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

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

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

<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