The FOXP2 gene is central to acquisition of speech and language in humans and vocal production in birds and mammals. Rodents communicate via ultrasonic vocalizations (USVs) and newborn pups emit distress USVs when separated from their dam, thereby facilitating their retrieval. We observed that isolated male rat pups emitted substantially more USV calls and these were characterized by a significantly lower frequency and amplitude compared to female rat pups. Moreover, the dam was more likely to first retrieve male pups back to the nest, then females. The amount of Foxp2 protein was significantly higher in multiple regions of the developing male brain compared to females and, a reduction of brain Foxp2 by siRNA eliminated the sex differences in USVs and altered the order of pup retrieval. Our results implicate Foxp2 as a component of the neurobiological basis of sex differences in vocal communication in mammals. We extended these observations to humans, a species reported to have gender differences in language acquisition, and found the amount of FOXP2 protein in the left hemisphere cortex of 4-year-old boys was significantly lower than in age-matched girls.

Microglia are being increasingly recognized as playing important roles in neurodevelopment. The cerebellum matures postnatally, undergoing major growth, but the role of microglia in the developing cerebellum is not well understood. Using the laboratory rat we quantified and morphologically categorized microglia throughout the vermis and across development using a design-based unbiased stereology method. We found that microglial morphology changed from amoeboid to ramified during the first 3 postnatal weeks in a region specific manner. These morphological changes were accompanied by the sudden appearance of phagocytic cups during the third postnatal week from P17 to P19, with an approximately fourfold increase compared with the first week, followed by a prompt decline at the end of the third week. The microglial phagocytic cups were significantly higher in the granular layer (∼69%) than in the molecular layer (ML; ∼31%) during a 3 d window, and present on ∼67% of microglia with thick processes and ∼33% of microglia with thin processes. Similar proportions of phagocytic cups associated to microglia with either thick or thin processes were found in the ML. We observed cell nuclei fragmentation and cleaved caspase-3 expression within some microglial phagocytic cups, presumably from dying granule neurons. At P17 males showed an approximately twofold increase in microglia with thin processes compared with females. Our findings indicate a continuous process of microglial maturation and a nonuniform distribution of microglia in the cerebellar cortex that implicates microglia as an important cellular component of the developing cerebellum.

Microglia are the primary immune cells of the brain and function in multiple ways to facilitate proper brain development. However, our current understanding of how these cells influence the later expression of normal behaviors is lacking. Using the laboratory rat, we administered liposomal clodronate centrally to selectively deplete microglia in the developing postnatal brain. We then assessed a range of developmental, juvenile, and adult behaviors. Liposomal clodronate treatment on postnatal days 0, 2, and 4 depleted microglia with recovery by about 10 days of age and induced a hyperlocomotive phenotype, observable in the second postnatal week. Temporary microglia depletion also increased juvenile locomotion in the open field test and decreased anxiety-like behaviors in the open field and elevated plus maze. These same rats displayed reductions in predator odor–induced avoidance behavior, but increased their risk assessment behaviors compared with vehicle-treated controls. In adulthood, postnatal microglia depletion resulted in significant deficits in male-specific sex behaviors. Using factor analysis, we identified two underlying traits—behavioral disinhibition and locomotion—as being significantly altered by postnatal microglia depletion. These findings further implicate microglia as being critically important to the development of juvenile and adult behavior.

Sex differences in vocal communication are prevalent in both the animals and humans. The mechanism(s) mediating gender differences in human language are unknown, although, sex hormones, principally androgens, play a central role in the development of vocalizations in a wide variety of animal species. The discovery of FOXP2 has added an additional avenue for exploring the origins of language and animal communication. The FOXP2 gene is a member of the forkhead box P (FOXP) family of transcription factors. Prior to the prenatal androgen surge in male fetuses, we observed no sex difference for Foxp2 protein levels in cultured cells. In contrast, 24 hours after the onset of the androgen surge, we found a sex difference for Foxp2 protein levels in cultured cortical cells with males having higher levels than females. Furthermore, we observed the potent nonaromatizable androgen dihydrotestosterone altered not only Foxp2 mRNA and protein levels but also Foxp1. Androgen effects on both Foxp2 and Foxp1 were found to occur in the striatum, cerebellar vermis, and cortex. Immunofluorescence microscopy and coimmunoprecipitation demonstrate Foxp2 and the androgen receptor protein interact. Databases for transcription factor binding sites predict a consensus binding motif for androgen receptor on the Foxp2 promoter regions. We also observed a sex difference in rat pup vocalization with males vocalizing more than females and treatment of females with dihydrotestosterone eliminated the sex difference. We propose that androgens might be an upstream regulator of both Foxp2 and Foxp1 expression and signaling. This has important implications for language and communication as well as neuropsychiatric developmental disorders involving impairments in communication.

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Valproic acid (VPA) is an anti-epileptic drug with teratogenicity activity that has been related to autism. In rodents, exposure to VPA in utero leads to brain abnormalities similar than those reported in the autistic brain. Particularly, VPA reduces the number of Purkinje neurons in the rat cerebellum parallel to cerebellar abnormalities found in autism. Thus, we injected pregnant females on embryonic day 12 either with VPA (600mg/kg, i.p.) or 0.9% saline solution and obtained the cerebellum from their offspring at different postnatal time points. Testosterone has been linked to autism and plays an important role during brain development. Therefore, we identified and analyzed the androgen receptor (AR) by immunohistochemistry and densitometry, respectively. We found VPA decreases AR density in the superficial Purkinje layer only in cerebellar lobule 8 at PN7, but increased it at PN14 compared to control in males. In females, VPA decreased AR density in the superficial Purkinje layer in cerebellar lobule 6 at PN14, but increased it in lobule 9 at the same time point. No differences were found in the deep Purkinje layer of any cerebellar lobule in terms of AR density neither in males nor females. We additionally found a particular AR density decreasing in both superficial and deep regions across development in the majority of cerebellar lobules in males, but in all cerebellar lobules in females. Thus, our results indicate that VPA disrupts the AR ontogeny in the developing cerebellum in an age and region specific manner in male and female rats. Future epigenetic studies including the evaluation of histone deacetylases (HDAC's) might shed light these results as HDAC's are expressed by Purkinje neurons, interact with the AR and are VPA targets. This work contributes to the understanding of the cerebellar development and it might help to understand the role of the cerebellum in neurodevelopmental disorders such as autism.

No abstract

Neuroscience has been transformed by the ability to genetically modify inbred mice, including the ability to express fluorescent markers specific to cell types or activation states. This approach has been put to particularly good effect in the study of the innate immune cells of the brain, microglia. These specialized macrophages are exceedingly small and complex, but also highly motile and mobile. To date, there have been no tools similar to those in mice available for studying these fundamental cells in the rat brain, and we seek to fill that gap with the generation of the genetically modified Sprague Dawley rat line: SD-Tg(Iba1-EGFP)Mmmc . Using CRISPR-Cas/9 technology, we knocked in EGFP to the promoter of the gene Iba1 . With four male and three female founders confirmed by quantitative PCR analysis to have appropriate and specific insertion, we established a breeding colony with at least three generations of backcrosses to obtain stable and reliable Iba1-EGFP expression. The specificity of EGFP expression to microglia was established by flow cytometry for CD45 low /CD11b + cells and by immunohistochemistry. Microglial EGFP expression was detected in neonates and persisted into adulthood. Blood macrophages and monocytes were found to express low levels of EGFP, as expected. Last, we show that EGFP expression is suitable for live imaging of microglia processes in acute brain slices and via intravital two-photon microscopy.