205 Thyroid hormone modulation of brain in vivo hydroxylase activity and kinetics in the female catfish Heteropneustes fossilis

R Chaube and K P Joy Department of Zoology, Banaras Hindu University, Varanasi-221005, India (Requests for offprints should be addressed toKPJoy;Email: [email protected])

Abstract In the female catfish Heteropneustes fossilis, administration than preparatory phase. Kinetic studies by Lineweaver– ff of thyroxine (T4), 1 µg/g body weight, i.p., in both Burk plots showed that the stimulatory e ect follow- gonadal resting and preparatory phases for 7, 14 and 21 ing T4 administration and T4 replacement on TH days caused hyperthyroidism, as evidenced from a activity was due to increased affinity of the enzyme duration-dependent significant increase in serum tri- for its cofactor (6,7-dimethyl-2-amino-4-hydroxy- iodothyronine (T3), and of tyrosine hydroxylase (TH) 5,6,7,8-tetrahydropteridine), as evident from a significant activity in telencephalon, hypothalamus–pituitary and decrease in apparent Michaelis–Menten constant (Km) and medulla oblongata (Newman–Keuls’ test; P,0·05). an increase in apparent velocity maximum (Vmax). The Hypothyroidism induced by adding 0·03% thiourea to TH inhibition due to the thiourea treatment can be related aquarium water holding the catfish for 7, 14 and 21 to decreased affinity of the enzyme for its cofactor, as days decreased serum T3 levels in a duration-dependent evident from a significant increase in apparent Km value manner (Newman–Keuls’ test; P,0·05) and inhibited and a significant decrease in Vmax. These data clearly show TH activity in the brain regions. T4 replacement in 21- that circulating levels of T4/T3 modulate brain TH day thiourea-treated fish restored and even elevated activity by altering the kinetic properties of the enzyme, significantly serum T3 levels as well as brain TH activity which, in turn, influence catecholaminergic activity and in a duration-dependent manner. In general, the dependent functions. changes in enzyme activity were higher in the forebrain Journal of Endocrinology (2003) 179, 205–215 regions than medulla oblongata and in the resting phase

Introduction distributed in neurons, glial and ependymal cells (Puymirat et al. 1991). Thyroid hormone (T3) is a primary epigenetic factor have been demonstrated to influ- influencing multiple events in neural development such as ence the maturation and maintenance of the brain cat- axonal maturation, neurite outgrowth, cell migration, echolamine (CA)-ergic system (Rastogi & Singhal 1976, myelin formation, etc. (Denver 1997, Oppenheimer & Claustre et al. 1996). Noradrenergic activity in the periph- Schwartz 1997). Apart from their well known feedback eral sympathetic nervous system and CA-ergic activity in actions in the brain related to the regulation of thyro- the adrenal medulla are also dependent on the thyroid trophin (TSH) secretion from the pituitary, thyroid hor- status; plasma noradrenaline increases in hypothyroid mones (thyroxine (T4) and (T3)) exert a subjects (Valens & Gripois 1990). Immunocytochemical crucial role in the overall neural activity of both central and studies have demonstrated the coexistence of both thyro- peripheral nervous systems in vertebrates (Rastogi & nergic (T3-containing) and noradrenergic systems (locus Singhal 1976, Ruiz-Marcos et al. 1994, Claustre et al. coeruleus neurons and their targets) and T3 can act as a 1996). T4 is transported into the brain, concentrated, neurotransmitter/neuromodulator (Rozanov & Dratman retained and metabolised in discrete neural systems 1996, Gordon et al. 1999). Tyrosine hydroxylase (TH) is (Dratman et al. 1987, Schreiber et al. 1990). T3, the the rate-limiting step in CA biosynthesis and is influenced functional thyroid hormone, accounts for the major part of by the thyroid status (Kizer et al. 1978, Wang et al. iodocompounds in the brain (80%) and its concentration is 1989, Claustre et al. 1996, Evans et al. 1999); surgical generally maintained at a relatively stable level despite thyroidectomy increases TH activity and T4 replacement thyroid hormone deficiency or excess (Dratman et al. restores it. However, hypothyroidism induced by pro- 1987). T3 receptors ( and  subtypes) are generally pylthiouracil (PTU) decreases TH activity in the anterior

