PKCε-dependent H-Ras activation encompasses the recruitment of the RasGEF SOS1 and of the RasGAP neurofibromin in the lipid rafts of embryonic neurons.

The spatial organization of plasma membrane proteins is a key factor in the generation of distinct signal outputs, especially for PKC/Ras/ERK signalling. Regulation of activation of the membrane-bound Ras, critical for neuronal differentiation and highly specialized functions, is controlled by exchanges in nucleotides catalyzed by nucleotide exchange factors (GEFs) for GTP loading and Ras activation, and by Ras GTPase Activated Proteins (RasGAPs) that lead to activation of the intrinsic GTPase activity of Ras and thus its inactivation. PKCs are potent Ras activators yet the mechanistic details of these interactions, or the involvement of specific PKC isoforms are now beginning to be addressed. Even less known is the topology where RasGAPs terminate Ras activation. Towards this aim, we isolated lipid rafts from chick embryo neural tissue and primary neuronal cultures when PKCε is the prominent isoform and in combination with in vitro kinase assays, we now show that, in response the PKCε-specific activating peptide ψεRACK, an activated PKCε is recruited to lipid rafts; similar mobility was established when PKCε was physiologically activated with the Cannabinoid receptor 1 (CB1) agonist methanandamide. Activation of H-Ras for both agents was then established for the first time using in vivo RasGAP activity assays, which showed similar temporal profiles of activation and lateral mobility. Moreover, we found that the GEF SOS1, and the major neuronal RasGAP neurofibromin, a specific PKCε substrate, were both transiently significantly enriched in the rafts. Finally, our in silico analysis revealed a highly probable, conserved palmitoylation site adjacent to a CARC motif on neurofibromin, both of which are included only in the RasGAP related domain type I (GRDI) with the known high H-RasGAP activity. Taken together, these results suggest that PKCε activation regulates the spatial plasma membrane enrichments of both SOS1 and neurofibromin, thus controlling the output of activated H-Ras available for downstream signalling in neurons.

Rapamycin blocks the neuroprotective effects of sex steroids in the adult birdsong system.

In adult songbirds, the telencephalic song nucleus HVC and its efferent target RA undergo pronounced seasonal changes in morphology. In breeding birds, there are increases in HVC volume and total neuron number, and RA neuronal soma area compared to nonbreeding birds. At the end of breeding, HVC neurons die through caspase-dependent apoptosis and thus, RA neuron size decreases. Changes in HVC and RA are driven by seasonal changes in circulating testosterone (T) levels. Infusing T, or its metabolites 5α-dihydrotestosterone (DHT) and 17 ß-estradiol (E2), intracerebrally into HVC (but not RA) protects HVC neurons from death, and RA neuron size, in nonbreeding birds. The phosphoinositide 3-kinase (PI3K)-Akt (a serine/threonine kinase)-mechanistic target of rapamycin (mTOR) signaling pathway is a point of convergence for neuroprotective effects of sex steroids and other trophic factors. We asked if mTOR activation is necessary for the protective effect of hormones in HVC and RA of adult male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii). We transferred sparrows from breeding to nonbreeding hormonal and photoperiod conditions to induce regression of HVC neurons by cell death and decrease of RA neuron size. We infused either DHT + E2, DHT + E2 plus the mTOR inhibitor rapamycin, or vehicle alone in HVC. Infusion of DHT + E2 protected both HVC and RA neurons. Coinfusion of rapamycin with DHT + E2, however, blocked the protective effect of hormones on HVC volume and neuron number, and RA neuron size. These results suggest that activation of mTOR is an essential downstream step in the neuroprotective cascade initiated by sex steroid hormones in the forebrain.

Aluminum affects neural phenotype determination of embryonic neural progenitor cells.

