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Catecholamine depletion modulates serum LH levels, GAD67 mRNA, and GABA synthesis in the goldfish
General and Comparative Endocrinology 140 (2005) 176–183 www.elsevier.com/locate/ygcen
Catecholamine depletion modulates serum LH levels, GAD67 mRNA, and GABA synthesis in the goldWsh Benjamin Hibbert, Irene Fung, Rebecca McAuley, Marwan Samia, Vance Trudeau¤ Department of Biology, Centre for Advanced Research in Environmental Genomics (CAREG), MacDonald Hall, University of Ottawa, Ottawa, Ont., Canada K1N 6N5 Received 9 July 2004; revised 28 October 2004; accepted 12 November 2004
Abstract It is established that dopamine inhibits while GABA stimulates LH release in goldWsh. In this study, we examine dopaminergic regulation of GABAergic activity in the hypothalamus of early recrudescent female goldWsh (Carassius auratus). We utilize a unique technique that permits concomitant quantiWcation and correlation of in vivo GAD65 and GAD67 mRNA with GABA synthesis rate in response to decreased dopamine levels. Catecholamine depletion was achieved by treatment with -methyl-para-tyrosine methyl ester (MPT; 240 g/g body weight), an inhibitor of tyrosine hydroxylase. Endogenous GABA levels were increased by intraperitoneal administration of -vinyl GABA (GVG; 300 g/g body weight), an inhibitor of the GABA catabolic enzyme GABA transaminase. Dual treatment of GVG + MPT increased serum LH levels 4-fold. However, LH mRNA levels in the pituitary remained stable, suggesting that treatments aVected secretion and not synthesis. In the hypothalamus, GABA synthesis rates increased 30% in response to MPT treatment. This was correlated (r D 0.61; p < 0.05) to increased levels of GAD67 mRNAs but not GAD65 (r D 0.14; p > 0.05). These observations suggest that catecholamines inhibit GABA synthesis in the goldWsh hypothalamus through isoform speciWc regulation of GAD67. 2004 Elsevier Inc. All rights reserved. Keywords: Gonadotropin-II; GABA synthesis rate; LH; Teleost
1. Introduction The goldWsh has an annual cycle of reproduction and in temperate climates spawning generally occurs in late April to early May. To ensure appropriate timing of seasonal sexual maturation and gonadal development in Wsh, release of lutenizing hormone (LH), and follicle stimulating hormone (FSH), also respectively known as gonadotropin-II (GtH-II) and gonadotropin-I (GtH-I) in teleosts, are regulated by an intricate network of neuroendocrine and hormonal feedback systems (Borg et al., 1998; Trudeau, 1997). While the principle stimulator of
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0016-6480/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2004.11.008
LH is gonadotropin-releasing hormone (GnRH), amino butyric acid (GABA) (Trudeau et al., 1993a,b), neuropeptide Y (NPY) (Peng et al., 1993a), noradrenaline (NA) (Chang et al., 1983), serotonin (5-hydroxytryptamine, 5-HT) (Somoza and Peter, 1991), and glutamate (Trudeau et al., 2000) have also been shown to increase LH release. In contrast, dopamine (DA) is the only known inhibitor of LH release in many Wsh species (Peter et al., 1986, 1988; Trudeau, 1997). Chang et al. (1983) Wrst showed that destruction of dopaminergic neurons or inhibition of catecholamine synthesis with -methyl-p-tyrosine (MPT) raised serum LH levels. In the same study, speciWc inhibition of NA synthesis failed to replicate the LH increase induced by MPT. Subsequently, DA has been shown to inhibit both basal and GnRH-stimulated LH release via DA
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type 2 receptors (Chang and Peter, 1983; Chang et al., 1984; Omeljaniuk et al., 1987) and reduce pituitary GnRH receptor binding (De Leeuw et al., 1989; Omeljaniuk et al., 1989). In addition, DA also inhibits GnRH at the level of the hypothalamus (Chang et al., 1990; Omeljaniuk et al., 1987) via a DA type 1 receptor (Yu and Peter, 1992). The central role of inhibitory DA systems is also supported by morphological studies showing that dopaminergic Wbres originating in the preoptic area innervate gonaodotrophs in the anterior goldWsh pituitary (Kah et al., 1987a,b). Another less well studied mechanism whereby DA may further inhibit LH involves interactions with the GABAergic neuroendocrine system. Treatment of Wsh with -vinyl GABA (GVG), an inhibitor of GABA catabolism, increases serum LH, upregulates pituitary expression of LH (Fraser et al., 2002) and also secretogranin-II, a marker of the regulated secretion pathway. Current evidence indicates that GABA stimulates LH release through activation of both A- and B-type GABA receptors, and by a dual action to enhance GnRH release and inhibit dopamine turnover in the goldWsh hypothalamus and pituitary (Trudeau et al., 1993a,b, 2000). Moreover, pretreatment with DA antagonists or DA depletion by MPT potentiates GABAstimulated LH release in goldWsh. Therefore, there appears to be a reciprocal inhibition of the DA and GABA systems involved in the control of LH release. GABA is synthesized from glutamate in a simple enzymatic step controlled by glutamic acid decarboxylase (GAD). There are two major forms of GAD in vertebrates, GAD67 and GAD65 (also known as GAD1 and GAD2), named for the molecular weight of the mature proteins (Martin and Rimvall, 1993). We have also found a third novel GAD in Wsh, which we called GAD3 (Bosma et al., 2001; Lariviere et al., 2002). In this study, we demonstrate that catecholamine depletion increases hypothalamic GABA synthesis rate and, when combined with GVG-treatment, results in LH release in vivo. The increase in GABA synthesis rate is correlated with an increase in the hypothalamic expression GAD67 but not the other GAD isoforms. We suggest DA inhibits GABA synthesis in the goldWsh hypothalamus by regulation of GAD67 mRNA levels.
2. Materials and methods 2.1. Animals and pharmacological treatments Common goldWsh (Carassius auratus) were purchased from ABC Aquarium Services (Montreal, Canada). Upon arrival, Wsh were separated by sex and kept in 18 °C de-chlorinated/oxygenated city of Ottawa tap water. Fish were fed standard Xake and pellet food and housed in a room with a simulated photoperiod for the
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Ottawa area. Female Wsh weighing between 13 and 37 g were selected and acclimated to tanks at least 2 weeks prior to any treatments. The experimental design consisted of four treatment groups CTRL, GVG, MPT, and a GVG/MPT group. The control group received 0.6% saline injections, The GVG (gift from Hoescht Marion Rousell) group received 300 g/g body weight intraperitoneally, the MPT (Sigma) group received 240 g MPT/g of body weight, and GVG/MPT group received both GVG and MPT at the doses indicated above. MPT treatments were given 6 and 1 day prior to sacriWce, a dose shown to achieve >50% DA depletion in target tissues (Trudeau et al., 1993a,b). GVG treatments were given one day prior to sacriWce. All injections were administered at a volume 5 l/g of body weight using a 21 gauge needle attached to a Hamilton microsyringe. 2.2. Dissections Fish were anesthetised in 0.5% MS222 (Sigma), weighed, decapitated, and the hypothalamus dissected, frozen immediately on dry ice, pooled, and stored at ¡80 °C till used for RNA isolation. For ribonuclease protection assay of GAD67, GAD65, and GAD3 mRNA levels, and HPLC analysis of GABA levels, brain tissue from 3 to 5 Wsh were pooled for one replicate. 2.3. GABA, protein, and RNA isolation Our laboratory has developed and validated an isolation procedure which enables concomitant quantiWcation of GABA synthesis rates and GAD mRNA levels from a unique sample (Hibbert et al., 2004). BrieXy, RNA was isolated using the GITC extraction method as described in Chomczynski and Sacchi (1987). We included homoserine (1 mg/ml) in the extraction solution to act as an internal control for subsequent HPLC analysis of GABA levels. The isopropanol phase of the RNA extraction contained the GABA and homoserine. Protein from each sample was isolated to correct for the varying amounts of tissue obtained during the dissection. Proteins were precipitated from the organic phase retained from the GITC RNA prep by addition of 1.5 ml of isopropanol. Samples were allowed to stand for 10 min at room temperature, and centrifuged at 12,000g to precipitate the proteins. The supernatant was discarded and the pellet washed three times for 20 min in 2 ml of 0.3 M guanidine hydrochloride/95% ethanol solution, followed by centrifugation (7500g) each time. The pellet was washed in 99% ethanol, vortexed, and incubated for 20 min at room temperature. Samples were centrifuged (7500g) for 5 min, the supernatant discarded, and samples air-dried for 5–10 min at room temperature. The protein samples were resuspended in 1.5 ml of 1% SDS/0.5 M NaOH. Protein concentrations were deter-
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mined (Smith et al., 1985) using a bicinchoninic acid protein assay kit (Sigma). 2.4. GABA quantiWcation by HPLC A modiWed version of the gradient reverse-phase HPLC technique with Xuorometric detection described by Sloley et al. (1992) was used to measure GABA concentrations. BrieXy, the isopropanol phases of the samples were put into pre-weighed (accurate to four decimal places) 2.0 ml centrifuge tubes. The volume of the samples was determined by dividing the mass of the isopropanol phase by the density of the isopropanol phase (1.0373 £ 10¡3 g/l). Before loading onto the HPLC machine, samples were Wrst Wltered with 1 ml syringes Wtted with Chromspec 0.2 l PTFE Wlters. Then, 20 l of the isopropanol phase sample were added to 60 l of ophthalaldahyde (OPA) (Pierce) and the solution was allowed to incubate for 3 min at room temperature. OPA binds the amine group of amino acids, yielding a derivatized molecule with high quantum eYciency, to allow for Xuorometric detection (Joseph and Davies, 1983). After incubation with OPA, 500 l of sodium acetate buVer (1.36 g/100 ml) were added, and the sample was loaded into the HPLC machine 2.5 min later. Then, 10 l of each preparation was injected onto a 4 m Superspher 100 RP-184 £ 75 mm analytical cartridge with a 5 m LiChrospher 100 C18 4 £ 4 mm guard cartridge (Merck). The linear gradient consisted of 9:1 acetonitrile buVer:methanol at t D 0 min (acetonitrile buVer is 9 parts 25 mmol NaH2PO4, 1 part acetonitrile, pH 6.0), 8:1 acetonitrile buVer:methanol at t D 10–12 min, and 9:1 acetonitrile buVer:methanol at t D 14 min, with a constant Xow rate of 1.0 ml/min. Fluorescence was detected with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The HPLC equipment comprised a Varian ProStar HPLC system consisting of models 410 autosampler with a 100 l loop, 230 pump, 360 Xuorescence detector and Star Workstation software (version 5.5).
