Néstor F. Díaz

Comparative analysis of immunoreactive cells for androgen receptors and oestrogen receptor alpha in copulating and non-copulating male rats.

In some species, including gerbils, guinea pigs, mice, rams and rats, some apparently normal males fail to mate. These kinds of animals have been named ‘noncopulating (NC)’. The cause of this behavioural deficit is unknown. The present study aimed to determine whether NC male rats have alterations in the amount of androgen (AR) and oestrogen receptor α (ERα) in a neuronal circuit important for the control of male sexual behaviour; the vomeronasal projection pathway. We evaluated the number of AR and ERα immunoreactive (AR‐IR and ERα‐IR) cells in the accessory olfactory bulb (AOB), the bed nucleus of the stria terminalis (BNST), the anterior‐dorsal medial amygdala (MeAD), the posterior dorsal amygdala (MePD) and the medial preoptic area (MPOA). The results demonstrate that the number of AR‐IR cells in NC males was significantly higher compared to copulating (C) males in the MePD, but no significant differences were found in any of the other structures analysed. ERα‐IR cells were more abundant in NC than in C males in the MeAD and the MePD. However, in the MPOA the number of ERα‐IR cells was significantly reduced in NC males. No significant differences were found in the AOB or in the BNST. A similar pattern of results was observed when regions within these structures that are activated by Fos expression, on mating or exposure to sexually relevant cues were analysed. The differences in the number of AR and ER in particular brain areas could be associated with alterations in sexual behaviour as well as partner and olfactory preference for receptive females seen in NC male rats.

Histamine in brain development.

J. Neurochem. (2012) 122, 872–882.

Estradiol promotes proliferation of dopaminergic precursors resulting in a higher proportion of dopamine neurons derived from mouse embryonic stem cells.

Estradiol protects dopamine neurons of the substantia nigra from toxic insults. Such neurons succumb in Parkinsons disease; one strategy for restoring dopamine deficiency is cell therapy with neurons differentiated from embryonic stem cells. We investigated the effects of 17β‐estradiol on dopaminergic induction of embryonic stem cells using the 5‐stage protocol. Cells were incubated with different steroid concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Estradiol added at nM concentrations only during stage 4 increases the proliferation of dopaminergic precursors expressing Lmx1a, inducing a higher proportion of dopamine neurons at stage 5. These actions were mediated by activation of estrogen receptors, because co‐incubation of cells with estradiol and ICI 182,780 completely abolished the positive effect on both proliferation of committed precursors, and subsequent differentiation to dopaminergic neurons. Our results suggest that estradiol should be useful in producing higher proportions of dopamine neurons from embryonic stem cells aimed for treating Parkinsons disease.

CHANGES IN THE CONTENT OF ESTROGEN α AND PROGESTERONE RECEPTORS DURING DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS TO DOPAMINE NEURONS

Embryonic stem cells (ESC) can differentiate to derivatives of the three embryonic germ layers. Dopamine neurons have been produced from mouse and human ESC. This in vitro induction mimics the developmental program followed by dopaminergic cells in vivo. Production of dopamine neurons might have clinical applications for Parkinsons disease, which has a higher incidence in men than in women, suggesting a protective role for sex hormones, particularly progesterone and estradiol. These hormones exert many of their effects through the interaction with their nuclear receptors. In this study, we used a described 5-stage protocol for dopamine neuron differentiation of ESC, allowing neuronal commitment as evidenced by specific markers and by behavioural recovery of hemiparkinsonian rats after grafting. We studied the expression of steroid hormone receptors by immunoblot during this procedure and found an increase in the content of both A and B isoforms of progesterone receptor (PR) and a decrease in estrogen receptor alpha (ER-alpha) when cells were at the neural/neuronal stages, when compared with the amount found in initial pluripotent conditions. We also found the same pattern of PR and ER-alpha expression by immunocytochemistry. Ninety-two percent of dopamine neurons expressed progesterone receptors and only 19% of these neurons co-expressed tyrosine hydroxylase and ER-alpha. These results show a differential expression pattern of ER-alpha and PR isoforms during neuronal differentiation of ESC.

