Disturbed sleep is associated with reduced verbal episodic memory and entorhinal cortex volume in younger middle-aged women with risk-reducing early ovarian removal.

Introduction: Women with early ovarian removal (<48 years) have an elevated risk for both late-life Alzheimer's disease (AD) and insomnia, a modifiable risk factor. In early midlife, they also show reduced verbal episodic memory and hippocampal volume. Whether these reductions correlate with a sleep phenotype consistent with insomnia risk remains unexplored. Methods: We recruited thirty-one younger middleaged women with risk-reducing early bilateral salpingo-oophorectomy (BSO), fifteen of whom were taking estradiol-based hormone replacement therapy (BSO+ERT) and sixteen who were not (BSO). Fourteen age-matched premenopausal (AMC) and seventeen spontaneously peri-postmenopausal (SM) women who were ~10y older and not taking ERT were also enrolled. Overnight polysomnography recordings were collected at participants' home across multiple nights (M=2.38 SEM=0.19), along with subjective sleep quality and hot flash ratings. In addition to group comparisons on sleep measures, associations with verbal episodic memory and medial temporal lobe volume were assessed. Results: Increased sleep latency and decreased sleep efficiency were observed on polysomnography recordings of those not taking ERT, consistent with insomnia symptoms. This phenotype was also observed in the older women in SM, implicating ovarian hormone loss. Further, sleep latency was associated with more forgetting on the paragraph recall task, previously shown to be altered in women with early BSO. Both increased sleep latency and reduced sleep efficiency were associated with smaller anterolateral entorhinal cortex volume. Discussion: Together, these findings confirm an association between ovarian hormone loss and insomnia symptoms, and importantly, identify an younger onset age in women with early ovarian removal, which may contribute to poorer cognitive and brain outcomes in these women.

Women's Brain Health: Midlife Ovarian Removal Affects Associative Memory.

Women with early bilateral salpingo-oophorectomy (BSO; removal of ovaries and fallopian tubes) have greater Alzheimer's disease (AD) risk than women in spontaneous/natural menopause (SM), but early biomarkers of this risk are not well-characterized. Considering associative memory deficits may presage preclinical AD, we wondered if one of the earliest changes might be in associative memory and whether younger women with BSO had changes similar to those observed in SM. Women with BSO (with and without 17ß-estradiol replacement therapy (ERT)), their age-matched premenopausal controls (AMC), and older women in SM completed a functional magnetic resonance imaging face-name associative memory task shown to predict early AD. Brain activation during encoding was compared between groups: AMC (n=25), BSO no ERT (BSO; n=15), BSO+ERT (n=16), and SM without hormone therapy (n=16). Region-of-interest analyses revealed AMC did not contribute to functional group differences. BSO+ERT had higher hippocampal activation than BSO and SM. This hippocampal activation correlated positively with urinary metabolite levels of 17ß-estradiol. Multivariate partial least squares analyses showed BSO+ERT had a different network-level activation pattern than BSO and SM. Thus, despite being approximately 10 years younger, women with BSO without ERT had similar brain function to those with SM, suggesting early 17ß-estradiol loss may lead to an altered functional brain phenotype which could influence late-life AD risk, making face-name encoding a potential biomarker for midlife women with increased AD risk. Despite similarities in activation, BSO and SM groups showed opposite within-hippocampus connectivity, suggesting menopause type is an important consideration when assessing brain function.

Scene memory and hippocampal volume in middle-aged women with early hormone loss.

The present study explored whether early midlife bilateral salpingo-oophorectomy (BSO), a female-specific risk factor for dementia, is associated with reduced medial temporal lobe structure and function. Younger middle-aged women with the BRCA1/2 mutation and a BSO prior to spontaneous menopause (SM) were recruited. We determined the performance of women with BSO not taking estradiol-based hormone therapy (n = 18) on a task measuring object and scene recognition and quantified medial temporal lobe subregion volumes using manually segmented high-resolution T2-weighted MRI scans. Comparisons were made to those with BSO taking estradiol-based hormone therapy (n = 20), age-matched premenopausal controls (n = 28), and older women in SM not taking hormone therapy matched for duration of hormone deprivation (n = 17). Reduced hippocampal integrity specific to the BSO group not taking hormone therapy was observed, reflected by significantly smaller dentate gyrus/CA2/CA3 volumes and lower scene recognition memory performance. These findings show that hippocampal subfield volume may be useful for identifying early midlife changes in women at elevated risk for dementia.

