Research project P7/17 (Research action P7)
In this project we address by a combination of in vivo imaging and molecular techniques the question how brain plasticity is regulated at the cellular level in two songbird models with different song learning and neuroplasticity characteristics. Our hypothesis is that hormones and the environment induce brain plasticity at least in part through epigenetic changes defined as alterations in gene expression that are self-perpetuating in the absence of the original signal that caused them. Recent evidence indicates that DNA methylation, histone modifications (acetylation, methylation) and noncoding RNAs (miRNA, long ncRNA) may serve as a contributing mechanism in memory formation and storage as well as neuronal plasticity. These mechanisms may be relevant for brain plasticity but also more generally in the control of a variety of clinical conditions such as Alzheimer's disease, depression, anxiety, schizophrenia and Rett syndrome. Their analysis is thus of broad interest.
The term neuroplasticity captures a wide diversity of phenomena ranging from activity-dependent changes in synaptic physiology (e.g., Long Term Potentiation and Long Term Depression) to morphological changes in organization of the motor cortex in response to deafferentation of peripheral effector organs or re-growth of spinal nerves after damage. The present research proposal focuses on another significant class of neuroplasticity, namely changes in brain morphology, physiology and organization that spontaneously occur during ontogeny and in adulthood under the influence of sex steroid and thyroid hormones.
Some of the most dramatic examples of plasticity in brain structure and function have been identified in the neural structures that control vocal production in songbirds. The brain of songbirds (oscines) displays an unusual plasticity both during ontogeny and, for most species, across seasons. This plasticity directly relates to the acquisition and expression of songs and thus provides a novel and useful model for understanding the neural plasticity and relationships between perception, cognition, behavior, and the underlying cellular and molecular processes in the nervous system. For historical and financial reasons, research on the song control system and its plasticity has developed mainly in the USA but in Belgium, two research groups have significantly contributed to this research field. Since the mid eighties, the laboratory of Jacques Balthazart (ULg) has in collaboration with Gregory F. Ball (Johns Hopkins University, Baltimore MD) analyzed the seasonal and ontogenetic plasticity of the songbird brain and its chemical neuroanatomy. Beginning in the late-nineties, the Bio-imaging group of Annemie Van der Linden in Antwerp (UA) has developed various MRI techniques (MRI, ME-MRI, DTI, fMRI, rsfMRI) that allow in vivo visualization and sequential studies of the songbird brain. They are to this date the world leaders in this field. J. Balthazart and A. Van der Linden have collaborated since 1998 and this has resulted in the publication of 13 papers co-signed by the two PIs but no specific funding has ever been obtained for this collaboration.
The time is now ripe to consolidate and expand this collaboration between Antwerp and Liège. Recognizing the unique value and potential of songbirds as a model for human biology and medicine, the NIH has supported the sequencing of the zebra finch genome which is now completed. Microarrays have also been developed and made available to the scientific community in order to analyze variation in gene expression in response to various endocrine or environmental factors. Adding to the already collaborating laboratories the expertise of excellent molecular biologists and epigeneticists will permit investigations of the genetic and epigenetic bases of neural plasticity with all new analytical tools developed during the last few years. For that reason they teamed up with Prof. W. Van Criekinge and Prof. T. De Meyer (BIOBIX) who founded in 2009 a next generation sequencing facility (NXTGNT) at UGent, and established a bioinformatics platform on DNA methylation biomarkers and RNA sequencing and also with the UA team of Prof. W. Vanden Berghe with whom the UGent team already had a longstanding collaboration on gene expression and MBD-based methylome profiling in several steroid-sensitive and insensitive cell models. In addition, literature data link thyroid hormones to several aspects of neuroplasticity but surprisingly this notion has never been investigated in relation with songbird neuroplasticity. One partner integrated in the network, Prof. Veerle Darras from KUL, has specialized for many years in the analysis of thyroid hormone effects on development in birds and will therefore provide expertise for the study of the role of these hormones in songbird neuroplasticity.