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part of the locus coeruleus and adrenal medulla and Varanasi. Female fish weighing 35–45 g were selected and hyperthyroidism by T4 injection elevates it (Claustre et al. acclimatised in flow-through aquarium tanks under nor- 1996). mal photoperiod and ambient temperature (resting phase: In teleosts, investigations on brain TH are largely related 10·5 h light:13·5 h darkness, 182 C; vitellogenic to its use as a phenotypic marker for the CA-ergic system phase: 11·5 h light:12·5 h darkness, 222 C). They (Hornby & Piekut 1990). In rainbow trout, TH activity were fed minced goat liver daily. After 15 days of accli- was demonstrated in forebrain regions (Linard et al. 1996). matisation, the fish were used for various experiments as Further, cDNA cloning and sequencing of TH have been follows. demonstrated in the rainbow trout (Linard et al. 1998) and eel (Boularand et al. 1998). In our earlier studies, we have demonstrated seasonal, diurnal, regional and sexual differ- Induction of hyperthyroidism ences in, and effects of environmental factors (photoperiod and temperature) as well as oestrogens on brain TH Fish were divided into two groups of 25 each. Group 1 activity and kinetics in the catfish (Chaube & Joy 2002, was injected i.p. on alternate days with T4 in a dose of 2003). To the best of our knowledge, there are no studies 1 µg/g body weight (BW). T4 was dissolved in alkaline relating to the role of thyroid hormones in the regulation saline (0·65% NaCl containing 5 M NaOH to adjust the of TH activity in non-mammals. In teleosts, the role pH to 8·1) as vehicle. Group 2 was treated with the of thyroid hormones in morphogenesis, development, vehicle as control. After 7, 14 and 21 days, five fish from growth, osmoregulation, migration, metabolism and each of the two groups were sampled at 1100–1200 h. reproduction are broadly defined (Eales 1993), but their Blood was collected by caudal puncture. The samples were involvement in specific functions of the brain is not centrifuged at 2000 g to collect serum, which was stored   demonstrated. T3 is the functional thyroid hormone and at 70 CforT3 assay. The fish were weighed, killed is formed extrathyroidally, while T4 is involved in the by decapitation and brains dissected out and stored at feedback regulation of TSH secretion (Eales et al. 1993). 70 C for TH assay. Ovaries were weighed and the  As in mammals, the teleost brain is also a site for T4 and T3 gonadosomatic index (GSI=ovary weight (g)/BW 100) deiodination (Plate et al. 2002). calculated. In the present study, we demonstrated the effects of hyperthyroidism (by T4 administration), hypothyroidism (induced by thiourea) and T4 replacement on in vivo TH activity and kinetics in the brain regions of the female Induction of hypothyroidism catfish Heteropneustes fossilis. Since surgical thyroidectomy is not feasible in catfish due to the diffuse distribution of thyroid follicles in the pharyngeal floor, hypothyroidism was induced by thio- Materials and Methods , a thyroid hormone inhibitor and . Fish were divided into two groups of 25 each. Group 1 was Chemicals maintained in water containing 0·03% thiourea, which  was replenished every day after feeding. Group 2 fish were Catalase, -tyrosine, 6,7-dimethyl-2-amino-4-hydroxy- maintained in dechlorinated tap water as control. At 5,6,7,8-tetrahydropteridine (DMPH4), BSA, Sephadex intervals of 7, 14 and 21 days, five fish each from the two G-25 and T4 were purchased from Sigma Chemical groups were sampled at 1100–1200 h. Blood was collected Company, St Louis, MO, USA. Sodium molybdate,   for serum separation and stored at 70 CforT3 assay. 2-mercaptoethanol, sodium nitrite, thiourea and Folin– The fish were weighed, killed by decapitation and brains Ciocalteu reagent were purchased from E-Merck, dissected out and stored at 70 C for 24 h for TH assay. Mumbai, India. RIA kits for T3 were purchased from Ovaries were weighed for calculation of GSI. Bhaba Atomic Research Centre, Mumbai, India.

Fish collection and acclimatisation Reversal of hypothyroidism by T4 replacement H. fossilis is a freshwater air-breathing catfish whose Fish were maintained in thiourea (0·03%) for 21 days, as reproductive cycle can be divided into five phases: resting described above, and then divided into two groups and (September–January); preparatory or early vitellogenic maintained in dechlorinated water. Group 1 was injected (February–April); prespawning or late vitellogenic (May– with T4 (1 µg/g BW), i.p., on alternate days. Group 2 was June); spawning (July–August); and post-spawning given vehicle only as control. Five fish from each of the (September–October). The study was conducted in go- two groups were sampled at intervals of 7, 14 and 21 days. nadal resting (December) and early vitellogenic (March) Blood was collected for serum separation and serum   phases. Fish were collected from local fish markets in stored at 70 CforT3 assay. The fish were weighed,

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killed by decapitation and brains dissected out and stored at Table 1 Effects of T4, thiourea and T4 replacement on 70 C for 24 h for TH assay. Ovaries were weighed for gonadosomatic index (meansS.E.M. in g %) in the female catfish Heteropneustes fossilis in the vitellogenic phase. Comparisons were calculation of GSI. made with respective controls and duration groups

