The C9ORF72 repeat expansion alters neurodevelopment.

Genetic mutations that cause adult-onset neurodegenerative diseases are often expressed during embryonic stages, but it is unclear whether they alter neurodevelopment and how this might influence disease onset. Here, we show that the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), a repeat expansion in C9ORF72, restricts neural stem cell proliferation and reduces cortical and thalamic size in utero. Surprisingly, a repeat expansion-derived dipeptide repeat protein (DPR) not known to reduce neuronal viability plays a key role in impairing neurodevelopment. Pharmacologically mimicking the effects of the repeat expansion on neurodevelopment increases susceptibility of C9ORF72 mice to motor defects. Thus, the C9ORF72 repeat expansion stunts development of the brain regions prominently affected in C9ORF72 FTD/ALS patients.

Oestrogen treatment modulates the impact of cognitive experience and task complexity on memory in middle-aged surgically menopausal rats.

Menopause has been linked to changes in memory. Oestrogen-containing hormone therapy is prescribed to treat menopause-related symptoms and can ameliorate memory changes, although the parameters impacting oestrogen-related memory efficacy are unclear. Cognitive experience and practice have been shown to be neuroprotective and to improve learning and memory during ageing, with the type of task playing a role in subsequent cognitive outcomes. Whether task complexity matters, and whether these outcomes interact with menopause and oestrogen status, remains unknown. To investigate this, we used a rat model of surgical menopause to systematically assess whether maze task complexity, as well as order of task presentation, impacts spatial learning and memory during middle age when rats received vehicle, low-17ß-oestradiol (E2 ) or high-E2 treatment. The direction, and even presence, of the effects of prior maze experience differed depending on the E2 dose. Surgical menopause without E2 treatment yielded the least benefit, as prior maze experience did not have a substantial effect on subsequent task performance for vehicle treated rats regardless of task demand level during the first exposure to maze experience or final testing. High-dose E2 yielded a variable benefit, and low-dose E2 produced the greatest benefit. Specifically, low-dose E2 broadly enhanced learning and memory in surgically menopausal rats that had prior experience on another task, regardless of the complexity level of this prior experience. These results demonstrate that E2 dose influences the impact of prior cognitive experience on learning and memory during ageing, and highlights the importance of prior cognitive experience in subsequent learning and memory outcomes.

Poly(lactic-co-glycolic Acid) Nanoparticle Encapsulated 17ß-Estradiol Improves Spatial Memory and Increases Uterine Stimulation in Middle-Aged Ovariectomized Rats.

Hormone therapy that contains 17ß-estradiol (E2) is used commonly for treatment of symptoms associated with menopause. E2 treatment has been shown to improve cognitive function following the decrease in ovarian hormones that is characteristic of menopause. However, once in circulation, the majority of E2 is bound to serum hormone binding globulin or albumin, becoming biologically inactive. Thus, therapeutic efficacy of E2 stands to benefit from increased bioavailability via sustained release of the hormone. Here, we focus on the encapsulation of E2 within polymeric nanoparticles composed of poly(lactic-co-glycolic) acid (PLGA). PLGA agent encapsulation offers several delivery advantages, including improved bioavailability and sustained biological activity of encapsulated agents. We hypothesized that delivery of E2 from PLGA nanoparticles would enhance the beneficial cognitive effects of E2 relative to free E2 or non-hormone loaded nanoparticle controls in a rat model of menopause. To test this hypothesis, spatial learning and memory were assessed in middle-aged ovariectomized rats receiving weekly subcutaneous treatment of either oil-control, free (oil-solubilized) E2, blank (non-hormone loaded) PLGA, or E2-loaded PLGA. Unexpectedly, learning and memory differed significantly between the two vehicle control groups. E2-loaded PLGA nanoparticles improved learning and memory relative to its control, while learning and memory were not different between free E2 and its vehicle control. These results suggest that delivery of E2 from PLGA nanoparticles offered cognitive benefit. However, when evaluating peripheral burden, E2-loaded PLGA was found to increase uterine stimulation compared to free E2, which is an undesired outcome, as estrogen exposure increases uterine cancer risk. In sum, a weekly E2 treatment regimen of E2 from PLGA nanoparticles increased cognitive efficacy and was accompanied with an adverse impact on the periphery, effects that may be due to the improved agent bioavailability and sustained biological activity offered by PLGA nanoparticle encapsulation. These findings underscore the risk of non-specific enhancement of E2 delivery and provide a basic framework for the study and development of E2's efficacy as a cognitive therapeutic with the aid of customizable polymeric nano-carriers.

Characterization of Activation of the Hypothalamic-Pituitary-Adrenal Axis by the Herbicide Atrazine in the Female Rat.

