Modeling glial contributions to seizures and epileptogenesis: cation-chloride cotransporters in Drosophila melanogaster.

Flies carrying a kcc loss-of-function mutation are more seizure-susceptible than wild-type flies. The kcc gene is the highly conserved Drosophila melanogaster ortholog of K+/Cl- cotransporter genes thought to be expressed in all animal cell types. Here, we examined the spatial and temporal requirements for kcc loss-of-function to modify seizure-susceptibility in flies. Targeted RNA interference (RNAi) of kcc in various sets of neurons was sufficient to induce severe seizure-sensitivity. Interestingly, kcc RNAi in glia was particularly effective in causing seizure-sensitivity. Knockdown of kcc in glia or neurons during development caused a reduction in seizure induction threshold, cell swelling, and brain volume increase in 24-48 hour old adult flies. Third instar larval peripheral nerves were enlarged when kcc RNAi was expressed in neurons or glia. Results suggest that a threshold of K+/Cl- cotransport dysfunction in the nervous system during development is an important determinant of seizure-susceptibility in Drosophila. The findings presented are the first attributing a causative role for glial cation-chloride cotransporters in seizures and epileptogenesis. The importance of elucidating glial cell contributions to seizure disorders and the utility of Drosophila models is discussed

Giant fiber and lateral pace-making neurons deficient in Kcc are enlarged.

<p>(<b>A1</b>) A 34 µm Z-stack projection in animals expressing membrane-bound GFP in the giant fiber system depicts the stereotyped morphology of the wild-type giant fiber soma, axon and dendrites (insets). (<b>A2</b>) Animals expressing GFP and kcc-RNAi-V in the giant fiber system have giant fiber neurons with enlarged somata and diffuse dendrites lacking defined spines (insets), as seen in this representative 34 µm Z-stack projection. (<b>B1</b>) Wild-type lateral pace-making neurons expressing membrane-bound GFP are nearly spherical, as shown in this representative 10 µm Z-stack projection. (<b>B2</b>) Lateral pace-making neurons expressing GFP and kcc-RNAi-B are enlarged, as seen in this larger (15 µm) representative encompassing Z-stack projection. (<b>C</b>) Quantification of soma volume for control and <i>kcc</i> RNAi expressing lateral pace-making neurons. Error bars are S.E.M. and significance for Student's <i>t</i>-test is: ***  = p<0.001. Scale bars are in microns. For orientation, A: anterior, D: dorsal, L: lateral, M: medial.</p

Reducing <i>kcc</i> expression by RNAi causes behavioral and electrophysiologically-recorded seizure-like activity.

<p>(<b>A</b>) Quantification of behavioral seizure-sensitivity for select GAL4/UAS genotypes, expressed as %bang-sensitive (%BS) paralysis on the y-axis, using two different UAS-kcc-RNAi transgenes. The GAL4 driver and expression domain for each genotype is shown on the x-axis. kcc-RNAi-B (black bars/text) is more effective than kcc-RNAi-V (grey bars/text) with respect to causing %BS paralysis and lethality phenotypes. (<b>B</b>) <i>in vivo</i> stimulation and recording from the giant fiber circuit of a fly mounted in dental wax for quantifying thresholds to evoked seizure-like activity. (<b>C</b>) High-frequency stimulus (200 Hz for 300 ms) seizure-like activity voltage thresholds, in volts high-frequency stimulus (V HFS), for select test and control genotypes. N-values are as noted for each genotype. Error bars are S.E.M. and significance for Student's <i>t</i>-tests is: ***  = p<0.001; n.s.  =  not significant. RNAi expression caused reduced V HFS thresholds relative to controls, thus indicating increased seizure-sensitivity. (<b>D</b>) Representative seizure-like discharges recorded in dorsal longitudinal muscles from flies of select genotypes, as indicated. Green insert is an enlargement of the region enclosed by green lines illustrating a muscle response following a giant fiber threshold stimulus pulse (∼2 V, 0.3 ms pulse-width). Red insert is an enlargement of the region enclosed by the first pair of red lines illustrating a failure following a single giant fiber threshold stimulus pulse. Remaining pairs of red lines indicated failures following seizures in other genotypes.</p

<i>kcc</i> knockdown causes swelling of third instar larval peripheral nerves.

