~28-3~8/90 %3.00+0.00

~e~~Qpburmuc~~~g~ Vol. 29,No.12,~~. 11614169, 1990

Copyright 0 1990 Pergamon Press plc

Printed in Great Britain. All rights reserved

DOPAMTNERGIC HYPOTHYROIDISM:

DYSFUNCTION DIFFERENTIAL GANGLIOSIDE

IN NEONATAL EFFECTS OF GM,

A. VACCARI,‘** E. STEFANINI,' G. DE MONTIS* and Z. L. ROSSETTI’ ‘Department of Neuroscience and %-rstitute of Pharmacology and Chemical Pathology, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy (Accepfed

6 August

1990)

Summary-The effects of daily treatment with GM, ganglioside (30 mg/kg s.c.) from birth to day 30, on striatal pre- and postsynaptic markers of the dopaminergic system in euthyroid- and 32 day-old hypothyroid rats were studied. The purpose was to assess whether GM, could prevent the extensive, hypothyroidism-provoked impairment of dopaminergic neurotransmission. Neonatal administration of GM, well counteracted the hypothyroidism-related deficits in striatal synaptosomal uptake of [3H]dopamine and in membrane binding of [3H]tyramine, a putative marker for the vesicular carrier of dopamine. In the hypothyroid striatum, the decrease of concentrations of DOPAC and HVA, the loss of [3H]SCH-23,390-labelled D,-receptors and the decrease of basal- or dopamine-stimulated, D,-mediated activity of adenylate cyclase were not prevented by GM,. Although somatic and neurobehavioural aberrations of hypothyroids were not at all or only partially ameliorated, a slight improvement of the thyroid status was suggested by less decreased levels of serum thyroxine (T4) after treatment with GM,. The ganglioside-driven selective recovery of the transport and storage process of [-‘Hfldopamine might result either from a chronically-exerted stimulation by GM, on the NA/K- and Mg-ATPase activities, thus reflecting on the ATPase-d~ndent neuronal and vesicular transport processes of dopamine or from a GM~-promoted maturation of the otherwise retarded f~ctionality of dopaminergic nerve endings in the neonatal hypothyroid striatum. Key NJords-hypothyroidism, ganglioside, dopamine and metabolites, pH]dopamine uptake, [‘Hjtyramine binding, D,-receptors, adenylate cyclase.

Thyroid deficiency through late foetal, and/or early postnatal age, when the mammalian central nervous system undergoes maturation, widely affects central

neurotransmission (see Vaccari, 1988), thus resulting in ncuro~havioural alterations of hypothyroid rats and perhaps also having clinical relevance in mentally retarded (cretinoid) hypothyroid children. In methimazole-treated, hypothyroid, newborn rats there is an extensive alteration of striatal dopaminergic pathways at both pre- and postsynaptic levels (Vaccari and Timiras, 1981; Vaccari, Rossetti, De Montis, Stefanini, Martin0 and Gessa, 1990). This may reflect either a diminished density of nigrostriatal dopaminergic-endings bearing an impaired reuptake and storage system for dopamine (Vaccari and Gessa, 1989) or a retarded maturation of synaptic processes (Lu and Brown, 1977). Increasing evidence shows that repeated administration of GM, ganglioside to adult rats, having been submitted to surgical or toxin-based lesions of the central nervous system, may facilitate “regeneration” of surviving neuronal processes, as well as behavioural recovery (Toffano, Agnati, Fuxe, Aldinio, Consolazione, Valenti and Savoini, 1984; Mahadik and Karpiak, 1988). In the developing animal, treat*To whom correspondence

should be addressed.

ment with GM, enhances maturational processes such as dendritogenesis, arborization and synaptogenesis, though having no effect on cell body proliferation (Mahadik and Karpiak, 1986b). Finally, admi~stration of ganglioside may be a tool to promote maturation where the growth and development of the brain have been impaired by pathological conditions like metabolic disorders, toxins or anoxia (Mahadik and Karpiak, 1986a,b). The purpose of the present study was to assess whether chronic administration of GM,, during the sensitive period of development of the brain, could prevent or attenuate the hypothyroidism-provoked impairment of the striatal dopamine-system in rats. Evidence will be reported that treatment with ganglioside induced a selective recovery of the otherwise deficient neuronal and vesicular transport processes for dopamine in the hypothyroid striatum, as assessed with measurement of synaptosomal uptake of [3H]dopamine and binding of [3H]tyramine to membrane preparations. METHODS Materials

[3H]Tyramine (26-39 Ci/mmol specactivity), [)H]dopamine (40.6 Ci/mmol spec.act.), [‘HI-(R)( + )-

