Behavior, ectopias and immunity in BD/DB reciprocal crosses

In a previous study, in which fertilized DBA ova were transferred into an autoimmune female, and NZB ova were transferred into a non-autoimmune female, we found that (1) the maternal environment affected the degree of autoimmunity, (2) the incidence of cortical ectopias was not affected by the maternal environment (3) DBA and NZB females had greater paw asymmetry if reared in an autoimmune uterus, and (4) avoidance learning scores were inversely related to degree of autoimmunity. In the present experiment, reciprocal crosses of DBA and BXSB mice were studied to confirm and extend the original findings. DB mice (DBA female × BXSB male) had greater immune activity than the BD animals, had poorer avoidance learning, but were better on black-white discrimination learning and the Lashley III maze. The BD mice had greater paw asymmetry. Only one of 38 animals had a cortical ectopia. The results lead to the following conclusions: (1) there is an inverse relationship between amount of immune activity and active avoidance learning; (2) some uterine factor in autoimmune mice causes females to have greater paw asymmetry; (3) cortical ectopias are under genetic control; and (4) the lesser immune activity of the BD mice suggests that they developed a suppressor system following early exposure to autoimmunity in the uterine/maternal environment.     

Spatial learning, discrimination learning, paw preference and neocortical ectopias in two autoimmune strains of mice

NZB and BXSB mice were given a battery of behavioral tests including paw preference, water escape, Lashley III maze, and discrimination learning. Their brains were then evaluated for cortical ectopias. The incidence of ectopias was 40.5% in NZBs and 48.5% in BXSBs. In the NZB strain left-pawed ectopic mice (both male and female) had the fastest swimming time in the water escape test, while right-pawed ectopics were the slowest. The same findings were obtained for left- and right-pawed ectopic BXSB males, but not for the females. However, on discrimination learning the BXSB males had the exact opposite pattern: right-pawed ectopics were the best learners while left-pawed ectopics were the worst. Male BXSBs and both male and female NZBs were manifesting autoimmune disease at the time of testing, while female BXSBs were not, suggesting that autoimmunity is a necessary background condition for the differential expression of ectopias and paw preference upon learning processes. The finding that the left-pawed ectopic BXSB mice, who were the poorest learners in the non-spatial discrimination learning test, learned best in the spatial water escape test is in agreement with the Geschwind hypothesis that pathological events during brain development may, in some instances, produce superiority of function.     

Environmental enrichment, neocortical ectopias, and behavior in the autoimmune NZB mouse.

New Zealand Black (NZB) mice have severe autoimmune disease and approximately 40% have cortical ectopias in layer I of sensorimotor cortex. Because the ectopias are similar to those found in dyslexics, NZB mice have been used as an animal model for developmental learning disorders. In addition, these mice have been used as a model of learning deficits associated with autoimmune disease. To determine whether early intervention would affect learning processes in NZB mice, they were reared after weaning in standard cages or enriched environments. They were given a battery of behavioral tests to measure learning, laterality, and activity, after which they were sacrificed and their brains examined for cortical ectopias. The tests sorted into two behavioral sets. Ectopia-associated behaviors included black-white discrimination learning and the Morris spatial maze. As a group, the mice performed well on these tasks. Ectopic mice had poorer performance than non-ectopics on these measures, and environmental enrichment countered the effects of the ectopias. Autoimmune-associated behavior involved two-way avoidance learning in a shuttlebox. Mice were uniformely poor on this task, ectopias did not affect behavior, and environmental enrichment was without benefit. Evidence from this and other studies shows that poor shuttlebox performance is related to the presence of autoimmune disease. Thus, autoimmune disease and cortical ectopias each appear to affect a separate set of behavioral processes. Environmental enrichment is most effective for behavioral impairments mediated via cortical ectopias, but is much less effective, if at all, if autoimmunity is the primary mediator of the impairments.     