7 days 14 days 21 days RIA of T3 Serum T3 level was assayed by RIA using the T3 kit. Experiment Serum (50 µl) was incubated in duplicate with 0·1 ml Vehicle control (VC) 1·460·081 1·500·121 1·440·091 125   2  3  4 I-T3 and 0·1 ml antiserum at 37 C for 3 h at room T4 1·74 0·18 2·16 0·24 2·44 0·18 temperature. After incubation, 1·0 ml of PEG–second VC 1·430·021 1·450·061 1·480·031 antibody (polyethylene glycol, 6% w/v+goat anti-rabbit Thiourea (TU) 0·700·012 0·910·043 0·420·024 antibody) was added, mixed by vortexing and centrifuged TU+VC 0·450·021 0·480·021 0·520·031   2  3  4 at 2000 g for 20 min at 4 C. The supernatant was TU+T4 1·34 0·15 1·78 0·02 1·92 0·12 decanted and the pellet dried. Radioactivity was measured in a gamma counter (Beckman DP5000; Beckman Instru- Values with the same superscripted numbers are not significant and those ments Inc., Fullerton, CA, USA). For standard curve with different numbers are significant in each experiment (two-way ANOVA; ff Newman–Keuls’ test). preparations, di erent concentrations of T3 (0·15, 0·3, 0·6, 1·2, 2·4 ng/ml) provided with the kit were processed in the same manner as the plasma samples. The percentage of B/B0 (B=count rate for each sample, B0=count rate for allowed to stand for 5 min. The colour was stable for non-specific binding sample) was calculated and the stan- 30 min. Half a millilitre of a 2 M NaOH solution was dard curve was plotted using different standard concen- quickly added and mixed. Absorption was immediately trations of T3 vs percentage of B/B0 on a logit–log scale. determined at 510 nm in a UV-VIS 118 spectro- From the standard curve, T3 concentration of the sample photometer (Systronics, Ahmedabad, India). To express was determined and expressed in ng/ml. All the samples enzyme activity, tissue protein content in each aliquot was were assayed from a single RIA kit. The minimum measured by the method of Lowry et al. (1951) using BSA sensitivity of the assay was 0·24 ng/ml. The intra-assay as standard. Enzyme activity was expressed as nmol -dopa coefficient of variation (determined from five standard formed/mg protein per h. curve assays) was 6%. Determination of kinetic parameters

TH activity The Michaelis–Menten constants (Km) and maximum Brains were thawed and dissected out immediately on ice. velocity (Vmax) of TH were determined from the inter- The telencephalon (excluding olfactory tract and bulb), cepts on the x- and y-axes respectively of double reciprocal and hypothalamus along with pituitary and medulla ob- Lineweaver–Burk plots with 1/[DMPH4] (1–8 mM) or  longata were separated as described earlier (Chaube & Joy 1/[ -tyrosine] (0·1–0·5 mM) as independent variable and 2002). Tissues were homogenised in 1 ml 30 mM sucrose 1/TH as dependent variable. containing 10 mM Tris–HCl buffer (pH 7·3) in a Potter– Elvehjem homogeniser with a loose-fitting Teflon pestle. Statistical analysis The rotor speed was 300–500 r.p.m. and the pestle was All data are expressed as meansS.E.M. The data were taken up and down four or five times. The homogenate analysed by two-way ANOVA followed by Newman– was centrifuged at 105 000 g for 1 h and passed through Keuls’ test (P,0·05). Sephadex G-25 column (1 ml column, flow rate 1 ml/  40 min) at 4 C to remove endogenous CAs. The eluate Results containing TH activity was stored up to 1 week at ff 20 C and used as the enzyme preparation for TH assay. E ects of T4 administration on GSI, serum T3 levels and Enzyme activity was not affected by storage up to 1 week brain TH (data not shown). The GSI registered an overall significant effect (two-way TH activity was measured by the method of Shiman ANOVA; P,0·001) after T4 administration in both et al. (1971). The incubation mixture contained 0·25 ml vitellogenic (Table 1) and resting (data not shown) phases. -tyrosine (2 mM), potassium phosphate-buffered saline The values increased significantly at all time points in both (PBS, 2·0 M, pH 6·2), 0·01 ml catalase (1 mg/3 ml in phases except on day 7 and 14 in the resting phase ff PBS bu er), 0·05 ml 0·28 M 2-mercaptoethanol, 0·05 ml (P,0·05; Newman–Keuls’ test). Serum T3 levels showed ff 6 mM DMPH4. The reaction mixture was incubated in a an overall significant e ect (two-way ANOVA; test tube at 30 C for 25 min. The reaction was stopped by P,0·001) in both vitellogenic (Fig. 1B) and resting (data adding 0·5 ml 0·5 M HCl. Freshly prepared nitrite- not shown) phases. The T3 levels increased significantly at molybdate reagent (1 ml) was added to the mixture and all time points (P,0·05; Newman–Keuls’ test). www.endocrinology.org Journal of Endocrinology (2003) 179, 205–215

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Figure 1 Effects of thiourea (TU) (A), T4 (B) and T4 replacement (C) on serum T3 levels in the female catfish Heteropneustes fossilis (meansS.E.M., n=5) in the vitellogenic phase. Data were analysed by two-way ANOVA (P,0·001) and Newman– Keuls’ test (P,0·05). Comparisons were made with respective controls and duration groups. Values with the same number are not significant and those with different numbers are significant in each experiment. VC, vehicle control.

