Linda L. Restifo

A Systematic Nomenclature for the Insect Brain

Despite the importance of the insect nervous system for functional and developmental neuroscience, descriptions of insect brains have suffered from a lack of uniform nomenclature. Ambiguous definitions of brain regions and fiber bundles have contributed to the variation of names used to describe the same structure. The lack of clearly determined neuropil boundaries has made it difficult to document precise locations of neuronal projections for connectomics study. To address such issues, a consortium of neurobiologists studying arthropod brains, the Insect Brain Name Working Group, has established the present hierarchical nomenclature system, using the brain of Drosophila melanogaster as the reference framework, while taking the brains of other taxa into careful consideration for maximum consistency and expandability. The following summarizes the consortiums nomenclature system and highlights examples of existing ambiguities and remedies for them. This nomenclature is intended to serve as a standard of reference for the study of the brain of Drosophila and other insects.

De Novo Pathogenic SCN8A Mutation Identified by Whole-Genome Sequencing of a Family Quartet Affected by Infantile Epileptic Encephalopathy and SUDEP

Individuals with severe, sporadic disorders of infantile onset represent an important class of disease for which discovery of the underlying genetic architecture is not amenable to traditional genetic analysis. Full-genome sequencing of affected individuals and their parents provides a powerful alternative strategy for gene discovery. We performed whole-genome sequencing (WGS) on a family quartet containing an affected proband and her unaffected parents and sibling. The 15-year-old female proband had a severe epileptic encephalopathy consisting of early-onset seizures, features of autism, intellectual disability, ataxia, and sudden unexplained death in epilepsy. We discovered a de novo heterozygous missense mutation (c.5302A>G [p.Asn1768Asp]) in the voltage-gated sodium-channel gene SCN8A in the proband. This mutation alters an evolutionarily conserved residue in Nav1.6, one of the most abundant sodium channels in the brain. Analysis of the biophysical properties of the mutant channel demonstrated a dramatic increase in persistent sodium current, incomplete channel inactivation, and a depolarizing shift in the voltage dependence of steady-state fast inactivation. Current-clamp analysis in hippocampal neurons transfected with p.Asn1768Asp channels revealed increased spontaneous firing, paroxysmal-depolarizing-shift-like complexes, and an increased firing frequency, consistent with a dominant gain-of-function phenotype in the heterozygous proband. This work identifies SCN8A as the fifth sodium-channel gene to be mutated in epilepsy and demonstrates the value of WGS for the identification of pathogenic mutations causing severe, sporadic neurological disorders.

Exome sequencing reveals new causal mutations in children with epileptic encephalopathies

The management of epilepsy in children is particularly challenging when seizures are resistant to antiepileptic medications, or undergo many changes in seizure type over time, or have comorbid cognitive, behavioral, or motor deficits. Despite efforts to classify such epilepsies based on clinical and electroencephalographic criteria, many children never receive a definitive etiologic diagnosis. Whole exome sequencing (WES) is proving to be a highly effective method for identifying de novo variants that cause neurologic disorders, especially those associated with abnormal brain development. Herein we explore the utility of WES for identifying candidate causal de novo variants in a cohort of children with heterogeneous sporadic epilepsies without etiologic diagnoses.

Discrete pulses of molting hormone, 20-hydroxyecdysone, during late larval development of Drosophila melanogaster: Correlations with changes in gene activity

Periodic pulses of the insect steroid molting hormone 20‐hydroxyecdysone (20E), acting via its nuclear receptor complex (EcR/USP), control gene expression at many stages throughout Drosophila development. However, during the last larval instar of some lepidopteran insects, subtle changes in titers of ecdysteroids have been documented, including the so‐called “commitment peak.” This small elevation of 20E reprograms the larva for metamorphosis to the pupa. Similar periods of ecdysteroid immunoreactivity have been observed during the last larval instar of Drosophila. However, due to low amplitude and short duration, along with small body size and staging difficulties, their timing and ecdysteroid composition have remained uncertain. Employing a rigorous regimen of Drosophila culture and a salivary gland reporter gene, Sgs3‐GFP, we used RP‐HPLC and differential ecdysteroid RIA analysis to determine whole body titers of 20E during the last larval instar. Three small peaks of 20E were observed at 8, 20, and 28 hr following ecdysis, prior to the well‐characterized large peak around the time of pupariation. The possible regulation of 20E levels by biosynthetic P450 enzymes and the roles of these early peaks in coordinating gene expression and late larval development are discussed. Developmental Dynamics 235:315–326, 2006.

