Low doses of malathion impair ovarian, uterine, and follicular integrity by altering oxidative profile and gene expression of rats exposed during the peripubertal period.

Malathion serves as a pivotal pesticide in agriculture and the management of the Aedes aegypti mosquito. Despite its widespread use, there is a notable absence of studies elucidating the mechanisms through which malathion may affect the female reproductive system. Consequently, the objective of this investigation was to assess whether exposing juvenile female rats to low doses of malathion during the juvenile and peripubertal periods could compromise pubertal onset, estradiol levels, and the integrity of the ovaries and uterus while also examining the underlying mechanisms of damage. To achieve this, thirty juvenile female rats were subjected to either a vehicle or malathion (10 mg/kg or 50 mg/kg) between postnatal days 22 and 60, with subsequent verification of pubertal onset. Upon completion of the exposure period, blood samples were collected for estradiol assessment. The ovaries and uterus were then examined to evaluate histological integrity, oxidative stress, and the expression of genes associated with cell proliferation, antiapoptotic responses, and endocrine pathways. Although estradiol levels and pubertal onset remained unaffected, exposure to malathion compromised the integrity and morphometry of the ovaries and uterus. This was evidenced by altered oxidative profiles and changes in the expression of genes regulating the cell cycle, anti-apoptotic processes, and endocrine pathways. Our findings underscore the role of malathion in inducing cell proliferation, promoting cell survival, and causing oxidative damage to the female reproductive system in rats exposed during peripubertal periods.

Revealing Sexual Dimorphism in Prolactin Regulation From Early Postnatal Development to Adulthood in Murine Models.

Serum prolactin (PRL) levels exhibit a gradual rise both in male and female rats from birth to adulthood, with females consistently displaying higher levels compared to age-matched males. This pattern has traditionally been attributed to the development and maturation of endocrine and neuroendocrine networks responsible for regulating PRL synthesis and secretion. However, the effect of dopamine (DA), which acts as an inhibitory factor on lactotroph function, also increases from birth to puberty, particularly in females. Nonetheless, the secretion of PRL remains higher in females compared to males. On the other hand, the observed sex differences in serum PRL levels during early postnatal development cannot be attributed to the influence of estradiol (E2). While serum E2 levels gradually increase after birth, only after 45 days of life do the disparities in E2 levels between females and males become evident. These observations collectively suggest that neither the maturation of hypothalamic DA regulation nor the rise in E2 levels can account for the progressive and sustained elevation in serum PRL levels and the observed sexual dimorphism during postnatal development. This review highlights the importance of recent discoveries in animal models that shed light on inhibitory mechanisms in the control of PRL secretion within the pituitary gland itself, that is intrapituitary mechanisms, with a specific emphasis on the role of transforming growth factor ß1 and activins in PRL secretion.

Sex-specific regulation of prolactin secretion by pituitary activins in postnatal development.

Serum prolactin increases from birth to adulthood in rats, being higher in females from birth. The maturation of hypothalamic/gonadal prolactin-releasing and -inhibiting factors does not explain some sex differences observed. During the first weeks of life, prolactin secretion increases, even when lactotrophs are isolated in vitro, in the absence of those controls, suggesting the participation of intra-pituitary factors in this control. The present work aimed to study the involvement of pituitary activins in the regulation of prolactin secretion during post-natal development. Sex differences were also highlighted. Female and male Sprague-Dawley rats at 11, 23 and 45postnatal days were used. Pituitary expression of activin subunits and activin receptors was maximum in p11 female pituitaries, being even higher than that observed in males. Those expressions decrease with age in females, and then the gender differences disappear at p23. Inhbb expression strongly increases at p45 in males, being the predominant subunit in this sex in adulthood. Activin inhibition of prolactin is mediated by the inhibition of Pit-1 expression. This action involves not only the canonical pSMAD pathway but also the phosphorylation of p38MAPK. At p11, almost all lactotrophs express p-p38MAPK in females, and its expression decreases with age with a concomitant increase in Pit-1. Our findings suggest that the inhibitory regulation of pituitary activins on prolactin secretion is sex specific; this regulation is more relevant in females during the first week of life and decreases with age; this intra-pituitary regulation is involved in the sex differences observed in serum prolactin levels during postnatal development.