2.6. Ribonuclease protection assay Regions of -actin, GAD3, GAD65, and GAD67 genes were ampliWed with PCR primers containing T7 (on antisense template) or SP6 (on sense template) RNA polymerase promoters. Primer sequences are as follows: KRPA65-FWD, att tag gtg aca cta tag ctc tga tcc gct ccg ttc; KRPA65-REV, taa tac gac tca cta tag cag cct ccc cga cat tta; KRPA67-FWD, att tag gtg aca cta tag acc aga gtc gct gga gca; KRPA67-REV, taa tac gac tca cta tag tcc atc gcc atc tcc att; KRPA3-FWD, att tag gtg aca cta tag agg cca agg gtc tgg ttc; KRPA3-REV, taa tac gac tca cta tag ttg tgg ggg ttc cat gtc; KRPABAC-FWD, att tag gtg aca cta tag gat ggt ggg aat ggg tca; and KRPABACREV, taa tac gac tca cta tag gga cag cac agc ctg gat. Promoter sequences are shown in italics. Product lengths were 304 bp for -actin, 252 bp for GAD3, 216 bp for GAD65, and 278 bp for GAD67. Protected probes were all 36 bp shorter than their respective initial probe lengths. Using T7 RNA polymerase (Ambion), in vitro transcription of antisense RNA molecules was performed incorporating [-32P]CTP (Amersham) for actin and [-32P]UTP (Amersham) for GAD65 and GAD67.The probe solution was then treated with DNase I for 30 min at 37 °C (Promega) and gel puriWed to eliminate traces of probe DNA template. Incubation of 10 g of total RNA from hypothalamus was then carried out with 3 £ 104 cpm of each RNA probe at 56 °C overnight. Non-protected regions of RNA were degraded with an RNase A/RNase T1 (Sigma) cocktail (7:5 ratio) at 30 °C for 45 min. Nucleases were degraded with proteinase K (Sigma) followed by phenol:chloroform extraction. Fragments were electrophoresed in a 5% denaturing polyacrylamide gel. Gels were dried and exposed to Kodak K-Screen phosphor screens for 18 h to visualize protected bands. A Molecular Imager FX (Bio-Rad) was used to scan the screens and bands were quantiWed using the Quantity One (Bio-Rad) software package. 2.7. Northern hybridizations
2.5. Calculation of GABA synthesis GABA synthesis rates in vivo were calculated as previously described (Fraser et al., 2002; Trudeau et al., 1993a). BrieXy, GABA levels in the dissected tissues were quantiWed 24 h after the Wnal saline or GVG injection in both the control and MPT-injected Wsh. Correction of GABA levels for variations in tissue size was based on total protein content per tissue. To obtain a net increase in GABA above basal levels, GABA levels in the salinetreated group in both control and MPT-treated animals are subtracted from those treated with GVG. This value is divided by 24 h to obtain an estimate of GABA synthesis rate per hour per milligram protein in control versus MPT-injected goldWsh.
To determine whether DA depletion or GVG injections aVected LH mRNA levels, 10 g of total RNA (as determined by OD260) from pituitary samples was electrophoresed on 1.5% denaturing agarose gels. Gels consisted of 3.0 g agarose B low EEO (electroendosmosis) (Biobasic), 170 ml DEPC-treated H2O, and 20 ml of 10£ Mops (morpholinopropanesulfonic acid; 41.8 g Mops, 4.1 g sodium acetate, and 2.9 g disodium EDTA per liter, pH adjusted to 7.0 with 10 N NaOH). 2.25 l of loading buVer (per 7 ml: 4 ml formamide, 0.8 ml of 10£ Mops, 0.4 ml glycerol, 1.4 ml formaldehyde, 0.1 ml of 0.5 M EDTA, pH 8.0, 0.4 ml DEPC treated water, 20 mg bromophenol blue, and 3.5:1 of 10 mg/ml ethidium bromide) was added for each l of sample volume. Samples were
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then denatured at 65 °C for 20 min and snap cooled on ice prior to loading. Running buVer consisted of 1£ MOPS and gels were run for 6 h at 100 V. Following electrophoresis, gels were washed twice in 5 volumes of ddH2O for 20 min. Subsequently, gels were washed once in 20£ standard sodium citrate buVer (SSC) for 20 min and concomitantly a piece of Hybond N+ nylon membrane (Amersham) was soaked with ddH2O and washed for 10 min in 2£ SSC. The gel was then transferred to the nylon membrane by capillary action with 20£ SSC overnight. Covalent cross-linking of RNA to the nylon membrane was achieved by exposure to UV-light for 5 min. Prior to each hybridization, membranes were washed with 0.1£ SSC/0.1£ SDS for 20 min at 65 °C, then prehybridized for >3 h in 10 ml of hybridization buVer (per 10 ml: 4.0 ml 25% dextran sulfate, 1.0 ml 10 % SDS, 3 ml 20£ SSC, 0.1 ml 10 mg/ml sheared herring sperm genomic DNA, 1.0 ml Denhardts’ solution, and 0.9 ml DEPC-treated H2O) per 100 cm2 of membrane. Radiolabeled probe ([32 P]dCTP) was synthesized using the Redi Prime II kit (Amersham) and the probe denatured and added to the hybridization buVer. Hybridization was allowed to proceed overnight. Membranes hybridized with LH were washed once in 1£ SSC/0.1% SDS and once in 0.5£ SSC/0.1% SDS. Membranes hybridized with ribosomal 18S (used as a loading control) were washed twice with 0.1£ SSC/0.1% SDS. All washes lasted 20 min and were conducted at 65 °C. Membranes were exposed to KScreen (phosphor screens) either overnight (LH) or for 2 h (18S). A Molecular Imager FX (Bio-Rad) was used to scan the phosphor screens and bands were quantiWed using the Quantity One (Bio-Rad) software package. Probe templates were prepared by RT-PCR from goldWsh total RNA and cloned into the pCRII-TOPO vector using the Invitrogen Topo-TA cloning kit. Primers for goldWsh LH and 18s were: (LH; forward primer 5⬘GAGCATGCGAGAGTTAGGCG-3⬘, reverse primer 5⬘-CGGACAAGGGACAGTATCGC-3⬘) and (18S; forward primer 5⬘-GAGCCTGAGAAACGGCTACC-3⬘, reverse primer 5⬘-GTATTCAGCGGCGACAGG-3⬘). Product sizes were 462 bp and 551 bp for LH (D88024) and 18S (AF047349), respectively. 2.8. Radioimmunoassay Serum gonadotropin-II (GTH-II) concentrations were determined using radioimmunoassay (RIA) elaborated by Peter et al. (1987). The assay was adapted to the solid phase format following the protocol by Merali et al. (1998). BrieXy, 96-well Dynex Immulon 4 polypropylene plates were coated with 100 l of 1.0 g/ml recombinant protein A/G (Calbiochem) in 0.1 M NaHCO3, pH 9, solution at 4 °C overnight. Plates were washed twice with wash buVer (25 mM Na-barbitone, 40 mM Na-acetate,
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0.25 mM thimerosal, 0.5% BSA, and 0.1% Tween 20, pH 8.6) for 2 min, once with diluent (wash buVer less Tween 20) for 20 min, and then treated at 4 °C overnight with 50 l of rabbit anti-carp LH antiserum at a dilution of 1:60,000 in diluent. Six wells were treated with normal rabbit serum at a dilution of 1:60,000 to determine nonspeciWc binding. Subsequently, plates were washed three times with wash buVer, and a standard curve using LH concentrations of 0 (100% binding), 0.19, 0.39, 0.79, 1.57, 3.13, 6.25, 12.5, 25, 50, and 100 ng/ml was prepared (25 l of diluent, 50 l of standard in diluent, and 25 l of I125GTH-II (»20,000 cpm)). Serum samples were similarly set up using 10 l per sample in triplicates (the diVerence in volume was made up with diluent). The plates were then placed at 4 °C for 48 h, washed three times with wash buVer, and 100 l of Optiphase Supermix (Wallac) scintillation Xuid was added to each well. Four hours later, the plates were counted in a Wallac Trilux Microbeta counter (1 min), and the assay evaluated using WiaCalc software. 2.9. Data analysis All statistical procedures were carried out using the SYSTAT8 (SPSS) software package. Two-way ANOVAs were used to examine the eVects of control, MPT, and GVG/MPT treatments on GAD mRNA levels, LH mRNA levels, and circulating LH levels. PostANOVA comparisons were done using Tukey’s multiple comparison procedure. GABA synthesis rates were compared using a T test. To examine the relationship between GAD mRNA levels and GABA synthesis rates, Pearson’s correlation analysis was performed and the probability determined using Bonferroni adjusted probabilities. In all statistical procedures p < 0.05 was considered signiWcant.