1162

A. ~ACCAR~et a!.

7-chloro-8-hydroxy-3-methyl-l-phenyl-2,3,4,5-tetrahydro-lH-3-benzazepine (SCH-23,390: 77 Ci/mmol spec.act.)J3*P]adenosine 5’-triphosphate (ATP: 28 Ci/ mmol specact.) and [3H]cyclic adenosine 3’,5’-monophosphate (CAMP: 38 Ci/mmol spec.act.) were purchased from New England Nuclear. The GM, ganghoside was obtained from Fidia (Padova, Italy). It was freshly diluted in saline in the absence or presence of the antithyroid drug methimazole and was administered in a volume of 3 ml/kg. All chemicals were of the highest quality commercially available. Neonatal hypothyroidism Female, Sprague-Dawley NOS (Nossan, Milano) rats were fed the antithyroid drug methimazole, approx. 50 mg/kg/day, in the drinking water during the last 6 days before delivery, thereafter they were returned on standard water supply. Male and female pups were injected subcutaneously (Vaccari, 1985) with 20 mg/kg/day methimazole from birth to day 10 of age and 30 mg/kg/day methimazole from then to postnatal day 30, in the absence or presence of 30 mgikg GM,. Control, euthyroid pups received an equal volume of saline or GM, alone. All rats were killed 48 hr after the last injection, in order to avoid possible direct effects of methimazole (Biassoni and Vaccari, 1985). The thyroid state of control and treated rats was assessed with standard RIA techniques (Spak Kits T, and T,: Byk-Mallinkrodt, Milano, Italy) for levels of thyroxine (T4) and triiodothyronine (T,) in serum. The striatum (caudatoputamen plus the globus pallidus) was quickly dissected over ice and soon after processed for uptake of [‘Hldopamine, binding of [‘H]tyramine and assays for adenylate cyclase. Samples to be assayed for measurement of dopamine and its metabolites, and for the binding of [3H]SCH-23,390, were stored at - 70°C until processed. ~~easure~~ent uf dapumine and metaba~ites of dopamine in the striatum Dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striaturn were assayed with high pressure liquid chromatography (HPLC), coupled with electrochemical detection (Rossetti, Mercuro and Rivano, 1983). Tissue (approx. 1Omg) was sonicated in 250~1 of 0.2 M HCIG, containing 0.1% of NazS,O, and 50 pg/l of 3,4-dihydroxybenzylamine as internal standard. After centrifugation at 12,000 g for 20 min, lo-20 ~1 of the clear supernatant were applied to the HPLC column (Supelco C-18 15 pm particle size) by using an automatic injector (Waters WISP 710B). The mobile phase was 50mM citrate-acetate buffer (pH 4.5) containing sodium octylsulphate 40 mg/l, 0.1 mM ethylenediaminetetraacetate (EDTA) and methanol 8% (v/v). The flow rate was 0.8 ml/min (Waters M 5 10 pump) and the electrochemical detector (Waters 460) was set to a potential of 0.6 V vs an

Ag/AgCl reference electrode. The signal was recorded with an electronic integrator (Waters 730 Data Module). Concentrations of dopamine and metabelites were calculated by peak area ratios. Uptake of ~3~~apamin~ Crude synaptosomes (P2) from the striata of individual rats were resuspended in 20 vol. of 0.32 M sucrose containing 10 mM glucose and 10 mM Tris-HC1 buffer, pH 7.4 (Buffer 1) (Shoemaker and Nickolson, 1983). Appropriate aliquots of this suspension were diluted 1:7 (v/v) with modified Krebs-Henseleit phosphate buffer (pH 7.4) of the following composition: 122 mM NaCi, 4.8 mM KCI, 1.3 mM CaCI,, 1.2 mM MgSO,, 15.8 mM NaH,PO,, 2.8 mM dithiothreitol (Buffer 2). Glucose (2 g/l) was added to buffer 2 just before use. Synaptosome aliquots were preincubated 5min at 37°C (Schoemaker and Nickolson, 1983), thereafter 350 ~1 of the preincubated suspensions were added to t-tubes containing 100~1 of [3H]dopamine plus 50~1 of unlabelled dopamine (total finai concentrations 5-320 nM), both diluted in buffer 2 supplemented with 54 yM EDTA, 1 mM ascorbic acid and 0.1 mM pargyiine (Buffer 3). Just before the addition of preincubated synaptosomes, the t-tubes with Buffer 3 had been moved from ice to a 37”C-warm waterbath. Samples in a final vol. of 0.5 ml were incubated for 5 min, then the reaction was interrupted by returning the t-tubes to ice. The samples were filtered on Whatman GF/F glass fibre filters, the t-tubes were rinsed once with 3.5 ml of ice-cold 0.9% NaCl, the rinse was filtered and the filters were further washed twice with 3.5 ml of cold saline. The uptake values were corrected for the nonspecific uptake, as measured by incubating the samples over ice. Binding of [31-iltyramine The striata from individual rats were homogenized (glass-glass grinders) in 10 ml of ice-cold 50 mM Tris-HC1 buffer (pH 7.8 at 4”C), containing 120 mM NaCl and 5 mM KCl. Homogenates were diluted to 40 ml of buffer and centrifuged at 48,000g for 10 min. The pellets resuspended with vortexing in 20 ml of buffer were incubated at 37°C for 10 min then further diluted to 40ml and centrifuged at 48,000g for 10 min. After an additional resuspension and centrifugation, the final pellets were manually homogenized 1: 40 (w/v) with ice-cold buffer supplemented with 10 b M pargyline and 50 /1M ascorbic acid (incubation buffer). Membrane aliquots (100 Al/ 100-150 pg protein), in a final vol of 1 ml, were incubated for 10min at 37°C with 0.5-32 nM 13H]tyramine in the absence or presence of 10pM unlabelled p-tyramine, as the displacer for nonspecific binding (Vaccari, 1986). Samples were then replaced on ice, rapidly filtered on Whatman GF/B filters and washed with 3 x 3.5 ml of ice-cold 0.9% NaCl.