Non-spatial water radial-arm maze learning in mice

Recently, we published a method for examining working and reference memory in mice using a spatial version of the water radial-arm maze. Here we describe a non-spatial version of the same maze. BXSB mice were able to learn the maze as shown by the decrease in the number of working and reference memory errors over sessions. This maze was used to examine learning differences between males and females and between mice with misplaced clusters of neurons in layer I of cortex (ectopias) and those without. In a prior study using the spatial version of the water radial-arm maze, male BXSB mice had poorer working memory than females during the acquisition phase. Similarly, in this study male BXSB mice demonstrated impaired working memory during the asymptotic phase of non-spatial radial-arm maze learning. Two prior studies showed that mice with neocortical ectopias demonstrated working memory impairments compared to non-ectopic littermates in the spatial version of the water radial-arm maze. Contrary to this, in the non-spatial radial-arm maze used here, ectopic mice were not impaired in working memory and showed better memory when the working memory 'load' was the highest. Overall, both versions of the maze can be useful tools to assess spatial and non-spatial working and reference memory in mice.     

Neuroanatomical anomalies in autoimmune mice

Summary The cerebral cortex was examined for sings of pathology in the NZB, BXSB, and MRL autoimmune strains of mice, crosses among these strains, and control mice. Previously, we reported that 20% of NZB mice had ectopic collections of neurons in layer I of the cortex. In this study we replicated this finding in the NZB, and extended it to the BXSB strain, and BXSB/NZB and MRL/NZB hybrids. The MRL strain, however, did not have a large number of individuals with brain anomalies. Thus, a number of autoimmune mice strains and hybrids develop brain anomalies, although at least one autoimmune strain does not. We suggest that in certain autoimmune strains maternal autoantibodies cross the placenta and damage the developing fetal brain, and that these strains may be useful experimental models for studying the development of brain anomalies seen in the dyslexic human.Supported in part by NIH grants 19819 and 20806 and grants from the Orton Dyslexia Society and Carl J. Herzog Foundation     

Abnormal architecture and connections disclosed by neurofilament staining in the cerebral cortex of autoimmune mice

Routine cell stains disclose abnormal, usually single, nests of ectopic neurons in neocortical layer I of New Zealand Black (NZB) and BXSB autoimmune mice. We have suggested that these anomalies represent only the most visible part of more widespread cortical disorganization. In an attempt to determine the true magnitude of cellular and fiber disruption associated with the presence of ectopias, we stained cortical sections containing ectopias in layer I from NZB and BXSB mice with an antibody directed against the 68 kDa subunit of neurofilament protein. The neurofilament-stained sections revealed substantial disruption of the cortical layers underlying the ectopic neurons. This resulted primarily from the existence of dense, radially oriented fiber bundles spanning the thickness of the cortex underlying the ectopias. In some instances these fiber bundles could be seen to join the corpus callosum. Even in a small ectopia, where standard stains show no associated cortical dysplasia, dense neurofilament staining was present in layers II and III. It was concluded that the brains of autoimmune mice have severe developmental cortical disorganization, which could account for the behavioral differences displayed by these animals.     

Learning and memory in the autoimmune BXSB mouse: effects of neocortical ectopias and environmental enrichment

Approximately 40–60% of BXSB mice have ectopic cell clusters in layer I of neocortex. Prior studies have shown distinct behavioral differences between those with ectopias and their non-ectopic littermates. In this study, female BXSB mice were reared after weaning in either enriched environments or standard cages. Following an initial round of behavioral testing, all mice were housed in standard cages and retested. Enriched cage mice (both ectopic and non-ectopic) showed increased activity, greater speed, and enhanced learning scores across a variety of tests. Additionally, prior test experience itself had significant positive effects on Hebb-Williams maze learning. The presence of ectopias resulted in better Morris maze learning for standard cage reared mice. Further, ectopic mice, regardless of their housing condition, showed better long-term retention in the Morris maze than did their non-ectopic counterparts. These findings show that abnormalities in corticogenesis need not always result in functional deficit.     

Neocortical ectopias are associated with attenuated neurophysiological responses to rapidly changing auditory stimuli

Developmental dyslexia has been separately associated with the presence of ectopic collections of neurons in layer I of neocortex (ectopias) and with alterations in processing rapidly changing stimuli. We have used BXSB/MpJ-Yaa mice, some of which have neocortical ectopias, to directly test the hypothesis that ectopias may alter auditory processing. Auditory event related potentials (AERPs) were elicited by pairs of 10.5 kHz tones separated by silence, 0.99 kHz, or 5.6 kHz tones of variable duration. Half of the mice tested had 1-3 ectopias in frontal or parietal cortex, and half had no ectopias. Mice with ectopias showed a reduced response to the second 10.5 kHz stimuli only when it was preceded by short duration 5.6 kHz tones. These results indicate that BXSB mice are an excellent model for determining how focal neocortical anomalies alter sensory processing.     