Aluminum (Al) is a neurotoxin and is associated with the etiology of neurodegenerative diseases, such as Alzheimer's disease (AD). The Al-free ion (Al3+) is the biologically reactive and toxic form. However, the underlying mechanisms of Al toxicity in the brain remain unclear. Here, we evaluated the effects of Al3+ (in the chloride form-AlCl3) at different concentrations (0.1-100 µM) on the morphology, proliferation, apoptosis, migration and differentiation of neural progenitor cells (NPCs) isolated from embryonic telencephalons, cultured as neurospheres. Our results reveal that Al3+ at 100 µM reduced the number and diameter of neurospheres. Cell cycle analysis showed that Al3+ had a decisive function in proliferation inhibition of NPCs during neural differentiation and induced apoptosis on neurospheres. In addition, 1 µM Al3+ resulted in deleterious effects on neural phenotype determination. Flow cytometry and immunocytochemistry analysis showed that Al3+ promoted a decrease in immature neuronal marker ß3-tubulin expression and an increase in co-expression of the NPC marker nestin and glial fibrillary acidic protein. Thus, our findings indicate that Al3+ caused cellular damage and reduced proliferation and migration, resulting in global inhibition of NPC differentiation and neurogenesis.

Na+/K+-ATPase is involved in the regulation of food intake in rainbow trout but apparently not through brain glucosensing mechanisms.

To assess the hypothesis that Na+/K+-ATPase (NKA) is involved in the central regulation of food intake in fish, we observed in a first experiment with rainbow trout (Oncorhynchus mykiss) that intracerebroventricular (ICV) treatment with ouabain decreased food intake. We hypothesized that this effect relates to modulation of glucosensing mechanisms in brain areas (hypothalamus, hindbrain, and telencephalon) involved in food intake control. Therefore, we evaluated in a second experiment, the effect of ICV administration of ouabain, in the absence or in the presence of glucose, on NKA activity, mRNA abundance of different NKA subunits, parameters related to glucosensing, transcription factors, and appetite-related neuropeptides in brain areas involved in the control of food intake. NKA activity and mRNA abundance of nkaα1a and nkaα1c in brain were inhibited by ouabain treatment and partially by glucose. The anorectic effect of ouabain is opposed to the orexigenic effect reported in mammals. The difference might relate to the activity of glucosensing as well as downstream mechanisms involved in food intake regulation. Ouabain inhibited glucosensing mechanisms, which were activated by glucose in hypothalamus and telencephalon. Transcription factors and neuropeptides displayed responses comparable to those elicited by glucose when ouabain was administered alone, but not when glucose and ouabain were administered simultaneously. Ouabain might therefore affect other processes, besides glucosensing mechanisms, generating changes in membrane potential and/or intracellular pathways finally modulating transcription factors and neuropeptide mRNA abundance leading to modified food intake.

Testosterone or Estradiol When Implanted in the Medial Preoptic Nucleus Trigger Short Low-Amplitude Songs in Female Canaries.

In male songbirds, the motivation to sing is largely regulated by testosterone (T) action in the medial preoptic area, whereas T acts on song control nuclei to modulate aspects of song quality. Stereotaxic implantation of T in the medial preoptic nucleus (POM) of castrated male canaries activates a high rate of singing activity, albeit with a longer latency than after systemic T treatment. Systemic T also increases the occurrence of male-like song in female canaries. We hypothesized that this effect is also mediated by T action in the POM. Females were stereotaxically implanted with either T or with 17ß-estradiol (E2) targeted at the POM and their singing activity was recorded daily during 2 h for 28 d until brains were collected for histological analyses. Following identification of implant localizations, three groups of subjects were constituted that had either T or E2 implanted in the POM or had an implant that had missed the POM (Out). T and E2 in POM significantly increased the number of songs produced and the percentage of time spent singing as compared with the Out group. The songs produced were in general of a short duration and of poor quality. This effect was not associated with an increase in HVC volume as observed in males, but T in POM enhanced neurogenesis in HVC, as reflected by an increased density of doublecortin-immunoreactive (DCX-ir) multipolar neurons. These data indicate that, in female canaries, T acting in the POM plays a significant role in hormone-induced increases in the motivation to sing.

Wnt-2b in the intermediate hyperpallium apicale of the telencephalon is critical for the thyroid hormone-mediated opening of the sensitive period for filial imprinting in domestic chicks (Gallus gallus domesticus).