Malathion exposure during juvenile and peripubertal periods downregulate androgen receptor and 17-ß-HSD testicular gene expression and compromised sperm quality in rats.

Malathion is an insecticide that is used to control arboviruses and agricultural pests. Adolescents that are exposed to this insecticide are the most vulnerable as they are in the critical period of postnatal sexual development. This study aimed to evaluate whether malathion damage can affect sperm function and its respective mechanisms when adolescents are exposed during postnatal sexual development. Twenty-four male Wistar rats (PND 25) were divided into three experimental groups and treated daily for 40 d: control group (saline 0.9%), 10 mg/kg (M10 group), or 50 mg/kg (M50 group) of malathion. At PND 65, the rats were anesthetized and euthanized. Testicles were collected for the evaluation of gene expression. Sperm cells from the epididymis were used for evaluation of the oxidative profile or spermatic function. Data showed that a lower dose of malathion downregulated the gene expression of androgen receptors and testosterone converter enzyme 17-ß-HSD in the testis. The acrosomal integrity of sperm cells was compromised in the M50 group, but not the M10 group. The mitochondrial activity was not impaired by exposure. Finally, although no alterations in malondialdehyde and glutathione levels were observed, malathion, at both doses, increased antioxidant enzyme catalase activity and, at a higher dose, superoxide dismutase activity. The present study showed that low doses of malathion considered to be inoffensive are capable of impairing sperm quality and function through the downregulation of testicular genic expression of AR enzyme 17-ß-HSD and can damage the spermatic antioxidant profile during critical periods of development.

Maternal Protein Restriction Alters the Expression of Proteins Related to the Structure and Functioning of the Rat Offspring Epididymis in an Age-Dependent Manner.

Nutrition is an environmental factor able to activate physiological interactions between fetus and mother. Maternal protein restriction is able to alter sperm parameters associated with epididymal functions. Since correct development and functioning of the epididymides are fundamental for mammalian reproductive success, this study investigated the effects of maternal protein restriction on epididymal morphology and morphometry in rat offspring as well as on the expression of Src, Cldn-1, AR, ER, aromatase p450, and 5α-reductase in different stages of postnatal epididymal development. For this purpose, pregnant females were allocated to normal-protein (NP-17% protein) and low-protein (LP-6% protein) groups that received specific diets during gestation and lactation. After weaning, male offspring was provided only normal-protein diet until the ages of 21, 44, and 120 days, when they were euthanized and their epididymides collected. Maternal protein restriction decreased genital organs weight as well as crown-rump length and anogenital distance at all ages. Although the low-protein diet did not change the integrity of the epididymal epithelium, we observed decreases in tubular diameter, epithelial height and luminal diameter of the epididymal duct in 21-day-old LP animals. The maternal low-protein diet changed AR, ERα, ERß, Src 416, and Src 527 expression in offspring epididymides in an age-dependent manner. Finally, maternal protein restriction increased Cldn-1 expression throughout the epididymides at all analyzed ages. Although some of these changes did not remain until adulthood, the insufficient supply of proteins in early life altered the structure and functioning of the epididymis in important periods of postnatal development.

Developmental differences in myocardial transmembrane Na+ transport: implications for excitability and Na+ handling.