Spatial and nonspatial Morris maze learning: impaired behavioral flexibility in mice with ectopias located in the prefrontal cortex

About half of BXSB/MpJ-Yaa (BXSB) mice have neocortical ectopias (misplaced clusters of neurons located in layer I of cortex). Previous behavioral studies have suggested that ectopic mice have superior spatial, but equivalent nonspatial, reference memory learning. However, since spatial and nonspatial learning were not assessed in the same apparatus and with the same testing procedure, it is unclear if this conclusion is accurate. We have created a new nonspatial Morris maze for mice that differs from the spatial task only in the type of cues that must be utilized to efficiently locate the platform (intra-maze black/white patterns vs. extra-maze room cues) and does not differ in the level of task complexity or the presence of objects within the maze. Ectopic mice were very good in utilizing extra-maze cues when learning the spatial version and in utilizing intra-maze cues when learning the nonspatial version of the Morris maze, while non-ectopics were not, suggesting that ectopics have superior spatial and nonspatial reference memory. Ectopias in BXSB mice are usually located in prefrontal and/or motor cortex. The prefrontal cortex is involved in behavioral flexibility (e.g. being able to easily switch from using spatial to nonspatial cues). Only ectopic mice with ectopias specifically located in the prefrontal region of cortex demonstrated difficulty switching from using extra-maze to intra-maze cues and vice versa. Thus, the presence of one or more ectopias in the prefrontal region of cortex disrupted one of the normal functions of the prefrontal cortex.     

The effect of developmental neuropathology on neocortical asymmetry in New Zealand black mice

Cerebral asymmetry can be considered along two continua-one based on direction (i.e., left or right) and another based on magnitude (i.e., symmetrical or asymmetrical). The possibility exists that these continua operate independently (Collins, 1981). To examine this possibility, the brains of 21 New Zealand Black (NZB) mice with molecular layer neuronal ectopias and 19 NZB mice without ectopias were studied. In NZB mice without ectopias, the magnitude of cerebral cortical asymmetry was negatively correlated to total cerebral cortical volume, a finding previously reported in both humans (Galaburda, Corsiglia, Rosen and Sherman, 1987) and rats (Galaburda, Aboitiz, Rosen and Sherman, 1986). NZB mice with ectopias showed no such relationship. However, both groups of mice had a consistent rightward bias in the direction of neocortical asymmetry, replicating previous results in rodents (Diamond, Johnson and Ingham, 1975; Diamond, Dowling and Johnson, 1981; Kolb, Sutherland, Nonneman and Whishaw, 1982; Ward and Collins, 1985). This suggests that the mechanisms underlying the magnitude of cerebral cortical asymmetry differ from those underlying the direction of this asymmetry.     

The midsagittal area of the corpus callosum and total neocortical volume differ in three inbred strains of mice

Differences in the midsagittal area of the corpus callosum have been reported between human males and females, between handled and nonhandled rats, and both within and between various strains of mice. This measure has, in addition, been related to handedness in humans and "pawedness" in certain strains of mice. The present study investigated the between- and within-strain differences in three inbred strains of mice, two with autoimmune disorders and spontaneously occurring developmental neuropathology, in the midsagittal area of the corpus callosum, the total neocortical volume, and the asymmetry of the neocortex. These morphometric measures were obtained from coronally sectioned celloidin-embedded material from New Zealand Black (NZB/BINJ), BXSB/MpJ, and DBA/2J mouse strains. NZB mice had a total neocortical volume larger than that of either the BXSB or DBA strains, whereas the BSXB mice had a midsagittal area of the corpus callosum larger than that of either of the other two strains. In addition, there was a positive correlation between these two measures. There was no correlation between total neocortical asymmetry and midsagittal area of the corpus callosum in any of the three strains. Finally, there were no differences in any morphometric measure between animals with or without developmental neuropathology in any given strain.     

