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.

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.

Metabotropic glutamate receptor subtype 7 ablation causes deficit in fear response and conditioned taste aversion.

Metabotropic glutamate receptors (mGluRs) consist of eight different subtypes and exert their effects on second messengers and ion channels via G-proteins. The function of individual mGluR subtypes in the CNS, however, largely remains to be clarified. We examined the fear response of freezing after electric shock in wild-type and mGluR7(-/-) knockout littermates. Wild-type mice displayed freezing immediately after and 1 d after footshock. In comparison, mGluR7(-/-) knockout mice showed significantly reduced levels in both immediate postshock and delayed freezing responses. However, the knockout mice exhibited no abnormalities in pain sensitivity and locomotor activity. To further examine amygdala-dependent behavior, we performed conditioned taste aversion (CTA) experiments. In wild-type mice, the administration of saccharin followed by intraperitoneal injection of the malaise-inducing agent LiCl resulted in an association between saccharin and LiCl. This association caused strong CTA toward saccharin. In contrast, mGluR7(-/-) knockout mice failed to associate between the taste and the negative reinforcer in CTA experiments. Again, the knockout mice showed no abnormalities in taste preference and in the sensitivity to LiCl toxicity. These results indicate that mGluR7 deficiency causes an impairment of two distinct amygdala-dependent behavioral paradigms. Immunohistochemical and immunoelectron-microscopic analyses showed that mGluR7 is highly expressed in amygdala and preferentially localized at the presynaptic axon terminals of glutamatergic neurons. Together, these findings strongly suggest that mGluR7 is involved in neural processes subserving amygdala-dependent averse responses.

Generation of chimeric monoclonal antibodies from mice that carry human immunoglobulin Cgamma1 heavy of Ckappa light chain gene segments.

Gene targeting in mouse embryonic stem (ES) cells was used to replace (i) the mouse immunoglobulin heavy chain (IgH) Cgamma2a gene segment (mCgamma2a) with the human Cgamma1 gene segment (hCgamma1), and (ii) the mouse immunoglobulin light chain (IgL) Ckappa gene segment (mC kappa) with its human counterpart (hC kappa). ES cells carrying these gene conversions were used to generate chimeric mice that transmitted the human alleles through the germ line. Mice homozygous for both gene alterations were generated by breeding. Serum from homozygous mutant mice contained comparable amounts of antibodies with chimeric kappa or mouse lambda light chains but only small fractions of basal serum IgG or antibodies elicited against immunizing agents contained chimeric heavy chains. A relative increase in immunogen-specific hCgamma1 antibodies was seen following immunization in combination with the saponin adjuvant QS-21. The effect of this was to shift the IgG1-dominated response to an IgG subclass profile that included significant amounts of IgG2a, IgG2b and IgG3 and chimeric IgG. The amounts of antibody secreted by hybridomas derived from mutant and wild-type mice were similar. Sequencing confirmed correct splicing of hCgamma1 and hCkappa gene segments to mouse J gene segments in hybridoma Ig gene transcripts. In conclusion, IgHhCgamma1/IgLhCkappa double mutant mice provide a useful animal model for deriving humanized antibodies with potential applications in immunotherapy and diagnostics in vivo as well as for investigating hCgamma1 associated functions.

Endogenous serine protease inhibitor modulates epileptic activity and hippocampal long-term potentiation.

Protease nexin-1 (PN-1), a member of the serpin superfamily, controls the activity of extracellular serine proteases and is expressed in the brain. Mutant mice overexpressing PN-1 in brain under the control of the Thy-1 promoter (Thy 1/PN-1) or lacking PN-1 (PN-1-/-) were found to develop epileptic activity in vivo and in vitro. Theta burst-induced long-term potentiation (LTP) and NMDA receptor-mediated synaptic transmission in the CA1 field of hippocampal slices were augmented in Thy 1/PN-1 mice and reduced in PN-1-/- mice. Compensatory changes in GABA-mediated inhibition in Thy 1/PN-1 mice suggest that altered brain PN-1 levels lead to an imbalance between excitatory and inhibitory synaptic transmission.

MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure.

MLP is a LIM-only protein of terminally differentiated striated muscle cells, where it accumulates at actin-based structures involved in cytoarchitecture organization. To assess its role in muscle differentiation, we disrupted the MLP gene in mice. MLP (-/-) mice developed dilated cardiomyopathy with hypertrophy and heart failure after birth. Ultrastructural analysis revealed dramatic disruption of cardiomyocyte cytoarchitecture. At birth, these hearts were not hypertrophic, but already abnormally soft, with cell-autonomous and MLP-sensitive alterations in cytoarchitecture. Thus, MLP promotes proper cardiomyocyte cytoarchitecture, whose perturbation can lead to dilated cardiomyopathy. In vivo analysis revealed that MLP-deficient mice reproduce the morphological and clinical picture of dilated cardiomyopathy and heart failure in humans, providing the first model for this condition in a genetically manipulatable organism.