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 Figure 2 Effects of T4 treatment on brain TH activity in the female catfish Heteropneustes fossilis (means S.E.M., n=5) in the vitellogenic phase. Data were analysed by two-way ANOVA (P,0·001) and Newman–Keuls’ test (P,0·05). Comparisons were made with respective controls and duration groups. Values with the same number are not significant and those with different numbers are significant in each experiment. VC, vehicle control.

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 Table 2 Effects of T4, thiourea and T4 replacement on apparent Km and Vmax (means S.E.M.) of tyrosine hydroxylase for cofactor in brain regions of the female catfish Heteropneustes fossilis in the vitellogenic phase. The Km and Vmax values were calculated from Lineweaver–Burk plots. Data were analysed by two-way ANOVA (P<0·001) and Newman–Keuls’ test (P<0·05). Comparisons were made with respective control groups and duration groups and were indicated by numbers (Km) and letters (Vmax) 7 days 14 days 21 days

Km Vmax Km Vmax Km Vmax (mM) (nmol/mg protein/h) (mM) (nmol/mg protein/h) (mM) (nmol/mg protein/h)

Hypothalamus 1. Vehicle control (VC) 0·190·0041 20·320·98a 0·220·0021 20·350·24a 0·200·0031 20·481·54a  1  a  2  b  2  c T4 0·18 0·005 20·86 1·80 0·10 0·003 22·14 0·72 0·09 0·001 24·60 1·28 2. Control 0·180·031 20·260·92a 0·190·011 20·301·86a 0·170·021 20·420·75a Thiourea (TU) 0·200·011 20·121·08a 0·580·032 14·651·05b 0·820·083 12·020·98c 3. TU+VC 0·780·121 11·621·42a 0·800·151 11·601·68a 0·820·081 11·651·72a  1  a  2  b  3  c TU+T4 0·70 0·08 12·08 0·86 0·42 0·04 14·83 2·02 0·30 0·06 18·12 0·28 Telencephalon 1. VC 0·150·0041 22·611·70a 0·140·0031 22·420·24a 0·140·0011 22·321·42a  1  a  1  b  1  c T4 0·14 0·002 22·86 1·82 0·11 0·002 24·42 0·62 0·05 0·001 26·45 1·18 2. Control 0·140·031 22·650·86a 0·150·011 22·542·02a 0·160·021 22·481·58a TU 0·350·052 22·580·40a 0·980·083 18·621·80b 1·280·154 10·251·02c 3. TU+VC 1·160·841 10·640·48a 1·100·021 10·660·78a 1·120·031 10·681·14a  2  a  3  b  4  c TU+T4 0·84 0·18 11·08 0·28 0·62 0·03 13·75 1·20 0·48 0·01 16·18 1·28 Medulla oblongata 1. VC 0·260·041 12·881·12a 0·240·0031 12·651·26a 0·220·0031 12·781·12a  1  a  1  b  1  c T4 0·20 0·01 13·24 0·61 0·15 0·002 16·24 0·78 0·12 0·001 18·68 0·82 2. Control 0·280·051 12·511·28a 0·260·021 12·581·34a 0·240·081 12·451·05a TU 0·300·031 11·221·01a 0·450·052 8·420·75b 0·580·063 5·420·86c 3. TU+VC 0·600·071 5·630·81a 0·560·081 5·611·06a 0·580·101 5·680·80a  1  a  2  b  2  c TU+T4 0·58 0·06 6·05 0·82 0·30 0·05 8·02 0·62 0·20 0·02 8·62 0·28

Values with the same superscripted numbers or letters are not significant and those with different numbers and letters are significant.