Differences in spatial learning comparing transgenic p75 knockout, New Zealand Black, C57BL/6, and Swiss Webster mice

Four strains of mice were compared regarding their relative abilities to solve the Morris water maze test of spatial memory. Members of the New Zealand Black (NZB) strain revealed steady improvement over the 6 days of training comparable to C57BL/6 mice. The neurotrophin low affinity receptor p75 knockout mouse, in which the binding region is rendered refractory to ligand, displayed profound deficits in the acquisition of this task. Members of the Swiss Webster strain performed intermediate between the poor performance of the p75 mice and the progressively improving learning curves of the NZB and C57 mice. The present results support the notion that interference with functioning of nerve growth factor (NGF) receptors on forebrain cholingergic neurons negatively impacts the animal's ability to utilize the spatial cues necessary for successful spatial navigation. This effect on NGF receptors was more behaviorally disrupting than the influence of neocortical and hippocampal ectopias as present in the NZB mice. These results support the use of the p75 knockout mouse as a model of forebrain cholinergic neuron dysfunction. On the other hand, these results do not support the use of the NZB mouse as an animal model of human learning disability and dementia.     

Perceptual auditory gap detection deficits in male BXSB mice with cerebrocortical ectopias

Underlying impairments in rapid auditory processing may contribute to disrupted phonological processing, which in turn characterizes developmental language impairment (LI). Identification of a neurobiological feature of LI that is associated with auditory deficits would further support this model. Accordingly, we found that adult male rats with induced cortical malformations were impaired in rapid auditory processing. Since 40-60% of BXSB mice exhibit spontaneous focal cerebrocortical ectopias (as seen in dyslexics brains), we assessed auditory gap detection in adult male BXSB mice. Ectopic mice were significantly worse than non-ectopics in detecting a 5 ms silent gap, but were not significantly impaired at longer gap durations (10-100 ms). Our results confirm that focal cortical malformations are associated with impairments in rapid auditory processing.     

Brief and long maternal separations decrease corticosterone secretion in a lupus-prone strain: dissociation from disease-related parameters

Neonatal manipulations are known to alter the activity of the immune system and the hypothalamus-pituitary-adrenal (HPA) axis. This study was performed in order to examine whether brief and long maternal separations (BMS and LMS, respectively) interfere with the onset and development of murine lupus in NZB/NZWF1 females, and to determine whether the pattern of corticosterone (CORT) secretion throughout life is associated to the expression of the disease. Maternal separation was performed daily during postnatal days 1-14, lasting 15 min in the BMS group and 3h in the LMS group. Blood was sampled from the retro-orbital plexus on the 9th week, and every other week, from 10th to 34th weeks of life, for detection of anti-nuclear antibodies (ANA) and anti-double-strand DNA (anti-dsDNA) antibodies, and for determination of CORT serum levels. Urine samples were collected on the 21st, 27th, 33rd and 37th weeks of life. There were no group differences in regard to disease-related parameters, but LMS females presented a tendency for late onset of anti-dsDNA antibodies. BMS and LMS mice exhibited reduced CORT levels compared to non-manipulated (NM) animals. There was a strong negative correlation between total mean CORT concentration and onset of ANA, and a strong positive correlation between total mean CORT concentration and life span only in the NM group. Neonatal manipulations appeared to eliminate these correlations; hence, both BMS and LMS modified basal CORT secretion and the association between glucocorticoids and immune activity in the NZB/NZWF1 mouse strain.     

Spatial and motor abilities during the course of autoimmune disease in (NZW x BXSB)F1 lupus-prone mice

In the systemic autoimmune/inflammatory lupus erythematosus disease, the involvement of the central nervous system is well recognized and frequently includes deficits in neurological function, cognition, and affect. The (NZW x BXSB)F1 lupus-prone mice are model of this pathology, in which a gene located on the Y chromosome provokes a sex specific morbidity in males. The present study examines whether autoimmune (NZW x BXSB)F1 mice develop impairments in learning and memory that correlate with severity of lupus-like disease. For this purpose, spatial and motor abilities were evaluated in 6- and 20-week-old male and female mice, and the immune status of these behaviorally tested mice was assessed by the presence of anti-nuclear antibodies (ANAbs) in the serum. The results showed that none of the animals had motor skill and motor learning disabilities, but that the older males were greatly impaired in their spatial abilities while the young ones and the females, whatever their age, were not. Besides, the ANAbs levels were similar and low in the young males, the young females and the old females, and very much higher in the old males, showing that spatial alterations were correlated to the anti-nuclear antibodies level.     

