Long-term potentiation and spatial memory training stimulate the hippocampal expression of RyR2 calcium release channels.

Introduction: Neuronal Ca2+ signals generated through the activation of Ca2+-induced Ca2+ release in response to activity-generated Ca2+ influx play a significant role in hippocampal synaptic plasticity, spatial learning, and memory. We and others have previously reported that diverse stimulation protocols, or different memory-inducing procedures, enhance the expression of endoplasmic reticulum-resident Ca2+ release channels in rat primary hippocampal neuronal cells or hippocampal tissue. Methods and Results: Here, we report that induction of long-term potentiation (LTP) by Theta burst stimulation protocols of the CA3-CA1 hippocampal synapse increased the mRNA and protein levels of type-2 Ryanodine Receptor (RyR2) Ca2+ release channels in rat hippocampal slices. Suppression of RyR channel activity (1 h preincubation with 20 µM ryanodine) abolished both LTP induction and the enhanced expression of these channels; it also promoted an increase in the surface expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR1 and GluR2 and caused a moderate but significant reduction of dendritic spine density. In addition, training rats in the Morris water maze induced memory consolidation, which lasted for several days after the end of the training period, accompanied by an increase in the mRNA levels and the protein content of the RyR2 channel isoform. Discussion: We confirm in this work that LTP induction by TBS protocols requires functional RyR channels. We propose that the increments in the protein content of RyR2 Ca2+ release channels, induced by LTP or spatial memory training, play a significant role in hippocampal synaptic plasticity and spatial memory consolidation.

The lack of Irs2 induces changes in the immunocytochemical expression of aromatase in the mouse retina.

Insulin receptor substrate (Irs) belongs to a family of proteins that mediate the intracellular signaling of insulin and IGF-1. Insulin receptor substrate 2 (Irs2) is necessary for retinal function, since its failure in Irs2-deficient mice in hyperglycemic situation promotes photoreceptor degeneration and visual dysfunction, like in diabetic retinopathy. The expression of P450 aromatase, which catalyzes androgen aromatization to form 17ß-estradiol, increases in some neurodegenerative diseases thus promoting the local synthesis of neuroestrogens that exert relevant neuroprotective functions. Aromatase is also expressed in neurons and glial cells of the central nervous system (CNS), including the retina. To further understand the role of Irs2 at the retinal level, we performed an immunocytochemical study in adult normoglycemic Irs2-deficient mice. For this aim, the retinal immunoexpression of neuromodulators, such as aromatase, glutamine synthetase (GS), and tyrosine hydroxylase (TH) was analyzed, joint to a morphometric and planimetric study of the retinal layers. Comparing with wild-type (WT) control mice, the Irs2-knockout (Irs2-KO) animals showed a significant increase in the immunopositivity to aromatase in almost all of the retinal layers. Besides, Irs2-KO mice exhibited a decreased immunopositive reaction for GS and TH, in Müller and amacrine cells, respectively; morphological variations were also found in these retinal cell types. Furthermore, the retina of Irs2-KO mice displayed alterations in the structural organization, and a generalized decrease in the retinal thickness was observed in each of the layers, except for the inner nuclear layer. Our findings suggest that the absence of Irs2 induces retinal neurodegenerative changes in Müller and amacrine cells that are unrelated to hyperglycemia. Accordingly, in the Irs2-KO mice, the increased retinal immunocytochemical reactivity of aromatase could be associated with an attempt to repair such neural retina injuries by promoting local neuroprotective mediators.

Cell Proliferation in the Piriform Cortex of Rats with Motor Cortex Ablation Treated with Growth Hormone and Rehabilitation.

Traumatic brain injury represents one of the main health problems in developed countries. Growth hormone (GH) and rehabilitation have been claimed to significantly contribute to the recovery of lost motor function after acquired brain injury, but the mechanisms by which this occurs are not well understood. In this work, we have investigated cell proliferation in the piriform cortex (PC) of adult rats with ablation of the frontal motor cortex treated with GH and rehabilitation, in order to evaluate if this region of the brain, related to the sense of smell, could be involved in benefits of GH treatment. Male rats were either ablated the frontal motor cortex in the dominant hemisphere or sham-operated and treated with GH or vehicle at 35 days post-injury (dpi) for five days. At 36 dpi, all rats received daily injections of bromodeoxyuridine (BrdU) for four days. We assessed motor function through the paw-reaching-for-food task. GH treatment and rehabilitation at 35 dpi significantly improved the motor deficit caused by the injury and promoted an increase of cell proliferation in the PC ipsilateral to the injury, which could be involved in the improvement observed. Cortical ablation promoted a greater number of BrdU+ cells in the piriform cortex that was maintained long-term, which could be involved in the compensatory mechanisms of the brain after injury.

