrast to controls; n.s. = not significant.

Table 2.

Summary of fold change IMF treatment differences in gene expression in the ARC, LH, and LC between male and female 12-month-old and 18-month-old mice. .

Region	Gene	12-month-old		18-month-old
Region	Gene	Male	Female	Male	Female
ARC	Agrp	↑	n.s.	n.s.	n.s.
	Npy	↑	n.s.	↑	n.s.
	Hcrtrl	n.s.	n.s.	n.s.	n.s.
	Adrla	↓	n.s.	↓	n.s.
	Adrlb	n.s.	n.s.	n.s.	n.s.
	Kcnq3	n.s.	n.s.	n.s.	n.s.
	Kcnq5	n.s.	n.s.	n.s.	n.s.
	Pomc	n.s.	n.s.	n.s.	n.s.
	Cart	n.s.	n.s.	n.s.	↓ trend
LH	Hcrt	n.s.	n.s.	n.s.	↓
	Npylr	↓	↓	n.s.	n.s.
	Npy5r	↓	n.s.	n.s.	n.s.
	Adrla	↓	n.s.	n.s.	n.s.
	Adrlb	↓	n.s.	n.s.	n.s.
LC	Slc6a2	n.s.	n.s.	n.s.	n.s.
	Dbh	n.s.	n.s.	n.s.	n.s.
	Hcrtrl	n.s.	n.s.	n.s.	n.s.

Open in a new tab

Adr1a, alpha-1A adrenergic receptor; Adr1b, alpha-1B adrenergic receptor; Agrp, agouti-related protein; ARC, arcuate nucleus; Cart, cocaine- and amphetamine-regulated transcript; Dbh, dopamine β-hydroxylase; Hcrt, hypocretin neuropeptide precursor; Hcrtr1, hypocretin receptor 1; Kcnq2, potassium voltage-gated channel subfamily Q member 2; Kcnq3, potassium voltage-gated channel subfamily Q member 3; Kcnq5, potassium voltage-gated channel subfamily Q member 5; LC, locus coeruleus; Npy, neuropeptide Y; Npy1r, neuropeptide Y receptor 1; Npy2r, neuropeptide Y receptor 2; Pomc, proopiomelanocortin; Slc6a2, solute carrier family 6 member 2. ↑ = increased fold change in gene expression IMF-treated mice in contrast to controls; ↓ = decreased fold change in gene expression in IMF-treated mice in contrast to controls; n.s. = not significant.

There is contradictory evidence to suggest that IMF improves cognition in humans and animals. For hippocampal-dependent memory, we found that IMF-treated females at 12 and 18 months, showed an improvement in spatial navigation in the Y-maze, although no differences were observed in males. Similar to our findings, one study has suggested in female C57BL6 mice, every other day IMF for 3 months starting at 8 weeks old improves long-term memory retention using the morris water maze (MWM), a similar test of spatial navigation [57]. In the MWM probe trial, 10 days after the last training trial in the acquisition phase, IMF mice spent up to 30 % more time in the target quadrant zone in contrast to ad libitum controls [57]. Interestingly, these mice were also tested 24 h after the last training trial, but no differences we observed [57]. Our IMF females had an improvement in memory after a short term (5 min) intertrial delay, collectively suggesting IMF may have a bimodal effect at least in female spatial navigation memory.

In support of our lack of findings in males, 6 weeks of every other day IMF in 8-month-old Wistar male rats did not improve radial arm water maze (RAWM) memory acquisition [58]. There were no differences in the number of errors across three short-term to long-term memory timepoints (30 min, 5 h, and 24 h) [58]. However, the authors did find that IMF animals that had the addition of exercise (access to a running wheel) did perform well on the RAWM as evidenced by a lower numbers of errors after 30 min on day 2, 5 h on day 3, and 24 h on day 2 [58]. In opposition to our findings that found no differences in males at the clinical level, Farooq and colleagues [26] used the Cambridge Neuropsychological Test Automated Battery, a computerized assessment tool, in order to oversee the Spatial Span test and the Stockings of Cambridge, a spatial planning assessment, between the hours of 8 am to 1 pm. The authors revealed in teenage boys practicing Ramadan that 4 weeks of IMF resulted in improved spatial cognition on both these assessments [26]. In animal models, 7-week-old CD-1 wild type male mice that underwent 11 months of IMF had improved memory performance in the Barnes maze, a test that also assesses spatial navigation [59]. Moreover, in a study that most closely can be compared to our aged mice, 20-month-old male and female C57BL/6 mice that were subjected to every other day IMF using the Y-maze forced alternation test, there was a spatial memory improvement in males, but not in females [60]. More specifically, IMF-treated males had a greater number of target arm entries in contrast with ad libitum control males. Singh and colleagues utilized aged male Wistar rats (21 mo) and conducted IMF every other day for 3 months resulting in significant improvements in memory on the Morris water maze [61]. These rats showed shorter latencies to find the platform and spent greater amounts of time in the target quadrant when compared to age-matched rats fed an ad libitum diet [61].

