Bilateral subcortical heterotopia with partial callosal agenesis in a mouse mutant.

Cognition and behavior depend on the precise placement and interconnection of complex ensembles of neurons in cerebral cortex. Mutations that disrupt migration of immature neurons from the ventricular zone to the cortical plate have provided major insight into mechanisms of brain development and disease. We have discovered a new and highly penetrant spontaneous mutation that leads to large nodular bilateral subcortical heterotopias with partial callosal agenesis. The mutant phenotype was first detected in a colony of fully inbred BXD29 mice already known to harbor a mutation in Tlr4. Neurons confined to the heterotopias are mainly born in midgestation to late gestation and would normally have migrated into layers 2-4 of overlying neocortex. Callosal cross-sectional area and fiber number are reduced up to 50% compared with coisogenic wildtype BXD29 substrain controls. Mutants have a pronounced and highly selective defect in rapid auditory processing. The segregation pattern of the mutant phenotype is most consistent with a two-locus autosomal recessive model, and selective genotyping definitively rules out the Tlr4 mutation as a cause. The discovery of a novel mutation with strong pleiotropic anatomical and behavioral effects provides an important new resource for dissecting molecular mechanisms and functional consequences of errors of neuronal migration.

The role of cerebellar FOXP1 in the development of motor and communicative behaviors in mice.

The gene FOXP2 is well established for a role in human speech and language; far less is known about FOXP1. However, this related gene has also been implicated in human language development as well as disorders associated with features of autism spectrum disorder (ASD). FOXP1 protein expression has also recently been identified in the cerebellum-a neural structure previously shown to express FOXP2 protein. The current study sought to elucidate the behavioral implications of a conditional knock-out of Foxp1 using an En1-Cre driver, which is active in the entirety of the cerebellum and a subset of neurons in the midbrain and spinal cord, in mice using a test battery including motor tasks associated with cerebellar dysfunction, as well as communicative and autistic-relevant behaviors. Male and female mice with a conditional knock-out (cKO, n = 31) and wildtype littermate controls (WT, n = 34) were assessed for gross and orofacial motor control, motor-coordination learning, locomotion, social behavior, anxiety, auditory processing and expressive vocalizations. Overall results suggest Foxp1 plays a specific role in the development of communicative systems, and phenotypic expression of disruptions may interact with sex. Robust motor deficits associated with Foxp1 protein loss may particularly affect vocalizations based on significant orofacial motor deficits in cKO subjects could also contribute to vocalization anomalies. In summary, the current study provides key insights into the role of Foxp1 in cerebellar function and associated behaviors in mice, with implications for an improved understanding of communicative and motor-based neurodevelopmental disabilities in humans.

Evaluation of the therapeutic benefit of delayed administration of erythropoietin following early hypoxic-ischemic injury in rodents.

Hypoxia-ischemia (HI) and associated brain injuries are seen in premature as well as term infants with birth complications. The resulting impairments involve deficits in many cognitive domains, including language development. Poor rapid auditory processing is hypothesized to be one possible underlying factor leading to subsequent language delays. Mild hypothermia treatment for HI injuries in term infants is widely used as an intervention but can be costly and time consuming. Data suggest that the effectiveness of hypothermia treatment following HI injury declines beyond 6 h following injury. Consequently, the availability of a therapeutic alternative without these limitations could allow doctors to treat HI-injured infants more effectively and thus reduce deleterious cognitive and language outcomes. Evidence from both human studies and animal models of neonatal HI suggests that erythropoietin (Epo), an endogenous cytokine hormone, may be a therapeutic agent that can ameliorate HI brain injury and preserve subsequent cognitive development and function. The current study sought to investigate the therapeutic effectiveness of Epo when administered immediately after HI injury, or delayed at intervals following the injury, in neonatal rodents. Rat pups received an induced HI injury on postnatal day 7, followed by an intraperitoneal injection of Epo (1,000 U/kg) immediately, 60 min, or 180 min following induction of injury. Subjects were tested on rapid auditory processing tasks in juvenile (P38-42) and adult periods (P80-85). Ventricular and cortical size was also measured from post mortem tissue. Results from the current study show a therapeutic benefit of Epo when given immediately following induction of HI injury, with diminished benefit from a 60-min-delayed injection of Epo and no protection following a 180-min-delayed injection. The current data thus show that the effectiveness of a single dose of Epo in ameliorating auditory processing deficits following HI injury decreases precipitously as treatment is delayed following injury. These data may have important implications for experimental human neonatal intervention with Epo.