Filial imprinting is the behavior observed in chicks during the sensitive or critical period of the first 2-3 days after hatching; however, after this period they cannot be imprinted when raised in darkness. Our previous study showed that temporal augmentation of the endogenous thyroid hormone 3,5,3'-triiodothyronine (T3) in the telencephalon, by imprinting training, starts the sensitive period just after hatching. Intravenous injection of T3 enables imprinting of chicks on days 4 or 6 post-hatching, even when the sensitive period has ended. However, the molecular mechanism of how T3 acts as a determinant of the sensitive period is unknown. Here, we show that Wnt-2b mRNA level is increased in the T3-injected telencephalon of 4-day old chicks. Pharmacological inhibition of Wnt signaling in the intermediate hyperpallium apicale (IMHA), which is the caudal area of the telencephalon, blocked the recovery of the sensitive period following T3 injection. In addition, injection of recombinant Wnt-2b protein into the IMHA helped chicks recover the sensitive period without the injection of T3. Lastly, we showed Wnt signaling to be involved in imprinting via the IMHA region on day 1 during the sensitive period. These results indicate that Wnt signaling plays a critical role in the opening of the sensitive period downstream of T3.

Endocrine disrupting effects of waterborne fluoxetine exposure on the reproductive axis of female goldfish, Carassius auratus.

Evidence suggests that pharmaceuticals and personal care products reach urban watersheds, bioconcentrate in fish, and potentially disrupt physiological homeostasis. These impairments often affect hormone functions. Selective serotonin reuptake inhibitors (SRRIs) are increasingly studied with regards to their endocrine disrupting effects on teleost physiological processes, including reproduction. To examine whether FLX effects on the endocrine regulation of reproductive physiology in goldfish are sex-specific, we exposed sexually recrudescent female goldfish to two waterborne concentrations of FLX (0.54µg/L and 54µg/L) using an experimental design previously used for sexually mature male goldfish. To evaluate possible endocrine disrupting effects, we quantified the gonadosomatic index, circulating hormone concentrations (luteinizing hormone, LH; growth hormone, GH; 17-ß estradiol, E2; and testosterone, T), and the expression of isotocin and vasotocin in the telencephalon, gonadotropin subunits and GH in the pituitary, and gonadotropin receptors, GH receptors, and aromatase in the ovary. Female goldfish exposed to 0.54µg/L FLX exhibited a significant decrease in circulating E2, and conversely, a significant increase in circulating LH and ovarian aromatase mRNA levels, suggesting disruption of E2-mediated feedback on LH release. These results, when compared with those previously observed in males, reveal that waterborne exposure to environmentally relevant levels of FLX sex-specifically disrupts the reproductive endocrine axis in goldfish, characterized by a decrease in E2 in females, and conversely, estrogen-like effects in males. These data emphasize the importance of studying the effect of endocrine disrupting chemicals on both sexes.

WNT-C59, a Small-Molecule WNT Inhibitor, Efficiently Induces Anterior Cortex That Includes Cortical Motor Neurons From Human Pluripotent Stem Cells.

UNLABELLED: The recapitulation of human neural development in a controlled, defined manner from pluripotent stem cells (PSCs) has considerable potential for studies of human neural development, circuit formation and function, and the construction of in vitro models of neurological diseases. The inhibition of Wnt signaling, often by the recombinant protein DKK1, is important for the induction of cortical neurons. Here, we report a novel differentiation method using a small-molecule WNT inhibitor, WNT-C59 (C59), to efficiently induce human anterior cortex. We compared two types of small molecules, C59 and XAV939 (XAV), as substitutes for DKK1 to induce cortical neurons from PSCs in serum-free embryoid body-like aggregate culture. DKK1 and XAV inhibited only the canonical pathway of Wnt signaling, whereas C59 inhibited both the canonical and noncanonical pathways. C59 efficiently induced CTIP2+/COUP-TF1- cells, which are characteristic of the cells found in the anterior cortex. In addition, when grafted into the cortex of adult mice, the C59-induced cells showed abundant axonal fiber extension toward the spinal cord. These results raise the possibility of C59 contributing to cell replacement therapy for motor neuron diseases or insults. SIGNIFICANCE: For a cell therapy against damaged corticospinal tract caused by neurodegenerative diseases or insults, cortical motor neurons are needed. Currently, their induction from pluripotent stem cells is considered very promising; however, an efficient protocol to induce motor neurons is not available. For efficient induction of anterior cortex, where motor neurons are located, various WNT inhibitors were investigated. It was found that one of them could induce anterior cortical cells efficiently. In addition, when grafted into the cortex of adult mice, the induced cells showed more abundant axonal fiber extension toward spinal cord. These results raise the possibility that this inhibitor contributes to a cell-replacement therapy for motor neuron diseases or insults.

