Neonatal treatments with the serotonin uptake inhibitors clomipramine and zimelidine, but not the noradrenaline uptake inhibitor desipramine, disrupt sleep patterns in adult rats

Kainic acid-induced status epilepticus results in delayed degeneration of CA3 hippocampal neurons in the mature but not immature rat hippocampus. In adult rats, the putative opioid precursor, preproenkephalin (PPE) mRNA increases in the dentate gyrus (DG), a region resistant to damage, following continuous limbic seizures. To explore why the immature brain is resistant to seizure-induced damage, the regional distribution of PPE mRNA expression and 45Ca2+ accumulation were compared in postnatal day 14 (P14) pup, and adult hippocampus at 5-6 h after kainate-induced status epilepticus. Inverted patterns of PPE expression and Ca2+ uptake were observed at the two ages. In P14 pups, PPE mRNA expression increased in DG and escalated in CA3, regions where 45Ca2+ accumulations were absent. In adult rats, PPE mRNA expression increased only in DG; 45Ca2+ labeling was predominant in CA3a,c and absent in DG. Pronounced increases in enkephalin neuropeptide synthesis in immature CA3 neurons may reduce glutamate release presynaptically and also prevent voltage-gated Ca2+ uptake into these neurons despite recurrent seizure activity. Opioid-mediated inhibition may provide an explanation for the resistance of the immature CA3 region to seizure-induced damage.     

Expression of the human antigen SPAG2 in the testis and localization to the outer dense fibers in spermatozoa

There is increasing evidence to suggest that opioid peptides may have widespread effects as regulators of growth. To evaluate the hypothesis that endogenous opioids control cellular proliferation during neural development, we have used in situ hybridization to examine opioid peptide and receptor mRNA expression in neuroepithelial zones of fetal rat brain and spinal cord. Our data show that proenkephalin mRNA is widely expressed in forebrain germinal zones and choroid plexus during the second half of gestation. In contrast, prodynorphin mRNA expression is restricted to the periventricular region of the ventral spinal cord. Little mu or delta receptor mRNA expression was detected in any regions of neuronal proliferation prior to birth. However, kappa receptor mRNA is widely expressed in hindbrain germinal zones during the 3rd week of gestation. Our present findings support the hypothesis that endogenous opioids may regulate proliferation of both neuronal and non-neuronal cells during central nervous system development. Given the segregated expression of proenkephalin mRNA in forebrain neuroepithelium and kappa receptor mRNA within hindbrain, different opioid mechanisms may regulate cell division in rostral and caudal brain regions.     

Perinatal delta9-tetrahydrocannabinol exposure reduces proenkephalin gene expression in the caudate-putamen of adult female rats

Perinatal delta9-tetrahydrocannabinol (delta9-THC) exposure in rats affects several behavioral responses, such as opiate self-administration behavior or pain sensitivity, that can be directly related to changes in opioidergic neurotransmission. In addition, we have recently reported that the administration of naloxone to animals perinatally exposed to delta9-THC produced withdrawal responses, that resemble those observed in opiate-dependent rats. The purpose of the present study was to examine the basal opioid activity in the brain of adult male and female rats that had been perinatally exposed to delta9-THC. To this aim, proenkephalin mRNA levels were measured, by using in situ hybridization histochemistry, in the caudate-putamen, nucleus accumbens, central amygdala and prefrontal cingulate cortex. The results showed a marked reduction in proenkephalin mRNA levels in the caudate-putamen of delta9-THC-exposed females as compared to oil-exposed females, whereas no changes were observed between delta9-THC- and oil-exposed males. There were no differences in proenkephalin mRNA levels in the nucleus accumbens, central amygdala and prefrontal cingulate cortex between males and females perinatally exposed to delta9-THC and their respective controls, although a certain trend to decrease was observed in delta9-THC-exposed females. In summary, perinatal exposure to delta9-THC exposure decreased proenkephalin gene expression in the caudate-putamen of adult rats, although this effect exhibited a marked sexual dimorphism since it was only seen in females. This result is in agreement with a previous observation from our laboratory that females, but not males, that had been perinatally exposed to delta9-THC, self-administered more morphine in adulthood. This suggests that low levels of proenkephalin mRNA may be used as a predictor of greater vulnerability to opiates.     

