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ABSTRACTMicroglia, resident myeloid immune cells of the central nervous system (CNS), actively shape the circuitry of the brain, maintain CNS homeostasis during the steady state and orchestrate immune responses upon CNS injury. Both canonical and non-canonical functions of the macroautophagy/autophagy-related protein ATG5 regulate myeloid cell survival and immune responses. Here, we report that loss of ATG5 in postnatal microglia does not perturb CNS tissue integrity, microglial cell survival, or immune activation. Learning task performances were unchanged in mutant mice. Furthermore, lack of ATG5 expression in microglia had no impact on the development of experimental autoimmune encephalomyelitis. These data indicate that, basal autophagy, identified to be essential for the survival and function of neuronal cells, is not required to maintain CNS homeostasis if absent in adult microglia and ATG5 expression is dispensable for the development of autoimmune neuroinflammation.AbbreviationsAg, antigen; APC, antigen presenting cell; ATG/Atg, autophagy-related; CD, cluster of differentiation; CNS, central nervous system; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; fl, floxed; LAP, LC3-associated phagocytosis; LC3, microtubule-associated protein 1 light chain 3; MFI, median fluorescence intensity; MHCII, major histocompatibility complex class II; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis

Although most nervous system diseases affect women and men differentially, most behavioral studies using mouse models do not include subjects of both sexes. Many researchers worry that data of female mice may be unreliable due to the estrous cycle. Here, we retrospectively evaluated sex effects on coefficient of variation (CV) in 5,311 mice which had performed the same place navigation protocol in the water‐maze and in 4,554 mice tested in the same open field arena. Confidence intervals for Cohen's d as measure of effect size were computed and tested for equivalence with 0.2 as equivalence margin. Despite the large sample size, only few behavioral parameters showed a significant sex effect on CV. Confidence intervals of effect size indicated that CV was either equivalent or showed a small sex difference at most, accounting for less than 2% of total group to group variation of CV. While female mice were potentially slightly more variable in water‐maze acquisition and in the open field, males tended to perform less reliably in the water‐maze probe trial. In addition to evaluating variability, we also directly compared mean performance of female and male mice and found them to be equivalent in both water‐maze place navigation and open field exploration. Our data confirm and extend other large scale studies in demonstrating that including female mice in experiments does not cause a relevant increase of data variability. Our results make a strong case for including mice of both sexes whenever open field or water‐maze are used in preclinical research.

The NONO protein has been characterized as an important transcriptional regulator in diverse cellular contexts. Here we show that loss of NONO function is a likely cause of human intellectual disability and that NONO-deficient mice have cognitive and affective deficits. Correspondingly, we find specific defects at inhibitory synapses, where NONO regulates synaptic transcription and gephyrin scaffold structure. Our data identify NONO as a possible neurodevelopmental disease gene and highlight the key role of the DBHS protein family in functional organization of GABAergic synapses.

Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aβ, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD.

Abstract.Phosphatidylinositol-3-kinase (PI3K) signaling influences susceptibility to virus infections, anoxia, obstetric complications, and cancer; which are changed in patients with schizophrenia and their first degree relatives. Therefore PI3K signaling might have impact on the pathophysiology of schizophrenia. PI3K signaling crucially involves phosphoinositide-dependent protein kinase (PDK1). Increased anxiety behavior is observed in PDK1 hypomorphic mice. Here we show enhanced prevalence of schizophrenia in carriers of the PDK1 CC genotype in human beings. Moreover, decreased parietal P300 amplitude, which is a well-studied schizophrenic endophenotype, was observed in PDK1 CC carriers. Glutamate and glutamine concentrations are increased in the frontal lobe of PDK1 dysmorphic mice and human CC individuals. Our results demonstrate that the PDK1 CC genotype is associated with increased risk to develop schizophrenia, a typical endophenotype profile observed in the disease and modified neurotransmitter concentrations in brain regions associated with the disease.

