Efferent projections of Vglut2, Foxp2, and Pdyn parabrachial neurons in mice.

The parabrachial nucleus (PB) is a complex structure located at the junction of the midbrain and hindbrain. Its neurons have diverse genetic profiles and influence a variety of homeostatic functions. While its cytoarchitecture and overall efferent projections are known, we lack comprehensive information on the projection patterns of specific neuronal subtypes in the PB. In this study, we compared the projection patterns of glutamatergic neurons here with a subpopulation expressing the transcription factor Foxp2 and a further subpopulation expressing the neuropeptide Pdyn. To do this, we injected an AAV into the PB region to deliver a Cre-dependent anterograde tracer (synaptophysin-mCherry) in three different strains of Cre-driver mice. We then analyzed 147 neuroanatomical regions for labeled boutons in every brain (n = 11). Overall, glutamatergic neurons in the PB region project to a wide variety of sites in the cerebral cortex, basal forebrain, bed nucleus of the stria terminalis, amygdala, diencephalon, and brainstem. Foxp2 and Pdyn subpopulations project heavily to the hypothalamus, but not to the cortex, basal forebrain, or amygdala. Among the few differences between Foxp2 and Pdyn cases was a notable lack of Pdyn projections to the ventromedial hypothalamic nucleus. Our results indicate that genetic identity determines connectivity (and therefore, function), providing a framework for mapping all PB output projections based on the genetic identity of its neurons. Using genetic markers to systematically classify PB neurons and their efferent projections will enhance the translation of research findings from experimental animals to humans.

Repetitive electroacupuncture causes prolonged increased met-enkephalin expression in the rVLM of conscious rats.

Enkephalinergic neurons in the rostral ventrolateral medulla (rVLM), an important presympathetic region in the brainstem, are activated by 30 min of low frequency (2 Hz) electroacupuncture (EA) at acupoints P5-P6, which overlie the median nerves. To more closely model the clinical application of acupuncture, we administered EA for 30 min twice over a 72 h period to unsedated conscious rats to examine its prolonged action. We hypothesized that repetitive EA would increase preproenkephalin mRNA and met-enkephalin in the rVLM of unsedated conscious rats. Rats received either EA (1-4 mA, 0.5 ms, 2 Hz) or sham stimulation (needle placement without electrical stimulation) twice at P5-P6 acupoints bilaterally. Preproenkephalin mRNA and its peptide met-enkephalin in the rVLM were measured 24 or 48 h after the final EA or sham procedure. Relative ratios of preproenkephalin mRNA levels (normalized with the 18S housekeeping gene) were almost doubled at 24h compared to sham (6.1 ± 0.79 vs. 3.1 ± 0.47). Met-enkephalin measured in rVLM tissue pooled from several rats exposed to the same treatment was increased by repeated EA by 68% after 24h and 51% after 48h, relative to sham. These findings suggest that repeated application of EA in the conscious rats enhances transcription and translation of enkephalin in rVLM for days. Since opioids in the rVLM contribute importantly to the action of EA on sympathetic outflow, this mechanism may contribute to the prolonged action of acupuncture on elevated blood pressure in patients.

Prenatal ethanol exposure stimulates neurogenesis in hypothalamic and limbic peptide systems: possible mechanism for offspring ethanol overconsumption.

