Proenkephalin Research Articles

Developmental regulation of preproenkephalin mRNA in the ovine paraventricular nucleus: effects of stress and glucocorticoids

The opioid peptides have profound effects at several levels within the hypothalamo-pituitary-adrenal (HPA) axis. Activation of fetal HPA function occurs during late gestation, and as part of the fetal adaptive response to stress. Changes in the relative levels, localization and distribution of hypothalamic preproenkephalin (PENK) mRNA in the ovine hypothalamic paraventricular nucleus (PVN) during development were examined by in situ hybridization histochemistry. The effects of fetal hypoxemia applied in the presence or absence of concomitant cortisol, to establish negative feedback potential in late gestation were also investigated. PENK mRNA was present at low levels within the PVN, by d60 (term d147). During mid to late gestation, there was an increase in PENK mRNA levels from d60–80 to d100–120, then reaching a peak at d130–140. Levels then decreased dramatically during the last 5–7 days prior to parturition, but increased again in the newborn lamb. Throughout gestation, PENK mRNA was confined exclusively to the parvocellular region of the PVN. Cortisol infusion induced significant decreases ( P < 0.05) in PENK mRNA, in normoxemic fetuses at d135 of gestation. The hypoxemic insult, which is known to stimulate plasma ACTH and cortisol, in these fetuses, did not significantly affect PENK mRNA. There was no significant difference in PENK mRNA between saline or cortisol infused hypoxemic fetuses. However, the combination of cortisol infusion and hypoxemia significantly decreased PENK mRNA compared to the saline-infused normoxemic fetuses. Together, these results indicate that the elevation of endogenous fetal cortisol, that occurs at the end of gestation, may act to inhibit expression of the PENK gene in the hypothalamic PVN of the developing ovine fetus.

Gene expression in the aging brown Norway rat epididymis.

The mammalian epididymis is the site where spermatozoa are matured and then stored. Though many studies have described epididymal functions and their regulation, little is known about how aging affects this tissue. The Brown Norway rat, which does not show the many age-related pathologies common to other rat strains, was used as a model to study aging of the epididymis. The present study was designed to determine the effect of aging on the mRNA levels for selected markers of epididymal function. Brown Norway rats ranging in age from 6 to 30 months were examined at 6-month intervals; epididymides were sectioned into caput-corpus and cauda regions. Relative mRNA concentrations were assessed using Northern blot analysis and specific cDNAs for the rat 5 alpha-reductase isozymes, types 1 and 2; proenkephalin; the androgen receptor; epididymal proteins B/C and D/E; and sulfated glycoprotein-2 (SGP-2, clusterin). Northern blots were quantitated by densitometric scanning. In the caput-corpus epididymidis, 5 alpha-reductase type 1 and type 2 mRNA levels decreased significantly by 43% and 33%, respectively, between 6 and 12 months and by 64% and 40%, respectively, between 6 and 30 months. No significant change, however, was found in the expression of the 5 alpha-reductase mRNAs in the cauda epididymidis. Interestingly, proenkephalin mRNA was only detected in the caput-corpus epididymidis of 6-month-old rats. In marked contrast to the 5 alpha-reductase isozymes and proenkephalin, no significant age-related changes were observed in the mRNA levels for the androgen receptor, protein B/C, or protein D/E. No age-related changes in mRNA expression for SGP-2 occurred in the caput-corpus epididymidis. However, in the cauda epididymidis, SGP-2 mRNA levels rose by twofold between 6 and 18 months and then decreased sharply by 75% between 18 and 30 months. We conclude that as the epididymis ages, the expression of genes for certain specific markers of epididymal function is affected in a region-specific manner. Further, the decrease in the concentrations of the mRNAs for the 5 alpha-reductase isozymes and proenkephalin in the epididymis between 6 and 12 months is thus far the earliest marker for aging in the male reproductive tract of the Brown Norway rat.

"Prohormone thiol protease" (PTP) processing of recombinant proenkephalin.