Estrogen receptor-beta messenger ribonucleic acid ontogeny in the prostate of normal and neonatally estrogenized rats

Neonatal exposure to estrogens permanently alters rat prostate growth and epithelial differentiation leading to prostatic dysplasia on aging. The effects are lobe-specific, with the greatest response observed in the ventral lobe. Recently, a novel estrogen receptor (ER) complementary DNA was cloned from the rat prostate and termed ER-beta (ER beta) due to its high homology with the classical ER alpha. The protein possesses high affinity for 17beta-estradiol, indicating that ER beta is an alternate molecule for mediating estrogenic effects. Importantly, ER beta messenger RNA (mRNA) was localized to rat prostatic epithelial cells, which contrasts with the stromal localization of ER alpha in the rat prostate. The present study was undertaken to determine the ontogeny of ER beta mRNA expression in the rat prostate lobes and to examine the effects of early estrogen exposure on prostatic ER beta expression. Male rat pups were given 25 microg estradiol or oil on days 1, 3, and 5; were killed on day 1, 3 (oils only), 6, 10, 30, or 90; and prostate lobes were frozen. Longitudinal sections were processed for in situ hybridization using an 35S-labeled antisense mRNA probe corresponding to a 400-bp EcoRI-AccI fragment in the 5' untranslated region of rat ER beta complementary DNA. Image analysis was used to quantitate silver grains. In addition, total RNA was isolated from the ventral prostate (VP) and used for semiquantitative RT-PCR. Results from in situ hybridization revealed that at birth, ER beta was equivalently expressed at low levels in both mesenchymal and epithelial cells in oil-treated rats. From day 1 onwards, expression in all stromal cells slowly and significantly declined, so that in the control adult prostate, stromal ER beta mRNA was slightly above background. In the oil-treated control rats, epithelial ER beta mRNA increased to moderate levels between days 6-10 in the VP and days 10-15 in the dorsal and lateral lobes as cells began differentiation and ducts lumenized. A further significant increase in ER beta message was observed at day 30, which indicates that full epithelial ER beta expression may require the completion of functional differentiation. By day 90, expression levels were maximal and similar between the lobes. RT-PCR substantiated this developmental increase in ER beta between days 1-90. Neonatal exposure to estrogens did not have an immediate effect on prostatic ER beta mRNA levels as determined by in situ hybridization and RT-PCR. However, the marked increase in epithelial cell expression at day 30 observed in the control VP was dampened in the VP of animals exposed neonatally to estrogens. By day 90, the VP of estrogenized rats possessed low ER beta message levels compared with the high expression in oil controls. In contrast, the dorsal and lateral lobes of neonatally estrogenized rats possessed high levels of ER beta mRNA at day 90, equivalent to controls. The present data demonstrate that ER beta mRNA expression in the rat prostate is developmentally regulated, and that neonatal estrogen can affect this expression in the adult VP. Because the effect of neonatal estrogens was not immediate, the data imply that early estrogen exposure may not directly autoregulate ER beta expression, and suggests that the adult effects on ER beta mRNA expression may be indirect. The differences in ER beta mRNA imprinting in the separate lobes may account for or reflect the lobe-specific neonatal estrogen imprints previously observed in the rat prostate.     

Effects of chronic nicotine treatment and withdrawal on hypothalamic proopiomelanocortin gene expression and neuroendocrine regulation

