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Implication of the endocannabinoid system in the locomotor hyperactivity associated with congenital hypothyroidism

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Implication of the Endocannabinoid System in the

Locomotor Hyperactivity Associated with Congenital

Hypothyroidism

Teresa Asu´ a, Ainhoa Bilbao, Miguel Angel Gorriti, Jose Antonio Lopez-Moreno, Maria del Mar A´ lvarez, Miguel Navarro, Fernando Rodrı´guez de Fonseca, Ana Perez-Castillo, and Angel Santos

Departamento de Bioquı´mica y Biologı´a Molecular (T.A., M.d.M.A., A.S.), Facultad de Medicina, Departamento de Psicobiologı´a (M.A.G., J.A.L.-M., M.N., F.R.d.F.), Facultad de Psicologı´a, Universidad Complutense de Madrid, 28040 Madrid, Spain; Instituto de Investigaciones Biome´dicas (A.P.-C.), Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid, 28049 Madrid, Spain; and Fundacio´n Instituto Mediterra´neo para el Avance de la Biomedicina y la Investigacio´n (A.B., F.R.d.F.), Hospital Carlos Haya, 29010 Ma´laga, Spain

Alterations in motor functions are well-characterized fea-tures observed in humans and experimental animals sub-jected to thyroid hormone dysfunctions during development. Here we show that congenitally hypothyroid rats display hy-peractivity in the adult life. This phenotype was associated with a decreased content of cannabinoid receptor type 1 (CB1) mRNA in the striatum and a reduction in the number of bind-ing sites in both striatum and projection areas. These findbind-ings suggest that hyperactivity may be the consequence of a thy-roid hormone deficiency-induced removal of the endocan-nabinoid tone, normally acting as a brake for hyperactivity at

the basal ganglia. In agreement with the decrease in CB1 re-ceptor gene expression, a lower cannabinoid response, mea-sured by biochemical, genetic and behavioral parameters, was observed in the hypothyroid animals. Finally, both CB1 receptor gene expression and the biochemical and behavioral dysfunctions found in the hypothyroid animals were im-proved after a thyroid hormone replacement treatment. Thus, the present study suggests that impairment in the en-docannabinoid system can underlay the hyperactive pheno-type associated with hypothyroidism. (Endocrinology 149: 2657–2666, 2008)

T

HYROID HORMONES (T3 and T4) play an important

role in the development and metabolism of many mammalian tissues. Most of the actions of these hormones are mediated by the binding of T3to specific nuclear

recep-tors that are ligand-dependent transcription facrecep-tors, which bind, in the target genes, to specific DNA sequences called thyroid hormone response elements, and regulate transcrip-tion initiatranscrip-tion (1). Hence, the primary actranscrip-tion of thyroid hor-mone is to control the expression of a limited number of genes in target cells, which ultimately will cause the phys-iological effects attributed to this hormone. Thyroid hormone receptors (TRs) are members of a large family of nuclear proteins that includes the receptors for steroids, retinoids, vitamin D3, peroxisomal proliferators, and receptors with no

recognized ligand known as orphan receptors (2).

The brain is an important target for thyroid hormone ac-tion, particularly during late fetal and early postnatal peri-ods, as shown by numerous clinical and experimental data (3). In the rat brain, the expression of thyroid hormone re-ceptors increases rapidly after birth, reaching the highest value by d 6 of postnatal life (4). Consistent with these data, thyroid hormone absence during the perinatal period leads

to a profound brain damage unless replacement therapy with thyroid hormone is started very soon after birth. The con-sequences of the lack of thyroid hormone include, among others, a reduction in dendritic arborization, poor connec-tivity among neurons, impaired myelin deposition, and al-tered cell migration and synaptogenesis (3, 5).