For hippocampal-dependent memory for spatial object orientation, in females at both ages, we found deficits in the spatial object recognition test, but did not find any differences in males. Our findings are somewhat consistent at the clinical level for similar spatial object recognition tests. In males and females undergoing Ramadan, a deficit in two spatial memory tests were detected; both for the verbal naming test which requires the participant to identify a three dimensional picture of an object and the visual spatial processing test which requires identification of differences in spatial relations within a scene [62]. This particular test did not conduct separate analysis of gender so those findings do not completely map on to our results. In opposition to our findings, the aforementioned work using 20-month-old male and female C57BL/6 mice that underwent IMF found that IMF enhanced spatial object recognition memory in males, and had a small positive effect in females [60].

Although spatial memory is one of the most widely evaluated cognitive functions in animal models, other forms of memory should be captured to elucidate the full range of alteration that occurs from IMF. For hippocampal-independent memory, we observed at 12 months, IMF-treated females exhibited a decrease and IMF-treated males displayed an enhancement in novel object recognition memory. There is very little prior work that evaluates IMF and recognition memory specifically, although one study found no difference between IMF (daily 18 h fast, 7 days per week, beginning 7 weeks of age) using the NOR test in male Sprague Dawley rats [63]. In the NOR, no differences were observed in the time spent with novel versus familiar objects between IMF and ad libitum controls in either obese or non-obese rats [63]. Although there is limited research using IMF, research using calorie restriction (CR) may provide some insight. One study using male Brown Norway × Fisher344 rats at four age groups (young: 8 mo; middle age: 12–15; old: 25–27 mo; very old: 35–38 mo) investigated a 40 % lifelong CR treatment [64]. The authors found a reduction in NOR memory performance in the CR group at all age groups such that they spent more time exploring both objects in the test trial in contrast to ad libitum controls [64]. This is contradictory with our findings as we observed males having an improvement, while females had an impairment. This can most likely be attributed to treatment and species differences. Another study found that 20-month-old ad libitum Sprague Dawley rats spent less time with the novel object after a 5 min or 24 h intertrial delay relative to 20-month-old CR (20 % CR for 2 weeks; then 40 % until death) rats. This indicates CR improved NOR memory in the old [65].

For the evaluation of anxiety-like behavior in the present study, we found in the OFT, 18-month-old IMF-treated females exhibited anxiolytic-like behavior and increased locomotion. We did not detect any differences in males at either age. Inconsistent with our lack of findings in males, Carteri and collegues [66] found that male C57BL/6 J mice (180 days old) undergoing IMF (10 cycles of 24 h food restriction followed by 24 h ad libitum access) displayed anxiolytic-like behavior in the Light/Dark box as evidenced by increased time in the light zone. In addition, these same mice spent more time in the open arms and less time in the closed arms of the elevated plus maze, suggesting an anxiolytic-like phenotype [66]. In the aforementioned work [60] that utilized 20-month-old male and female C57BL/6 mice that were subjected to every other day IMF, the authors also evaluated anxiety-like behavior using the OFT. Although no differences were observed in OFT center measurements, a difference was observed in rearing, a behavior which may reflect an anxiolytic phenotype [67]. Rearing was maintained in the IMF males, but not in the ad libitum males, although no differences were observed in females; suggesting late-life IMF thwarts aging-induced anxiety-like behavior [67]. Clinically, in males aged 20 to 28 years practicing Ramadan IMF, the Visual Analogue Scale was used to assess global mood, which is a mixture of a depressed and anxiety phenotype [68]. On this measure, compared to the baseline day prior to IMF, global mood significantly decreased in these males, potentially indicating an anxiogenic phenotype [68]. Although another study combining analysis of men and women revealed an anxiolytic phenotype [21]. When pre-Ramadan anxiety scores were compared to post-Ramadan scores, anxiety was found to be lower at the end of Ramadan, when evaluated by the Depression Anxiety Stress Scale [21].