Our experimental approach will be based on the skills and expertise of the teams in the network.. Plasticity in a variety of contexts will first be identified by in vivo brain imaging techniques and this approach will allow us to determine where and when plasticity takes places. Studies will then follow by a variety of tools ranging from molecular biology, transcriptomics, epigenomics, in situ hybridization, immunohistochemistry and tract tracing to define more precisely the cellular and biochemical events underlying and mediating the observed plasticity.
Specifically, 16 tasks (T) organized in 4 independent but nevertheless related work packages (WP) are outlined in this grant proposal.
In WP1, we shall investigate the genetic and epigenetic mechanisms that control the development of the connections between HVC and RA in the caudal motor pathway of the song control system in zebra finches. Male and female brains will be collected at the adequate developmental ages (T1.1.) and screened by RNA sequencing followed by custom microarray or QPCR for differentially expressed genes as a function of sex and developmental age (T1.2.). The differential expression of selected genes will then be confirmed by in situ hybridization (ISH) or immunohistochemistry (IHC) (T1.3.) and then unbiased DNA methylation profiling of gDNA will be used to establish full epigenome DNA methylation maps during HVC and RA development that will subsequently be validated for selected differentially expressed genes by CpG pyrosequencing (T1.4.). Histone modification specific antibodies will then be tested in immunofluorescence assays on brain sections to provide additional evidence of localized epigenetic mechanisms (T1.5.).
WP2 will analyze the effects of steroid and thyroid hormones on genetic and epigenetic controls of axonal outgrowth during ontogeny in zebra finches. In vivo MRI techniques will first be developed to allow sequential visualization of the HVC to RA connection in developing zebra finches (T2.1.). The effects of the sex steroid estradiol (T2.2.) and of thyroid hormones (T2.3.) on the development of this pathway will then be described by the MRI tools established in T2.1 and by ISH/IHC. Brains will be collected at the critical ages for genetic and epigenetic profiling (T2.4.) as described in WP1. Finally a selection of critical genes that are significantly affected in their expression in parallel with epigenetic modifications will be selected for causal analysis performed by local infusion in the brain of epigenetic drugs or of cofactor specific LNA or siRNA against differentially expressed epigenetic enzymes (T2.5.).
WP3 will be modeled on WP2 but will analyze the genetic and epigenetic control of seasonal plasticity of the HVC to RA connection in adult starlings. This plasticity will first be monitored by MRI tools in birds subjected to photoperiodic manipulations to place them in a physiological stage corresponding to the fall and then switched to long days to simulate the vernal increase in daylength (T3.1.). Brains will be collected at critical time points and we shall investigate the changes in gene expression and their epigenetic control during the development of the HVC-RA connection (T3.2.). The effects of thyroid hormones on these processes will be determined by treatments inducing hypo- or hyperthyroidism (goitrogen/antagonist treatment or T3/T4 administration; T3.3.) and finally the expression of selected genes will be manipulated by antisense technology to test in a causal manner their implication in this type of adult neural plasticity (T3.4.).
Finally in WP4, we will initiate investigations of the brain changes that take place during song learning in adult starlings. As in WP3 photoperiodic manipulations will be used to place male birds in a physiological stage corresponding to the fall when they will be exposed to defined song learning programs. The degree to which they have learned and they express new vocalizations will be quantified by MRI tools and direct sound recording (T4.1.). If reproducible changes in brain activity in response to learned songs are detected by MRI, the underlying genetic and epigenetic mechanisms will be studied by methods similar to those used in WP2 and 3 (T4.2.).
Together these studies will lead to major advances in our understanding of molecular (genetic and epigenetic) mechanisms underlying brain plasticity as it occurs spontaneously during ontogeny or during the annual cycle under the influence of steroid and thyroid hormones. This knowledge is important for the understanding of brain functioning in general (control of behavior, learning, effects of hormones, …) but it should also have an impact on the understanding of the mechanisms that could promote brain repair in pathological conditions such as brain trauma or neurodegenerative diseases.