GM, and dopamine-system in hypothyroidism Binding of [‘H]SCH-23,390

Pooled striata from 2 rats were homogenized 1: 100 (w/v) with teflon-glass grinder in ice-cold 50mM Tris-HCl buffer, pH 7.4 and centrifuged at 35,000g for 20 min. The final pellets were resuspended 1: 666 (w/v) in 50mM Tris-HCl, pH 7.4, containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl, and 1 mM MgCl, (incubation buffer) (Kilkpatrick, Jenner and Marsden, 1986). Membrane aliquots (1 ml, 15@ 200 pg protein) in a final vol of 3 ml of buffer were incubated at 37°C for 30min with 0.2-4nM [3H]SCH-23,390 in the absence or presence of 5 PM cis-flupenthixol. The reaction was terminated by rapid filtration through Whatman GFjC filters. Filters were immediately washed with 3 x 5 ml of ice cold buffer. Assay of adenylate cyclase

The striata from individual rats were homogenized (teflon-glass grinders) 1: 10 (w/v) in 5 mM Tris-HCl buffer (pH 7.4) containing 1 mM dithiothreitol, 1 mM ethyleneglycol-bis-aminoethylether-tetra-acetate (EGTA) and 10% sucrose. Homogenates were then centrifuged at 9OOg for 10 min. The supernatants (S,) were centrifuged at 9000 g for 20 min. The resuspended Pz fraction was immediately assayed for activity of adenylate cyclase (Salomon, Londos and Rodbell, 1974). The reaction mixture contained 75 mM Tris-HCl (pH 7.4), 0.33 mM EGTA, 2 mM MgCl,, 0.5 mM 3-isobutyl-l-methyl-xanthine, 1 mM CAMP, 100 pm [cL-~~P] ATP, 5 mM phosphocreatine, 5 units of creatine phosphokinase, 2 PM guanosine S-triphosphate (GTP), 50 pg bovine serum albumin, 0.33 mM dithiothreitol, in the absence (basal adenylate cyclase activity) or presence (dopaminestimulated activity) of l-100 PM dopamine in a final vol of 150~1. The reaction was started by adding 50 ~1 of membrane preparation (3040 pg protein) and continued for 5 min at 37°C. The incubation was stopped by adding 200 ~1 of a solution containing 2% sodium dodecylsulphate, 45 mM ATP and 1.3 mM CAMP, pH 7.5. After the addition of [3H]cAMP to monitor recovery of CAMP, the samples were placed in a boiling water bath for 3 min and CAMP was then isolated. In the kinetic analysis for dopaminestimulated activity, basal values for the activity of adenylate cyclase were subtracted. Protein assay

Proteins were estimated using a modified Folin phenol procedure (Peterson, 1977) with bovine serum albumin as the standard. Data analysis

Binding parameters for saturation kinetics were calculated with the LIGAND program (Munson and Rodbard, 1980). Parameters for uptake of [3H]dopamine and activity of adenylate cyclase were calculated with Eadie-Hofstee analysis (Zivin and

