Hyperactivity,startle reactivity and cell‐proliferation deficits are resistant to chronic lithium treatment in adult Nr2e1frc/frc mice

The NR2E1 region on Chromosome 6q21‐22 has been repeatedly linked to bipolar disorder (BP) and NR2E1 has been associated with BP, and more specifically bipolar I disorder (BPI). In addition, patient sequencing has shown an enrichment of rare candidate‐regulatory variants. Interestingly, mice carrying either spontaneous (Nr2e1^frc) or targeted (Tlx^?) deletions of Nr2e1 (here collectively known as Nr2e1‐null) show similar neurological and behavioral anomalies, including hypoplasia of the cerebrum, reduced neural stem cell proliferation, extreme aggression and deficits in fear conditioning; these are the traits that have been observed in some patients with BP. Thus, NR2E1 is a positional and functional candidate for a role in BP. However, no Nr2e1‐null mice have been fully evaluated for behaviors used to model BP in rodents or pharmacological responses to drugs effective in treating BP symptoms. In this study we examine Nr2e1^frc/frc mice, homozygous for the spontaneous deletion, for abnormalities in activity, learning and information processing, and cell proliferation; these are the phenotypes that are either affected in patients with BP or commonly assessed in rodent models of BP. The effect of lithium, a drug used to treat BP, was also evaluated for its ability to attenuate Nr2e1^frc/frc behavioral and neural stem cell‐proliferation phenotypes. We show for the first time that Nr2e1‐null mice exhibit extreme hyperactivity in the open field as early as postnatal day 18 and in the home cage, deficits in open‐field habituation and passive avoidance, and surprisingly, an absence of acoustic startle. We observed a reduction in neural stem/progenitor cell proliferation in Nr2e1^frc/frc mice, similar to that seen in other Nr2e1‐null strains. These behavioral and cell‐proliferation phenotypes were resistant to chronic‐adult‐lithium treatment. Thus, Nr2e1^frc/frc mice exhibit behavioral traits used to model BP in rodents, but our results do not support Nr2e1^frc/frc mice as pharmacological models for BP.     

Prepulse inhibition （PPI） of tactile startle response in recombinant congenic strains of mice： QTL mapping and comparison with acoustic PPI

Prepulse inhibition (PPI) of the startle response is a psychophysiological measure of sensorimotor gating believed to be cross-modal between different sensory systems.We analyzed the tactile startle response (TSR) and PPI of TSR (tPPD,using light as a prepulse stimulus,in the mouse strains A/J and C57BL/6J and 36 recombinant congenic strains derived from them.Parental strains were significantly different for TSR,but were comparable for tPPI.Among the congenic strains,variation for TSR was significant in both genetic backgrounds,but that of tPPI was significant only for the C57BL/6J background.Provisional mapping for loci modulating TSR and tPPI was carded out.Using mapping data from our previous study on acoustic startle responses (ASR) and PPI of ASR (aPPI),no common markers for aPPI and tPPI were identified.However,some markers were significantly associated with both ASR and TSIL at least in one genetic background.These results indicate cross-modal genetic regulation for the startle response but not for PPI,in these mouse strains.     

Difference in anxiety and sensitization of the acoustic startle response between the two inbred mouse strains BALB/cAN and DBA/2N

The inbred mouse strain BALB has been proposed to be an animal model for pathological anxiety. BALB exhibits a stronger acoustic startle response (ASR) than the 'less emotional' inbred strain DBA. Four experiments were conducted to determine whether this strong ASR is due to a higher anxiety level and/or to greater sensitization in BALB than in DBA, with the following results: (1) The ASR to the very first startle stimulus was found to be much stronger in BALB than in DBA, and freezing behavior evoked by startle stimuli was more pronounced in BALB than in DBA. These findings indicate a higher level of anxiety in this strain. (2) ASR amplitudes of BALB initially rose much higher during consecutive startle stimuli and remained at a high level much longer than in DBA. Thereafter, ASR amplitude dropped more slowly and to a lesser degree than in DBA. Startle amplitudes decreased similarly in both strains (strong exponential decrease) only when a low sound pressure level (SPL) was used which elicited approximately the same low ASR in both strains. These results can only be explained by increased sensitization in BALB. (3) The slope of the i/o-function, which represents the relation between sensory input and motor output, was steeper in BALB than in DBA. As it has been shown recently, sensitization increases the slope of the startle i/o-function indicating increased sensitization in BALB. It is discussed, however, whether anxiety also contributes to this effect. (4) Footshocks increased the ASR much less in BALB than in DBA, again showing increased sensitization in BALB. Both a higher level of anxiety and greater sensitization therefore determined the greater strength of the ASR in BALB than in DBA.     