Inhibition of X-linked inhibitor of apoptosis with embelin differentially affects male versus female behavioral outcome following neonatal hypoxia-ischemia in rats.

Hypoxia-ischemia (HI; concurrent oxygen/blood deficiency) and associated encephalopathy represent a common cause of neurological injury in premature/low-birth-weight infants and term infants with birth complications. Resulting behavioral impairments include cognitive and/or sensory processing deficits, as well as language disabilities, and clinical evidence shows that male infants with HI exhibit more severe cognitive deficits compared to females with equivalent injury. Evidence also demonstrates activation of sex-dependent apoptotic pathways following HI events, with males preferentially activating a caspase-independent cascade of cell death and females preferentially activating a caspase-dependent cascade following neonatal hypoxic and/or ischemic insults. Based on these combined data, the 'female protection' following HI injury may reflect the endogenous X-linked inhibitor of apoptosis (XIAP), which effectively binds effector caspases and halts downstream cleavage of effector caspases (thus reducing cell death). To test this theory, the current study utilized neonatal injections of vehicle or embelin (a small molecule inhibitor of XIAP) in male and female rats with or without induced HI injury on postnatal day 7 (P7). Subsequent behavioral testing using a clinically relevant task revealed that the inhibition of XIAP exacerbated HI-induced persistent behavioral deficits in females, with no effect on HI males. These results support sex differences in mechanisms of cell death following early HI injuries, and suggest a potential clinical benefit from the development of sex-specific neuroprotectants for the treatment of HI.

Behavioral and neuroanatomical outcomes following altered serotonin expression in a hypoxic-ischemic injury neonate rodent model.

BACKGROUND: Children born prematurely (<37 gestational weeks) are at risk for a variety of adverse medical events. They may experience ischemic and/or hemorrhagic events leading to negative neural sequelae. They are also exposed to repeated stressful experiences as part of life-saving care within the neonatal intensive care unit (NICU). These experiences have been associated with methylation of SLC6A4, a gene which codes for serotonin transport proteins, and is associated with anxiety, depression, and increased incidence of autism spectrum disorders.The purpose of this study was to examine the effects of altered serotonin levels on behavioral and neuroanatomical outcomes in a neonatal rodent model with or without exposure to hypoxic-ischemic (HI) injury. METHODS: Wistar rat pups were randomly assigned to either HI injury or sham groups. Pups within each group were treated with a chronic SSRI (Citalopram HBr) to simulate the effects of SLC6A4 methylation, or saline (NS). Subjects were assessed on behavioral tasks and neuropathologic indices. RESULTS: HI injured subjects performed poorly on behavioral tasks. SSRI subjects did not display significantly greater anxiety. HI + SSRI subjects learned faster than HI+NS. Histologically, SSRI subjects had predominantly larger brain volumes than NS. CONCLUSION: SSRI treated subjects without injury showed patterns of increased anxiety, consistent with theories of SLC6A4 methylation. The paradoxical trend to improved cognition in HI+SSRI subjects relative to HI alone, may reflect an unexpected SSRI neuroprotective effect in the presence of injury, and may be related to serotonin-induced neurogenesis.

Evaluation of visual motion perception ability in mice with knockout of the dyslexia candidate susceptibility gene Dcdc2.

Developmental dyslexia is a heritable disability characterized by difficulties in learning to read and write. The neurobiological and genetic mechanisms underlying dyslexia remain poorly understood; however, several dyslexia candidate risk genes have been identified. One of these candidate risk genes-doublecortin domain containing 2 (DCDC2)-has been shown to play a role in neuronal migration and cilia function. At a behavioral level, variants of DCDC2 have been associated with impairments in phonological processing, working memory and reading speed. Additionally, a specific mutation in DCDC2 has been strongly linked to deficits in motion perception-a skill subserving reading abilities. To further explore the relationship between DCDC2 and dyslexia, a genetic knockout (KO) of the rodent homolog of DCDC2 (Dcdc2) was created. Initial studies showed that Dcdc2 KOs display deficits in auditory processing and working memory. The current study was designed to evaluate the association between DCDC2 and motion perception, as these skills have not yet been assessed in the Dcdc2 KO mouse model. We developed a novel motion perception task, utilizing touchscreen technology and operant conditioning. Dcdc2 KOs displayed deficits on the Pairwise Discrimination task specifically as motion was added to visual stimuli. Following behavioral assessment, brains were histologically prepared for neuroanatomical analysis of the lateral geniculate nucleus (LGN). The cumulative distribution showed that Dcdc2 KOs exhibited more small neurons and fewer larger neurons in the LGN. Results compliment findings that DCDC2 genetic alteration results in anomalies in visual motion pathways in a subpopulation of dyslexic patients.