Stimulatory effect of the secretogranin-ll derived peptide secretoneurin on food intake and locomotion in female goldfish (Carassius auratus).

Secretoneurin (SN) is a conserved peptide derived by proteolytic processing from the middle domain of the ∼600 amino acid precursor secretogranin-II (SgII). Secretoneurin is widely distributed in secretory granules of endocrine cells and neurons and has important roles in reproduction as it stimulates luteinizing hormone release from the pituitary. A potential new role of SN in goldfish feeding is the subject of this study. Firstly, we established that acute (26 h; p<0.0001) and short-term (72 h; p=0.016) fasting increased SgIIa precursor mRNA levels 1.25-fold in the telencephalon, implicating SN in the control of feeding. Secondly, we determined that intracerebroventricular injections of the type A SN (SNa; 0.2 and 1 ng/g BW) increased food intake and locomotor behavior by 60 min. Fish injected with the lower and higher doses of SNa (0.2 and 1 ng/g) respectively exhibited significant 1.77- and 2.58-fold higher food intake (p<0.0001) than the saline-injected control fish. Locomotor behavior was increased by 1.35- and 2.26-fold for 0.2 ng/g SNa (p=0.0001) and 1 ng/g SNa (p<0.0001), respectively. Injection of 1 ng/g SNa increased mRNA levels of hypothalamic neuropeptide Y 1.36-fold (p=0.038) and decreased hypothalamic cocaine-and amphetamine-regulated transcript by 33% (p=0.01) at 2h and 5h post-injection, respectively. These data suggest interactions of SNa with stimulatory and inhibitory pathways of food intake control in fish.

Deletion of Gαq in the telencephalon alters specific neurobehavioral outcomes.

G(αq) -coupled receptors are ubiquitously expressed throughout the brain and body, and it has been shown that these receptors and associated signaling cascades are involved in a number of functional outputs, including motor function and learning and memory. Genetic alterations to G(αq) have been implicated in neurodevelopmental disorders such as Sturge-Weber syndrome. Some of these associated disease outcomes have been modeled in laboratory animals, but as G(αq) is expressed in all cell types, it is difficult to differentiate the underlying circuitry or causative neuronal population. To begin to address neuronal cell type diversity in G(αq) function, we utilized a conditional knockout mouse whereby G(αq) was eliminated from telencephalic glutamatergic neurons. Unlike the global G(αq) knockout mouse, we found that these conditional knockout mice were not physically different from control mice, nor did they exhibit any gross motor abnormalities. However, similarly to the constitutive knockout animal, G(αq) conditional knockout mice demonstrated apparent deficits in spatial working memory. Loss of G(αq) from glutamatergic neurons also produced enhanced sensitivity to cocaine-induced locomotion, suggesting that cortical G(αq) signaling may limit behavioral responses to psychostimulants. Screening for a variety of markers of forebrain neuronal architecture revealed no obvious differences in the conditional knockouts, suggesting that the loss of G(αq) in telencephalic excitatory neurons does not result in major alterations in brain structure or neuronal differentiation. Taken together, our results define specific modulation of spatial working memory and psychostimulant responses through disruptions in G(αq) signaling within cerebral cortical glutamatergic neurons.

Emotions and motivated behavior converge on an amygdala-like structure in the zebrafish.

