Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: Psychoneuroendocrinology. 2018 Feb 19;90:92–101. doi: 10.1016/j.psyneuen.2018.02.013

Corticotrophin releasing factor receptor 1antagonists prevent chronic stress-induced behavioral changes and synapse loss in aged rats

Hongxin Dong 1, Jack M Keegan 1, Ellie Hong 1, Christopher Gallardo 1, Janitza Montalvo-Ortiz 1, Becky Wang 1, Kenner C Rice 2, John Csernansky 1
PMCID: PMC5864558  NIHMSID: NIHMS945693  PMID: 29477954

Abstract

Mounting evidence suggests that chronic stress can alter brain structure and function and promote the development of neuropsychiatric disorders, such as depression and Alzheimer’s disease. Although the results of several studies have indicated that aged brains are more vulnerable to chronic stress, it remains unknown whether antagonists of a key stress regulator, the corticotrophin releasing factor receptor 1 (CRF1), can prevent stress-induced anxiety and memory deficits in animal models. In this study, we evaluated the potential benefits of two CRF1 antagonists, R121919 and antalarmin, for preventing stress-induced anxiety-related behavioral and memory deficits and neurodegeneration in aged rats. We stressed rats using isolation-restraint for 3 months starting from the 18 months of age. Subsets of animals were administrated either R121919 or antalarmin through food chow for 3 months, followed by a series of behavioral, biochemical and morphological analyses. We found that stressed aged rats displayed body weight losses and increased corticosterone levels, as well as anxiety-related behaviors and memory deficits. Additionally, chronic stress induced a loss of cortical dendritic spines and synapses. However, R121919 and antalarmin both prevented stress-induced behavioral changes including anxiety-related behaviors and memory deficits and prevented synapse loss, perhaps through reversing HPA axis dysfunction. These results suggest that CRF1 antagonists may hold promise as a potential therapy for preventing stress-induced anxiety and memory deficits in aged individuals.

Keywords: Stress, Aging, Corticotrophin releasing factor receptor 1 antagonist, Memory, Neurodegeneration, Rats

1. Introduction

There is widespread evidence that chronic stress can affect both the structure and function of the brain (Oliveira et al., 2016; Prenderville et al., 2015) with important consequences for learning, memory, decision-making and emotional responses (Scott et al., 2015). Moreover, stress has been linked to the development of neuropsychiatric disorders, such as depression and Alzheimer’s disease (AD) (Carroll et al., 2011; Csernansky et al., 2006; Daulatzai, 2014; Dong et al., 2004; Greenberg et al., 2014; Kang et al., 2007). Moreover, the impact of stress on the brain may be dependent on the stage of brain development (Barnum et al., 2012; Kingsbury et al., 2016; Lesse et al., 2016; Lin et al., 2016; Naninck et al., 2015). For instance, neonatal exposure to stress has been observed to affect brain structure and function later in life, and increase the risk of developing age-related neuropsychiatric disorders (Barnum et al., 2012; Kingsbury et al., 2016; Lesse et al., 2016; Sousa et al., 2014). Surprisingly, the behavioral consequences of chronic stress in aged animals has not been studied in similar detail. The aged brain may show decreased resiliency in response to environmental stressors (Prenderville et al., 2015). Moreover, aging may be seen as a stressor itself, since the effects of aging can mimic the effects of chronic stress in regard to hyperactivation of the hypothalamic–pituitary–adrenal (HPA) axis and increases in basal glucocorticoid release, impaired HPA negative feedback and reduced central glucocorticoid receptor expression (Gaffey et al., 2016; Holmes et al., 2010).

Corticotrophin releasing factor (CRF) and its receptor 1 (CRF1) are critical components of a signaling cascade that regulates the HPA axis in response to stressors that vary depending on changes in the internal and external environment (Bao et al., 2008; Johnson et al., 1992). In addition to its central role in regulation of the HPA axis, CRF/CRF1 signaling pathway has a second, interrelated function in the brain (Birnbaum et al., 2004; Blank et al., 2002; Grammatopoulos et al., 2001; Hains et al., 2009). CRF1 is distributed extensively throughout the cortex, hippocampus and amygdala, where it modulates neural activity related to stress-sensitive cognitive processes, such as learning and memory (De Souza and Battaglia, 1988; Gallagher et al., 2008; Orozco-Cabal et al., 2006).

Some stressors, such as physical illness and psychosocial loss, may be unavoidable during aging. Therefore, decreasing the effects of such stressors through the manipulation of stress-related signaling pathways may help to prevent or reduce stress-related disorders. To date, there has been relatively little study on the effects of chronic stress on CRF1 activation in aged animals and age-related declines in brain structure and function. In this study, we evaluated the effects of isolation-restraint stress in aged rats by measuring body weight and plasma corticosterone changes, as well as dendritic spine and synapse density and anxiety-related behaviors and memory deficits. Then, we examined the ability of CRF1 antagonists to block the effects of stress-related changes in these biological and behavioral measures.

2. Material and Methods

2.1. Animals

A total of 48 rats (Sprague Dawley, 24 males and 24 females) at 18 months (n=8 per group) of age from the Charles River Laboratories (Bar Harbor, Maine) were used for this study. Animals were housed on a 12-hour light/dark cycle and given food and water ad libitum. All procedures were performed according to NIH guidelines for the treatment of animals and the Current Guide for the Care and Use of Laboratory Animals (2011, 8th edition) under a protocol approved by the Northwestern University Animal Care and Use Committee.

2.2. Isolation-restraint stress

Isolation-restraint stress was performed by individually housing rat at 18 months of age in a cage one-third smaller (27 × 14 × 11 cm3) than standard-sized rat cages (43.2 × 34.0 × 19.8 cm 3), in which individual rats (body weight >500g) could still freely move and reach food and water. The isolation-restraint rat cages were placed in a separate area (a satellite facility next to the main laboratory) from other animals to prevent visual contact. Control animals were group-housed with three animals per standard cage for the same period of time. The body weight and food consumption of each rat was measured weekly.

2.3. CRF1 Antagonist administration

CRF1 antagonists, R121919 and antalarmin (20 mg/kg, from Dr. Rice’s laboratory at NIDA and NIAAA), were administrated by mixing them in food chow (LabDiet, St Louis, MO 64144) to stressed and non-stressed rats for 3 months; i.e., from 18 to 21 months of age. We selected R121919 and antalarmin because they were commonly used for preclinical studies (Kehne and Cain, 2010). Further, R121919 has been used in patients for clinical trials up to phase II (Saunders et al. 2001; Zorrilla and Koob, 2010). Both drugs pass through the blood brain barrier (BBB) reasonably well (Marinelli et al., 2007; Sabino et al., 2013). We selected a daily doses of 20 mg/kg based on our previous work (Dong et al., 2014) and dosages used in previous animal studies from other laboratories (Marinelli et al., 2007; Sabino et al., 2013). Using measure of average food intake/day (FI, 129.75 ± 10.32g)/7), average body weight at 18 months of age (BW, 606.82 ± 69.16g), dosage = mg of compound per kg of body weight (CK, 20mg/kg), we were able to calculate the concentration of CRF1 antagonist to be mixed in the food chow based on the formula ((CK × BW)/FI)/10,000 = concentration of the drug in the diet). In our study, our concentration in the food chow was 0.06%.