In the present study in the DH, IMF-treated males (12 mo) exhibited a greater amount of NE content and IMF-treated males (18 mo) had a reduction. We did not see any differences in males or females at either age in the PFC. We predicted that IMF would improve memory, in part, by elevating NE signaling in the hippocampus. Although we observed differences in hippocampal NE content in males, in both hippocampal-dependent tests, Y-maze and SOR, we detected no differences in males. In addition, although we saw no differences in HPC NE content for females, we did observe an improved hippocampal-dependent memory for spatial navigation, but a deficit for spatial object orientation. These findings suggest that NE content in the hippocampus did not translate to these particular hippocampal-dependent behavioral tests. Perhaps such tests such as the Barnes maze or Morris water maze could have detected such effects. Even though we did not have a direct translation of behavior to neurotransmitter signaling we did observe that IMF alters NE content through our proposed circuitry. Ghrelin, a gastrointestinal peptide that stimulates feeding [69] exerts its orexigenic response by stimulating NPY neurons [70]. Our results suggest that IMF activates NPY neurons in the ARC, NPY neurons from the ARC send projections to the LH, and these orexigenic neurons in the LH project to the LC. In the LC, IMF-induced NE neurons are excited and send signals toward the HPC, but not robust enough to affect the PFC. Thus, modulation of the ARC → LH → LC circuit may underly the cognitive and anxiolytic effects of IMF.

The ARC contains neurons that are critical to the regulation of energy homeostasis and is a target for several peripheral signals [71,72]. In the ARC, both 12- and 18-month male mice had an increase in Npy expression. This finding is consistent with our previous work such that Npy relative mRNA levels were elevated in response to an every other day IMF protocol in two groups of male mice either fed a low-fat diet or a high-fat diet compared to diet-matched ad libitum controls [17]. Npy, is one of the most abundant peptides seen in mammalian central nervous system [73] and is known to increase appetite/food intake, but also increase energy stores [73]. Acute bouts of food deprivation in adult male mice fasted for 48 h, have been shown to increase hypothalamic Npy mRNA expression by 23 % [74]. Moreover, in male and female rats that underwent food restriction for 2 weeks and food deprivation for 4 days, Brady and colleagues [75] observed an increase in Npy mRNA. Npy and Agrp appear to be coexpressed in the ARC. Using fluorescent in situ hybridization of mRNA, one study found close to all Npy neurons in the ARC coexpressed Agrp mRNA [74]. Agrp mRNA was observed in 94 ± 1.4 % of ARC NPY neurons in males fed an ad libitum diet, and in 99 ± 0.4 % in IMF-treated mice [74]. When we evaluated ARC Agrp expression, we only observed an increase in 12-month-old male mice. If in the ARC, Npy and Agrp are coexpressed, there likely would be parallel regulation of expression among them during IMF [74]. Perhaps this expression is extinguished later in life as we did not detect an increase in Agrp in 18-month-old males. Moreover, these findings were in an acute 48 h fast, this effect may potentially be augmented by the repetitive nature of an IMF protocol. In addition, we did not see this pattern emerge in females which may be consistent with aforementioned work [74], as it did not include female mice in its analysis.

In the hypothalamus, the actions of NE are mediated by adrenergic receptors such as, Adr1a and Adr1b, and are involved in the regulation of cardiometabolic control [76,77]. Nucleus of solitary tract tyrosine hydroxylase-expressing neurons innervate the ARC and activation promotes feeding via NE release onto ARC Adr1a Agrp neurons [78]. In the current study, in the ARC we observed males at both ages exhibit a decrease fold change in Adr1a, suggesting that our IMF protocol facilitated this effect. In females at both ages, we did not observe this effect. Although in the aforementioned work [78], 6–12 week old male and female mice were utilized in a group analysis, so determinations based on sex are unknown.