Atrazine (ATR) is a commonly used pre-emergence and early postemergence herbicide. Rats gavaged with ATR and its chlorometabolites desethylatrazine (DEA) and deisopropylatrazine (DIA) respond with a rapid and dose-dependent rise in plasma corticosterone, whereas the major chlorometabolite, diaminochlorotriazine (DACT), has little or no effect on corticosterone levels. In this study, we investigated the possible sites of ATR activation of the hypothalamic-pituitary-adrenal (HPA) axis. ATR treatment had no effect on adrenal weights but altered adrenal morphology. Hypophysectomized rats or rats under dexamethasone suppression did not respond to ATR treatment, suggesting that ATR does not directly stimulate the adrenal gland to induce corticosterone synthesis. Immortalized mouse corticotrophs (AtT-20) and primary rat pituitary cultures were treated with ATR, DEA, DIA, or DACT. None of the compounds induced an increase in ACTH secretion or potentiated ACTH release in conjunction with CRH on ACTH release. In female rats gavaged with ATR, pretreatment with the CRH receptor antagonist astressin completely blocked the ATR-induced rise in corticosterone concentrations, implicating CRH release in ATR-induced HPA activation. Intracerebroventricular infusion of ATR, DEA, and DIA but not DACT at concentrations equivalent to peak plasma concentrations after gavage dosing resulted in an elevation of plasma corticosterone concentrations. However, ATR did not induce c-Fos immunoreactivity in the paraventricular nucleus of the hypothalamus. These results indicate that ATR activates the HPA axis centrally and requires CRH receptor activation, but it does not stimulate cellular pathways associated with CRH neuronal excitation.

Contrasting effects of individual versus combined estrogen and progestogen regimens as working memory load increases in middle-aged ovariectomized rats: one plus one does not equal two.

Most estrogen-based hormone therapies are administered in combination with a progestogen, such as Levonorgestrel (Levo). Individually, the estrogen 17ß-estradiol (E2) and Levo can improve cognition in preclinical models. However, although these hormones are often given together clinically, the impact of the E2 + Levo combination on cognitive function has yet to be methodically examined. Thus, we investigated E2 + Levo treatment on a cognitive battery in middle-aged, ovariectomized rats. When administered alone, E2 and Levo treatments each enhanced spatial working memory relative to vehicle treatment, whereas the E2 + Levo combination impaired high working memory load performance relative to E2 only and Levo only treatments. There were no effects on spatial reference memory. Mitogen-activated protein kinases/extracellular signal-regulated kinases pathway activation, which is involved in memory formation and estrogen-induced memory effects, was evaluated in 5 brain regions implicated in learning and memory. A distinct relationship was seen in the E2-only treatment group between mitogen-activated protein kinases/extracellular signal-regulated kinases pathway activation in the frontal cortex and working memory performance. Collectively, the results indicate that the differential neurocognitive effects of combination versus sole treatments are vital considerations as we move forward as a field to develop novel, and to understand currently used, exogenous hormone regimens across the lifespan.

Cognitive changes across the menopause transition: A longitudinal evaluation of the impact of age and ovarian status on spatial memory.

Cognitive changes that occur during mid-life and beyond are linked to both aging and the menopause transition. Studies in women suggest that the age at menopause onset can impact cognitive status later in life; yet, little is known about memory changes that occur during the transitional period to the postmenopausal state. The 4-vinylcyclohexene diepoxide (VCD) model simulates transitional menopause in rodents by depleting the immature ovarian follicle reserve and allowing animals to retain their follicle-deplete ovarian tissue, resulting in a profile similar to the majority of perimenopausal women. Here, Vehicle or VCD treatment was administered to ovary-intact adult and middle-aged Fischer-344 rats to assess the trajectory of cognitive change across time with normal aging and aging with transitional menopause via VCD-induced follicular depletion, as well as to evaluate whether age at the onset of follicular depletion plays a role in cognitive outcomes. Animals experiencing the onset of menopause at a younger age exhibited impaired spatial memory early in the transition to a follicle-deplete state. Additionally, at the mid- and post- follicular depletion time points, VCD-induced follicular depletion amplified an age effect on memory. Overall, these findings suggest that age at the onset of menopause is a critical parameter to consider when evaluating learning and memory across the transition to reproductive senescence. From a translational perspective, this study illustrates how age at menopause onset might impact cognition in menopausal women, and provides insight into time points to explore for the window of opportunity for hormone therapy during the menopause transition period. Hormone therapy during this critical juncture might be especially efficacious at attenuating age- and menopause- related cognitive decline, producing healthy brain aging profiles in women who retain their ovaries throughout their lifespan.

Distribution and estrogen regulation of membrane progesterone receptor-ß in the female rat brain.