A new angle on mental rotation ability in transgender men: Modulation by ovarian milieu.

Organizational/activational theory posits that transgender individuals should perform in the direction of their gender, not their sex, on cognitive tasks that show sex differences-the largest of which are observed on visuospatial tasks. Yet, tests of this hypothesis have been mixed for transgender men (TM). One possible reason is that performance shifts associated with the hormonal milieu at testing have not been fully considered in TM. Although "activating" influences, like gender-affirming hormone therapy (GAHT), are well-characterized in this population, endogenous ones, like ovarian cycling, have gone unaddressed. To provide a more complete picture of hormonal activation, we explored an influence of ovarian milieu on visuospatial performance of TM, and its potential contributions toward effects of sex and GAHT. We administered two male-favoring mental rotation tests (MRTs), and a sex-neutral control task to 22 TM naïve to GAHT (TM-), 29 TM receiving GAHT (TM+), and cisgender men (CM; n = 24) and women (CW; n = 43), testing cycling men (TM-) and women (CW) in either early follicular phase (Follicular) or midluteal phase (Luteal). On MRTs, performance of TM- varied across the menstrual cycle, and matched that of menstrual phase-matched CW. Additionally, cycling individuals in Follicular performed as strongly as TM+ and CM, all of whom performed above individuals in Luteal. Effects did not extend to a verbal control task, on which TM+ performed below others. Rather than conforming to static categories that suggest sex- or gender-typical organization of cognitive circuits, our findings support dynamic shifts in visuospatial ability of TM, and illustrate the need to consider activating effects of hormones beyond GAHT.

A review of the current state of knowledge on sex differences in sleep and circadian phenotypes in rodents.

Sleep is a vital part of our lives as it is required to maintain health and optimal cognition. In humans, sex differences are relatively well-established for many sleep phenotypes. However, precise differences in sleep phenotypes between male and female rodents are less documented. The main goal of this article is to review sex differences in sleep architecture and electroencephalographic (EEG) activity during wakefulness and sleep in rodents. The effects of acute sleep deprivation on sleep duration and EEG activity in male and female rodents will also be covered, in addition to sex differences in specific circadian phenotypes. When possible, the contribution of the female estrous cycle to the observed differences between males and females will be described. In general, male rodents spend more time in non-rapid eye movement sleep (NREMS) in comparison to females, while other differences between sexes in sleep phenotypes are species- and estrous cycle phase-dependent. Altogether, the review illustrates the need for a sex-based perspective in basic sleep and circadian research, including the consideration of sex chromosomes and gonadal hormones in sleep and circadian phenotypes.

Role of Ovarian Hormones in the Modulation of Sleep in Females Across the Adult Lifespan.

Ovarian hormones, including 17ß-estradiol, are implicated in numerous physiological processes, including sleep. Beginning at puberty, girls report more sleep complaints than boys, which is maintained throughout the reproductive life stage. Sleep problems are exacerbated during the menopausal transition, evidenced by greater risk for sleep disorders. There is emerging evidence that menopause-associated hormone loss contributes to this elevated risk, but age is also an important factor. The extent to which menopause-associated sleep disturbance persists into postmenopause above and beyond the effects of age remains unknown. Untreated sleep disturbances have important implications for cognitive health, as they are emerging as risk factors for dementia. Given that sleep loss impairs memory, an important knowledge gap concerns the role played by menopause-associated hormone loss in exacerbating sleep disturbance and, ultimately, cognitive function in aging women. In this review, we take a translational approach to illustrate the contribution of ovarian hormones in maintaining the sleep-wake cycle in younger and middle-aged females, with evidence implicating 17ß-estradiol in supporting the memory-promoting effects of sleep. Sleep physiology is briefly reviewed before turning to behavioral and neural evidence from young females linking 17ß-estradiol to sleep-wake cycle maintenance. Implications of menopause-associated 17ß-estradiol loss is also reviewed before discussing how ovarian hormones may support the memory-promoting effects of sleep, and why menopause may exacerbate pathological aging via effects on sleep. While still in its infancy, this research area offers a new sex-based perspective on aging research, with a focus on a modifiable risk factor for pathological aging.

Cognitive markers of dementia risk in middle-aged women with bilateral salpingo-oophorectomy prior to menopause.

