Simple and robust monitoring of ethanol fermentations by capillary electrophoresis.

Free-solution capillary electrophoresis (CE), or capillary zone electrophoresis, with direct UV detection was used for the first time for the determination of mono- and disaccharides, sugar alcohols, and ethanol in fermentation broths. Sample preparation proved to be minimal: no derivatization or specific sample purification was needed. The CE conditions can be adapted to the type of fermentation by simply altering the background electrolyte (BGE). KOH (130 mM) or NaOH (130 mM) as the BGE led to the fastest analysis time when monitoring simple fermentations. A mixture of 65 mM NaOH and 65 mM LiOH led to a 19% improvement in resolution for a complex mixture of carbohydrates. Quantification of a simple carbohydrate fermentation by CE showed values in close agreement with that of high-performance anion exchange chromatography and high-performance liquid chromatography (HPLC) on a cation exchange resin. For complex fermentations, quantification of carbohydrates by HPLC and CE led to similar results, whereas CE requires an injection volume of only 10-20 nL. Analysis of an ethanol fermentation of hydrolyzed plant fiber demonstrated the robustness of the separation and detection of carbohydrates, as well as ethanol. Ethanol determination is achieved by coupling the CE method to pressure mobilization, using the same instrument and the same sample.

Periadolescent maturation of the prefrontal cortex is sex-specific and is disrupted by prenatal stress.

The prefrontal cortex (PFC) undergoes dramatic, sex-specific maturation during adolescence. Adolescence is a vulnerable window for developing mental illnesses that show significant sexual dimorphisms. Gestational stress is associated with increased risk for both schizophrenia, which is more common among men, and cognitive deficits. We have shown that male, but not female, rats exposed to prenatal stress develop postpubertal deficits in cognitive behaviors supported by the prefrontal cortex. Here we tested the hypothesis that repeated variable prenatal stress during the third week of rat gestation disrupts periadolescent development of prefrontal neurons in a sex-specific fashion. Using Golgi-Cox stained tissue, we compared dendritic arborization and spine density of prelimbic layer III neurons in prenatally stressed and control animals at juvenile (day 20), prepubertal (day 30), postpubertal (day 56), and adult (day 90) ages (N = 115). Dendritic ramification followed a sex-specific pattern that was disrupted during adolescence in prenatally stressed males, but not in females. In contrast, the impact of prenatal stress on the female PFC was not evident until adulthood. Prenatal stress also caused reductions in brain and body weights, and the latter effect was more pronounced among males. Additionally, there was a trend toward reduced testosterone levels for adult prenatally stressed males. Our findings indicate that, similarly to humans, the rat PFC undergoes sex-specific development during adolescence and furthermore that this process is disrupted by prenatal stress. These findings may be relevant to both the development of normal sex differences in cognition as well as differential male-female vulnerability to psychiatric conditions.

NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy.

NADH is a naturally fluorescent metabolite associated with cellular respiration. Exploiting the different fluorescence lifetime of free and bound NADH has the potential to quantify the relative amount of bound and free NADH, enhancing understanding of cellular processes including apoptosis, cancer pathology, and enzyme kinetics. We use the phasor-fluorescence lifetime image microscopy approach to spatially map NADH in both the free and bound forms of live undifferentiated and differentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. Comparison of the spatial distribution of NADH in the nucleus of cells induced to differentiate through serum starvation and undifferentiated cells show differing distributions of bound and free NADH. Undifferentiated cells displayed a short lifetime indicative of free NADH in the nucleus and a longer lifetime attributed to the presence of bound NADH outside of the nucleus. Differentiating cells displayed redistribution of free NADH with decreased relative concentration of free NADH within the nucleus whereas the majority of NADH was found in the cytoplasm.

Sex steroids and schizophrenia.

The peak in incidence for schizophrenia is during late adolescence for both sexes, but within this time frame the peak is both earlier and steeper for males. Additionally, women have a second peak in incidence following menopause. Two meta-analyses have reported that men have an overall ∼40% greater chance of developing schizophrenia than do women (Aleman et al., 2003; McGrath et al., 2004). These and other findings have led to the suggestion that ovarian hormones may be protective against schizophrenia. Less explored is the potential role of testosterone in schizophrenia, although disruptions in steroid levels have also been reported in men with the illness. The relationship between increased gonadal hormone release per se and peri-adolescent vulnerability for psychiatric illness is difficult to tease apart from other potentially contributory factors in clinical studies, as adolescence is a turbulent period characterized by many social and biological changes. Despite the obvious opportunity provided by animal research, surprisingly little basic science effort has been devoted to this important issue. On the other hand, the animal work offers an understanding of the many ways in which gonadal steroids exert a powerful impact on the brain, both shaping its development and modifying its function during adulthood. Recently, investigators using preclinical models have described a greater male vulnerability to neurodevelopmental insults that are associated with schizophrenia; such studies may provide clinically relevant insights into the role of gonadal steroids in psychiatric illness.