Evidences for Expression and Location of ANGPTL8 in Human Adipose Tissue.

The metabolism of triglycerides (TGs) is regulated, among others, by the lipoprotein lipase (LPL) that hydrolyses the TGs on endothelial cells. In turn, LPL is inhibited by the ANGPTLs family of proteins, such as ANGPTL3, 4, and, 8; the latter is the least known. In this work, we have tried to establish the expression and localisation of the Angiopoietin-like 8 (ANGPTL8) protein in the visceral adipose tissue (VAT) of morbid-obese and non-obese patients. 109 subjects (66 women and 43 men) undergoing laparoscopic surgery participated in this study. A blood sample and a portion of the VAT were obtained, and the patients were classified according to their Body Mass Index (BMI) as non-obese (19.5-30 kg/m2) and morbid-obese (40-50 kg/m2). No significant changes in ANGPTL8 plasma levels were determined by EIA in obese patients. The immunocytochemistry and Western blotting showed the presence of increased ANGPTL8 in morbid-obese patients (p < 0.05). In-situ hybridisation and a real time polymerase chain reaction (RT-PCR) confirmed that the mRNA that encodes ANGPTL8 was present in adipocytes, without differences in their nutritional state (p = 0.89), and even in the endothelial cells. Our data suggests that ANGPT8 plasmatic levels do not change significantly in patients with morbid obesity, although there is a modest difference related to gender. Besides, we demonstrate that in visceral adipose tissue, ANGPTL8 is well defined in the cytoplasm of adipocytes coexisting with perilipin-1 and its mRNA, also is present in endothelial cells. These findings suggest the possibility that among other functions, ANGPTL8 could perform either a paracrine and/or an endocrine role in the adipose tissue.

Factors Involved in the Functional Motor Recovery of Rats with Cortical Ablation after GH and Rehabilitation Treatment: Cortical Cell Proliferation and Nestin and Actin Expression in the Striatum and Thalamus.

Previously we demonstrated, in rats, that treatment with growth hormone (GH) and rehabilitation, carried out immediately after a motor cortical ablation, significantly improved the motor affectation produced by the lesion and induced the re-expression of nestin in the contralateral motor cortex. Here we analyze cortical proliferation after ablation of the frontal motor cortex and investigate the re-expression of nestin in the contralateral motor cortex and the role of the striatum and thalamus in motor recovery. The rats were subjected to ablation of the frontal motor cortex in the dominant hemisphere or sham-operated and immediately treated with GH or the vehicle (V), for five days. At 1 dpi (days post-injury), all rats received daily injections (for four days) of bromodeoxyuridine and five rats were sacrificed at 5 dpi. The other 15 rats (n = 5/group) underwent rehabilitation and were sacrificed at 25 dpi. GH induced the greatest number of proliferating cells in the perilesional cortex. GH and rehabilitation produced the functional recovery of the motor lesion and increased the expression of nestin in the striatum. In the thalamic ventral nucleus ipsilateral to the lesion, cells positive for nestin and actin were detected, but this was independent on GH. Our data suggest that GH-induced striatal nestin is involved in motor recovery.

The influence of the lack of insulin receptor substrate 2 (IRS2) on the thyroid gland.

Involvement of IRS2 in the proliferative effects of IGF-I of follicular thyroid cells has been described, but there are no evidences for in vivo participation of IRS2. This study aimed to analyse the in vivo relevance of IRS2 in the proliferation and apoptosis of thyroid cells by immunocytochemical studies for PCNA, Ki67, and active-caspase-3 in thyroid cells of IRS2 knockout (IRS2-KO) mice, jointly to TUNEL assay. Thyroid hormones were lower in IRS2-KO mice than in their wild-type (WT) counterparts. Increases in the area, perimeter and diameter of thyroid follicles of IRS2-KO mice were observed, which also showed increased proliferation rate of follicular cells and decreased percentage of apoptotic cells that was more evident in the central than in the marginal region of the gland. Sex-related differences were also found, since the follicular epithelium height was higher in male than in female mice. The percentage of proliferating cells showed significant changes in male but not in female mice, and apoptotic cells were more abundant in female than in male IRS2-KO animals, without significant differences between WT-animals. Therefore, our results suggest that IRS2 could be involved in the maintenance of thyroid cells population and in the normal physiology of the thyroid gland.

Prolactin system in the hippocampus.

