During my PhD research with HHMI Investigator Michael J. Welsh, we established CFTR, the gene mutated to cause the most common Caucasian lethal genetic disease cystic fibrosis (CF), encodes the epithelial apical membrane chloride channel essential for trans-epithelial ion and fluid secretion. CFTR cDNA induced chloride channels in diverse cell types (Anderson et al., Science, 1991a) and amino acids mutations in transmembrane domains altered pore properties (Anderson et al., Science, 1991b). Our work also established CFTR displays novel channel regulation by intracellular, hydrolysable, nucleoside triphosphates through the nucleotide binding domains (Anderson et al., Cell, 1991; Anderson et al., Science, 1992), clarifying the enigma that CFTR was not a known member of any ion channel gene family, but instead resembled a family of ATP hydrolyzing ABC transporter proteins (pumps). More importantly, the results explained how CF mutations cause disease.
During the interval from 1993-2000, I had a hiatus from research, finishing my MD-PhD training with clinical rotations in medical school, residency clinical training in anatomic pathology (University of Iowa College of Medicine), subspecialty fellowship training in neuropathology (Brigham and Women’s Hospital; Boston Children’s Hospital; and Massachusetts General Hospital), and board certification in both clinical fields. From 1998-2003, I practiced medicine as a Senior Staff Physician at Brigham and Women’s Hospital, Boston Children’s Hospital, and then at Massachusetts General Hospital with neuropathology case sign out responsibilities. Partially overlapping this clinical practice period from 2000-2005, I initiated my postdoctoral research fellowship training to expand my research skill set, and transition my biomedical research from epithelial transport/ion channel biophysics to neuroscience/molecular mouse genetics. This phase of training took place with Nobel Laureate and HHMI Investigatory Susumu Tonegawa at MIT where I acquired expertise in the following areas: neuroscience, conditional mouse genetics, bacterial artificial chromosome gene molecular engineering, mouse sleep physiology, mouse behavioral analysis, and brain slice whole cell patch clamp electrophysiology. During this time, I also trained in the implantation of microdrives onto mice for in vivo multi-unit tetrode wire electrophysiology recording of hippocampus in awake behaving mice with the laboratory Professor Matt Wilson at MIT. As is typical and expected of trainees in the Tonegawa lab, a single seminal study was published after 5-6 years of personally engineering two new conditional mouse genetic lines, developing slice whole cell patch clamp of thalamus, and performing in vivo electrophysiologic recordings to publish this work. The subjects and methods were novel to the Tonegawa Lab that had primarily been focused on learning and memory and synaptic plasticity.
My studies examined the function of ion channels mediating burst firing, prominent in thalamus during sleep. Cre/loxP-based conditional mouse genetics were developed to identify a novel form of insomnia produced when T-type Ca2+ channel Cav3.1 is selectively inactivated in thalamus. This was the first and only example of selective gene manipulation in thalamus, now available at JAXS lab (Tg(Kcnc2-cre)K128Stl). In aggregate, the studies identified a brain region and cellular mechanism for a novel sleep disorder (Anderson et al., PNAS, 2005). The brain slice electrophysiology data in the manuscript were actually performed in my own laboratory at Harvard Medical School and Beth Israel Deaconess Medical Center.
In my own laboratory, beginning in 2005/2006, I reconstituted the techniques of my two former mentor’s laboratories. Using this repertoire of techniques, my lab then generated a new mouse model of a human epilepsy disorder due to mutations in LGI1, performed brain slice whole cell patch clamp electrophysiology to interrogate hippocampal circuits, and utilized in vivo extracellular recordings of cortical and hippocampal neuronal activity to examine seizure susceptibility. We discovered that the human epilepsy gene, LGI1, is mutated to arrest postnatal development of glutamatergic circuits (Zhou et al. Nature Medicine, 2009; NINDS R01NS057444-01), revealing a novel mechanism for human epilepsy and uncovering a key molecular pathway in childhood brain development (News and Views, Nature Medicine, 2009; Anderson. Epilepsy Currents, 2010). In manuscripts under revision or submitted, we have subsequently established that LGI1 regulates axonal pruning in the thalamic retinogeniculate circuit during postnatal childhood brain (Zhou et al. J Neuroscience 2011) and regulates the adaptive homeostatic response of thalamic neuronal circuits to a seizure (Smith et al. J Neurochemistry 2011), both providing further insights into the pathophysiological basis of this human epilepsy.
In other work currently also under revision, we created a mouse transgenic model of human epilepsy associated with T-type Ca2+ channel Cav3.2 mutations (NINDS K02 NS054674) and report an age-dependent promotion of spontaneous network discharge that is magnified by a prior seizure (environment) and an epilepsy-associated mutation (gene) leading to a gene-environment-developmental age interaction model of human childhood absence epilepsy.
In a manuscript developed through a collaborative study on hypothalamic circuit repair under revision (Science 2011, collaborating with Drs. Jeffrey Flier and Jeffrey Mackliss), my lab used fluorescence-guided whole-cell, patch-clamp to analyze fluorescent progenitor transplanted neurons in hypothalamic brain slices. The study evaluates whether transplanted progenitors can functionally incorporate into the adult hypothalamus to rescue obesity due to leptin receptor deficiency. GFP-positive neural progenitors incorporate into hypothalamic circuitries, again as a large variety of neuronal subtypes. Furthermore, reconstituting the leptin responsive functions of the local hypothalamic circuitry partially ameliorated the obesity phenotype. In a second collaborative study with Dr. Jeffrey Flier’s lab, we evaluated whether functional neurogenesis continues in adult hypothalamus. Adult neurogenesis is currently only well established in dentate gyrus (one neuronal subtype) and olfactory bulb (two neuronal subtypes). Using retroviral (GFP) labeling, newly generated cells in the adult hypothalamus were shown to be electrophysiologically active neurons. Unlike these other two adult neurogenesis zones, hypothalamus generated at least three distinct neuronal subtypes (electrophysiological properties). Furthermore, newborn hypothalamic neurons form both excitatory and inhibitory synaptic connection with other neurons within native hypothalamus established via paired neuron recordings.
Currently, a major effort of my laboratory is to uncover the cellular mechanisms of human autism and schizophrenia, focusing on recently reported genomic copy number variations. We recently succeeded in reconstituting all three core autism behavioral traits (impaired social interaction, reduced vocal communication, and repetitive self grooming behavior) in a model of the most common human genetic autism disorder due to copy number variations, 15q11-13 duplication (inv dup15) and triplication (isodicentric chromosome, idic15) (NINDS R21 ARRA Heterogeneity in Autism Disorder Grant 1R21NS070295-01; Science Translational Medicine 2011; patent filed). We also identified underlying circuit defects that could be important in generating some of the behavioral deficits. We are also developing mouse models of schizophrenia due to the most common copy number variation, 16p11.2 duplication, and to investigate the underlying circuit defects.
In summary, my laboratory now merges these diverse training experiences combining mouse genetic engineering, behavioral analysis, in vivo electrophysiology, and ultrastructural, biochemical, and in vitro patch clamp electrophysiological analysis of neuronal circuitries to gain insights into the pathophysiological mechanisms of neurodevelopmental disorders including autism, schizophrenia, and epilepsy.