Sex-specific neuroendocrine and behavioral phenotypes in hypomorphic Type II Neuregulin 1 rats.

Neuregulin 1 (NRG1) is an important growth factor involved in the development and plasticity of the central nervous system. Since its identification as a susceptibility gene for schizophrenia, several transgenic mouse models have been employed to elucidate the role NRG1 may play in the pathogenesis of psychiatric disease. Unfortunately very few studies have included females, despite the fact that some work suggests that the consequences of disrupted NRG1 expression may be sex-specific. Here, we used Nrg1 hypomorphic (Nrg1(Tn)) Fischer rats to demonstrate sex-specific changes in neuroendocrine and behavioral phenotypes as a consequence of reduced Type II NRG1 expression. We have previously shown that male Nrg1(Tn) rats have increased basal corticosterone levels, and fail to habituate to an open field despite normal overall levels of locomotor activity. The current studies show that, in contrast, female Nrg1(Tn) rats exhibit enhanced suppression of corticosterone levels following an acute stress, reduced locomotor activity, and enhanced habituation to novel environments. Furthermore, we also show that female, but not male, Nrg1(Tn) rats have impaired prepulse inhibition. Finally, we provide evidence that sex-specific changes are not likely attributable to major disruptions in the hypothalamic-pituitary-gonadal axis, as measures of pubertal onset, estrous cyclicity, and reproductive capacity were unaltered in female Nrg1(Tn) rats. Our results provide further support for both the involvement of NRG1 in the control of hypothalamic-pituitary-adrenal axis function and the sex-specific nature of this relationship.

Concurrent upregulation of postsynaptic L-type Ca(2+) channel function and protein kinase A signaling is required for the periadolescent facilitation of Ca(2+) plateau potentials and dopamine D1 receptor modulation in the prefrontal cortex.

Further understanding of how prefrontal cortex (PFC) circuit change during postnatal development is of great interest due to its role in working memory and decision-making, two cognitive abilities that are refined late in adolescence and become altered in schizophrenia. While it is evident that dopamine facilitation of glutamate responses occurs during adolescence in the PFC, little is known about the cellular mechanisms that support these changes. Among them, a developmental facilitation of postsynaptic Ca(2+) function is of particular interest given its role in coordinating neuronal ensembles, a process thought to contribute to maturation of PFC function. Here we conducted whole-cell patch clamp recordings of deep-layer pyramidal neurons in PFC brain slices and determined how somatic-evoked Ca(2+)-mediated plateau depolarizations change throughout postnatal day (PD) 25 (juvenile) to adulthood (PD 80). Postsynaptic Ca(2+) potentials in the PFC increase in duration throughout postnatal development. A remarkable shift from short to prolonged depolarizations was observed after PD 40. This change is reflected by an enhancement of L-type Ca(2+) channel function and postsynaptic PKA signaling. We speculate that such a protracted developmental facilitation of Ca(2+) response in the PFC may contribute to improvement of working memory performance through adolescence.

Prenatal stress: role in psychotic and depressive diseases.

RATIONALE: The birth of neurons, their migration to appropriate positions in the brain, and their establishment of the proper synaptic contacts happen predominately during the prenatal period. Environmental stressors during gestation can exert a major impact on brain development and thereby contribute to the pathogenesis of neuropsychiatric illnesses, such as depression and psychotic disorders including schizophrenia. OBJECTIVE: The objectives here are to present recent preclinical studies of the impact of prenatal exposure to gestational stressors on the developing fetal brain and discuss their relevance to the neurobiological basis of mental illness. The focus is on maternal immune activation, psychological stresses, and malnutrition, due to the abundant clinical literature supporting their role in the etiology of neuropsychiatric illnesses. RESULTS: Prenatal maternal immune activation, viral infection, unpredictable psychological stress, and malnutrition all appear to foster the development of behavioral abnormalities in exposed offspring that may be relevant to the symptom domains of schizophrenia and psychosis, including sensorimotor gating, information processing, cognition, social function, and subcortical hyperdopaminergia. Depression-related phenotypes, such as learned helplessness or anxiety, are also observed in some model systems. These changes appear to be mediated by the presence of proinflammatory cytokines and/or corticosteroids in the fetal compartment that alter the development the neuroanatomical substrates involved in these behaviors. CONCLUSION: Prenatal exposure to environmental stressors alters the trajectory of brain development and can be used to generate animal preparations that may be informative in understanding the pathophysiological processes involved in several human neuropsychiatric disorders.