The endocannabinoid system is a neuromodulatory sys-tem, which plays an important role in cognitive, affective, and motor functions in the brain (6). The basic components of this system are the cannabinoid receptor type 1 (CB1) and

its endogenous ligands, anandamide and 2-arachidonoylg-lycerol, which act as retrograde signaling molecules to reg-ulate synaptic plasticity (reviewed in Ref. 7). The CB1

recep-tor is a highly abundant G protein-coupled receprecep-tor, which regulates, through multiple subtypes of Gi/o proteins, the activity of adenylyl cyclase, voltage-activated calcium chan-nels, potassium chanchan-nels, and mitogen-activated kinases (8). In addition to CB1receptors, another closely related

canna-binoid receptor, the cannacanna-binoid receptor type 2 (9), has been cloned. The cannabinoid receptor type 2 receptor is mainly expressed in immune cells and consequently mediates the immunomodulating activities of the cannabinoids. The ex-istence of additional, not-yet-characterized cannabinoid re-ceptors in the brain has been postulated to fully understand the activity of these substances (10). In addition to these basic constituents of the endocannabinoid system, other important components include enzymes and transporters implicated in the metabolism of anandamide and 2-arachidonoylglycerol (6).

The motor system is a target of cannabinoids as shown by First Published Online January 24, 2008

Abbreviations: ADHD, Attention deficit with hyperactivity disorder; BW, body weight; CB1receptor, cannabinoid receptor type 1; RTH,

resistance to thyroid hormone; TR, thyroid hormone receptor. Endocrinologyis published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

doi: 10.1210/en.2007-1586

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the profound impact that cannabinoid agonists have on mo-tor behavior and the high level of expression of CB1receptor

in the basal ganglia and cerebellum (11). In the basal ganglia, the CB1 receptor gene is expressed in the medium spiny

neurons of the striatum and the protein accumulates in the presynaptic terminals of these neurons in the output nuclei, globus pallidus, and reticular part of the substantia nigra (12, 13). In the cerebellum, both the mRNA and protein are abun-dant (12, 13). It has been shown that the dopaminergic-mediated hyperactivity is modulated by the endogenous cannabinoid system (14 –16). Removal of endogenous can-nabinoid tone by selective blockade of CB1receptor

poten-tiates dopaminergic-mediated hyperactivity. In addition, po-tentiation of endocannabinoid transmission with the anandamide uptake blocker AM404 reduces hyperlocomo-tion mediated by dopamine D2 receptors (14).

Alterations in motor activities are also well-characterized features of thyroid hormone dysfunctions during develop-ment, in both humans and experimental animals (17). We focused our attention in one of these features, motor hyper-activity. In humans, between 50 and 70% of the patients with resistance to thyroid hormone (RTH), a syndrome caused by the dominant inheritance of a mutated form of the TR␤ isoform (18), fulfill during childhood the diagnosis criteria for attention deficit with hyperactivity disorder (ADHD) (19, 20). In contrast, in the general population, only 2–5% of school-age children meet these criteria. In experimental mod-els, transient perinatal hypothyroidism (from the end of pregnancy to weaning) causes attention-deficit and hyper-active neurobiological symptoms in adult animals (21–24). In congenital hypothyroidism (hypothyroidism induced in the perinatal life and maintained chronically), conflicting evi-dences regarding motor hyperactivity have been published (25, 26). In the case of perinatal hyperthyroidism, both tran-sient and chronic models have been associated with hyper-activity in the adult life (26, 27). However, very little is known relative to the possible genes regulated by thyroid hormone, which could be implicated in the appearance of the hyper-active phenotype, although it has been suggested that alter-ations in the dopaminergic and noradrenergic systems can underlay the effects of thyroid hormone (25, 28, 29).

All these data have prompted us to hypothesize that the hyperactivity associated with hypothyroidism during devel-opment may be derived from alterations of the endocan-nabinoid system, which exerts a negative feedback that limits dopaminergic hyperactivity. To test this premise, we ana-lyzed the locomotor activity, the expression of the CB1

re-ceptor gene, and the response to cannabinoids in congeni-tally hypothyroid rats. Our results clearly show that chronic congenitally hypothyroid animals are hyperactive in the adult life and that this phenotype correlates with a decreased expression of the CB1receptor gene in the basal ganglia, as

shown by a lower content of transcripts in the striatum and a lower ligand binding in the striatum and midbrain. Con-sequently, a lower biochemical, genetic, and behavioral re-sponse to cannabinomimetic drugs is observed in these an-imals. Finally, the biochemical and behavioral parameters altered in the hypothyroid rats can be clearly improved by the administration of therapeutic doses of thyroid hormones.