Food consumption was measured weekly. Given that the drug concentration in food chow was calculated using average food consumption, and each rat’s daily consumption of CRF1 antagonists was different, we calculated daily drug administration for each individual rat using the formula (((weekly food chow consumption/7) X 0.06%)/body weight).

To determine whether the administration of CRF1 antagonists through food chow was comparable to our previous study (Dong et al 2014), the concentration of R121919 and antalarmin in the plasma was measured by liquid-liquid extraction with methyl tert-butyl ether (MTBE), using a previously published method (Dong et al, 2014). Plasma concentrations of R121919 were 32.4±5.34ng/ml and antalarmin were 37.825±6.56 ng/ml, which are comparable to plasma concentrations achieved by delivering the drugs through drinking water and 12 hours following IP injection (Dong et al., 2014).

After 3 months of CRF antagonist administration, tests of anxiety- and memory-related behaviors were performed. During behavioral testing, animals were still exposed to isolation-restraint stress and/or CRF1 administration in their food chow. After behaviors testing was complete, blood from each animal was collected, and brain tissues including the cortex and hippocampus were prepared for measurements of dendritic spine and synapse density.

2.4. Corticosterone levels

Blood was collected by rapid retro-orbital phlebotomy in the early morning (7:00am) after CRF1 antagonist treatment and after behavioral testing was finished. Plasma corticosterone levels were measured using an EIA kit (Cayman Chemical Company, Ann Arbor, MI) according to the instruction manual. The final corticosterone concentration was calculated in nanograms per milliliter (ng/mL) according to the manufacturer’s instructions.

2.5. Open field

General locomotion and anxiety-related behavior were measured using the Open Field Test. An automated tracking system (Any-Maze, Stoelting Co. Wood Dale, IL 60191) tracked the locomotion of each subject in an open box (72 cm × 72cm × 36 cm) for 5 minutes. Anxiety-related behaviors were determined by comparing the number of times the animals went to the center zone, the amount of time the animals spent in the center zone, and the amount of time the animals stayed in the periphery zone (wall-hugging) of the open field.

2.6. Novel object recognition (NOR)

NOR test is commonly used to assess memory-related behavior in rodents. The test was modified from previously published methods (Horiguchi et al., 2012; Horiguchi et al., 2011). The apparatus for this test consists of an evenly illuminated Plexiglas box (72 cm × 72 cm × 36 cm). The procedure included three phases: habituation, acquisition and retention. During habituation (Day 1–3), each individual mouse was allowed to freely explore the box absent of objects for 10 minutes. During the acquisition trial, two identical objects were placed in the box and positioned 6 cm away from the walls in opposite corners. The rats were allowed to explore the identical objects for 10 min and then returned to their home cages. During the retention trial, one of the two familiar objects was replaced with a novel object. Following a one-hour gap, rats were returned to the box and allowed to explore for 10 minutes. All objects used in this study were different in shape and color but identical in size. To minimize olfactory cues, the box and objects were cleaned with 70% ethanol after each trial. Object exploration was defined as sniffing, licking or touching the objects using the forepaw, but not leaning against, turning around, standing, or sitting on the objects. Behavioral data were video-recorded for further analysis by an experimenter blind to the treatments. The exploration time (in seconds) was recorded manually using 2 stopwatches. Total exploration time of both objects in the acquisition and retention trials and total exploratory activity was calculated. Discrimination index (DI) was used as the memory-related measure, and was calculated as the proportion of the total time spent exploring the novel versus the familiar object during the retention trial.

2.7. Morris water maze (MWM)

We used a modified MWM (delayed matching to place) to test working memory-related behavior (Lindner et al., 1992). Working memory is commonly assessed as a cognitive index of aging (Hertzog et al., 2003) and of neuropsychiatric disorders (Abi-Dargham et al., 2002; Bird et al., 2010). The Morris water maze test was performed in a water tank with a moveable platform equipped with a video camera and computerized data analysis software (Any Maze) for 5 days. The water temperature was maintained at 23°C. Prior to testing, rats were acclimated to the Morris water maze testing room for 30 minutes. Rats then underwent acquisition and test trials as described below. Upon completion of these trials, rats were removed from the tank and put into a drying cage.

To assess working memory, acquisition trials were conducted on Days 1–2, with two sessions in the AM and 2 sessions in the PM for a total of 4 sessions/day. For each of these sessions, rats were given a maximum of 60 seconds to find a hidden platform with no inter-trial interval (ITI) between the two AM trials or PM trials. On Days 3–5, rats underwent test trials identical to the acquisition trial with the exception of a one minute ITI. Trial 1 was the acquisition trial while trials 2–4 were the test trials. The swimming distance and time during trial 2–4 was used for memory measurement. On each day, the location of the platform and the animal’s start position remained constant for all trials; between days, the location of the hidden platform was changed.

2.8. Dendritic spine density (Golgi staining)

After behavioral testing was complete, rats were deeply anesthetized using an overdose of pentobarbital sodium solution (150mg/kg) and perfused transcardially with 0.1 M phosphate buffer. Then, the brain was removed, and one hemisphere of each brain was collected and subjected to Golgi staining (FD Rapid GolgiStain Kit; FD Neurotechnologies) according to the manufacturer’s instructions. Another hemisphere was prepared for electron microscopic study. For Golgi study, the brain tissues were immersed in a Golgi-Cox solution. The mixture of solutions was replaced once after 12 hours of initial immersion, with storage at room temperature in darkness for 2–3 weeks. Then, the brains were transferred to a cryoprotectant solution and stored at 4°C for at least 48 hours in the dark. The brains were rapidly frozen with dry ice and cut in the coronal plane at approximately 150 μm thickness on a cryostat. The sections were transferred onto gelatin-coated slides. Cut sections were air dried at room temperature in the dark. After drying, sections were rinsed with distilled water, stained in a developing solution and dehydrated, cleared and cover-slipped. Pyramidal neurons from layer V of the frontal cortex (up to the dorsal hippocampal area, from Bregma -2.12 to -4.30 mm) and pyramidal neurons of the CA1 in the dorsal hippocampus were selected for dendritic analysis. Twenty neurons (10 from the cortex and 10 from the dorsal hippocampus) from each animal were selected by stereological methods. Four neurons (2 from the cortex and 2 from the hippocampus) were randomly selected from 5 sections, out of a possible 12–15 sections, that included the frontal cortex and dorsal hippocampal areas for dendritic branch and spine density measurement. Then, the dendritic projections were tracked and the spines that were located in the outer band of the Ballarger in Layers IV and striatum orient-alveus of CA1 in the dorsal hippocampus were imaged. Spines were identified and characterized based on previous description (Hering and Sheng, 2001). Spine density was expressed as the number of spines per 10 μm of dendrite length.