Characterization of the cognitive impairments induced by prenatal exposure to stress in the rat.

We have previously shown that male rats exposed to gestational stress exhibit phenotypes resembling what is observed in schizophrenia, including hypersensitivity to amphetamine, blunted sensory gating, disrupted social behavior, impaired stress axis regulation, and aberrant prefrontal expression of genes involved in synaptic plasticity. Maternal psychological stress during pregnancy has been associated with adverse cognitive outcomes among children, as well as an increased risk for developing schizophrenia, which is characterized by significant cognitive deficits. We sought to characterize the long-term cognitive outcome of prenatal stress using a preclinical paradigm, which is readily amenable to the development of novel therapeutic strategies. Rats exposed to repeated variable prenatal stress during the third week of gestation were evaluated using a battery of cognitive tests, including the novel object recognition task, cued and contextual fear conditioning, the Morris water maze, and iterative versions of a paradigm in which working and reference memory for both objects and spatial locations can be assessed (the "Can Test"). Prenatally stressed males were impaired relative to controls on each of these tasks, confirming the face validity of this preclinical paradigm and extending the cognitive implications of prenatal stress exposure beyond the hippocampus. Interestingly, in experiments where both sexes were included, the performance of females was found to be less affected by prenatal stress compared to that of males. This could be related to the finding that women are less vulnerable than men to schizophrenia, and merits further investigation.

Myelination of the corpus callosum in male and female rats following complex environment housing during adulthood.

Myelination is an important process in brain development, and delays or abnormalities in this process have been associated with a number of conditions including autism, developmental delay, attention deficit disorder, and schizophrenia. Myelination can be sensitive to developmental experience; however, although the adult brain remains highly plastic, it is unknown whether myelination continues to be sensitive to experience during adulthood. Male and female rats were socially housed until four months of age, at which time they were moved into either a complex or "enriched" environment (EC) or an isolated condition (IC). Although the area of the splenium (posterior 20% of the callosum, which contains axons from visual cortical neurons) increased by about 10% following two months of EC housing, the area occupied by myelinated axons was not influenced by adult housing condition. Instead, it was the area occupied by glial cell processes and unmyelinated axons which significantly increased following EC housing. Neither the size nor the myelin content of the genu (anterior 15% of the callosum) was sensitive to manipulations of adult housing condition, but males had more area occupied by myelinated axons in both callosal regions. Finally, the inability of two months of complex environment housing during adulthood to impact the number of myelinated axons in the splenium was confirmed in a subset of animals using quantitative electron microscopy. We conclude that the sensitivity of myelination to experience is reduced in adulthood relative to development in both sexes.

Architectural changes to CA1 pyramidal neurons in adult and aged mice after peripheral immune stimulation.

The expression of several inflammatory cytokines that inhibit synaptic plasticity and hippocampal-dependent learning and memory is higher in the brains of aged mice compared to young adults after peripheral injection of lipopolysaccharide (LPS). In this study we investigated whether the exaggerated inflammatory cytokine response in the hippocampus of aged mice after IP injection of LPS is associated with architectural changes to dendrites of pyramidal neurons in the dorsal CA1 hippocampus. Compared to young adults, aged mice had higher basal expression of MHC class II, lower basal expression of two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and a decrease in total dendritic length in both the basal and apical tree. After IP LPS administration, expression of IL-1beta, IL-6, and TNFalpha mRNA was higher in hippocampus of aged mice compared to young adults whereas NGF and BDNF mRNA was reduced similarly in both age groups. The basal dendritic tree was not affected by LPS in either adult or aged mice 72h after treatment; however, length and branching of the apical tree was reduced by LPS in aged but not adult mice. The present findings indicate that a peripheral infection in the aged can cause a heightened inflammatory cytokine response in the hippocampus and atrophy of hippocampal neurons. Architectural changes to dorsal CA1 hippocampal neurons may contribute to cognitive disorders evident in elderly patients with an infection.