The "prohormone thiol protease" (PTP) from adrenal medullary chromaffin granules has been demonstrated as a novel cysteine protease that converts the model enkephalin precursor, ([35S]Met)-preproenkephalin, to appropriate enkephalin related peptide products [Krieger, T. J., & Hook, V. Y. H. (1991) J. Biol. Chem. 266, 8376-8383; Kreiger, T. J., Mende-Mueller, L., & Hook, V. Y. H. (1992) J. Neurochem. 59, 26-31; Azaryan, A. V., & Hook, V. Y. H. (1994) FEBS Lett. 341, 197-202]. In this report, PTP processing of authentic proenkephalin (PE) was examined with respect to production of appropriate intermediate products, and kinetics of PE processing were assessed. Recombinant PE was obtained by high level expression in Escherichia coli, with the pET3c expression vector; PE was then purified from E. coli by DEAE-Sepharose chromatography, preparative gel electrophoresis, and reverse-phase HPLC. Authentic purified PE was confirmed by amino acid composition analyses and peptide microsequencing. In time course studies, PTP converted PE (12 microM) to intermediates of 22.5, 21.7, 12.5, and 11.0 kDa that represented NH2-terminal fragments of PE, as assessed by peptide microsequencing. Differences in molecular masses of the 22.5, 21.7, 12.5, and 11.0 kDa products reflect PTP processing of PE within the COOH-terminal region of PE, which resembles PE processing in vivo [Liston, D. L., Patey, G., Rossier, J., Verbanck, P., & Vanderhaeghen, J. (1983) Science 225, 734-737; Udenfriend, S., & Kilpatrick, D. L. (1983) Arch. Biochem. Biophys. 221, 309-314]. Products of 12.5, 11.0, and 8.5 kDa were generated by PTP cleavage between Lys-Arg at the COOH-terminus of (Met)enkephalin-Arg6-Gly7-Leu8.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of restraint or hypertonic saline stress on corticotrophin-releasing factor, arginine vasopressin, and proenkephalin A mRNAs in the CFY, Sprague-Dawley and Wistar strains of rat

It is generally assumed that the stress response of different strains of rat will be identical following exposure to acute stress. In the present study we have examined the activation of the hypothalamo-pituitary-adrenal axis in the Wistar, Sprague-Dawley and CFY strains of rat following exposure to either the predominantly psychological stress of restraint or the physical stress of i.p. hypertonic saline injection. We have investigated the hypothalamic activation of corticotrophin-releasing factor (CRF) and proenkephalin A (PEA) mRNAs in the parvocellular cells of the paraventricular nucleus (PVN) and arginine vasopressin (AVP) in both the magnocellular and parvocellular regions in the PVN following acute stress. In addition we have measured corticosterone as an index of end-point activation. Circulating corticosterone and CRF mRNA were increased in all three strains following either stress. AVP and PEA mRNAs were increased following hypertonic saline but only in the CFY strain following restraint. Overall the relative increase in the parameters measured was greater in the CFY strain of rat than the other strains. These data demonstrate marked differences in response to acute stress in the three strains of rat examined. These varying responses must be taken into consideration when designing or interpreting any study investigating the stress response.

Proenkephalin and prodynorphin mRNA level in brain of rats with absence epilepsy

An in situ hybridization method was used to estimate the proenkephalin (PENK) and prodynorphin (PDYN) mRNA levels in the brain of epileptic 6-month-old WAG Rij rats in comparison with non-epileptic: 3-month-old WAG Rij rats, 3-month-old ACl rats and 6-month-old ACl rats. The epileptic rats had a significantly higher level of PENK mRNA in the striatum as compared to non-epileptic controls. The PDYN mRNA level was significantly elevated only in the hippocampus of epileptic rats, whereas age- or strain-related changes in the striatal and cortical PDYN mRNA levels were found in both epileptic and non-epileptic rats. The changes in the biosynthetic activity of endogenous opioid peptide systems may be important for the occurrence of epileptic discharges in these animals.