Considerable evidence suggest that some responses to smoking and nicotine are mediated by forebrain beta-endorphinergic opioid mechanisms. It has also been demonstrated that nicotine stimulates rat tuberoinfundibular dopaminergic activity. Since we have proposed that interactions between mediobasohypothalamic (MBH) dopaminergic and beta-endorphinergic mechanisms have a key role in neuroendocrine integration, we investigated the effects of chronic nicotine treatment and withdrawal on: (1) MBH concentrations of proopiomelanocortin (POMC, precursor for beta-endorphin biosynthesis) mRNA; (2) MBH concentrations of tyrosine hydroxylase (TH, rate limiting enzyme in catecholamine biosynthesis) mRNA; (3) corresponding serum prolacin, corticosterone, luteinizing hormone (LH), and testosterone concentrations. POMC and TH mRNA levels were measured by RNase protection/solution hybridization assay; serum hormone levels were measured by radioimmunoassay. Adult male rats received subcutaneous injections of either nicotine or saline during the dark period of each day on an increasing frequency (1-3 injections/day) and dosage (0.4-0.5 mg nicotine/kg body weight) schedule over 4 weeks. The rats were sacrificed after 4 weeks treatment and at 1, 3, 7, 14 and 21 days withdrawal. Chronic daily nicotine administration induced significant changes in serum corticosterone, serum prolactin, MBH TH mRNA, and MBH POMC mRNA concentrations that tended to persist through day 3 of withdrawal; serum prolactin and MBH POMC mRNA concentrations were suppressed whereas serum corticosterone and MBH TH mRNA concentrations were stimulated. None of the parameters were significantly different from control levels following 7 or more days of withdrawal from nicotine, except for a significant decrease of MBH POMC mRNA concentrations on day 21. Chronic daily nicotine or withdrawal did not significantly alter serum LH or testosterone concentrations. These results suggest that chronic nicotine inhibited POMC gene expression and thus, probably, biosynthesis of beta-endorphin and other opiomelanocortins. We hypothesize that suppression of forebrain beta-endorphin synthesis in response to long-term nicotine exposure produces a chronically opioid deficient condition which may play an important role in maintaining nicotine self-administration and in mediating some changes during the nicotine withdrawal syndrome.     

Specific reductions of striatal prodynorphin and D1 dopamine receptor messenger RNAs during cocaine abstinence

It is well established that the opioid neuropeptide and dopamine systems are altered following the use of cocaine. However very little information is available about their possible involvement during cocaine abstinence. In the present study, the mRNA expression of the dopamine receptors, D1 and D2, and the opioid peptides, prodynorphin and proenkephalin, were analyzed in the rat striatum using in situ hybridization histochemistry. Saline or cocaine (30 mg/kg, i.p.) were administered to rats once daily for 1 or 10 days. To examine cocaine abstinence, animals were treated for 10 days as described followed by a 10-day drug free period. Acute and intermittent cocaine administration elevated the prodynorphin mRNA expression in the dorsal striatum, consistent with previous reports, while the abstinent phase resulted in a significant reduction of prodynorphin mRNA levels in the ventrorostral striatum. The D1-receptor mRNA was decreased in the caudorostral striatum during cocaine withdrawal, a finding opposite to the increase observed following a single injection of the drug. Proenkephalin and the D2-receptor mRNAs were not altered during cocaine abstinence, though proenkephalin was elevated following acute but not repeated cocaine administration. These results show long-term suppression on prodynorphin and D1-receptor systems in specific striatal populations localized mainly in rostral areas during withdrawal from cocaine.     

Hepatic concentrations of proenkephalin-derived opioids are increased in a rat model of cholestasis

The liver of adult rats with cholestasis secondary to bile duct resection has been shown to express the proenkephalin gene and, by immunohistochemical stains, to contain met-enkephalin. To further study hepatic opioids in cholestasis, concentrations of proenkephalin-derived endogenous opioids were measured in a rat model of cholestasis by the use of radioimmunoassays. The specificity of the immunoreactivity detected by the assays was confirmed by high performance liquid chromatography (HPLC). In adult male rats with cholestasis due to BDR, the concentrations of three proenkephalin-derived opioid peptides were increased. Specifically, the mean hepatic concentrations of met-enkephalin, Met-Enk-Arg6-Phe7 and leu-enkephalin were 2.5 (p < 0.005), 2.1 (p < 0.005) and 2.5 (p < 0.01) fold higher than the corresponding mean for controls. These findings provide further independent evidence that opioid peptides accumulate in the liver in a model of cholestasis and are consistent with de novo synthesis of opioid peptides occurring in the cholestatic liver. This phenomenon may have relevance to the altered function of the opioid system in cholestasis and to the role of the liver as a neuroendocrine organ.     