Although several studies have reported the localization of membrane progesterone (P(4)) receptors (mPR) in various tissues, few have attempted to describe the distribution and regulation of these receptors in the brain. In the present study, we investigated expression of two mPR subtypes, mPRα and mPRß, within regions of the brain, known to express estradiol (E(2))-dependent [preoptic area (POA) and hypothalamus] and independent (cortex) classical progestin receptors. Saturation binding and Scatchard analyses on plasma membranes prepared from rat cortex, hypothalamus, and POA demonstrated high-affinity, specific P(4)-binding sites characteristic of mPR. Using quantitative RT-PCR, we found that mPRß mRNA was expressed at higher levels than mPRα, indicating that mPRß may be the primary mPR subtype in the rat brain. We also mapped the distribution of mPRß protein using immunohistochemistry. The mPRß-immunoreactive neurons were highly expressed in select nuclei of the hypothalamus (paraventricular nucleus, ventromedial hypothalamus, and arcuate nucleus), forebrain (medial septum and horizontal diagonal band), and midbrain (oculomotor and red nuclei) and throughout many areas of the cortex and thalamus. Treatment of ovariectomized female rats with E(2) benzoate increased mPRß immunoreactivity within the medial septum but not the medial POA, horizontal diagonal band, or oculomotor nucleus. Together, these findings demonstrate a wide distribution of mPRß in the rodent brain that may contribute to functions affecting behavioral, endocrine, motor, and sensory systems. Furthermore, E(2) regulation of mPRß indicates a mechanism through which estrogens can regulate P(4) function within discrete brain regions to potentially impact behavior.

Perinatal dexamethasone-induced alterations in apoptosis within the hippocampus and paraventricular nucleus of the hypothalamus are influenced by age and sex.

Exposure to high levels of glucocorticoids (GCs) during development leads to long-term changes in hypothalamic-pituitary-adrenal (HPA) axis regulation, although little is known about the neural mechanisms that underlie these alterations. In this study, we investigated the effects of late gestational (days 18-22) or postnatal (days 4-6) administration of the GC receptor agonist dexamethasone (DEX) on an apoptosis marker in two brain regions critical to HPA axis regulation, the hippocampus and the hypothalamic paraventricular nucleus (PVN). One day after the final DEX injection, male and female rats were sacrificed, and brains were processed for immunohistochemical detection of cleaved caspase-3, an apoptotic cell death indicator. DEX increased cleaved caspase-3 immunoreactivity in the CA1 hippocampal region of both sexes following prenatal but not postnatal treatment. Prenatal DEX also increased caspase-3 immunoreactivity in the CA3 region, an elevation that tended to be greater in females. In contrast, postnatal DEX resulted in a much smaller, albeit significant, induction in CA3 caspase-3 compared with prenatal treatment. Quantitative real-time PCR analysis revealed that prenatal but not postnatal DEX-induced hippocampal cleaved caspase-3 correlated with elevated mRNA of the proapoptotic gene Bad. Few caspase-3-ir cells were identified within the PVN regardless of treatment age, although postnatal but not prenatal DEX increased this number. However, the region immediately surrounding the PVN (peri-PVN) showed significant increases in caspase-3-ir cells following pre- and postnatal DEX. Together these findings indicate that developmental GC exposure increases apoptosis in HPAaxis-associated brain regions in an age- and sex-dependent manner.

The differential effect of atrazine on luteinizing hormone release in adrenalectomized adult female Wistar rats.

High doses of atrazine (ATR), administered for 4 days, suppress luteinizing hormone (LH) release and increase adrenal hormones levels. Considering the known inhibitory effects of adrenal hormones on the hypothalamo-pituitary-gonadal axis, we investigated the possible role the adrenal gland has in mediating ATR inhibition of LH release. To determine the extant and duration of adrenal activation, ovariectomized Wistar rats were given a single dose of ATR (0, 50, or 200 mg/kg), and corticosterone (CORT) levels were assayed at multiple time points posttreatment. CORT levels were increased within 20 min and remained elevated over 12 h postgavage in 200-mg/kg animals. To determine the effects of adrenalectomy on ATR inhibition of the LH surge and pulsatile LH release, adrenalectomized (ADX) or sham-operated ovariectomized rats were treated for 4 days with ATR (0, 10, 100, or 200 mg/kg), and an LH surge was induced with hormone priming. In the afternoon following the last dose of ATR, blood was sampled hourly for 9 h. Another cohort of ovariectomized rats was examined for pulsatile patterns of LH secretion after ATR (0, 50, or 200 mg/kg) and sampled every 5 min for 3 h. ADX had no effect on ATR inhibition of the LH surge but prevented the ATR disruption of pulsatile LH release. These data indicate that ATR selectively affects the LH pulse generator through alterations in adrenal hormone secretion. Adrenal activation does not play a role in ATR's suppression of the LH surge, and therefore ATR may work centrally to alter the preovulatory LH surge in female rats.

Electrophoretic exclusion for the selective transport of small molecules.

A novel method capable of differentiating and concentrating small molecules in bulk solution termed "electrophoretic exclusion" is described and experimentally investigated. In this technique, the hydrodynamic flow of the system is countered by the electrophoretic velocity to prevent a species from entering into the channel. The separation can be controlled by changing the flow rate or applied electric field in order to exclude certain species selectively while allowing others to pass through the capillary. Proof of principle studies employed a flow injection regime of the method and examined the exclusion of Methyl Violet dye in the presence of a neutral species. Methyl Violet was concentrated almost 40 times the background concentration in 30 s using 6 kV. Additionally, a threshold voltage necessary for exclusion was determined. The establishment of a threshold voltage enabled the differentiation of two similar cationic species: Methyl Green and Neutral Red.