Receptor‐mediated regulation of neuropeptide gene expression in astrocytes

One of the functions of glial receptors is to regulate synthesis and release of a variety of neuropeptides and growth factor peptides, which in turn act on neurons or other glia. Because of the potential importance of these interactions in injured brain, we have examined the role of two different receptors in the regulation of astrocyte neuropeptide synthesis. Stimulation of beta-adrenergic receptors on type 1 astrocytes resulted in increased mRNA and protein for the proenkephalin (PE) and somatostatin genes. This receptor also increased expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The potential role of opiate receptors was examined in several ways. Treatment of newborn rats for 7 days with the opiate antagonist naltrexone, prior to preparation of astrocytes, had no effect on PE mRNA or met-enkephalin content but resulted in a significant increase in NGF content. However, treatment of astrocytes in culture with met-enkephalin, morphine, or naltrexone had no effect on any of these parameters. No opiate binding could be detected, using either etorphine or bremazocine, to membranes of astrocytes prepared from cortex, cerebellum, striatum, or hippocampus of 1-day, 7-day, or 14-day postnatal rats. Thus we conclude that type 1 astrocytes do not express opiate receptors and that the in vivo effects of naltrexone are mediated indirectly via some other cell type/receptor.

Aging, Cardiac Proenkephalin mRNA and Enkephalin Peptides in the Fisher 344 Rat

Cardiac proenkephalin (PENK) mRNA, methionine-enkephalin (ME) and leucine-enkephalin (LE) were determined from 2 days of age through senescence in Fisher 344 rats. Tissues were collected at 2 days, 2 weeks, 1, 2, 3, 7, 19, and either 22 or 27 months of age. Hearts were dissected, extracted and assaysed for ME and LE by radioimmunoassay (RIA) or for PENK mRNA by Northern blot analysis with a cDNA probe. Relative left ventricular (LV) PENK mRNA was low in 2 day animals and increased slowly between 2 weeks and 3 months of age. LV PENK mRNA then rose five to six-fold between 3 and 27 months of age. LV ME measurements were high in neonatal animals, declined to a nadir during development and then rose again as the animals matured and advanced in age. The pattern for right ventricular (RV) ME was similar. Atrial ME, also high at 2 days, declined thereafter and remained low. LE measurements in LV, RV and the atrial followed patterns similar to those described for ME. To evaluate for peptides contributed by cardiac nerves, 3, 7 and 22-month-old animals were acutely sympathectomized for 24 h with 6-hydroxydopamine. No decline in LV ME and LE was observed in the 6-hydroxydopamine treated animals. These data suggest several conclusions regarding mycardial enkephalinergic systems: (a) tissue enkephalin and PENK mRNA with advancing age, (b) tissue enkephalins may not strictly correlate with the relative abundance of PENK mRNA, and (c) most myocardial enkephalins are non-adrenergic in origin. The age-associated patterns in both PENK mRNA, ME and LE suggest that physiological, maturational or behavioral events between 3 and 7 months of age initiate the up-regulation and subsequent expansion of cardiac enkephalinergic systems.

Differential influence of haloperidol and sulpiride on dopamine receptors and peptide mRNA levels in the rat striatum and pituitary

We examined the effect of chronic administration (14 days) of haloperidol (2 mg/kg/day) or sulpiride (100 mg/kg/day), on the mRNA levels of various genes in the rat striatum and pituitary by quantitative in situ and Northern blot hybridizations. In the pituitary, haloperidol and sulpiride induced similar increases of mRNAs of pro-opiomelanocortin (POMC) (+65% and +73%), prolactin (PRL) (+821% and +840%) and growth hormone (GH) (+32% and +47%), but sulpiride induced a greater increase of D2R mRNA (+125%) than haloperidol (+92%). In the striatum, sulpiride and haloperidol had different effects: sulpiride induced a higher increase than haloperidol of both preproenkephalin A (PPA) mRNA (+67% versus +47%) and D 2 dopamine receptor (D 2R) mRNAs (+72% versus +40%). Moreover, haloperidol and sulpiride had opposite effects on substance P (SP) mRNA. Haloperidol decreased the amount of SP mRNA by 20% while sulpiride increased it by 20%. The D 1 dopamine receptor (D 1R) mRNA level was not significantly modified after either treatment. Our results demonstrate that the effect of a chronic haloperidol treatment on striatal dopamine receptors and neuropeptide mRNA levels is different to that of sulpiride, whereas it is similar on pituitary hormones mRNA levels.