Aberrant early-phase ERK inactivation impedes neuronal function in fragile X syndrome.

Fragile X syndrome (FXS) has so far resisted efforts to define the basic cellular defects caused by the absence of a single protein, fragile X mental retardation protein (FMRP), because the patients have a wide variety of symptoms of varying severity. Immature-appearing dendritic spines on neurons found in FXS patients and fmr1-KO mice suggest a role for FMRP in modulating production of synaptic structural proteins. We isolated cortical synaptoneurosomes from WT and KO mice and studied MAPK pathway activation after group I metabotropic glutamate receptor (mGluR) stimulation. Here, we show that ERK in KO synaptoneurosomes is rapidly dephosphorylated upon mGluR1/5 stimulation, whereas it is phosphorylated in WT mice, suggesting that aberrant activation of phosphatases occurs in KO synapses in response to synaptic stimulation. In KO synapses, protein phosphatase 2A (PP2A) is overactivated after mGluR1 stimulation, and tyrosine phosphatase is overactivated after mGluR5 stimulation, causing the rapid deactivation of ERK. ERK activation can be restored in KO by pretreatment with phosphatase blockers; blocking of PP2A by okadaic acid could successfully restore normal ERK activation in KO synaptoneurosomes. We propose that overactivation of phosphatases in synapses may be a key deficit in FXS, which affects synaptic translation, transcription, and synaptic receptor regulation.

Social recognition memory: influence of age, sex, and ovarian hormonal status.

Social recognition memory underlies many forms of rodent interaction and can be easily tested in the laboratory. Sex differences in aspects of this memory have been reported among young adults, and some studies indicate an age-related decline among male rats. In contrast, neither the impact of natural fluctuations in ovarian hormones nor the performance of aged female rats on social recognition memory has been previously evaluated. In experiments 1 and 2, the social recognition memory of young adult female Long-Evans rats (age 3-5 months) was compared during proestrus and estrus, and performance was found to be stable across estrous cycle phases. In experiment 3, the social recognition memory of young adults as compared to aged (16.5-19.5 months) rats was tested using the social discrimination procedure, following delays of 15, 45, 90 or 120 min. The estropausal status of aged female rats was tracked during the experiment but was not found to influence memory ability. Males of both ages investigated juveniles (both novel and familiar) more than did females, although despite this difference, both sexes demonstrated robust memory. Interestingly, only young adult females were capable of demonstrating memory following the longest delay. Collectively, our findings indicate that the pattern of age-related changes in social recognition memory is subtle and that aging does not greatly alter the behavioral sex differences observed among young adults.

Motor learning induces astrocytic hypertrophy in the cerebellar cortex.

Motor skill learning, but not mere motor activity, is associated with an increase in both synapse number and glial cell volume within the cerebellar cortex. The increase in synapse number has been shown to persist for at least 4 weeks in the absence of continued training. The present experiment similarly examined how a prolonged interruption in training affects the training-induced increase in astrocytic volume. Adult female rats were randomly allocated to either an acrobatic motor learning condition (AC) or a motor control condition (MC). The AC animals were trained to traverse a complex series of obstacles and each AC animal was pair matched with an MC animal that traversed an obstacle-free runway. These groups were further assigned to one of three training conditions. Animals in the early condition were trained for 10 consecutive days, animals in the delay condition received the same 10 days of training followed by a 28-day period without training, and animals in the continuous condition were trained for the entire 38 days. Unbiased stereological techniques were used to determine that AC animals had a significantly greater volume of astrocytes per Purkinje cell in the cerebellar paramedian lobule than the MC animals, a difference which was reduced (and not statistically detectable) among animals in the delay condition. These findings demonstrate that learning triggers the hypertrophy of astrocytic processes and furthermore that, unlike learning-induced synaptogenesis, astrocytic growth is reduced in the absence of continued training.

