Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 µm dendrite

Age-Related Deficits in Spatial Memory and Hippocampal Spines in Virgin, Female Fischer 344 Rats

Effects of aging on memory and brain morphology were examined in aged, 21-month-old, and young, 4-month-old, Fischer 344 female rats. Spatial memory was assessed using the object placement task, and dendritic spine density was determined on pyramidal neurons in the hippocampus following Golgi impregnation. Consistent with previous studies, aged females showed poorer object placement performance than young subjects. Young subjects significantly discriminated the location of objects with a 1.5-hour intertrial delay while aged subjects did not. Spine density of basal dendrites on CA1 pyramidal cells was 16% lower in the aged subjects as compared to the young subjects. No differences in spine density were found between young and aged subjects in basal dendrites of CA1 or in either dendritic field of CA3 pyramidal neurons. Thus, decreased hippocampal CA1 dendritic spine density in aged rats may contribute to poorer spatial memory as compared to young rats. The possibility that the neuroplastic changes observed in this study may pertain only to female subjects having had a specific set of life experiences is discussed. Different factors, such as reproductive status, diet, and handling may contribute to neuroplasticity of the brain during aging; however, this view requires further examination

Adolescent Bisphenol-A Exposure Decreases Dendritic Spine Density: Role of Sex and Age

Bisphenol-A (BPA), a common environmental endocrine disruptor, modulates estrogenic, androgenic, and anti-androgenic effects throughout the lifespan. We recently showed that low dose BPA exposure during adolescence increases anxiety and impairs spatial memory independent of sex. In the current study, six week old Sprague Dawley rats (n=24 males, n=24 females) received daily subcutaneous injections (40 µg/kg bodyweight) of BPA or vehicle for one week. Serum corticosterone levels in response to a 1 h restraint stress and spine density were examined at age 7 (cohort 1) and 11 (cohort 2) weeks. Adolescent BPA exposure did not alter stress dependent corticosterone responses but decreased spine density on apical and basal dendrites of pyramidal cells in the medial prefrontal cortex (mPFC) and hippocampal CA1 region (CA1). Sex differences in spine density were observed on basal dendrites of the mPFC and CA1 with females having greater spine density than males. This sex difference was further augmented by both age and treatment, with results indicating that BPA-dependent decreases in spine density were more pronounced in males than females on mPFC basal dendrites. Importantly, the robust neuronal alterations were observed in animals exposed to BPA levels below the current U.S.E.P.A. recommended safe daily limit. These results are the first demonstrating that BPA given during adolescence leads to enduring effects on neural morphology at adulthood. Given that humans are routinely exposed to low levels of BPA through a variety of sources, the decreased spine density reported in both male and female rats after BPA exposure warrants further investigation

Bisphenol-A Exposure During Adolescence Leads to Enduring Alterations in Cognition and Dendritic Spine Density in Adult Male and Female Rats

We have previously demonstrated that adolescent exposure of rats to bisphenol-A (BPA), an environmental endocrine disrupter, increases anxiety, impairs spatial memory, and decreases dendritic spine density in the CA1 region of the hippocampus (CA1) and medial prefrontal cortex (mPFC) when measured in adolescence in both sexes. The present study examined whether the behavioral and morphological alterations following BPA exposure during adolescent development are maintained into adulthood. Male and female, adolescent rats received BPA, 40 μg/kg/bodyweight, or control treatments for one week. In adulthood, subjects were tested for anxiety and locomotor activity, spatial memory, non-spatial visual memory, and sucrose preference. Additionally, stress-induced serum corticosterone levels and dendritic spine density in the mPFC and CA1 were measured. BPA-treated males, but not females, had decreased arm visits on the elevated plus maze, but there was no effect on anxiety. Non-spatial memory, object recognition, was also decreased in BPA treated males, but not females. BPA exposure did not alter spatial memory, object placement, but decreased exploration during the tasks in both sexes. No significant group differences in sucrose preference or serum corticosterone levels in response to a stress challenge were found. However, BPA exposure, regardless of sex, significantly decreased spine density of both apical and basal dendrites on pyramidal cells in CA1 but had no effect in the mPFC. Current data are discussed in relation to BPA dependent changes, which were present during adolescence and did, or did not, endure into adulthood. Overall, adolescent BPA exposure, below the current reference safe daily limit set by the U.S.E.P.A., leads to alterations in some behaviors and neuronal morphology that endure into adulthood