Oophorectomy prior to menopause is associated with late-life dementia. Memory decline may start within 6 months after oophorectomy in middle-aged women, suggested by lower verbal and working memory performance. Unknown is whether such changes persist beyond 6 months, and whether they are reversed by estradiol. Short-term benefits of estradiol on verbal memory following oophorectomy were observed in one study, but longer term effects remain unknown. In the present study, middle-aged BRCA1/2 mutation carriers with early oophorectomy at least 1 year prior to study onset were tested on verbal and working memory with results stratified by (1) current estradiol use (n = 22) or (2) no history of estradiol use (n = 24), and compared to age-matched premenopausal controls (n = 25). Both memory abilities were adversely affected by oophorectomy, but only working memory was maintained by estradiol. Estrogen metabolite levels correlated with working memory, suggesting a role for estradiol in preserving this ability. Memory decline appears to persist after early oophorectomy, particularly for women who do not take estradiol.

Sex difference or hormonal difference in mental rotation? The influence of ovarian milieu.

Sex differences in visuospatial cognition have long been reported, with men being advantaged on the Mental Rotations Test (MRT). The data, however, are variable, and sensitive to design parameters. When men and women are compared directly, with women in different hormonal milieus combined, there seem to be sex differences. When women alone are studied, taking into account different ovarian steroid concentrations and treatments, MRT performance varies with these changes. Indeed, several reports describe better performance among women with reduced estrogens. To better understand whether the sex difference in MRT persists once hormonal status is considered, we recruited reproductive age adults designated male and female at birth (MAB, FAB), and administered the Vandenberg-Kuse (V/K) MRT-comparing performance among MAB (n = 169) and FAB (n = 219). For FAB combined, we found a sex difference with MAB performing better than FAB. However, when FAB were analyzed by current menstrual cycle phase (Early Follicular (EF), Periovulatory (PO), Midluteal (ML)) or by hormone therapy (transmasculine testosterone administration (TM+), oral contraceptive (OC) ingestion prior to (OC+) or after cognitive testing (OC-)), low-estradiol groups (EF, OC-, TM+) performed as strongly as MAB, and had better MRT than cycling FAB in high-estradiol menstrual cycle phases (PO, ML). On a verbal memory control task, neither a sex difference nor a low estrogen advantage was detected, although performance varied with hormonal milieu. Our findings support a dynamic model of spatial performance and suggest that both MAB and FAB perform strongly on MRT, contingent on hormonal status.

Adverse Effects of Aromatase Inhibition on the Brain and Behavior in a Nonhuman Primate.

Breast cancer patients using aromatase inhibitors (AIs) as an adjuvant therapy often report side effects, including hot flashes, mood changes, and cognitive impairment. Despite long-term use in humans, little is known about the effects of continuous AI administration on the brain and cognition. We used a primate model of human cognitive aging, the common marmoset, to examine the effects of a 4-week daily administration of the AI letrozole (20 µg, p.o.) on cognition, anxiety, thermoregulation, brain estrogen content, and hippocampal pyramidal cell physiology. Letrozole treatment was administered to both male and female marmosets and reduced peripheral levels of estradiol (E2), but unexpectedly increased E2 levels in the hippocampus. Spatial working memory and intrinsic excitability of hippocampal neurons were negatively affected by the treatment possibly due to increased hippocampal E2. While no changes in hypothalamic E2 were observed, thermoregulation was disrupted by letrozole in females only, indicating some impact on hypothalamic activity. These findings suggest adverse effects of AIs on the primate brain and call for new therapies that effectively prevent breast cancer recurrence while minimizing side effects that further compromise quality of life.SIGNIFICANCE STATEMENT Aromatase inhibitors (AIs) are used as an adjuvant therapy for estrogen-receptor-positive breast cancer and are associated with side effects, including hot flashes, depression/anxiety, and memory deficits severe enough for many women to discontinue this life-saving treatment. AIs are also used by men, yet sex differences in the reported side effects have not been systematically studied. We show that AI-treated male and female marmosets exhibit behavioral changes consistent with these CNS symptoms, as well as elevated hippocampal estradiol and compromised hippocampal physiology. These findings illustrate the need for (1) a greater understanding of the precise mechanisms by which AIs impact brain function and (2) the development of new treatment approaches for breast cancer patients that minimize adverse effects on the brain.

Ovarian hormones, sleep and cognition across the adult female lifespan: An integrated perspective.