1163

Table 1. Thyroid state in GM, ganglioside-treated, hypothyroid rats

euthyroid and

Thyroid state

Treatment

Euthyroid Euthyroid

Saline GM,

53.2 f 2.3 56.3 + 3.0

1.39 * 0.05 1.45 f 0.04

Hypothyroid Hypothyroid

MM1 MM1 + GM,

16.0 f 2.2** 25.1 f 2.1**5

1.41 f 0.08 1.49kO.13

T,

T,

The serum levels of thyroxine (T,) and triiodothyronine (T,) are expressed as nmol/litre and were assayed in 32 day-old, male and female rats. Values are means &-SEM from 13-30 rats, which were injected subcutaneously from birth to postnatal day 30 with saline or methimazole (MMI) (20-30 mg/kg/day), in the absence or presence of 30 mg/kg/day of GM,. **f’ < 0.001, MM1 and MM1 + GM,-treated, compared with salinetreated rats. §P < 0.02, MM1 + GM,-treated, compared with MMI-treated rats. One-way ANOVA.

Waud, 1982). Statistical significance of differences was determined using one-way analysis of variance (ANOVA). -

RESULTS

General effects of hypothyroidism and the influence of administration of GM,

The combined prenatal and neonatal treatment with antithyroid drug decreased by 70% the serum levels of T,, while leaving unchanged levels of Tj, as compared to euthyroid counterparts (Table l), a hormonal status similar to that found in congenitally hypothyroid children (Thilly, Delange, Lagasse, Bourdoux, Ramioul, Berquist and Ermans, 1978). The neonatal administration of GM, to hypothyroid pups enhanced by 57% levels of T, above those of hypothyroid controls, though not normalizing them (Table 1); GM, did not affect hormonal levels in euthyroids (Table 1). The somatic features of GM,-treated and untreated, hypothyroid rats were similar: the gain in body weight in both groups was markedly depressed, as compared to euthyroid controls (Data not presented) and all rats displayed the same poor appearance, with sparse hair. On the contrary, euthyroid rats treated with GM, displayed a very healthy appearance, though their gain in body weight was similar to that in saline-treated controls (data not presented). Finally, administration of GM, did not correct the marked hypothyroidism-provoked delay in the complete opening of the eyes, which occurred at postnatal day 16 in euthyroid or euthyroid-GM, rats and only at day 22 in hypothyroids. Although it was not quantified, GM,-treated hypothyroids were clearly more aggressive and rective to handling than saline-treated hypothyroid counterparts, through the last days of the antithyroid treatment. Both hypothyroid-saline and hypothyroid-GM, rats displayed the characteristic “highstepping, hopping gait”, i.e. rather than walking, they moved with frequent, short jumps (Schwark, 1977).

A. VACCARI et al.

1164

Table 2. Effects of neonatal administration of GM, ganglioside on concentrations of dooamine and metabolites of doeamine in the striatum of euthvroid and hypoihyroid rats Treatment Saline

GM, MM1 MM1 +GM,

Dopamine 354.5 398.4 400.8 327.6

f + + +

DOPAC

16.7 25.2 16.3’ 21.3**

150.1 * 5.4 162.7 i_ 1I.9 68.8 i 3.8”. 68.0 + 5.3”’

HVA 24.6 f 26.9 + 12.8 f II.8 +

N

I .8 1.8 0.9*** 1.5’:’

I7 19 30 I3

Newborn rats were injected subcutaneously daily, from birth to postnatal day 30, with 20-30 mg/kg methimazole (MMI) or saline, in the absence or presence of 30 mg/kg GM,. Values are expressed as pmol/mg protein and are means ? SEM from N male and female, 32 day-old rats. *P = 0.072, N.S., as compared to saline-treated rats. l*P < 0.02 as compared with MMI-treated hypothyroids. ***P < 0.001. as cornoared with saline-treated euthyroids. One-way AN&A. .

compared to euthyroid controls; at the same time, there was a decrease (by 38%) in the K,-values, corresponding to a 1.6-fold enhancement in the binding affinity (Table 4). Neonatal administration of GM, ganglioside to euthyroid pups did not influence binding characteristics of [3H]tyramine, while it completely counteracted the hypothyroidism-provoked decrease and K. values (Table 4). of B,,x

Effects qf treatment with GM, ganglioside on the content of dopamine and dopamine-metabolites in the striatum of euthyroid and hypothyroid rats

Congenital-like hypothyroidism did not affect the content of dopamine in the striatum and severely decreased concentrations of DOPAC (by 54%) and HVA (by 48%) an effect that was not counteracted by neonatal administration of GM, ganglioside (Table 2). Levels of dopamine in the striatum of GM, treated hypothyroids were slightly (by 18%) though significantly (P < 0.02), lower than in salineinjected hypothyroids (Table 2). The administration of GM, did not modify the content of dopamine, DOPAC or HVA in the striatum of euthyroid of euthyroid rats (Table 2).

