Drina Vurbic

Brain orexins and wake regulation in rats exposed to maternal deprivation.

Maternal deprivation (MD) is a neonatal stressor that leads to behavioral and molecular manifestations of chronic stress in adulthood. Recent evidence has suggested that stress may impact wake regulation through corticotropin-releasing hormone (CRH) and the orexinergic system. We studied the wake/sleep features and brain levels of orexin and orexin receptors in adult rats neonatally subjected to either ten days of MD or a control procedure from postnatal day 4. At 3 months of age, one set of rats from both groups underwent 48 h of polysomnographic recording. All rats (including those that did not undergo surgery) were subsequently sacrificed for ELISA, radioimmunoassay and western blot measurement of orexins, orexin receptors and CRH in multiple brain regions. Neonatal MD induced an increase of total wake time (decreased total sleep) during the light period, which corresponds to human night time. This increase was specifically composed of quiet wake, while a small but significant decrease of active wake was observed during the dark period. At the molecular level, MD led to increased hypothalamic CRH and orexin A, and frontal cortical orexin 1 receptors (OX1R). However, hippocampal orexin B was reduced in the MD group. Our study discovered for the first time that the adult MD rat has sleep and neurobiological features of hyperarousal, which is typical in human insomnia. We concluded that neonatal MD produces adult hyperarousal in sleep physiology and neurobiology, and that the adult MD rat could be a model of insomnia with an orexinergic mechanism.

Changes in brain orexin levels in a rat model of depression induced by neonatal administration of clomipramine.

Depression is associated with a deficiency of serotonergic neurons that have been found to suppress orexinergic neurons, which in turn activate these neurons in a feedback loop. This evidence suggests that orexins may be involved in the pathology of depression. Long Evans rats were treated with clomipramine (CLI) and saline (SAL) from postnatal days 8 through 21. One set of rats from both groups was sacrificed at 35 days of age for quantification of orexins in multiple brain regions. At 3—4 months of age a second set of rats was tested for immobility in a forced swim procedure, a common test for depressive signs in rats, and a third set was sacrificed for the quantification of orexins. Compared with the control rats, adult rats with neonatal CLI treatment had (1) increased forced swim immobility and (2) increased orexins A and B in the hypothalamus. However, both orexins A and B levels were decreased in multiple brain regions in the juvenile CLI rats compared with same-age controls. We concluded that although orexin levels were decreased in juvenile CLI rats, adult CLI rats with features of depression had significantly higher levels of hypothalamic orexins compared with adult controls. These results imply that orexins are likely to be involved in the pathological regulation of depression.

Lower urogenital tract anatomical and functional phenotype in lysyl oxidase like-1 knockout mice resembles female pelvic floor dysfunction in humans

Female pelvic floor dysfunction (FPFD) is a complex group of conditions that include urinary incontinence and pelvic organ prolapse (POP). In humans, elastin homeostasis has been implicated in the pathophysiology of FPFD. Lysyl oxidase-like 1 knockout (LOXL1-KO) mice demonstrate abnormal elastic fiber homeostasis and develop FPFD after parturition. We compared the lower urogenital tract (LUT) anatomy and function in LOXL1-KO mice with and without POP. LUT anatomy was assessed in LOXL1-KO mice over 28 wk. Pelvic visceral anatomy in LOXL1-KO was evaluated with a 7-Tesla magnetic resonance imaging (MRI) scanner. LUT function was assessed using conscious cystometry and leak point pressure (LPP) testing. Quantitative histological analysis of elastic fibers was performed on external urethral sphincter (EUS) cross sections. By 25 wk of age, 50% of parous LOXL1-KO mice developed POP. LOXL1-KO mice with POP had greater variability in the size and location of the bladder on MRI compared with mice without POP. Parity and POP were associated with lower LPP. Elastin clusters were significantly increased in the EUS of LOXL1-KO mice with POP. Because parity triggers POP in LOXL1-KO mice, LOXL1-KO mice with POP have variable internal pelvic anatomy, and both parity and POP are associated with a decrease in LPP, we conclude that LOXL1 LUT anatomical and functional phenotype resembles FPFD in humans. The increase in elastin clusters in the urethra of LOXL1-KO mice with POP suggests that elastin disorganization may lead to functional abnormalities. We conclude that LOXL1 warrants further investigation in the pathphysiology of FPFD.