Chronic administration of fluoxetine impairs inhibitory avoidance in male but not female mice

The effects of chronic administration of fluoxetine (20 mg/kg/day i.p.) on a one-trial step-through inhibitory avoidance task were investigated in male and female CD1 mice. In Experiment 1, treatment was administered for 21 days before the training session, whereas in Experiment 2, other subjects were subjected to the same treatment starting 24 h after the training session. The comparison of test versus training latencies showed memory deterioration with pre-training administration of fluoxetine (Experiment 1), which affected males but not females. Sex differences in this task were also observed in Experiment 1, with females showing a better performance. Sex differences were evident in controls as well as in treated animals. The locomotor activity of the animals was also tested in Experiment 1. Due to the absence of sex differences in the drug effects on this measure, the sex differences in the effects of fluoxetine on inhibitory avoidance were hardly attributable to non specific effects on locomotor activity. The lack of effect of post-training administration of fluoxetine (Experiment 2) constitutes additional support of the idea that the observed effect on inhibitory avoidance in Experiment 1 is specifically related to learning and memory.     

Sex differences in rapid auditory processing deficits in ectopic BXSB/MpJ mice

Prior research with rodent models, performed predominantly in males, has demonstrated a significant association between focal neocortical malformations (e.g. ectopias and microgyria) and rate-specific auditory processing deficits. In the current study and consistent with prior findings, we report that ectopic male BXSB/MpJ mice exhibit impairments in detecting a two-tone oddball stimulus at short but not long inter-stimulus interval durations when compared to non-ectopic male littermates. However, ectopic female littermates showed no rapid auditory processing deficit when compared to non-ectopic females on this same task. Current results add growing support to: (1) an association between focal cortical malformations and impaired auditory processing in males; and (2) the existence of sex differences in the behavioral consequences of focal cortical malformations.     

Strain and sex differences in the degree of paw preference in mice.

The direction and degree of paw preference was studied in male and female mice from the C3H/He, C3H/OuJIco and New Zealand Black (NZB) strains, using the method previously described by Collins. All the populations of mice tested exhibited a low degree of lateralization, which varied among strains: NZB mice were more strongly lateralized than both C3H substrains; C3H/He had a higher degree of lateralization than C3H/OuJIco mice. Females tended to be more strongly lateralized than males, particularly in the C3H/He mice. No direction bias was observed in the strains tested, as lateralized mice were equally distributed into left-and right-handers. The distributions of paw preference found in the strains tested in this study are different from those obtained by Collins in C57BL/6J mice, in that most of his animals were strongly lateralized and only a few were ambidextrous. These strain differences in the degree of paw preference may be explained by genetic factors or differential rearing histories, which are known to influence the development of brain asymmetries.     

Water version of the radial-arm maze: Learning in three inbred strains of mice

The conventional land radial-arm maze has several disadvantages, including requiring a complicated automated apparatus, the elimination of odors as cues, and the use of food deprivation. We have created a water version of the maze, based on the principles of the land version, which maintains the advantages and excludes some of the disadvantages. In our maze, BXSB and C57BL/6 mice significantly reduced the number of working and reference memory errors committed over sessions, while NZB mice did not. For each strain, as the working memory ‘load' increased during a session, the number of errors increased. However, with practice the BXSB and C57BL/6 strains were able to handle this memory load more effectively. Mice were able to learn the maze without extensive adaptation, training, or testing and they did not exhibit ‘chaining'. This maze can also be considered to be an example of a water win-shift task that mice can easily learn. Therefore, the water version of the radial-arm maze can be a simple and useful tool for studying rodent learning and memory.     

B and T cells in the brains of autoimmune mice

Systemic lupus erythematosus (SLE) is an autoimmune disorder that can involve the central nervous system (CNS). Recently, we reported the presence of autoantibodies bound to the brain tissue of murine models of lupus; MRL/lpr and BXSB. We postulated that the source of these autoantibodies was in part due to in situ production, caused by the entry of B and T cells. Frozen brain sections of MRL/lpr and BXSB at 1 and 4 months of age were stained for CD3 (T cells) and CD19 (B cells) markers using an immunofluorescent antibody binding assay. Confocal fluorescence microscopy showed both CD3(+) and CD19(+) cells at 4 months of age only in MRL/lpr mice. There were no lymphocytes seen in the other autoimmune model, BXSB. Results suggest a difference in the mechanisms by which autoantibodies access the brain in these two autoimmune models of lupus.