Spontaneous seizures and altered gene expression in GABA signaling pathways in a mind bomb mutant zebrafish

Abstract

Disruption of E3 ubiquitin ligase activity in immature zebrafish mind bomb mutants leads to a failure in Notch signaling, excessive numbers of neurons, and depletion of neural progenitor cells. This neurogenic phenotype is associated with defects in neural patterning and brain development. Because developmental brain abnormalities are recognized as an important feature of childhood neurological disorders such as epilepsy and autism, we determined whether zebrafish mutants with grossly abnormal brain structure exhibit spontaneous electrical activity that resembles the long-duration, high-amplitude multispike discharges reported in immature zebrafish exposed to convulsant drugs. Electrophysiological recordings from agar immobilized mind bomb mutants at 3 d postfertilization confirmed the occurrence of electrographic seizure activity; seizure-like behaviors were also noted during locomotion video tracking of freely behaving mutants. To identify genes differentially expressed in the mind bomb mutant and provide insight into molecular pathways that may mediate these epileptic phenotypes, a transcriptome analysis was performed using microarray. Interesting candidate genes were further analyzed using conventional reverse transcriptase-PCR and real-time quantitative PCR, as well as whole-mount in situ hybridization. Approximately 150 genes, some implicated in development, transcription, cell metabolism, and signal transduction, are differentially regulated, including downregulation of several genes necessary for GABA-mediated signaling. These findings identify a collection of gene transcripts that may be responsible for the abnormal electrical discharge and epileptic activities observed in a mind bomb zebrafish mutant. This work may have important implications for neurological and neurodevelopmental disorders associated with mutations in ubiquitin ligase activity.

Figure 1.

Seizures in immature mind bomb zebrafish. A1, Representative image of an agar immobilized wild-type sibling zebrafish at 3 dpf. Note the patch recording electrode, at right, placed in the forebrain. A2, Gap-free extracellular field recordings from two different WT sibling zebrafish recorded in normal embryo medium. Top trace represents a typical recording from the zebrafish forebrain; same fish as in A1. Bottom trace represents a typical recording from the zebrafish optic tectum. Note the absence of large-amplitude multispike activity in these recordings. B1, Representative image of an agar immobilized mib^hi904 mutant zebrafish at 3 dpf. Note the curled body, small and misshapen tectum and forebrain, and smaller eyes. B2, Gap-free extracellular field recordings from two different mib^hi904 mutant zebrafish recorded in normal embryo medium. Top trace represents a typical recording from the zebrafish forebrain; same fish as in B1. Bottom trace represents a typical recording from the zebrafish optic tectum. Note the presence of large-amplitude spontaneous burst discharges. C, High-resolution traces from the gap-free recordings shown in A and B: a, brief burst of synchronized activity; b, long-duration high-amplitude burst; c, long-duration high-amplitude burst with clear multispike activity; and d, typical long-duration high-amplitude burst with multispike activity recorded in a 3 dpf WT zebrafish exposed to 15 mm PTZ. D, Extracellular field responses obtained after paired-pulse stimulation (10–90 s interpulse interval) of the contralateral eye. Paired-pulse facilitation at brief interpulse intervals (10–40 s) can be seen in traces from WT sibling and mib^hi904 mutant zebrafish recorded in normal embryo medium at 3 dpf. A plot of percentage facilitation (second population spike amplitude/first population spike amplitude × 100) at all interpulse intervals is shown at right. Stimulus intensity was set at three times the threshold for eliciting a detectable population response. Filled circles, WT control; open circles, mib. Data are shown as the mean ± SEM.

Figure 2.

Seizure behaviors in immature mind bomb zebrafish. A, Sample locomotion tracking plots are shown for mib^hi904 mutant zebrafish in normal bathing medium. Blue dots indicate movement; lines indicate rapid convulsive-like activity. Plots are shown for typical stage 0 (S0; no swim activity), stage 1 (S1; increased swim activity), and stage 3 (S3; full-body convulsions) recording epochs. For an example of a stage 3 seizure, see supplemental Video 1 (available at www.jneurosci.org as supplemental material). B, Bar plot showing the percentage of all fish recorded during locomotion tracking. Plots were scored by individuals blind to the status of the zebrafish and show a clear trend toward spontaneous seizure-like behavior in mib^hi904 mutant zebrafish.

Figure 3.