<p>(<b>A1</b>) Membrane-bound glial GFP illuminating peripheral nerves at a low magnification 40 µm slice. (<b>A2</b>) Peripheral nerve glia GFP in a high magnification 2 µm slice colocalizes with the pervasive Kcc (<b>A3</b>) that also marks the bundled neuronal processes (<b>A4</b>). (<b>A5</b>) Orthogonal view of A4 at indicated position (A4, arrowhead). (<b>B1</b>) Glial GFP of swollen peripheral nerves in a low magnification 40 µm slice of animals also expressing glial kcc-RNAi-B. High magnification 2 µm slice of nerve glial GFP (<b>B2</b>) and Kcc (<b>B3</b>) showing whole nerve enlargement (<b>B4</b>). (<b>B5</b>) Orthogonal view of B4 at indicated position (B4, arowhead). (<b>C1</b>) Glial GFP of peripheral nerve in low magnification 40 µm slice of animals also expressing glial ncc69-RNAi-V. High magnification 2 µm slice of nerve glial GFP (<b>C2</b>) and Kcc (<b>C3</b>) showing swelling and fraying in a peripheral nerve bulge (<b>C4</b>), as shown previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101117#pone.0101117-Leiserson1" target="_blank">[26]</a>. (<b>C5</b>) Orthogonal view of C4 at indicated position (C4, arrowhead). (<b>D1</b>) Neuronal GFP of peripheral nerves in low magnification 40 µm slice. (<b>D2</b>) Wild-type neuronal processes in animals expressing neuronal membrane-bound GFP are tightly bundled within peripheral nerve with pervasive Kcc (<b>D3</b>). (<b>D4</b>) Kcc is within and surrounding nerve GFP+ neuronal processes of wild-type larvae. (<b>D5</b>) Orthogonal view of D4 at indicated position (D4, arrowhead). (<b>E1</b>) Neuronal GFP of peripheral nerve in low magnification 40 µm slice of animals also expressing neuronal kcc-RNAi-V. Neuronal processes within peripheral nerve (<b>E2</b>) and Kcc mostly on the surface (<b>E3</b>) do not extensively overlap (<b>E4</b>). (<b>E5</b>) Orthogonal view of E4 at indicated position (E4, arrowhead). (<b>F</b>) and (<b>G</b>) Quantification of average cross-sectional areas of peripheral nerves in control and RNAi genotypes. Error bars are S.E.M. and significance for Student's <i>t</i>-tests is: **  = p<0.01. White numbers indicate abdominal nerve number. Scale bars are in microns.</p

<i>kcc</i> knockdown leads to brain volume increases in 24–48 h-old adults.

<p>Shown are representative brain enlargements via <i>kcc</i> RNAi in 0.5 µm confocal slices, (<b>A2</b>), (<b>B2</b>), (<b>C2</b>), with respect to controls, (<b>A1</b>), (<b>B1</b>), (<b>C1</b>), for the repo, A307, and OK107 GAL4 drivers. *: optic lobes of test or control brains were often severed to distinguish genotypes stained in same solutions. (<b>D</b>) Quantification of mean %volume increases for test genotype brains compared to their respective controls. Expressing <i>kcc</i> RNAi in glia or neurons causes whole-brain swelling. High magnification of glia with membrane-bound GFP in the wild-type mushroom body region (<b>E2</b>) wraps and interlaces neuronal somata and the calyx neuropile (<b>E4</b>). Dorsal Kcc in this same region (<b>E3</b>) colocalizes with cortex and surface glia (<b>E1</b>) and with glomeruli of the calyx (<b>E5</b>). Glia expressing membrane-bound GFP and kcc-RNAi-B in similar mushroom body regions (<b>F2</b>) are largely absent; defined cortex glia, glia wrapping the calyx, and stereotyped surface glia are few, and thus, Kcc colocalization is reduced (<b>F1</b>). Kcc in this region (<b>F3</b>) mostly colocalizes with the neuronal calyx (<b>F4</b>) neuropile (<b>F5</b>). Neuronal expression of membrane-bound GFP in the mushroom body (<b>G1</b>) confirms Kcc localization (<b>G2</b>) in the somata and calyx neuropile (<b>G3</b>). (<b>H1</b>) <i>kcc</i> RNAi and membrane-bound GFP expression in the mushroom body caused a significant reduction of Kcc (<b>H2</b>) in the calyx (<b>H3</b>). Moreover, brain surface Kcc is further from the calyx in this loss-of-function genotype, as seen in other genotypes with neuronal <i>kcc</i> RNAi expression (data not shown). (<b>I</b>) Quantification of Kcc knockdown in the mushroom body calyx due to <i>kcc</i> RNAi expression. Significance for Student's <i>t</i>-tests is: **  = p<0.01; ***  = p<0.001. Scale bars are in microns.</p

The Drosophila surface glia transcriptome: evolutionary conserved blood-brain barrier processes.

AbstractCentral nervous system (CNS) function is dependent on the stringent regulation of metabolites, drugs, cells, and pathogens exposed to the CNS space. Cellular blood-brain barrier (BBB) structures are highly specific checkpoints governing entry and exit of all small molecules to and from the brain interstitial space, but the precise mechanisms that regulate the BBB are not well understood. In addition, the BBB has long been a challenging obstacle to the pharmacologic treatment of CNS diseases; thus model systems that can parse the functions of the BBB are highly desirable. In this study, we sought to define the transcriptome of the adult Drosophila melanogaster BBB by isolating the BBB surface glia with FACS and profiling their gene expression with microarrays. By comparing the transcriptome of these surface glia to that of all brain glia, brain neurons, and whole brains, we present a catalog of transcripts that are selectively enriched at the Drosophila BBB. We found that the fly surface glia show high expression of many ABC and SLC transporters, cell adhesion molecules, metabolic enzymes, signaling molecules, and components of xenobiotic metabolism pathways. Using gene sequence-based alignments, we compare the Drosophila and Murine BBB transcriptomes and discover many shared chemoprotective and small molecule control pathways, thus affirming the relevance of invertebrate models for studying evolutionary conserved BBB properties. The Drosophila BBB transcriptome is valuable to vertebrate and insect biologists alike as a resource for studying proteins underlying diffusion barrier development and maintenance, glial biology, and regulation of drug transport at tissue barriers