Effects of sex and MK-801 on auditory-processing deficits associated with developmental microgyric lesions in rats.

Neonatally induced microgyric lesions produce defects in rapid auditory processing in adult male rats. Given that females across species are less susceptible to the deleterious effects of neural injury and that treatment with neuroprotective agents at the time of injury can reduce neural damage, the authors tested the effects of sex and neuroprotectant exposure on the behavioral consequences of microgyric lesions in rats. Results showed that sham but not microgyric males were able to perform the task at the fastest rate of stimulus presentation. Microgyric females, in contrast, discriminated at all stimulus conditions and did not differ from female shams. Microgyric males treated with MK-801 had reduced cortical damage and performed the discrimination at the fastest condition. Results suggest that females are less susceptible to the behavioral effects of neocortical microgyria and that MK-801 may ameliorate the behavioral consequences of these lesions in male rats.

Functional lateralization for auditory temporal processing in male and female rats.

In 3 studies adult male rats showed significantly better discrimination of tone sequences with the right ear than with the left ear. This result parallels similar findings of left-hemisphere specialization for auditory temporal processing in humans and nonhuman primates. Furthermore, because clinical evidence supports a link between specialization of the left hemisphere for auditory temporal processing and for speech processing, these results may reflect evolutionary precursors to left-hemisphere language specialization. Because male rats showed a stronger ear advantage than female rats, the findings may relate to evidence of a stronger right ear advantage in men than in women. Finally, results suggest that neonatal handling enhances lateralization for auditory temporal processing in both sexes. Combined results implicate neuroendocrine mechanisms as important factors in the development of lateralization for auditory temporal processing.

Corpus callosum: interactive effects of infantile handling and testosterone in the rat.

Previous research found that the corpus callosum of male rats is larger than that of females; handling rats in infancy enhances this sex difference; and female rat pups, when handled in infancy and given 1 injection of testosterone propionate (TP) on Day 4 of life, will have callosa as large as those of males. In 2 experiments, male pups were castrated on Day 1 or received sham surgery; female pups were injected with TP on Day 4 or received an oil injection. Litters were handled or nonhandled. The previous finding that females, when handled and given TP in infancy, have a larger callosum was confirmed; however, a TP effect when administered to nonhandled females was not found. Because handling is known to cause a corticosterone release, these findings were interpreted as evidence of a developmental interaction between adrenal and gonadal hormones at the cortical level.

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.

Impaired detection of variable duration embedded tones in ectopic NZB/BINJ mice.

Utilizing rodent models, prior research has demonstrated a significant association between focal neocortical malformations (i.e. induced microgyria, molecular layer ectopias), which are histologically similar to those observed in human dyslexic brains, and rate-specific auditory processing deficits as seen in language impaired populations. In the current study, we found that ectopic NZB/BINJ mice exhibit significant impairments in detecting a variable duration 5.6 kHz tone embedded in a 10.5 kHz continuous background, using both acoustic reflex modification and auditory event-related potentials (AERP). The current results add further support to the association between focal cortical malformations and impaired auditory processing, and the notion that these auditory effects may occur regardless of the cortical location of the anomaly.

A factor analysis of the rat's corpus callosum.

Previous work from our laboratory (Berrebi et al., Brain Research, 438 (1988) 216-224) demonstrated region-specific sexual dimorphisms in the size of the rat's corpus callosum, which are modifiable by extra stimulation in early life. These differences are assumed to reflect regional corticocortical fibers of passage which are altered differentially by gender and our experimental manipulations. In this paper, we report our findings when the original data are reanalyzed using a newly developed computer program. This program not only reproduced, with very high accuracy, the original means, but also permitted us to examine computer generated callosal width scores via a factor analysis procedure. Such a procedure yields useful information concerning the clustering of callosal fibers and thus contributes significantly to our hypothesis that discrete cortical regions are selectively sensitive to experimental variables. Factor analyses of the callosal variables and brain weight of 155 rats found 7 width factors, and an eighth factor which contained the variables of brain weight, callosal length, and callosal perimeter. Callosal area did not load significantly on any of these factors. The percentile locations of the width factors, starting at the anterior (genu) end were: widths 1-5, 6-17, 24-38, 46-57, 62-72, 79-95 and 96-99. Use of these factor scores in analyses of variance revealed that the male callosum is wider than the female's, with the differences most pronounced in the genu and the most posterior portion of the splenium. Both age and early handling experience influenced the callosal width factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired two-tone processing at rapid rates in male rats with induced microgyria.