EfSects of GM, on binding of [-‘H]SCH-23,390 to D,-type dopamine-receptors in the striatum of euthyvoid and hypothyroid rats

Effects of GM, on striatal uptake of [3H]dopamine into synaptosomes from euthyroid and hypothyroid rats

Neonatal hypothyroidism decreased the F’,,,,, for synaptosomal uptake of [3H]dopamine by 60%, as compared to age-matched controls, an effect which coincided with a 44% decrease in the K,-values or, in other words, with a 1.8-fold increase in the affinity for the substrate (Table 3). While administration of GM, to euthyroids did not influence the uptake parameters for [3H]dopamine, it completely prevented the hypothyroidism-provoked impairment of uptake of [3H]dopamine, both in terms of V,,,,, and K, (Table 3). Eflects of GM, on the binding of [‘Hltyramine striatum of euthyroid and hypothyroid rats

in the

Congenital-like hypothyroidism decreased by 42% the B,,, for membrane binding of [3H]tyramine,

and KD values for the binding of The &,, [3H]-SCH-23,390 to striatal membranes of hypothyroid rats were 49% and 35% smaller than those in euthyroid controls, respectively (Table 5) the latter effect corresponding to a 1.5-fold increase in the binding affinity. Neonatal administration of GM, to euthyroids did not affect binding characteristics of [3H]SCH-23,390 and it did not significantly counteract the hypothyroidism-provoked impairment of D,receptors, although a trend towards recovery was observed (Table 5). Effects of administration of GM, on basal and dopamine-stimulated activity of adenylate cyclase in the striatum of euthyroid and hypothyroid rats

Basal, steady-state activity of adenylate cyclase in terms of production of CAMP was decreased by 32% in striatal tissues of congenital-like hypothyroids, as compared to controls (Table 6). This effect was not counteracted by neonatal administration of GM, ; similarly, GM, did not affect the activity of adenylate

Table 3. Neonatal administration ofGM, ganglioside corrected the hypothyroidismprovoked loss of uptake of [‘Hldopamine into striatal synaptosomes of 32 day-old rats V nlax

Thyroid state

Treatment

Euthyroid Euthyroid

Saline GM, MM1 MM1 + GM,

Hypothyroid Hypothyroid

(pmoiimg proteinimin)

(nKG)

14.6 ? I .5 17.7 k 2.8

48.4 f I .9 48.2 f 5.9

5.8 + 0.9** 14.2 f I.74

27.0 + 2.6*’ 48.6 f 3.35

Synaptosomal suspensions were incubated with [jH]dopamine, plus unlabelled dopamine (5-320 nM). For further details see the Methods section. Values are means f SEM from 7 separate experiments, performed in triplicate on 7 (4 male, 3 female) rats. **P < 0.001, as compared with saline-treated rats. §P < 0.003, as compared with MMI-treated rats. One-way ANOVA.

GM,

and dopamine-system in hypothyroidism

1165

Table 4. Neonatal administration of GM, ganglioside restored the hypothy roidism-provoked decrease of binding of [‘Hltyramine in the striatum of 32 day-old rats Emax

Thyroid state

Treatment

(pmol/mg protein)

Euthyroid Euthyroid

(n%)

Saline GM,

3.8 f 0.2 3.9 + 0.3

7.0 f 0.5 6.5 F 0.3

Hypothyroid Hvoothvroid

MM1 MM1 + GM,

2.2 * 0.2” 3.7 + 0.36

4.3 * 0.4” 6.1 + 0.7 #

Striatal membranes were incubated with [3H]tyramine (0.5-32 nM) in the absence or presence of 10 PM p-tyramine as the displacer. For further details see the Methods section. Values are means +_SEM from 8 separate experiments, performed in triplicate on 8 (4 male, 4 female) rats. l*P i 0.001, as compared with saline euthyroids. 6P < 0.005, and # P < 0.05 as compared with MMI-treated rats. One-way ANOVA.

cyclase in euthyroid rats (Table 6). In a kinetic study, the V,,,,, for dopamine-stimulated activity of adenylate cyclase in striatal tissues of hypothyroid rats was 36% less than that in euthyroid controls, an effect that was not prevented by neonatal administration of GM, (Table 6). The K, values were almost significantly reduced in hypothyroidism, a trend that was no longer evident after treatment with ganglioside (Table 6). The GM, ganglioside did not influence the dopamine-induced production of CAMP in striatal tissues of euthyroid rats (Table 6). In vitro effects of GM, on the uptake of [‘Hjdopamine and binding of [‘Hltyramine

The addition of 0.075pg GM, to the incubation mixtures for assays of uptake of dopamine or binding of tyramine did not affect the two processes. The presence of 1 pg GM, increased by 11% and 29% Vmaxand B,,, values for the uptake and binding processes, respectively, as compared to GM,-free controls (Table 7).