Exposure to ethanol during the prenatal period contributes to increased alcohol consumption and preference in rodents and increased risk for alcoholism in humans. With studies in adult animals showing the orexigenic peptides, enkephalin (ENK), galanin (GAL) and orexin (OX), to stimulate ethanol consumption, the question addressed here is whether prenatal ethanol alters the development in utero of specific neurons that express these peptides. With reports describing suppressive effects of high doses of ethanol, we examined the offspring of dams gavaged from embryonic day 9 to parturition with a control solution or lower ethanol doses, 1 and 3g/kg/day, known to promote ethanol consumption in the offspring. To understand underlying mechanisms, measurements were taken in postnatal offspring of the expression of ENK in the hypothalamic paraventricular nucleus (PVN) and nucleus accumbens (NAc), GAL in the PVN, and OX in the perifornical lateral hypothalamus (PFLH) using real-time qPCR and in situ hybridization, and also of the cell proliferation marker, 5-bromo-2-deoxyuridine (BrdU), and its double-labeling with either neuronal nuclei (NeuN), a marker of mature neurons, or the peptides. On postnatal day 15 (P15), after two weeks without ethanol, the offspring showed increased expression of ENK in the PVN and NAc core but not shell, GAL in the PVN, and OX in the PFLH. In these same areas, prenatal ethanol compared to control increased the density at birth (P0) of neurons expressing these peptides and at P0 and P15 of neurons double-labeling BrdU and NeuN, indicating increased neurogenesis. These BrdU-positive neurons were found to express ENK, GAL and OX, indicating that prenatal ethanol promotes neurogenesis in these specific peptide systems. There were no changes in gliogenesis or apoptosis. This increase in neurogenesis and density of peptide-expressing neurons suggests the involvement of these hypothalamic and accumbal peptide systems in mediating the increased alcohol consumption observed in prenatal ethanol-exposed offspring.

Predictors of ethanol consumption in adult Sprague-Dawley rats: relation to hypothalamic peptides that stimulate ethanol intake.

To investigate mechanisms in outbred animals that increase the propensity to consume ethanol, it is important to identify and characterize these animals before or at early stages in their exposure to ethanol. In the present study, different measures were examined in adult Sprague-Dawley rats to determine whether they can predict long-term propensity to overconsume ethanol. Before consuming 9% ethanol with a two-bottle choice paradigm, rats were examined with the commonly used behavioral measures of novelty-induced locomotor activity and anxiety, as assessed during 15 min in an open-field activity chamber. Two additional measures, intake of a low 2% ethanol concentration or circulating triglyceride (TG) levels after a meal, were also examined with respect to their ability to predict chronic 9% ethanol consumption. The results revealed significant positive correlations across individual rats between the amount of 9% ethanol ultimately consumed and three of these different measures, with high scores for activity, 2% ethanol intake, and TGs identifying rats that consume 150% more ethanol than rats with low scores. Measurements of hypothalamic peptides that stimulate ethanol intake suggest that they contribute early to the greater ethanol consumption predicted by these high scores. Rats with high 2% ethanol intake or high TGs, two measures found to be closely related, had significantly elevated expression of enkephalin (ENK) and galanin (GAL) in the hypothalamic paraventricular nucleus (PVN) but no change in neuropeptide Y (NPY) in the arcuate nucleus (ARC). This is in contrast to rats with high activity scores, which in addition to elevated PVN ENK expression showed enhanced NPY in the ARC but no change in GAL. Elevated ENK is a common characteristic related to all three predictors of chronic ethanol intake, whereas the other peptides differentiate these predictors, with GAL enhanced with high 2% ethanol intake and TG measures but NPY related to activity.

Electroacupuncture enhances preproenkephalin mRNA expression in rostral ventrolateral medulla of rats.

Electroacupuncture (EA) causes prolonged suppression of reflex elevations in blood pressure for at least 60min in anesthetized preparations. Thus, EA can modify sympathetic outflow and elevated blood pressure through actions in a number of hind brain regions, including the rostral ventrolateral medulla (rVLM). Since our previous data show that the opioid system plays a role in EA-related prolonged inhibition of presympathetic neuronal activity in the rVLM, we postulated that EA increases preproenkephalin (PPE) mRNA in this region, possibly for prolonged periods of time. Under alpha-chloralose anesthesia, rats received EA (1-2mA, 2Hz, 0.5ms) at P5-P6 acupoints (overlying median nerves) or sham (needle placement without electrical stimulation) for 30min. PPE mRNA in the rVLM also was evaluated in control rats that received surgery but no EA, or sham treatment. 20min, 1.5h or 4h following EA or sham treatment, PPE mRNA in the rVLM was analyzed by reverse transcription and quantitative real-time PCR. Relative ratios of PPE mRNA levels (normalized with 18s house keeping gene) were increased 1.5h after EA stimulation (7.77+/-1.39, n=6) relative to sham (2.84+/-0.37, n=5) but were unchanged both 20min and 4h after EA, compared to the sham or surgery groups at the same time points. Thus, 30min of EA transiently stimulates the production of enkephalin in a region of the brain that importantly regulates sympathetic outflow suggesting that even a single brief acupuncture treatment can increase the expression of this modulatory neuropeptide.

