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Brain Development Laboratory

Research Interests

1. Abnormal brain development in a rat model of Hirschsprung’s disease caused by endothelin receptor B mutation
   (detailed below).
2. Changes in the enteric nervous system of a rat model of Hirschsprung's disease (with Dr. David  Croaker)
3. Roles of adrenergic receptors in cerebral neuronal development (detailed below).

Research project on brain abnormalities in Hirschsprung’s disease

(with Dr. David  Croaker, Canberra Hospital, Canberra; Dr. Daniel Cass, Westmead Hospital, Sydney).
Hirschsprung’s disease (HSCR) is a congenital malformation characterized by the absence of enteric ganglia (aganglionosis) and is associated with a variety of neurological disorders. HSCR in human, rat and mouse is polygenic, and the endothelin receptor B (ETRB) gene mutation is involved. ETRB has important roles in brains including the increase in neuronal and astrocytic proliferation and anti-apoptosis. In addition, ETRB mediates increases in cytoskeletal proteins in astrocytes associated with injury, and it increases the production of neurotrophic factors.
One of our main scientific goals is to understand the cellular, molecular and functional abnormalities in the brains of HSCR patients, using a rat model of HSCR caused by an ETRB mutation (spotting lethal rat, sl rat). Our initial studies showed that substantially fewer proliferating cells but more apoptotic cells in many brain regions in spotting lethal rat, compared with normal littermates. We also expect to reveal significant changes in the biochemistry of sl rat brains, including altered levels of cytoskeletal proteins and production of neurotrophic factors and endothelins. We are conducting a battery of molecular biological and morphological tests in fetal and postnatal sl rats. Because the endothelin system interacts with the GDNF signalling system commonly involved in HSCR, our findings in the sl rat will contribute to the understanding of HSCR etiology in human. New understanding of the cellular and molecular changes in the brains of HSCR patients will allow investigation of functional defects outside the gut (e.g. autonomic and cognitive defects), and will contribute to the development of therapies for these neurological defects.

Research project on the effects of alpha 2A adrenergic receptors on spine growth.

s Catecholamines (adrenaline and noradrenaline) have been implicated in regulating neuronal maturation through various adrenoceptors. Our study (Song et al. 2004, Neuroscience 123:405-418) demonstrates that application of alpha2 adrenoceptor agonists significantly increases dendritic growth in primary neuronal cultures. We further discovered that alpha2A adrenoceptors regulate dendrite growth through alteration of the phosphorylation of microtubule-associated protein in cortical neurons. We expanded this study to determine the effects of alpha2-ARs on the development of dendritic spines, the small protrusions extending from the dendritic shafts of neurons. Our results showed that application of alpha2-AR agonists, UK 14304 or guanfacine, to cultured neurons caused significant increase in the length and density of dendritic spines. In this project we will examine the underlying molecular mechanisms, which may involve (1) cAMP / cAMP response element binding protein (CREB) pathway (2) direct interaction between adrenoceptors and spinophilin, a key protein in the spines. We will use cell culture, immunocytochemistry and morphometric analysis, together with Western blot quantification of the levels and phosphorylation states of CREB and spinophilin. The results will elucidate the molecular mechanisms of adrenoceptor mediated spine development.

Laboratory Techniques

Retrograde tracing with fluorescent dyes and Neurobiotin
Organ culture, slice culture and primary cell culture
Enzyme histochemistry
Multiple-labelling immunohistochemistry
Fluorescence microscopy
Laser scanning confocal microscopy
Transmission electron microscopy, immuno-electron microscopy
Intracellular recording and whole cell recording
Brain morphometric analysis
Brain apoptosis study
Brain neurogenesis study
Protein analysis (Western blot, slot blot, immunoprecipitation, phosphorylation, ELISA)
DNA analysis (PCR, RT-PCR)