Little is currently known about possible developmental changes in myocardial Na+ handling, which may have impact on cell excitability and Ca2+ content. Resting intracellular Na+ concentration ([Na+ ]i ), measured in freshly isolated rat ventricular myocytes with CoroNa green, was not significantly different in neonates (3-5 days old) and adults, but electrical stimulation caused marked [Na+ ]i rise only in neonates. Inhibition of L-type Ca2+ current by CdCl2 abolished not only systolic Ca2+ transients, but also activity-dependent intracellular Na+ accumulation in immature cells. This indicates that the main Na+ influx pathway during activity is the Na+ /Ca2+ exchanger, rather than voltage-dependent Na+ current (INa ), which was not affected by CdCl2 . In immature myocytes, INa density was two-fold greater, inactivation was faster, and the current peak occurred at less negative transmembrane potential (Em ) than in adults. Na+ channel steady-state activation and inactivation curves in neonates showed a rightward shift, which should increase channel availability at diastolic Em , but also require greater depolarization for excitation, which was observed experimentally and reproduced in computer simulations. Ventricular mRNA levels of Nav 1.1, Nav 1.4 and Nav 1.5 pore-forming isoforms were greater in neonate ventricles, while a decrease was seen for the ß1 subunit. Both molecular and biophysical changes in the channel profile may contribute to the differences in INa density and voltage-dependence, and also to the less negative threshold Em , in neonates compared to adults. The apparently lower excitability in immature ventricle may confer protection against the development of spontaneous activity in this tissue. KEY POINTS: Previous studies showed that myocardial preparations from immature rats are less sensitive to electrical field stimulation than adult preparations. Freshly isolated ventricular myocytes from neonatal rats showed lower excitability than adult cells, e.g. less negative threshold membrane potential and greater membrane depolarization required for action potential triggering. In addition to differences in mRNA levels for Na+ channel isoforms and greater Na+ current (INa ) density, Na+ channel voltage-dependence was shifted to the right in immature myocytes, which seems to be sufficient to decrease excitability, according to computer simulations. Only in neonatal myocytes did cyclic activity promote marked cytosolic Na+ accumulation, which was prevented by abolition of systolic Ca2+ transients by blockade of Ca2+ currents. Developmental changes in INa may account for the difference in action potential initiation parameters, but not for cytosolic Na+ accumulation, which seems to be due mainly to Na+ /Ca2+ exchanger-mediated Na+ influx.

Spatiotemporally differential expression of HER (human epithelial growth factor receptor)-2 with sexual dimorphism in submandibular gland of mice

SUMMARY: Considering that the submandibular gland (SMG) of postnatal mice performs active cell proliferation, apoptosis and differentiation which are regulated by proto-oncogene products in cancerous cells, the expression and localization of a proto-oncogene product HER (human epidermal growth factor receptor)-2 was examined in SMG of postnatal mice. In Western blot analysis, the expression for HER-2 was high until pre-puberty, and it decreased from puberty to young adult stages with male SMG more dominant. In immunohistochemistry, the immunoreactivity was positive in acinar and ductal cells of newborn SMG with distinct localization at the intercellular apposition sites. The immunoreactivity in acinar cells progressively decreased to negligible levels by pre-pubertal stage, while it remained positive in most ductal cells throughout the postnatal time-course. The immunoreactivity in cells of terminal tubules and intercalated ducts, both of which have a high potential to produce cells, were seen at levels similar to those of more proximal ducts, while the immunoreactivity in ductal basal cells was significantly high, but the granular convoluted tubule cells were seen at negligible levels in male and at faint levels in female. In immuno-electron microscopy of excretory ducts, the immunoreactivity was dominantly localized on the basal infolding membranes as well as vesicles and vacuoles of various sizes, but rarely in Golgi apparatus and mitochondria. The immunoreactivity without association to any membranous structures were also seen, though not numerous. The relation of expression levels of HER-2 in various portions of normal SMG to those in their cancerous ones is briefly discussed.

RESUMEN: Considerando que la glándula submandibular (GSM) de ratones postnatales realiza la proliferación celular activa, apoptosis y diferenciación que están reguladas por productos protooncogénicos en células cancerosas, la expresión y localización de un producto protooncogénico HER (receptor del factor de crecimiento epidérmico humano) - 2 se examinó en GSM de estos ratones. En el análisis de Western blot, la expresión de HER-2 fue alta hasta la prepubertad, y disminuyó desde la pubertad hasta las etapas de adultos jóvenes con GSM macho más dominante. En inmunohistoquímica, la inmunorreactividad fue positiva en las células acinares y ductales de GSM de recién nacido con una localización distinta en los sitios de aposición intercelular. La inmunorreactividad en las células acinares disminuyó progresivamente a niveles insignificantes en la etapa prepuberal, mientras que permaneció positiva en la mayoría de las células ductales durante el transcurso del tiempo posnatal. La inmunorreactividad en las células de los túbulos terminales y los conductos intercalados, los cuales tienen un alto potencial para producir células, se obser- vó a niveles similares a los de los conductos más proximales, mientras que la inmunorreactividad en las células basales ductales fue significativamente alta, pero en el túbulo contorneado granular las células se observaron en niveles insignificantes en los machos y en niveles débiles en las hembras. En la microscopía inmunoelectrónica de los conductos excretores, la inmunorreactividad se localizó de manera predominante en las membranas de pliegues basales, así como en vesículas y vacuolas de varios tamaños, pero raramente en el aparato de Golgi y en las mitocondrias. También se observó la inmunorreactividad sin asociación a ninguna estructura membranosa, aunque no numerosa. Se discute brevemente la relación de los niveles de expresión de HER-2 en varias porciones de GSM normal con aquellos en sus cancerosos.