Materials and Methods

Materials

HU210 [(⫺)-11-hydroxy-⌬8-tetrahydrocannabinol-dimethylheptyl],

Win55212-2 [R-(⫹ )-(2,3-dihydro-5-methyl)-3-[(morpholinyl)meth-yl[pyrrolo[1,2,3-de]-1,4-benzoxazinyl] (1-naphthalenyl)methanone me-sylate], and AM404 [N-(4-hydroxyphenyl] arachidonylamide) were purchased from Tocris (Cookson, UK). SR141716A [N -(piperidin-1-yl)- 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4methyl-1H-pyrazole-3-carboxamide hydrochloride] was a gift from Sanofi-Aventis (Montpel-lier, France). [␣-32P]dCTP (3000 Ci/mmol) and [3H]SR141716A (53 Ci/

mmol) were purchased from Amersham Corp. (Buckinghamshire, UK) and [35S]GTPS (1000 Ci/mmol) from NEN Life Science Products

(Boston, MA). GTP␥S, T4, T3, and 2-mercapto-1-methylimidazole were

purchased from Sigma Chemical Co. (St. Louis, MO). All other chem-icals were reagent grade or molecular biology grade.

Animals

All procedures were carried out in accordance with the European Communities Council, directive 86/609/EEC. Special care was taken to minimize animal suffering. Female Wistar rats in proestrus were mated during the night, and d 0 of fetal age was considered the following morning if spermatozoa were found in a vaginal frotis. To induce fetal and neonatal hypothyroidism, dams were given 0.02% 2-mercapto-1-methylimidazole in the drinking water starting from the 12th day after conception and throughout the whole experimental period including weaning. This protocol ensures that the animals are hypothyroid during all the neonatal age, as shown by noticeable physiological landmarks of hypothyroidism, such as a decreased growth rate, which is clearly ev-ident from neonatal d 15, and low levels of T4and T3(30, 31). N70 rats

are animals between 10 and 12 wk of age. For thyroid hormone treat-ment, hypothyroid animals were ip injected once daily according to the following protocols: 1) a high dose of T3[20␮g per 100 g body weight

(BW)] to analyze the kinetics of the response to this hormone and 2) a more physiological combination of T4(0.9␮g/100 g BW) and T3(0.2␮g

per 100 g BW). The corresponding hypothyroid controls received an equivalent volume of physiological saline. Two groups of animals re-ceived ip injections of quinpirole (0.5 mg/kg) or HU210. At the indicated times, the animals were killed by decapitation, and the brain was quickly removed, dissected, frozen, and kept at⫺80 C until used.

Behavioral tests

The motor activity of the animals in an open-field device (100⫻100 cm) was recorded with a digital video tracking system (SMART; Panlab, Barcelona, Spain), and the time of immobility and horizontal activity were analyzed at intervals of 5 min. Twenty-four hours before any drug treatment, including normalization of thyroid hormone levels, all the animals remained 10 min in the open field to facilitate context habituation.

Real-time PCR and Northern analysis

Total RNA was purified according to the method of Chomczynski and Sacchi (32), and samples (2␮g) from the indicated rat brains areas were used for the synthesis of cDNA with the RETRoscript kit (Ambion, Austin, Tx). Real-time PCR was performed in an ABI Prism equipment using the SYBR Green PCR master mix (Applied Biosystems, War-rington, UK) and 300 nmconcentrations of specific primers. The primers used for the determination of the concentration of the different tran-scripts were as follows: for CB1receptor, two different sets of primers

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Primers were CCA GTA AGT GCG GGT CAT AAG C and CCT CAC TAA ACC ATC CAA TCG G (18S rRNA) and GGC AAG TCC ATC TAC GGA GAG A and GCC AGG ACC TGT ATG CTT CAG (cyclophilin A) that synthesize a fragment of 92 and 65 bp, respectively (35, 36). In all runs, melting curves were performed to make sure that only the cor-responding DNA fragment was amplified. Each RNA sample was re-verse transcribed at least one time with oligo-dT and one time with pd(N)6 random hexamer and each specific sequence measured at least twice in triplicate. In adult control striatum, random primer cDNA (dilution 1:10) gave cycle threshold values of around 20, 19, and 23 for CB1receptor, cyclophilin A, and c-fostranscripts, respectively. In the case

of 18S rRNA, a dilution of 1:1000 gave cycle threshold values between 20 and21. Northern analyses were performed as previously described (31) using a labeled rat CB1receptor probe expanding from nt 89 to 3876

(13).