The LH is a central regulator of food intake and when activated increases food intake [71,79]. The gut hormone ghrelin, an energy regulator, stimulates orexin neurons in the LH [80]. The LH is involved in feeding and is a downstream target of ARC Npy/Pomc neurons [71]. When Npy is overexpressed in the LH, meal sizes, but not meal number is increased [81]. In the present study, in the LH we observed a decrease fold change in G-protein coupled NPY receptor subtypes, Npy1r and Npy5r, in 12-month-old IMF male mice, but not in female mice or in either group at 18 months. There is an increase in calorie intake from central activation of Npy1r and Npy5r [82,83]. One prominent study observed that ghrelin’s control of food reward in the LH is sexually dimorphic [84]. Females have greater growth hormone secretagogue receptor (Ghsr) levels in the LH in contrast to males, and blockade of the Ghsr in the LH significantly reduced food intake [84]. Accordingly, although we detected several differences in males, the only fold change that we detected in the LH of females was a decrease in the anorectic peptide, Cart, at 18-months-old.

Collectively, our outcomes indicate that IMF may alter ARC → LH → LC circuitry. Although we cannot directly establish causation, we propose a means by which IMF alters these neural connections (Fig. 17) and subsequent behavior. We found the greatest number of differences in gene expression, neurotransmitter content, and behavior in male mice at 12-months of age. The increase in ARC Npy gene expression by IMF in 12-month-old male mice is offset by the suppression of Npy1r and Npy5r expression in the LH leading to a disinhibition of the Npy tone which results in an increase in activity of LH neurons. The fold change decrease of Adr1b suggests activation of which produces satiation [85]. Although we did not find gene expression differences in the LC, we did find an increase in DH NE content in the IMF-treated 12-month-old males, which implies activation of this circuit. One caveat; however, is we did not see differences in hippocampal-dependent memory, but we did find differences in hippocampal-independent memory on the NOR test. Because the LC also has noradrenergic connections to the cingulate cortex, a brain region we did not include, and this region is also involved in NOR, it may be possible that differences in NE content in that region would have been altered. We also found differences in gene expression, neurotransmitter content, and behavior in female mice at 18-months of age. Although we only found a trend for a fold change decrease in ARC Cart expression and a significant decrease in LH Hcrt, the combination of the decrease in DH NE content implies an alteration of our proposed circuitry. This decrease in DH NE content translates to differences observed in behavior. In these females we found an improvement in hippocampal-dependent memory on the Y maze, but an impairment in hippocampal-dependent memory in the SOR test. Differences between these two behavior tests implies that IMF effects this type of hippocampal-dependent memory depending on if it is memory of spatial navigation or memory of object orientation.

Fig. 17. — Our results suggest that IMF may alter arcuate nucleus (ARC) neuropeptide Y (NPY) activity and subsequently the reciprocal projections of arcuate NPY neurons (dark gray arrow) and lateral hypothalamus (LH) orexin neurons (light gray arrow). Orexin neurons then project to the locus coeruleus (LC) to activate norepinephrine neurons (black arrow). These NE neurons project to the prefrontal cortex (PFC) and hippocampus (HPC) to control cognition and memory.

5. Conclusion

Our findings suggest that IMF improves and impairs cognition and anxiety-like behvaior depending on age and sex. For hippocampal-dependent memory, in IMF-treated females regardless of age, we observed an improvement in spatial navigation and an impairment in spatial object orientation. For hippocampal-independent memory, we only observed differences at the younger age, such that females had an impairment and males had an improvement in this type of memory. In regard to anxiety, we only observed older females display differences, such that of an anxiolytic phenotype and increased locomotion. In the DH, younger IMF-treated males had a greater amount of NE content and older IMF-treated males had a reduction. Treatment differences in gene expression were observed in the ARC and LH depending on age and sex. In the LC, despite seeing no changes in genes involved in NE synthesis, we did see a suggested increase in LC release that indicates activity of the neurons have changed. Our findings indicate that modulation of the ARC → LH → LC circuit may underly the cognitive and anxiolytic effects of IMF. IMF produces alterations in mood, cognition, DH NE content, and ARC, LH, and LC gene expression depending on sex and age.