Tissue inhibitors of metalloproteinases (TIMPs) are a family of closely related proteins that inhibit matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMPs 2, 3, and 4 are constitutively expressed at high levels, whereas TIMP1 can be induced by various stimuli. Here, we studied the effects of constitutive expression of TIMP1 in the CNS in transgenic mice. Transgene expression started prenatally and persisted throughout lifetime at high levels. Since MMP activity has been implicated in CNS development, in proper function of the adult CNS, and in inflammatory disorders, we investigated Timp1-induced CNS alterations. Despite sufficient MMP inhibition, high expressor transgenic mice had a normal phenotype. The absence of compensatory up-regulation of MMP genes in the CNS of Timp1transgenic mice indicates that development, learning, and memory functions do not require the entire MMP arsenal. To elucidate the effects of strong Timp1 expression in CNS inflammation, we induced experimental allergic encephalomyelitis. We observed a Timp1 dose-dependent mitigation of both experimental allergic encephalomyelitis symptoms and histological lesions in the CNS of transgenic mice. All in all, our data demonstrate that (1) long-term CNS expression of TIMP1 with complete suppression of gelatinolytic activity does not interfere with physiological brain function and (2) TIMP1 might constitute a promising candidate for long-term therapeutic treatment of inflammatory CNS diseases such as multiple sclerosis.

P revious behavioral studies (Minichiello et al., Neuron 1999;24:401–414) showed that mice deficient for the TrkB receptor in the forebrain were unable to learn a swimming navigation task with an invisible platform and were severely impaired in finding a visible platform in the same setup. Likewise, additional behavioral deficits suggested a malfunction of the hippocampus and proximally connected forebrain structures. In order to discriminate whether the behavioral impairment was caused either by deficits in spatial memory and learning, or alternatively by loss of behavioral flexibility, 8 trkBmutant, 13 wild‐type, and 22 heterozygous mice were implanted with transponders and released for 21 days into a large outdoor pen (10 × 10 m). The enclosure contained 2 shelters and 8 computer‐controlled feeder boxes, delivering food portions for every mouse only during their first visit. Every third day, mice received food ad libitum inside the shelters. All mice learned to patrol the boxes correctly within a few days. However, significant differences emerged during those days with free food available. Wild‐type mice remained inside the shelters, while all homozygous mutants continued to patrol the boxes in their habitual way, the heterozygous mutants showing intermediate scores. These and previous data suggest that one of the natural functions of the mouse hippocampus is to comediate behavioral flexibility, and that TrkB receptors might play an essential role in maintaining the neuronal short‐term plasticity necessary for this capacity. Hippocampus 2002;12:27–38. © 2002 Wiley‐Liss, Inc.

No abstract.

We have mapped the spatio-temporal expression of the multidomain serine protease neurotrypsin in the developing mouse by in situhybridization. On embryonic day (E) 8, mRNA is detected in giant trophoblast cells, later in embryonic mesenchymal tissues. On E11, expression begins in Schwann cell precursors, olfactory epithelium, trigeminal ganglion, and midbrain. The floor plate shows strong expression on E12. Further prenatal development is characterized by rising neurotrypsin mRNA in sensory ganglia and motor neurons. Staining in cerebral cortex emerges around birth and culminates toward the end of the first week with a complex laminar and areal pattern. Expression in peripheral nerves and nonneural tissues vanishes soon after birth and the adult neuronal distribution is gradually established until weaning age. This developmental expression pattern suggests roles of neurotrypsin in morphogenesis of nonneural tissues, as well as in neural development, in particular in axonal target invasion, synaptogenesis, and Schwann cell differentiation.

Abstract.: Several lines of mice with targeted deletion of the prion protein gene (Prnp) have been produced, some of them appearing phenotypically normal, others developing late-onset ataxia. This has been tentatively attributed to the size of the targeted deletion in the Prnp gene, but a masking role of genetic background could not be excluded. Thus, we have crossed an ataxic mutant line with large deletion of Prnp (Ngsk Prnp 0/0) with a knockout line showing only partial deletion of Prnp and no neurological deficits (Zrch1 Prnp 0/0). A F2 generation was then studied for up to 70 weeks for co-segregation of lesion size and behavioral phenotype, including cognitive and neurological anomalies. These mice were later crossed with a recently generated PrP-deficient line also having a large deletion and late-onset ataxia (Zrch2 Prnp 0/0). They underwent similar testing for up to 90 weeks. The ataxic phenotype always co-segregates with large homozygous deletions involving either the Ngsk or the Zrch2 allele, independent of genetic background or sex. Compound heterozygous Zrch1/Ngsk mice or Zrch1/Zrch2 mice showed intermediate neurological phenotypes, suggesting a gene-dosage effect of large deletions. At 12 weeks of age, large deletions were also associated with minor non-cognitive impairments in water maze learning, and hyperactivity in open field and elevated zero maze. These impairments were not predictive for the development of ataxia. Thus, the neurological deficits are closely associated with large deletions, which entail an upregulation of the recently discovered prion Doppel protein (Dpl), while genetic background factors seem to be responsible for shifting the onset of neurological symptoms.