Among the more than 300 biological actions described for prolactin, its role in the neurogenic capacity of the hippocampus, which increases synaptogenesis and neuronal plasticity, consolidates memory and acts as a neuronal protector against excitotoxicity-effects mediated through its receptors are more recently known. The detection of prolactin in the hippocampus and its receptors, specifically in the Ammon's horn and dentate gyrus, opened up a new field of study on the possible neuroprotective effect of hormones in a structure involved in learning and memory, as well as in emotional and behavioral processes. It is currently known, although controversial, that prolactin may be related to sex and age and that the hormone could be synthesized in the hippocampus itself. However, the regulatory mechanisms of changes in prolactin or in its hippocampal receptors still remain unknown. This review introduces the reader to general aspects concerning prolactin and its receptors and to what is currently known about the role prolactin plays in the brain and, in particular, in the hippocampus.

Sequential testicular atrophy involves changes in cellular proliferation and apoptosis associated with variations in aromatase P450 expression levels in Irs-2-deficient mice.

Insulin receptor substrate 2 (Irs-2) is an intracellular protein susceptible to phosphorylation after activation of the insulin receptor. Its suppression affects testis development and its absence induces peripheral resistance to insulin. The aim of this study was to identify changes induced by the deletion of Irs-2 in the testicular structure and by the altered expression of cytochrome P450 aromatase, a protein necessary for the development and maturation of germ cells. Adult knockout (KO) mice (Irs-2-/- , 6 and 12 weeks old) and age-matched wild-type (WT) mice were used in this study. Immunohistochemistry and Western blot analyses were performed to study proliferation (PCNA), apoptosis (active caspase-3) and P450 aromatase expression in testicular histological sections. Deletion of Irs-2 decreased the number of epithelial cells in the seminiferous tubule and rete testis. Aberrant cells were frequently detected in the epithelia of Irs-2-/- mice, accompanied by variations in spermatogonia, which were shown to exhibit small hyperchromatic nuclei as well as polynuclear and anuclear structures. The amount of cell proliferation was significantly lower in Irs-2-/- mice than in WT mice, whereas apoptotic processes were more common in Irs-2-/- mice. Aromatase P450 reactivity was higher in 6-week-old KO mice than in WT mice of the same age and was even higher at 12 weeks. Our results suggest that Irs-2 is a key element in spermatogenesis because silencing Irs-2 induces the sequential development of testicular atrophy. The effects are observed mainly in germ cells present in the seminiferous tubule, which may be due to changes in cytochrome P450 aromatase expression.

Variations in adrenal gland medulla and dopamine effects induced by the lack of Irs2

The adrenomedullary chromaffin cells' hormonal pathway has been related to the pathophysiology of diabetes mellitus. In mice, the deletion of insulin receptor substrate type 2 (Irs2) causes peripheral insulin resistance and reduction in Beta-cell mass, leading to overt diabetes, with gender differences on adrenergic signaling. To further unravel the relevance of Irs2 on glycemic control, we analyzed in adult Irs2 deficient (Irs2-/-) mice, of both sexes but still normoglycemic, dopamine effects on insulin secretion and glycerol release, as well as their adrenal medulla by an immunohistochemical and morphologic approach. In isolated islets, 10 μM dopamine significantly inhibited insulin release in wild-type (WT) and female Irs2−/− mice; however, male Irs2−/− islets were insensitive to that catecholamine. Similarly, on isolated adipocytes, gender differences were observed between WT and Irs2-/- mice in basal and evoked glycerol release with crescent concentrations of dopamine. By immunohistochemistry, reactivity to tyrosine hydroxylase (TH) in female mice was significantly higher in the adrenal medulla of Irs2-/- compared to WT; although no differences for TH-immunopositivity were observed between the male groups of mice. However, compared to their corresponding WT animals, adrenomedullary chromaffin cells of Irs2-/- mice showed a significant decrease in the cellular and nuclear areas, and even in their percentage of apoptosis. Therefore, our observations suggest that, together with gender differences on dopamine responses in Irs2-/- mice, disturbances in adrenomedullary chromaffin cells could be related to deficiency of Irs2. Accordingly, Irs2 could be necessary for adequate glucose homeostasis and maintenance of the population of the adrenomedullary chromaffin cells

Variations in adrenal gland medulla and dopamine effects induced by the lack of Irs2.

