The effect of mGlu8 deficiency in animal models of psychiatric diseases.

The metabotropic glutamate receptor subtype 8 (mGlu(8)) is presynaptically located and regulates the release of the transmitter. Dysfunctions of this mechanism are involved in the pathophysiology of different psychiatric disorders. mGlu(8) deficient mice have been previously investigated in a range of studies, but the results are contradictory and there are still many open questions. Therefore, we tested mGlu(8)-deficient animals in different behavioral tasks that are commonly used in neuropsychiatric research. Our results show a robust contextual fear deficit in mGlu(8)-deficient mice. Furthermore, novel object recognition, chlordiazepoxide-facilitated extinction of operant conditioning and the acoustic startle response were attenuated by mGlu(8) deficiency. We found no changes in sensory processing, locomotor activity, prepulse inhibition, phencyclidine-induced changes in locomotion or prepulse inhibition, operant conditioning, conditioned fear to a discrete cue or in animal models of innate fear and post-traumatic stress disorder. We conclude that mGlu(8) might be a potential target for disorders with pathophysiological changes in brain areas where mGlu(8) modulates glutamate and gamma-amino butyric acid (GABA) transmission. Our data especially point to anxiety disorders involving exaggerated contextual fear, such as generalized anxiety disorders, and to conditions with disturbed declarative memory.

Metabotropic glutamate 7 receptor subtype modulates motor symptoms in rodent models of Parkinson's disease.

Metabotropic glutamate (mGlu) receptors modulate synaptic transmission in the central nervous system and represent promising therapeutic targets for symptomatic treatment of Parkinson's disease (PD). Among the eight mGlu receptor subtypes, mGlu7 receptor is prominently expressed in the basal ganglia, but its role in restoring motor function in animal models of PD is not known. The effects of N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), the first selective allosteric activator of mGlu7 receptors, were thus tested in different rodent models of PD. Here, we show that oral (5 mg/kg) or intrastriatal administration (0.1 and 0.5 nmol) of AMN082 reverses haloperidol-induced catalepsy in rats. AMN082 (2.5 and 5 mg/kg) reduces apomorphine-induced rotations in unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats. In a more complex task commonly used to evaluate major akinetic symptoms of PD patients, 5 mg/kg AMN082 reverses the increased reaction time to respond to a cue of bilateral 6-OHDA-lesioned rats. In addition, AMN082 reduces the duration of haloperidol-induced catalepsy in a mGlu7 receptor-dependent manner in wild-type but not mGlu7 receptor knockout mice. Higher doses of AMN082 (10 and 20 mg/kg p.o.) have no effect on the same models of PD. Overall these findings suggest that mGlu7 receptor activation can reverse motor dysfunction associated with reduced dopamine activity. Selective ligands of mGlu7 receptor subtypes may thus be considered as promising compounds for the development of antiparkinsonian therapeutic strategies.

mGluR7 facilitates extinction of aversive memories and controls amygdala plasticity.

Formation and extinction of aversive memories in the mammalian brain are insufficiently understood at the cellular and molecular levels. Using the novel metabotropic glutamate receptor 7 (mGluR7) agonist AMN082, we demonstrate that mGluR7 activation facilitates the extinction of aversive memories in two different amygdala-dependent tasks. Conversely, mGluR7 knockdown using short interfering RNA attenuated the extinction of learned aversion. mGluR7 activation also blocked the acquisition of Pavlovian fear learning and its electrophysiological correlate long-term potentiation in the amygdala. The finding that mGluR7 critically regulates extinction, in addition to acquisition of aversive memories, demonstrates that this receptor may be relevant for the manifestation and treatment of anxiety disorders.

Functional role of mGluR1 and mGluR4 in pilocarpine-induced temporal lobe epilepsy.