[3H]SR141716A-binding assay

Membranes from different parts of the brain were prepared as pre-viously described (37), resuspended in buffer B [HEPES 20 mm(pH 7.4) and MgCl21 mm] and stored at⫺80 C until use. Radioligand binding

was initiated by the addition of 25␮g of protein to tubes containing 0.5 ml of buffer C (buffer B plus fatty acid free BSA 0.5%) and [3H]SR141716A (2.5 nm) and incubated at 30 C during 90 min. The

reaction was stopped with ice-chilled buffer C and rapidly filtered through GF/C filters (Whatman, Brentford, Middlesex, UK) (37). Non-specific binding was assessed in the presence of 5 ⫻ 10⫺7 m cold

SR141716A.

[35S]GTPS-membrane binding assay

Frozen membranes were thawed and 7␮g of proteins were incubated at 30 C for 20 min in buffer D [50 mmTris-HCl, 10 mmMgCl2, 1 mm

EDTA, 100 mmNaCl, 60␮mGDP, 0.2 mg/ml BSA and 50␮m phenyl-methylsulfonyl fluoride (pH 7.4)]. CB1receptor agonist was added

to-gether with [35S]GTPS (0.05 nm) and further incubated at 30 C during

1 h. Reaction was stopped with ice-cold buffer E [50 mmTris-HCl, 10 mm

MgCl2, 1 mmEDTA, 100 mmNaCl, 0.5% BSA (pH 7.4)] and rapidly

filtered through Whatman GF/C filters. Nonspecific binding was de-termined in the absence of CB1receptor agonist and the presence of 10

␮munlabeled GTP␥S.

Western blot analysis

Tissues were homogenized in radioimmunoprecipitation assay buffer, and equal quantities of total protein were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes, and Western analysis was performed with primary anti CB1receptor (Alexis

Bio-chemicals, Lausen, Switzerland) and anticyclophilin A polyclonal anti-bodies (Calbiochem, San Diego, CA).

Statistical analysis

Data were analyzed by ANOVA, followed by Newman-Keul’s test as post hocor Studentttest. Statistical significance was considered when P⬍0.05.

Results

Motor activity

We first determined motor activity in open field in control and hypothyroid N70 rats. As shown in Fig. 1A, the loco-motor score in control rats, as expected, decreases with time. In contrast, in the hypothyroid group, barely any decrease in locomotor activity with time was observed, indicating a lack of habituation and a hyperactivity-like pattern. Cumulative scores showed a 2-fold increase in the number of crossings in the hypothyroid group, compared with controls. The do-paminergic D2 agonist, quinpirole, further increased hori-zontal locomotion in both groups of animals. In the hypo-thyroid group, the number of crossings increases 5-fold

between 5 and 120 min, in comparison with controls, in which only a 2-fold increase was observed (Fig. 1B). These results suggest that the activity of the endocannabinoid sys-tem could be decreased in the hypothyroid animals because it is well established that this system reduces dopaminergic-induced hyperactivity (14, 15).

Effect of hypothyroidism on the expression of CB1 receptor gene

We first determined whether hypothyroidism has any effect on the expression of CB1receptor gene in the brain

of developing animals. For that purpose, the content of CB1receptor transcripts and [3H]SR141716A binding were

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control animals the highest amount of CB1receptor

tran-scripts were found in the striatum and cerebellum, fol-lowed by the cerebral cortex and hippocampus. The lowest concentration was observed in the thalamus, midbrain, and brain stem. Relative to the effect of hypothyroidism, a significant decrease on CB1receptor mRNA levels (49%

of controls) in the striatum and a slight increase in the hippocampus of hypothyroid animals were observed.