3. Results 3.1. Sexual maturity of experimental Wsh, GSI Previous studies have shown that steroid hormone levels, related to the sexual maturity of the Wsh, can modulate GABA synthesis (Bosma et al., 2001). To ensure that diVerences observed in GAD mRNA levels and GABA synthesis levels were due to MPT treatment and not diVerences in between groups, sexual maturity of the Wsh was determined by measuring the gonadosomatic index (GSI; gonadal weight/body weight). No statistical diVerence was observed between control Wsh and those in the various treatment groups (data not shown). Means varied between 2.6 and 3.6%, with an average of 3.3% for all Wsh used in the experiment. This is consistent with previous data reported for early recrudescent female goldWsh (Sohn et al., 1999; Trudeau, 1997).
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3.2. EVect of GVG and MPT on LH The eVects of GVG and MPT alone and in combination on serum LH levels were evaluated by radioimmunoassay. Relative to the saline injected control Wsh, GVG or MPT treatments did not aVect LH release. In contrast, Wsh receiving the combination treatment of GVG/MPT had a signiWcant 4-fold increase in serum LH relative to the other treatment groups (Fig. 1; p < 0.01). Pituitary levels of LH mRNA were not altered as assessed by Northern blot (Fig. 2).
3.3. EVects of pharmacological treatments on GABAergic systems of the hypothalamus Pretreatment with MPT increased (p < 0.05) hypothalamic GABA synthesis rate by approximately 30% relative to control (Fig. 3A). To determine which GAD isoform was responsible for the increased GAD synthesis rate, GAD67, GAD65, and GAD3 mRNA levels where measured by RPA. GAD65 mRNA levels were found to remain relatively constant between all of the experimental groups (Fig. 3C). However, GAD67 mRNA levels increased signiWcantly (p < 0.05) in the GVG/MPT group when compared to the other treatments (Fig. 3D). When correlational analysis was performed between GAD isoform mRNA levels and GABA synthesis rate, a signiWcant Pearson correlation coeYcient of 0.605 was obtained only for GAD67 (Fig. 3B). In contrast, no signiWcant correlation was found between GAD65 and GABA synthesis rate in the groups that were studied. Notably, the GAD3 isoform mRNA levels measured by RPA were low and did not to vary signiWcantly with any of the treatment groups or correlate with GABA synthesis rates in the hypothalamus (data not shown).
Fig. 1. EVects of GVG, MPT, or GVG/MPT injections on serum LH levels in early recrudescent female goldWsh. CT is the saline/saline injected group; G is the GVG/saline injected group; M is the MPT/ saline injected group; and M/G is the MPT/GVG injected group. Data are expressed as means § SEM. The (¤) indicates signiWcant diVerence from all other treatments (p < 0.01); n D 7–13.
Fig. 2. EVects of GVG, MPT, or MPT/GVG injections on pituitary LH subunit mRNA levels adjusted for ribosomal 18S in early recrudescent female goldWsh. CT is the saline/saline injected group; G is the GVG/saline injected group; M is the MPT/saline injected group; and M/G is the MPT/GVG injected group. Values are means § SEM. No signiWcant diVerence between experimental groups was observed (p < 0.05); n D 6–7.
Fig. 3. EVects of pharmacological treatments on GABAergic systems in the hypothalamus of early recrudescent female goldWsh. (A) GABA synthesis rates at baseline (control) and in catecholamine depleted Wsh (MPT); n D 6–7. (B) Pearson correlation coeYcients between GAD isoform mRNA levels and GABA synthesis rates; n D 13. (C) GAD65 mRNA levels adjusted for -actin; n D 6–7. (D) GAD67 relative mRNA levels adjusted for -actin. CT is the saline/saline injected group; G is the GVG/saline injected group; M is the MPT/saline injected group; and M/G is the MPT/GVG injected group. Data are expressed as means § SEM. The (¤) indicates signiWcant diVerence from all other treatments (p < 0.05).
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4. Discussion In this study, we demonstrate that catecholamine depletion by MPT increased GABA synthesis in the goldWsh hypothalamus. GABA is an important stimulator of LH release in the goldWsh and other Wsh species. Moreover, our previous work indicated that the catecholamine DA inhibits LH release induced by GABA drugs (Trudeau et al., 1993a). This is especially important given that DA is the single known inhibitor of LH release in goldWsh, acting to inhibit GnRH-induced LH release, sperm release and ovulation in many Wsh species (Peter et al., 1986, 1988). Blockage of catecholamine synthesis by MPT is not necessarily enough to increase LH release. Previously, we demonstrated that similar MPT injection protocols deplete brain and pituitary DA by approximately 50– 90% (Trudeau et al., 1993a,b). In one study of sexually regressed goldWsh, MPT treatment alone was not suYcient to stimulate LH release, but it did potentiate GnRH-stimulated LH release by 2.4-fold (Trudeau et al., 1993b). In the current experiments, GVG injection alone did not stimulate LH release. It is known that the stimulatory eVects of GABA on LH decrease with gonadal development or estradiol treatment in goldWsh (Kah et al., 1992; Peng et al., 1993b). Therefore, the lack of eVect of GVG alone was expected in sexually recrudescent females. However, treatment with both MPT and GVG stimulated a 4-fold increase in LH in dual treated Wsh. The combined inhibition of the DA system and inhibition of GABA catabolism increased LH levels to those observed during both natural and hormonally induced ovulation in female goldWsh (Kobayashi et al., 1986; Peter et al., 1986). There are several possible mechanisms and sites for DA–GABA interactions as related to the control of LH release. Our previous work demonstrated DA depletion by MPT potentiated GVG-stimulated LH release in sexually regressed goldWsh (Trudeau et al., 1993a) Furthermore, pretreatment with the DA type 2 receptor antagonist domperidone enhanced LH release induced by the GABA-A receptor agonist muscimol in sexually recrudescent female goldWsh. Thus, there appears to be a reciprocal inhibition of the DA–GABA system in the brain. We questioned whether increased LH release was a result of increase LH synthesis and to partially address this we assessed the level of LH subunit mRNA in the pituitaries of our experimental Wsh. Both gonadotropins possess a common subunit and derive speciWcity by unique subunits speciWc to FSH and LH that are encoded by distinct genes (Kobayashi et al., 1997; Sohn et al., 1998; Yoshiura et al., 1997). We found that LH mRNA levels were unaVected by MPT and GVG alone or in combination. This suggests that there was enhanced release of LH but no upregulation of LH synthesis as assessed using north-
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ern blots of LH mRNA. Previous work on tilapia dispersed pituitary cells in vitro indicated that dopamine did not aVect LH mRNA levels (Melamed et al., 1998), in accordance with the lack of eVect of MPT on LH mRNA in vivo. We hypothesized that catecholamine depletion might increase GABA synthesis in addition to potentiating GABA-induced LH release. Therefore, we developed a method to isolate amino acids in the isopropanol fraction of the RNA isolation protocol we use routinely. The power of this approach is that GABA synthesis rates and the levels of GAD mRNAs can be determined in the same hypothalamic sample. Catecholamine depletion led to a 30% increase in GABA synthesis rate. A signiWcant correlation of GABA synthesis rate with GAD67 mRNA levels suggests that GAD67 is mediating this increase in GABA synthesis following DA depletion with MPT. Modulation of GAD isoform levels is a crucial target for the regulation of GABAergic activity (Martin and Rimvall, 1993), especially as it pertains to regulation of reproduction (Trudeau et al., 2000). In the hypothalamus of the deep sea armed grenadier, GAD65 expression is sexually dimorphic (Trudeau et al., 2000). As well, sex steroids have been shown to diVerentially modulate GAD65 and GAD67 mRNA levels in the hypothalamus and telencephalon of sexually regressed goldWsh of both sexes (Bosma et al., 2001). For example, estradiol increased hypothalamic GAD65 in females but decreased it in males (Bosma et al., 2001). These studies suggest that transcriptional activity of GAD65 and GAD67 is sensitive to reproductive hormones. Our experiment supports this hypothesis, demonstrating regulation of the GAD67 isoform and GABA synthesis rate by dopamine, a potent inhibitor of GnRH and LH release in Wsh. This type of dopaminergic regulation of GABA is also found in mammalian systems. For example, dopaminergic deaVerentation of rat striatum results in increased levels of GAD67 mRNA but not the GAD65 isoform (Lindefors, 1993; Vernier et al., 1988). Similarly, dopamine has also been implicated in the selective regulation of alternative splicing of the GAD67 isoform mRNA during development of the rat brain (Kuppers et al., 2000). An important question is the signiWcance of a 30% change in GABA synthesis. In previous studies we showed that physiological levels of estradiol increase hypothalamic GABA synthesis rates by 30% (Trudeau et al., 1993a). In addition, a 13 °C change in water temperature from 11 to 24 °C increases hypothalamic GABA synthesis rates by approximately 40%, which is also accompanied by a 3-fold increase in serum LH levels in female goldWsh (Fraser et al., 2002). Therefore, changes in GABA synthesis induced by dopamine depletion are within a physiologically relevant range and would likely contribute to increased LH release.