Brain TH activity showed an overall significant effect genic phase (Table 1) and in the resting phase (data not (two-way ANOVA; P,0·001) in the vitellogenic phase shown). Newman–Keuls’ analysis indicated significant (Fig. 2A–C) and resting phase (data not shown). In both inhibition (P,0·05) at all times except on day 7 in the phases, enzyme activity increased significantly on day 14 resting phase. The treatment resulted in an overall signifi- ff (except medulla oblongata) and on day 21 in all brain cant e ect (two-way ANOVA; P,0·001) on serum T3 regions (P,0·05; Newman–Keuls’ test). The percentage levels in both vitellogenic (Fig. 1A) and resting (data not increase on day 21 was lower in the vitellogenic phase shown) phases. The T3 levels decreased significantly at all (hypothalamus 27%, telencephalon 21%, and medulla time points compared with control values (P,0·05; oblongata 37%) than resting phase (76, 69 and 53% Newman–Keuls’ test). The inhibition on day 7, 14 and 21 respectively). was 36, 54·5 and 60% respectively in the vitellogenic The T4 administration produced overall significant phase and 47, 49 and 72% respectively in the resting phase. ff e ects on both apparent Km and Vmax of the enzyme for The thiourea treatment produced an overall significant the cofactor in different brain regions (Table 2; two-way effect (two-way ANOVA; P,0·001) on TH activity in ff ANOVA; P,0·001). The Km and Vmax values did not di erent regions of the brain in both vitellogenic (Fig. vary significantly in the vehicle control groups over the 3A–C) and resting (data not shown) phases. In both duration in any of the brain regions. The Km values hypothalamus and telencephalon, enzyme activity de- decreased, and the Vmax increased significantly in all brain creased at all time points, but in the medulla oblongata, the regions on day 14 and 21 (P,0·05; Newman–Keuls’ test). decrease was significant only on day 14 and 21 (P,0·05; In the resting phase, similar changes were found (data not Newman–Keuls’ test). The percentage decrease on day 21 shown) (two-way ANOVA; P,0·001; Newman–Keuls’ was 54% (hypothalamus), 33% (telencephalon) and 20% test; P,0·05). (medulla oblongata) in the vitellogenic phase and 54·5, 61 and 21% respectively in the resting phase. Apparent K and V of the enzyme for the cofactor ff levels and m max E ects of thiourea treatment on GSI, serum T3 showed overall significant effects (two-way ANOVA; brain TH P,0·001) in both vitellogenic (Table 2) and resting (data The GSI showed an overall significant effect after thiourea not shown) phases. Since the treatment influenced both treatment (two-way ANOVA; P,0·001) in the vitello- Km and Vmax, the inhibition appeared to be of the mixed

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Figure 3 Effects of thiourea treatment on brain TH activity in the female catfish Heteropneustes fossilis (meansS.E.M., n=5) in the vitellogenic phase. Data were analysed by two-way ANOVA (P,0·001) and Newman–Keuls’ test (P,0·05). Comparisons were made with respective controls and duration groups. Values with the same number are not significant and those with different numbers are significant in each experiment.