Effect of chronic ethanol on enkephalin in the hypothalamus and extra-hypothalamic areas.

BACKGROUND: Ethanol may be consumed for reasons such as reward, anxiety reduction, or caloric content, and the opioid enkephalin (ENK) appears to be involved in many of these functions. Previous studies in Sprague-Dawley rats have demonstrated that ENK in the hypothalamic paraventricular nucleus (PVN) is stimulated by voluntary consumption of ethanol. This suggests that this opioid peptide may be involved in promoting the drinking of ethanol, consistent with our recent findings that PVN injections of ENK analogs stimulate ethanol intake. To broaden our understanding of how this peptide functions throughout the brain to promote ethanol intake, we measured, in rats trained to drink 9% ethanol, the expression of the ENK gene in additional brain areas outside the hypothalamus, namely, the ventral tegmental area (VTA), nucleus accumbens shell (NAcSh) and core (NAcC), medial prefrontal cortex (mPFC), and central nucleus of the amygdala (CeA). METHODS: In the first experiment, the brains of rats chronically drinking 1 g/kg/d ethanol, 3 g/kg/d ethanol, or water were examined using real-time quantitative polymerase chain reaction (qRT-PCR). In the second experiment, a more detailed, anatomic analysis of changes in gene expression, in rats chronically drinking 3 g/kg/d ethanol compared to water, was performed using radiolabeled in situ hybridization (ISH). The third experiment employed digoxigenin-labeled ISH (DIG) to examine changes in the density of cells expressing ENK and, for comparison, dynorphin (DYN) in rats chronically drinking 3 g/kg/d ethanol versus water. RESULTS: With qRT-PCR, the rats chronically drinking ethanol plus water compared to water alone showed significantly higher levels of ENK mRNA, not only in the PVN but also in the VTA, NAcSh, NAcC, and mPFC, although not in the CeA. Using radiolabeled ISH, levels of ENK mRNA in rats drinking ethanol were found to be elevated in all areas examined, including the CeA. The experiment using DIG confirmed this effect of ethanol, showing an increase in density of ENK-expressing cells in all areas studied. It additionally revealed a similar change in DYN mRNA in the PVN, mPFC, and CeA, although not in the NAcSh or NAcC. CONCLUSIONS: While distinguishing the NAc as a site where ENK and DYN respond differentially, these findings lead us to propose that these opioids, in response to voluntary ethanol consumption, are generally elevated in extra-hypothalamic as well as hypothalamic areas, possibly to carry out specific area-related functions that, in turn, drive animals to further consume ethanol. These functions include calorie ingestion in the PVN, reward and motivation in the VTA and NAcSh, response-reinforcement learning in the NAcC, stress reduction in the CeA, and behavioral control in the mPFC.

Striatal opioid peptide gene expression differentially tracks short-term satiety but does not vary with negative energy balance in a manner opposite to hypothalamic NPY.