2.9. Synaptic density (quantitative electron microcopy analysis)

A block of the tissue containing frontal cortex and dorsal hippocampus in another half hemisphere was trimmed and fixed with 4% paraformaldehyde, 2.5% glutaraldehyde, 1% sucrose in 0.1M sodium cacodylate buffer for 30 minutes. The tissue was then sectioned at 250 mm thick in the coronal plane using a vibratome. Twelve sections encompassing the frontal cortex were selected, trimmed, and rinsed 3 times with 0.1M sodium cacodylate, post-fixed in 2% osmium tetroxide in 0.1M sodium cacodylate buffer, pH 7.3 for 1 hour. The sections were rinsed with distilled water 3 times, stained with 3% uranyl acetate for 1.5 hours, and rinsed in dH2O and dehydrated in ascending grades (50%-100%) of ethanol. The sample was transitioned from ethanol to infiltration with 3 changes of propylene oxide and embedded in resin mixture of Embed-812 and Araldite. Semi-thin (1 mm) sections were cut (Leica UCT ultramicrotome) and stained with toluidine blue and served as reference sections for ultra-thin section. The sections were then trimmed and ultra-cut into thin (75 nm) sections containing the frontal cortex, followed by mounting on 400-mesh grids (every mesh grid is 62x62 mm2, Electron Microscopy Sciences, Hatfield, PA). The sections were stained using 3% uranyl acetate for 10 min followed by lead citrate for 10 min and then examined under FEI Tecnai Spirit G2 transmission electron microscopy with 3D tomography capability (FEI company, Creek Drive, Hillsboro, OR). For the synaptic density analysis, 8–15 photographs from each electron section were taken systematically in the Inner band and Outer band in both Layers IV and V at 4800x magnification using alternate grid squares. Two sections from the frontal cortex area in each animal were assessed. A total of 30 photographs/frontal cortex/per animal were taken for analysis. Synapses were identified on photographs by the presence of synaptic vesicles and postsynaptic densities. The area of the unbiased counting frame is 81 μm2 for each photograph, the dissector height is 0.075 μm, and the dissector volume is 6.075μm3. The latter value was used to calculate the density of synapses (Dong et al., 2007; Dong et al., 2008).

2.10. Statistical analysis

We used one-way and two-way analyses of variance (ANOVA) to assess the effects of stress (i.e., isolation-restraint vs. group housing) and drug (i.e., antalarmin or R121919 vs. controls), on measures of corticosterone levels, behavioral measures, and spine and synaptic density measures. Repeated measures ANOVA was used to assess the effects of these same factors on body weight (Prism statistic program, SPSS). When significant effects of stress, age or drug, as well as significant interactions among these factors were found, post-hoc (Bonferroni/Dunn tests) testing was employed. Data represent mean values ± S.E.M.

3. Results

3.1. CRF1 antagonist administration by food chow

We found that the average dose of CRF1 antagonists administered by food chow was between 17.24±1.61 mg/kg and 19.80±0.59mg/kg, which was slightly lower than the dosage we expected of 20mg/kg but still comparable (p>0.05). Additionally, there was no significant difference in CRF1 antagonist intake between the stress and control groups of animals (Figure 1A).

Figure 1. CRF1 antagonist intake and body weight in aged rats.

Figure 1

Open in a new tab

A: CRF1 antagonist intake by food chow was arranged between 17.24±1.61 mg/kg and 19.80±0.59mg/kg and there was no difference between groups. B: The body weight of aged rats measured before and after 3 months of isolation-restraint stress. Stressed rats showed significant loss of body weight in all stressed groups with or without CRF1 antagonist administration as compared to non-stressed rats (**p < 0.01; ***p< 0.001). C: Stressed aged rats decreased food chow intake in the third month of isolation-restraint stress (*p < 0.05). D: There was no difference in the food intake between vehicle and CRF1 antagonist administration in the stressed groups.

3.2. Food intake and Body weight

After 3 months of isolation-restraint stress, stressed aged rats showed significant body weight loss [-80.571 ± 27.56g, from 614.14 ± 82.58g to 533.86 ± 64.76g] as compared to non-stressed aged rats [50.00 ± 9.032g, from 599.5 ± 55.75g to 648.75 ± 47.11g] (Figure 1B). CRF1 antagonists did not prevent body weight loss induced by chronic stress (Figure 1B).

To investigate whether body weight loss was due to differences in food consumption caused by stress or CRF antagonists mixed in the food chow, we measured the amount of food chow consumed weekly by individual rats. Calculations of total and mean volume of food chow consumed were determined for the stressed and/or drug conditions, and adjusted by body weight. Chronic isolation-restraint stress led to a decrease in food intake by the third month in aged rats as compared to control aged rats (Figure 1C, p<0.05). However, the mean amount of total food consumption between groups with or without CRF1 antagonist treatment did not differ (Figure 1C and D).

3.3. Plasma corticosterone

Two-way ANOVA indicated significant effects of stress (F1,40 = 28.49, p<0.0001) and drug (F2,40 = 3.98, p<0.05), but no stress by drug interaction on plasma corticosterone levels in aged rats. Post-hoc analysis indicated that chronic isolation-restraint stress significantly increased corticosterone levels as compared to non-stressed aged rats (Figure 2A, p<0.01). CRF1 antagonist, antalarmin but not R121919 showed a significant decrease in corticosterone levels (p<0.05) in stressed aged rats.

Figure 2. Preventative effects of CRF1 antagonists on plasma corticosterone levels and anxiety-related behavior in aged rats.

Figure 2

Open in a new tab

A: Corticosterone levels between control and experimental groups in aged rats after 3 months of isolation-restraint stress. Stressed rats showed significantly higher corticosterone levels as compared to non-stressed rats (**p < 0.01 non-stressed vs. stressed control groups). Both R121919 and antlarmin showed a decrease in corticosterone levels but antlarmin reached significant levels (*p< 0.05). B: Tracking plots in an open field showed the locomotion of non-stressed (upper panels) and stressed (lower panels) rats with or without CRF1 antagonist administration. C: Isolation-restraint stressed rats showed significantly increased time in the peripheral zone as compared to non-stressed rats (***p < 0.001, as compared to non-stressed controls; * p< 0.05, as compared to non-stressed R121919 and antalarmin groups). R121919 and antalarmin decreased time in the peripheral zone as compared to controls in stressed rats. (*p < 0.05). D: Isolation-restraint stress rats showed significantly decreased time in the center zone as compared to non-stressed rats (#p < 0.05, as compared to non-stressed controls). Both R121919 and antalarmin increased time spent in the center zone as compared controls in stressed rats. (**p < 0.01).

3.4. Anxiety-related behaviors

Two-way ANOVA analysis found a significant effect of stress (F2, 39 = 19.38, p<0.001), but not drug (F2, 39 = 2.63, p>0.05), and a significant stress by drug interaction (F2, 39 = 13.91, p<0.01) in time spent in the peripheral zone (Figure 2B and C). Two-way ANOVA analysis found a significant effect of stress (F1, 37 = 11.23, p<0.01), but not drug or a stress by drug interaction on the entries of animals into the center area. Also, significant effects of stress (F2, 37 = 10.27, p<0.01) and drug (F2, 37 = 4.69, p<0.05), but no stress by drug interaction were found in the time spent in the center zone (Figure 2B, and D). Post-hoc analysis revealed that stressed rats spent significantly more time in the peripheral zone (Figure C, p<0.001) and decreased the amount of time spent in the center zone (p<0.05), as compared to control rats. However, both R211919 and antalarmin decreased the time in the peripheral zone (p<0.05) and increased the time spent in the center zone (p<0.01) in stressed rats (Figure 2C and D). These results suggest that stressed aged rats displayed an increase in anxiety-related behaviors as compared to non-stressed rats, and that CRF1 antagonists mitigated these increases in stress-related behaviors.