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Interestingly, MPT treatment alone was unable to independently augment GAD67 mRNA. However, GAD67 mRNA did increase in animals co-treated with MPT and GVG. We hypothesize that this is likely due to reciprocal inhibition (Trudeau et al., 1993a) of dopaminergic and GABAergic systems in the hypothalamus. GVG treatment further inhibits dopaminergic input on GABAergic neurons, resulting in the increase of GAD67 mRNA and LH release in the GVG/MPT group. However, the current technique measures ongoing GABA synthesis in a dynamic assay following GVG treatment. Assessment of GABA synthesis rates in Wsh treated with MPT alone is not possible with our technique. Further testing of this hypothesis will require development of alternative methods to calculate ongoing GABA synthesis rates in vivo. One limitation of the current study is the lack of information regarding GAD isoform protein levels. Studies in mammals have shown that regulation of GABA release by dopamine could occur independent of transcriptional regulation (Lindefors, 1993; O’Connor et al., 1991) and that GAD67 can also be regulated at the post-transcriptional level (Martin and Rimvall, 1993). We were unable to quantify GAD protein levels because speciWc antibodies diVerentiating between Wsh GAD65, GAD67 or GAD3 are not yet available. In conclusion, our study is the Wrst to demonstrate that dopaminergic regulation of teleost GABAergic neuroendocrine systems occurs through isoform speciWc regulation of GAD67 transcriptional activity. Future experimentation investigating the close relationship between GAD mRNA, GAD protein, and in vivo GABA synthesis rates should lead to a better understanding of dopamine-GABA interactions and their roles in regulation of the goldWsh reproductive axis. Acknowledgments This research was supported by NSERC-Canada Discovery Grants to VTL. B.H., I.F., and R.M., were recipients of NSERC Summer Studentships. Dr. J. Arnason provided access to HPLC facilities. The animal care assistance of Bill Fletcher and Jennifer Keyte is acknowledged and appreciated. References Borg, B., Antononopuolou, E., Mayer, I., Andersson, E., Berglund, I., Swanson, P., 1998. EVects of gonadectomy and androgen treatments on pituitary and plasma levels of gonadotropins in mature male Atlantic salmon, Salmo salar, parr-positive feedback control of both gonadotropins. Biol. Reprod. 58 (3), 814–820. Bosma, P.T., Blasquez, M., Fraser, E.J., Schulz, R.W., Docherty, K., Trudeau, V.L., 2001. Sex steroid regulation of glutamate decarboxylase mRNA expression in goldWsh brain is sexually dimorphic. J. Neurochem. 76 (4), 945–956.
Chang, J.P., Cook, A.F., Peter, R.E., 1983. InXuence of catecholamines on gonadotropin secretion in goldWsh, Carassius auratus. Gen. Comp. Endocrinol. 49, 22–31. Chang, J.P., Peter, R.E., 1983. EVects of dopamine on gonadotropin release in female goldWsh, Carassius auratus. Neuroendocrinology 36 (5), 351–357. Chang, J.P., Peter, R.E., Nahorniak, C.S., Sokolowska, M., 1984. EVects of catecholaminergic agonists and antagonists on serum gonadotropin concentrations and ovulation in goldWsh: evidence for speciWcity of dopamine inhibition of gonadotropin secretion. Gen. Comp. Endocrinol. 55 (3), 351–360. Chang, J.P., Yu, K.L., Wong, A.O., Peter, R.E., 1990. DiVerential actions of dopamine receptor subtypes on gonadotropin and growth hormone release in vitro in goldWsh. Neuroendocrinology 51 (6), 664–674. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156–159. De Leeuw, R., Habibi, H.R., Nahorniak, C.S., Peter, R.E., 1989. Dopaminergic regulation of pituitary gonadotrophin-releasing hormone receptor activity in the goldWsh (Carassius auratus). J. Endocrinol. 121 (2), 239–247. Fraser, E.J., Bosma, P.T., Trudeau, V.L., Docherty, K., 2002. The eVect of water temperature on the GABAergic and reproductive systems in female and male goldWsh (Carassius auratus). Gen. Comp. Endocrinol. 125 (2), 163–175. Hibbert, B.M., Fung, I., McAuley, R., Lariviere, K., MacNeil, B., BaWYeboa, N., Livesey, J., Trudeau, V.L., 2004. Increased GAD67 mRNA levels are correlated with in vivo GABA synthesis in the MPTP-treated catecholamine-depleted goldWsh brain. Brain Res. Mol. Brain Res. 128 (2), 121–130. Joseph, M.H., Davies, P., 1983. Electrochemical activity of o-phtalaldehyde-mercaptoethanol derivatives of amino acids. Application to high-performance liquid chromatographic determination of amino acids in plasma and other biological materials. J. Chromatogr. 277, 125–136. Kah, O., Dubourg, P., Martinoli, M.G., Rahbi, M., Gonnet, F., GeVard, M., Calas, A., 1987a. Central GABAergic innervation of the pituitary in the goldWsh: a radioautographic and immunocytochemical study at the electron microscope level. Gen. Comp. Endocrinol. 67, 324–332. Kah, O., Dubourg, P., Thibault, J., Peter, R.E., 1987b. Neuroanatomical substrate for the inhibition of gonadotrophin secretion in goldWsh: existence of a dopaminergic proptico-hypophyseal pathway. Neuroendocrinology 45 (6), 451–458. Kah, O., Trudeau, V.L., Sloley, B.D., Chang, J.P., Dubourg, P., Yu, K.L., Peter, R.E., 1992. InXuence of GABA on gonadotropin release in the goldWsh. Neuroendocrinology 55, 396–404. Kobayashi, M., Aida, K., Hanyu, I., 1986. Gonadotropin surge during spawning in male goldWsh. Gen. Comp. Endocrinol. 62, 70–79. Kobayashi, M., Kato, Y., Yoshiura, Y., Aida, K., 1997. Molecular cloning of cDNA encoding two types of pituitary gonadotropin alpha subunit from the goldWsh, Carassius auratus. Gen. Comp. Endocrinol. 105 (3), 372–378. Kuppers, E., Sabolek, M., Anders, U., Pilgrim, C., Beyer, C., 2000. Developmental regulation of glutamic acid decarboxylase mRNA expression and splicing in the rat striatum by dopamine. Brain Res. Mol. Brain Res. 81 (1–2), 19–28. Lariviere, K., Maceachern, L., Greco, V., Majchrzak, G., Chiu, S., Drouin, G., Trudeau, V.L., 2002. GAD(65) and GAD(67) isoforms of the glutamic acid decarboxylase gene originated before divergence of cartilaginous Wshes. Mol. Biol. Evol. 19 (12), 2325–2329. Lindefors, N., 1993. Dopaminergic regulation of glutamic acid decarboxylase mRNA expression and GABA release in the striatum: a review. Prog. Neuropsychopharmacol. Biol. Psychiatry 17 (6), 887–903. Martin, D.L., Rimvall, K., 1993. Regulation of -aminobutyric acid synthesis in the brain. J. Neurochem. 60, 395–407.