Biosynthesis of proenkephalin and prodynorphin in the brain of rats with genetic absence epilepsy

Endogenous opioid peptides are involved in the genesis or/and control of an epileptic activity in WAG/Rij rats - a genetic model of absence epilepsy. An in situ hybridization study showed that epileptic rats had a significantly higher level of proenkephalin (PENK) mRNA in the striatum and frontal cortex as compared with non-epileptic controls. In contrast to PENK, only age-related changes in PDYN mRNA in the cortex and, to the lesser extent in the striatum were detected in both epileptic and non-epileptic rats. In the hippocampus of epileptic rats, an elevated PDYN mRNA level and no differences in hippocampal PENK mRNA level were found

Regulation of proenkephalin gene expression in cultured bovine chromaffin cells

In cultured bovine chromaffine cells proenkephalin gene expression is induced by a variety of stimuli, such as membrane depolarization by high potassium ions, activation of adenylate cyclase by forskolin or activation of protein kinase C by phorbol esters. In this study the regulatory elements responsible for the proenkephalin (PENK) gene activation have been studied by transfection of rat PENKCAT reporter genes in primary cultured bovine chromaffin cells. It is shown that the ENKCRE-2 element confers the inducibility of the PENK gene in response to high potassium ions, forskolin and phorbolesters

Neurotrophic factor gene expression in astrocytes during development and following injury

Astrocyte cultures were utilized to examine regulation of the expression of several trophic factor genes. Regulation by the β-adrenergic receptor was demonstrated by exposure of striatal and cortical astrocytes to isoproterenol, which resulted in increased content of mRNAs for nerve growth factor (NGF), brain-derived neurotrophic factor, and proenkephalin (PE), as well as NGF and Met-enkephalin. Developmental regulation was analyzed by preparing cortical astrocytes from animals of four different ages—embryonic day 20, postnatal days 3 and 8, and adult. Because both the PE and NGF genes showed developmental downregulation, we asked whether we could prepare reactive astrocytes from lesioned adult brain and see expression turned back on. Astrocytes prepared from 6-hydroxydopamine-lesioned rat striatum or MPTP-lesioned mouse striatum contained increased GFAP and NGF mRNA. Comparable changes in GFAP and NGF could be achieved by treatment of control cultures with interferon-gamma or interleukin-1β. These results suggest that locus coeruleus neurons could control astrocyte synthesis of neurotrophic factors through release of norepinephrine, but that in injured brain other factors, such as cytokines, may become equally important.

Genetic linkage analysis of manic depression in Iceland.

Genetic linkage analysis has been used to study five Icelandic pedigrees multiply affected with manic depression. Genetic markers were chosen from regions which had been implicated by other studies or to which candidate genes had been localized. The transmission model used was of a dominant gene with incomplete penetrance and allowing for a large number of phenocopies, especially for unipolar rather than bipolar cases. Multipoint analysis with linked markers enabled information to be gained from regions spanning large distances. Using this approach we have excluded regions of chromosome 11p, 11q, 8q, 5q, 9q and Xq. Candidate genes excluded include those for tyrosine hydroxylase, the dopamine type 2 receptor, proenkephalin, the 5HT1A receptor and dopamine beta hydroxylase. Nevertheless, we remain optimistic that this approach will eventually identify at least some of the genes predisposing to manic depression.