Developmental expression of genes involved in progesterone synthesis, metabolism and action during the post-natal cerebellar myelination.

Progesterone is involved in dendritogenesis, synaptogenesis and maturation of cerebellar Purkinge cells, major sites of steroid synthesis in the brain. To study a possible time-relationship between myelination, neurosteroidogenesis and steroid receptors during development of the postnatal mouse cerebellum, we determined at postnatal days 5 (P5),18 (P18) and 35 (P35) the expression of myelin basic protein (MBP), components of the steroidogenic pathway, levels of endogenous steroids and progesterone's classical and non-classical receptors. In parallel with myelin increased expression during development, P18 and P35 mice showed higher levels of cerebellar progesterone and its reduced derivatives, higher expression of steroidogenic acute regulatory protein (StAR) mRNA, cholesterol side chain cleavage enzyme (P450scc) and 5α-reductase mRNA vs. P5 mice. Other steroids such as corticosterone and its reduced derivatives and 3ß-androstanodiol (ADIOL) showed a peak increase at P18 compared to P5. Progesterone membrane receptors and binding proteins (PGRMC1, mPRα, mPRß, mPRγ, and Sigma1 receptors) mRNAs levels increased during development while that of classical progesterone receptors (PR) remained invariable. PRKO mice showed similar MBP levels than wild type. Thus, these data suggests that progesterone and its neuroactive metabolites may play a role in postnatal cerebellar myelination.

Intrauterine growth restriction: screening and diagnosis using animal models.

Intrauterine growth restriction (IUGR) is a serious condition of multifactorial origin, mainly caused by maternal malnutrition, multiple gestation associated with nutrient competition, abuse of nocive substances and infections. The diagnosis of such syndrome is complex, as its own manifestations can mask its occurrence, requiring a thorough assessment of body weight and size. Moreover, it is not responsive to any kind of treatment. There is evidence that IUGR may predispose the individual to several pathologies, such as diabetes, hypertension and metabolic syndrome in adulthood, and it has also been linked to thrifty phenotype hypothesis. Thus, a healthy lifestyle is needed to better prevent those pathologies. Given the world high prevalence and importance of IUGR, mainly in developing countries, this review is focused on discussing how different animal models contribute to the biological screening and diagnosis of this condition.

Impairment of postnatal epididymal development and immune microenvironment following administration of low doses of malathion during juvenile and peripubertal periods of rats.

Malathion is an organophosphate pesticide widely used for agricultural crops and for vector control of Aedes aegypti. Humans are exposed to this environmental contaminant by ingesting contaminated food. The juvenile and peripubertal periods are critical for the postnatal development of the epididymis and are when animals are most vulnerable to toxic agents. Since juveniles and adolescents are developing under exposure to the insecticide malathion, the aim of the present study was to evaluate the effects of exposure to low doses of malathion on postnatal epididymal development in rats. Male Wistar rats were exposed to malathion daily via gavage at doses of 10 mg kg-1 (M10 group) or 50 mg kg-1 (M50 group) for 40 days (postnatal days (PNDs) 25-65). The control group received the vehicle (0.9% saline) under the same conditions. On PND 40, the epididymides were removed, weighed and used for histological analysis and determination of the inflammatory profile and sperm count. Sperm from the vas deferens were subjected to sperm motility analysis. The M50 group showed tissue remodelling in the caput and cauda epididymides and increased neutrophil and macrophage migration in the caput epididymis. The M10 group showed decreased motile spermatozoa and IL-6 levels in the caput epididymis. Both doses decreased the IL-1ß level and altered the morphology of the same region. These results show that malathion exposure may impair postnatal epididymal development. Furthermore, alterations of the immune system in the epididymal environment are presented as new findings regarding the action of malathion on the epididymis.

TGFß1 regulates prolactin secretion during postnatal development: gender differences.