A novel splice variant of a metabotropic glutamate receptor, human mGluR7b.

Two splice variants of the human metabotropic glutamate receptor 7, named hmGluR7a and hmGluR7b, were isolated from a human brain cDNA library. The isoforms differ by an out-of-frame insertion of 92 nucleotides close to the C-terminus of the hmGluR7 coding region, hmGluR7a has a length of 915 amino acids and represents the human homolog of the recently cloned rat mGluR7. hmGluR7b is seven amino acids longer and exhibits a novel C-terminus of 23 amino acids in length. RT-PCR analysis demonstrated the existence of mGluR7b transcripts in wild-type mouse brain and its absence in mGluR7 knockout mice. Northern blot analysis indicate that mGluR7 expression is developmentally regulated. It is expressed at high levels in human fetal brain and at a lower level in many regions of adult human brain. Stimulation of hmGluR7b with L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP) or L-glutamate in stably transfected Chinese hamster ovary (CHO) cells depressed forskolin-induced cAMP accumulation, whereas (1S,3R)-1-aminocyclopentane-1,3,-dicarboxylic acid ((1S,3R)-ACPD) and quisqualate (both at 1mM) had no significant effects. As described for rat mGluR7, the rank order of agonist potencies is: L-SOP, L-AP4 > L-glutamate > (1S,3R)-ACPD, quisqualate.

Neurite outgrowth on non-permissive substrates in vitro is enhanced by ectopic expression of the neural adhesion molecule L1 by mouse astrocytes.

Axonal regrowth in the lesioned central nervous system (CNS) of adult mammals is, in part, prevented by non-permissive properties of glial cells and myelin. To test if ectopic expression of the neurite outgrowth promoting recognition molecule L1 will overcome these non-permissive influences and promote neurite outgrowth, L1 was expressed in astrocytes of transgenic mice using regulatory sequences of the glial fibrillary acidic protein (GFAP) gene. Northern blot analysis of different transgenic lines revealed different levels of transgenically expressed L1. Cultured astrocytes derived from transgenic animals displayed L1 immunoreactivity at the cell surface and in situ hybridization and immunocytochemical analysis of optic nerves from adult transgenic mice localized L1 expression to astrocytes. Expression of L1 protein by transgenic astrocytes was significantly upregulated in lesioned optic nerves. When mouse small cerebellar neurons or chick dorsal root ganglion neurons were cultured on cryosections of lesioned optic nerves or astrocyte monolayers from transgenic mice, respectively, neurite outgrowth was increased up to 400% on tissue sections and 50% on astrocytes compared with similar preparations from non-transgenic mice. The increase in neurite outgrowth on tissue sections or astrocyte monolayers from different transgenic lines was proportional to the different levels of L1 expression. Moreover, increased neurite outgrowth on these substrates was specifically inhibited by polyclonal L1 antibodies. In vivo, rescue of severed axons was enhanced in transgenic versus wild type animals, while regrowth of axons was slightly, but not significantly, increased. Together, our observations demonstrate that L1 promotes neurite outgrowth when expressed ectopically by astrocytes and that L1 is able to overcome, at least partially, the non-permissive substrate properties of differentiated CNS glial cells in vitro.

The pattern of prothymosin alpha gene expression coincides with that of myc proto-oncogenes during mouse embryogenesis.

The nuclear protein prothymosin alpha is thought to play a critical role in cellular proliferation. Transcription of the gene encoding prothymosin alpha has been shown to be activated by the proto-oncogene c-myc. Also, prothymosin alpha mRNA expression correlates with that of c-myc in human colon cancer. We compared the previously reported embryonic expression pattern of the proto-oncogene c-myc and the pattern of the prothymosin alpha gene by in situ hybridization. Prothymosin alpha is transcribed in all tissues expressing c-myc, including brown adipose tissue, salivary gland, thymus and liver. In addition, we show that the prothymosin alpha gene is active in tissues expressing specifically N-myc such as the neuronal anlage and hair follicles in skin. Therefore, during mouse foetal development the temporal, spatial and tissue-specific expression patterns of both myc proto-oncogenes coincide with the pattern of prothymosin alpha.

Mice deficient in CD23 reveal its modulatory role in IgE production but no role in T and B cell development.