Loss of ovarian function in women is associated with sleep disturbances and cognitive decline, which suggest a key role for estrogens and/or progestins in modulating these symptoms. The effects of ovarian hormones on sleep and cognitive processes have been studied in separate research fields that seldom intersect. However, sleep has a considerable impact on cognitive function. Given the tight connections between sleep and cognition, ovarian hormones may influence selective aspects of cognition indirectly, via the modulation of sleep. In support of this hypothesis, a growing body of evidence indicates that the development of sleep disorders following menopause contributes to accelerated cognitive decline and dementia in older women. This paper draws from both the animal and human literature to present an integrated view of the effects of ovarian hormones on sleep and cognition across the adult female lifespan.

The middle-aged ovariectomized marmoset (Callithrix jacchus) as a model of menopausal symptoms: Preliminary evidence.

Menopausal women often suffer from hot flashes and sleep disturbances that significantly impact their quality of life. Both human and animal studies suggest that loss of estrogens during menopause contribute to these symptoms. In the female rat, both core body temperature (CBT) and sleep are sensitive to 17ß-estradiol (E2) levels, but important differences between the rat and the human patterns limit the interpretation of the results. The sleep and thermoregulation of the common marmoset (Callithrix jacchus) more closely resemble human patterns. However, no study to date has examined whether E2 influences sleep and thermoregulation in this species. The main goal of the present study was to investigate the suitability of the ovariectomized (OVX) marmoset for studying two major menopausal symptoms experienced by women, sleep disturbance and thermodysregulation. Two middle-aged OVX marmosets (6years old) were implanted with a telemeter that records electroencephalograms (EEG), electromyograms (EMG), and CBT. Sleep patterns and CBT were recorded under baseline, two E2 replacement (6 and 12µg/kg/day, p.o.) conditions and two E2 withdrawal conditions. Relative to both baseline and withdrawal, high E2 replacement was associated with lower nighttime CBT. In addition, fewer nighttime arousals were observed under low E2 replacement compared to baseline. Higher delta power was observed under both E2 replacement conditions suggesting enhanced sleep quality. These preliminary results suggest that E2 modulates sleep and thermoregulation in the OVX marmoset, making it a promising model for studying menopausal symptoms.

Intra-perirhinal cortex administration of estradiol, but not an ERß agonist, modulates object-recognition memory in ovariectomized rats.

Intra-rhinal cortical infusion of 17-ß estradiol (E2, 244.8pg/µl) enhances performance on the Novel-Object Preference (NOP) test and impairs accuracy on the delayed nonmatching-to-sample (DNMS) task in the same set of ovariectomized rats (Gervais, Jacob, Brake, & Mumby, 2013). These results appear paradoxical, as normal performance on both tests require intact object-recognition memory (ORM) ability. While demonstrating a preference for the novel object requires recognizing the sample object, rodents can recognize the sample object and still fail to demonstrate a preference. Therefore, enhanced NOP test performance is consistent with both improved ORM and increased novel-object exploration independent of memory processes. There is some evidence suggesting that estrogen receptor (ER) ß agonists enhance NOP test performance (Jacome et al., 2010), but no study to date has examined the role of this receptor in DNMS task performance in rodents. The aim of the present study was to determine whether intra-PRh infusion of an ER ß agonist, diarylpropionitrile (DPN, 2µg/µl), has divergent effects on novel-object preference (i.e. novelty preference) and accuracy on the DNMS task. Ovariectomized (OVX) rats (n=7) received chronic low E2 (∼22pg/ml serum) replacement, then intra-PRh infusion of DPN (2µg/µl), E2 (244.8pg/µl), or vehicle before each mixed-delay session (0.5-5min) of the DNMS task. A different set of OVX rats (n=10) received the same infusions before each NOP test trial, and were tested either 4 or 72h later. Consistent with Gervais et al. (2013), intra-PRh E2 reduced accuracy on the DNMS task following a 5-min retention delay and enhanced novelty preference on both tests. Intra-PRh DPN was associated with accuracy that was similar to the vehicle-infusion condition, despite enhancing novelty preference on both tests. The accuracy results suggest that while intra-PRh E2 impairs ORM, ERß does not play a role. However, ERß in the PRh appears to be important for the expression of novelty preference, in a manner that is unaffected by retention delay. These findings suggest that the modulation of novelty preference by intra-PRh E2/ERß may be due to factors unrelated to ORM.