The brain reward circuitry plays a key role in emotional and motivational behaviors, and its dysfunction underlies neuropsychiatric disorders such as schizophrenia, depression and drug addiction. Here, we characterized the neuronal activity pattern induced by acute amphetamine administration and during drug-seeking behavior in the zebrafish, and demonstrate the existence of conserved underlying brain circuitry. Combining quantitative analyses of cfos expression with neuronal subtype-specific markers at single-cell resolution, we show that acute d-amphetamine administration leads to both increased neuronal activation and the recruitment of neurons in the medial (Dm) and the lateral (Dl) domains of the adult zebrafish pallium, which contain homologous structures to the mammalian amygdala and hippocampus, respectively. Calbindin-positive and glutamatergic neurons are recruited in Dm, and glutamatergic and γ-aminobutyric acid (GABAergic) neurons in Dl. The drug-activated neurons in Dm and Dl are born at juvenile stage rather than in the embryo or during adulthood. Furthermore, the same territory in Dm is activated during both drug-seeking approach and light avoidance behavior, while these behaviors do not elicit activation in Dl. These data identify the pallial territories involved in acute psychostimulant response and reward formation in the adult zebrafish. They further suggest an evolutionarily conserved function of amygdala-like structures in positive emotions and motivated behavior in zebrafish and mammals.

Aromatase inhibitor letrozole downregulates steroid receptor coactivator-1 in specific brain regions that primarily related to memory, neuroendocrine and integration.

As one of the third generation of aromatase inhibitors, letrozole is a favored drug for the treatment of hormone receptor-positive breast cancer with some adverse effects on the nervous system, but the knowledge is limited and the results are controversial, the mechanism underlying its central action is also unclear. Accumulated evidences have demonstrated that estrogens derived from androgens by aromatase play profound roles in the brain through their receptors, which needs coactivator for the transcription regulation, among which steroid receptor coactivator-1 (SRC-1) has been shown to be multifunctional potentials in the brain, but whether it is regulated by letrozole is currently unknown. In this study, we examined letrozole regulation on SRC-1 expression in adult mice brain using immunohistochemistry. The results showed that letrozole induced dramatic decrease of SRC-1 in the medial septal, hippocampus, medial habenular nucleus, arcuate hypothalamic nucleus and superior colliculus (p<0.01). Significant decrease was detected in the dorsal lateral septal nucleus, bed nucleus of stria terminalis, ventral taenia tecta, dorsomedial and ventromedial hypothalamic nuclei, dorsomedial periaqueductal gray, superior paraolivary nucleus and pontine nucleus (p<0.05). In the hippocampus, levels of estradiol content, androgen receptor, estrogen receptor α and ß also decreased significantly after letrozole injection. The above results demonstrated letrozole downregulation of SRC-1 in specific regions that are primarily related to learning and memory, cognition and mood, neuroendocrine as well as information integration, indicating that SRC-1 may be one important downstream central target of letrozole. Furthermore, these potential central adverse effects of letrozole should be taken into serious considerations.

Exposure to benzidine caused apoptosis and malformation of telencephalon region in zebrafish.

Exposure to benzidine has been known to induce human cancers, particularly bladder carcinomas. In this study, the zebrafish model was used to investigate the developmental toxicity of benzidine. Embryos at 6 h postfertilization (hpf) that were exposed to benzidine exhibited embryonic death in a dose- and time-dependent manner. Benzidine induced malformations in zebrafish, such as small brain development, shorter axes, and a slight pericardial edema. High concentrations (50, 100, and 200 µM) of benzidine triggered widespread apoptosis in the brain and dorsal neurons, as evidenced by acridine orange and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays. Real-time polymerase chain reaction analysis also showed that benzidine treatment affected p53, bax, and noxa expression. Decreases in specific brain markers, such as emx1 in the telencephalon, ngn1 in differentiated neurons, and otx2 in the midbrain, were observed in benzidine-treated embryos at 24 hpf. Conversely, no overt changes to pax2.1 expression in the midbrain-hindbrain boundary were found. Moreover, the use of Tg(HuC:GFP) zebrafish showed that benzidine caused a malformation of the telencephalon region. Our findings show that benzidine exposure triggers widespread apoptosis in the zebrafish brain and dorsal neurons, resulting in the development of an abnormal telencephalon.

Adenosine receptor antagonists including caffeine alter fetal brain development in mice.