Optimization of the touchscreen paired-associate learning (PAL) task for mice and its dorsal hippocampal dependency

Two versions of the touchscreen paired-associate learning (PAL) task have been developed for rodents: same PAL (sPAL) and different PAL (dPAL). These tasks are very important in studying murine models of Alzheimer’s disease and schizophrenia, and have also been used to test object-location memory in various studies. However, the relatively long time needed for the tasks (approx. 50 days for mice) limits their widespread use. By giving training that was more intensive with a higher number of trials, we shortened the time required for learning saturation in sPAL and dPAL to about one-third of the time required for the generally used protocol. Furthermore, by applying a reduced number of objects and trial types for sPAL, we developed a simplified version of sPAL, termed 2-object sPAL, in which mice could reach the fully learned level in 6 days. Our pharmacological experiments indicate that the dorsal hippocampal CA1 region is crucial for the performance of the two PAL tasks with the new protocols and the new 2-object sPAL. This work has significantly enhanced the usefulness of the touchscreen PAL tasks to increase the speed of learning, but they remain highly hippocampus-dependent object-location memory tasks.     

Reduced programmed cell death in the retina and defects in lens and cornea of Tgfbeta2(-/-) Tgfbeta3(-/-) double-deficient mice

We have previously shown that immunoneutralization of transforming growth factor- (TGF-) in the chick embryo significantly reduces programmed cell death (PCD) in peripheral neurons, spinal cord, and retina. In order to validate these results we have begun to analyze PCD in mice with targeted ablations of the TGF-2 and TGF-3 genes. Recent analyses of mice lacking TGF-3 had failed to reveal an overt eye phenotype, while retinae of TGF-2-deficient mice showed retinal hypercellularity. We report now that eyes of Tgf2/Tgf3 double-deficient mice display severe alterations in the morphology of the retina, lens, and cornea. The inner neural retina—the region where TGF- receptor (TR) I and II immunoreactivities are most prominent—is significantly thickened, and numbers of TUNEL-positive cells are significantly reduced compared to wild-type littermates. In Tgf2^–/–Tgf3^–/– and Tgf2^–/–Tgf3^+/– littermates the retina was consistently detached from the underlying pigment epithelium. Cornea, corneal stroma, and lens epithelium were significantly thinner in these mutants. In contrast, retinal morphology in Tgf2^+/–Tgf3^–/– mutant littermates resembles the situation observed in wild-type retinae except for the retinal detachment. Thus, regression in the thickness of cornea and corneal stroma seems to be TGF- isoform and gene dose dependent. Our results substantiate the notion based on previous analyses of chick embryos with reduced levels of endogenous TGF- that TGF-, most notably TGF-2, is required to mediate PCD in developing retinal cells in vivo. Moreover, our data indicate that TGF-s play essential roles in cornea and lens development.This work was supported by grants from the Deutsche Forschungsgemeinschaft.     

Hyposensitivity to the amnesic effects of scopolamine or amyloid β25–35 peptide in heterozygous acetylcholinesterase knockout (AChE) mice

We examined the sensitivity of AChE^+/− mice to the amnesic effects of scopolamine and amyloid β peptide. AChE^+/− and AChE^+/+ littermates, tested at 5–9 weeks of age, failed to show any difference in locomotion, exploration and anxiety in the open-field test, or in-place learning in the water-maze. However, when treated with the muscarinic receptor antagonist scopolamine (0.5, 5 mg/kg s.c.) 20 min before each water-maze training session, learning impairments were observed at both doses in AChE^+/+ mice, but only at the highest dose in AChE^+/− mice. The central injection of Aβ_25–35 peptide (9 nmol) induced learning deficits only in AChE^+/+ but not in AChE^+/− mice. Therefore, the hyper-activity of cholinergic systems in AChE^+/− mice did not result in increased memory abilities, but prevented the deleterious effects of muscarinic blockade or amyloid toxicity.     

Induction of T helper 1 response by immunization of BALB/c mice with the gene encoding the second subunit of Echinococcus granulosus antigen B (EgAgB8/2)

A pre-designed plasmid containing the gene encoding the second subunit of Echinococcus granulosus AgB8 (EgAgB8/2) was used to study the effect of the immunization route on the immune response in BALB/c mice. Mice were immunized with pDRIVEEgAgB8/2 or pDRIVE empty cassette using the intramuscular (i.m.), intranasal (i.n.) or the epidermal gene gun (g.g.) routes. Analysis of the antibody response and cytokine data revealed that gene immunization by the i.m. route induced a marked bias towards a T helper type 1 (Th1) immune response as characterized by high IFN-γ gene expression and a low IgG1/IgG2a reactivity index (R.I.) ratio of 0.04. The i.n. route showed a moderate IFN-γ expression but a higher IgG1/IgG2a R.I. ratio of 0.25 indicating a moderate Th1 response. In contrast, epidermal g.g. immunization induced a Th2 response characterized by high IL-4 expression and the highest IgG1/IgG2a R.I. ratio of 0.58. In conclusion, this study showed the advantage of genetic immunization using the i.m. route and i.n. over the epidermal g.g. routes in the induction of Th1 immunity in response to E. granulosus AgB gene immunization.