It has long been known that central opioid systems play an important role in certain aspects of appetite and food intake, particularly with regard to the hedonic or rewarding impact of calorically dense food, such as fat and sugar. Ventral striatal enkephalin may be a key component of this system, as infusions of mu-opiate agonists into this region strongly increase feeding, whereas infusions of opiate antagonists decrease food intake. While pharmacological analysis has consistently supported such a role, direct measurement of enkephalin gene expression in relation to differing food motivational conditions has not been examined. In this study, the effects of a restricted laboratory chow diet (resulting in negative energy balance) as well has recent consumption of chow (short-term satiety) on striatal preproenkephalin (PPE) and prodynorphin (PD) mRNA expression were measured in rats, using both Northern blot analysis and in situ hybridization methods. As a comparison, hypothalamic (arcuate nucleus) neuropeptide Y (NPY) was also measured in these conditions. PPE expression was broadly downregulated throughout the striatum in animals that had recently consumed a meal, whereas it was unaffected by negative energy balance. Expression of an additional striatal peptide gene, PD, did not follow this pattern, although diet restriction caused a decrease in accumbens core dynorphin mRNA. Conversely, as expected, arcuate nucleus NPY mRNA expression was markedly upregulated by negative energy balance, but was unchanged by recent food consumption. This double dissociation between striatal and hypothalamic peptide systems suggests a specific role for striatal PPE in relatively short-term food motivational states, but not in long-term metabolic responses to diet restriction.

Role of serotonin in the regulation of the dynorphinergic system by a kappa-opioid agonist and cocaine treatment in rat CNS.

It has been shown that chronic cocaine increases prodynorphin mRNA in the caudate putamen and decreases it in the hypothalamus. In addition, treatment with a kappa-opioid receptor agonist produced the opposite effect on prodynorphin gene expression in these brain regions and also evoked a decrease in the hippocampus. It is already known that kappa-opioid receptor agonists decrease the development of sensitization to some of the behavioral effects of cocaine. The serotonin system has also been shown to regulate dynorphin gene expression and a continuous infusion of fluoxetine induced prodynorphin gene expression in the same pattern as the kappa-opioid agonist (+)(5a,7a,8b)-N-methyl-N-[7-(1-pyrrolidinyl)-1 oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U-69593) in the brain regions investigated. It is interesting to note that treatment with a continuous infusion of cocaine produced different effects on this parameter. To determine whether serotonin plays a role in the regulation of prodynorphin mRNA by kappa-opioid agonists or cocaine, rats were treated with the serotonin depleter parachloroamphetamine (PCA). Beginning 24 h later, rats were treated with the selective kappa-opioid agonist U-69593 for 5 days or continuously with cocaine for 7 days and prodynorphin mRNA was measured. Prodynorphin mRNA was decreased significantly in the hypothalamus, caudate putamen, and hippocampus of rats treated with a single injection of PCA. Subsequent to PCA administration the effects of U-69593 or cocaine on prodynorphin mRNA were differentially affected across brain regions. Prodynorphin gene expression was still increased by U-69593 treatment in the hypothalamus and decreased in the caudate putamen. Cocaine treatment still produced a decrease in this parameter in the hypothalamus and an increase in the caudate putamen. In contrast, in the hippocampus, the decrease in prodynorphin mRNA produced by U-69593 was no longer evident after PCA and cocaine, which previously had no effect, now increased it in the serotonin-depleted group. These findings suggest that serotonin is necessary to maintain normal levels of dynorphin mRNA in all of the investigated brain areas and that the regulation of prodynorphin mRNA expression by chronic treatment with a kappa-opioid receptor agonist or cocaine requires serotonin in the hippocampus, but not in the hypothalamus or caudate putamen.

Spinal NF-kB activation induces COX-2 upregulation and contributes to inflammatory pain hypersensitivity.

Cyclooxygenase-2 (COX-2) is a major contributor to the elevation of spinal prostaglandin E2, which augments the processing of nociceptive stimuli following peripheral inflammation, and dynorphin has been shown to have an important role in acute and chronic pain states. Moreover, the transcription factor, nuclear factor-kappa B (NF-kB), regulates the expressions of both COX-2 and dynorphin. To elucidate the role of spinal NF-kB in the induction of inflammatory pain hypersensitivity, we examined whether activated NF-kB affects pain behavior and the expressions of the mRNAs of COX-2 and prodynorphin following peripheral inflammation. Intrathecal pretreatment with different NF-kB inhibitors, namely, NF-kB decoy or pyrrolidine dithiocarbamate, significantly reduced mechanical allodynia and thermal hyperalgesia following unilateral hindpaw inflammation evoked by complete Freund's adjuvant (CFA). These NF-kB inhibitors also suppressed the activation of spinal NF-kB and the subsequent remarkable elevation of spinal COX-2 mRNA, but not that of prodynorphin mRNA. In addition, the activation of spinal NF-kB following CFA injection was inhibited by intrathecal pretreatments with interleukin-1 beta receptor antagonist or caspase-1 inhibitor. In view of the fact that interleukin-1 beta (IL-1 beta) is the major inducer of spinal COX-2 upregulation following CFA injection, our results suggest that IL-1 beta-induced spinal COX-2 upregulation and pain hypersensitivity following peripheral inflammation are mediated through the activation of the NF-kB-associated pathways.