The distinction between the different classes of glycolipids is conditioned by the action of specific core transferases. The entry point for lacto-series glycolipids is catalyzed by the β1,3N-acetylglucosaminyltransferase GlcNAc(β1,3)Gal(β1,4)Glc-ceramide (Lc3) synthase enzyme. The Lc3 synthase activity has been shown to be regulated during development, especially during brain morphogenesis. Here, we report the molecular cloning of a mouse gene encoding an Lc3 synthase enzyme. The mouse cDNA included an open reading frame of 1131 base pairs encoding a protein of 376 amino acids. The Lc3 synthase protein shared several structural motifs previously identified in the members of the β1,3 glycosyltransferase superfamily. The Lc3 synthase enzyme efficiently utilized the lactosyl ceramide glycolipid acceptor. The identity of the reaction products of Lc3 synthase-transfected CHOP2/1 cells was confirmed by thin-layer chromatography immunostaining using antibodies TE-8 and 1B2 that recognize Lc3 and Gal(β1,4)GlcNAc(β1,3)Gal(β1,4)Glc-ceramide (nLc4) structures, respectively. In addition to the initiating activity for lacto-chain synthesis, the Lc3 synthase could extend the terminalN-acetyllactosamine unit of nLc4 and also had a broad specificity for gangliosides GA1, GM1, and GD1b to generate neolacto-ganglio hybrid structures. The mouse Lc3 synthase gene was mainly expressed during embryonic development. In situhybridization analysis revealed that that the Lc3 synthase was expressed in most tissues at embryonic day 11 with elevated expression in the developing central nervous system. Postnatally, the expression was restricted to splenic B-cells, the placenta, and cerebellar Purkinje cells where it colocalized with HNK-1 reactivity. These data support a key role for the Lc3 synthase in regulating neolacto-series glycolipid synthesis during embryonic development.

Increasingly sophisticated and precise molecular genetic tools are applied to mice in order to study the cellular mechanisms underlying higher brain functions, including learning and memory. However, several studies have produced unclear or conflicting results. One reason for this is that performance in the behavioural tests used to assess learning and memory is influenced by various non-cognitive phenomena and can thus easily be affected by mutations through mechanisms unrelated to memory function. We conducted principal component analysis on data from 3003 mice tested using a standardized protocol to demonstrate this for the Morris swimming navigation test, one of the most widely used paradigms to assess memory and hippocampal function. In addition, we present a meta-analysis showing that genetic background and environment alone produce sufficient variation to span the range of most, if not all, behavioural variables and can thus easily mask or fake mutation effects if genetic studies are not designed properly. We suggest that the chance of obtaining useful results is maximized if behavioural deficits are differentiated by combining complementary behavioural protocols and by analysing multiple complementary parameters in each of them. Mutation effects must be contrasted statistically against the influences of genetic background and environment. In many situations, this is most efficiently achieved if (i) mutations are backcrossed to and maintained in one or (preferably) two well-characterized, commonly available inbred strains and (ii) if mutant and wild-type littermates are analysed on a hybrid or mixed genetic background, that is in F1 or F2 generations derived from the inbred stocks

Increasingly sophisticated and precise molecular genetic tools are applied to mice in order to study the cellular mechanisms underlying higher brain functions, including learning and memory. However, several studies have produced unclear or conflicting results. One reason for this is that performance in the behavioural tests used to assess learning and memory is influenced by various non‐cognitive phenomena and can thus easily be affected by mutations through mechanisms unrelated to memory function. We conducted principal component analysis on data from 3003 mice tested using a standardized protocol to demonstrate this for the Morris swimming navigation test, one of the most widely used paradigms to assess memory and hippocampal function. In addition, we present a meta‐analysis showing that genetic background and environment alone produce sufficient variation to span the range of most, if not all, behavioural variables and can thus easily mask or fake mutation effects if genetic studies are not designed properly. We suggest that the chance of obtaining useful results is maximized if behavioural deficits are differentiated by combining complementary behavioural protocols and by analysing multiple complementary parameters in each of them. Mutation effects must be contrasted statistically against the influences of genetic background and environment. In many situations, this is most efficiently achieved if (i) mutations are backcrossed to and maintained in one or (preferably) two well‐characterized, commonly available inbred strains and (ii) if mutant and wild‐type littermates are analysed on a hybrid or mixed genetic background, that is in F1 or F2 generations derived from the inbred stocks