Voiding function in obese and type 2 diabetic female rats

The effects of obesity and type 2 diabetes (DMII) on the lower urinary tract (LUT) were characterized by evaluating voiding function and anatomy in female Zucker diabetic fatty (ZDF) rats. Age-matched female virgin rats were separated into three experimental groups: Zucker lean rats (control; normal diet, n = 22), ZDF rats (obese+nondiabetic; low-fat diet, n = 22), and ZDF rats (obese+diabetic; high-fat diet, n = 20). Rats were placed on their specified diet for 10 wk before urodynamic LUT evaluation. A suprapubic catheter was implanted 2 days before urodynamic studies. Voiding function was evaluated by cystometric and leak point pressure (LPP) testing. The bladder, urethra, and vagina were immediately excised for qualitative histological evaluation. Compared with control rats, obese+nondiabetic and obese+diabetic rats had significantly decreased contraction pressure (P = 0.003) and increased cystometric filling volume (P < 0.001). Both obese groups exhibited significantly higher voided volumes (P = 0.003), less frequent urinary events (P < 0.001), and increased residual volumes (P = 0.039). LPP studies showed a nonsignificant decrease in LPP (P = 0.075) and baseline pressure (P = 0.168) in both obese groups compared with control. Histology of the external urethral sphincter in obese rats showed increased fibrosis, leading to disruption of the skeletal muscle structure compared with control. Additionally, the bladder wall of the obese+nondiabetic and obese+diabetic rats demonstrated edema and vasculopathy. Voiding dysfunction was evident in both obese groups but with no significant differences due to DMII, suggesting that voiding dysfunction in DMII may be attributable at least in part to chronic obesity.

The impact of cesarean delivery on pelvic floor dysfunction in lysyl oxidase like-1 knockout mice.

Objective: Lysyl oxidase like-1 (LOXL1) knockout mice have abnormal elastic fiber homeostasis and frequently develop pelvic floor dysfunction after pregnancy and delivery. The objective of this study was to test the hypothesis that tissue changes associated with vaginal delivery lead to pelvic floor dysfunction as a result of abnormal elastic fiber homeostasis. Methods: Female LOXL1 knockout mice delivered either spontaneously or by cesarean delivery. Mice were assessed weekly for pelvic organ prolapse (POP). At 12 weeks postpartum, lower urinary tract function was assessed by cystometry and leak-point pressure testing. Urethrovaginal cross-sections were analyzed using a histologic grading scale to assess elastin fiber disorganization. Results: A total of 39 mice delivered by spontaneous vaginal delivery and 36 by cesarean delivery. Twelve weeks after spontaneous vaginal delivery or cesarean delivery, 23 (59%) and 11 (31%) mice had developed POP, respectively. The mean time to develop POP was 7.2 weeks after spontaneous vaginal delivery and 10.5 weeks after cesarean delivery (log rank, P = 0.0008). The Cox proportional hazard ratio was 0.55 (95% confidence interval, 0.38–0.79). Mice with POP had increased frequency of bladder contractions not associated with voiding during cystometry (P = 0.02). POP, but not mode of delivery, was associated with increased elastic fiber disorganization. Conclusions: Cesarean delivery delays the development of POP in LOXL1 knockout mice. POP is associated with increased bladder contraction frequency and increased elastic fiber disorganization in the urethra and vagina. The mechanisms underlying these findings warrant further investigation.

Maternal stress induces adult reduced REM sleep and melatonin level.

We have previously reported that neonatal maternal deprivation (MD) resulted in a decrease of total sleep and an increase of orexin A in adult rats. Now, we characterized features of sleep, activity, and melatonin levels in rats neonatally treated with MD and control (MC) procedures. Adult male Sprague–Dawley rats were treated with either MD or MC procedures for 10 days starting at postnatal day 4. At 3 months of age, sleep was recorded for 48 h in one set of MD and MC rats, while another set of MD and MC rats was measured for locomotor activity (under LD = 12:12). Melatonin levels in the blood, pineal gland, and hypothalamus were measured as well as clock protein level in the hypothalamus. Compared to the MC rats, REM sleep in the MD rats was significantly reduced in the light periods but not in the dark periods. Both quiet wake and total wake in the MD rats were significantly increased during the light period compared to the MC rats. The weight of the pineal gland of the MD rats was significantly smaller than in MC rats. Melatonin levels of the MD group were significantly reduced in the pineal gland and hypothalamus compared to the MC group. No significant difference was identified between groups in the expression of the clock protein in the hypothalamus. Neonatal MD resulted in reduced REM sleep and melatonin levels, without changes of circadian cycle of locomotor activity and levels of clock protein.

The effect of clomipramine on wake/sleep and orexinergic expression in rats.