Attenuation of dendritic spine density in the perirhinal cortex following 17ß-Estradiol replacement in the rat.

Intraperirhinal cortex infusion of 17-ß estradiol (E2) impairs object-recognition memory. However, it is not currently known whether this hormone modulates synaptic plasticity in this structure. Most excitatory synapses in the central nervous system are located on dendritic spines, and elevated E2 levels influence the density of these spines in several brain areas. The goal of the present study was to determine whether differences in dendritic spine density in the perirhinal cortex are observed following high E2 replacement in ovariectomized rats. The density of total spines, and mushroom-shaped (i.e. mature) spines were compared between a high E2 replacement (10 µg/kg/day, s.c.) and a no replacement condition. The perirhinal cortex is subdivided into Broadmann's area 35 and 36 and so group comparisons were made within each sub-region separately. High E2 replacement resulted in lower density of mushroom-shaped spines in area 35 relative to no replacement. There was no effect of high E2 replacement on dendritic spine density in area 36. These findings are consistent with the idea that higher E2 levels reduce dendritic spine density in area 35, which may result from spine shrinkage, or reduced synapse formation. This study provides preliminary evidence for a mechanism through which E2 may impair object-recognition memory.

Retrograde and anterograde memory following selective damage to the dorsolateral entorhinal cortex.

Anatomical and electrophysiological evidence suggest the dorsolateral entorhinal cortex (DLEC) is involved in processing spatial information, but there is currently no consensus on whether its functions are necessary for normal spatial learning and memory. The present study examined the effects of excitotoxic lesions of the DLEC on retrograde and anterograde memory on two tests of allocentric spatial learning: a hidden fixed-platform watermaze task, and a novelty-preference-based dry-maze test. Deficits were observed on both tests when training occurred prior to but not following n-methyl d-aspartate (NMDA) lesions of DLEC, suggesting retrograde memory impairment in the absence of anterograde impairments for the same information. The retrograde memory impairments were temporally-graded; rats that received DLEC lesions 1-3 days following training displayed deficits, while those that received lesions 7-10 days following training performed like a control group that received sham surgery. The deficits were not attenuated by co-infusion of tetrodotoxin, suggesting they are not due to disruption of neural processing in structures efferent to the DLEC, such as the hippocampus. The present findings provide evidence that the DLEC is involved in the consolidation of allocentric spatial information.

Modulatory effect of 17-ß estradiol on performance of ovariectomized rats on the Shock-Probe test.

17-ß estradiol (E2) has been shown to modulate fear conditioning by influencing freezing behavior following re-exposure to either the conditioning context or a cue associated with shock. Fear-related behaviors other than freezing may be influenced differently by E2 replacement. Accordingly, the present study examined whether E2 modulates fear conditioning using the Shock-Probe test, which allows for the observation of multiple fear responses. Ovariectomized (OVX) rats were divided into three groups: no E2 replacement (Shock-Oil; 0.1ml/kg sesame oil), high E2 replacement (Shock-E2; 3 daily doses of 10µg/kg, s.c.), and a no shock, no E2 replacement (Naïve-oil) group. During the acquisition phase, the two shock groups (Shock-Oil and Shock-E2) demonstrated comparable fear expression on all measures. During the retention tests, rats without E2 replacement demonstrated intact contextual-fear conditioning but impaired cued-fear conditioning, while rats with E2 replacement demonstrated the opposite pattern. In the context paired with shock, the Shock-Oil group spent more time burying the probe than both the Naïve-oil group and Shock-E2 group. The Shock-E2 group avoided the probe area relative to the other two groups when tested in the context unpaired with shock. The present findings demonstrate the importance of using multiple behaviors to measure fear conditioning and suggest that high E2 replacement impairs contextual and facilitates cued fear conditioning.

Systemic and intra-rhinal-cortical 17-ß estradiol administration modulate object-recognition memory in ovariectomized female rats.