DISCUSSION

The present results show that in a model of neonatal hypothyroidism, where there is an extensive pre- and postsynaptic impairment of the striatal dopaminergic system (Vaccari, 1988; Vaccari et al., 1990), the early administration of GM,-ganglioside induced a selective recovery of the hypofunctioning

striatal transport process for dopamine. Treatment with ganglioside, however, did not counteract the hypothyroidism-provoked loss of the content of metabolites of dopamine, in the number of D,-receptors and in basal- or dopamine-stimulated production of CAMP, triggered by D,-sites. Since the evaluation of the uptake of [3H]dopamine into synaptosomes is thought to be a biochemical estimate for the presence of functional dopaminergic nerve endings in the striatum (Hadjiconstantinou and Neff, 1988), it can be inferred that early administration of GM, to congenital hypothyroids can counteract the effects of either a loss of nigro-striatal innervation, or, more likely, the presence of abnormally abundant immature dopaminergic neurones at that age, as compared to euthyroid counterparts. As a matter of fact, it is reasonable to expect that early hypothyroidism may retard the ontogenetic development of synapticallylocated events, as a direct consequence of the retarded synaptogenesis in the striatum (Lu and Brown, 1977). Furthermore, since the vesicular transporter for dopamine, as putatively labelled by [3H]tyramine (Vaccari, 1986), was particularly affected by hypothyroidism, while the neuronal transport system for dopamine, as labelled by [‘Hlmazindol (Javitch, Blaustein and Snyder, 1983) showed little sensitivity to hypothyroidism (Vaccari and Gessa, 1989), it might be concluded that neonatal administration of GM, could well prevent the hypothyroidismprovoked impairment in the vesicular transport of dopamine.

Table 5. Effects of neonatal administration of GM, ganglioside on the hypothyroidism-provoked loss of D,-type dopamine-receptors in the striatum B InSl

Thvroid state

Treatment

(fmol/ma vrotein)

Euthyroid Euthyroid

Saline GM,

304.8 f 56. I 310.5 + 57.8

0.53 + 0.08 0.57 * 0.09

Hypothyroid Hypothyroid

MMI MM1 + GM,

156.4 & 23.5” 281.4 f 6O.q

0.34 * 0.04” 0.47 f o.osg

Striatal membranes of 32 day-old rats were incubated with [‘HISCH-23,390 (0.2-S nM), in the absence or presence of 5 pM cis-flupenthixol as the displacer. Values are means f SEM from 7 or 8 separate experiments, each performed with the pooled striata from I male and 1 female rat. For further details see the Methods section. **P i 0.05, as compared with saline-treated euthyroids. §P > 0.05, N.S. as compared wit\ saline- or MMI-treated rats. One-way ANOVA.

1166

A. VACCARI et al. Table 6. Effects of administration of GM, ganglioside on hypothyroidism-provoked decrease in basal- and dopamine-stimulated (D,-coupled) activity of adenylate cyclase in the striatum of 32 day-old rats CAMP produced

Thyroid state

Treatment

Dopamine-stimulated Basal steady-state (pmol/mg proteinjmZ7 ($f,

Euthyroid Euthyroid

Saline GM,

83.3 + 1.3 80.2 k 3.2

79.4 f 4.2 15.2 f 8.9

3.7 * 0.9 3.2 5 0.4

Hypothyroid Hvoothvroid

MM1 MM1 + GM,

56.3 k 1.2’ 44.8 f 3.2”

50.6 & 4.3” 49.5 * 4.6.’

1.9I 0.35 3.3 + 0.7

Samples were incubated with IOOpM [‘2P]ATP, in the absence (basal) or presence (D,-coupled) of l-100 PM dopamine (for further details see the Methods section). Values are means f SEM from 6 separate experiments performed on 6 (3 male. 3 female) rats. ‘P < 0.03; **P < 0.001; IP = 0.079, N.S., as compared with saline-treated euthyroids. One-way ANOVA.