Defective neuronal development in the mushroom bodies of Drosophila fragile X mental retardation 1 mutants.

Fragile X mental retardation 1 (Fmr1) is a highly conserved gene with major roles in CNS structure and function. Its product, the RNA-binding protein FMRP, is believed to regulate translation of specific transcripts at postsynaptic sites in an activity-dependent manner. Hence, Fmr1 is central to the molecular mechanisms of synaptic plasticity required for normal neuronal maturation and cognitive ability. Mutations in its Drosophila ortholog, dfmr1, produce phenotypes of brain interneurons and axon terminals at the neuromuscular junction, as well as behavioral defects of circadian rhythms and courtship. We hypothesized that dfmr1 mutations would disrupt morphology of the mushroom bodies (MBs), highly plastic brain regions essential for many forms of learning and memory. We found developmental defects of MB lobe morphogenesis, of which the most common is a failure of β lobes to stop at the brain midline. A similar recessive β-lobe midline-crossing phenotype has been previously reported in the memory mutant linotte. The dfmr1 MB defects are highly sensitive to genetic background, which is reminiscent of mammalian fragile-X phenotypes. Mutations of dfmr1 also interact with one or more third-chromosome loci to promote α/β-lobe maturation. These data further support the use of the Drosophila model system for study of hereditary cognitive disorders of humans.

Remodeling of the insect nervous system

Our nervous systems and behavior are shaped by hormonally driven developmental changes that continue beyond the embryonic period. Key insights into this process have emerged from studies of the insect nervous system. During insect metamorphosis, the nervous system is remodeled through postembryonic neurogenesis, programmed cell death and the modification of persistent neurons. These changes are regulated to a large degree by gene cascades that are triggered by steroid hormones, the ecdysteroids. Current studies are attempting to reveal the molecular mechanisms involved in regulating these dramatic examples of developmental plasticity.

The Steroid Hormone 20-Hydroxyecdysone Enhances Neurite Growth of Drosophila Mushroom Body Neurons Isolated during Metamorphosis

Mushroom bodies (MBs) are symmetrically paired neuropils in the insect brain that are of critical importance for associative olfactory learning and memory. In Drosophila melanogaster, the MB intrinsic neurons (Kenyon cells) undergo extensive reorganization at the onset of metamorphosis. A phase of rapid axonal degeneration without cell death is followed by axonal regeneration. This re-elaboration occurs as levels of the steroid hormone 20-hydroxyecdysone (20E) are rising during the pupal stage. Based on the known role of 20E in directing many features of CNS remodeling during insect metamorphosis, we hypothesized that the outgrowth of MB axonal processes is promoted by 20E. Using a GAL4 enhancer trap line (201Y) that drives MB-restricted reporter gene expression, we identified Kenyon cells in primary cultures dissociated from early pupal CNS. Paired cultures derived from single brains isolated before the 20E pupal peak were incubated in medium with or without 20E for 2–4 d. Morphometric analysis demonstrated that MB neurons exposed to 20E had significantly greater total neurite length and branch number compared with that of MB neurons grown without hormone. The relationship between branch number and total neurite length remained constant regardless of hormone treatment in vitro, suggesting that 20E enhances the rate of outgrowth from pupal MB neurons in a proportionate manner and does not selectively increase neuritic branching. These results implicate 20E in enhancing axonal outgrowth of Kenyon cells to support MB remodeling during metamorphosis.

