Daria S. Hekmat-Scafe

Mutations in the K+/Cl− Cotransporter Gene kazachoc (kcc) Increase Seizure Susceptibility in Drosophila

During a critical period in the developing mammalian brain, there is a major switch in the nature of GABAergic transmission from depolarizing and excitatory, the pattern of the neonatal brain, to hyperpolarizing and inhibitory, the pattern of the mature brain. This switch is believed to play a major role in determining neuronal connectivity via activity-dependent mechanisms. The GABAergic developmental switch may also be particularly vulnerable to dysfunction leading to seizure disorders. The developmental GABA switch is mediated primarily by KCC2, a neuronal K+/Cl− cotransporter that determines the intracellular concentration of Cl− and, hence, the reversal potential for GABA. Here, we report that kazachoc (kcc) mutations that reduce the level of the sole K+/Cl− cotransporter in the fruitfly Drosophila melanogaster render flies susceptible to epileptic-like seizures. Drosophila kcc protein is widely expressed in brain neuropil, and its level rises with developmental age. Young kcc mutant flies with low kcc levels display behavioral seizures and demonstrate a reduced threshold for seizures induced by electroconvulsive shock. The kcc mutation enhances a series of other Drosophila epilepsy mutations indicating functional interactions leading to seizure disorder. Both genetic and pharmacological experiments suggest that the increased seizure susceptibility of kcc flies occurs via excitatory GABAergic signaling. The kcc mutants provide an excellent model system in which to investigate how modulation of GABAergic signaling influences neuronal excitability and epileptogenesis.

Control of Drosophila perineurial glial growth by interacting neurotransmitter-mediated signaling pathways

Drosophila peripheral nerves, similar structurally to the peripheral nerves of mammals, comprise a layer of axons and inner glia, surrounded by an outer perineurial glial layer. Although it is well established that intercellular communication occurs among cells within peripheral nerves, the signaling pathways used and the effects of this signaling on nerve structure and function remain incompletely understood. Here we demonstrate with genetic methods that the Drosophila peripheral nerve is a favorable system for the study of intercellular signaling. We show that growth of the perineurial glia is controlled by interactions among five genes: ine, which encodes a putative neurotransmitter transporter; eag, which encodes a potassium channel; push, which encodes a large, Zn2+-finger-containing protein; amn, which encodes a putative neuropeptide related to the pituitary adenylate cyclase activator peptide; and NF1, the Drosophila ortholog of the human gene responsible for type 1 neurofibromatosis. In other Drosophila systems, push and NF1 are required for signaling pathways mediated by Amn or the pituitary adenylate cyclase activator peptide. Our results support a model in which the Amn neuropeptide, acting through Push and NF1, inhibits perineurial glial growth, whereas the substrate neurotransmitter of Ine promotes perineurial glial growth. Defective intercellular signaling within peripheral nerves might underlie the formation of neurofibromas, the hallmark of neurofibromatosis.

A high-enrollment course-based undergraduate research experience improves student conceptions of scientific thinking and ability to interpret data.

The authors developed and assessed an innovative course-based undergraduate research experience that emphasized collaboration among students and focused on data analysis.

Seizure sensitivity is ameliorated by targeted expression of K+-Cl- cotransporter function in the mushroom body of the Drosophila brain.

The kccDHS1 allele of kazachoc (kcc) was identified as a seizure-enhancer mutation exacerbating the bang-sensitive (BS) paralytic behavioral phenotypes of several seizure-sensitive Drosophila mutants. On their own, young kccDHS1 flies also display seizure-like behavior and demonstrate a reduced threshold for seizures induced by electroconvulsive shock. The product of kcc shows substantial homology to KCC2, the mammalian neuronal K+–Cl− cotransporter. The kccDHS1 allele is a hypomorph, and its seizure-like phenotype reflects reduced expression of the kcc gene. We report here that kcc functions as a K+–Cl− cotransporter when expressed heterologously in Xenopus laevis oocytes: under hypotonic conditions that induce oocyte swelling, oocytes that express Drosophila kcc display robust ion transport activity observed as a Cl−-dependent uptake of the K+ congener 86Rb+. Ectopic, spatially restricted expression of a UAS-kcc+ transgene was used to determine where cotransporter function is required in order to rescue the kccDHS1 BS paralytic phenotype. Interestingly, phenotypic rescue is largely accounted for by targeted, circumscribed expression in the mushroom bodies (MBs) and the ellipsoid body (EB) of the central complex. Intriguingly, we observed that MB induction of kcc+ functioned as a general seizure suppressor in Drosophila. Drosophila MBs have generated considerable interest especially for their role as the neural substrate for olfactory learning and memory; they have not been previously implicated in seizure susceptibility. We show that kccDHS1 seizure sensitivity in MB neurons acts via a weakening of chemical synaptic inhibition by GABAergic transmission and suggest that this is due to disruption of intracellular Cl− gradients in MB neurons.

Olfactory Coding in a Compound Nose: Coexpression of Odorant-Binding Proteins in Drosophilaa

ABSTRACT: Odorant‐binding proteins (OBPs) are small, soluble proteins present in the aqueous medium surrounding olfactory receptor neurons. Their function in olfaction is unknown: they have been proposed to facilitate the transit of hydrophobic molecules to olfactory receptors, to deactivate the odorant stimulus, and/or to play a role in chemosensory coding. We have examined the genomic organization and expression patterns of two olfactory‐specific genes (OS‐E and OS‐F) of Drosophila melanogaster, the products of which are members of a protein family in Drosophila sharing sequence similarity with moth OBPs. We found that the OS‐E and OS‐F transcription units are located <1 kb apart. They are oriented in the same direction and display a similar intron‐exon organization. Expression of both OS‐E and OS‐F proteins is spatially restricted to the ventro‐lateral region of the Drosophila antenna. Within this region, both OS‐E and OS‐F proteins are expressed within two different types of sensory hairs: in most, if not all, sensilla trichodea and in ∼40% of the interspersed small sensilla basiconica. We consistently observe that OS‐E and OS‐F are coexpressed, indicating that an individual sensillum can contain more than one odorant‐binding protein. This finding has potential implications for the roles of odorant‐binding proteins in olfactory coding.

Using yeast to determine the functional consequences of mutations in the human p53 tumor suppressor gene: An introductory course-based undergraduate research experience in molecular and cell biology

The opportunity to engage in scientific research is an important, but often neglected, component of undergraduate training in biology. We describe the curriculum for an innovative, course‐based undergraduate research experience (CURE) appropriate for a large, introductory cell and molecular biology laboratory class that leverages students′ high level of interest in cancer. The course is highly collaborative and emphasizes the analysis and interpretation of original scientific data. During the course, students work in teams to characterize a collection of mutations in the human p53 tumor suppressor gene via expression and analysis in yeast. Initially, student pairs use both qualitative and quantitative assays to assess the ability of their p53 mutant to activate expression of reporter genes, and they localize their mutation within the p53 structure. Through facilitated discussion, students suggest possible molecular explanations for the transactivation defects displayed by their p53 mutants and propose experiments to test these hypotheses that they execute during the second part of the course. They use a western blot to determine whether mutant p53 levels are reduced, a DNA‐binding assay to test whether recognition of any of three p53 target sequences is compromised, and fluorescence microscopy to assay nuclear localization. Students studying the same p53 mutant periodically convene to discuss and interpret their combined data. The course culminates in a poster session during which students present their findings to peers, instructors, and the greater biosciences community. Based on our experience, we provide recommendations for the development of similar large introductory lab courses.