Abstract We have previously found that neonatal treatment with clomipramine (CLI) induced a decrease in brain orexins during the juvenile period and that these changes were reversed at adulthood. This study investigated the effect of CLI on the orexinergic component and sleep/wake states. Two groups of adult male rats were conducted for 48-h polysomnographic recording. One group of rats was treated with CLI (20 mg/kg every 12 h), and a second group was treated with equivolume of saline (SAL) simultaneously after the first 24 h of polysomnographic recording. Rats were killed 2 h after the third dose of treatment. Brain tissues were collected for radioimmunoassay quantification of orexins and real-time PCR analysis of prepro-orexin and orexin receptor mRNA. The CLI group had significantly shorter rapid eye movement (REM) sleep and longer REM latency compared with both the baseline day and the SAL group and had significantly less active wake and more quiet wake. Compared with the control rats, the CLI rats had significantly higher mRNA expression of prepro-orexin in the hypothalamus and the frontal cortex, but not in the hippocampus. The CLI rats also had significantly less orexin B in the hypothalamus than the control rats. These results suggest that suppression of active wake and orexin B by CLI may be a factor responsible for CLI-induced depression and that the increase of prepro-orexin mRNA may be a sign of increased brain orexins found in this model.

Sexual development and fertility of Loxl1-/- male mice

Our objective was to investigate the genitourinary defects and fertility of the male lysyl oxidase-like 1 gene (Loxl1) knockout (Loxl1(-/-)) mouse, with particular attention to fecundity and testicular, epididymal, gubernacular, and penile histopathology, which may lead us to a better understanding of the role of the elastin-homeostasis gene, LOXL1, in male sexual development. Genital morphometric evaluation of 6- to 9-month-old male Loxl1(-/-) mice (n = 26) was compared with C57Bl/6 controls (n = 24). Measurements included: body weight, scrotal development, evidence of feminization (nipples or vaginal pouch), penile malformations, anogenital distance, and absence/presence and size of perineal bulge. Sperm production was estimated using a standardized technique. A breeding program was conducted to determine how much of the infertility observed in Loxl1(-/-) pairs was due to the male factor. Finally, we performed histopathologic comparison of the genitourinary organs of Loxl1(-/-) and control mice. Loxl1(-/-) mice weighed less than their age-matched C57Bl/6 counterparts (P < .001). Size-adjusted perineal bulge was larger (P < .001), and resting location of the gonads was higher intra-abdominally (P = .048) in the Loxl1(-/-) mice. Estimates of daily sperm counts revealed that the Loxl1(-/-) mice had lower sperm production (P = .048). Loxl1(-/-) males bred with control females demonstrated relative fecundity values intermediate between Loxl1(-/-) pairs (lowest fecundity) and control pairs (highest fecundity), suggesting a component of male-factor infertility. No histologic differences were noted using hematoxylin-eosin or specialized elastin staining of the gonads, gubernaculum, and penis. Although further studies are warranted, these findings suggest a subtle and likely multifactorial role of the LOXL1 protein in male sexual development and fertility.

Neonatal REM sleep is regulated by corticotropin releasing factor

Sleep/wake regulation is quite different during the neonatal and adult periods. Although cholinergic neurons have been recognized to be the major source of rapid eye movement (REM) sleep regulation in adulthood, their effect on neonatal REM sleep remains to be discovered. Current evidence suggests that corticotropin-releasing factor (CRF) may play a role in REM promotion during the neonatal period. We conducted the following study to test our hypothesis that blocking CRF R1 receptor would reduce REM sleep in developing rat pups. First, rat pups were surgically implanted with electrodes on postnatal day (PN) 13. On PN 14, six hours of polysomnographic (PSG) data were collected before and after administration of three different doses of NBI 27914 (NBI), a CRF R1 receptor antagonist. Compared with baseline, REM sleep was significantly reduced in all groups treated with NBI but not with dimethyl sulfoxide/saline. The reduction of REM sleep was dose-related and was replaced primarily by non-REM (NREM) sleep. Second, two groups of rat pups were given a single dose of either NBI or vehicle on PN 14 for quantification of ACTH and acetylcholine without PSG recording. NBI induced no change of either ACTH or acetylcholine. Third, the effect of administering atropine (6 mg/kg) on sleep/wake in two-week-old rats was investigated. Atropine suppressed REM sleep significantly and increased wakefulness simultaneously. Our data revealed that blockage of CRF R1 receptors deprives neonatal REM sleep. The mechanism for CRF in enhancing REM sleep may be associated with but not be similar to the cholinergic mechanism.