Altered expression and distribution of neurotransmitter receptors, including metabotropic glutamate receptors (mGluRs), constitute key aspects in epileptogenesis, impaired hippocampal excitability and neuronal degeneration. mGluR1 mediates predominantly excitatory effects, whereas mGluR4 acts as inhibitory presynaptic receptor. Increased hippocampal expression of mGluR1 and mGluR4 has been observed in human temporal lobe epilepsy (TLE). In this study, we address whether genetic mGluR1 upregulation and mGluR4 knock-down influence seizure susceptibility and/or vulnerability of hippocampal neurons by analyzing transgenic animals in the pilocarpine TLE model. Therefore, we generated transgenic mice expressing mGluR1-enhanced green fluorescent protein (EGFP) fusion protein under control of the human cytomegalovirus (CMV) immediate early promoter. Status epilepticus (SE) was induced in (a) mice overexpressing mGluR1-EGFP and (b) mice deficient for mGluR4 (mGluR4 KO) as well as littermate controls. In the acute epileptic stage after pilocarpine application, mGluR4 KO mice showed a significant increase of severe seizure activity, in contrast to mGluR1 transgenics. Analysis of both transgenic mouse lines in the chronic epileptic phase, using a telemetric EEG-/video-monitoring system, revealed a significant increase in seizure frequency only in mGluR1-EGFP mice. In contrast, enhanced neuronal cell loss was only present in the hippocampus of epileptic mGluR4 KO mice. Our results suggest a role for mGluR1 in promoting seizure susceptibility as well as for mGluR4 to counteract excitatory activity and seizure-associated vulnerability of hippocampal neurons. Therefore, our data strongly recommend both mGluRs as potential drug targets to interfere with the development of hippocampal damage and seizure activity in TLE.

siRNA-mediated knockdown of the serotonin transporter in the adult mouse brain.

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressant drugs that increase the extracellular levels of serotonin by blocking the reuptake activity of the serotonin transporter (SERT). Although SSRIs elevate brain serotonergic neurotransmission acutely, their full therapeutic effects involve neurochemical adaptations that emerge following chronic drug administration. The adaptive downregulation of SERT has recently been implicated in the therapeutic response of SSRIs. Interestingly, studies using SERT-knockout mice reveal somewhat paradoxical depression-related effects, probably specific to the downregulation of SERT during early development. However, the behavioral significance of SSRI-mediated downregulation of SERT during adulthood is still unknown. We investigated whether somatic gene manipulation, triggered by infusing short interfering RNA (siRNA) into the ventricular system, would enable the downregulation of SERT in the adult mouse brain. Infusing the SERT-targeting siRNA, for 2 weeks, significantly reduced the mRNA levels of SERT in raphe nuclei. Further, a significant, specific and widespread downregulation of SERT-binding sites was achieved in the brain. In contrast, 2-week infusion of the SSRI, citalopram, produced a widespread downregulation of SERT-binding sites, independent of any alterations at the mRNA level. Irrespective of their mechanisms for downregulating SERT in the brain, infusions of SERT-siRNA or citalopram elicited a similar antidepressant-related behavioral response in the forced swim test. These results signify a role for the downregulation of SERT in mediating the antidepressant action of SSRIs in adults. Further, these data demonstrate that siRNA-induced widespread knockdown of gene expression serves as a powerful tool for assessing the function of endogenous genes in the adult brain.

Adnexal masses in pregnancy.

With the widespread use of routine abdominal ultrasound examination during pregnancy, adnexal masses are observed with increasing frequency. Most patients are clinically asymptomatic at the time of presentation, and most of the adnexal masses detected during early pregnancy disappear during the first 16 weeks of pregnancy. Ovarian tumors are estimated to occur in about 1 in 1,000 pregnancies and of these 3% are malignant. Here we present an overview about frequency, diagnostic procedures and pathological characteristics of these ovarian tumors. Moreover, current modalities for treatment during pregnancy are summarized. Surgical treatment of the adnexal masses has to be performed with adequate staging and debulking equal to the treatment of non-pregnant women. However, whereas during organogenesis abortion has to be considered prior to chemotherapy, later in pregnancy surgical debulking as complete as possible, followed by taxol-platinum chemotherapy is indicated. If the fetus is not viable at the time of primary surgery, neoadjuvant chemotherapy and complementation of surgery after delivery of the baby should be performed. It should be stressed that chemotherapy for ovarian cancer applied during pregnancy appears to be safe. However, no studies have evaluated the long-term consequences for children exposed to intra-uterine chemotherapy. Aspiration of cysts should be avoided, as the correlation between the histological evaluation of an ovarian malignancy and the cytological evaluation of aspirates is poor. Moreover, spillage of malignant cysts is hazardous for the patient.