Serum prolactin levels gradually increase from birth to puberty in both male and female rats, with higher levels observed in female since the first days of life. The increase in lactotroph secretion was attributed to the maturation of prolactin-inhibiting and prolactin-releasing factors; however, those mechanisms could not fully explain the gender differences observed. Prolactin secretion from isolated lactotrophs, in the absence of hypothalamic control, also increases during the first weeks of life, suggesting the involvement of intra-pituitary factors. We postulate that pituitary transforming growth factor beta 1 (TGFß1) is involved in the regulation of prolactin secretion as well as in the gender differences observed at early postnatal age. Several components of the local TGFß1 system were evaluated during postnatal development (11, 23, and 45 days) in female and male Sprague-Dawley rats. In vivo assays were performed to study local TGFß1 activation and its impact on prolactin secretion. At day 11, female pituitaries present high levels of active TGFß1, concomitant with the highest expression of TGFß1 target genes and the phospho-Smad3 immunostaining in lactotrophs. The steady increase in prolactin secretion inversely correlates with active TGFß1 levels only in females. Dopamine and estradiol induce TGFß1 activation at day 11, in both genders, but its activation induces the inhibition of prolactin secretion only in females. Our findings demonstrate that: (1) TGFß1 activation is regulated by dopamine and estradiol; (2) the inhibitory regulation of local TGFß1 on prolactin secretion is gender specific; and (3) this mechanism is responsible, at least partially, for the gender differences observed being relevant during postnatal development.

Postnatal intermittent hypoxia enhances phrenic and reduces vagal upper airway motor activities in rats by epigenetic mechanisms.

NEW FINDINGS: What is the central question of this study? What are the alterations in respiratory motor activity that may underlie ventilatory dysfunctions in juvenile and adult animals exposed to postnatal chronic intermittent hypoxia? What is the main finding and its importance? Postnatal chronic intermittent hypoxia modifies the motor activity to pumping and upper airway respiratory muscles in rats, mediated by epigenetic DNA hypermethylation, enhancing resting pulmonary ventilation and predisposing to collapse of the upper airways in juvenile and adult life. ABSTRACT: Periods of apnoea, commonly observed in prematures and newborns, are an important risk factor for the development of cardiorespiratory diseases in adulthood. In the present study, we evaluated changes in pulmonary ventilation and respiratory motor pattern in juvenile and adult rats exposed to postnatal chronic intermittent hypoxia (pCIH). Newborn male Holtzman rats (P1) were submitted to pCIH (6% O2 for 30 s, every 9 min, 8 h a day (09.30-17.30 h)) during their first 10 days of life, while control animals were maintained under normoxic conditions (20.8% O2 ). Thereafter, animals of both groups were maintained under normoxia until the experiments. Unanaesthetized juvenile pCIH rats (n = 27) exhibited elevated tidal volume and respiratory irregularities (P < 0.05) compared to control rats (n = 7). Decerebrate, arterially perfused in situ preparations of juvenile pCIH rats (n = 11) displayed augmented phrenic nerve (PN) burst amplitude and reduced central vagus nerve activity in comparison to controls (n = 10). At adulthood, pCIH rats (n = 5) showed enhanced tidal volume (P < 0.05) and increased respiratory variability compared to the control group (n = 5). The pCIH-induced changes in ventilation and respiratory motor outputs were prevented by treatment with the DNA methyltransferase inhibitor decitabine (1 mg kg-1 , i.p.) during the exposure to pCIH. Our data demonstrate that pCIH in rats impacts, in a persistent way, control of the respiratory pattern, increasing PN activity to the diaphragm and reducing the vagal-related activity to laryngeal muscles, which, respectively, may contribute to improve resting pulmonary ventilation and predispose to collapse of the upper airways during quiet breathing.

Maternal Supplementation With Avocado (Persea americana Mill.) Pulp and Oil Alters Reflex Maturation, Physical Development, and Offspring Memory in Rats.