Sexually Dimorphic Effects of Prenatal Stress on Cognition, Hormonal Responses, and Central Neurotransmitters

Exposure to stress during gestation results in physiological and behavioral alterations that persist into adulthood. This study examined the effects of prenatal stress on the postnatal expression of sexually differentiated cognitive, hormonal, and neurochemical profiles in male and female rats. Pregnant dams were subjected to restraint stress three times daily for 45 min during d 14-21 of pregnancy. The offspring of control and prenatally stressed dams were tested for anxiety-related and cognitive behaviors, stress and gonadal steroid hormone levels, as well as monoamines and metabolite levels in selected brain regions. Postnatal testosterone levels (measured at 1 and 5 d) did not differ between controls and prenatally stressed animals. In adulthood, the serum corticosterone response to stress was attenuated in prenatally stressed females, eliminating the sex difference normally observed in this parameter. Prenatally stressed females exhibited higher anxiety levels, evidenced by longer open field entry latencies. Prenatal stress had no effect on object recognition memory, but eliminated the advantage normally seen in the male performance of a spatial memory task. Neurochemical profiles of prenatally stressed females were altered toward the masculine phenotype in the prefrontal cortex, amygdala, and hippocampus. Thus, prenatal stress altered subsequent cognitive, endocrine, and neurochemical responses in a sex-specific manner. These data reinforce the view that prenatal stress affects multiple aspects of brain development, interfering with the expression of normal behavioral, neuroendocrine, and neurochemical sex differences. These data have implications for the effects of prenatal stress on the development of sexually dimorphic endocrine and neurological disorders

High-Affinity Naloxone Binding to Filamin A Prevents Mu Opioid Receptor–Gs Coupling Underlying Opioid Tolerance and Dependence

Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [3H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [3H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A2561-2565 as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A2561-2565 abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor–Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence

Sex Differences in Cognition Following Variations in Endocrine Status

Spatial memory, mediated primarily by the hippocampus, is responsible for orientation in space and retrieval of information regarding location of objects and places in an animal\u27s environment. Since the hippocampus is dense with steroid hormone receptors and is capable of robust neuroplasticity, it is not surprising that changes in spatial memory performance occur following a variety of endocrine alterations. Here, we review cognitive changes in both spatial and nonspatial memory tasks following manipulations of the hypothalamic–pituitary–adrenal and gonadal axes and after exposure to endocrine disruptors in rodents. Chronic stress impairs male performance on numerous behavioral cognitive tasks and enhances or does not impact female cognitive function. Sex-dependent changes in cognition following stress are influenced by both organizational and activational effects of estrogen and vary depending on the developmental age of the stress exposure, but responses to gonadal hormones in adulthood are more similar than different in the sexes. Also discussed are possible underlying neural mechanisms for these steroid hormone-dependent, cognitive effects. Bisphenol A (BPA), an endocrine disruptor, given at low levels during adolescent development, impairs spatial memory in adolescent male and female rats and object recognition memory in adulthood. BPA\u27s negative effects on memory may be mediated through alterations in dendritic spine density in areas that mediate these cognitive tasks. In summary, this review discusses the evidence that endocrine status of an animal (presence or absence of stress hormones, gonadal hormones, or endocrine disruptors) impacts cognitive function and, at times, in a sex-specific manner

Age-Related Deficits in Spatial Memory and Hippocampal Spines in Virgin, Female Fischer 344 Rats

Effects of aging on memory and brain morphology were examined in aged, 21-month-old, and young, 4-month-old, Fischer 344 female rats. Spatial memory was assessed using the object placement task, and dendritic spine density was determined on pyramidal neurons in the hippocampus following Golgi impregnation. Consistent with previous studies, aged females showed poorer object placement performance than young subjects. Young subjects significantly discriminated the location of objects with a 1.5-hour intertrial delay while aged subjects did not. Spine density of basal dendrites on CA1 pyramidal cells was 16% lower in the aged subjects as compared to the young subjects. No differences in spine density were found between young and aged subjects in basal dendrites of CA1 or in either dendritic field of CA3 pyramidal neurons. Thus, decreased hippocampal CA1 dendritic spine density in aged rats may contribute to poorer spatial memory as compared to young rats. The possibility that the neuroplastic changes observed in this study may pertain only to female subjects having had a specific set of life experiences is discussed. Different factors, such as reproductive status, diet, and handling may contribute to neuroplasticity of the brain during aging; however, this view requires further examination