We previously reported that adult male rats with bilateral induced microgyria exhibit deficits in rapid auditory processing, which appear similar to auditory processing deficits seen in individuals with developmental language disabilities. The current study was designed to further elaborate that finding using an improved paradigm in which stimulus duration was uncoupled from testing experience and learning effects. Specifically, two-tone stimuli with durations of 540, 390, 332 and 249 ms were all presented within a single test session in a modified operant conditioning paradigm. Subjects were tested over a period of 12 days using this variable-stimulus format. Results confirmed microgyric male rats were impaired only in processing two-tone stimuli presented at rapid rates (i.e., 249 ms duration). Thus the current results support the previously observed link between focal malformations and deficits in rapid auditory processing.

Corpus callosum: effects of neonatal hormones on sexual dimorphism in the rat.

The rat's corpus callosum is sexually dimorphic, with the male's being larger. In addition, giving rats extra stimulation in infancy via handling increases callosal area in males, but not in females. To determine if this dimorphism is testosterone-dependent, male pups were castrated on Day 1 of life while females received an injection of testosterone propionate (TP) on Day 4. Control males had sham surgery and control females received an injection of sesame oil. All animals were handled daily from birth until weaning. Animals were sacrificed at 110 days and a mid-sagittal section of the callosum was obtained. From this section measures of callosal area, perimeter, length, and 99 widths were derived. We verified our previous finding that the male callosum is larger than that of the female. Neonatal TP treatment masculinized the callosa of the females, but castration did not affect the males. TP treatment affected the width dimension of the callosum but not callosal length or brain weight. In a related study the synthetic estrogen DES did not increase callosal size for castrated males or for intact females, while the estrogen blocker, tamoxifen, had a defeminizing effect on females' callosa. These findings suggest that there is an estrogen-dependent active process of feminization of cortical tissue in the female brain.

Corpus callosum: ovarian hormones and feminization.

The rat's corpus callosum is sexually dimorphic with the male's being larger. This difference appears to depend in part on the neonatal presence of testosterone in the male and ovarian hormones in the female. To further investigate the possibility that ovarian hormones participate in the differentiation of the rat's callosum, females received one of the following treatments on postnatal day 8, 12 or 16: (1) ovariectomy (Ovx); (2) 1 mg of testosterone propionate (TP); or (3) sham surgery. All animals were handled daily from birth until weaning. They were sacrificed at 110 days and a mid-sagittal section of the callosum was obtained. From this section measures of callosal area, perimeter, length, and 99 widths were derived. Widths were averaged into 7 factors as defined by prior factor analysis. Ovariectomy, whether on day 8, 12 or 16, enlarged callosal area and 3 of the callosal width factors. TP had no effect on any callosal variable when administered on day 8, 12 or 16. A comparison of control males and females replicated our prior findings of sexual dimorphism. We conclude that ovarian hormones act to feminize the female callosum, and that their removal results in defeminization. Furthermore, the fact that ovariectomy was effective as late as day 16, while TP treatment on day 8 or later had no effect, suggests that masculinization and feminization of this structure constitute separate processes with distinct sensitive periods.

Corpus callosum: region-specific effects of sex, early experience and age.

In infancy, rats were provided handling stimulation and compared at 110 and 215 days of age with non-handled controls. Measurements were made of corpus callosum area, perimeter and length; and width measures were taken at 7 points along the longitudinal axis of the callosum. Callosal size was larger in males than in females, even when adjusted for the larger brain weight of the male. At 110 days handling stimulation increased callosal parameters and resulted in a more regular callosum in males, but this effect was no longer apparent by 215 days. Within the callosum, region-specific effects were found, suggesting that certain callosal fiber populations were involved. Handled males have previously been shown to be more lateralized than non-handled males; thus at least in this experimental system, increased callosal size and regularity is associated with greater hemispheric specialization.