We showed previously for mice that size differences of the infrapyramidal hippocampal mossy fiber projection (IIP-MF) correlate with spatial learning abilities. In order to clarify the role of the IIP-MF in a natural environment, we studied the bank vole (Clethrionomys glareolus), adapted to a wide range of different habitats, and the root vole (Microtus oeconomus), living in homogenous grassland habitats with small home ranges. Morphometry on Timm-stained horizontal brain sections of six C. glareolus and six M. oeconomus revealed that the size of the entire mossy fiber projection was 42% larger in C. glareolus than M. oeconomus. C. glareolus had also an IIP-MF projection about 230% larger than that of the root vole. A sample of captured animals was then transferred to the laboratory (C. glareolus, n = 23; M. oeconomus, n = 15) and underwent testing for swimming navigation according to a standardized protocol used to assess water maze learning in about 2,000 normal and transgenic mice. Both species learned faster than laboratory mice. Overall escape times showed no differences, but path length was significantly reduced in C. glareolus, which also showed superior performance in a variety of scores assessing spatial search patterns. On the other hand, M. oeconomus showed faster swimming speed, and strong thigmotaxis combined with circular swimming. M. oeconomus also scored at chance levels during the probe trial, about as poorly as mutant knockout mice considered to be deficient in spatial memory. These differences probably reflect differential styles of water maze learning rather than spatial memory deficits: C. glareolus appears to be superior in inhibiting behavior interfering with proper spatial search behavior, while M. oeconomus succeeds in escaping by using rapid circular swimming. We assume that size variations of the IIP-MF correspond to a mechanism stabilizing hippocampal processing during spatial learning or complex activities. This corresponds to the ecological lifestyle of the two species and is in line with previous observations on the role of the IIP-MF. Hippocampus 2000;10:17–30. © 2000 Wiley-Liss, Inc.

We showed previously for mice that size differences of the infrapyramidal hippocampal mossy fiber projection (IIP‐MF) correlate with spatial learning abilities. In order to clarify the role of the IIP‐MF in a natural environment, we studied the bank vole (Clethrionomys glareolus), adapted to a wide range of different habitats, and the root vole (Microtus oeconomus), living in homogenous grassland habitats with small home ranges.

We have cloned a novel murine cDNA encoding a multidomain serine protease, termed neurotrypsin, which exhibits an unprecedented domain composition. The deduced amino acid sequence defines a mosaic protein of 761 amino acids consisting of a kringle domain, followed by three scavenger receptor cysteine-rich repeats, and a serine protease domain. Based on comparisons of the primary structure, the protease domain belongs to the subfamily of trypsin-like serine proteases.In situhybridization revealed that the expression of neurotrypsin in the adult murine nervous system is confined to distinct subsets of neurons. The most prominent expression was found in the cerebral cortex, the hippocampus, and the amygdala, i.e., structures engaged in the processing and storage of learned behaviors and memories. Together with the recently obtained evidence that extracellular serine proteases play a role in neural plasticity, this expression pattern suggests that the extracellular proteolytic action of neurotrypsin subserves structural reorganizations associated with learning and memory operations.

Spatial learning of transgenic mice is often assessed in the Morris watermaze, where mice must use distant cues to locate a submerged platform. Such learning is confounded by species-specific noncognitive swimming strategies. Factor analysis permits cognitive and noncognitive strategies to be disentangled and their association with electrophysiological phenomena to be investigated.

Prenatally administered oxazepam (OX) impairs adult radial maze performance in mice, possibly by permanent hippocampal changes. CDI mice were tested in swimming navigation, a sensitive indicator for hippocampal damage. Ten males and ten females were exposed to OX on fetal days 12–16 by maternal administration PO of 30 mg/kg/day and fostered at birth to untreated dams, while control mice received vehicle solution. All mice were tested at 8–9 weeks for ability to find a submerged platform in a fixed location (acquisition: 18 trials, 6 trials per day) and for capacity to re-orient towards a new platform position (reversal: 12 trials, 6 trials per day). OX mice showed a slight but significant impairment of swimming navigation during the initial part of training, as indicated by longer swimming paths during the fourth and fifth trial (day 1), an impairment due both to delayed habituation to the novel stressfull condition and acquisition of platform climbing but unrelated to navigational abilities. No treatment-dependent differences were observed in the reversal phase. During reversal, both OX and control females spent significantly more time in swimming across the location of the old platform. Unrelated to navigational performance, females showed a slightly but significantly higher swimming speed than males. Due to the absence of any navigational impairment, data suggest that prenatal exposure to oxazepam exerts long-term influence on adult learning capacities primarily through interaction with brain systems located outside the hippocampus.

Female mice from inbred strains carrying a Robertsonian translocation (nine CBARb and eight C57BL/6Rb) were compared with animals from their respective strains (seven CBA and nine C57BL/6) first in open-field activity (two exposures of 10-min duration), then during 5 days (with six trials each) in Morris' swimming navigation test, and finally, in their ability to extrapolate the future position of a food reward being moved slowly out of their reach. ANOVA (strain and translocation) revealed significant effects of Robertsonian translocations (Rb) in swimming navigation,Rb mice being impaired primarily in the initial phases of acquisition and during the first trials of platform reversal and the impairment being stronger in C57BL/6 mice. In the open field,Rb mice were as active as the normal strains but showed significantly increased path tortuosity and moved slightly faster. In the extrapolation task,Rb mice showed above-chance levels in moving to the target indicated by the disappearance of the stimulus, while normal mice chose at chance levels, but the translocation effects were not statistically significant. These data indicate that telocentric fusion of chromosomes may entail behavioral alterations, perhaps by subtle changes in neurotransmitters or limbic circuitry. The expression of such alterations, however, can be remarkably strain dependent.

Spatial learning of transgenic mice is often assessed in the Morris watermaze, where mice must use distant cues to locate a submerged platform. Such learning is confounded by species-specific noncognitive swimming strategies. Factor analysis permits cognitive and noncognitive strategies to be disentangled and their association with electrophysiological phenomena to be investigated.

The size of the intra-/infrapyramidal mossy fiber projections (IIP-MF) and their left/right asymmetry were assessed in 86 mice of either sex, including 26 animals from two mouse lines bred for strong or weak paw preference, 38 mice of a randomly bred F3generation derived from an eight-way cross, and 22 mice with variably sized corpora callosa in which only the left hippocampus was measured. Prior to morphometry, all mice were tested for paw preference. In addition, we compared the strain means in paw preference as observed in nine inbred mouse strains with known differences in their IIP-MF distribution. Mice bred for strong paw preference had a 70% larger IIP-MF projection than weakly lateralized and dyscallosal mice; random-bred mice fell in-between the extremes. The individual scores of the strength of paw preference were positively correlated with the extent of the IIP-MF. Among the inbred strains, the extent of the IIP-MF was similarly correlated with the strength of paw preference. The acallosal mice showed a significant negative correlation between extent of the IIP-MF projection and test-retest reliability of paw use. The left-right asymmetry of the IIP-MF was significantly and positively correlated with the direction of paw preference in the entire sample. We conclude that size and asymmetry of the IIP-MF projection are some of the many factors influencing the direction of paw preference and its strength, albeit moderately. We hypothesize that mice with larger IIP-MF projections use a given paw more consistently, being perhaps more resistant to interferences, and that left-right asymmetries of the IIP-MF may bias and/or reinforce an initial choice of a paw. In addition, the data provide another example of correlations between IIP-MF variations and nonspatial behavior.

The brains of mice selectively bred for differential locomotor activity in an open field (DeFrieset al., Behav. Genet.8:3–13, 1978) were analyzed for selection-dependent changes in the size of synaptic fields at the midseptotemporal level of the hippocampus. Timm-stained areas of all hippocampal fields from both left and right hippocampi were measured on five horizontal sections from the midseptotemporal level. The sample included 25 mice from two replicate lines, each one consisting of a high (HI); a low (LO), and a control line (CTL). The main selection effect was an enlargement of the intrainfrapyramidal mossy fiber (IIP-MF) projection in both HI lines by about 70% compared to LO and CTL mice (p<.0001), while other mossy fiber fields did not show differences. These findings confirm that genetic variations of the IIP-MF projection influence hippocampal processes mediating exploratory activities.