Distribution of glutamic acid decarboxylase, neurotensin, enkephalin, neuropeptide Y, and cholecystokinin neurons in the septo-preoptic region of male rats.

Neurons in the lateral septum (LS) and preoptic area (POA) are known to play an inhibitory role in feminine sexual behavior regulation in male rats. In this study, the distribution of neurons containing glutamic acid decarboxylase (GAD) and of the peptidergic neurotransmitters neurotensin (NT), enkephalin (ENK), neuropeptide Y (NPY), and cholecystokinin (CCK), was examined immunohistochemically in the LS and POA of castrated male rats subcutaneously implanted with estrogen-containing Silastic tubes. Colchicine was injected into the lateral ventricle of the animals. The forebrain sections were immunostained for each substance. A large number of GAD-immunoreactive (ir) cells were found in the LS. Many NT-ir cells were seen in the intermediate and ventral parts of the LS at the rostral and middle levels. A considerable number of ENK-ir cells were scattered over the LS at the rostral and middle levels and were observed in the ventral part of the caudal LS. There were only a few NPY-ir cells in the LS. No CCK-ir cells were observed in the LS. In the POA, GAD-ir cells were observed in abundance. Many NT-ir cells were seen, especially in the medial preoptic nucleus. Some ENK-ir cells and a few NPY-ir cells were found in the medial POA. CCK-ir cells of the POA were restricted to the periventricular and paraventricular hypothalamic nuclei.

Gender differences in proenkephalin gene expression response to delta9-tetrahydrocannabinol in the hypothalamus of the rat.

Chronic exposure to delta9-tetrahydrocannabinol (delta9-THC) produces an activation of preproenkephalin (PENK) gene expression in the rat hypothalamus. The levels of circulating gonadal steroids concurrently modulate this neuropeptide in male and female rats. However, whether gonadal steroids regulate delta9-THC effects on PENK gene expression in the hypothalamus of male and female rats remains unknown. To test this hypothesis, experiments were carried out on intact, 2-week-gonadectomized, 1-week-gonadectomized, 1-week-dihydrotestosterone (DHT) replaced male rats, and 2-week-gonadectomized, 1-week-gonadectomized, 1-week-oestradiol replaced female rats. One week after hormonal replacement, animals were treated with vehicle or delta9-THC (5 mg/kg/day, i.p. 7 days). In males, delta9-THC administration to intact animals induced PENK mRNA in the paraventricular nucleus (PVN) and ventromedial nucleus (VMN) of the hypothalamus. Orchidectomy did not affect basal PENK mRNA levels in the PVN, but reduced PENK mRNA levels in the VMN. However, delta9-THC treatment induced PENK gene expression to the same extent in both hypothalamic nuclei of intact, castrated and DHT-replaced males. In females, ovariectomy decreased PENK gene expression in PVN and VMN. delta9-THC administration increased PENK gene expression in castrated females, but had no effect in the oestradiol-replaced group. Taken together, these results suggest gender differences in the response of chronic exposure to cannabinoids on PENK gene expression in the hypothalamus. Furthermore, it appears that alterations in opioid gene expression induced by cannabinoids in female rats depend upon the presence or absence of circulating oestradiol.

Peripubertal ontogeny and estrogen stimulation of cholecystokinin and preproenkephalin mRNA in the rat hypothalamus and limbic system.