Previous studies using the novel-object-preference (NOP) test suggest that estrogen (E) replacement in ovariectomized rodents can lead to enhanced novelty preference. The present study aimed to determine: 1) whether the effect of E on NOP performance is the result of enhanced preference for novelty, per se, or facilitated object-recognition memory, and 2) whether E affects NOP performance through actions it has within the perirhinal cortex/entorhinal cortex region (PRh/EC). Ovariectomized rats received either systemic chronic low 17-ß estradiol (E2; ~20 pg/ml serum) replacement alone or in combination with systemic acute high administration of estradiol benzoate (EB; 10 µg), or in combination with intracranial infusions of E2 (244.8 pg/µl) or vehicle into the PRh/EC. For one of the intracranial experiments, E2 was infused either immediately before, immediately after, or 2 h following the familiarization (i.e., learning) phase of the NOP test. In light of recent evidence that raises questions about the internal validity of the NOP test as a method of indexing object-recognition memory, we also tested rats on a delayed nonmatch-to-sample (DNMS) task of object recognition following systemic and intra-PRh/EC infusions of E2. Both systemic acute and intra-PRh/EC infusions of E enhanced novelty preference, but only when administered either before or immediately following familiarization. In contrast, high E (both systemic acute and intra-PRh/EC) impaired performance on the DNMS task. The findings suggest that while E2 in the PRh/EC can enhance novelty preference, this effect is probably not due to an improvement in object-recognition abilities.

Enhanced adolescent learning and hippocampal axonal projections following preadolescent spatial exposure to a water or dry maze.

The present work sought to determine whether preadolescent exposure to a different task in the same spatial environment would lead to enhancement of water-maze performance and changes in hippocampal connectivity. Separate groups of preadolescent (p16-p26) Long Evans rats (LER) were exposed to the same room and arena using either a water-maze (WM) or a dry-maze (DM), while a third group received no exposure to the spatial cues (NT) but were handled. Three weeks later, rats were tested on the WM or DM task in the same room where preadolescent exposure took place. This set up conditions where the DM/WM and WM/DM groups were exposed to the same spatial cues during the preadolescent and adolescent (p40-p44) phases but performed different tests allowing for the dissociation of spatial cue exposure and task familiarity on adolescent performance. When animals experienced similar preadolescent and adolescent conditions (WM/WM or DM/DM), there was improved performance over animals with no preadolescent spatial exposure (NT/WM or NT/DM). In group DM/WM, there was enhanced adolescent performance compared to group NT/WM. In contrast, group WM/DM did not show enhanced adolescent performance. Compared to groups with no preadolescent spatial exposure, groups with both preadolescent and adolescent spatial exposure showed elevated synaptophysin staining in the hippocampal CA3 region indicating an expanded axonal projection in this region. These data suggest the possibility that exposure to spatial cues during the preadolescent period, independent from task-specific requirements, contributes to enhanced adolescent spatial performance on the WM. This appears to be linked with the reorganization of axonal inputs to the CA3 region.

A randomized clinical trial of cognitive-behavioral therapy and applied relaxation for adults with generalized anxiety disorder.

This randomized clinical trial compared cognitive-behavioral therapy (CBT), applied relaxation (AR), and wait-list control (WL) in a sample of 65 adults with a primary diagnosis of generalized anxiety disorder (GAD). The CBT condition was based on the intolerance of uncertainty model of GAD, whereas the AR condition was based on general theories of anxiety. Both manualized treatments were administered over 12 weekly 1-hour sessions. Standardized clinician ratings and self-report questionnaires were used to assess GAD and related symptoms at pretest, posttest, and at 6-, 12-, and 24-month follow-ups. At posttest, CBT was clearly superior to WL, AR was marginally superior to WL, and CBT was marginally superior to AR. Over follow-up, CBT and AR were equivalent, but only CBT led to continued improvement. Thus, direct comparisons of CBT and AR indicated that the treatments were comparable; however, comparisons of each treatment with another point of reference (either waiting list or no change over follow-up) provided greater support for the efficacy of CBT than AR.

The impact of written exposure on worry: a preliminary investigation.

The main goal of this study was to examine the effect of written exposure on generalized anxiety disorder (GAD)-related symptoms in high worriers. Thirty nonclinical high worriers were randomly assigned to either a written exposure condition or a control writing condition. Self-report measures were used to assess worry, GAD somatic symptoms, depression, and intolerance of uncertainty at four time points during the study. Using hierarchical linear modeling (HLM), the authors found that all symptoms (i.e., worry, GAD somatic symptoms, and depression) significantly decreased over time in the written exposure group (although GAD somatic symptoms also decreased in the control group). Moreover, consistent with previous findings that intolerance of uncertainty preceded changes in worry over the course of treatment, intolerance of uncertainty scores predicted subsequent scores on all symptom measures in the experimental group. In contrast, worry and depression scores predicted subsequent intolerance of uncertainty scores in the control group.