Histamine is required during neural stem cell proliferation to increase neuron differentiation

Histamine in the adult central nervous system (CNS) acts as a neurotransmitter. This amine is one of the first neurotransmitters to appear during development reaching its maximum concentration simultaneously with neuron differentiation peak. This suggests that HA plays an important role in neurogenesis. We have previously shown that HA is able to increase neuronal differentiation of neural stem cells (NSCs) in vitro, by activating the histamine type 1 receptor. However the mechanism(s) by which HA has a neurogenic effect on NSCs has not been explored. Here we explore how HA is able to increase neuron phenotype. Cortex neuroepithelium progenitors were cultured and at passage two treatments with 100 μM HA were given during cell proliferation and differentiation or only during differentiation. Immunocytochemistry was performed on differentiated cultures to detect mature neurons. To explore the expression of certain important transcriptional factors involved on asymmetric cell division and commitment, RT-PCR and qRT-PCR were performed. Results indicate that HA is required during cell proliferation in order to increase neuron differentiation and suggest that this amine increases neuron commitment during the proliferative phase probably by rising prospero1 and neurogenin1 expression.

Axon responses of embryonic stem cell-derived dopaminergic neurons to Semaphorins 3A and 3C

Class 3 Semaphorins are a subfamily of chemotropic molecules implicated in the projection of dopaminergic neurons from the ventral mesencephalon and in the formation of the nigrostriatal pathway (NSP) during embryonic development. In humans, loss of mesencephalic dopaminergic neurons leads to Parkinsons disease (PD). Cell replacement therapy with dopaminergic neurons generated from embryonic stem cells (ES‐TH+) is being actively explored in models of PD. Among several requisites for this approach to work are adequate reconstruction of the NSP and correct innervation of normal striatal targets by dopaminergic axons. In this work, we characterized the response of ES‐TH+ neurons to semaphorins 3A, 3C, and 3F and compared it with that of tyrosine hidroxylase‐positive neurons (TH+) obtained from embryonic ventral mesencephalon (VM‐TH+). We observed that similar proportions of ES‐TH+ and VM‐TH+ neurons express semaphorin receptors neuropilins 1 and 2. Furthermore, the axons of both populations responded very similarly to semaphorin exposure: semaphorin 3A increased axon length, and semaphorin 3C attracted axons and increased their length. These effects were mediated by neuropilins, insofar as addition of blocking antibodies against these proteins reduced the effects on axonal growth and attraction, and only TH+ axons expressing neuropilins responded to the semaphorins analyzed. The observations reported here show phenotypic similarities between VM‐TH+ and ES‐TH+ neurons and suggest that semaphorins 3A and 3C could be employed to guide axons of grafted ES‐TH+ in therapeutic protocols for PD.

Suckling-induced activation of neural c-fos expression at lower thoracic rat spinal cord segments.

Suckling stimulation is essential for neuroendocrine and sympathetic reflex activation during lactation. In the present study, the induction of c-fos gene expression was used to identify neuronal populations in the spinal cord activated by acute 5 min suckling or by electrical stimulation of the central stump of the first abdominal mammary nerve in lactating rats previously separated from their litters for 6 or 18 h. In addition, to investigate whether spinal sympathetic preganglionic neurons are activated by suckling, dual immunostaining (Fos and choline acetyltransferase) was performed. Fos was expressed at low levels in continuously suckled and 6 h nonsuckled mothers, but no expression was found after 18 h of nonsuckling. On the other hand, in 6 h nonsuckled rats, significant increments in Fos expression occurred in several regions after acute suckling and after electrical stimulation. Also, the pattern of Fos expression in each spinal laminae was different for the two stimuli, i.e. more intense effects of suckling in deep laminae V-X and more intense effects in laminae I-IV with electrical stimulation. Double-labeling after suckling was found only in sympathetic preganglionic neurons from the intermedio-medial cell column, whereas after electrical stimulation, double label was observed only in neurons from the intermedio-lateral cell column. On the other hand, no effect upon Fos protein expression was observed after suckling and only a minor effect after electrical stimulation of mammary nerve in 18 h nonsuckled rats. These results are consistent with previous findings on the sympathetic reflex regulation of the mammary gland, as well as on the importance of the nonsuckling interval for optimal functioning of lactation.