The neuropeptide cholecystokinin (CCK) is expressed in limbic system and hypothalamic nuclei that form a circuit that regulates the display of the female rodent reproductive behavior, lordosis. CCK mRNA and peptide levels fluctuate across the estrous cycle and have been shown to be modulated by estrogen exposure. The objective of these experiments was to examine the expression of CCK mRNA during postnatal development of this limbic-hypothalamic, lordosis regulating circuit, and to determine the age at which CCK mRNA expression becomes responsive to estrogen stimulation, by using quantitative in situ hybridization histochemistry. CCK mRNA levels were below the level of detectability within the circuit during the postnatal period, but increased during the peripubertal period. Rats were injected with either estradiol benzoate (EB), EB and progesterone, progesterone, or oil, and were killed 48 hours later on postnatal day (PND) 15, 20, and 25. Alternate brain sections were processed for CCK and preproenkephalin (PPE) mRNA in situ hybridization histochemistry. EB treatment induced CCK mRNA expression in the central portion of the medial preoptic nucleus and posterodorsal medial amygdala at PND 20 and 25, respectively. However, EB treatment increased PPE mRNA levels within the nuclei of the circuit at all ages examined. Progesterone had neither an independent nor additive effect on the EB induction of these neuropeptide messages. The estrogenic induction of CCK mRNA appears to be dependent on estrogen sensitive pathways of neurotransmission, or components of second messenger pathways which regulate CCK mRNA expression in the adult limbic-hypothalamic lordosis regulating circuit, which are not functional before PND 18-25.

Proenkephalin gene regulation in the paraventricular nucleus by GABA: interactions with opioid systems in a transgenic model.

GABA, which is present at high levels within the paraventricular nucleus (PVN) of the hypothalamus, has been implicated in neuroendocrine regulation. Here we use a transgenic mouse model expressing a human proenkephalin-beta-galactosidase fusion gene, in which both up-regulation and down-regulation of gene expression can be measured easily, to study the effects of GABA on basal and stress-induced gene expression within the PVN. This model has been shown previously to be appropriately physiologically regulated by stress within the PVN. Acute systemic administration of GABA, of aminooxyacetic acid, and of the selective GABA(B) receptor agonist, baclofen, induces transgene expression in the PVN. Chronic administration of aminooxyacetic acid inhibits both basal and stress-induced transgene expression. Acute or chronic administration of the selective GABA(A) agonist, muscimol, inhibits both basal and stress-induced expression of the transgene. Muscimol also inhibits transgene expression in hypothalamic slice cultures in which extrahypothalamic afferents are removed. It is surprising that, following chronic aminooxyacetic acid administration, the opiate antagonist naltrexone induces transgene expression in a manner similar to that observed with naloxone-precipitated opioid withdrawal and that expression is accompanied by a behavioral syndrome that partially mimicks opioid withdrawal. Taken together with our previous work, and using gene expression as our read-out, we conclude that transgene-expressing PVN neurons receive tonic inhibitory inputs mediated via GABA(A) receptors. Moreover, these inhibitory inputs can themselves be inhibited via GABA(B) and mu-opioid receptors.

Opiate receptors modulate estrogen-induced cholecystokinin and tachykinin but not enkephalin messenger RNA levels in the limbic system and hypothalamus.