B. Hibbert et al. / General and Comparative Endocrinology 140 (2005) 176–183 Melamed, P., Rosenfeld, H., Elizur, A., Yaron, Z., 1998. Endocrine regulation of gonadotropin and growth hormone gene transcription in Wsh. Comp. Biochem. Physiol. C 119, 325–358. Merali, Z., Mcintosh, J., Kent, P., Michaud, D., Anisman, H., 1998. Aversive and appetitive events evoke the release of corticotropinreleasing hormone and bombesin-like peptides at the central nucleus of the amygdala. J. Neurosci. 18, 4758–4766. O’Connor, W.T., Linderfors, N., Brene, S., Herrera-Marschitz, M., Persson, H., Ungerstedt, U., 1991. Short-term dopaminergic regulation of GABA release in dopamine deaVerented caudate-putamen is not directly associated with glutamic acid decarboxylase gene expression. Neurosci Lett. 128 (1), 66–70. Omeljaniuk, R.J., Shih, S.H., Peter, R.E., 1987. In-vivo evaluation of dopamine receptor-mediated inhibition of gonadotrophin secretion from the pituitary gland of the goldWsh, Carassius auratus. J. Endocrinol. 114 (3), 449–458. Omeljaniuk, R.J., Habibi, H.R., Peter, R.E., 1989. Alterations in pituitary GnRH and dopamine receptors associated with the seasonal variation and regulation of gonadotropin release in the goldWsh (Carassius auratus). Gen. Comp. Endocrinol. 74 (3), 392–399. Peng, C., Humphries, S., Peter, R.E., Rivier, J.E., Blomqvist, A.G., Larhammar, D., 1993a. Actions of goldWsh neuropeptide Y on the secretion of growth hormone and gonadotropin-II in female goldWsh. Gen. Comp. Endocrinol. 90 (3), 306–317. Peng, C., Trudeau, V.L., Peter, R.E., 1993b. Seasonal variation of neuropeptide Y actions on growth hormone and gonadotropin-II secretion in the goldWsh: eVects of sex steroids. J. Neuroendocrinol. 5 (3), 273–280. Peter, R.E., Chang, J.P., Nahorniak, C.S., Omeljaniuk, R.J., Sokolowska, M., Shih, S.H., Billard, R., 1986. Interactions of catecholamines and GnRH in regulation of gonadotropin secretion in teleost Wsh. Recent Prog. Horm. Res. 42, 513–548. Peter, R.E., Lin, H.R., Vand der Kraak, G., 1988. Induced ovulation and spawning of cultured freshwater Wsh in China: advances in application of GnRH analogues and dopamine antagonists. Aquaculture 74, 1–10. Peter, R.E., Nahorniak, C.S., Shih, S., King, J.A., Millar, R.P., 1987. Activity of position-8-substituted analogs of mammalian gonadotropin-releasing hormone (mGnRH) and chicken and lamprey gonadotropin-releasing hormones in goldWsh. Gen. Comp. Endocrinol. 65, 385–393. Sloley, B.D., Kah, O., Trudeau, V.L., Joseph, G.D., Peter, R.E., 1992. Amino acid neurotransmitters and dopamine in brain and pituitary
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of the goldWsh: involvement in the regulation of gonadotropin secretion. J. Neurochem. 58, 2254–2262. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. Sohn, Y.C., Yoshiura, Y., Kobayashi, M., Aida, K., 1999. Seasonal changes in mRNA levels of gonadotropin and thyrotropin subunits in the goldWsh, Carassius auratus. Gen. Comp. Endocrinol. 113, 436–444. Sohn, Y.C., Suetake, H., Yoshiura, Y., Kobayashi, M., Aida, K., 1998. Structural and expression analyses of gonadotropin Ibeta subunit genes in goldWsh (Carassius auratus). Gene 222 (2), 257–267. Somoza, G.M., Peter, R.E., 1991. EVects of serotonin on gonadotropin and growth hormone release from in vitro perifused goldWsh pituitary fragments. Gen. Comp. Endocrinol. 82 (1), 103–110. Trudeau, V.L., Sloley, B.D., Peter, R.E., 1993a. GABA stimulation of gonadotropin-II release in goldWsh: involvement of GABAA receptors, dopamine, and sex steroids. Am. J. Physiol. 265, R348– R355. Trudeau, V.L., Sloley, B.D., Peter, R.E., 1993b. Noradrenaline turnover in the goldWsh brain is modulated by sex steroids and GABA. Brain Res. 624, 29–34. Trudeau, V.L., 1997. Neuroendocrine regulation of gonadotrophin-II release and gonadal growth in the goldWsh, Carassius auratus. Rev. Reprod. 2, 55–68. Trudeau, V.L., Spanswick, D., Fraser, E.J., Lariviere, K., Crump, D., Chiu, S., Macmillan, M., Schulz, R., 2000. The role of amino acid neurotransmitters in the regulation of pituitary gonadotropin release in Wsh. Biochem. Cell. Biol. 78, 241–259. Vernier, P., Julien, J.F., Rataboul, P., Fourrier, O., Feuerstein, C., Mallet, J., 1988. Similar time course changes in striatal levels of glutamic acid decarboxylase and proenkephalin mRNA following dopaminergic deaVerentation in the rat. J. Neurochem. 51, 1375– 1380. Yoshiura, Y., Kobayashi, M., Kato, Y., Aida, K., 1997. Molecular cloning of the cDNAs encoding two gonadotropin beta subunits (GTH-I beta and -II beta) from the goldWsh, Carassius auratus. Gen. Comp. Endocrinol. 105 (3), 379–389. Yu, K.L., Peter, R.E., 1992. Adrenergic and dopaminergic regulation of gonadotropin-releasing hormone release from goldWsh preopticanterior hypothalamus and pituitary in vitro. Gen. Comp. Endocrinol. 85 (1), 138–146.
Catecholamine depletion modulates serum LH levels, GAD67 mRNA, and GABA synthesis in the goldWsh Benjamin Hibbert, Irene Fung, Rebecca McAuley, Marwan Samia, Vance Trudeau¤ Department of Biology, Centre for Advanced Research in Environmental Genomics (CAREG), MacDonald Hall, University of Ottawa, Ottawa, Ont., Canada K1N 6N5 Received 9 July 2004; revised 28 October 2004; accepted 12 November 2004
Abstract It is established that dopamine inhibits while GABA stimulates LH release in goldWsh. In this study, we examine dopaminergic regulation of GABAergic activity in the hypothalamus of early recrudescent female goldWsh (Carassius auratus). We utilize a unique technique that permits concomitant quantiWcation and correlation of in vivo GAD65 and GAD67 mRNA with GABA synthesis rate in response to decreased dopamine levels. Catecholamine depletion was achieved by treatment with -methyl-para-tyrosine methyl ester (MPT; 240 g/g body weight), an inhibitor of tyrosine hydroxylase. Endogenous GABA levels were increased by intraperitoneal administration of -vinyl GABA (GVG; 300 g/g body weight), an inhibitor of the GABA catabolic enzyme GABA transaminase. Dual treatment of GVG + MPT increased serum LH levels 4-fold. However, LH mRNA levels in the pituitary remained stable, suggesting that treatments aVected secretion and not synthesis. In the hypothalamus, GABA synthesis rates increased 30% in response to MPT treatment. This was correlated (r D 0.61; p < 0.05) to increased levels of GAD67 mRNAs but not GAD65 (r D 0.14; p > 0.05). These observations suggest that catecholamines inhibit GABA synthesis in the goldWsh hypothalamus through isoform speciWc regulation of GAD67. 2004 Elsevier Inc. All rights reserved. Keywords: Gonadotropin-II; GABA synthesis rate; LH; Teleost
1. Introduction The goldWsh has an annual cycle of reproduction and in temperate climates spawning generally occurs in late April to early May. To ensure appropriate timing of seasonal sexual maturation and gonadal development in Wsh, release of lutenizing hormone (LH), and follicle stimulating hormone (FSH), also respectively known as gonadotropin-II (GtH-II) and gonadotropin-I (GtH-I) in teleosts, are regulated by an intricate network of neuroendocrine and hormonal feedback systems (Borg et al., 1998; Trudeau, 1997). While the principle stimulator of
*
Corresponding author. Fax: +1 613 562 5486. E-mail address: [email protected] (V. Trudeau).