Uptake of tri-iodothyronine and thyroxine in myoblasts and myotubes of the embryonic heart cell line H9c2(2-1).

Uptake of tri-iodothyronine (T(3)) was compared with that of thyroxine (T(4)) in the embryonic heart cell line H9c2 (2-1). These cells propagate as myoblasts and form differentiated myotubes upon reduction of the serum concentration, as indicated by a 31-fold increase in creatine kinase activity. Protein and DNA content per well were around 2-fold higher in myotubes than in myoblasts. When expressed per well, T(3) and T(4) uptake were, compared with myoblasts, 1.9- to 2-fold and 3.1- to 4-fold higher in myotubes respectively. On the other hand, the characteristics of T(3) and T(4) uptake were similar in myoblasts and myotubes. At any time-point, T(4) uptake was 2-fold higher than that of T(3), and both uptakes were energy but not Na(+) dependent. T(3) and T(4) uptake exhibited mutual inhibition in myoblasts and myotubes: 10 microM unlabeled T(3) reduced T(4) uptake by 51-60% (P<0.001), while 10 microM T(4) inhibited T(3) uptake by 48-51% (P<0.001). Furthermore, T(3) and T(4) uptake in myoblasts was dose-dependently inhibited by tryptophan (maximum inhibition around 70%; P<0.001). Exposure of the cells to T(3) or T(4) during differentiation significantly increased the fusion index (35 and 40%; P < 0.01). Finally, both myoblasts and myotubes showed a small deiodinase type I activity, while deiodinase type II activity was undetectable. In conclusion, T(3) and T(4) share a common energy-dependent transport system in H9c2(2-1) cells, that may be important for the availability of thyroid hormone during differentiation.

Age-specific differences in treatment and survival of patients with cervical cancer in the southeast of The Netherlands, 1986-1996.

Age at diagnosis has been proven to be an important determinant of the choice of initial treatment for several sites of cancer. Elderly patients are more likely to receive no treatment or less intensive treatment modalities. This study analysed the influence of age on treatment choice and survival in patients diagnosed with cervical cancer. This population-based study used data on 1176 new cases of invasive cervical cancer diagnosed in the period of 1986-1996 from three regional cancer registries in the Netherlands. All available information on treatment and survival (on 1 January 1998) was recorded. Relative survival rates were calculated according to the Hakulinen method. Relative risks (RR) for excess mortality due to the diagnosis of cervical cancer were calculated with a regression model for relative survival rates. Only 5% of the patients aged 70 years and older (n=224) were diagnosed with stage IA disease, compared with 11 and 30% of the patients aged 50-69 years and 49 years and younger, respectively. Almost 50% of the 70+ patients with stage IB-IIA were treated with radiotherapy as a single treatment modality, whereas 64% of the patients aged < or =49 years were treated with surgery alone. In all age groups, treatment for advanced stage disease (stage > or =IIB) was radiotherapy alone. No treatment was given to 10% of the patients aged 70 years and older, 5% of those aged 50-69 years and 1% of those aged 49 years and younger. Five-year relative survival was 69% (95% Confidence Interval (CI): 66-72%) and differed significantly (P=0.001) with age (70+ years: 49%; 50-69 years 58%; < or =49 years: 81%). Multivariate analyses on a subset of patients showed that age was not an independent prognostic factor, whereas stage and treatment modality were very important prognostic factors. Although elderly cancer patients were sometimes treated differently from younger patients, this was in accordance with the guidelines. Relative survival rates differed significantly by age. The multivariate analyses on the subset of patients also revealed that excess mortality increased with age. However, when adjustment was made for stage and treatment, this difference disappeared. The influence of treatment on survival is likely to be due to the selection of patients based on other characteristics, such as tumour volume, comorbidity and performance status.