Age-associated increase in rat cardiac opioid production

Several lines of evidence suggest that opioid peptide production in the heart may increase during aging from adulthood through senescence. We tested the hypothesis that cardiac opioid peptides and preproenkephalin (PNK) mRNA would increase with advancing age. Ventricles and atria from male Wistar rats, aged 2, 6, 18, and 22-24 mo of age, were acid extracted and assayed for methionine enkephalin (ME) and leucine enkephalin (LE). Total RNA was extracted from hearts of age-matched rats and probed for PNK mRNA by Northern blot analysis using a cDNA probe. ME and LE peptides were significantly elevated with advancing age in both ventricles [P < 0.001; by analysis of variance (ANOVA)]. Ventricular ME concentration exhibited a biphasic increase with approximately twofold higher peaks at 6 and 22-24 mo of age compared with the 2 mo value of 1.04 +/- 0.05 (SE) pmol/g wet wt. In contrast, ventricular LE concentration was largely unchanged until 22-24 mo of age when it increased approximately threefold over the 2-mo value of 2.19 +/- 0.14 pmol/g wet wt. Left ventricular PNK mRNA increased approximately fivefold between 2 and 18 mo of age (P < 0.01; ANOVA). Thus both enkephalins and the mRNA coding for them were increased in hearts of older vs. younger rats. Because opioid receptor stimulation can negatively modulate several characteristics of cardiac myocyte contraction, these results may have important functional implications for the senescent heart.

Estrogenic regulation of proenkephalin mRNA expression in the ventromedial hypothalamus of the adult male rat

Endogenous opioid peptides and their receptors are regulated by gonadal steroid hormones in the rat hypothalamus. Recent evidence suggests that gonadal steroids are capable of regulating the expression of proenkephalin (PE) mRNA in the ventromedial hypothalamus (VMH) of female, but not male rats. Therefore, we examined the effect of gonadectomy with or without four weeks of hormone treatment on PE mRNA expression in adult male Fisher 344 rats using quantitative in situ hybridization histochemistry. Gonadectomy reduced plasma testosterone and dihydrotestosterone (DHT) levels compared to intact rats, while subsequent estradiol (E 2) or DHT treatment increased plasma E 2 and DHT levels, respectively. Furthermore, gonadectomy reduced PE mRNA expression in the VMH, but not in the striatum nor the olfactory tubercle compared to intact rats, and this reduction was prevented in the presence of E 2, but not DHT. The results suggest that the activation of estrogen receptors normally functions to maintain the level of VMH PE mRNA expression, which is sensitive to hormonal regulation in adult male rats. Thus, gonadal steroid hormones might regulate those reproductive functions which are modulated by PE-derived opioid peptides in the male rat brain.

Basic fibroblast growth factor (bFGF) regulates tyrosine hydroxylase and proenkephalin mRNA levels in adrenal chromaffin cells

bFGF is a neurotrophic protein expressed in various regions of the adult peripheral and central nervous system. The present study was undertaken to examine the role of bFGF in multihormonal, catecholaminergic and enkephalinergic cells of the adrenal medulla (AM). Western blot analysis revealed the presence of at least three bFGF isoforms (18, 22/23, and 24 kDa) in cultured bovine AM cells. Incubation of AM cells with the exogenous 18 kDa bFGF produced time-dependent increases in tyrosine hydroxylase (TH) and proenkephalin (PEK) mRNA, with maximal changes occurring at 12 h (TH) or 24 h (PEK) of bFGF exposure. Effects of bFGF on TH and PEK mRNA were non-additive with increases induced by exposure of AM cells to nicotine, the depolarizing agent veratridine, or the adenylate cyclase activator forskolin. These data indicate that bFGF effects may occur through intracellular pathways accessed during transsynaptic induction of TH and PEK genes. The increases in PEK mRNA induced by nicotine or bFGF were inhibited by the calcium antagonist TMB-8 also inhibited bFGF-induced increases in TH mRNA as well. However, treatment with TMB-8 increased basal levels of TH mRNA. The addition of bFGF increased endogenous levels of c- fos mRNA, c-Fos and c-Fos-related proteins, suggesting that bFGF may active TH and PEK gene expression through a calcium-AP1 transcriptional regulatory pathway. Immunohistochemical analysis revealed the presence of bFGF-immunoreactivity (bFGF-IR) in the cytoplasm and in the nucleus of AM cells. Incubation of cells with exogenous bFGF produced time-dependent increases of nuclear bFGF-IR. The intensity of nuclear bFGF-IR was also enhanced in cells treated with veratridine, indicating that in both cases nuclear translocation of bFGF occurs. The present study indicates that bFGF could serve as a paracrine or autocrine factor regulating the expression of TH and PEK genes in AM cells. It also suggests that nuclear translocation of bFGF in AM cells may play a role in transcriptional effects of bFGF in those cells.