Avocado (Persea americana Mill.) is an oleaginous fruit source of fatty acids with high levels of neuroprotective phytocomplexes. The objective of this study was to evaluate the development of reflex and somatic maturation, fatty acid profiles in the brain, and memory in different stages of life in the offspring of dams supplemented with avocado pulp and oil during gestation and lactation. The dams were randomly divided into three groups (n = 15 pups/group), and recieved by gavage supplementation: control group (CG)-distilled water; Avocado Oil (AO)-3,000 mg avocado oil/kg animal weight, and Avocado Pulp (AP)-3,000 mg avocado pulp/kg animal weight. We performed the following tests: Analysis of Somatic Development and Ontogeny of Postnatal Reflex (T0 to T21), the Open Field Habituation Test and the Object Recognition Test (ORT) in the adolescent (T45) and adult (T90) phases. The cerebral fatty acids content was evaluated at times T0, T21, T45, and T90. The results were analyzed using the statistical program GraphPad Prism and significant statistics were considered when p < 0.05. Acceleration of reflex maturation and reflex ontogeny was observed in the offspring of AO and AP fed dams, with the results being more pronounced in the pulp fed group (p < 0.05). All groups presented a decrease in the ambulation parameter in the second exposure to the Open Field Habituation Test, at T45 and T90 (p < 0.05). In the ORT, the AO and AP offspring presented memory improvements in the short and long term in the adult and adolescent phases (p < 0.05). The results of the brain fatty acid profiles presented higher polyunsaturated fatty acids (PUFA) content in the AO and AP groups at T21, T45, and T90. The docosahexaenoic fatty acid (DHA) content was higher at T21 (AO and AP), at T45 (AO and AP), and at T90 (AP) (p < 0.05). The arachidonic acid (ARA) content was higher at T45 (AO and AP), and at T90 (AO) (p < 0.05). Maternal supplementation with avocado oil and pulp anticipates reflex maturation and somatic postnatal development, and improves memory during the adolescent and adult phases.

Novel cellular mechanism that mediates the collecting duct formation during postnatal renal development.

We have previously demonstrated that kidney embryonic structures are present in rats, and are still developing until postnatal Day 20. Consequently, at postnatal Day 10, the rat renal papilla contains newly formed collecting duct (CD) cells and others in a more mature stage. Performing primary cultures, combined with immunocytochemical and time-lapse analysis, we investigate the cellular mechanisms that mediate the postnatal CD formation. CD cells acquired a greater degree of differentiation, as we observed that they gradually lose the ability to bind BSL-I lectin, and acquire the capacity to bind Dolichos biflorus. Because CD cells retain the same behavior in culture than in vivo, and by using DBA and BSL-I as markers of cellular lineage besides specific markers of epithelial/mesenchymal phenotype, the experimental results strongly suggest the existence of mesenchymal cell insertion into the epithelial CD sheet. We propose such a mechanism as an alternative strategy for CD growing and development.

Intrauterine growth restriction: screening and diagnosis using animal models

Intrauterine growth restriction (IUGR) is a serious condition of multifactorial origin, mainly caused by maternal malnutrition, multiple gestation associated with nutrient competition, abuse of nocive substances and infections. The diagnosis of such syndrome is complex, as its own manifestations can mask its occurrence, requiring a thorough assessment of body weight and size. Moreover, it is not responsive to any kind of treatment. There is evidence that IUGR may predispose the individual to several pathologies, such as diabetes, hypertension and metabolic syndrome in adulthood, and it has also been linked to thrifty phenotype hypothesis. Thus, a healthy lifestyle is needed to better prevent those pathologies. Given the world high prevalence and importance of IUGR, mainly in developing countries, this review is focused on discussing how different animal models contribute to the biological screening and diagnosis of this condition.

Intrauterine growth restriction: screening and diagnosis using animal models

Intrauterine growth restriction (IUGR) is a serious condition of multifactorial origin, mainly caused by maternal malnutrition, multiple gestation associated with nutrient competition, abuse of nocive substances and infections. The diagnosis of such syndrome is complex, as its own manifestations can mask its occurrence, requiring a thorough assessment of body weight and size. Moreover, it is not responsive to any kind of treatment. There is evidence that IUGR may predispose the individual to several pathologies, such as diabetes, hypertension and metabolic syndrome in adulthood, and it has also been linked to thrifty phenotype hypothesis. Thus, a healthy lifestyle is needed to better prevent those pathologies. Given the world high prevalence and importance of IUGR, mainly in developing countries, this review is focused on discussing how different animal models contribute to the biological screening and diagnosis of this condition.(AU)

Calcium Channels in Postnatal Development of Rat Pancreatic Beta Cells and Their Role in Insulin Secretion.