3.5 Novel objective recognition (NOR)

During the acquisition trial of the NOR test, there were no effects of stress on the exploration time of left and right objects between stressed and control rats, nor did the administration of CRF1 antagonists affect the exploration time of either left or right objects. However, during the retention trial, isolation-restraint stress significantly decreased the novel object exploration time (Figure 3A), and CRF1 antagonists increased the time to spent exploring the novel object (Figure 3A, stressed group). The discrimination index (DI) indicated a significant effects of stress (F1,40 = 18.36, p<0.001) and drug (F1,40 = 6.722, p<0.05), but not stress by drug interaction. Post-hoc analysis indicated that CRF1 antagonists, especially R121919, significantly increased the time spent exploring the novel object over the familiar object in stressed rats (Figure 3B).

Figure 3. Preventative effects of CRF1 antagonists on memory-related behaviors in aged rats.

Figure 3

Open in a new tab

A and B: Effects of CRF1 antagonists in aged rats on the novel object recognition test. A: Time spent in exploration of familiar and novel objects was measured during the retention trial (*p < 0.05, as compared with the time spent on familiar objects within group). B: Discrimination index (DI) assessed during the retention trial (normalization of individual performances for group comparison, *p < 0.05 as compared to non-stressed controls; ##p < 0.01 as compared to stressed R121919 and Antalarmin groups). C: Effects of CRF1 antagonists in aged rats on Morris water maze test. Latency to platform was measured for each trial and indicated that CRF1 antagonists decreased the time to find the platform in stressed but not non-stressed aged rats (**p < 0.01 and ***p < 0.001 between stressed R121919 and stressed controls of corresponding trials; ##p < 0.01 and ###p < 0.001 between stressed antalarmin and stressed controls of corresponding trials).

3.6 Morris Water Maze (MWM)

In the MWM, significant effects of stress (F7,168 =18.76, p<0.001) and drug (F2,168 =15.62), as well as a stress by drug interaction (F14,168 = 2.067, p<0.05) were found on the latency to find the platform. Subsequent post-hoc analyses showed that stressed rats required a longer time to find the target platform compared to non-stressed rats (p<0.01), and that both R121919 and antalarmin treatment significantly mitigated this impairment (Figure 3C, p<0.001 in both groups). R121919 and antalarmin administration did not influence MWM performance in non-stressed aged rats (Figure 3C).

3.7 Spine density

We measured the spine density of dendrites on the pyramidal cells at layer V of the frontal cortex and in CA1 area of the hippocampus using Golgi staining sections and compared them in the stressed and non-stressed groups with and without drug treatment. The representative images from stressed and non-stressed rats are presented in the Figure 4 (Figure 4A, B and C).

Figure 4. Evaluation of the dendritic spines in aged rats after chronic stress and CRF1 antagonist administration.

Figure 4

Open in a new tab

A and B: Golgi staining showed similar gross visual morphology of neuron and dendritic branching in non-stressed and chronic isolation-restraint stressed rats (Scale bar = 10 μm inB also applied to A). C: Higher magnification of dendritic spines in non-stressed and stressed groups with and without CRF1 antagonist administration. D: Quantitative analysis of spine density in the cortex indicated that stressed rats have the lowest number of spine density as compared to non-stressed rats (*p < 0.05 as compared to non-stressed controls). CRF antagonists restored spine density in stressed rats (#p < 0.05, ##p < 0.01 as compared to stressed controls). E: Stressed rats showed a trend of decreased spine density in the hippocampus as compared to non-stressed rats but did not reach significance. There was also no difference between the CRF antagonists and control groups in stressed rats. Spine density is expressed as number of spines per 10 μm dendrite length.

Two-way ANOVA indicated an effect of drug (F2, 202 = 8.78, p<0.001), but no effect of stress nor a drug by stress interaction (F2,202 =7.43, p<0.001) on spine density in the frontal cortex. Stressed rats displayed significant decreases in spine densities compared to control rats (Figure 4D, p<0.01). CRF1 antagonists restored decreases in spine density observed after isolation-restraint stress (Figure 4D). Two-way ANOVA indicated a significant effect of stress (F1,178=8.56, p< 0.01), but no effect of drug nor a drug by stress interaction on spine density in the hippocampus. Post-hoc analysis indicated no significant differences between groups (Figure 4E).

3.8 Synaptic density

We conducted a quantitative electron microscopy analysis focusing on the cortex (Figure 5A–D). At higher magnification, synapses were identified and counted (Figure 5E). One-way ANOVA indicated a group effect (F 3,142 = 6.07. p<0.001) on synaptic density in the frontal cortex. Post-hoc analysis indicated that there was a significant decrease in synaptic density in stressed rats as compared to control rats (Figure 5F, (p<0.01),), and that both CRF1 antagonists (R121919 and antalarmin) were able to mitigate synaptic loss in the cortex of stressed rats (Figure 5F, p<0.05).

Figure 5. Evaluation of synaptic density in aged rats after chronic stress and CRF1 antagonist administration.

Figure 5

Open in a new tab

A, B, C and D: Electron microscope photographs taken in the pyramidal layer of the cortex in non-stressed (A) and stressed rats (B), stressed rats with R121919 (C) and stressed rats with antalarmin (D). The white arrows in the images indicate synapses that have been counted. E: Higher magnification of the image in panel A (red box) to display the synapses that we counted. F: Quantification data indicates that stressed rats have decreased synaptic density as compared to non-stressed rats (*p < 0.05), and CRF1 antagonists restored synaptic density in the stressed aged rats (#p < 0.05 as compared to stressed control). Scale bar = 1 μm in D, also applied to A, B and C).

4. Discussion

In this study, we investigated the effects of chronic isolation-restraint stress on anxiety- and memory-related behaviors in aged rats, and sought to determine whether CRF1 antagonists could mitigate any observed effects of such stress. We found that stressed rats displayed increases anxiety-related behaviors and memory deficits as compared to control rats. Stressed rats also had higher corticosterone levels, and decreased densities of dendritic spines and synapses as compared to non-stressed rats. Moreover, administration of two CRF1 antagonists, R121919 and antalarmin blocked the effects of isolation-restraint stress on anxiety-related behaviors and memory deficits, and also reversed the effects of stress on spine and synapse densities. While our results confirmed our main hypotheses, we also found that CRF antagonists tended to increase the time in the open field and improve performance in the NOR test in control animals that were not subjected to isolation-restraint stress. These results suggest that CRF1 antagonists may have beneficial effects on aged rats in general, and is consistent with the idea that aging may be associated with changes in CRF1 activation even in the absence of a defined stressor. HPA axis dysfunction has been reported with advancing age (Gaffey et al., 2016; Holmes et al., 2010), and changes in CRF1 signaling may contribute to aging-related anxiety and memory deficits in non-stressed aged rats.