Corticosterone response to acute stress in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS), the most common form of inherited mental retardation, results from the silencing of the Fmr1 gene that encodes the Fragile X mental retardation protein (FMRP). Because (1) mRNA for the glucocorticoid receptor is bound by FMRP and (2) the response to acute stress is elevated in children with FXS, we examined whether this heightened response is characteristic of a mouse model of FXS. Fmr1 knockout (KO) and wildtype (WT) control mice were exposed to 30 min of acute restraint; serum corticosterone levels were assayed from unstressed animals and those examined either immediately following stress or after a 15 or 60 min recovery period. Under unstressed conditions, KOs and WTs did not differ in serum corticosterone, although both genotype and sex affected corticosterone levels observed following exposure to acute stress. Similar to FXS patients, serum glucocorticoid levels of KO mice exhibited a protracted return to baseline following acute stress. This suggests that the stress response is misregulated in Fmr1 KO mice as in FXS patients and provides the first evidence for a link between a particular FMRP-binding mRNA and a functional phenotype of FXS (impaired glucocorticoid negative feedback).

Antioxidant activity in Australian native sarsaparilla (Smilax glyciphylla).

A hot water extract of the Australian native sarsaparilla Smilax glyciphylla Sm. (Smilaceae) inhibited peroxidation of phosphatidylcholine liposomes initiated by Fe(2+)/ascorbate (IC50, 10 microg/mL) and AAPH (IC50, 33 microg/mL) in vitro. It also inhibited deoxyribose degradation and quenched chemically generated superoxide anion (IC50, 50 microg/mL). Reactivity towards ABTS (2,2'-azinobis(3-ethylbenzothiazoline 6-sulphonate) radical cation was equivalent to 48.4 mM TROLOX, the water soluble alpha-tocopherol analogue. Smilax glyciphylla is a rich source of the dihydrochalcone glycyphyllin. Given the reported level of activity it is unlikely that glycyphyllin would provide direct antioxidant protection in tissues affected by oxidative stress. However, consuming Smilax glyciphylla as a tea may be sufficient to reduce oxidative damage in the gastrointestinal tract. It is also possible that glycyphyllin is metabolised and adsorbed as phloretin, a compound with known anticancer properties. These findings indicate that further studies of the chemopreventative properties of Smilax glyciphylla is warranted.

The cellular basis for volume changes in the rat cortex during puberty: white and gray matter.

We have found that developmental changes through the adolescent period in the rat cerebral cortex provide parallels to those seen in the human cortex. Like humans, the rat cerebral white matter increases during this time due to increases in the number of axons that become myelinated even while the total number of axons decreases. We have preliminary evidence that estrogen decreases the rate of myelination, which results in a sex difference in adult rats. Another parallel to the human cortex is the nonlinear changes in the size of the cortex. We have found that in some cortical regions, female rats show decreases in cortical volume and number of neurons across the time of puberty, and removal of the ovaries stops these decreases. The rat cortex may serve as a model for the cellular changes underlying the volume changes seen in adolescent humans.

Experience-driven brain plasticity: beyond the synapse.

The brain is remarkably responsive to its interactions with the environment, and its morphology is altered by experience in measurable ways. Histological examination of the brains of animals exposed to either a complex ('enriched') environment or learning paradigm, compared with appropriate controls, has illuminated the nature of experience-induced morphological plasticity in the brain. For example, this research reveals that changes in synapse number and morphology are associated with learning and are stable, in that they persist well beyond the period of exposure to the learning experience. In addition, other components of the nervous system also respond to experience: oligodendrocytes and axonal myelination might also be permanently altered, whereas changes in astrocytes and cerebrovasculature are more transient and appear to be activity- rather than learning-driven. Thus, experience induces multiple forms of plasticity in the brain that are apparently regulated, at least in part, by independent mechanisms.

Aging and sex influence the anatomy of the rat anterior cingulate cortex.

Cognitive processes supported by the prefrontal cortex undergo an age-related decline. Until very recently, nonhuman animal models of aging have relied on the exclusive use of male subjects. This study was designed to investigate the influence of age, sex, and ovarian hormonal state on anatomy of the rat medial prefrontal cortex (anterior cingulate cortex). Dendritic tree extent and spine density were examined in young adult (3-5 mos.) and aged (20-24 mos.) male and female rats. Young adult females were examined either at proestrus or estrus, and aged females were examined in one of two reproductively senescent (estropausal) phases, persistent estrus or persistent diestrus. Neither the estrous cycle nor state of estropause influenced spine density or dendritic tree extent. However, the anatomy of the anterior cingulate cortex of young adult rats was sexually dimorphic, with males having greater dendritic spine density as well as arborization. While there was a reduction in density and tree extent with age for both sexes, this reduction was more pronounced for males, resulting in a disappearance of most sex differences with age. Thus the results of this study suggest that aging of the rodent cerebral cortex may follow a sexually dimorphic pattern.