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or uncompetitive type. The Km values increased, and activation detected after 2 weeks of treatment. These the Vmax decreased significantly over the duration of the observations are in agreement with studies in mammals of ff treatment. In the thiourea-treated groups, the Km and the a stimulatory e ect of T4/T3 on the enzyme in developing Vmax values did not change significantly in the hypo- or adult brains and adrenals (Rastogi & Singhal 1976, Kato thalamus and medulla oblongata on day 7 compared with et al. 1982, Gripois & Valens 1984, Valens & Gripois the control groups (P,0·05; Newman–Keuls’ test). In the 1990). However, conflicting reports of a lack of enzyme ff telencephalon, the Vmax also did not vary significantly on response, perhaps due to brain regional di erences, short day 7. In the resting phase, similar changes were noticed. vs long treatment, developmental stage at which exposure was made, dosage, etc., are also available (Valens & ff Gripois 1990, Claustre et al. 1996). In the catfish, T4 E ects of T4 replacement on GSI, serum T3 and brain TH administration resulted in hyperthyroidic conditions, as The administration of T4 in 21-day thiourea-treated fish evident from the duration-dependent rise in serum T3 for 7, 14 and 21 days produced an overall significant effect levels, which may be responsible for the elevated brain TH (two-way ANOVA; P,0·001) on GSI in the vitellogenic activity. The response varied with the brain region, (Table 1; treatment F=6·25, duration F=16·85 and in- duration of the treatment and season. The magnitude of teraction of both F=26·65) and resting (data not shown) the response was higher in the forebrain regions than phases. The GSI increased significantly at all time points medulla oblongata and in the resting phase than vitello- ff (P,0·05; Newman–Keuls’ test). Serum T3 levels showed genic phase. These di erences may be due to regional a significant effect in both vitellogenic (Fig. 1C) and differences in enzyme activity (Chaube & Joy 2003) or the resting (data not shown) phases. The T3 levels increased interplay of other factors like gonadal oestrogens on the significantly on day 7, 14 and 21 and the percentage brain–pituitary axis. The forebrain regions contain oestro- increase was 119·5, 250 and 400% respectively in the gen feedback sites and the oestradiol (E2) feedback is vitellogenic phase and 215, 335 and 418% respectively in stronger in the vitellogenic phase than resting phase the resting phase. In the control (thiourea+vehicle) (Senthilkumaran & Joy 1995). The increase in GSI sug- groups, the T3 levels remained low even up to 21 days of gests that hyperthyroidism caused a stimulation of ovarian the withdrawal. activity. Since thyroid hormones modulate gonadal ster- Brain TH activity showed an overall significant effect oidogenesis (Cyr & Eales 1988, Timmermans et al. 1997), (two-way ANOVA; P,0·001) after the replacement the resulting strong E2 feedback might have lessened the ff treatment in the vitellogenic and resting (data not shown) otherwise full-blown e ect of T4/T3 in the vitellogenic phases. The T4 replacement caused a significant increase phase (Chaube & Joy 2002). in TH activity in all brain regions at all times in the Hypothyroidism has long been linked to retardation of vitellogenic (Fig. 4A–C) and resting (data not shown) development, maturation and functions of the nervous phases. The percentage increase on day 21 was much system and causes mental ailment, motor dysfunction, lower in the vitellogenic phase (124%, hypothalamus; behavioural changes, early neurodegenerative activity, etc. 58·9%, telencephalon; 21%, medulla oblongata) than rest- – some of these disorders have been related to impairment ing phase (213%, hypothalamus; 310%, telencephalon; and of CA metabolism (Evans et al. 1999, Kincaid 2001). 50%, medulla oblongata). Hypothyroidism caused tyrosinaemia (tyrosine accumu- ff T4 replacement produced overall significant e ects (two- lation) in the brain, adrenal, heart, etc. (Diarra et al. 1989) way ANOVA; P,0·001) on both apparent Km and Vmax and the genetically hypothyroid mouse (non-functional values of the enzyme for the cofactor in the vitellogenic thyroid due to defective TSH receptor) showed sig- (Table 2) and resting (data not shown) phases. The values nificantly fewer (40%) dopamine (TH-positive) neurons did not change significantly in the vehicle groups during the in the substantia nigra and adjacent ventral tegmental area duration of the treatment. In the replacement groups, the (Kincaid 2001). The reported effects of hypothyroidism on values altered (Km decreased and Vmax increased) signifi- TH are at variance and seem to be influenced by several cantly in both phases on day 14 and 21 (P,0·05; Newman– factors including its nature of induction. Hypothyroidism Keuls’ test). On day 7, both the values are significantly by surgical thyroidectomy resulted in an increase in TH ff di erent in the hypothalamus (resting phase) and the Km function (activity and mass) in the median eminence values in the medulla oblongata (resting phase) and telen- (Wang et al. 1989) and in some hypothalamic nuclei (Kizer cephalon (vitellogenic phase). et al. 1978) but did not alter it in the preoptic nucleus (Kizer et al. 1978), substantia nigra (Nakahara et al. 1976) and superior cervical ganglia (Wang et al. 1989). Hypothy- Discussion roidism induced by PTU treatment caused an increase in TH activity in the median eminence of the rat (Kizer et al. The present study demonstrates clearly the involvement of 1978), a decrease in the anterior locus coeruleus and thyroid hormones in the modulation of brain TH activity. adrenal, and no changes in the posterior locus coeruleus The administration of T4 evoked significant brain TH and substantia nigra (Claustre et al. 1996). In the rat,

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Figure 4 Effects of T4 replacement in the 21-day thiourea (TU)-treated female catfish Heteropneustes fossilis (meansS.E.M., n=5) in the vitellogenic phase. Data were analysed by two-way ANOVA (P,0·001) and Newman–Keuls’ test (P,0·05). Comparisons were made with respective controls and duration groups. Values with the same number are not significant and those with different numbers are significant in each experiment.