The adrenomedullary chromaffin cells' hormonal pathway has been related to the pathophysiology of diabetes mellitus. In mice, the deletion of insulin receptor substrate type 2 (Irs2) causes peripheral insulin resistance and reduction in ß-cell mass, leading to overt diabetes, with gender differences on adrenergic signaling. To further unravel the relevance of Irs2 on glycemic control, we analyzed in adult Irs2 deficient (Irs2-/-) mice, of both sexes but still normoglycemic, dopamine effects on insulin secretion and glycerol release, as well as their adrenal medulla by an immunohistochemical and morphologic approach. In isolated islets, 10 µM dopamine significantly inhibited insulin release in wild-type (WT) and female Irs2-/- mice; however, male Irs2-/- islets were insensitive to that catecholamine. Similarly, on isolated adipocytes, gender differences were observed between WT and Irs2-/- mice in basal and evoked glycerol release with crescent concentrations of dopamine. By immunohistochemistry, reactivity to tyrosine hydroxylase (TH) in female mice was significantly higher in the adrenal medulla of Irs2-/- compared to WT; although no differences for TH-immunopositivity were observed between the male groups of mice. However, compared to their corresponding WT animals, adrenomedullary chromaffin cells of Irs2-/- mice showed a significant decrease in the cellular and nuclear areas, and even in their percentage of apoptosis. Therefore, our observations suggest that, together with gender differences on dopamine responses in Irs2-/- mice, disturbances in adrenomedullary chromaffin cells could be related to deficiency of Irs2. Accordingly, Irs2 could be necessary for adequate glucose homeostasis and maintenance of the population of the adrenomedullary chromaffin cells.

Relation among Aromatase P450 and Tumoral Growth in Human Prolactinomas.

The pituitary gland is part of hypothalamic-pituitary-gonadal axis, which controls development, reproduction, and aging in humans and animals. In addition, the pituitary gland is regulated mainly by hormones and neurotransmitters released from the hypothalamus and by systemic hormones secreted by target glands. Aromatase P450, the enzyme responsible for the catabolization of aromatizable androgens to estrogens, is expressed in different parts of body, including the pituitary gland. Moreover, aromatase P450 is involved in sexual dimorphism where alteration in the level of aromatase can initiate a number of diseases in both genders. On the other hand, the direct actions of estrogens, mainly estradiol, are well known for stimulating prolactin release. Numerous studies have shown that changes in the levels of estrogens, among other factors, have been implicated in the genesis and development of prolactinoma. The pituitary gland can produce estradiol locally in several types of endocrine cells, and it is possible that aromatase could be responsible for the maintenance of the population of lactotroph cells and the modulation of the action of central or peripheral regulators. Aromatase overexpression due to inappropriate gene regulation has clinical effects such as the pathogenesis of prolactinomas. The present study reports on the synthesis of pituitary aromatase, its regulation by gonadal steroids, and the physiological roles of aromatase on pituitary endocrine cells. The involvement of aromatase in the pathogenesis of pituitary tumors, mainly prolactinomas, through the auto-paracrine production of estradiol is reviewed.

Endothelial immunocytochemical expression of pituitary IL-1ß and its relation to ACTH-positive cells is regulated by corticosterone in the male rat.

Interleukin-1 beta (IL-1ß) is a cytokine linking the neuroendocrine system and metabolic homeostasis. We have previously demonstrated the relevance of IL-1ß for maintaining the pituitary ACTH-producing cells by immuno-blocking its effects in pituitary cultures. However, the morphological characteristics and the intimate relationship of the pituitary cells expressing IL-1ß and ACTH remain unknown. For determining pituitary variations of immunoreactivity for IL-1ß and its relation with ACTH-positive cells under stress situations, we performed an immunohistochemical analysis of the expression of IL-1ß and ACTH in the pituitary gland of adult rats, in the absence or presence of corticosterone, by establishing different groups: untreated, sham-operated, and bilaterally adrenalectomized animals. In the rats subjected to surgery, the glucocorticoid was administered on the same day of the intervention and on the third day post-surgery. Interestingly, it was observed that IL-1ß was located in the pituitary endothelial cells at the hypophyseal portal vessels, regardless of the treatment schedule. When comparing the pituitary immunoreactive surface to IL-1ß expression without corticosterone, adrenalectomized animals displayed a significantly greater area than the sham-operated animals. Corticosterone significantly inhibited the effect of adrenalectomy depending on the time interval it was administered. By in situ hybridization, IL-1ß mRNA expression was also correlated with immnunocytochemical expression of pituitary IL-1ß. Our results demonstrate that IL-1ß is a constitutive element in endothelial portal pituitary vessels and under stress experimental conditions IL-1ß increases its expression and its relation with ACTH-positive cells, suggesting that IL-1ß could participate in an autocrine-paracrine fashion thereby modulating the pituitary population of ACTH-positive cells.