Aging is a process that is too often accompanied by a decline in physical and mental function (Finch and Shams, 2016; Geokas et al., 1990; Yin, 2016). Moreover, the aging brain may be particularly sensitive to common stressors, such as physical illness and psychosocial losses (Hidalgo et al., 2015; Prenderville et al., 2015; Scott et al., 2015). In the elderly, chronic stress due to physical illness or psychosocial loss can significantly accelerate age-related declines in cognition and increase sensitivity to psychiatric illnesses, such as anxiety and depression (Boldy and Grenade, 2011; Buechel et al., 2014; Cruces et al., 2014; Glass et al., 2006; Steptoe et al., 2013) (Cacioppo and Hawkley, 2009; Lupien et al., 2009; Prenderville et al., 2015). Several studies have reported increases in anxiety-related behaviors in aged rats (Shoji and Mizoguchi, 2010). The precise mechanisms underlying the behavioral effects of aging remain unclear, although changes in the HPA axis are among the most accepted explanations (Revesz et al., 2014). Our results demonstrated higher corticosterone levels in stressed, aged rats as compared to non-stressed rats, and so provide further support for the hypothesis that HPA activity is irregular in aged animals (Merrett et al., 2010). In fact, aging and chronic stress may have parallel effects on cognition and emotion (Prenderville et al., 2015). More specifically, with reduced HPA axis plasticity, aged animals may be less capable of adapting to unpredictable stressors (Khlebnikov et al., 2015). Others have observed that stress-induced reductions in the densities of neuronal dendrites in the prefrontal cortex can be reversed in young, but not in aged, animals (Bloss et al., 2010), which is also consistent with the general idea that the aged brain is less able to recover from the deleterious effects of stress.

Unfortunately, there is no known strategy for blocking the effects of stress of the aging brain. In this study, we assessed the therapeutic potential of CRF1 antagonists in stress and non-stressed aged rats, and found evidence that both R121919 and antalarmin could block stress induced changes in anxiety-related behaviors and memory deficits. R121919 appeared to display more robust benefits on behaviors, but not markers of spine and synapse densities, in both stressed and non-stressed rats. Overall, these results suggest that manipulating CRF/CRF1 signaling may help to mitigate stress-related effects on behavior and neural architecture in age animals.

CRF1 antagonists have been used in clinical trials of mood disorders for many years, but with disappointing results (Nielsen, 2006; Zorrilla and Koob, 2004). The inconsistency of results of CRF1 antagonist administration between preclinical studies and clinical trials may due to many factors, including: 1) the doses used (Kehne and Cain, 2010), which may have failed to achieve necessary levels of CRF1 receptor occupancy in clinical trials, 2) differences in the symptoms experienced by patients and the behaviors assessed in rodents, and 3) differences in the involvement of different CRF1 receptor populations and/or different functional states of the CRF1 in patients versus animal subjects (Kehne and Cain, 2010). Additionally, CRF1 antagonists may have achieved a poor record of success as anti-depressants because of problems with bioavailability (Binneman et al., 2008; Cruces et al., 2014; Nielsen, 2006; Zorrilla and Koob, 2004, 2010). Notably, R121919 showed significant efficacy in the treatment of MDD in a small proof-of-concept, open-label, clinical trial. Unfortunately, further development of this drug was halted because of observed side-effects on liver function (Saunders et al. 2001). Therefore, new CRF1 antagonists need to being developed for clinical testing that have improved bioavailability and better safety profiles (Zorrilla and Koob, 2010).

The therapeutic potential of CRF1 antagonists as treatments for neurodegenerative disorders, such as Alzheimer’s disease, has been evaluated by both our group and others and there is growing evidence that CRF1 antagonists can also block stress-induced increases in amyloid-β (Aβ) accumulation (Dong et al., 2014; Kang et al., 2007; Lee et al., 2009). Additionally, CRF1 antagonists may have beneficial effects on tau phosphorylation (Carroll et al., 2011; Rissman et al., 2012). Recent work (Bangasser et al., 2016; Zhang et al., 2015) also suggests that CRF/CRF1 signaling may regulate AD neuropathology and synaptic function in a sex-biased manner. While our current study focused on the effects of stress and CRF1 antagonists in both males and females, we were able to preliminarily investigate sex difference in response to chronic stress and CRF1 antagonist administration. Although the numbers of subjects were very small (n=4), we found a trend toward female rats displaying higher corticosterone levels as compared to male rats, but no differences in stress-related changes in behaviors. In future studies of the effects of stress and CRF1 antagonists in aged rats, the numbers of animals should be increased so that sex differences can be carefully studied.

Isolation-restraint stress induced weight loss in aged rats. The mechanisms underlying the age-dependent effects of stress on body weight are not known, but may be related to stress-induced changes in the effects of the HPA axis on feeding behavior. In support of this hypothesis, we found that average food consumption decreased during the 3rd month of stress in both stressed and non-stressed aged rats. However, while CRF1 antagonists decreased corticosterone levels, corticosterone levels were still higher in the stressed groups as compared to the non-stressed groups, and CRF1 antagonists were unable to block stress-induced changes in body weight. In one study that compared the body weight of young and aged mice after two weeks of stress, the investigators also found that aged mice, but not young mice, lost weight, and associated these effects with age-related changes in ghrelin-related factors (Yamada et al., 2015).

In summary, our results demonstrate that aging is associated with an increased sensitivity to chronic isolation-restraint stress, as demonstrated by changes in anxiety- and memory-related behaviors, along with increases in HPA axis activity and synapse loss. CRF1 antagonists were able to decrease the anxiety-related behaviors and improve memory-related performance in aged rats under both stressed and non-stressed conditions. On the basis of an analysis of synaptic measures, our results also suggest that CRF1 antagonists may exert their beneficial effects on behavior by preserving synapses. These studies suggest the possibility that CRF1 antagonists, by virtue of their ability to mitigate the effects of biological impact of stress on aging, should be considered as prevention and treatments to preserve or brain function in older individuals when they are exposed to unavoidable environmental stressors.

Highlights.

  • Aged rats display severe behavior deficits and synaptic loss after chronic stress.

  • CRF1 antagonists prevent anxiety-related behaviors and memory deficits in aged rats.

  • CRF1 antagonists restore HPA axis function and prevent synapse loss in stressed aged rats.

Acknowledgments

This work was supported by the Alzheimer’s Drug Discovery Foundation (grant 20111208, JGC) and NIH (1R56AG053491-01, HXD). A portion of this research was supported by the Intramural Research Programs of the National Institute on Drug Abuse and the National Institute of Alcohol Abuse and Alcoholism, NIH, US Department of Health and Human Services (KCR).

Footnotes

Disclosure statement

Drs. Dong and Csernansky have received research grants from the NIMH, NIA, and Dr. John G. Csernansky has served as a Data Safety and Monitoring Board (DSMB) member for Eli Lilly and Sanofi-Aventis, and has received funding for his research from Genentech. The rest of the authors declare that they have no competing financial interests.