Differential posttranslational processing of proenkephalin in rat bone marrow and spleen mononuclear cells: evidence for synenkephalin cleavage.

Proenkephalin (PENK) messenger RNA was reported to be present in bone marrow mononuclear cells (BMMC) and spleen mononuclear cells (SMC). Nevertheless, the pattern of PENK products in normal cells of the rat immune system, which is important for defining the physiological role of PENK gene expression, has not been well established. In this work we have characterized the processing of the opioid portion (met-enkephalin-containing peptides) and nonopioid portion (synenkephalin-derived peptides) of PENK in rat BMMC and SMC. Met-enkephalin-containing peptides were detected in mononuclear cells of both hematopoietic tissues. In BMMC, free immunoreactive (IR)-met-enkephalin corresponded only to the 15% of total met-enkephalin-IR, whereas in SMC it represented the 66.5%. Gel filtration chromatography showed that BMMC contained partially processed PENK-derived peptides of high and intermediate molecular weight, whereas SMC displayed fully processed products containing met-enkephalin and/or the carboxyterminal portion of synenkephalin. HPLC purification of low molecular weight products showed that free IR-met-enkephalin in SMC mainly corresponded to met-enkephalin and oxidized met-enkephalin. In addition we have characterized in SMC three peptides lower than 3.0 kilodalton containing the C-terminal sequence of synenkephalin. These peptides were purified by gel filtration, affinity chromatography, ion exchange chromatography, and HPLC. These results show that PENK was processed in mononuclear cells of the primary (bone marrow) and secondary (spleen) organs of the rat hematopoietic system, as occurs in neural and endocrine tissues. Nevertheless, the precursor was cleaved only in the latter tissue to low molecular weight peptides. Furthermore we demonstrated that synenkephalin (proenkephalin 1-70) in SMC was processed to low molecular weight peptides containing the C-terminus free. This last result suggests that a dibasic Lys-Lys and monobasic (Lys) sites were cleaved.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) effects on enkephalinergic neurons in various regions of mouse brain

In order to elucidate the effects of MPTP on enkephalinergic neurons, dopamine (DA), norepinephrine (NE), proenkephalin (PE) mRNA and met-enkephalin (ME) were measured in striatum, olfactory tubercle, and prefrontal cortex of C57/B16 mice 1 day–2 weeks following treatment with 96 mg/kg MPTP-HCl (24 mg/kg i.p., twice/day for 2 days). DA and its metabolites were depleted 70% in striatum and 40% in olfactory tubercle within 1 day. In cortex, DA was unchanged, whereas homovanillic acid and NE were depleted 50 and 40% respectively by 3 days. ME increased in all three brain regions at different times whereas PE mRNA showed a different pattern in each region, with an increase in olfactory tubercle, a decrease in cortex, and in striatum, a decrease at 1 day followed by an increase at 3 days. Thus enkephalinergic neurons in each region respond differently to MPTP treatment. In striatum and olfactory tubercle, DA is depeleted sufficiently to release its tonic inhibition on the enkephalinergic neurons, thereby leading to increased enkephalin synthesis. In cortex, the change in NE metabolism appears to cause a decrease of ME release and thereby a depression of PE synthesis. The possible relationship between these results and the changes observed in Parkinson's disease are discussed.