Cholecystokinin, substance P and methionine enkephalin all regulate the display of reproductive behaviour. Their expression is exquisitely regulated by estrogen in the limbic-hypothalamic circuit, a circuit that regulates the display of estrogen-sensitive female reproductive behavior. Relatively little is known, however, about the interaction of endogenous opioid peptides with cholecystokinin and substance P in the limbic-hypothalamic circuit. Opiates antagonize the release of cholecystokinin and substance P in the hypothalamus and periaqueductal gray and stimulate cholecystokinin messenger RNA levels in the amygdala. To determine the effect of endogenous opioid input on estrogen-induced cholecystokinin, enkephalin and substance P expression, in situ hybridization histochemistry was used to examine estrogen-induced messenger RNA levels of these neuropeptides in specific nuclei of the limbic system and hypothalamus in the presence of opiate receptor antagonists. Estrogen treatment of ovariectomized rats significantly elevated cholecystokinin messenger RNA levels in the central portion of the medial preoptic nucleus, the encapsulated portion of the bed nucleus of the stria terminalis and the posterodorsal medial amygdala, as well as increased preproenkephalin and preprotachykinin messenger RNA levels in the ventromedial hypothalamic nucleus and the posterodorsal medial amygdala. The universal opiate receptor antagonist naltrexone and the delta-opiate receptor antagonist naltrindole each potentiated the estrogen-induced increase and elevated cholecystokinin messenger RNA levels an additional 1.9- to 2.8-fold depending on the nucleus examined, but had no effect on the estrogen-induced expression of either preproenkephalin or preprotachykinin messenger RNA. beta-Funaltrexamine, a mu-opiate receptor antagonist, had no effect on the medial preoptic or medial amygdaloid cholecystokinin messenger RNA levels or on the estrogen-induced expression of preproenkephalin messenger RNA but did cause a decrease in estrogen-induced cholecystokinin messenger RNA levels in the bed nucleus of the stria terminalis and a decrease in the preprotachykinin messenger RNA levels in the ventromedial hypothalamic nucleus. These results indicate that endogenous opioids, acting on the delta-opiate receptor within nuclei of the limbic-hypothalamic circuit, restrain the estrogen-induced increase of cholecystokinin messenger RNA expression. Activation of the mu-opiate receptor, however, may facilitate cholecystokinin messenger RNA expression in the bed nucleus of the stria terminalis and preprotachykinin messenger RNA expression in the ventromedial hypothalamic nucleus. Thus, endogenous opioid peptides may act in a site- and receptor-specific manner to modulate estrogen-induced neuropeptide levels in the limbic system and hypothalamus.

[2 Hz and 100 Hz electroacupuncture accelerate the expression of genes encoding three opioid peptides in the rat brain].

Previous findings from this laboratory have shown that low (2 Hz) and high (100 Hz)-frequency electroacupuncture (EA) accelerated the release of different kinds of opioid peptides in the CNS. In the present study, we tried to elucidate whether EA of different frequencies would affect the transcription of genes encoding different opioid peptides. Digoxin-labeled antisense cRNA probes were used for in situ hybridization to detect the mRNA encoding preproenkephalin (PPE), preprodynorphin (PPD) and proopiomelanocortin (POMC) in the rat brain. The results showed that: (1) Neither 2 Hz nor 100 Hz EA altered the POMC mRNA level in the rat brain. (2) EA of the two frequencies induced a similar degree of increase of PPE mRNA in rostromedial reticular formation (gigantocellular, paragigantocellular and lateral reticular nucleus); whereas in supraoptic nucleus, suprachiasmatic nucleus, arcuate nucleus, paraventricular hypothalamic nucleus, ventromedial nucleus and the nucleus of lateral lemniscus, 2 Hz EA induced a higher PPE mRNA expression than 100 Hz EA. (3) 100 Hz EA markedly increased the PPD mRNA levels in supraoptic nucleus, paraventricular hypothalamic nucleus, ventromedial nucleus and parabrachial nucleus, while 2 Hz was without effect. Since de novo peptide synthesis is regarded as a natural outcome following accelerated peptide release, the present results substantiate our previous observation that EA of different frequencies exert different acceleratory effects on the release and synthesis of different opioid peptides in the central nervous system.

cDNA sequence and expression of bovine prodynorphin.

Prodynorphin (ProDYN) in the anterior pituitary gland appears to be processed differently from the brain, and the ProDYN-derived peptides may function differently in the anterior pituitary than in the brain. To further investigate the roles of ProDYN-derived peptides in the anterior pituitary, we have determined the nucleotide (nt) sequence of the cDNA encoding bovine ProDYN. This is the first time a complete cDNA sequence for ProDYN has been reported. The nt and deduced amino acid (aa) sequences were compared to the known ProDYN of other species. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) combined with Southern blot analysis demonstrated that the expression of ProDYN in both the anterior and posterior pituitary glands was much lower than that in the neural tissues of the striatum and hypothalamus.