0016-6480/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2004.11.008
LH is gonadotropin-releasing hormone (GnRH), amino butyric acid (GABA) (Trudeau et al., 1993a,b), neuropeptide Y (NPY) (Peng et al., 1993a), noradrenaline (NA) (Chang et al., 1983), serotonin (5-hydroxytryptamine, 5-HT) (Somoza and Peter, 1991), and glutamate (Trudeau et al., 2000) have also been shown to increase LH release. In contrast, dopamine (DA) is the only known inhibitor of LH release in many Wsh species (Peter et al., 1986, 1988; Trudeau, 1997). Chang et al. (1983) Wrst showed that destruction of dopaminergic neurons or inhibition of catecholamine synthesis with -methyl-p-tyrosine (MPT) raised serum LH levels. In the same study, speciWc inhibition of NA synthesis failed to replicate the LH increase induced by MPT. Subsequently, DA has been shown to inhibit both basal and GnRH-stimulated LH release via DA
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type 2 receptors (Chang and Peter, 1983; Chang et al., 1984; Omeljaniuk et al., 1987) and reduce pituitary GnRH receptor binding (De Leeuw et al., 1989; Omeljaniuk et al., 1989). In addition, DA also inhibits GnRH at the level of the hypothalamus (Chang et al., 1990; Omeljaniuk et al., 1987) via a DA type 1 receptor (Yu and Peter, 1992). The central role of inhibitory DA systems is also supported by morphological studies showing that dopaminergic Wbres originating in the preoptic area innervate gonaodotrophs in the anterior goldWsh pituitary (Kah et al., 1987a,b). Another less well studied mechanism whereby DA may further inhibit LH involves interactions with the GABAergic neuroendocrine system. Treatment of Wsh with -vinyl GABA (GVG), an inhibitor of GABA catabolism, increases serum LH, upregulates pituitary expression of LH (Fraser et al., 2002) and also secretogranin-II, a marker of the regulated secretion pathway. Current evidence indicates that GABA stimulates LH release through activation of both A- and B-type GABA receptors, and by a dual action to enhance GnRH release and inhibit dopamine turnover in the goldWsh hypothalamus and pituitary (Trudeau et al., 1993a,b, 2000). Moreover, pretreatment with DA antagonists or DA depletion by MPT potentiates GABAstimulated LH release in goldWsh. Therefore, there appears to be a reciprocal inhibition of the DA and GABA systems involved in the control of LH release. GABA is synthesized from glutamate in a simple enzymatic step controlled by glutamic acid decarboxylase (GAD). There are two major forms of GAD in vertebrates, GAD67 and GAD65 (also known as GAD1 and GAD2), named for the molecular weight of the mature proteins (Martin and Rimvall, 1993). We have also found a third novel GAD in Wsh, which we called GAD3 (Bosma et al., 2001; Lariviere et al., 2002). In this study, we demonstrate that catecholamine depletion increases hypothalamic GABA synthesis rate and, when combined with GVG-treatment, results in LH release in vivo. The increase in GABA synthesis rate is correlated with an increase in the hypothalamic expression GAD67 but not the other GAD isoforms. We suggest DA inhibits GABA synthesis in the goldWsh hypothalamus by regulation of GAD67 mRNA levels.
2. Materials and methods 2.1. Animals and pharmacological treatments Common goldWsh (Carassius auratus) were purchased from ABC Aquarium Services (Montreal, Canada). Upon arrival, Wsh were separated by sex and kept in 18 °C de-chlorinated/oxygenated city of Ottawa tap water. Fish were fed standard Xake and pellet food and housed in a room with a simulated photoperiod for the
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Ottawa area. Female Wsh weighing between 13 and 37 g were selected and acclimated to tanks at least 2 weeks prior to any treatments. The experimental design consisted of four treatment groups CTRL, GVG, MPT, and a GVG/MPT group. The control group received 0.6% saline injections, The GVG (gift from Hoescht Marion Rousell) group received 300 g/g body weight intraperitoneally, the MPT (Sigma) group received 240 g MPT/g of body weight, and GVG/MPT group received both GVG and MPT at the doses indicated above. MPT treatments were given 6 and 1 day prior to sacriWce, a dose shown to achieve >50% DA depletion in target tissues (Trudeau et al., 1993a,b). GVG treatments were given one day prior to sacriWce. All injections were administered at a volume 5 l/g of body weight using a 21 gauge needle attached to a Hamilton microsyringe. 2.2. Dissections Fish were anesthetised in 0.5% MS222 (Sigma), weighed, decapitated, and the hypothalamus dissected, frozen immediately on dry ice, pooled, and stored at ¡80 °C till used for RNA isolation. For ribonuclease protection assay of GAD67, GAD65, and GAD3 mRNA levels, and HPLC analysis of GABA levels, brain tissue from 3 to 5 Wsh were pooled for one replicate. 2.3. GABA, protein, and RNA isolation Our laboratory has developed and validated an isolation procedure which enables concomitant quantiWcation of GABA synthesis rates and GAD mRNA levels from a unique sample (Hibbert et al., 2004). BrieXy, RNA was isolated using the GITC extraction method as described in Chomczynski and Sacchi (1987). We included homoserine (1 mg/ml) in the extraction solution to act as an internal control for subsequent HPLC analysis of GABA levels. The isopropanol phase of the RNA extraction contained the GABA and homoserine. Protein from each sample was isolated to correct for the varying amounts of tissue obtained during the dissection. Proteins were precipitated from the organic phase retained from the GITC RNA prep by addition of 1.5 ml of isopropanol. Samples were allowed to stand for 10 min at room temperature, and centrifuged at 12,000g to precipitate the proteins. The supernatant was discarded and the pellet washed three times for 20 min in 2 ml of 0.3 M guanidine hydrochloride/95% ethanol solution, followed by centrifugation (7500g) each time. The pellet was washed in 99% ethanol, vortexed, and incubated for 20 min at room temperature. Samples were centrifuged (7500g) for 5 min, the supernatant discarded, and samples air-dried for 5–10 min at room temperature. The protein samples were resuspended in 1.5 ml of 1% SDS/0.5 M NaOH. Protein concentrations were deter-
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mined (Smith et al., 1985) using a bicinchoninic acid protein assay kit (Sigma). 2.4. GABA quantiWcation by HPLC A modiWed version of the gradient reverse-phase HPLC technique with Xuorometric detection described by Sloley et al. (1992) was used to measure GABA concentrations. BrieXy, the isopropanol phases of the samples were put into pre-weighed (accurate to four decimal places) 2.0 ml centrifuge tubes. The volume of the samples was determined by dividing the mass of the isopropanol phase by the density of the isopropanol phase (1.0373 £ 10¡3 g/l). Before loading onto the HPLC machine, samples were Wrst Wltered with 1 ml syringes Wtted with Chromspec 0.2 l PTFE Wlters. Then, 20 l of the isopropanol phase sample were added to 60 l of ophthalaldahyde (OPA) (Pierce) and the solution was allowed to incubate for 3 min at room temperature. OPA binds the amine group of amino acids, yielding a derivatized molecule with high quantum eYciency, to allow for Xuorometric detection (Joseph and Davies, 1983). After incubation with OPA, 500 l of sodium acetate buVer (1.36 g/100 ml) were added, and the sample was loaded into the HPLC machine 2.5 min later. Then, 10 l of each preparation was injected onto a 4 m Superspher 100 RP-184 £ 75 mm analytical cartridge with a 5 m LiChrospher 100 C18 4 £ 4 mm guard cartridge (Merck). The linear gradient consisted of 9:1 acetonitrile buVer:methanol at t D 0 min (acetonitrile buVer is 9 parts 25 mmol NaH2PO4, 1 part acetonitrile, pH 6.0), 8:1 acetonitrile buVer:methanol at t D 10–12 min, and 9:1 acetonitrile buVer:methanol at t D 14 min, with a constant Xow rate of 1.0 ml/min. Fluorescence was detected with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The HPLC equipment comprised a Varian ProStar HPLC system consisting of models 410 autosampler with a 100 l loop, 230 pump, 360 Xuorescence detector and Star Workstation software (version 5.5).
2.6. Ribonuclease protection assay Regions of -actin, GAD3, GAD65, and GAD67 genes were ampliWed with PCR primers containing T7 (on antisense template) or SP6 (on sense template) RNA polymerase promoters. Primer sequences are as follows: KRPA65-FWD, att tag gtg aca cta tag ctc tga tcc gct ccg ttc; KRPA65-REV, taa tac gac tca cta tag cag cct ccc cga cat tta; KRPA67-FWD, att tag gtg aca cta tag acc aga gtc gct gga gca; KRPA67-REV, taa tac gac tca cta tag tcc atc gcc atc tcc att; KRPA3-FWD, att tag gtg aca cta tag agg cca agg gtc tgg ttc; KRPA3-REV, taa tac gac tca cta tag ttg tgg ggg ttc cat gtc; KRPABAC-FWD, att tag gtg aca cta tag gat ggt ggg aat ggg tca; and KRPABACREV, taa tac gac tca cta tag gga cag cac agc ctg gat. Promoter sequences are shown in italics. Product lengths were 304 bp for -actin, 252 bp for GAD3, 216 bp for GAD65, and 278 bp for GAD67. Protected probes were all 36 bp shorter than their respective initial probe lengths. Using T7 RNA polymerase (Ambion), in vitro transcription of antisense RNA molecules was performed incorporating [-32P]CTP (Amersham) for actin and [-32P]UTP (Amersham) for GAD65 and GAD67.The probe solution was then treated with DNase I for 30 min at 37 °C (Promega) and gel puriWed to eliminate traces of probe DNA template. Incubation of 10 g of total RNA from hypothalamus was then carried out with 3 £ 104 cpm of each RNA probe at 56 °C overnight. Non-protected regions of RNA were degraded with an RNase A/RNase T1 (Sigma) cocktail (7:5 ratio) at 30 °C for 45 min. Nucleases were degraded with proteinase K (Sigma) followed by phenol:chloroform extraction. Fragments were electrophoresed in a 5% denaturing polyacrylamide gel. Gels were dried and exposed to Kodak K-Screen phosphor screens for 18 h to visualize protected bands. A Molecular Imager FX (Bio-Rad) was used to scan the screens and bands were quantiWed using the Quantity One (Bio-Rad) software package. 2.7. Northern hybridizations
2.5. Calculation of GABA synthesis GABA synthesis rates in vivo were calculated as previously described (Fraser et al., 2002; Trudeau et al., 1993a). BrieXy, GABA levels in the dissected tissues were quantiWed 24 h after the Wnal saline or GVG injection in both the control and MPT-injected Wsh. Correction of GABA levels for variations in tissue size was based on total protein content per tissue. To obtain a net increase in GABA above basal levels, GABA levels in the salinetreated group in both control and MPT-treated animals are subtracted from those treated with GVG. This value is divided by 24 h to obtain an estimate of GABA synthesis rate per hour per milligram protein in control versus MPT-injected goldWsh.