Pituitary Aromatase P450 May Be Involved in Maintenance of the Population of Luteinizing Hormone-Positive Pituitary Cells in Mice.

As aromatase P450 is located in several pituitary cells, testosterone can be transformed into 17ß-estradiol in the gland by the enzyme. The possible role of this transformation in pituitary function remains to be elucidated, but some evidence suggests a physiological and pathophysiological role for pituitary aromatase. To determine its relevance in the modulation of pituitary function, mainly associated with reproduction, luteinizing hormone (LH)-positive cells in the hypophysis of female and male aromatase knockout (ArKO) mice were studied. In all LH-positive cells, significant increases in the cellular (p < 0.01) and nuclear (p < 0.05) areas were found in the ArKO mice compared to the wild-type mice. In the ArKO mice, LH-positive cells were more abundant (p < 0.01); they were characterized by a stronger cytoplasmic reaction and the cells were more polygonal and exhibited more short, thick cytoplasmic prolongations than those in the wild-type mice. Moreover, LH-positive cells showed a greater proliferation rate in the ArKO mice compared to the wild-type mice (p < 0.01). These findings suggest that the local production of estradiol mediated by pituitary aromatase is necessary for the regulation of LH-positive gonadotropic cells, exerting an autoparacrine inhibitory regulation. These results could underlie the higher pituitary aromatase expression observed in male versus female mice. Similar effects were found in ArKO male and female mice, suggesting that in both sexes the effects of estrogens on maintenance of the LH-positive pituitary cell population could be related to the local aromatization of testosterone to estradiol inside the hypophysis. Therefore, aromatase could modulate pituitary LH-positive cells in males through local estradiol synthesis.

Relevance of pituitary aromatase and estradiol on the maintenance of the population of prolactin-positive cells in male mice.

In previous studies we demonstrated the expression of aromatase in pituitary cells. This expression is gender related, and is also associated with the presence of prolactinomas. To ascertain the relevance of aromatase in modulating the populations of prolactin-positive pituitary cells an immunocytochemical and morphometric study of prolactin-positive pituitary cells was carried out using the pituitary glands of adult male and female aromatase-knockout (ArKO) mice. Additionally has been determined if pituitary aromatase is involved in a gender-linked differentiated regulation of the prolactin-producing pituitary cells. Compared to wild-type mice, the knockout animals of both genders showed a significant decrease (p<0.01) in the cellular and nuclear areas of their prolactin cells, as well as in the percentages of the prolactin-positive cells and the proliferating prolactin cells. Our results suggest that estradiol is responsible for the maintenance of the population of prolactin cell in males and, so as not to disturb the endocrine reproductive environment, estradiol is synthesized inside the pituitary by circulating testosterone via means of aromatase P450, which acts in paracrine way. This new role for pituitary aromatase may well explain the previous findings establishing that the pituitary expression of aromatase is higher in males than in females, and the association between the development of prolactinomas and the increased expression of aromatase in tumours.

Dopamine modulates insulin release and is involved in the survival of rat pancreatic beta cells.

The local synthesis of dopamine and its effects on insulin release have been described in isolated islets. Thus, it may be accepted that dopamine exerts an auto-paracrine regulation of insulin secretion from pancreatic beta cells. The aim of the present study is to analyze whether dopamine is a regulator of the proliferation and apoptosis of rat pancreatic beta cells after glucose-stimulated insulin secretion. Glucose stimulated pancreatic islets obtained from male Wistar rats were cultured with 1 or 10 µM dopamine from 1 to 12 h. Insulin secretion was analyzed by RIA. The cellular proliferation rate of pancreatic islets and beta cells was studied with immunocytochemical double labelling for both insulin and PCNA (proliferating cell nuclear antigen), and active caspase-3 was detected to evaluate apoptosis. The secretion of insulin from isolated islets was significantly inhibited (p<0.01), by treatment with 1 and 10 µM dopamine, with no differences between either dose as early as 1 h after treatment. The percentage of insulin-positive cells in the islets decreased significantly (p<0.01) after 1 h of treatment up to 12 h. The proliferation rate of insulin-positive cells in the islets decreased significantly (p<0.01) following treatment with dopamine. Apoptosis in pancreatic islets and beta cells was increased by treatment with 1 and 10 µM dopamine along 12 h. In conclusion, these results suggest that dopamine could modulate the proliferation and apoptosis of pancreatic beta cells and that dopamine may be involved in the maintenance of pancreatic islets.