Phenotypes of Drosophila Brain Neurons in Primary Culture Reveal a Role for Fascin in Neurite Shape and Trajectory

Subtle cellular phenotypes in the CNS may evade detection by routine histopathology. Here, we demonstrate the value of primary culture for revealing genetically determined neuronal phenotypes at high resolution. Gamma neurons of Drosophila melanogaster mushroom bodies (MBs) are remodeled during metamorphosis under the control of the steroid hormone 20-hydroxyecdysone (20E). In vitro, wild-type γ neurons retain characteristic morphogenetic features, notably a single axon-like dominant primary process and an arbor of short dendrite-like processes, as determined with microtubule-polarity markers. We found three distinct genetically determined phenotypes of cultured neurons from grossly normal brains, suggesting that subtle in vivo attributes are unmasked and amplified in vitro. First, the neurite outgrowth response to 20E is sexually dimorphic, being much greater in female than in male γ neurons. Second, the γ neuron-specific “naked runt” phenotype results from transgenic insertion of an MB-specific promoter. Third, the recessive, pan-neuronal “filagree” phenotype maps to singed, which encodes the actin-bundling protein fascin. Fascin deficiency does not impair the 20E response, but neurites fail to maintain their normal, nearly straight trajectory, instead forming curls and hooks. This is accompanied by abnormally distributed filamentous actin. This is the first demonstration of fascin function in neuronal morphogenesis. Our findings, along with the regulation of human Fascin1 (OMIM 602689) by CREB (cAMP response element-binding protein) binding protein, suggest FSCN1 as a candidate gene for developmental brain disorders. We developed an automated method of computing neurite curvature and classifying neurons based on curvature phenotype. This will facilitate detection of genetic and pharmacological modifiers of neuronal defects resulting from fascin deficiency.

Interaction Between Hormonal Signaling Pathways in Drosophila melanogaster as Revealed by Genetic Interaction Between Methoprene-tolerant and Broad-Complex

Juvenile hormone (JH) regulates insect development by a poorly understood mechanism. Application of JH agonist insecticides to Drosophila melanogaster during the ecdysone-driven onset of metamorphosis results in lethality and specific morphogenetic defects, some of which resemble those in mutants of the ecdysone-regulated Broad-Complex (BR-C). The Methoprene-tolerant (Met) bHLH–PAS gene mediates JH action, and Met mutations protect against the lethality and defects. To explore relationships among these two genes and JH, double mutants were constructed between Met alleles and alleles of each of the BR-C complementation groups: broad (br), reduced bristles on palpus (rbp), and 2Bc. Defects in viability and oogenesis were consistently more severe in rbp Met or br Met double mutants than would be expected if these genes act independently. Additionally, complementation between BR-C mutant alleles often failed when MET was absent. Patterns of BRC protein accumulation during metamorphosis revealed essentially no difference between wild-type and Met-null individuals. JH agonist treatment did not block accumulation of BRC proteins. We propose that MET and BRC interact to control transcription of one or more downstream effector genes, which can be disrupted either by mutations in Met or BR-C or by application of JH/JH agonist, which alters MET interaction with BRC.

Animal models of autism spectrum disorders: information for neurotoxicologists.

Recent findings derived from large-scale datasets and biobanks link multiple genes to autism spectrum disorders. Consequently, novel rodent mutants with deletions, truncations and in some cases, overexpression of these candidate genes have been developed and studied both behaviorally and biologically. At the Annual Neurotoxicology Meeting in Rochester, NY in October of 2008, a symposium of clinicians and basic scientists gathered to present the behavioral features of autism, as well as strategies to model those behavioral features in mice and primates. The aim of the symposium was to provide researchers with up-to-date information on both the genetics of autism and how they are used in differing in vivo and in vitro animal models as well as to provide a background on the environmental exposures being tested on several animal models. In addition, researchers utilizing complementary approaches, presented on cell culture, in vitro or more basic models, which target neurobiological mechanisms, including Drosophila. Following the presentation, a panel convened to explore the opportunities and challenges of using model systems to investigate genetic and environment interactions in autism spectrum disorders. The following paper represents a summary of each presentation, as well as the discussion that followed at the end of the symposium.