To assess roles of CD23 in lymphocyte development and immune function in vivo, CD23-deficient mice (CD23-/-) were generated. Mice heterozygous with respect to the defective allele (CD23+/-) display 50% reduced levels of CD23 expression on CD23+ cell types. This pattern is consistent with a lack of parental or tissue-specific imprinting of the CD23 gene. Neither a 50% reduced level nor a complete lack of CD23 caused profound changes in lymphocyte compartments (thymocytes, peripheral T cells, and B-1 and B-2 B cells). The lack of CD23 also did not significantly alter in vitro the proliferative response of B cells triggered via the Ag receptor in combination with CD40 ligand, IL-2, and/or IL-4. Effects on polyclonal Ig production were tested in a Th2 cell-driven immune response in vivo after infection with Nippostrongylus brasiliensis, a parasite that dramatically enhances CD23 expression on B cells. In both primary and secondary immune responses, heterozygous CD23+/- mice developed slightly higher and CD23-/- mice similar serum IgE and IgG1 levels as compared with CD23+/+ (wild-type) mice. The increase in blood eosinophil counts was similar in all three types of mice. These findings show that after N. brasiliensis infection 1) a complete lack of CD23 in vivo neither prohibits nor significantly alters quantitatively polyclonal IgE levels in serum, and 2) a 50% reduction in cell-surface CD23 expression (CD23+/- mice) correlates with slightly increased serum IgE levels.

Transgenes encoding mutant simian virus 40 large T antigens unmask phenotypic and functional constraints in thymic epithelial cells.

Transgenes encoding simian virus 40 (SV40) T antigen (Tag) can cause hyperplastic or tumorigenic lesions of desired but also of unforeseen cellular origin. Unexpectedly the human growth hormone-releasing factor (GRF) gene promoter directs expression of SV40 Tag specifically in thymic epithelial (TE) cells. Expression starts in the neonate, in which GRF-Tag+ cells display strict numerical and spatial constraints. Tag supersedes mechanisms that constrain these features and GRF-Tag mice develop thymic hyperplasia. To characterize GRF-Tag+ TE cells and their putative normal counterparts we compared phenotypic and functional effects caused by transgenes encoding mutant large T antigens. This strategy is applicable to any situation in which T antigen is used to alter development. One large Tag mutant (K1 + 5080) does not cause thymic hyperplasia. GRF-Tag (K1 + 5080)+ TE cells display strict temporal and spatial constraints throughout life. TE cells expressing other mutant large T antigens that cause thymic hyperplasia do not obey these rules and reveal that phenotypically distinct GRF-Tag+ TE-cell stages exist in vivo. Analysis of conditional immortal GRF-Tag(tsA58)+ TE cells expressing a temperature-sensitive large Tag shows that large Tag blocks differentiation in these cells. Phenotype and functions in these cells are regulated by cellular differentiation and interleukin 4 (IL-4).

Thymic hyperplasia in transgenic mice caused by immortal epithelial cells expressing c-kit ligand.

To dissect mechanisms that co-ordinate specific events in thymopoiesis we have characterized alterations in thymic structure and function caused by expression of a transgene. This gene encodes SV40Tag and is specifically expressed in a subset of thymic epithelial (TE) cells around birth. As a result the number of immortal TE cells increases, thymic mass increases (up to 3 g), and thymopoiesis is expanded. The latter is reflected by a approximately 100-fold increase of the major thymocyte subsets and increased peripheral T cell counts. Grossly hyperplastic thymi retain many but not all morphological features of a normal thymus. Also in grafts, SV40Tag+ TE cells steer expansion (up to 8 g) and organize a tissue with mainly cortex-like features that includes mainly SV40Tag+ TE cells, thymocytes, and macrophages. To investigate expression of specialized gene functions in the immortal TE cells, a cell line was derived. The Epi-A1 cell line expresses the genes for major histocompatibility complex class I and II, Thy-1, interleukin (IL)-6, IL-7, macrophage-colony-stimulating factor, and transforming growth factor-beta 3. Most importantly, Epi-A1 cells also express the IL-4 receptor and the c-kit ligand (KL), a factor that, in concert with commitment factors, channels progenitors into hemopoietic lineages. The expression of low constitutive levels of KL mRNA does not require IL-4, but KL mRNA levels are increased dramatically in response to IL-4. Since constitutive expression of KL mRNA in vivo is restricted to a small subset of TE cells in the thymus, our findings reveal a novel specific interaction between thymocytes and a specialized subset of TE cells.

The murine rac1 gene: cDNA cloning, tissue distribution and regulated expression of rac1 mRNA by disassembly of actin microfilaments.

A 886 bp cDNA encoding the murine rac1 protein has been isolated. The abundance of rac1 mRNA was determined in fourteen tissues from both mouse and pig. The mRNA 5' non-coding sequence is very rich in G + C, and has the potential to form several stable secondary structures. In addition, this region contains a putative open reading frame of 57 amino acids. Disruption of the actin microfilament network by cytochalasin B in LLC-PK1 cells results in down regulation of rac1 mRNA. In agreement with the proposed role of rac1 in exocytosis, these results could explain the inhibitory effect of cytochalasin B on secretory processes.