Developmental expression of alpha 9 acetylcholine receptor mRNA in the rat cochlea and vestibular inner ear

Expression of alpha9 acetylcholine receptor (AChR) mRNA was studied by in situ hybridization in the rat adult and developing cochlea and vestibular inner ear. Alpha9 AChR mRNA was first observed in cochlear hair cells (HCs) at embryonic day 18 (E18), increased markedly after birth, stayed high until postnatal day 10 (P10), and decreased to substantially lower adult levels by P14. High levels of alpha9 AChR mRNA expression were also noted in the developing nonneuronal structures of the inner sulcus, chondrocytes, and/or osteoblasts in the cochlear capsule and interscalar laminae. Both developing and adult bone marrow cells also expressed intense alpha9 AChR mRNA. In the vestibular system, alpha9 AChR mRNA was first observed in HCs at E16 in all sensory epithelia, increased to its highest levels by P0-P4, then decreased slightly to reach adult levels by P10. The results are consistent with the alpha9 AChR subserving efferent neurotransmission to both cochlear and vestibular HCs. The observation of alpha9 AChR mRNA in cochlear HCs 2 weeks prior to functional onset in the cochlea further suggests that expression of this gene is not related to HC activity. The observation of substantial nonneuronal expression of alpha9 AChR mRNA suggests that this receptor also has functions separate from its role in neurotransmission.     

Initial processing of human proenkephalin in bovine chromaffin cells

The opioid peptide precursor preproenkephalin (PPE) contains seven enkephalin sequences and is synthesized by epinephrine-producing adrenal chromaffin cells and various peripheral and central neurons. After removal of its signal peptide, PPE undergoes processing at dibasic amino acid sites to yield its final opioid products-Met-enkephalin, Leu-enkephalin, and various larger, enkephalin-containing peptides. Processing of PPE was examined in bovine chromaffin cells using a plasmid containing the human PPE (hPPE) cDNA under the control of the cytomegalovirus immediate early enhancer/promoter. Following transfection of this hPPE-containing plasmid into bovine chromaffin cells, several proenkephalin-immunoreactive bands were observed on western blots with monoclonal antibodies that recognize human, but not bovine, proenkephalin sequences. The pattern of hPPE-derived peptides observed was similar to that of bovine PPE processing products. A series of recombinant plasmids containing mutations in the hPPE sequence at putative processing sites was then constructed. Conversion of Lys-Lys and Lys-Arg sequences to Lys-Gln and of Arg-Arg to Arg-Gln altered initial hPPE processing at only three of the putative processing sites. When hPPE cDNA containing mutations at all of these initially processed sites was expressed, one or more alternative processing sites were revealed. These data suggest the importance of structural features in addition to the dibasic sequences that limit the processing of proenkephalin.     

Differential induction of nerve growth factor and basic fibroblast growth factor mRNA in neonatal and aged rat brain

Stimulation of glucocorticoid or beta-adrenergic receptors (BAR) has been shown to increase nerve growth factor (NGF) biosynthesis in adult rat brain. Little is known about the role of these receptors in the regulation of NGF expression in neonatal and aged brain. We have examined the effect of the synthetic glucocorticoid dexamethasone (DEX) and the BAR agonist clenbuterol (CLE) on the levels of NGF mRNA in neonatal (8 day old), adult (3 month old) and aged (24 month old) rats. By 3 h, DEX (0.5 mg/kg, s.c.) evoked a comparable increase in NGF mRNA in the cerebral cortex and hippocampus in both 8-day and 3-month-old rats. In contrast, CLE (10 mg/kg, i.p.) failed to change NGF mRNA levels in neonatal rats, while increasing (2-3-fold) NGF mRNA levels in the cerebral cortex of adult rats. In 24-month-old rats, both DEX and CLE elicited only a modest increase in NGF mRNA. This increase was, however, anatomically and temporally similar to that observed in adult animals. The weak effect of DEX or CLE was not related to a down-regulation of receptor function because both DEX and CLE were able to elicit a comparable increase in the mRNA levels for basic fibroblast growth factor (FGF2) in neonatal, adult and aged rat brain. Our data demonstrate that induction of NGF expression by neurotransmitter/hormone receptor activation varies throughout life and suggest that pharmacological agents might be useful tools to enhance trophic support in aging.     