To determine whether DA depletion or GVG injections aVected LH mRNA levels, 10 g of total RNA (as determined by OD260) from pituitary samples was electrophoresed on 1.5% denaturing agarose gels. Gels consisted of 3.0 g agarose B low EEO (electroendosmosis) (Biobasic), 170 ml DEPC-treated H2O, and 20 ml of 10£ Mops (morpholinopropanesulfonic acid; 41.8 g Mops, 4.1 g sodium acetate, and 2.9 g disodium EDTA per liter, pH adjusted to 7.0 with 10 N NaOH). 2.25 l of loading buVer (per 7 ml: 4 ml formamide, 0.8 ml of 10£ Mops, 0.4 ml glycerol, 1.4 ml formaldehyde, 0.1 ml of 0.5 M EDTA, pH 8.0, 0.4 ml DEPC treated water, 20 mg bromophenol blue, and 3.5:1 of 10 mg/ml ethidium bromide) was added for each l of sample volume. Samples were
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then denatured at 65 °C for 20 min and snap cooled on ice prior to loading. Running buVer consisted of 1£ MOPS and gels were run for 6 h at 100 V. Following electrophoresis, gels were washed twice in 5 volumes of ddH2O for 20 min. Subsequently, gels were washed once in 20£ standard sodium citrate buVer (SSC) for 20 min and concomitantly a piece of Hybond N+ nylon membrane (Amersham) was soaked with ddH2O and washed for 10 min in 2£ SSC. The gel was then transferred to the nylon membrane by capillary action with 20£ SSC overnight. Covalent cross-linking of RNA to the nylon membrane was achieved by exposure to UV-light for 5 min. Prior to each hybridization, membranes were washed with 0.1£ SSC/0.1£ SDS for 20 min at 65 °C, then prehybridized for >3 h in 10 ml of hybridization buVer (per 10 ml: 4.0 ml 25% dextran sulfate, 1.0 ml 10 % SDS, 3 ml 20£ SSC, 0.1 ml 10 mg/ml sheared herring sperm genomic DNA, 1.0 ml Denhardts’ solution, and 0.9 ml DEPC-treated H2O) per 100 cm2 of membrane. Radiolabeled probe ([32 P]dCTP) was synthesized using the Redi Prime II kit (Amersham) and the probe denatured and added to the hybridization buVer. Hybridization was allowed to proceed overnight. Membranes hybridized with LH were washed once in 1£ SSC/0.1% SDS and once in 0.5£ SSC/0.1% SDS. Membranes hybridized with ribosomal 18S (used as a loading control) were washed twice with 0.1£ SSC/0.1% SDS. All washes lasted 20 min and were conducted at 65 °C. Membranes were exposed to KScreen (phosphor screens) either overnight (LH) or for 2 h (18S). A Molecular Imager FX (Bio-Rad) was used to scan the phosphor screens and bands were quantiWed using the Quantity One (Bio-Rad) software package. Probe templates were prepared by RT-PCR from goldWsh total RNA and cloned into the pCRII-TOPO vector using the Invitrogen Topo-TA cloning kit. Primers for goldWsh LH and 18s were: (LH; forward primer 5⬘GAGCATGCGAGAGTTAGGCG-3⬘, reverse primer 5⬘-CGGACAAGGGACAGTATCGC-3⬘) and (18S; forward primer 5⬘-GAGCCTGAGAAACGGCTACC-3⬘, reverse primer 5⬘-GTATTCAGCGGCGACAGG-3⬘). Product sizes were 462 bp and 551 bp for LH (D88024) and 18S (AF047349), respectively. 2.8. Radioimmunoassay Serum gonadotropin-II (GTH-II) concentrations were determined using radioimmunoassay (RIA) elaborated by Peter et al. (1987). The assay was adapted to the solid phase format following the protocol by Merali et al. (1998). BrieXy, 96-well Dynex Immulon 4 polypropylene plates were coated with 100 l of 1.0 g/ml recombinant protein A/G (Calbiochem) in 0.1 M NaHCO3, pH 9, solution at 4 °C overnight. Plates were washed twice with wash buVer (25 mM Na-barbitone, 40 mM Na-acetate,
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0.25 mM thimerosal, 0.5% BSA, and 0.1% Tween 20, pH 8.6) for 2 min, once with diluent (wash buVer less Tween 20) for 20 min, and then treated at 4 °C overnight with 50 l of rabbit anti-carp LH antiserum at a dilution of 1:60,000 in diluent. Six wells were treated with normal rabbit serum at a dilution of 1:60,000 to determine nonspeciWc binding. Subsequently, plates were washed three times with wash buVer, and a standard curve using LH concentrations of 0 (100% binding), 0.19, 0.39, 0.79, 1.57, 3.13, 6.25, 12.5, 25, 50, and 100 ng/ml was prepared (25 l of diluent, 50 l of standard in diluent, and 25 l of I125GTH-II (»20,000 cpm)). Serum samples were similarly set up using 10 l per sample in triplicates (the diVerence in volume was made up with diluent). The plates were then placed at 4 °C for 48 h, washed three times with wash buVer, and 100 l of Optiphase Supermix (Wallac) scintillation Xuid was added to each well. Four hours later, the plates were counted in a Wallac Trilux Microbeta counter (1 min), and the assay evaluated using WiaCalc software. 2.9. Data analysis All statistical procedures were carried out using the SYSTAT8 (SPSS) software package. Two-way ANOVAs were used to examine the eVects of control, MPT, and GVG/MPT treatments on GAD mRNA levels, LH mRNA levels, and circulating LH levels. PostANOVA comparisons were done using Tukey’s multiple comparison procedure. GABA synthesis rates were compared using a T test. To examine the relationship between GAD mRNA levels and GABA synthesis rates, Pearson’s correlation analysis was performed and the probability determined using Bonferroni adjusted probabilities. In all statistical procedures p < 0.05 was considered signiWcant.
3. Results 3.1. Sexual maturity of experimental Wsh, GSI Previous studies have shown that steroid hormone levels, related to the sexual maturity of the Wsh, can modulate GABA synthesis (Bosma et al., 2001). To ensure that diVerences observed in GAD mRNA levels and GABA synthesis levels were due to MPT treatment and not diVerences in between groups, sexual maturity of the Wsh was determined by measuring the gonadosomatic index (GSI; gonadal weight/body weight). No statistical diVerence was observed between control Wsh and those in the various treatment groups (data not shown). Means varied between 2.6 and 3.6%, with an average of 3.3% for all Wsh used in the experiment. This is consistent with previous data reported for early recrudescent female goldWsh (Sohn et al., 1999; Trudeau, 1997).
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3.2. EVect of GVG and MPT on LH The eVects of GVG and MPT alone and in combination on serum LH levels were evaluated by radioimmunoassay. Relative to the saline injected control Wsh, GVG or MPT treatments did not aVect LH release. In contrast, Wsh receiving the combination treatment of GVG/MPT had a signiWcant 4-fold increase in serum LH relative to the other treatment groups (Fig. 1; p < 0.01). Pituitary levels of LH mRNA were not altered as assessed by Northern blot (Fig. 2).
3.3. EVects of pharmacological treatments on GABAergic systems of the hypothalamus Pretreatment with MPT increased (p < 0.05) hypothalamic GABA synthesis rate by approximately 30% relative to control (Fig. 3A). To determine which GAD isoform was responsible for the increased GAD synthesis rate, GAD67, GAD65, and GAD3 mRNA levels where measured by RPA. GAD65 mRNA levels were found to remain relatively constant between all of the experimental groups (Fig. 3C). However, GAD67 mRNA levels increased signiWcantly (p < 0.05) in the GVG/MPT group when compared to the other treatments (Fig. 3D). When correlational analysis was performed between GAD isoform mRNA levels and GABA synthesis rate, a signiWcant Pearson correlation coeYcient of 0.605 was obtained only for GAD67 (Fig. 3B). In contrast, no signiWcant correlation was found between GAD65 and GABA synthesis rate in the groups that were studied. Notably, the GAD3 isoform mRNA levels measured by RPA were low and did not to vary signiWcantly with any of the treatment groups or correlate with GABA synthesis rates in the hypothalamus (data not shown).
Fig. 1. EVects of GVG, MPT, or GVG/MPT injections on serum LH levels in early recrudescent female goldWsh. CT is the saline/saline injected group; G is the GVG/saline injected group; M is the MPT/ saline injected group; and M/G is the MPT/GVG injected group. Data are expressed as means § SEM. The (¤) indicates signiWcant diVerence from all other treatments (p < 0.01); n D 7–13.
