Disorders of the inhibitory glycine receptor: the spastic mouse

The mutant mouse spastic suffers from a motor disorder of autosomal recessive inheritance which is characterized by tremor, myoclonic episodes, and a disturbed righting response. The most prominent alteration in the mutant is a substantial deficit of postsynaptic glycine receptor channels resulting in a dramatic reduction of glycinergic synaptic inhibition. Function and structure of the glycine receptor protein appear unaffected, which argues for a regulatory rather than a structural effect of the spastic mutation. It appears that other alterations in the spastic mouse are secondary to this fundamental disturbance in the balance of excitatory and inhibitory impulses. In particular, a significant increase in GABAA receptors of the lower parts of the CNS may serve a compensatory function, counteracting in part losses of glycinergic inhibition. Pharmacological experiments indeed show that facilitation of GABAA receptor-mediated inhibition alleviates symptoms of the spastic motor disorder. The recent cDNA cloning of glycine receptor subunits should help define the molecular mechanism by which the spastic gene causes the glycine receptor deficit.     

Decreased agonist affinity and chloride conductance of mutant glycine receptors associated with human hereditary hyperekplexia.

Hereditary hyperekplexia is a dominant neurological disorder associated with point mutations at the channel-forming segment M2 of the glycine receptor alpha 1 subunit. Voltage-clamp recordings from the heterologously expressed mutants (alpha 1R271L or alpha 1R271Q) revealed 146- to 183-fold decreased potencies of glycine to activate the chloride channel, and significantly reduced maximal whole-cell currents as compared with wild-type receptors. In contrast, the ability of the competitive antagonist strychnine to block glycine-induced currents was similar in all cases. Radioligand binding assays showed a 90- to 1365-fold reduction in the ability of glycine to displace [3H]strychnine from its binding site on the mutant receptors. Paralleling the reductions in whole-cell current, the elementary main-state conductances of the mutants (alpha 1R271L, 64 pS; alpha 1R271Q, 14 pS) were lower than that of the wild-type receptor (86 pS). The decreased agonist affinities and chloride conductances of the mutants are likely to cause neural hyperexcitability of affected patients by impairing glycinergic inhibition. In addition, our data reveal that structural modifications of the ion-channel region can affect agonist binding to the glycine receptor.     

Distribution and characterization of the gaba receptors in the CNS of ataxic mutant mouse

In an attempt to elucidate molecular pathogenesis of ataxia without cytological abberations in the cerebellum, Rolling Mouse Nagoya (C3Hf/Nem-rol) was used to study distribution of GABA receptors in membrane fractions. Among muscimol binding sites of various regions in the ataxic CNS, those in pons and medulla were significantly decreased (P<0.001) compared with control and non-ataxic heterozygote CNS, followed by cerebellum at a lower degree of significance (P<0.01). The kinetic studies demonstrated that dissociation constants of high- and low-affinity binding sites of muscimol of each control and those of ataxic mutant mouse were similar, i.e.,K _H=41 nM andK _L=1.1 M, respectively.GAD in the various regions was assayed, and it showed higher activity in the thalamus and hypthalamus, and lower activity in the cerebellum, of the ataxic mutant mouse as compared to that of the control mouse.     

Distribution and characterization of the glutamate receptors in the CNS of ataxic mutant mouse

In a search for neurochemical involvement in cerebellar ataxia, Rolling Mouse Nagoya (C3Hf/Nem-rol), which shows only hypoplasia of the cerebellum but no pathological configuration of the cerebellar structure, was used to study glutamate receptors in the CNS. Kainic acid binding sites were significantly decreased in the thalamus, hypothalamus, pons, and cerebellum, and in the frontal cortex of both the ataxic mutant mouse and the non-ataxic heterozygote. Only spinal cord and midbrain of the ataxic mutant mouse showed decreased distribution of kainate binding sites in the membrane fraction.Among enzymes responsible for supplying glutamate to the receptor, GDH showed higher activity in the spinal cord of the ataxic mutanat mouse.     

Distribution and characterization of the TRH receptors in the CNS of ataxic mutant mouse

The distribution of TRH receptors in the membrane fraction of the CNS in ataxic mutant mice (C3Hf/Nem-rol and C57BL/6j-tg) was studied. TRH binding sites in cerebellum and frontal lobe of the ataxic form and the non-ataxic heterozygotes of Rolling Mouse Nagoya were decreased in comparison with the controls, whereas those in the spinal cord of Rolling Mouse Nagoya and cerebellum of Tottering Mouse were increased in the ataxic mice over the controls. Kinetic studies were performed on cerebrum and cerebellum of the different ataxic mutant mice. Such species differences in the distribution of the TRH receptors have to be considered in the action of TRH in individual ataxia cases.     

Modulation of motoneuron N-methyl-D-aspartate receptors by the inhibitory neurotransmitter glycine.

Previous studies in the central nervous system have shown that glycine is a co-agonist with glutamate at central N-methyl-D-aspartate receptors (NMDA-Rs). However, there is considerable controversy as to whether the glycine site on NMDA-Rs is saturated. If this site were not saturated then glycine released from glycinergic synaptic terminals might 'spill-over' and activate NMDA-Rs. Since motoneurons have both NMDA and glycine synapses these neurons present an optimal substrate for testing whether the glycine binding site of NMDA-Rs is activated by transmitter released from glycine synaptic terminals. Using an in vitro brainstem slice preparation we report on initial experiments to investigate whether the glycine binding site of NMDA-Rs is saturated in motoneurons. Specifically, we investigated the question of whether the response of neonatal rat hypoglossal motoneurons (HMs) to a brief application of NMDA is enhanced by the presence of exogenous glycine. We found that exogenously applied glycine (1 mM) enhanced the NMDA activated membrane current. We conclude that in brainstem slices the glycine site at motoneuronal NMDA-Rs is not saturated, and that synaptically-released glycine may modulate NMDA-Rs mediated responses.     