Uptake of triiodothyronine and triiodothyroacetic acid in neonatal rat cardiomyocytes: effects of metabolites and analogs.

Cellular and nuclear uptake of [125I]tri-iodothyronine (T3) and [125I]triiodothyroacetic acid (Triac) were compared in cardiomyocytes of 2-3 day old rats, and the effect of thyroid hormone analogs on cellular T(3) uptake was measured. Cells (5-10 x 10(5) per well) were cultured in DMEM-M199 with 5% horse serum and 5% FCS. Incubations were performed for from 15 min to 24 h at 37 degrees C in the same medium, 0.5% BSA and [125I]T3 (100 pM), or [125I]Triac (240 pM). Expressed as % dose, T(3) uptake was five times Triac uptake, but expressed as fmol/pM free hormone, Triac uptake was at least 30% (P<0.001) greater than T3 uptake, whereas the relative nuclear binding of the two tracers was comparable. The 15 min uptake of [125I]T3 was competitively inhibited by 10 microM unlabeled T3 (45-52%; P<0.001) or 3,3'- diiodothyronine (T2) (52%; P<0.001), and to a smaller extent by thyroxine (T(4)) (27%; 0.05

Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7.

To study the role of mGlu7 receptors (mGluR7), we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7(-/-)). After the serendipitous discovery of a sensory stimulus-evoked epileptic phenotype, we tested two convulsant drugs, pentylenetetrazole (PTZ) and bicuculline. In animals aged 12 weeks and older, subthreshold doses of these drugs induced seizures in mGluR7(-/-), but not in mGluR7(+/-), mice. PTZ-induced seizures were inhibited by three standard anticonvulsant drugs, but not by the group III selective mGluR agonist (R,S)-4-phosphonophenylglycine (PPG). Consistent with the lack of signs of epileptic activity in the absence of specific stimuli, mGluR7(-/-) mice showed no major changes in synaptic properties in two slice preparations. However, slightly increased excitability was evident in hippocampal slices. In addition, there was slower recovery from frequency facilitation in cortical slices, suggesting a role for mGluR7 as a frequency-dependent regulator in presynaptic terminals. Our findings suggest that mGluR7 receptors have a unique role in regulating neuronal excitability and that these receptors may be a novel target for the development of anticonvulsant drugs.

Characterization of uptake and compartmentalization of 3,5,3'-tri-iodothyronine in cultured neonatal rat cardiomyocytes.

The uptake of tri-iodothyronine (T(3)) in cultured neonatal rat cardiomyocytes was investigated and compared with the uptake of reverse T(3 )(rT(3)) and thyroxine (T(4)). Cellular compartmentalization of T(3) was studied by distinguishing T(3) activity associated with the plasma membrane from that in the cytosol or incorporated in the cell nucleus. T(3) and T(4) uptake displayed similar temperature dependencies which, in magnitude, differed from that of rT(3) uptake. T(3) uptake was Na(+ )independent, and sensitive to oligomycin and monodansylcadaverine (42-49% and 25% inhibition of 15-min cellular uptake respectively). Furthermore, T(3) uptake could be inhibited by tryptophan (20%) and tyrosine (12%), while 2-aminobicyclo[2,2,1]heptane-carboxylic acid had no effect. Co-incubation with tryptophan and oligomycin resulted in an additive inhibition of T(3) uptake (77%). We therefore conclude that (i) T(3) uptake is energy dependent, (ii) receptor-mediated endocytosis may be involved and (iii) the aromatic amino acid transport system T may play a role, while system L is not involved in T(3) transport in cardiomyocytes. Co-incubation with unlabeled iodothyronines showed that 3,3'-di-iodothyronine and T(3) itself were the most effective inhibitors of T(3) uptake (30% and 36% inhibition of 15-min cellular uptake respectively). At 15-min incubation time, 38% of the total cell-associated T(3) was present in the cytosol and nucleus, and 62% remained associated to the plasma membrane. Unidirectional uptake rates did not saturate over a free T(3) concentration range up to 3.9 microM. We have concluded that T(3) uptake in neonatal rat cardiomyocytes occurs by an energy- and temperature-dependent mechanism that may include endocytosis and amino acid transport system T, and is not sensitive to the Na(+) gradient. Elucidation of the molecular basis for the T(3) transporter is the subject of current investigation.

Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABA(B) responses in mice lacking GABA(B(1)).

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus).

A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish FSH-R (cfFSH-R) mRNA expression was observed in testis and ovary; abundant mRNA expression was also detected in seminal vesicles. The isolated cDNA encoded a functional receptor since its transient expression in human embryonic kidney (HEK-T) 293 cells resulted in ligand-dependent cAMP production. Remarkably, African catfish LH (cfLH; the catfish FSH-like gonadotropin has not been purified yet) had the highest potency in this system. From the other ligands tested, only human recombinant FSH (hrFSH) was active, showing a fourfold lower potency than cfLH, while hCG and human TSH (hTSH) were inactive. Human CG (as well as cfLH, hrFSH, eCG, but not hTSH) stimulated testicular androgen secretion in vitro but seemed to be unable to bind to the cfFSH-R. However, it was known that hCG is biologically active in African catfish (e.g., induction of ovulation). This indicated that an LH receptor is also expressed in African catfish testis. We conclude that we have cloned a cDNA encoding a functional FSH-R from African catfish testis. The cfFSH-R appears to be less discriminatory for its species-specific LH than its avian and mammalian counterparts.

Chemotherapy resistant ovarian cancer in carriers of an hMSH2 mutation?

Hereditary Non-Polyposis Colorectal Cancer (HNPCC, Lynch syndrome) is an autosomal dominant condition of cancer susceptibility with high penetrance, characterised by early onset of colon tumours as well as a variety of extracolonic tumours including ovarian cancer and, in particular, cancer of the endometrium. Germline mutations in one of five DNA-mismatch repair (MMR) genes (hMLH1, hMSH2, hMSH6, PMS1, PMS2) are known to cause HNPCC. To date, mutations in two of these genes (hMSH2 and hMLH1) are found in the majority of mutation positive families. Recent literature suggests that especially hMSH2 mutations are associated with extracolonic tumours. We describe two women from an HNPCC family carrying an hMSH2 mutation (deletion of exon 6 of this gene) who developed ovarian cancer. In these patients (full cousins) the ovarian cancers were noted for their aggressive development and rapid recurrence after surgical debulking and during regular multichemotherapy including Cisplatin. This report strengthens recent in vitro studies suggesting an involvement of MMR-gene mutations in ovarian cancer cell biology with decreased susceptibility to Cisplatin therapy. The possible implications for the therapy of ovarian cancer, the screening and genetic counselling of family members are discussed.

Subcellular localization of wild-type and Parkinson's disease-associated mutant alpha -synuclein in human and transgenic mouse brain.