Neonatal prazosin exposure reduces ovarian weight and estrogen receptor binding in adult female rats.

Exposure of estrogen treated adult female guinea pigs to the alpha-1 antagonist prazosin has been shown to reduce levels of estrogen binding in the hypothalamus and preoptic area. To further investigate this interaction between the noradrenergic and neuroendocrine axes, newborn female rat pups received an s.c. implant of prazosin (0.0125 mg/day for 5 days) or placebo. In adulthood, subjects were sacrificed by perfusion with DMSO on the morning of proestrous. Tissue analysis of the medial preoptic area, corticomedial amygdala, and mediobassal hypothalamus revealed that cytosolic estrogen binding was significantly reduced in all three areas for the prazosin treated group as compared to controls. Ovarian weight was also significantly reduced in the prazosin treated group, although uterine weight was unaffected. Interestingly, prazosin treated females showed a post-pubertal increase in body weight characteristic of ovariectomized females, while controls showed no such increase. These results support the existence of a significant developmental interaction between the noradrenergic system and the neuroendocrine axis as measured by ovarian weight and estrogen binding in the brain.

Corpus callosum: demasculinization via perinatal anti-androgen.

The male rat's corpus callosum is significantly larger than the female's. This dimorphism depends in part on the early presence of testosterone, since postnatal administration of testosterone to female pups enlarges their callosa in adulthood to the size of males. However, castrating males on day 1 is ineffective in reducing (demasculinizing) the size of their callosa as adults. We then addressed the question as to whether testosterone acts prior to day 1 to enlarge the callosa of males. To investigate this hypothesis pregnant rats were administered a non-steroidal androgen blocker, flutamide, during the last 5 days of pregnancy, while controls received vehicle only. Male pups from flutamide litters were castrated on day 3 to prevent postnatal recovery following clearance of flutamide, while others received sham surgery. Callosal sex differences were found between males and females of control litters, but not between males and females from flutamide litters. The absence of sex effects among flutamide litters was a consequence of small callosal size in flutamide-castrated males as compared to control males. We concluded that the prenatal production of testosterone in the male rat pup contributes to sexual dimorphism in the callosa of adult rats.

Laterality in animals: relevance to schizophrenia.

Anomalies in the laterality of numerous neurocognitive dimensions associated with schizophrenia have been documented, but their role in the etiology and early development of the disorder remain unclear. In the study of normative neurobehavioral organization, animal models have shed much light on the mechanisms underlying and the factors affecting adult patterns of both functional and structural asymmetry. Nonhuman species have more recently been used to investigate the environmental, genetic, and neuroendocrine factors associated with developmental language disorders in humans. We propose that the animal models used to study the basis of lateralization in normative development and language disorders such as dyslexia could be modified to investigate lateralized phenomena in schizophrenia.

Neonatal estrogen blockade prevents normal callosal responsiveness to estradiol in adulthood.

The rat corpus callosum (CC) is larger in males than females, and is responsive to hormone manipulations during development. We previously demonstrated that P25 ovariectomy (Ovx) enlarged (defeminized) adult CC, while P70 ovary transfer (OvT) counteracted this enlarging effect, resulting in smaller (feminized) CC. Since OvT females were not Ovx'd until P25, they received some neonatal estrogen (E) exposure. Behavioral data suggest that adult responsiveness to ovarian hormones depends upon prior organization by neonatal E. It has not been determined whether a similar phenomenon occurs for the feminization of brain morphology. The current experiment examined whether our previous finding of adult CC responsiveness to ovarian hormones depended upon neonatal E exposure. We investigated this by assessing the effects of P70 ovarian hormone replacement (via ovary transfer or E pellet) in females that received either (1) normal ovarian hormone exposure until P25 Ovx, or (2) the E receptor blocker tamoxifen from birth to P25 Ovx. Females receiving normal neonatal hormone exposure responded to P70 E in the female-typical manner: E reduced CC size. In contrast, females receiving neonatal E blockade responded to adult E in the opposite manner: E increased CC size. As far as we are aware, this is the first report suggesting that neonatal E exposure organizes the female brain so that it responds normally to the organizing actions of E when later exposure occurs. These findings further challenge the traditional model of female brain development, which asserts that normal female brain organization occurs by default, in the absence of gonadal hormone exposure.