Pancreatic beta cells during the first month of development acquire functional maturity, allowing them to respond to variations in extracellular glucose concentration by secreting insulin. Changes in ionic channel activity are important for this maturation. Within the voltage-gated calcium channels (VGCC), the most studied channels are high-voltage-activated (HVA), principally L-type; while low-voltage-activated (LVA) channels have been poorly studied in native beta cells. We analyzed the changes in the expression and activity of VGCC during the postnatal development in rat beta cells. We observed that the percentage of detection of T-type current increased with the stage of development. T-type calcium current density in adult cells was higher than in neonatal and P20 beta cells. Mean HVA current density also increased with age. Calcium current behavior in P20 beta cells was heterogeneous; almost half of the cells had HVA current densities higher than the adult cells, and this was independent of the presence of T-type current. We detected the presence of α1G, α1H, and α1I subunits of LVA channels at all ages. The Cav 3.1 subunit (α1G) was the most expressed. T-type channel blockers mibefradil and TTA-A2 significantly inhibited insulin secretion at 5.6 mM glucose, which suggests a physiological role for T-type channels at basal glucose conditions. Both, nifedipine and TTA-A2, drastically decreased the beta-cell subpopulation that secretes more insulin, in both basal and stimulating glucose conditions. We conclude that changes in expression and activity of VGCC during the development play an important role in physiological maturation of beta cells.

Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3.

TRPM4 is a Ca2+-activated non-selective cationic channel that conducts monovalent cations. TRPM4 has been proposed to contribute to burst firing and sustained activity in several brain regions, however, the cellular and subcellular pattern of TRPM4 expression in medial prefrontal cortex (mPFC) during postnatal development has not been elucidated. Here, we use multiplex immunofluorescence labeling of brain sections to characterize the postnatal developmental expression of TRPM4 in the mouse mPFC. We also performed electrophysiological recordings to correlate the expression of TRPM4 immunoreactivity with the presence of TRPM4-like currents. We found that TRPM4 is expressed from the first postnatal day, with expression increasing up to postnatal day 35. Additionally, in perforated patch clamp experiments, we found that TRPM4-like currents were active at resting membrane potentials at all postnatal ages studied. Moreover, TRPM4 is expressed in both pyramidal neurons and interneurons. TRPM4 expression is localized in the soma and proximal dendrites, but not in the axon initial segment of pyramidal neurons. This subcellular localization is consistent with a reduction in the basal current only when we locally perfused 9-Phenanthrol in the soma, but not upon perfusion in the medial or distal dendrites. Our results show a specific localization of TRPM4 expression in neurons in the mPFC and that a 9-Phenanthrol sensitive current is active at resting membrane potential, suggesting specific functional roles in mPFC neurons during postnatal development and in adulthood.

Differential Expression of Ion Channels in Adult and Neonatal Rat Ventral Respiratory Column.

In mammals, the neural control of breathing is attributed to circuits distributed along the ventral respiratory column (VRC) in the ventrolateral medulla. The VRC contains the kernel for generation of the inspiratory phase of respiratory rhythm and nuclei involved in central chemoreception. During development, the respiratory rhythm, as well as central chemosensitivity, adjusts to meet the changing physiological requirements associated with increased body weight and size. Gene expression in VRC ontogeny is well characterized. However, little is known about gene expression in the VRC during postnatal development. Here, we sought to characterize the changes in gene expression that occur in the VRC of the adult rat (5-6 months of age) in comparison with the VRC of neonate rat (1-4 days old). We isolated total RNA from VRC tissue punches collected from thick transversal slices. We hybridized cDNA to a 5000-oligonucleotide rat microarray. We found that 218 genes (4.4%) of the 5000 genes in the microarray changed their expression in adult VRC with respect to that from neonate. To further analyze the modified expression of specific genes, we quantified the differential expression of 84 genes of neuronal ion channels using a quantitative RT-PCR array. This analysis confirmed the overexpression of 68 genes and the underexpression of 14 genes in the VRC from adult compared with that from neonate. Our findings may help to explain the functional changes in respiratory rhythm and chemosensitivity occurring throughout life.