Consumption of certain substances during pregnancy can interfere with brain development, leading to deleterious long-term neurological and cognitive impairments in offspring. To test whether modulators of adenosine receptors affect neural development, we exposed mouse dams to a subtype-selective adenosine type 2A receptor (A2AR) antagonist or to caffeine, a naturally occurring adenosine receptor antagonist, during pregnancy and lactation. We observed delayed migration and insertion of γ-aminobutyric acid (GABA) neurons into the hippocampal circuitry during the first postnatal week in offspring of dams treated with the A2AR antagonist or caffeine. This was associated with increased neuronal network excitability and increased susceptibility to seizures in response to a seizure-inducing agent. Adult offspring of mouse dams exposed to A2AR antagonists during pregnancy and lactation displayed loss of hippocampal GABA neurons and some cognitive deficits. These results demonstrate that exposure to A2AR antagonists including caffeine during pregnancy and lactation in rodents may have adverse effects on the neural development of their offspring.

Aging and substitutive hormonal therapy influence in regional and subcellular distribution of ERα in female rat brain.

Estrogens are not only critical for sexual differentiation it is well-known for the role of 17ß-estradiol (E2) in the adult brain modulating memory, learning, mood and acts as a neuroprotector. E2 exerts its actions through two classical receptors: estrogen receptor alpha (ERα) and estrogen receptor beta (ERß). The distribution of both receptors changes from one brain area to another, E2 being able to modulate their expression. Among the classical features of aging in humans, we find cognitive impairment, dementia, memory loss, etc. As estrogen levels change with age, especially in females, it is important to know the effects of low E2 levels on ERα distribution; results from previous studies are controversial regarding this issue. In the present work, we have studied the effects of long-term E2 depletion as well as the ones of E2 treatment on ERα brain distribution of ovariectomized rats along aging in the diencephalon and in the telencephalon. We have found that ovariectomy causes downregulation and affects subcellular localization of ERα expression during aging, meanwhile prolonged estrogen treatment produces upregulation and overexpression of the receptor levels. Our results support the idea of the region-specific neuroprotection mechanisms mediated by estradiol.

Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions.

To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM) identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease.

CHOP potentially co-operates with FOXO3a in neuronal cells to regulate PUMA and BIM expression in response to ER stress.

Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in various neurodegenerative diseases including Parkinson Disease, Alzheimer Disease and Huntington Disease. PUMA (p53 upregulated modulator of apoptosis) and BIM (BCL2 interacting mediator of cell death), pro-apoptotic BH3 domain-only, BCL2 family members, have previously been shown to regulate ER stress-induced cell death, but the upstream signaling pathways that regulate this response in neuronal cells are incompletely defined. Consistent with previous studies, we show that both PUMA and BIM are induced in response to ER stress in neuronal cells and that transcriptional induction of PUMA regulates ER stress-induced cell death, independent of p53. CHOP (C/EBP homologous protein also known as GADD153; gene name Ddit3), a critical initiator of ER stress-induced apoptosis, was found to regulate both PUMA and BIM expression in response to ER stress. We further show that CHOP knockdown prevents perturbations in the AKT (protein kinase B)/FOXO3a (forkhead box, class O, 3a) pathway in response to ER stress. CHOP co-immunoprecipitated with FOXO3a in tunicamycin treated cells, suggesting that CHOP may also regulate other pro-apoptotic signaling cascades culminating in PUMA and BIM activation and cell death. In summary, CHOP regulates the expression of multiple pro-apoptotic BH3-only molecules through multiple mechanisms, making CHOP an important therapeutic target relevant to a number of neurodegenerative conditions.

Endosulfan and flutamide, alone and in combination, target ovarian growth in juvenile catfish, Clarias batrachus.