Expression pattern of BDNF in WT and mib^hi904 zebrafish (3 dpf). A, Detection of gene expression in 2% ethidium bromide agarose gel electrophoresis. B, Levels of mRNA, measured by quantitative PCR and normalized to β-act. Error bars indicate ±SEM. Student's t test, p < 0.05. C, Whole-mount in situ hybridization, lateral view. Note a diffuse but prominent upregulation in the telencephalon, optic tectum, midbrain, and hindbrain of age-matched mib^hi904 mutants. Ba, Branchial arches; DC, diencephalon; HB, hindbrain; MHB, midbrain hindbrain boundary; Tel, telencephalon; TeO, optic tectum.

Figure 4.

Expression pattern of PYYa in WT and mib^hi904 zebrafish (3 dpf). A, Detection of gene expression in 2% ethidium bromide agarose gel electrophoresis. B, Levels of mRNA, measured by quantitative PCR and normalized to β-act. Error bars indicate ±SEM. Student's t test, p < 0.05. C, Whole-mount in situ hybridization, lateral view. Note a low-level expression of PYYa near the olfactory bulb, locus ceruleus, ventral thalamic cluster, and hindbrain in wild-type sibling zebrafish and a prominent upregulation of gene expression in hindbrain extending into the spinal cord in mib^hi904 mutants. Ce, Cerebellum; LC, locus ceruleus; HB, hindbrain; MO, medulla oblongata; OB, olfactory bulb; Pr, pretectum; SC, spinal cord; TeO, optic tectum; Tel, telencephalon; VT, ventral thalamic cluster.

Figure 5.

Expression pattern of GAD1 in WT and mib^hi904 zebrafish (3 dpf). A, Detection of gene expression in 2% ethidium bromide agarose gel electrophoresis. B, Levels of mRNA, measured by quantitative PCR and normalized to β-act. Error bars indicate ±SEM. Student's t test, p < 0.05. C, Whole-mount in situ hybridization, ventral view. Note prominent reduction of GAD1 expression in mib^hi904 mutants (near absence) in the boundary of the olfactory pit and diencephalon. Some nonspecific staining can be seen in the eyes of the mib^hi904 mutants. DC, Diencephalon; HB, hindbrain; OP, olfactory pit; Ret, retina; Tel, telencephalon.

Figure 6.

Expression pattern of Gabra1 in WT and mib^hi904 zebrafish (3 dpf). A, Detection of gene expression in 2% ethidium bromide agarose gel electrophoresis. B, Levels of mRNA, measured by quantitative PCR and normalized to β-act. Error bars indicate ±SEM. Student's t test, p < 0.05. C, Whole-mount in situ hybridization, lateral view. Note a dramatic reduction in the spatial expression pattern of Gabra1 in mib^hi904 mutants in the telencephalon, optic tectum, and hindbrain. Aac, Arch associated cells; Ce, cerebellus; HB, hindbrain; MHB, midbrain hindbrain boundary; TeO, optic tectum; Tel, telencephalon.

Figure 7.

Expression pattern of Calb2 in WT and mib^hi904 zebrafish (3 dpf). A, Detection of gene expression in 2% ethidium bromide agarose gel electrophoresis. B, Levels of mRNA, measured by quantitative PCR and normalized to β-act. Error bars indicate ±SEM. Student's t test, p < 0.05. C, Whole-mount in situ hybridization, lateral view. Note a dramatically reduced Calb2 expression pattern in the telencephalon, optic tectum, cerebellus, and hindbrain in mib^hi904 mutants. Ce, Cerebellus; DC, diencephalon; HB, hindbrain; MHB, midbrain hindbrain boundary; TeO, optic tectum; Tel, telencephalon.

Figure 8.

Expression pattern of Pvalb5 in WT and mib^hi904 zebrafish (3 dpf). A, Detection of gene expression in 2% ethidium bromide agarose gel electrophoresis. B, Levels of mRNA, measured by quantitative PCR and normalized to β-act. Error bars indicate ±SEM. Student's t test, p < 0.05. C, Whole-mount in situ hybridization, lateral view. Note a clear reduction in Pvalb5 expression across the entire brain in mib^hi904 mutants. DC, Diencephalon; hy, hypothalamus; HB, hindbrain; MHB, midbrain hindbrain boundary; OP, olfactory pits; Tel, telencephalon; TeO, optic tectum.