Verification:
  1. Drs. Csernansky and Dong have received research grants from the NIMH, NIA, and Dr. John G. Csernansky has served as a Data Safety and Monitoring Board (DSMB) member for Eli Lilly and Sanofi-Aventis, and has received funding for his research from Genentech. The rest of the authors declare that they have no competing financial interests.
  2. This work was supported by the Alzheimer’s Drug Discovery Foundation (grant 20111208, JGC) and NIH (1R56AG053491-01, HXD). A portion of this research was supported by the Intramural Research Programs of the National Institute on Drug Abuse and the National Institute of Alcohol Abuse and Alcoholism, NIH, US Department of Health and Human Services (KCR).
  3. The data contained in the manuscript have not been previously published and have not been submitted elsewhere. These data also will not been submitted elsewhere while under consideration at Neurobiology of Aging.
  4. All authors have reviewed the contents of the manuscript being submitted, approve of its contents and validate the accuracy of the data.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Abi-Dargham A, Mawlawi O, Lombardo I, Gil R, Martinez D, Huang Y, Hwang DR, Keilp J, Kochan L, Van Heertum R, Gorman JM, Laruelle M. Prefrontal dopamine D1 receptors and working memory in schizophrenia. J Neurosci. 2002;22:3708–3719. doi: 10.1523/JNEUROSCI.22-09-03708.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bangasser DA, Dong H, Carroll J, Plona Z, Ding H, Rodriguez L, McKennan C, Csernansky JG, Seeholzer SH, Valentino RJ. Corticotropin-releasing factor overexpression gives rise to sex differences in Alzheimer’s disease-related signaling. Mol Psychiatry. 2016:185. doi: 10.1038/mp.2016.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bao AM, Meynen G, Swaab DF. The stress system in depression and neurodegeneration: focus on the human hypothalamus. Brain Res Rev. 2008;57:531–553. doi: 10.1016/j.brainresrev.2007.04.005. [DOI] [PubMed] [Google Scholar]
  4. Barnum CJ, Pace TW, Hu F, Neigh GN, Tansey MG. Psychological stress in adolescent and adult mice increases neuroinflammation and attenuates the response to LPS challenge. J Neuroinflammation. 2012;9:9. doi: 10.1186/1742-2094-9-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Binneman B, Feltner D, Kolluri S, Shi Y, Qiu R, Stiger T. A 6-week randomized, placebo-controlled trial of CP-316,311 (a selective CRH1 antagonist) in the treatment of major depression. Am J Psychiatry. 2008;165:617–620. doi: 10.1176/appi.ajp.2008.07071199. [DOI] [PubMed] [Google Scholar]
  6. Bird CM, Chan D, Hartley T, Pijnenburg YA, Rossor MN, Burgess N. Topographical short-term memory differentiates Alzheimer’s disease from frontotemporal lobar degeneration. Hippocampus. 2010;20:1154–1169. doi: 10.1002/hipo.20715. [DOI] [PubMed] [Google Scholar]
  7. Birnbaum SG, Yuan PX, Wang M, Vijayraghavan S, Bloom AK, Davis DJ, Gobeske KT, Sweatt JD, Manji HK, Arnsten AF. Protein kinase C overactivity impairs prefrontal cortical regulation of working memory. Science. 2004;306:882–884. doi: 10.1126/science.1100021. [DOI] [PubMed] [Google Scholar]
  8. Blank T, Nijholt I, Eckart K, Spiess J. Priming of long-term potentiation in mouse hippocampus by corticotropin-releasing factor and acute stress: implications for hippocampus-dependent learning. J Neurosci. 2002;22:3788–3794. doi: 10.1523/JNEUROSCI.22-09-03788.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bloss EB, Janssen WG, McEwen BS, Morrison JH. Interactive effects of stress and aging on structural plasticity in the prefrontal cortex. J Neurosci. 2010;30:6726–6731. doi: 10.1523/JNEUROSCI.0759-10.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Boldy D, Grenade L. Loneliness and social isolation among older people: the views of community organisations and groups. Aust N Z J Public Health. 2011;35:583. doi: 10.1111/j.1753-6405.2011.00795.x. [DOI] [PubMed] [Google Scholar]
  11. Buechel HM, Popovic J, Staggs K, Anderson KL, Thibault O, Blalock EM. Aged rats are hypo-responsive to acute restraint: implications for psychosocial stress in aging. Front Aging Neurosci. 2014;6:13. doi: 10.3389/fnagi.2014.00013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cacioppo JT, Hawkley LC. Perceived social isolation and cognition. Trends Cogn Sci. 2009;13:447–454. doi: 10.1016/j.tics.2009.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Carroll JC, Iba M, Bangasser DA, Valentino RJ, James MJ, Brunden KR, Lee VM, Trojanowski JQ. Chronic Stress Exacerbates Tau Pathology, Neurodegeneration, and Cognitive Performance through a Corticotropin-Releasing Factor Receptor-Dependent Mechanism in a Transgenic Mouse Model of Tauopathy. J Neurosci. 2011;31:14436–14449. doi: 10.1523/JNEUROSCI.3836-11.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cruces J, Venero C, Pereda-Perez I, De la Fuente M. A higher anxiety state in old rats after social isolation is associated to an impairment of the immune response. J Neuroimmunol. 2014;277:18–25. doi: 10.1016/j.jneuroim.2014.09.011. [DOI] [PubMed] [Google Scholar]
  15. Csernansky JG, Dong H, Fagan AM, Wang L, Xiong C, Holtzman DM, Morris JC. Plasma cortisol and progression of dementia in subjects with Alzheimer-type dementia. Am J Psychiatry. 2006;163:2164–2169. doi: 10.1176/appi.ajp.163.12.2164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Daulatzai MA. Role of stress, depression, and aging in cognitive decline and Alzheimer’s disease. Curr Top Behav Neurosci. 2014;18:265–296. doi: 10.1007/7854_2014_350. [DOI] [PubMed] [Google Scholar]
  17. De Souza EB, Battaglia G. Corticotropin-releasing hormone (CRH) receptors in brain. Adv Exp Med Biol. 1988;245:123–136. doi: 10.1007/978-1-4899-2064-5_9. [DOI] [PubMed] [Google Scholar]
  18. Dong H, Goico B, Martin M, Csernansky CA, Bertchume A, Csernansky JG. Modulation of hippocampal cell proliferation, memory, and amyloid plaque deposition in APPsw (Tg2576) mutant mice by isolation stress. Neuroscience. 2004;127:601–609. doi: 10.1016/j.neuroscience.2004.05.040. [DOI] [PubMed] [Google Scholar]