EMMA--the European mouse mutant archive.

The European Mouse Mutant Archive (EMMA) offers the worldwide scientific community a free archiving service for its mutant mouse lines and access to a wide range of disease models and other research tools. EMMA is currently comprised of seven partners who operate as the primary mouse repository in Europe. EMMA' s primary objectives are to establish and manage a unified repository for maintaining mouse mutations and to make them available to the scientific community. In addition to these core services, the consortium can generate germ-free (axenic) mice for its customers and also hosts courses in cryopreservation. EMMA is a founder member of the Federation of International Mouse Resources (FIMRe). The EMMA network is funded by the participating institutes, national research programmes and the European Commission Research Infrastructures Programme.     

Binding of mouse choriomammotropin to prolactin receptors in the mouse mammary gland.

The interaction between mouse choriomammotropin and mouse mammary glands was examined by radioreceptor assays using ovine prolactin (NIH-P-S9) iodinated by lactoperoxidase as a tracer. Mouse pituitary extracts and placental extracts were subjected to 10% acrylamide gel electrophoresis. Gels were cut into 2-mm segments after electrophoresis, and stored in 1 ml 0.05 M phosphate buffer (pH 7.4) containing 0.05 M NaCl overnight for elution. Lactating mammary tissues from D strain mice were incubated for 120 min in 1 ml Medium 199 containing 6 ng of 125I-prolactin and 0.1 ml of each eluate. Pituitary extracts displaced 125I-prolactin only at the position which coincides with the prolactin band. Displacement was observed at two positions of the gel when placental extracts were used. Relative mobilities (Rm) were 0.21 and 0.71, respectively. The slowly migrating component of choriomammotropin inhibited the binding of 125-I-prolactin more strongly that the rapidly migrating one. Neither of them was identified as a distinct band in stained gels. The molecular weight of ovine prolactin, mouse pituitary prolactin and the slowly migrating component of mouse choriomammotropin was estimated to be 23000 using disc electrophoresis but the ion charges of these hormones were considerably different.     

Active domains in wild-type and mutant glucocorticoid receptors.

[3H]Triamcinolone acetonide was used to tag covalently specific glucocorticoid receptors by photoaffinity labelling at lambda greater than or equal to 320 nm. Receptors of wild-type mouse lymphoma cells and two glucocorticoid resistant mutants of "nuclear transfer deficient" (nt-) and "increased nuclear transfer" (nti) phenotypes, respectively, were used. Wild-type and nt- receptors yielded radiolabelled polypeptide bands of mol. wt. 98 000 as revealed by gel electrophoresis under denaturing conditions and fluorography. In contrast, the nti receptor had a mol. wt. of 42 000. Partial proteolysis of the wild-type receptor with alpha-chymotrypsin resulted in a fragment of mol. wt. 39 000 which still contained the steroid binding site but had increased affinity for DNA indistinguishable from that of the nti receptor. Chymotrypsin thus removed a domain from the wild-type receptor polypeptide which is involved in modulating DNA binding. The same domain is missing from the nti receptor.     

Abnormal elevation of the neural folds in the loop-tail mutant mouse.

The processes of elevation and convergence of the spinal neural folds were analyzed in normal (+/+; Lp/+) and abnormal (Lp/Lp) embryos of the loop-tail mutant mouse in order to determine possible mechanisms underlying the dysraphic defect characterized by a failure of the neural fold to close in this mutant. The results indicate that the neural folds are already defective during very early phases of elevation, with greater distances between the apical points of the paired walls of the neural groove, larger ventral angles and higher ratios of luminal/basal linear distances occurring in the abnormal embryos relative to those in normal embryos. The cross-sectional area of the neuroepithelium is also greater in abnormals, suggesting that faulty elongation of the neuraxis may contribute to the dysraphic condition.     

Aberrant convergence of the neural folds in the mouse mutant vl.

Progressive changes in the dorsolateral angles (DA) and ventral angle (VA) during elevation and convergence of the caudal neural folds were morphometrically analyzed in normal and dysraphic abnormal embryos of the mouse mutant vacuolated lens (vl), and correlations with the configuration of microfilaments in the apices of neuroepithelial cells were made by means of ultrastructural cytochemistry. In 22-28 somite stage abnormal (vl/vl) embryos, the DA and VA are larger than those in their normal counterparts at each comparable level of the caudal neural folds, suggesting that defective convergence involves both the DA and VA in this mutant. In 30-35 somite stage abnormal embryos, the VA is likewise larger than that in normal embryos in which the neural folds have converged and closed; however, the DAs are much smaller, indicating that a medial collapse of the dorsal ends of the neural folds may occur secondary to the closure failure. At the DA, the ultrastructural configuration of microfilaments is similar in abnormal and normal embryos in terms of their circumferential arrangement around the perimeters of the neuroepithelial cell apices. In abnormal embryos, however, the bundles of microfilaments are more delicate and less prominent than in normal embryos; thus it is possible that a quantitative and/or functional deficiency in these elements may be involved in the failure of the abnormal neuroepithelium to bend properly during convergence of the neural folds.     

N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express.

In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.