Chronic intracerebroventricular cocaine differentially affects prodynorphin gene expression in rat hypothalamus and caudate-putamen.

We investigated the effects of sustained administration of cocaine on the regulation of prodynorphin gene expression in rat brain. Intracerebroventricular (i.c.v.) infusion of cocaine hydrochloride (30 micrograms/day) for 7 days, by means of osmotic minipumps, elicited a significant 35% decrease of prodynorphin mRNA levels in rat hypothalamus and increase (22%) in caudate-putamen. At the same time and in the same animals, no significant changes were detected in the hippocampus or in the nucleus accumbens. These results indicate that continuously infused cocaine is able to modulate expression of the prodynorphin gene in opposite directions or has no effect on prodynorphin expression, depending on the brain region analysed. Cocaine, as well as opiates, might activate specific neuronal pathways, shared by different classes of drugs of abuse, involving, at least in part, the endogenous opioid system.

[Comparative study on the expression and interaction of oncogene c-fos/c-jun and three opioid genes induced by low and high frequency electroacupuncture].

The present work was designed (a) to study comparatively the effect of 2Hz and 100Hz electroacupuncture (EA) on the expression of oncogene c-fos/c-jun and three opioid (preproenkephalin-PPE; preprodynorphin-PPD; proopiomelanocortin-POMC) genes in the rat brain; (b) to clarify the role of Fos/Jun (AP-1) on opioid genes expression induced by EA stimulation through specific blockade of EA-induced Fos/Jun expression using antisense oligodeoxynucleotides (ODNs) of c-fos/c-jun. The results were: (a) 2Hz and 100Hz EA induced differential Fos expression in different brain areas; (b) EA of both frequencies accelerated PPE gene transcription, but 2Hz EA was more effective than 100Hz EA; (c) PPD expression was accelerated by 100Hz EA, but not by 2Hz EA; (d) the blockade of Fos/Jun expression by c-fos/c-jun antisense ODNs prevented EA from accelerating PPD but not PPE mRNA expression.

Prenatal morphine exposure differentially alters expression of opioid peptides in striatum of newborns.

The biochemical and cellular mechanisms involved in the development and/or maintenance of morphine tolerance remain unclear. In the adult central nervous system (CNS) results are contradictory. For the neonate, a variety of drug induced deficits have been observed following prenatal addiction to opioids, although very little work on the biochemical and molecular level has been done. Therefore, the present study was carried out to investigate the effects of prenatal morphine treatment on the levels and expression of endogenous opioid peptides in brain regions of newborns. Dams were implanted with one morphine pellet (75 mg each) 1 week prior to the birth of pups. Changes in mRNA levels for the opioid peptides were determined by Northern blot analysis. Alterations in opioid peptide levels were determined by radioimmunoassays. Prenatal morphine treatment significantly increased proenkephalin mRNA levels and decreased met-enkephalin levels in striatum of newborns. These data are in contrast to what is observed in the adult CNS. These data indicate that prenatal morphine treatment may increase met-enkephalin release and/or cause inhibition at the level of translation. In addition, increased transcription may be necessary to maintain equilibrium in the system when there is an increase in met-enkephalin release.

[Expression and localization of immediate early genes and preproenkephalin gene in central nervous system following electroacupuncture stimulation].

Using the techniques of immunohistochemistry, Northern blotting and in situ hybridization, we have found that electroacupuncture (2/15Hz) accelerated the expression of mRNA and protein synthesis of immediate early genes c-fos, c-jun as well as proenkephalin gene in rat central nervous system (CNS). Northern hybridization revealed that EA stimulation induced c-fos gene expression within 1-2 h, followed by PENK gene expression which peaked at 48h. Morphological study demonstrated that the sites of c-fos, c-jun and PENK mRNA expression and protein synthesis coincide with each other in the CNS. The results suggest that Fos/Jun may function as the transcription regulator of PENK gene.