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neonatal hypothyroidism induced by PTU impaired turn, thyroid hormones can influence central CA-ergic TH activation in the adrenal and was reversed by T3 activity and the dependent physiological processes such replacement (Valens & Gripois 1990). The varied effects as osmoregulation, growth and reproduction in the catfish. suggest that modulation of TH activity by T4/T3 is influenced by the interplay of other regulatory/modulatory signals as well. In the present study, catfish were rendered Funding hypothyroidic by thiourea treatment, which resulted in a time-dependent significant decrease in serum T3 levels. In R C is grateful to the Council of Scientific and Industrial the hypothyroidic catfish, a duration-dependent signifi- Research, New Delhi for the award of a senior research cant decrease in TH activity was noticed in all brain fellowship. regions in both seasons. The inhibition was higher in the forebrain regions than medulla oblongata with the telen- cephalic activity showing marked seasonal difference. The decrease in the GSI following hypothyroidism suggests References decreased gonadal and E2 feedback activity and this may account for the higher inhibition of the enzyme in the Blouquit MF, Valens M, BagayokoA&Gripois D 1990 Adrenal vitellogenic phase. Claustre et al. (1996) reported de- tyrosine hydroxylase activation in the developing rat: influence of the thyroid. Journal of Developmental Physiology 14 325–329. creased TH activity after PTU treatment, but Kizer et al. Boularand S, Biguet NF, Vidal B, Veron M, Mallet J, Vincent JD, (1978) reported TH activation in rat brain nuclei. The DufourS&VernierP1998 Tyrosine hydroxylase in the European eel (Anguilla anguilla): cDNA cloning, brain distribution and thiourea-induced TH inhibition could be reversed by T4 replacement, but not by withdrawal of the treatment alone phylogenetic analysis. Journal of Neurochemistry 71 460–470. ChaubeR&JoyKP2002Effects of ovariectomy and oestradiol-17 (vehicle control group). The data also showed that fore- replacement on brain tyrosine hydroxylase in the catfish brain TH activity was more sensitive to the replacement Heteropneustes fossilis: changes in in vivo activity and kinetic treatment. parameters. Journal of Endocrinology 175 329–342. The kinetic data presented in this study may explain the ChaubeR&JoyKP2003Brain tyrosine hydroxylase in the catfish Heteropneustes fossilis: annual and circadian variations and sex and changes in TH activity in relation to thyroid hormone ff ff regional di erences in enzyme activity and some kinetic properties. excess and deficiency. The stimulatory e ect of T4 General and Comparative Endocrinology 130 29–40. appeared to be caused by a significant lowering of the Claustre J, BalendeC&PujolJF1996Influence of the thyroid hormone status on tyrosine hydroxylase in central and peripheral apparent Km value of the enzyme for the cofactor with concomitant increase in the apparent V , as reported in catecholaminergic structures. Neurochemistry International 28 max 277–281. thyroidectomised rats (Kizer et al. 1978). Thus, the stimu- ff ff ffi Cyr DG & Eales JG 1988 In vitro e ects of thyroid hormones on latory e ect of T4 can be correlated to an increased a nity gonadotropin-induced estradiol-17 secretion by ovarian follicles of of the enzyme for the cofactor. In contrast, thiourea rainbow trout, Salmo gairdneri. General and Comparative Endocrinology treatment (hypothyroidism) produced kinetic changes in 69 80–87. the reverse manner and the TH inhibition could be due to Denver RJ 1997 Thyroid hormone action in brain development: ffi molecular biological aspects. In Advances in Comparative alowa nity of the enzyme for the cofactor (high Km and Endocrinology: Proceedings of the XIIIth International Congress of low Vmax). The thiourea-induced changes in the enzyme Comparative Endocrinology, pp 1071–1078. Eds S Kawashima & S Kikuyama. Bologna, Italy: Monduzzi Editore SPA. kinetics could be reversed by T4 replacement, resulting in a significant decrease in the K values and a significant Diarra A, Lefauconnler JN, Valens M, GeorgesP&Gripois D 1989 m Tyrosine content, influx and accumulation rate, and catecholamine increase in the Vmax. In contrast, Kizer et al. (1978) biosynthesis measured in vivo, in the central nervous system and in reported that PTU treatment decreased the Km value for peripheral organs of the young rat, influence of neonatal hypo- and the cofactor and increased the Vmax, like surgical thyroid- hyperthyroidism. Archives of International Physiology and Biochemistry ectomy. In hypothyroidic rat adrenal, the decreased TH 97 317–332. activity was associated with an increase in K value in Dratman MB, Crutchfield FL, Futaesaku Y, Goldberger ME & m Murray M 1987 125I triiodothyronine in the rat brain: evidence for comparison with euthyroid animals (Blouquit et al. 1990). neural localization and axonal transport derived from thaw-mount Thus, the stimulatory effect of thyroid hormones on TH film autoradiography. Journal of Comparative Neurology 260 392–408. activity may be mediated by the activation of the enzyme Eales JG, Maclatchy DL & Sweeting RM 1993 Thyroid hormone by kinetic changes. An increase in enzyme synthesis also deiodinase system in salmonids and their involvement in the regulation of thyroidal status. Fish Physiology and Biochemistry 11 may lead to increased TH activity. The thiourea treatment 313–321. might have interfered with the enzyme activation and Evans IM, Sinha AK, Pickard MR, Edwards PR, Leonard AJ & Ekins synthesis by decreasing the T4/T3 levels. Further studies RP 1999 Maternal hypo-thyroxinemia disrupts neurotransmitter are required to understand the molecular mechanisms metabolic enzymes in developing brain. Journal of Endocrinology 161 involved in the modulation of TH activity by T /T 273–279. 4 3. Gordon JT, Kamins , DM, Rozanov CB & Dratman MB 1999 In conclusion, brain TH is sensitive to the thyroid Evidence that 3,3,5- triiodothyronine is concentrated in and status; hormone excess activates and deficiency retards delivered from the locus coeruleus to its noradrenergic targets via the enzyme activity by modifying its kinetic function. In anterograde axonal transport. Neuroscience 93 943–954.