  19. Dong H, Martin MV, Chambers S, Csernansky JG. Spatial relationship between synapse loss and beta-amyloid deposition in Tg2576 mice. J Comp Neurol. 2007;500:311–321. doi: 10.1002/cne.21176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Dong H, Wang S, Zeng Z, Li F, Montalvo-Ortiz J, Tucker C, Akhtar S, Shi J, Meltzer HY, Rice KC, Csernansky JG. Effects of corticotrophin-releasing factor receptor 1 antagonists on amyloid-beta and behavior in Tg2576 mice. Psychopharmacology (Berl) 2014;231:4711–4722. doi: 10.1007/s00213-014-3629-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Dong H, Yuede CM, Coughlan C, Lewis B, Csernansky JG. Effects of memantine on neuronal structure and conditioned fear in the Tg2576 mouse model of Alzheimer’s disease. Neuropsychopharmacology. 2008;33:3226–3236. doi: 10.1038/npp.2008.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Finch CE, Shams S. Apolipoprotein E and Sex Bias in Cerebrovascular Aging of Men and Mice. Trends Neurosci. 2016;39:625–637. doi: 10.1016/j.tins.2016.07.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gaffey AE, Bergeman CS, Clark LA, Wirth MM. Aging and the HPA axis: Stress and resilience in older adults. Neurosci Biobehav Rev. 2016;68:928–945. doi: 10.1016/j.neubiorev.2016.05.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gallagher JP, Orozco-Cabal LF, Liu J, Shinnick-Gallagher P. Synaptic physiology of central CRH system. Eur J Pharmacol. 2008;583:215–225. doi: 10.1016/j.ejphar.2007.11.075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Geokas MC, Lakatta EG, Makinodan T, Timiras PS. The aging process. Ann Intern Med. 1990;113:455–466. doi: 10.7326/0003-4819-113-6-455. [DOI] [PubMed] [Google Scholar]
  26. Glass TA, De Leon CF, Bassuk SS, Berkman LF. Social engagement and depressive symptoms in late life: longitudinal findings. J Aging Health. 2006;18:604–628. doi: 10.1177/0898264306291017. [DOI] [PubMed] [Google Scholar]
  27. Grammatopoulos DK, Randeva HS, Levine MA, Kanellopoulou KA, Hillhouse EW. Rat cerebral cortex corticotropin-releasing hormone receptors: evidence for receptor coupling to multiple G-proteins. J Neurochem. 2001;76:509–519. doi: 10.1046/j.1471-4159.2001.00067.x. [DOI] [PubMed] [Google Scholar]
  28. Greenberg MS, Tanev K, Marin MF, Pitman RK. Stress, PTSD, and dementia. Alzheimers Dement. 2014;10:S155–165. doi: 10.1016/j.jalz.2014.04.008. [DOI] [PubMed] [Google Scholar]
  29. Hains AB, Vu MA, Maciejewski PK, van Dyck CH, Gottron M, Arnsten AF. Inhibition of protein kinase C signaling protects prefrontal cortex dendritic spines and cognition from the effects of chronic stress. Proc Natl Acad Sci U S A. 2009;106:17957–17962. doi: 10.1073/pnas.0908563106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hering H, Sheng M. Dendritic spines: structure, dynamics and regulation. Nat Rev Neurosci. 2001;2:880–888. doi: 10.1038/35104061. [DOI] [PubMed] [Google Scholar]
  31. Hertzog C, Dixon RA, Hultsch DF, MacDonald SW. Latent change models of adult cognition: are changes in processing speed and working memory associated with changes in episodic memory? Psychol Aging. 2003;18:755–769. doi: 10.1037/0882-7974.18.4.755. [DOI] [PubMed] [Google Scholar]
  32. Hidalgo V, Pulopulos MM, Puig-Perez S, Espin L, Gomez-Amor J, Salvador A. Acute stress affects free recall and recognition of pictures differently depending on age and sex. Behav Brain Res. 2015;292:393–402. doi: 10.1016/j.bbr.2015.07.011. [DOI] [PubMed] [Google Scholar]
  33. Holmes MC, Carter RN, Noble J, Chitnis S, Dutia A, Paterson JM, Mullins JJ, Seckl JR, Yau JL. 11beta-hydroxysteroid dehydrogenase type 1 expression is increased in the aged mouse hippocampus and parietal cortex and causes memory impairments. J Neurosci. 2010;30:6916–6920. doi: 10.1523/JNEUROSCI.0731-10.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Horiguchi M, Hannaway KE, Adelekun AE, Jayathilake K, Meltzer HY. Prevention of the phencyclidine-induced impairment in novel object recognition in female rats by co-administration of lurasidone or tandospirone, a 5-HT(1A) partial agonist. Neuropsychopharmacology. 2012;37:2175–2183. doi: 10.1038/npp.2012.64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Horiguchi M, Huang M, Meltzer HY. The role of 5-hydroxytryptamine 7 receptors in the phencyclidine-induced novel object recognition deficit in rats. The Journal of pharmacology and experimental therapeutics. 2011;338:605–614. doi: 10.1124/jpet.111.180638. [DOI] [PubMed] [Google Scholar]
  36. Johnson EO, Kamilaris TC, Chrousos GP, Gold PW. Mechanisms of stress: a dynamic overview of hormonal and behavioral homeostasis. Neurosci Biobehav Rev. 1992;16:115–130. doi: 10.1016/s0149-7634(05)80175-7. [DOI] [PubMed] [Google Scholar]
  37. Kang JE, Cirrito JR, Dong H, Csernansky JG, Holtzman DM. Acute stress increases interstitial fluid amyloid-beta via corticotropin-releasing factor and neuronal activity. Proc Natl Acad Sci U S A. 2007;104:10673–10678. doi: 10.1073/pnas.0700148104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kehne JH, Cain CK. Therapeutic utility of non-peptidic CRF1 receptor antagonists in anxiety, depression, and stress-related disorders: evidence from animal models. Pharmacol Ther. 2010;128:460–487. doi: 10.1016/j.pharmthera.2010.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Khlebnikov VV, Kuznetsov SL, Chernov DA, Agrytskov AM, Ahmad A, Nor-Ashikin MN, Ullah M, Kapitonova MY. Age-related peculiarities of the hypothalamo-hypophyseo-adrenal system in chronic heterotypic stress. Morfologiia. 2015;147:15–20. [PubMed] [Google Scholar]
  40. Kingsbury M, Weeks M, MacKinnon N, Evans J, Mahedy L, Dykxhoorn J, Colman I. Stressful Life Events During Pregnancy and Offspring Depression: Evidence From a Prospective Cohort Study. J Am Acad Child Adolesc Psychiatry. 2016;55:709–716. e702. doi: 10.1016/j.jaac.2016.05.014. [DOI] [PubMed] [Google Scholar]
  41. Lee KW, Kim JB, Seo JS, Kim TK, Im JY, Baek IS, Kim KS, Lee JK, Han PL. Behavioral stress accelerates plaque pathogenesis in the brain of Tg2576 mice via generation of metabolic oxidative stress. J Neurochem. 2009;108:165–175. doi: 10.1111/j.1471-4159.2008.05769.x. [DOI] [PubMed] [Google Scholar]
  42. Lesse A, Rether K, Groger N, Braun K, Bock J. Chronic Postnatal Stress Induces Depressive-like Behavior in Male Mice and Programs second-Hit Stress-Induced Gene Expression Patterns of OxtR and AvpR1a in Adulthood. Mol Neurobiol. 2016:15. doi: 10.1007/s12035-016-0043-8. [DOI] [PubMed] [Google Scholar]