Next, we determined the specific binding of [3H]SR141716A in membranes prepared from the same

brain areas of control and hypothyroid N15 rats. As shown in Fig. 2B, differences between control and hypothyroid animals were observed in the cerebellum and midbrain but not the other areas analyzed. The lack of differences in binding observed in the hippocampus is in contrast with the moderate increase detected in the amount of CB1

re-ceptor transcripts. In the cerebellum and midbrain of hy-pothyroid rats, [3H]SR141716A binding was only 80 and

74% of the control values. This effect of hypothyroidism is

apparently in contrast with its lack of effect on the content of CB1receptor transcripts in the same areas. However, in

the midbrain, the decrease in [3H]SR141716A binding in

hypothyroid animals can be explained by the decrease in the number of CB1receptor transcripts observed in the

striatum of the same animals. On the other hand, the described discrepancies between binding and the levels of transcripts observed in the cerebellum and hippocampus of hypothyroid neonates suggest a complex action of thy-roid hormone on the expression of this gene.

In view of the previous results showing a decrease in CB1 receptor mRNA in the striatum and a decrease in

[3H]SR141716 binding in the midbrain and cerebellum of

hypothyroid 15-d-old animals, we next analyzed the de-velopmental profile of CB1 receptor gene expression in

these areas. In the striatum of control animals, the amount of CB1 receptor transcripts increased after birth (8-fold

from N5 to N15) (Fig. 3A). This increase was markedly reduced in hypothyroid animals, and therefore, significant differences were observed between control and hypothy-roid rats from N15 to adult life (Fig. 3A). Regarding [3H]SR141716A binding in the striatum of control animals, a 2.6-fold increase was observed between N15 and N70 in contrast with the earlier increase detected in transcripts levels (Fig. 3A). These results suggest a higher efficiency of translation or a longer half-life of the receptors in the adult animals, compared with N15. In agreement with the observed decrease in the level of transcripts, a clear re-duction in [3H]SR141716A binding was observed in striatal

(Fig. 3A) and midbrain membranes (Fig. 3B) isolated from both N30 and N70 hypothyroid animals. In contrast, the lack of effect of thyroid hormone on transcript number observed in N15 midbrain was also observed in N70 (Fig. 3B). In the cerebellum, no effect of thyroid hormone on CB1

receptor mRNA content was observed (Fig. 3C). In con-trast, the binding of [3H]SR141716A to cerebellar

mem-branes was decreased in N30, as previously observed for N15, although no differences were observed at N70, in-dicating that the effect of hypothyroidism was transient and occurred at a posttranscriptional level (Fig. 3C).

We next tested the response of hypothyroid animals to the acute injection of T3(20␮g per 100 g BW). As can be

observed in Fig. 4A, the administration of T3rapidly

in-creases the content of CB1receptor mRNA in the striatum

of N15 and N70 hypothyroid rats. In the neonates, 12 h after the administration of T3, the content of transcripts is

similar to the one observed in the control animals, and after 72 h these values were twice that of the control animals. Similar results were obtained with N70 animals, indicating a rapid response of this gene to thyroid hor-mone administration. This response to T3administration

correlates with the changes observed in binding in the striatum and mid brain (Fig. 4B) of N30 hypothyroid rats after the administration of the hormone.

To further substantiate these results, we next analyzed the mRNA and protein levels of CB1receptor in the striatum by

Northern and Western blot analysis, respectively, in N70 animals. As shown in Fig. 5, a clear decrease in the amount of mRNA was observed in the striatum of hypothyroid rats. In contrast, in the midbrain the levels of CB1receptor mRNA FIG. 2. Effect of hypothyroidism on CB1 mRNA content and

[3H]SR141716A binding. A, CB

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were lower than in the striatum, and no differences between control and hypothyroid animals were found (Fig. 5A). Also, a lower amount of CB1 receptor protein was detected in

hypothyroid striatum (Fig. 5C). All these results are in agree-ment with the RT-PCR and ligand binding results. No

changes in ligand affinity were detected when control and hypothyroid membranes were compared (dissociation con-stant 0.9⫹0.2 and 0.91⫹0.15 nmin control and hypothyroid animals, respectively, data not shown).