Mutations in the alpha-synuclein (alphaSYN) gene are associated with rare cases of familial Parkinson's disease, and alphaSYN is a major component of Lewy bodies and Lewy neurites. Here we have investigated the localization of wild-type and mutant [A30P]alphaSYN as well as betaSYN at the cellular and subcellular level. Our direct comparative study demonstrates extensive synaptic colocalization of alphaSYN and betaSYN in human and mouse brain. In a sucrose gradient equilibrium centrifugation assay, a portion of betaSYN floated into lower density fractions, which also contained the synaptic vesicle marker synaptophysin. Likewise, wild-type and [A30P]alphaSYN were found in floating fractions. Subcellular fractionation of mouse brain revealed that both alphaSYN and betaSYN were present in synaptosomes. In contrast to synaptophysin, betaSYN and alphaSYN were recovered from the soluble fraction upon lysis of the synaptosomes. Synaptic colocalization of alphaSYN and betaSYN was directly visualized by confocal microscopy of double-stained human brain sections. The Parkinson's disease-associated human mutant [A30P]alphaSYN was found to colocalize with betaSYN and synaptophysin in synapses of transgenic mouse brain. However, in addition to their normal presynaptic localization, transgenic wild-type and [A30P]alphaSYN abnormally accumulated in neuronal cell bodies and neurites throughout the brain. Thus, mutant [A30P]alphaSYN does not fail to be transported to synapses, but its transgenic overexpression apparently leads to abnormal cellular accumulations.

Selective activation of mGlu4 metabotropic glutamate receptors is protective against excitotoxic neuronal death.

Activation of group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, and mGluR8) has been established to be neuroprotective in vitro and in vivo. To disclose the identity of the receptor subtype(s) that exert(s) the protective effect, we have used group III agonists in combination with mGluR4 subtype-deficient mice (-/-). In cortical cultures prepared from wild-type (+/+) mice and exposed to a toxic pulse of NMDA, the selective group III agonist (+)-4-phosphonophenylglycine [(+)-PPG] reversed excitotoxicity with an EC(50) value of 4.9 microm, whereas its enantiomer (-)-PPG was inactive. This correlated closely with the potency of (+)-PPG in activating recombinant mGluR4a. In cortical neurons from -/- mice, (+)-PPG showed no protection against the NMDA insult up to 300 microm, whereas group I/II mGluR ligands still retained their protective activity. Classical group III agonists (l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate) were also substantially neuroprotective against NMDA toxicity in +/+ and heterozygous (+/-) cultures but were inactive in -/- cultures. Interestingly, -/- cultures were more vulnerable to low concentrations of NMDA and showed higher extracellular glutamate levels compared with +/+ cultures. We have also examined neurodegeneration induced by intrastriatal infusion of NMDA in wild-type or mGluR4-deficient mice. Low doses of (R,S)-PPG (10 nmol/0.5 microl) substantially reduced NMDA toxicity in +/+ mice but were ineffective in -/- mice. Higher doses of (R,S)-PPG were neuroprotective in both strains of animals. Finally, microdialysis studies showed that intrastriatal infusion of NMDA increased extracellular glutamate levels to a greater extent in -/- than in +/+ mice, supporting the hypothesis that the mGluR4 subtype is necessary for the maintenance of the homeostasis of extracellular glutamate levels.

Neuropathology in mice expressing human alpha-synuclein.

The presynaptic protein alpha-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinson's disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimer's disease. alpha-Synuclein accumulates in Lewy bodies and Lewy neurites, and two missense mutations (A53T and A30P) in the alpha-synuclein gene are genetically linked to rare familial forms of Parkinson's disease. Under control of mouse Thy1 regulatory sequences, expression of A53T mutant human alpha-synuclein in the nervous system of transgenic mice generated animals with neuronal alpha-synucleinopathy, features strikingly similar to those observed in human brains with Lewy pathology, neuronal degeneration, and motor defects, despite a lack of transgene expression in dopaminergic neurons of the substantia nigra pars compacta. Neurons in brainstem and motor neurons appeared particularly vulnerable. Motor neuron pathology included axonal damage and denervation of neuromuscular junctions in several muscles examined, suggesting that alpha-synuclein interfered with a universal mechanism of synapse maintenance. Thy1 transgene expression of wild-type human alpha-synuclein resulted in similar pathological changes, thus supporting a central role for mutant and wild-type alpha-synuclein in familial and idiotypic forms of diseases with neuronal alpha-synucleinopathy and Lewy pathology. These mouse models provide a means to address fundamental aspects of alpha-synucleinopathy and test therapeutic strategies.