Juvenile Catfish(es), Clarias batrachus of 50 days post hatch (dph) were exposed to endosulfan (2.5 parts per billion [ppb]) and flutamide (33 ppb), alone and in combination for 50 days to access their impact on ovarian development. The doses used in this study were nominal considering pervious reports. Sampling was done at 100 dph to perform histology and measurement of various transcripts, estradiol-17ß and aromatase activity. In general, treatments enhanced expression of ovary-specific transcription factors, steroidogenic enzymes steroidogenic acute regulatory protein and aromatases while transcripts of tryptophan hydroxylase2 (tph2) and catfish gonadotropin-releasing hormone declined in the brain of all treated groups with maximum reduction in the endosulfan group. Significant reduction of tph2 immunoreactivity in the forebrain/telencephalon-preoptic area endorsed our results. Increased number of pre-vitellogenic and less immature oocytes in the treated groups indicated hastened ovarian growth. Elevated ovarian aromatase activity and plasma estradiol-17ß levels were noticed in the treated groups with maximum being in the endosulfan group. These data together demonstrate that the exposure of endosulfan causes synchronous precocious ovarian development better than flutamide, alone or in combination. Our results suggest that both endosulfan and flutamide alter ovarian growth by triggering precocious development in catfish.

Chronic fluoxetine treatment in middle-aged rats induces changes in the expression of plasticity-related molecules and in neurogenesis.

BACKGROUND: Antidepressants promote neuronal structural plasticity in young-adult rodents, but little is known of their effects on older animals. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) may mediate these structural changes through its anti-adhesive properties. PSA-NCAM is expressed in immature neurons and in a subpopulation of mature interneurons and its expression is modulated by antidepressants in the telencephalon of young-adult rodents. RESULTS: We have analyzed the effects of 14 days of fluoxetine treatment on the density of puncta expressing PSA-NCAM and different presynaptic markers in the medial prefrontal cortex, hippocampus and amygdala of middle-aged (8 months old) rats. The density of puncta expressing PSA-NCAM increased in the dorsal cingulate cortex, as well as in different hippocampal and amygdaloid regions. In these later regions there were also increases in the density of puncta expressing glutamic acid decarboxylase 65/67 (GAD6), synaptophysin (SYN), PSA-NCAM/SYN and PSA-NCAM/GAD6, but a decrease of those expressing vesicular glutamate transporter 1 (VGluT1). Since there is controversy on the effects of antidepressants on neurogenesis during aging, we analyzed the number of proliferating cells expressing Ki67 and that of immature neurons expressing doublecortin or PSA-NCAM. No significant changes were found in the subgranular zone, but the number of proliferating cells decreased in the subventricular zone. CONCLUSIONS: These results indicate that the effects of fluoxetine in middle-aged rats are different to those previously described in young-adult animals, being more restricted in the mPFC and even following an opposite direction in the amygdala or the subventricular zone.

Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals.

During early telencephalic development, the major portion of the ventral telencephalic (subpallial) region becomes subdivided into three regions, the lateral (LGE), medial (MGE), and caudal (CGE) ganglionic eminences. In this study, we systematically recapitulated subpallial patterning in mouse embryonic stem cell (ESC) cultures and investigated temporal and combinatory actions of patterning signals. In serum-free floating culture, the dorsal-ventral specification of ESC-derived telencephalic neuroectoderm is dose-dependently directed by Sonic hedgehog (Shh) signaling. Early Shh treatment, even before the expression onset of Foxg1 (also Bf1; earliest marker of the telencephalic lineage), is critical for efficiently generating LGE progenitors, and continuous Shh signaling until day 9 is necessary to commit these cells to the LGE lineage. When induced under these conditions and purified by fluorescence-activated cell sorter, telencephalic cells efficiently differentiated into Nolz1(+)/Ctip2(+) LGE neuronal precursors and subsequently, both in culture and after in vivo grafting, into DARPP32(+) medium-sized spiny neurons. Purified telencephalic progenitors treated with high doses of the Hedgehog (Hh) agonist SAG (Smoothened agonist) differentiated into MGE- and CGE-like tissues. Interestingly, in addition to strong Hh signaling, the efficient specification of MGE cells requires Fgf8 signaling but is inhibited by treatment with Fgf15/19. In contrast, CGE differentiation is promoted by Fgf15/19 but suppressed by Fgf8, suggesting that specific Fgf signals play different, critical roles in the positional specification of ESC-derived ventral subpallial tissues. We discuss a model of the antagonistic Fgf8 and Fgf15/19 signaling in rostral-caudal subpallial patterning and compare it with the roles of these molecules in cortical patterning.