Response to cannabinoid agonists

The results presented so far suggest that the striatum of hypothyroid animals could be less sensitive to the action of cannabinoids than control animals. We tested this hypothesis by determining the induction by WIN55212–2 of GTP␥ bind-ing to G proteins in striatal membranes isolated from N70 control and hypothyroid rats. We also analyzed the response to HU210 of the immediate response gene c-fos in both groups of animals. As shown in Fig. 6A, a lower GTP binding response to WIN55212–2 (3⫻10⫺8and 10⫺5m) was observed

in hypothyroid membranes (69% of the control values). This decrease in GTP binding was observed with all the concen-tration tested (Fig. 6B), and a similar EC50(0.9⫹0.12 10⫺7m FIG. 3. Effect of hypothyroidism on the developmental profile of CB1

receptor gene expression in the striatum, midbrain, and cerebellum. Total RNA and membranes were isolated from striatum (A), midbrain (B), and cerebellum (C) of control (C) and hypothyroid (Tx) rats at the indicated ages and the amount of CB1 receptor transcripts and [3H]SR141716A binding determined. The relative abundance of CB

1 transcripts was normalized to the 18S rRNA signal and is expressed relative to the value in N15 controls. Values are the average of trip-licate determinations in at least five different samples, and thebars represent theSEs. *,P0.05; **,P0.01; ***,P0.001vs.the corresponding control.

FIG. 4. T3induces the expression of the CB1receptor gene. Hypo-thyroid rats (N15, N30, and N70) were injected with T3(20␮g per 100 g BW) and killed at the indicated times. Total RNA from the striatum (N15 and N70; A) and membranes from the striatum and midbrain (N30; B) were isolated and CB1receptor mRNA content and [3H]SR141716A binding determined. The relative abundance of CB

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and 0.92⫹0.14 10⫺7m) was obtained for both control and

hypothyroid membranes. These results are clearly in agree-ment with the lower number of CB1receptors observed in the

hypothyroid animals.

As commented above, we also analyzed the induction of the early gene c-fos by the cannabinoid receptor agonist HU210. The time course of c-fosgene in response to HU210 in the striatum and cerebellum of N70 control and hypo-thyroid animals is shown in Fig. 7. In the striatum of control animals (Fig. 7A), a rapid a transient increase (3-fold) was observed 30 min after administration of the drug, which was completely absent in hypothyroid rats. On the contrary, in the cerebellum, in which no differences in CB1

receptor content were observed between N70 control and hypothyroid animals, the same response was observed in both groups after the injection of the cannabinoid.

Based on the biochemical results described above, it could be anticipated that the hypothyroid animals are less responsive to cannabinoid treatment. To test this hypoth-esis, we studied the inhibition of locomotor activity in response to HU210 in control and hypothyroid animals. As shown in Fig. 8, HU210 clearly decreased locomotor activity in the control group but not the hypothyroid group.

Effect of thyroid hormone treatment

As shown before in the kinetic studies, T3injection increased

CB1receptor gene expression in the hypothyroid animals in a

relatively short time, suggesting that thyroid hormone could also normalize the biochemical and behavioral parameters an-alyzed in this study. To verify this premise, N70 hypothyroid rats were injected with physiological doses of thyroid hormones and, after 5 d of treatment, biochemical and behavioral param-eters were analyzed. This treatment, as expected, normalized the CB1receptor mRNA in the striatum of hypothyroid animals

(Fig. 9A). In accordance with the normalization of CB1receptor

mRNA content, the response of the c-fosgene to cannabinoid administration was also normalized in the striatum of hypo-thyroid animals treated with hypo-thyroid hormones, as shown by the transient 3-fold increase observed in c-fostranscript content (Fig. 9B). Finally, horizontal locomotor activity was clearly de-creased by this treatment (Fig. 9C), and the behavioral response to cannabinoid agonist was restored in treated animals as shown by the appearance of typical motor inactivity in response to HU210 (Fig. 9D).

FIG. 5. Hypothyroidism decreases CB1receptor mRNA and protein con-tent. A, Total RNA was extracted from striatum and midbrain of control (C) and hypothyroid (Tx) N70 rats and the content of CB1 receptor mRNA was measured by Northern analysis, as indicated inMaterials and Methods. B, The same blot stained with methylene blue for RNA loading control. C, Membranes were isolated from the striatum of control (C) and hypothyroid (Tx) N70 rats, and the amount of CB1receptor protein was determined by Western blot analysis, as indicated in Ma-terials and Methods. D, Same blot probed with a cyclophilin A antibody.

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Discussion

In this work we analyzed the role of the endocannabinoid system in the hyperactive phenotype observed in congeni-tally hypothyroid animals. Our results clearly show that the postnatal increase in CB1 receptor gene expression in the

basal ganglia circuitry of control rats is thyroid hormone dependent. Thus, lower levels of CB1receptors and a lower

biochemical and behavioral response to cannabinomimetic drugs were observed in adult congenitally hypothyroid an-imals. These changes correlate with higher spontaneous and dopaminergic-induced locomotor activity. Because it is gen-erally accepted that the endocannabinoid system in the basal ganglia circuitry acts as a brake in motor activity initiation (11, 15), these results suggest that the CB1receptor gene could

be an important mediator in this phenotypic manifestation of hypothyroidism, as suggested in a former study in juvenile spontaneous hypertensive rats, a putative model of attention deficit hyperactivity disorder (14).

In humans, the clearest correlation between dysthyroid-ism and hyperactivity comes from studies with RTH pa-tients, which inherit a mutant form of the TR␤gene (18, 39, 40). The mutated protein has normal DNA binding activity but lower T3 binding and transactivation capabilities and

antagonizes the function of normal receptors in a dominant negative manner (41). RTH patients have elevated levels of thyroid hormones and usually they are in an euthyroid or mildly hypothyroid metabolic state, yet also symptoms of hyperthyroidism can be observed in specific organs, prob-ably as a consequence of the different levels of expression of TR isoforms in the different tissues (42). Thus, it is unclear whether the hyperactivity of these individuals is the conse-quence of hypo- or hyperthyroidism. In this regard, different observations (19, 43– 46) indicate that the administration of supraphysiological doses of thyroid hormone, and particu-larly the use of high affinity ligands of the TR␤isoform, such

as triiodothyroacetic acid (47), could be beneficial for reduc-ing hyperactivity in these individuals (44, 45), suggestreduc-ing that the brain of these patients is in a hypothyroid state. Consisting with these results, a long-term prospective study reported a high rate of ADHD in children from areas of moderate iodine deficiency (48). These results are in agree-ment with the biochemical and behavior data presented here, showing that congenitally hypothyroid animals are hyper-active and that they significantly improved after thyroid hormone administration.

Also, in experimental animals, transient perinatal hypo-thyroidism is associated to motor hyperactivity and other ADHD symptoms in the adult life (21–24). However, in con-genitally hypothyroid rats, conflicting results have been pub-lished. In line with the data presented here, Overstreetet al.

(25) have shown increased locomotor activity in iodine-de-ficient congenitally hypothyroid rats. In contrast, other au-thors did not find any increase in motor activity (26). In the hyt/hyt mouse model of hypothyroidism, no changes in motor activity have been reported (49). The causes under-lying this apparent contradiction are unclear. Although the hyt/hyt model is not entirely comparable with the one we used here, the experimental model used by Sala-Rocaet al.

(26) is essentially the same. One possible explanation is that the degree of hypothyroidism in the animals used in the study of Sala-Rocaet al.could be very low or almost negli-gible during the critical developmental period because, as they point out, their animals grew normally during the first 5 wk of postnatal life. In this regard it is known that hyper-activity is a phenotypic manifestation of perinatal hypothy-roidism, but it is not observed when adult rats are made hypothyroid (data not shown). Interestingly, and in agree-ment with the data obtained from the study of human RTH, when a mutated human TR␤(TR␤PV) is expressed in mice by transgenic or knockin techniques, male animals become clearly hyperactive (50, 51).