Acknowledgments

The authors thank Dr. Kristie Conde, Sarah Paladino, Johanna Trinidad, Kenneth Morales, and Dhristi Raval for their assistance. This work was supported by R21ES027119; R01MH123544; and USDA-NIFA NJ06195 to T.A.R.; and K.W. was funded by T32ES007148 and 1K99ES033256-01A1.

Abbreviations:

Adr1a: alpha-1A adrenergic receptor
Adr1b: alpha-1B adrenergic receptor
Agrp: agouti-related protein
ARC: arcuate nucleus
Cart: cocaine- and amphetamine-regulated transcript
Dbh: dopamine β-hydroxylase
DH: dorsal hippocampus
Hcrt: hypocretin neuropeptide precursor
Hcrtr1: hypocretin receptor 1
HPC: hippocampus
IMF: intermittent fasting
Kcnq2: potassium voltage-gated channel subfamily Q member 2
Kcnq3: potassium voltage-gated channel subfamily Q member 3
Kcnq5: potassium voltage-gated channel subfamily Q member 5
LH: lateral hypothalamus
LC: locus coeruleus
NE: norepinephrine
NOR: novel object recognition
Npy: neuropeptide Y
Npy1r: neuropeptide Y receptor 1
Npy2r: neuropeptide Y receptor 2
OFT: open field test
Pomc: proopiomelanocortin
PFC: prefrontal cortex
qPCR: quantitative real-time polymerase chain reaction
Slc6a2: solute carrier family 6 member 2
SOR: spatial object recognition

Footnotes

CRediT authorship contribution statement

Kimberly Wiersielis: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing, Supervision. Ali Yasrebi: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. Thomas J. Degroat: Data curation, Formal analysis. Nadja Knox: Data curation, Methodology. Catherine Rojas: Data curation, Investigation, Methodology. Samantha Feltri: Data curation, Investigation, Methodology. Troy A. Roepke: Conceptualization, Formal analysis, Funding acquisition, Project administration, Resources, Supervision.

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PERMALINK

Intermittent fasting disrupts hippocampal-dependent memory and norepinephrine content in aged male and female mice

Kimberly Wiersielis

Ali Yasrebi

Thomas J Degroat

Nadja Knox

Catherine Rojas

Samantha Feltri

Troy A Roepke

Abstract

1. Introduction

2. Methods

2.1. Animals and housing conditions

2.2. Behavioral analysis

Fig. 1.

2.2.1. Y-maze

2.2.2. Open field test (OFT)

2.2.3. Spatial object recognition (SOR)

2.2.4. Novel object recognition (NOR)

2.3. Norepinephrine ELISA

2.3.1. Tissue processing

2.3.2. ELISA procedure

2.4. RNA extraction and quantitative real-time PCR

2.5. Statistical analysis

2.5.1. Outliers

2.5.1.1. Behavior.

2.5.1.2. ELISA.

2.5.1.3. qPCR.

3. Results

3.1. Y-Maze

3.1.1. 12 months

Fig. 2.

3.1.2. 18 months

Fig. 3.

3.2. Open field test

3.2.1. 12 months

Fig. 4.

3.2.2. 18 months

Fig. 5.

3.2.3. Comparison of the tested and calculated results with non-penetrating impact effect

Fig. 8.

Fig. 9.

Fig. 10.

3.3. Spatial object recognition

3.3.1. 12 months

Fig. 6.

3.3.2. 18 months

Fig. 7.

3.4. Novel object recognition

3.4.1. 12 months

3.4.2. 18 months

3.5. Norepinephrine assay

3.5.1. 12 months

3.5.2. 18 months

3.6. qPCR

3.6.1. ARC

3.6.1.1. 12 months.

Fig 11.

3.6.1.2. 18 months.

Fig. 12.

3.6.2. LH

3.6.2.1. 12 months.

Fig. 13.

3.6.2.2. 18 months.

Fig. 14.

3.6.3. LC

3.6.3.1. 12 months.

Fig. 15.

3.6.3.2. 18 months.

Fig. 16.

4. Discussion

Table 1.

Table 2.

Fig. 17.

5. Conclusion

Acknowledgments

Abbreviations:

Footnotes

References

ACTIONS