Fig. 2. EVects of GVG, MPT, or MPT/GVG injections on pituitary LH subunit mRNA levels adjusted for ribosomal 18S in early recrudescent female goldWsh. CT is the saline/saline injected group; G is the GVG/saline injected group; M is the MPT/saline injected group; and M/G is the MPT/GVG injected group. Values are means § SEM. No signiWcant diVerence between experimental groups was observed (p < 0.05); n D 6–7.
Fig. 3. EVects of pharmacological treatments on GABAergic systems in the hypothalamus of early recrudescent female goldWsh. (A) GABA synthesis rates at baseline (control) and in catecholamine depleted Wsh (MPT); n D 6–7. (B) Pearson correlation coeYcients between GAD isoform mRNA levels and GABA synthesis rates; n D 13. (C) GAD65 mRNA levels adjusted for -actin; n D 6–7. (D) GAD67 relative mRNA levels adjusted for -actin. CT is the saline/saline injected group; G is the GVG/saline injected group; M is the MPT/saline injected group; and M/G is the MPT/GVG injected group. Data are expressed as means § SEM. The (¤) indicates signiWcant diVerence from all other treatments (p < 0.05).
B. Hibbert et al. / General and Comparative Endocrinology 140 (2005) 176–183
4. Discussion In this study, we demonstrate that catecholamine depletion by MPT increased GABA synthesis in the goldWsh hypothalamus. GABA is an important stimulator of LH release in the goldWsh and other Wsh species. Moreover, our previous work indicated that the catecholamine DA inhibits LH release induced by GABA drugs (Trudeau et al., 1993a). This is especially important given that DA is the single known inhibitor of LH release in goldWsh, acting to inhibit GnRH-induced LH release, sperm release and ovulation in many Wsh species (Peter et al., 1986, 1988). Blockage of catecholamine synthesis by MPT is not necessarily enough to increase LH release. Previously, we demonstrated that similar MPT injection protocols deplete brain and pituitary DA by approximately 50– 90% (Trudeau et al., 1993a,b). In one study of sexually regressed goldWsh, MPT treatment alone was not suYcient to stimulate LH release, but it did potentiate GnRH-stimulated LH release by 2.4-fold (Trudeau et al., 1993b). In the current experiments, GVG injection alone did not stimulate LH release. It is known that the stimulatory eVects of GABA on LH decrease with gonadal development or estradiol treatment in goldWsh (Kah et al., 1992; Peng et al., 1993b). Therefore, the lack of eVect of GVG alone was expected in sexually recrudescent females. However, treatment with both MPT and GVG stimulated a 4-fold increase in LH in dual treated Wsh. The combined inhibition of the DA system and inhibition of GABA catabolism increased LH levels to those observed during both natural and hormonally induced ovulation in female goldWsh (Kobayashi et al., 1986; Peter et al., 1986). There are several possible mechanisms and sites for DA–GABA interactions as related to the control of LH release. Our previous work demonstrated DA depletion by MPT potentiated GVG-stimulated LH release in sexually regressed goldWsh (Trudeau et al., 1993a) Furthermore, pretreatment with the DA type 2 receptor antagonist domperidone enhanced LH release induced by the GABA-A receptor agonist muscimol in sexually recrudescent female goldWsh. Thus, there appears to be a reciprocal inhibition of the DA–GABA system in the brain. We questioned whether increased LH release was a result of increase LH synthesis and to partially address this we assessed the level of LH subunit mRNA in the pituitaries of our experimental Wsh. Both gonadotropins possess a common subunit and derive speciWcity by unique subunits speciWc to FSH and LH that are encoded by distinct genes (Kobayashi et al., 1997; Sohn et al., 1998; Yoshiura et al., 1997). We found that LH mRNA levels were unaVected by MPT and GVG alone or in combination. This suggests that there was enhanced release of LH but no upregulation of LH synthesis as assessed using north-
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ern blots of LH mRNA. Previous work on tilapia dispersed pituitary cells in vitro indicated that dopamine did not aVect LH mRNA levels (Melamed et al., 1998), in accordance with the lack of eVect of MPT on LH mRNA in vivo. We hypothesized that catecholamine depletion might increase GABA synthesis in addition to potentiating GABA-induced LH release. Therefore, we developed a method to isolate amino acids in the isopropanol fraction of the RNA isolation protocol we use routinely. The power of this approach is that GABA synthesis rates and the levels of GAD mRNAs can be determined in the same hypothalamic sample. Catecholamine depletion led to a 30% increase in GABA synthesis rate. A signiWcant correlation of GABA synthesis rate with GAD67 mRNA levels suggests that GAD67 is mediating this increase in GABA synthesis following DA depletion with MPT. Modulation of GAD isoform levels is a crucial target for the regulation of GABAergic activity (Martin and Rimvall, 1993), especially as it pertains to regulation of reproduction (Trudeau et al., 2000). In the hypothalamus of the deep sea armed grenadier, GAD65 expression is sexually dimorphic (Trudeau et al., 2000). As well, sex steroids have been shown to diVerentially modulate GAD65 and GAD67 mRNA levels in the hypothalamus and telencephalon of sexually regressed goldWsh of both sexes (Bosma et al., 2001). For example, estradiol increased hypothalamic GAD65 in females but decreased it in males (Bosma et al., 2001). These studies suggest that transcriptional activity of GAD65 and GAD67 is sensitive to reproductive hormones. Our experiment supports this hypothesis, demonstrating regulation of the GAD67 isoform and GABA synthesis rate by dopamine, a potent inhibitor of GnRH and LH release in Wsh. This type of dopaminergic regulation of GABA is also found in mammalian systems. For example, dopaminergic deaVerentation of rat striatum results in increased levels of GAD67 mRNA but not the GAD65 isoform (Lindefors, 1993; Vernier et al., 1988). Similarly, dopamine has also been implicated in the selective regulation of alternative splicing of the GAD67 isoform mRNA during development of the rat brain (Kuppers et al., 2000). An important question is the signiWcance of a 30% change in GABA synthesis. In previous studies we showed that physiological levels of estradiol increase hypothalamic GABA synthesis rates by 30% (Trudeau et al., 1993a). In addition, a 13 °C change in water temperature from 11 to 24 °C increases hypothalamic GABA synthesis rates by approximately 40%, which is also accompanied by a 3-fold increase in serum LH levels in female goldWsh (Fraser et al., 2002). Therefore, changes in GABA synthesis induced by dopamine depletion are within a physiologically relevant range and would likely contribute to increased LH release.
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Interestingly, MPT treatment alone was unable to independently augment GAD67 mRNA. However, GAD67 mRNA did increase in animals co-treated with MPT and GVG. We hypothesize that this is likely due to reciprocal inhibition (Trudeau et al., 1993a) of dopaminergic and GABAergic systems in the hypothalamus. GVG treatment further inhibits dopaminergic input on GABAergic neurons, resulting in the increase of GAD67 mRNA and LH release in the GVG/MPT group. However, the current technique measures ongoing GABA synthesis in a dynamic assay following GVG treatment. Assessment of GABA synthesis rates in Wsh treated with MPT alone is not possible with our technique. Further testing of this hypothesis will require development of alternative methods to calculate ongoing GABA synthesis rates in vivo. One limitation of the current study is the lack of information regarding GAD isoform protein levels. Studies in mammals have shown that regulation of GABA release by dopamine could occur independent of transcriptional regulation (Lindefors, 1993; O’Connor et al., 1991) and that GAD67 can also be regulated at the post-transcriptional level (Martin and Rimvall, 1993). We were unable to quantify GAD protein levels because speciWc antibodies diVerentiating between Wsh GAD65, GAD67 or GAD3 are not yet available. In conclusion, our study is the Wrst to demonstrate that dopaminergic regulation of teleost GABAergic neuroendocrine systems occurs through isoform speciWc regulation of GAD67 transcriptional activity. Future experimentation investigating the close relationship between GAD mRNA, GAD protein, and in vivo GABA synthesis rates should lead to a better understanding of dopamine-GABA interactions and their roles in regulation of the goldWsh reproductive axis. Acknowledgments This research was supported by NSERC-Canada Discovery Grants to VTL. B.H., I.F., and R.M., were recipients of NSERC Summer Studentships. Dr. J. Arnason provided access to HPLC facilities. The animal care assistance of Bill Fletcher and Jennifer Keyte is acknowledged and appreciated. References Borg, B., Antononopuolou, E., Mayer, I., Andersson, E., Berglund, I., Swanson, P., 1998. EVects of gonadectomy and androgen treatments on pituitary and plasma levels of gonadotropins in mature male Atlantic salmon, Salmo salar, parr-positive feedback control of both gonadotropins. Biol. Reprod. 58 (3), 814–820. Bosma, P.T., Blasquez, M., Fraser, E.J., Schulz, R.W., Docherty, K., Trudeau, V.L., 2001. Sex steroid regulation of glutamate decarboxylase mRNA expression in goldWsh brain is sexually dimorphic. J. Neurochem. 76 (4), 945–956.
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