  43. Lin LY, Zhang J, Dai XM, Xiao NA, Wu XL, Wei Z, Fang WT, Zhu YG, Chen XC. Early-life stress leads to impaired spatial learning and memory in middle-aged ApoE4-TR mice. Mol Neurodegener. 2016;11:51. doi: 10.1186/s13024-016-0107-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Lindner MD, Balch AH, VanderMaelen CP. Short forms of the “reference-“ and “working-memory” Morris water maze for assessing age-related deficits. Behav Neural Biol. 1992;58:94–102. doi: 10.1016/0163-1047(92)90303-l. [DOI] [PubMed] [Google Scholar]
  45. Lupien SJ, McEwen BS, Gunnar MR, Heim C. Effects of stress throughout the lifespan on the brain, behaviour and cognition. Nat Rev Neurosci. 2009;10:434–445. doi: 10.1038/nrn2639. [DOI] [PubMed] [Google Scholar]
  46. Marcuzzo S, Dall’oglio A, Ribeiro MF, Achaval M, Rasia-Filho AA. Dendritic spines in the posterodorsal medial amygdala after restraint stress and ageing in rats. Neurosci Lett. 2007;424:16–21. doi: 10.1016/j.neulet.2007.07.019. [DOI] [PubMed] [Google Scholar]
  47. Marinelli PW, Funk D, Juzytsch W, Harding S, Rice KC, Shaham Y, Le AD. The CRF1 receptor antagonist antalarmin attenuates yohimbine-induced increases in operant alcohol self-administration and reinstatement of alcohol seeking in rats. Psychopharmacology (Berl) 2007;195:345–355. doi: 10.1007/s00213-007-0905-x. [DOI] [PubMed] [Google Scholar]
  48. Merrett DL, Kirkland SW, Metz GA. Synergistic effects of age and stress in a rodent model of stroke. Behav Brain Res. 2010;214:55–59. doi: 10.1016/j.bbr.2010.04.035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Naninck EF, Hoeijmakers L, Kakava-Georgiadou N, Meesters A, Lazic SE, Lucassen PJ, Korosi A. Chronic early life stress alters developmental and adult neurogenesis and impairs cognitive function in mice. Hippocampus. 2015;25:309–328. doi: 10.1002/hipo.22374. [DOI] [PubMed] [Google Scholar]
  50. Nielsen DM. Corticotropin-releasing factor type-1 receptor antagonists: the next class of antidepressants? Life Sci. 2006;78:909–919. doi: 10.1016/j.lfs.2005.06.003. [DOI] [PubMed] [Google Scholar]
  51. Oliveira TG, Chan RB, Bravo FV, Miranda A, Silva RR, Zhou B, Marques F, Pinto V, Cerqueira JJ, Di Paolo G, Sousa N. The impact of chronic stress on the rat brain lipidome. Mol Psychiatry. 2016;21:80–88. doi: 10.1038/mp.2015.14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Orozco-Cabal L, Pollandt S, Liu J, Shinnick-Gallagher P, Gallagher JP. Regulation of synaptic transmission by CRF receptors. Rev Neurosci. 2006;17:279–307. doi: 10.1515/revneuro.2006.17.3.279. [DOI] [PubMed] [Google Scholar]
  53. Prenderville JA, Kennedy PJ, Dinan TG, Cryan JF. Adding fuel to the fire: the impact of stress on the ageing brain. Trends Neurosci. 2015;38:13–25. doi: 10.1016/j.tins.2014.11.001. [DOI] [PubMed] [Google Scholar]
  54. Revesz D, Verhoeven JE, Milaneschi Y, de Geus EJ, Wolkowitz OM, Penninx BW. Dysregulated physiological stress systems and accelerated cellular aging. Neurobiol Aging. 2014;35:1422–1430. doi: 10.1016/j.neurobiolaging.2013.12.027. [DOI] [PubMed] [Google Scholar]
  55. Rissman RA, Staup MA, Lee AR, Justice NJ, Rice KC, Vale W, Sawchenko PE. Corticotropin-releasing factor receptor-dependent effects of repeated stress on tau phosphorylation, solubility, and aggregation. Proc Natl Acad Sci U S A. 2012;109:6277–6282. doi: 10.1073/pnas.1203140109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sabino V, Kwak J, Rice KC, Cottone P. Pharmacological characterization of the 20% alcohol intermittent access model in Sardinian alcohol-preferring rats: a model of binge-like drinking. Alcohol Clin Exp Res. 2013;37:635–643. doi: 10.1111/acer.12008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Saunders J, Williams JP. New developments in the study of corticotropin releasing factor. Annu Rep Med Chem. 2001;36:21–30. [Google Scholar]
  58. Scott SB, Graham-Engeland JE, Engeland CG, Smyth JM, Almeida DM, Katz MJ, Lipton RB, Mogle JA, Munoz E, Ram N, Sliwinski MJ. The Effects of Stress on Cognitive Aging, Physiology and Emotion (ESCAPE) Project. BMC Psychiatry. 2015;15:146. doi: 10.1186/s12888-015-0497-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Shoji H, Mizoguchi K. Acute and repeated stress differentially regulates behavioral, endocrine, neural parameters relevant to emotional and stress response in young and aged rats. Behav Brain Res. 2010;211:169–177. doi: 10.1016/j.bbr.2010.03.025. [DOI] [PubMed] [Google Scholar]
  60. Sousa VC, Vital J, Costenla AR, Batalha VL, Sebastiao AM, Ribeiro JA, Lopes LV. Maternal separation impairs long term-potentiation in CA1-CA3 synapses and hippocampal-dependent memory in old rats. Neurobiol Aging. 2014;35:1680–1685. doi: 10.1016/j.neurobiolaging.2014.01.024. [DOI] [PubMed] [Google Scholar]
  61. Steptoe A, Shankar A, Demakakos P, Wardle J. Social isolation, loneliness, and all-cause mortality in older men and women. Proc Natl Acad Sci U S A. 2013;110:5797–5801. doi: 10.1073/pnas.1219686110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Vyas S, Rodrigues AJ, Silva JM, Tronche F, Almeida OF, Sousa N, Sotiropoulos I. Chronic Stress and Glucocorticoids: From Neuronal Plasticity to Neurodegeneration. Neural Plast. 2016;2016:6391686. doi: 10.1155/2016/6391686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Yamada C, Saegusa Y, Nahata M, Sadakane C, Hattori T, Takeda H. Influence of Aging and Gender Differences on Feeding Behavior and Ghrelin-Related Factors during Social Isolation in Mice. PLoS One. 2015;10:e0140094. doi: 10.1371/journal.pone.0140094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yin D. The Essential Mechanisms of Aging: What Have We Learnt in Ten Years? Curr Top Med Chem. 2016;16:503–510. doi: 10.2174/1568026615666150813142854. [DOI] [PubMed] [Google Scholar]
  65. Zhang C, Kuo CC, Moghadam SH, Monte L, Campbell SN, Rice KC, Sawchenko PE, Masliah E, Rissman RA. Corticotropin-releasing factor receptor-1 antagonism mitigates beta amyloid pathology and cognitive and synaptic deficits in a mouse model of Alzheimer’s disease. Alzheimers Dement. 2015:007. doi: 10.1016/j.jalz.2015.09.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zorrilla EP, Koob GF. The therapeutic potential of CRF1 antagonists for anxiety. Expert Opin Investig Drugs. 2004;13:799–828. doi: 10.1517/13543784.13.7.799. [DOI] [PubMed] [Google Scholar]
  67. Zorrilla EP, Koob GF. Progress in corticotropin-releasing factor-1 antagonist development. Drug Discov Today. 2010;15:371–383. doi: 10.1016/j.drudis.2010.02.011. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES