Mouse Strain and Sex-Dependent Differences in Long-term Behavioral Abnormalities and Neuropathologies after Developmental Zika Infection.

Exposure of the developing fetus to Zika virus (ZIKV) results in a set of brain abnormalities described as the congenital Zika syndrome. Although microcephaly is the most obvious outcome, neuropathologies, such as intracranial calcifications and polymicrogyria, can occur in the absence of microcephaly. Moreover, the full impact of exposure on motor, social, and cognitive skills during development remains uncharacterized. We examined the long-term neurobehavioral consequences of neonatal ZIKV exposure in four genetically divergent inbred mouse strains (C57BL/6J, 129S1/SvImJ, FVB/NJ, and DBA/2J). Male and female mice were infected on postnatal day 1, considered comparable with exposure late in the second trimester of humans. We demonstrate strain differences in early susceptibility to the virus and the time course of glial reaction in the brain. These changes were associated with strain- and sex-dependent differences in long-term behavioral abnormalities that include hyperactivity, impulsiveness, and motor incoordination. In addition, the adult brains of susceptible mice exhibited widespread calcifications that may underlie the behavioral deficits observed. Characterization of the neuropathological sequelae of developmental exposure to the Zika virus in different immunocompetent mouse strains provides a foundation for identifying genetic and immune factors that contribute to long-term neurobehavioral consequences in susceptible individuals.SIGNIFICANCE STATEMENT Developmental Zika virus (ZIKV) infection is now known to cause brain abnormalities in infants that do not display microcephaly at birth, and the full impact of these more subtle neuropathologies has yet to be determined. We demonstrate in a mouse model that long-lasting behavioral aberrations occur after developmental ZIKV exposure. We compare four divergent mouse strains and find that the effects of Zika infection differ greatly between strains, in terms of behavioral changes, sex differences, and the intracranial calcifications that develop in the brains of susceptible mice. These findings provide a foundation for identifying susceptibility factors that lead to the development of abnormal behaviors secondary to ZIKV infection early in life.

Staufen2 deficiency leads to impaired response to novelty in mice.

Staufen2 (Stau2) is a double-stranded RNA-binding protein (RBP) involved in posttranscriptional gene expression control in neurons. In flies, staufen contributes to learning and long-term memory formation. To study the impact of mammalian Stau2 on behavior, we generated a novel gene-trap mouse model that yields significant constitutive downregulation of Stau2 (Stau2GT). In order to investigate the effect of Stau2 downregulation on hippocampus-dependent behavior, we performed a battery of behavioral assays, i.e. open field, novel object recognition/location (NOR/L) and Barnes maze. Stau2GT mice displayed reduced locomotor activity in the open field and altered novelty preference in the NOR and NOL paradigms. Adult Stau2GT male mice failed to discriminate between familiar and newly introduced objects but showed enhanced spatial novelty detection. Additionally, we observed deficits in discriminating different spatial contexts in a Barnes maze assay. Together, our data suggest that Stau2 contributes to novelty preference and explorative behavior that is a driver for proper spatial learning in mice.

Oxytocin receptor knockout mice display deficits in the expression of autism-related behaviors.

A wealth of studies has implicated oxytocin (Oxt) and its receptors (Oxtr) in the mediation of social behaviors and social memory in rodents. It has been suggested that failures in this system contribute to deficits in social interaction that characterize autism spectrum disorders (ASD). In the current analyses, we investigated the expression of autism-related behaviors in mice that lack the ability to synthesize the oxytocin receptor itself, Oxtr knockout (KO) mice, as compared to their wild-type (WT) littermates. In the visible burrow system, Oxtr KO mice showed robust reductions in frontal approach, huddling, allo-grooming, and flight, with more time spent alone, and in self-grooming, as compared to WT. These results were corroborated in the three-chambered test: unlike WT, Oxtr KO mice failed to spend more time in the side of the test box containing an unfamiliar CD-1 mouse. In the social proximity test, Oxtr KO mice showed clear reductions in nose to nose and anogenital sniff behaviors oriented to an unfamiliar C57BL/6J (B6) mouse. In addition, our study revealed no differences between Oxtr WT and KO genotypes in the occurrence of motor and cognitive stereotyped behaviors. A significant genotype effect was found in the scent marking analysis, with Oxtr KO mice showing a decreased number of scent marks, as compared to WT. Overall, the present data indicate that the profile for Oxtr KO mice, including consistent social deficits, and reduced levels of communication, models multiple components of the ASD phenotype. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.

Behavioral and genetic investigations of low exploratory behavior in Il18r1(-/-) mice: we can't always blame it on the targeted gene.

The development of gene-targeting technologies has enabled research with immune system-related knockout mouse strains to advance our understanding of how cytokines and their receptors interact and influence a number of body systems, including the central nervous system (CNS). A critical issue when we are interpreting phenotypic data from these knockout strains is the potential role of genes other than the targeted one. Although many of the knockout strains have been made congenic on a C57BL/6 (B6) genetic background, there remains a certain amount of genetic material from the129 substrain that was used in the development of these strains. This genetic material could result in phenotypes incorrectly attributed to the targeted gene. We recently reported low-activity behavior in Il10(-/-) mice that was linked to this genetic material rather than the targeted gene itself. In the current study we confirm the generalizability of those earlier findings, by assessing behavior in Il18(-/-) and Il18r1(-/-) knockout mice. We identified low activity and high anxiety-like behaviors in Il18r1(-/-) mice, whereas Il18(-/-) mice displayed little anxiety-like behavior. Although Il18r1(-/-) mice are considered a congenic strain, we have identified substantial regions of 129P2-derived genetic material not only flanking the ablated Il18r1 on Chromosome 1, but also on Chromosomes 4, 5, 8, 10, and 14. Our studies suggest that residual 129-derived gene(s), rather than the targeted Il18r1 gene, is/are responsible for the low level of activity seen in the Il18r1(-/-) mice. Mapping studies are necessary to identify the gene or genes contributing to the low-activity phenotype.

Brain Iron Accumulation and the Formation of Calcifications After Developmental Zika Virus Infection.

Intracranial calcifications (ICC) are the most common neuropathological finding in the brains of children exposed in utero to the Zika virus (ZIKV). Using a mouse model of developmental ZIKV infection, we reported widespread calcifications in the brains of susceptible mice that correlated in multiple ways with the behavioral deficits observed. Here, we examined the time course of ICC development and the role of iron deposition in this process, in 3 genetically distinct inbred strains of mice. Brain iron deposits were evident by Perls' staining at 2 weeks post infection, becoming increasingly dense and coinciding with calcium buildup and the formation of ICCs. A regional analysis of the brains of susceptible mice (C57BL/6J and 129S1/SvImJ strains) revealed the presence of iron initially in regions containing many ZIKV-immunoreactive cells, but then spreading to regions containing few infected cells, most notably the thalamus and the fasciculus retroflexus. Microglial activation was widespread initially and later delineated the sites of ICC formation. Behavioral tests conducted at 5-6 weeks of age revealed greater deficits in mice with the most extensive iron deposition and calcification of subcortical regions, such as thalamus. These findings point to iron deposition as a key factor in the development of ICCs after developmental ZIKV infection.

Intrasession and intersession habituation in mice: from inbred strain variability to linkage analysis.

When placed in a novel environment, mice tend to explore for a period of time, and then reduce the level of exploration. This reduction in locomotor or exploratory behavior is known as habituation and can occur within a single session or across sessions, respectively, termed intrasession and intersession habituation. Recent research indicates that there is a genetic component to habituation behavior and that some of the genes involved differ between the two types of habituation. The genetic evidence also suggests that intrasession habituation and intersession habituation are measuring somewhat different conceptual entities and with more such evidence may eventually help us understand the different pathways involved. Some of the genetic methods and tools used to unravel the roles of specific genes in both types of habituation are outlined here, with examples from the literature, as well as new data, to illustrate that this seemingly simple behavior is actually very complicated in terms of genetics. Evidence to date suggests that a number of genetic regions play roles in one or both types of habituation, and further research will be necessary to determine the specific genes involved.

Cautionary insights on knockout mouse studies: the gene or not the gene?

Gene modification technologies play a vital role in the study of biological systems and pathways. Although there is widespread and beneficial use of genetic mouse models, potential shortcomings of gene targeting technology exist, and are not always taken into consideration. Oversights associated with the technology can lead to misinterpretation of results; for example, ablation of a gene of interest can appear to cause an observed phenotype when, in fact, residual embryonic stem cell-derived genetic material in the genetic background or in the area immediately surrounding the ablated gene is actually responsible. The purpose of this review is to remind researchers, regardless of scientific discipline, that the background genetics of a knockout strain can have a profound influence on any observed phenotype. It is important that this issue be appropriately addressed during data collection and interpretation.

Antibody induction of lupus-like neuropsychiatric manifestations.

Although systemic lupus erythematosus (SLE) is usually evaluated with regard to autoimmune reactivity toward the kidney, there are multiple psychiatric abnormalities associated with this autoimmune disease. Lupus-prone male NZM88 mice, derived from NZB/NZW F1 mice, develop early neuropsychiatric manifestations without any signs of nephritis. In addition to the usual repertoire of antibody specificities, including autoantibodies to dsDNA and renal antigens, mice of this inbred strain express autoantibodies to numerous brain antigens. Here, we show that autoantibodies to brain antigens, assessed by Western analysis, are as individually varied as are the diverse neuropsychiatric manifestations observed in SLE patients. Additionally, a monoclonal antibody derived from the spleen of an untreated NZM88 male when injected into healthy BALB/cByJ, but not C57BL/6J, mice induced behaviors similar to those of lupus-prone NZM88 mice. This monoclonal antibody, which is specific to dynamin-1, binds preferentially in BALB/cByJ cortex and induces substantial expression of cytokines mainly in the hypothalamus. Thus, an antibody to just one brain antigen can induce multiple behavioral changes, and multiple autoantibodies to different brain antigens exist in lupus-prone mice; however, susceptibility to the induction of neurobehavioral deficits is dependent on host genetics.

Assessing autism-like behavior in mice: variations in social interactions among inbred strains.

Autism is a pervasive developmental disorder, with characteristics including impairments in reciprocal social interaction, impaired communication, and repetitive/stereotyped behaviors. Despite decades of research, the etiology of autism remains elusive. Thus, it is important that we pursue all avenues, in attempting to understand this complicated disorder. One such avenue is the development of animal models. While autism may be uniquely human, there are behavioral characteristics of the disorder that can be established in animal models. Evidence supports a genetic component for this disorder, and over the past few decades the mouse has been a highly valuable tool for the elucidation of pathways involved in many human disorders (e.g., Huntington's disease). As a first step toward establishing a mouse model of autism, we studied same-sex social behavior in a number of inbred mouse strains. In Study 1, we examined intra-strain social behavior of male pairs after one mouse had 15 min prior exposure to the testing chamber. In Study 2, we evaluated intra-strain and inter-strain social behavior when both mice were naive to the testing chamber. The amount and type of social behavior seen differed between these studies, but overall there were general inbred strain differences in social behavior. Some strains were highly social, e.g., FVB/NJ, while others displayed low levels of social behavior (e.g., A/J, BTBR T+tf/J). These strains may be useful in future genetic studies to determine specific genes involved in mouse social behavior, the findings of which should in turn help us to determine some of the genes involved in human social behavior and its disorders (e.g., autism).

Divergence and inheritance of neocortical heterotopia in inbred and genetically-engineered mice.

Cortical function emerges from the intrinsic properties of neocortical neurons and their synaptic connections within and across lamina. Neurodevelopmental disorders affecting migration and lamination of the neocortex result in cognitive delay/disability and epilepsy. Molecular layer heterotopia (MLH), a dysplasia characterized by over-migration of neurons into layer I, are associated with cognitive deficits and neuronal hyperexcitability in humans and mice. The breadth of different inbred mouse strains that exhibit MLH and inheritance patterns of heterotopia remain unknown. A neuroanatomical survey of numerous different inbred mouse strains, 2 first filial generation (F1) hybrids, and one consomic strain (C57BL/6J-Chr 1A/J/NaJ) revealed MLH only in C57BL/6 mice and the consomic strain. Heterotopia were observed in numerous genetically-engineered mouse lines on a congenic C57BL/6 background. These data indicate that heterotopia formation is a weakly penetrant trait requiring homozygosity of one or more C57BL/6 alleles outside of chromosome 1. These data are relevant toward understanding neocortical development and disorders affecting neocortical lamination.

Habituation in rodents: a review of behavior, neurobiology, and genetics.

Habituation to a novel environment in rodents is commonly defined as a change in exploratory or locomotor activity over time (intrasession) or with repeated exposures (intersession). While numerous neuroactive substances are known to influence habituation, neurotransmitters that play particularly important roles are serotonin, acetylcholine, dopamine and glutamate. Although habituation is a complex process, studies over the past two decades have demonstrated that there is a genetic component. At present, although researchers are still attempting to isolate key genes that control habituation, findings in mutant mice have begun to highlight some of the genes that could play a role. The challenge will be in deciphering what genes are directly involved in the process of habituation, what genes indirectly influence habituation through a secondary mechanism, and what genes have no role in habituation but are only affected as part of the downstream cascade.

Behavioral differences among fourteen inbred mouse strains commonly used as disease models.

We compared the behavior of 14 inbred mouse strains and an F1 hybrid commonly used in transgenic and knockout production. These strains were 129P3/J, 129S1/SvImJ, 129S6/SvEvTac, 129T2/SvEmsJ, 129X1/SvJ (formerly 129/J, 129/Sv-p+Tyr+Kitl+/J, 129/SvEvTac, 129SvEmsJ, and 129/SvJ, respectively), A/JCrTac, BALB/cAnNTac, C3H/HeNTac, C57BL/6J, C57BL/6NTac, DBA/2NTac, FVB/NTac, NOD/MrkTac, SJL/JCrNTac, and the hybrid B6129S6F1Tac. Performance in three behavioral tests (rotorod, open-field activity-habituation, and contextual and cued fear conditioning) was determined. On the rotorod assay, SJL/JCrNTac mice had the shortest latencies to fall on the first day of testing, and DBA/2NTac mice showed impaired motor learning. Open-field behavior was analyzed using the parameters total distance, center distance, velocity, and vertical activity. 129T2/EvEmsJ and A/JCrTac were least active in the open field, whereas NOD/MrkTac mice were most active. Contrary to earlier studies, we found that all strains habituated to the open field in at least one of these parameters. In contextual and cued fear conditioning, all strains displayed activity suppression. However, FVB/NTac mice reacted less strongly to both context and cue than did most of the other strains. There were no significant behavioral differences between C57BL/6J and C57BL/6NTac, except for higher open-field activity in C57BL/6J female mice. These findings illustrate the importance of the appropriate selection of background strain for transgenic, gene targeting, or drug research.

Exploratory activity and fear conditioning abnormalities develop early in R6/2 Huntington's disease transgenic mice.

The Huntington's disease R6/2 transgenic mouse model, containing exon 1 of the human huntingtin gene with a greatly increased CAG repeat length, shows multiple effects of the altered polyglutamine in the resultant protein. The authors report that exploratory and fear conditioning behavioral changes appear well before the onset of obvious pathology. The first differences in exploratory and fear conditioning behavior emerge by 4 and 5 weeks of age, respectively. These behaviors correlate with the earliest neurochemical and molecular changes previously reported and provide insight into functional mechanisms by which cellular and subcellular disease changes may mediate neurological symptoms. These studies provide behavioral protocols suitable for high-throughput screening of therapeutic agents.

Behavioral differences among 129 substrains: implications for knockout and transgenic mice.

Most knockout (KO) mice are produced with embryonic stem cells derived from a 129 strain. Because most KO strains are backcrossed to B6 yet retain a portion of their genome from 129, especially around the ablated target locus, phenotypes previously attributed to the ablated locus may be due to passenger 129 genes. Thus, the authors decided to test several 129 substrains for their behavioral characteristics. Seven 129 substrains were put through a battery of tasks to determine their behavioral profiles. Differences were found in anxiety-related behaviors in the zero-maze, habituation to the open field, and cued fear conditioning. All strains successfully performed the rotorod task. The behavioral differences observed may have important implications for the interpretation of data and show divergence of behavioral performance in these 129 substrains.

The development of behavioral abnormalities in the motor neuron degeneration (mnd) mouse.

The motor neuron degeneration (mnd) mouse, which has widespread abnormal accumulating lipoprotein and neuronal degeneration, has a mutation in CLN8, the gene for human progressive epilepsy with mental retardation (EPMR). EPMR is one of the neuronal ceroid lipofuscinoses (NCLs), a group of neurological disorders characterized by autofluorescent lipopigment accumulation, blindness, seizures, motor deterioration, and dementia. The human phenotype of EPMR suggests that, in addition to the motor symptoms previously categorized, various types of progressive behavioral abnormalities would be expected in mnd mice. We have therefore examined exploratory behavior, fear conditioning, and aggression in 2-3 month and 4-5 month old male mnd mice and age-matched C57BL/6 (B6) controls. The mnd mice displayed increased activity with decreased habituation in the activity monitor, poor contextual and cued memory, and heightened aggression relative to B6 controls. These behavioral deficits were most prominent at 4-5 months of age, which is prior to the onset of gross motor symptoms at 6 months. Our results provide a link from the mutation via pathology to a quantifiable multidimensional behavioral phenotype of this naturally occurring mouse model of NCL.

Early exploratory behavior abnormalities in R6/1 Huntington's disease transgenic mice.

The Huntington's disease (HD) R6/1 transgenic mouse model, containing a human huntington gene exon-1 with approximately 115 CAG repeats, has multiple biochemical and neuroanatomical abnormalities. Overt neurological symptoms have a relatively late onset (15-21 weeks of age). In this paper, we report exploratory behavior abnormalities that appear well before the onset of obvious pathology. The first differences in exploratory behaviors were evident by 4 weeks of age, when R6/1 mice were hyperactive relative to wild-type controls. However, by 6-7 weeks of age, R6/1 mice were less active than controls. R6/1 mice traveled less in the activity monitor, engaged in fewer stereotypic movements, spent more time resting, and traveled less distance per movement than did wild-type controls. R6/1 mice also displayed intersession habituation abnormalities over the 3 days of testing. These behavioral abnormalities precede the earliest neurochemical and molecular changes reported in the literature to date, and thus indicate subtle early pathology that has not yet been documented. These behavioral abnormalities also occur prior to weight loss in the transgenic mice. Since we were able to detect an abnormal phenotype at an early age in R6/1 mice, this assay may be a useful tool for evaluating therapeutic agents.

Maternal exposure to mercury chloride during pregnancy and lactation affects the immunity and social behavior of offspring.

Developmental HgCl2 exposures of F1 offspring (H-2(q/s)) from unsociable SJL/J (H-2(s)) dams with high susceptibility to Hg-induced autoimmunity (SFvF1) and from highly sociable FVB/NJ (FVB; H-2(q)) dams with lower susceptibility to Hg-induced autoimmunity (FvSF1) were investigated. Hg exposure increased the serum IgG levels of all offspring at postnatal day 21 (pnd21) and of SJL/J dams but not of FVB dams. Serum IgG anti-brain antibody (Ab) levels of pnd21 SFvF1 offspring and SJL dams were higher than those of the FvSF1 offspring and FVB dams, but Hg only increased the titers of the FVB dams and their offspring. Hg significantly elevated the presence of IgG in all brain regions of the pnd21 SFvF1 offspring, and the SFvF1 offspring had greater amounts of IgG in the brain than the FvSF1 offspring, which had Hg-induced increases in only two brain regions. Cytokine levels were elevated in the brain regions of Hg-treated pnd21 SFvF1 but not of FvSF1 offspring, and SFvF1 females had more brain regions expressing cytokines than the males. At pnd70, the serum IgG, serum antibrain Abs, amounts of brain IgG, and brain cytokine levels of all of the Hg-treated offspring were equivalent to those of their appropriate controls, suggesting that developmental Hg exposure did not induce chronic immunological effects. However, the social behaviors of Hg-exposed SFvF1 offspring at pnd70 were significantly impaired, and SFvF1 females displayed greater decline in social behaviors than males, suggesting that the higher neuroinflammation of SFvF1 females earlier in life is associated with the altered behavior. Thus, developmental Hg exposure induces long-lasting effects on social behavior of offspring, which is dependent on sex and genetics and the induction of neuroinflammation.

The maternal autoimmune environment affects the social behavior of offspring.

Autism spectrum disorders (ASD) are neurodevelopmental disorders with unknown etiology. BTBR-T(+)tf/J (BTBR) mice, a mouse strain with behaviors that resemble autism and with elevated levels of anti-brain antibodies (Abs), have enhanced activation of peripheral B cells and CD4(+) T cells and an expanded percentage of CD4(+) T cells expressing Vß6 chains. The CD4(+)CD25(+)Vß6(+) and Vß6-splenic cells of BTBR mice have elevated levels of IL-4, IFN-γ and IL-17, but there appears to be no preferential CD4(+) T subset skewing/polarization. The high level of IgG production by BTBR B cells was dependent on T cells from BTBR mice. The CD4(+) T cells of BTBR mice, especially those expressing Vß6 become spontaneously activated and expanded in an autoimmune-like manner, which occurred in both BTBR and B6 hosts that received an equal number of BTBR and B6 bone marrow cells. BTBR mice also have an elevated percentage of peripheral blood neutrophils, which may represent their elevated inflammatory state. B6 offspring derived from B6 dams that were gestationally injected with purified IgG from sera of BTBR mice, but not IgG of B6 mice, developed significantly impaired social behavior. Additionally, B6 offspring that developed in BTBR dams had impaired social behavior, while BTBR offspring that developed in B6 dams had improved social behavior. All of the immunological and behavioral parameters of BTBR mice were compared with those of B6 mice, which have relatively normal behaviors. The results indicate maternal Abs and possibly other maternal influences affect the social behavior of offspring.

The BTBR T+ tf/J mouse model for autism spectrum disorders-in search of biomarkers.

Autism spectrum disorders (ASD) form a common group of neurodevelopmental disorders appearing to be under polygenic control, but also strongly influenced by multiple environmental factors. The brain mechanisms responsible for ASD are not understood and animal models paralleling related emotional and cognitive impairments may prove helpful in unraveling them. BTBR T+ tf/J (BTBR) mice display behaviors consistent with the three diagnostic categories for ASD. They show impaired social interaction and communication as well as increased repetitive behaviors. This review covers much of the data available to date on BTBR behavior, neuroanatomy and physiology in search for candidate biomarkers, which could both serve as diagnostic tools and help to design effective treatments for the behavioral symptoms of ASD.

Developmental exposure to mercury chloride does not impair social behavior of C57BL/6 × BTBR F(1) mice.

The effects of mercury (Hg) on social behavior and the mechanisms involved remain unknown. This study shows that Hg chloride (HgCl(2)) exposure during fetal development does not impair social behavior of a mouse strain susceptible to environment-induced autistic-like behavior based on the parental phenotype. On the contrary, Hg exposure elevated the sociability of females. Since B6 mice are behaviorally normal and BTBR mice display low levels of sociability, the F(1) offspring (B6BF(1)) of female B6 mice and male BTBR mice were used to investigate their social behavior and the effects of Hg. Developmental Hg-treatment increased the serum IgG levels of the post-natal day (pnd) 21 offspring, but not pnd70 offspring or the B6 dams. After Hg treatment, there were negligible levels of serum IgG anti-brain antibodies (Ab) in the pnd21 and pnd70 offspring as well as their dams. However, Hg did elevate IgG deposition in multiple assayed brain regions of the pnd21 offspring, but the higher levels were no longer present at pnd70. Cytokine levels were not changed in pnd21 or pnd70 brain by Hg exposure, suggesting neuroinflammation was not induced. Social behavior was assayed at pnd70. Surprisingly, Hg-treatment significantly enhanced sociability of female B6BF(1) offspring, but not that of the male offspring. Our data indicates that developmental exposure to HgCl(2) did not impair social behavior of B6BF(1) offspring, but it enhanced the sociability of females, which was significantly lower in adult B6BF(1) females than B6BF(1) males in the absence of any Hg exposure.