Sexual activity increases the number of newborn cells in the accessory olfactory bulb of male rats

In rodents, sexual behavior depends on the adequate detection of sexually relevant stimuli. The olfactory bulb (OB) is a region of the adult mammalian brain undergoing constant cell renewal by continuous integration of new granular and periglomerular neurons in the accessory (AOB) and main (MOB) olfactory bulbs. The proliferation, migration, survival, maturation, and integration of these new cells to the OB depend on the stimulus that the subjects received. We have previously shown that 15 days after females control (paced) the sexual interaction an increase in the number of cells is observed in the AOB. No changes are observed in the number of cells when females are not allowed to control the sexual interaction. In the present study we investigated if in male rats sexual behavior increases the number of new cells in the OB. Male rats were divided in five groups: (1) males that did not receive any sexual stimulation, (2) males that were exposed to female odors, (3) males that mated for 1 h and could not pace their sexual interaction, (4) males that paced their sexual interaction and ejaculated one time and (5) males that paced their sexual interaction and ejaculated three times. All males received three injections of the DNA synthesis marker bromodeoxyuridine at 1h intervals, starting 1 h before the beginning of the behavioral test. Fifteen days later, males were sacrificed and the brains were processed to identify new cells and to evaluate if they differentiated into neurons. The number of newborn cells increased in the granular cell layer (GrCL; also known as the internal cell layer) of the AOB in males that ejaculated one or three times controlling (paced) the rate of the sexual interaction. Some of these new cells were identified as neurons. In contrast, no significant differences were found in the mitral cell layer (also known as the external cell layer) and glomerular cell layer (GlCL) of the AOB. In addition, no significant differences were found between groups in the MOB in any of the layers analyzed. Our results indicate that sexual behavior in male rats increases neurogenesis in the GrCL of the AOB when they control the rate of the sexual interaction.

Progesterone Increases Dopamine Neurone Number in Differentiating Mouse Embryonic Stem Cells

Progesterone participates in the regulation of several functions in mammals, including brain differentiation and dopaminergic transmission, but the role of progesterone in dopaminergic cell differentiation is unknown. We investigated the effects of progesterone on dopaminergic differentiation of embryonic stem cells using a five‐stage protocol. Cells were incubated with different progesterone concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Progesterone added at 1, 10 and 100 nm during stage 4 increased the number of dopamine neurones at stage 5 by 72%, 80% and 62%, respectively, compared to the control group. The administration of progesterone at stage 5 did not induce significant changes in the number of dopamine neurones. These actions were not mediated by the activation of intracellular progesterone receptors because RU 486 did not block the positive effects of progesterone on differentiation to dopaminergic neurones. The results obtained suggest that progesterone should prove useful with respect to producing higher proportions of dopamine neurones from embryonic stem cells in the treatment of Parkinson’s disease.

MicroRNAs in central nervous system development.

Abstract During early and late embryo neurodevelopment, a large number of molecules work together in a spatial and temporal manner to ensure the adequate formation of an organism. Diverse signals participate in embryo patterning and organization synchronized by time and space. Among the molecules that are expressed in a temporal and spatial manner, and that are considered essential in several developmental processes, are the microRNAs (miRNAs). In this review, we highlight some important aspects of the biogenesis and function of miRNAs as well as their participation in ectoderm commitment and their role in central nervous system (CNS) development. Instead of giving an extensive list of miRNAs involved in these processes, we only mention those miRNAs that are the most studied during the development of the CNS as well as the most likely mRNA targets for each miRNA and its protein functions.