Regulation of proenkephalin expression in C6 rat glioma cells

The expression of proenkephalin (PENK) mRNA in C6 rat glioma cells was stimulated by norepinephrine (a β-adrenergic agonist) and markedly enhanced by the addition of dexamethasone (a glucocorticoid agonist) to the culture medium, although dexamethasone alone exhibited no significant increase in PENK mRNA. Furthermore, no induction of glucocorticoid-response-element (GRE)-binding proteins was detectable. In contrast, the stimulation of PENK mRNA expression was not observed with a protein kinase C activator, 12-tetradecanoylphorbol-13-acetate (TPA), which stimulated the expression of c-fos and c-jun mRNA and their proto-oncoproteins (c-Fos and c-Jun). In addition, an AP-1 activity was induced by TPA and an induction of a κB-like binding activity was found with TPA plus cycloheximide-treated cells. Together, they suggest that activation of PENK gene in C6 cells is probably mediated mainly through the, β-adrenergic agonist-elicited cyclic AMP signal pathway, and induction of AP-1 and κB-like binding activities appear not to participate in gene activation. Interestingly, the Western blot data showed no increase in intracellular levels of proenkephalin between control and treated cells. However, a marked increase in immunoreactivities for proenkephalin and its derivative, [Met 5]-enkephalin was detected in medium and a lesser elevation in cells from modulator-treated cell culture through the time course. These results indicated that there was an association between an increase in PENK mRNA expression and an elevation of proenkephalins, and subsequently, the synthesized proenkephalins were released into the medium.

Reserpine treatment stimulates enkephalin and D 2 dopamine receptor gene expression in the rat striatum

We investigated the effect of catecholamine depletion on gene expression for preproenkephalin A (PPA) and D 2 dopamine receptor (D 2R) in the rat nigrostriatal complex, using quantitative Northern blot analysis. The D 2R probe indifferently recognizes the two mRNA isoforms generated by alternative splicing from the same gene. Short-term and chronic reserpine treatment increase the level of PPA and D 2R mRNA in the striatum in a complex manner. For short-term treatment, we injected 10 mg/kg of reserpine the first day, 5 mg/kg 24 h later and sacrificed the rats at various times after the last injection. This treatment resulted in an increase of the level of PPA mRNA by 50% and D 2R mRNA up to 150%. For chronic treatment, we injected 0.5 mg/kg of reserpine for 21 days, sacrificed the rats one day after the last injection and observed an increase in PPA and D 2R mRNA levels by 100%. Statistical analysis revealed that the PPA mRNA level after chronic treatment was significantly higher from the one obtained after short-term treatment while no such difference was observed for the D 2R mRNA. In contrast, reserpine treatment does not modify the level of D 2R mRNA in the substantia nigra suggesting that catecholamine depletion has postsynaptic but not presynaptic consequences in the rat nigrostriatal complex. These results demonstrate that reserpine acts at the gene or the mRNA level to induce dopamine supersensitivity in striatal dopaminoceptive neurons.

GABAergic afferents modulate proenkephalin mRNA expression in the guinea pig hypothalamic magnocellular dorsal nucleus

The proenkephalin (PE) gene expression within Met-enkephalin (ME) neurons of the guinea pig magnocellular dorsal nucleus (MDN) was measured following activation of GABAergic transmission by aminooxyacetic acid treatment (AOAA). A combination of preembedding ME immunocytochemistry and PE in situ hybridization was used to investigate PE mRNA and enkephalin immunoreactivity levels. In perikarya of the MDN, chronic AOAA-treatment resulted in an important decline in PE mRNA signal (−128%). In double-labeled neurons, the PE mRNA decrease was associated to an increase in ME immunoreactivity. These results indicate that the increase in ME immunoreactivity in the MDN following AOAA-treatment is not due to a rise in PE gene trasncription. This study provides additional morphological evidence supporting a role for GABA in modulating the enkephalinergic hypothalamoseptal tract of the guinea pig.