Additionally, our data show that both hyperactivity and the abnormal response of the endocannabinoid system found in hypothyroid animals are clearly improved by physiolog-FIG. 7. Hypothyroidism decreases HU210 induction of c-fosgene in

the striatum but not the cerebellum. Total RNA was extracted from striatum (A) and cerebellum (B) of control (C) and hypothyroid (Tx) N70 rats injected with vehicle or HU210 (50␮g/kg BW), killed at the indicated times, and the amount of c-fosmRNA measured by RT-PCR. The relative abundance of c-fostranscripts was normalized to the cyclophylin A signal and expressed relative to the values at time 0. Values are the average of at least four different samples, and thebars represent theSEs. *,P0.05; **,P0.01vs.the value at time 0.

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ical doses of thyroid hormones, suggesting that the brain response to these hormones, in this particular system, is maintained in adult congenitally hypothyroid rats. The rea-sons that in the transient model of perinatal hypothyroidism (21–24), the hyperactive phenotype remains despite thyroid hormone normalization are unclear. However, there are many reports suggesting that perinatal manipulation of the endocannabinoid system with drugs leads to permanent changes in its functionality, resulting, among others, in hy-peractivity (52), altered emotional responses (53), and mem-ory deficits (54).

The CB1receptor is broadly expressed in the brain;

how-ever, we show here that it is mainly in the basal ganglia in which its expression is targeted by thyroid hormones. In contrast, in other areas of the brain, known to contain active thyroid hormone receptors, no effect or even an opposite effect of thyroid hormone on the expression of the CB1

re-ceptor gene was observed (Fig. 2). These apparently

contra-dictory results are in agreement with many reports showing that the action of thyroid hormones on gene expression in the developing brain is especially dependent on the age and the brain area analyzed (38, 55). The molecular bases for this variability in thyroid hormone action are not clear, although it is well established that, in addition to the receptors, many other different proteins are required for gene regulation by thyroid hormones (56).

Our results are in agreement with those reported by other authors showing an inverse relationship between the activity of the endocannabinoid system and locomotor activity. The administration of cannabinoid agonists and inhibitors of anandamide and 2-arachidonoylglycerol uptake reduce hy-peractivity in spontaneously hypertensive rats (14, 57) and hyperactivity induced by psychostimulants and D2 agonists (58). On the other hand, CB1receptor antagonists favor

do-pamine receptor-mediated induction of motor activity (15) and stereotyped behaviors (59). Impulsive spontaneously FIG. 9. Thyroid hormone

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hypertensive rats have lower levels of CB1receptors in the

prefrontal cortex (57). Also, in reserpine-treated rats, an an-imal model of Parkinson’s disease, an increase in 2-AG has been described in the globus pallidus, which has been related to the suppression of locomotion in these animals, and CB1

receptor antagonists are required for a better normalization of motor activity (60). These results may apparently be in contradiction with the lack of a similar phenotype in can-nabinoid CB1receptor knockouts. However, although these

animals are not hyperactive, they display abnormal dopam-ine-mediated behavior such us acquisition of operant re-sponding for cocaine and opiates (61). Moreover, the selec-tive reduction of CB1receptors exhibited by hypothyroid rats

with the global lack of CB1receptors in different species and

throughout the whole developmental period cannot be fully compared.

Collectively, our results suggest, but do not unequivocally show, that a deficit in the endocannabinoid system function in the basal ganglia could cause, at least in part, the motor hyperactivity associated with the congenital hypothyroid state, and open new research expectations in ADHD.

Acknowledgments

Received November 19, 2007. Accepted January 14, 2008.

Address all correspondence and requests for reprints to: A. Santos, Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Medi-cina, Universidad Complutense 28040 Madrid, Spain. E-mail: piedras3@med.ucm.es; or A. Perez-Castillo, Instituto de Investigaciones Biome´dicas, Consejo Superior de Investigaciones Cientificas-Univer-sidad Autonoma, Arturo Duperier 4, 28029 Madrid, Spain. E-mail: aperez@iib.uam.es.

This work was supported by Ministerio de Educacion y Ciencia Grants SAF2004-06263-CO2-02 (to A.S.), SAF2004-06263-CO2-01, and SAF2007-62811 and Comunidad de Madrid Grants GR/SAL/0033/2004 (to A.P.-C.) and SAF004-07762 (to FRdF). T.A. was a predoctoral fellow from the Ministerio de Educacio´n y Ciencia.

Disclosure Statement: The authors have nothing to disclose.

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