There is evidence that systemic administration of GM, can promote recovery of dopamine function after neurotoxin-induced or mechanical lesions of the nigrostriatal system, depending on the type of procedure used to provoke brain damage (Toffano et al., 1984). Thus, large, knife-provoked hemitransection of nigro-striatal pathways would induce collateral “sprouting” and dopaminergic reinnervation to the striatum, whereas more discrete, electrolytic or neurotoxin-based lesions of nigrostriatal fibers would be less effective (see Jackson, Jenner and Marsden, 1989). A reason for such discrepancy might be a differential release of those neurotrophic or neuritepromoting agents, first of all the Nerve Growth Factor (N.G.F.), which are claimed to mediate the “regenerative” effects of GM, after having interacted with gangliosides (Nieto-Sampedro, Lewis and Cotman, 1982; Ledeen, Byrne, Roisen, Yorke and Scalfani, 1983; Cuello, Kenigsberg, Maysinger, Pioro and Garofalo, 1988). Thus, larger lesions could release more abundant factors than more discrete lesions and result in more effective promotion of dopaminergic recovery.

Table 7. In oifro effects of GM, ganglioside on uptake (‘Hldopamine and binding of (jH]tyramine in the striatum

of

Uptake of [3H]dopamine V max

GM, added 0 0.075 /lg I.Ovg

(pmol/mg proteiwmin)

(nKfi)

16.5 f 0.8 69.5 ? 1.8 16.4 + 0.8 69.3 + I.5 18.4i l.l* 74.1 + 1.6” Binding of [‘Hltyramine B mm

0 0.075 fig 1.0 tig

(pmolimg protein)

(nKh)

3.98 + 0.2 4.61 F 0.3 5.16 f 0.25

9.0 * 0.9 7.4 It 0.7 7.4 * 0.5

GM, was added to synaptosomal or membrane suspensions obtained from 30 day-old, euthyroid rats, at the start of the 5 min of preincubation at 37’C (uptake assay) or of the IOmin of incubation at 37’C (binding assay). Uptake and binding reactions were then run according to the standard procedures. Values are means + SEM from 3 separate experiments performed in triplicate, with 3 male rats. *P < 0.025; gP < 0.002; **P c 0.001, as compared to respective, GM,-free controls. Paired. two-tailed I-test.

There is no information available as to whether hypothyroidism may affect the content in brain or lesion-induced release of neurotrophic or neuritepromoting factors. If it is true that the presence of those factors is a requisite for the “regenerative” activity of GM,, it must be hypothesized that, in the hypothyroid CNS, there is enough of those endogenous agents to warrant the activity of exogenous GM,. Otherwise it must be concluded that GM, can directly affect dopaminergic innervation, even in the absence of cofactors. The present finding that administration of GM, increased the uptake capacity of the striatum for [‘Hldopamine, an index for a significant preservation or recovery in dopamine-projections to the striatum and did not unequivocally raise back to normal the number of D,-receptors or the activity of D,-coupled activity of adenylate cyclase is of interest. D,-Type dopamine-receptors are primarily located on neurones intrinsic to the caudoputamen, including striatonigral projection neurones, rather than on terminals of the dopamine-containing nigrostriatal neurones innervating the striatum (Leff, Adams, Hyttel and Creese, 1981; Filloux, Wamsley and Dawson, 1987). This might indicate that the “regenerative” effect of GM,-ganglioside was primarily directed to the dopamine-innervation towards striatal neurones, while postsynaptically-located cell bodies were less sensitive to GM,, as suggested by the slightly less than significant trend to recovery of changes in D, receptors. Treatment with GM, could only induce recovery of the transport function for dopamine in hypothyroids, while not improving the levels of metabolites of dopamine. In this respect it is of interest to note that profound changes in the in vice or in vitro metabolism of dopamine cannot generally be predicted even after severe manipulations of the reuptake function for dopamine (Westerink, Damsma, De Vries and Konig, 1987). To the purpose of explaining the presynaptic selectivity of GM,, it is important to consider the influence of gangliosides on the neuronal Na/K- and Mg- pumps, the activities of which are, of course, fundamental for the dopaminereuptake and storage processes. Firstly, there is clear