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GripoisD&Valens M 1984 Tyrosine hydroxylase induction in the Puymirat J, Miehe M, Marchand R, Sarlieve L & Dussault JH 1991 young rat: control by thyroid hormones. Molecular and Cellular Immunocytochemical localization of thyroid hormone receptors in Endocrinology 38 87–89. the adult rat brain. Thyroid 1 173–184. Hornby PJ & Piekut DT 1990 Distribution of catecholamine- Rastogi B & Singhal RL 1976 Influence of neonatal and adult synthesizing enzymes in goldfish brains: presumptive dopamine and hyperthyroidism on behavior and biosynthetic capacity for norepinephrine neuronal organization. Brain, Behavior and Evolution norepinephrine, dopamine and 5-hydroxytryptamine in rat 35 49–64. brain. Journal of Pharmacology and Experimental Therapeutics 198 Kato T, Yamaguchi T, Togari A, Nagatsu T, Yajima T, Maeda N & 609–618. Kumegawa M 1982 Ontogenesis of monoamine-synthesizing Rozanov CB & Dratman MB 1996 Immunohistochemical mapping of enzyme activities and biopterin levels in rat brain or salivary glands, brain triiodothyronine reveals prominent localization in central and the effects of thyroxine administration. Journal of Neurochemistry noradrenergic systems. Neuroscience 74 897–915. 38 896–901. Ruiz-Marcos A, Cartagena-Abella P, Martinez-Galan JR, Calvo R, Kincaid AE 2001 Spontaneous circling behavior and dopamine Morreale de EscobarG&EscobardelReyF1994 Thyroxine neuron loss in a genetically hypothyroid mouse. Neuroscience 105 treatment and the recovery of pyramidal cells of the cerebral cortex 891–898. from changes induced by juvenile-onset hypothyroidism. Journal of Kizer JS, Humm J, Nicholson G, GreeleyG&Youngblood W 1978 Neurobiology 25 808–818. The effect of castration, thyroidectomy and haloperidol upon the turnover rates of dopamine and norepinephrine and the kinetic Schreiber G, Aldred AR, Jaworowski A, Nilsson C, Achen MG & properties of tyrosine hydroxylase in discrete hypothalamic nuclei of Segal MB 1990 Thyroxine transport from blood to brain via the male rat. Brain Research 146 95–107. synthesis in choroid plexus. American Journal of 258 Linard B, Bennani S, Jego P & Saligaut C 1996 Tyrosine hydroxylase Physiology 338–345. activity and dopamine turnover of rainbow trout (Oncorhynchus SenthilkumaranB&JoyKP1995Changesinhypothalamic mykiss) brain : the special status of the hypothalamus. Fish Physiology catecholamines, dopamine--hydroxylase, and phenylethanolamine- and Biochemistry 15 41–48. N-methyltransferase in the catfish, Heteropneustes fossilis in relation Linard B, Pakdel F, Marmignon MH & Saligaut C 1998 Cloning to season, raised photoperiod and temperature, ovariectomy and of a cDNA coding for active tyrosine hydroxylase in the rainbow estradiol-17 replacement. General and Comparative Endocrinology 97 trout (Oncorhynchus mykiss): comparison with other hydroxylases 121–134. and enzymatic expression. Journal of Neurochemistry 71 920–928. Shiman R, Akino M & Kaufman S 1971 Solublization and partial Lowry OH, Rosenbrough NJ, Farr AL & Randall RJ 1951 Protein purification of tyrosine hydroxylase from bovine adrenal medulla. measurement with Folin–phenol reagent. Journal of Biological Journal of Biological Chemistry 246 1330–1340. Chemistry 193 265–275. Timmermans LP, Chmilevsky DA, Komen H & Schipper H 1997 Nakahara T, Uchimura H, Hirano M, Saito M & Ito M 1976 Effects Precocious onset of spermatogenesis in juvenile carp (Cyprinus carpio of gonadectomy and thyroidectomy on the tyrosine hydroxylase L., Teleostei) following treatment with low doses of -thyroxine. activity in individual hypothalamic nuclei and lower brain stem European Journal of Morphology 35 344–353. catecholaminergic cell groups of the rat. Brain Research 117 Valens M & Gripois D 1990 Influence of neonatal hypothyroidism on 351–356. adrenal tyrosine hydroxylase activation in the young rat. Clinical and Oppenheimer JH & Schwartz HL 1997 Molecular basis of thyroid Experimental Pharmacology and Physiology 17 371–376. hormone-dependent brain development. Endocrine Reviews 18 Wang PS, Gonzalez HA, Reymond MJ & Porter JC 1989 Mass and 462–475. in situ molar activity of tyrosine hydroxylase in the median Plate E, Adams B, Allison W, Martens G, HawryshynC&Eales J ff ff eminence. E ect of thyroidectomy and thyroid hormone 2002 The e ects of thyroxine or a GnRH analogue on thyroid replacement. Endocrinology 49 659–663. hormone deiodination in the olfactory epithelium and retina of rainbow trout, Oncorhynchus mykiss and sockeye salmon, Oncorhynchus nerka. General and Comparative Endocrinology 127 Received in final form 21 July 2003 59–67. Accepted 24 July 2003

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