Downregulation of polo-like kinase correlates with loss of proliferative ability of cardiac myocytes

Cardiac myocytes rapidly increase the cell number during the fetal and early neonatal period, but they lose their proliferative ability soon after birth. To understand the mechanism of how cardiac myocytes exit from the cell cycle, we examined the role of a newly identified serine/threonine kinase, polo-like kinase (Plk), in the process of proliferation of cardiac myocytes. Northern blot analysis revealed that Plk gene was abundantly expressed in cardiac myocytes and non-myocytes of fetal and neonatal rats but not in cardiocytes of adult rats. Western blot analysis showed that Plk protein was also detected only in fetal and neonatal hearts. During the early stage of cardiac differentiation. Plk expression was well correlated with the proliferative ability of cardiocytes. Plk mRNA was most abundant in undifferentiated embryonic stem (ES) cells and the mRNA levels decreased along with cardiac differentiation in the developing ES cell system. Once serum was deprived from the culture media, expression levels of Plk were markedly decreased and DNA was not synthesized in both cardiac myocytes and non-myocytes of neonatal rats. Re-addition of serum stimulated Plk gene expression and DNA synthesis in non-myocytes but not in cardiomyocytes. All these results taken together with the critical role of Plk in DNA synthesis in many cell types suggest that downregulation of Plk is important for the permanent withdrawal of cardiomyocytes from the cell cycle.     

Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes

Neuregulins (i.e. neuregulin-1 (NRG1), also called neu differentiation factor, heregulin, glial growth factor, and acetylcholine receptor-inducing activity) are known to induce growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells. Unexpectedly, mice with loss of function mutations of NRG1 or of either of two of their cognate receptors, ErbB2 and ErbB4, die during midembryogenesis due to the aborted development of myocardial trabeculae in ventricular muscle. To examine the role of NRG and their receptors in developing and postnatal myocardium, we studied the ability of a soluble NRG1 (recombinant human glial growth factor 2) to promote proliferation, survival, and growth of isolated neonatal and adult rat cardiac myocytes. Both ErbB2 and ErbB4 receptors were found to be expressed by neonatal and adult ventricular myocytes and activated by rhGGF2. rhGGF2 (30 ng/ml) provoked an approximate 2-fold increase in embryonic cardiac myocyte proliferation. rhGGF2 also promoted survival and inhibited apoptosis of subconfluent, serum-deprived myocyte primary cultures and also induced hypertrophic growth in both neonatal and adult ventricular myocytes, which was accompanied by enhanced expression of prepro-atrial natriuretic factor and skeletal alpha-actin. Moreover, NRG1 mRNA could be detected in coronary microvascular endothelial cell primary cultures prepared from adult rat ventricular muscle. NRG1 expression in these cells was increased by endothelin-1, another locally acting cardiotropic peptide within the heart. The persistent expression of both a neuregulin and its cognate receptors in the postnatal and adult heart suggests a continuing role for neuregulins in the myocardial adaption to physiologic stress or injury.     

The localisation of TPPS4 in some organs and its possible nephrotoxicity in rats

Endogenous opioids have been implicated in the mechanism of action of antidepressant drugs. In this study, we evaluated effects of single and repeated imipramine administration on proenkephalin and prodynorphin gene expression in the rat nucleus accumbens and striatum. In situ hybridization study showed that single imipramine injection (10 mg/kg, i.p.) decreased the level of proenkephalin (by ca. 20%) and prodynorphin (by ca. 25%) mRNA to the same extent in both structures. Repeated imipramine administration (10 mg/kg i.p. twice daily for 10 days) had no effect on proenkephalin mRNA level, whereas the prodynorphin gene expression was regulated differently. At 3 h after the last dose of imipramine the prodynorphin mRNA level was decreased (by ca. 25%) in the striatum, but not in the nucleus accumbens, whereas at 24 h an up-regulation (by ca. 27%) of prodynorphin mRNA level could be observed in the nucleus accumbens only. In the light of involvement of opioids in mood regulation, these adaptive changes may participate in neurochemical mechanisms of antidepressant therapy.