GM,

and dopamine-system in hypothyroidism

evidence that thyroxine regulates both the activity and concentration of Na/K-ATPase in the synaptic plasma membrane in the developing rat brain (Lindholm, 1984), where experimental hypothyroidism reduces the activity of ATPase (Valcana and Timiras, 1969). Secondly, mixed treatments with ganglioside or GM, have been shown to prevent lesion- or disease-provoked impairments of the activity of Na/K-ATPase in tissues of the central or peripheral nervous system (Leon, Facci, Toffano, Sonnino and Tettamanti, 1981; Mahadik, Hawver, Li and Karpiak, 1987; Calcutt, Tomlisson and Willars, 1988). Furthermore, the sialic acid units in gangliosides, located on the external surface of the neuronal membrane, and contributing to the charge- and chemical conformations (Bretscher, 1973) are involved in the high affinity uptake process of dopamine by synaptosomes from the brain of the rat, as suggested by the inhibition of the uptake of DA after the neuraminidase-provoked loss of synaptosomal sialic acids (Zaleska and Erecinska, 1987). On the other hand, the rate of synthesis and turnover of major gangliosides in brain undergo neonatal development (Suzuki, 1967; Quarles and Brady, 1971) the content being markedly decreased by neonatallyprovoked hypothyroidism (Farina de Raveglia, Gomez and Ghittoni, 1972; Vitiello, Clos, Di Benedetta and Gombos, 1989). Last but not least, N.G.F., a putative cofactor for the “regenerative” activity of GM,, plays by itself a crucial role in the young target neurones, where it controls the performance of the Na/K-pump. The pump is inhibited or operates in the absence or presence of N.G.F., respectively (see Varon, Manthorpe and Williams, 1983/1984), though there is no direct evidence that N.G.F. can affect dopamine neurones. Based on the previous consideration, it might be hypothesized that the GM,-induced recovery of dopamine transport, rather than truly reflecting a “sprouting” effect, i.e. an increased number of dopaminergic nerve endings in the hypothyroid striatum, may also result from the chronic incorporation and storage of exogenous GM, into ganglioside-deficient, hypothyroid neuronal membranes. This would enhance the proton translocating ATPase activities in the immature or scantier nerve endings available and thus, stimulate pump-related dopamine-transport processes (Bianchi, Janigro, Milan, Giudici and Gorio, 1986). If this is true, it cannot be excluded that GM, may to a certain extent directly affect the neuronal membrane and thereby stimulate the transport of dopamine. As a matter of fact, exogenous GM, enhances the uptake of [‘Hldopamine (increase in I’,,,,,, decrease in Km values) in dissociated mesencephalic cultures of the foetal mouse (Leon, Dal Toso, Presti, Benvegnu, Facci, Kirschner, Tettamanti and Toffano, 1988). The incubation of striatal synaptosomes in the absence or presence of 0.075 pg GM,, a concentration estimated to correspond roughly to

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the content of ganglioside in each aliquot of hypothyroid tissue, used for uptake assays after an individual injection of 30mg/kg GM, to 30 day-old pups, did not significantly affect the uptake parameters for [3H]dopamine. The same was true for the binding of [3H]tyramine in the present experiments. Incubating the samples with 1 pg GM,, a very large concentration, increased by 11% the I’,,,,, for dopamineuptake and by 29% the B,,,,. for the binding of tyramine (Table 7). Similarly, Cumar, Maggio and Caputto (1980) showed a 23% increase in the uptake of [3H]dopamine, after incubating striatal synaptosomes with 260 PM GM,. Of course, it must be taken into account that after peripheral administration, only a small fraction of the injected ganglioside enters the brain (Ghidoni, Trinchera, Venerando, Fiorilli and Tettamanti, 1986). Most probably, the recovery of dopamine transport processes in the hypothyroid striatum results from both GM,-promoted maturation of otherwise retarded dopaminergic terminals where reuptake of dopamine takes place and selective induction of the proton-translocating ATPase, that energizes the transport system for dopamine. In more general terms, the repeated, neonatal administration of GM, ganglioside did not improve the grossly evident somatic aberrations of hypothyroid rats (see the Results section), however, rendered them clearly more aggressive and reactive to handling, as compared with saline-treated hypothyroids. Serum levels of T, , though consistently higher than in untreated, hypothyroid counterparts (Table 1), were still 53% lower than in euthyroids. This finding might reflect a modest recovery from the dysthyroid state. The mechanism by which GM, could increase levels of T, is unknown and the possibility that GM, interferes with the biodistribution of methimazole and its thyreostatic activity, deserves further investigation. Furthermore, more attention must be paid to find any possible, functional implication of the major effect displayed here by treatment with GM, ganglioside on the recovery of the otherwise deficient transport process for dopamine. Acknowledgements-This work was supported by grants from the Italian Ministry of Education to A.V. (National Research Projects 40% 1987 and 1988). The GM,-ganglioside was kindly supplied by Fidia (Abano Terme, Italy).

REFERENCES

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Dopaminergic dysfunction in neonatal hypothyroidism: differential effects of GM1 ganglioside.

The effects of daily treatment with GM1 ganglioside (30 mg/kg s.c.) from birth to day 30, on striatal pre- and postsynaptic markers of the dopaminergi...
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