Non-invasive recordings using a patch pipette on the surface of the optic tectum.

<p>(A) Photo of the non-invasive EEG ZF recording set-up. To the left is a surface recording pipette, filled with 1 M NaCl. In the upper right is the reference electrode. Both are connected to the amplifier, as shown in the schematic in (B). The mounted fish is positioned on a microscope with which ZF viability can be monitored continuously. (C), long-term EEG recording of a kcnj10a morphant fish initially paralyzed in 20 mM D-tubocurarine. Fish were generally viable for over an hour, but paralysis appeared to wear off after 50 to 60 min. Movement artifacts ensued, associated with electrical activity and visible twitching (arrowhead). A 5 s period indicated by a box in C is represented above the trace in higher resolution.</p

<i>cln3</i> ATG MO morphants have abnormal brain and heart morphology.

(A, A’) 32 hpf normal development of the WT forebrain, midbrain, hindbrain, retina and fourth ventricle. (B, B’) 32 hpf 1.6 ng cln3 ATG MO morphants display abnormal development of all parts of the brain, a smaller retina, and enlargement of the fourth ventricle. (C, C’) normal development of the WT tail, yolk, pericardial sac and heart at 4 dpf. The WT brain completely fills the cranium. (D, D’) 1.6 ng cln3 ATG MO morphants have a curved tail, and a larger yolk and pericardial sac at 4 dpf. The heart has an elongated appearance and is lacking pigmented erythrocytes. The fourth ventricle is enlarged and the mid- and hindbrain appear smaller. Lateral views. Anterior is to left. Dorsal is up. Abbreviations: v, fourth ventricle; hb, hindbrain; mhb, mid-hindbrain boundary; mb, midbrain, fb, forebrain, r, retina; t, tail; y, yolk; ps, pericardial sac; h, heart; (n = 4 per group). Lateral views. Scale bars: A-D 250 μm; A’-D’ 100 μm.</p

Invasive recordings using a patch pipette inserted into the optic tectum of 120 hpf old ZF larvae.

<p>(A), 22 min recording of a buffer-injected control larva. Frequent spiking and also low level activity is present throughout the trace. (B), higher temporal and voltage resolution of the area marked in A. (C), kcnj10a morphant larva, showing genuine epileptic activity (marked with stars). Low-level activity is followed by a spontaneous large transient (arrowhead). Similar transients were seen while the fish showed brief total body contractions and could also be produced by a light tap on the recording setup (vertical arrow). The square marked “D” in C is shown in (D) at higher resolution.</p

Analysis of surface EEGs.

<p>(A-C) Original traces (top) and averaged frequency spectra (below) obtained in WT ZF, kcnj10a morphant ZF and WT ZF pretreated with pentylenetetrazol. (D) The averaged amplitudes (mean over 2–4 Hz) from (A-C) are compared. Note the significant epileptic activity in kcnj10a morphant (MO) and pentylenetetrazol-treated (PTZ) ZF. (E-G) Original traces (top) and averaged frequency spectra (below) obtained in kcnj10a morphant fish, and kcnj10a morphants treated with pentobarbitone (PB) or diazepam (DZP), respectively. (H) Averaged amplitudes (mean over 2–4 Hz) from (E-G) are compared. Note the significant suppression of epileptic activity by PB treatment, and lack of suppression by DZP.</p

Neurons and glia are disrupted in <i>cln3</i> ATG MO morphants.

<p>(A-A”, B-B”) Immunohistochemical staining for axons (acetylated α-tubulin) at 4 dpf. (A-A”) normal development of axons in WT larvae. (B-B”) 1.6 ng <i>cln3</i> ATG MO morphants have a complete absence of axonal organisation throughout the brain, with axonal accumulation (B, dashed arrows), loss of the optic tectum and a narrowing of the optic nerve (B'', dashed arrow). (A-A”, B-B”), anterior to the left; A, B, lateral view, dorsal up; A’, B’, dorsal view; A”, B” ventral view. (C-C’, D-D’) Immunohistochemistry using antibodies to glia (glial fibrillary acidic protein, GFAP) at 4 dpf. (C-C”) Normal staining in WT larvae. (D-D’) Ectopic GFAP is observed in the notochord in 1.6 ng <i>cln3</i> ATG MO morphants (dashed arrow). Lateral view. Anterior to the left. Dorsal up. (E-E’, F-F’, G-G’) Transgenic zebrafish expressing GFP under the control of the <i>HuC</i> promoter in neurons were injected with 1.6 ng <i>cln3</i> ATG MO and observed at 3 dpf. (E-E’) In WT zebrafish, the normal structure of the developing brain and retina can be observed. (F-F’) In morphants, there appear to be fewer neurons and the normal brain structure is lost. Many GFP-positive cells were enlarged and found nearer the surface of the brain (F’, dashed arrows). (G-G’) When the morphology of these enlarged cells was examined further, they lacked typical neuronal morphology. Lateral view. Anterior to the left. Dorsal up. Abbreviations: cb, cerebellum, fb, forebrain; hb, hindbrain; ot, optic tectum; on, optic nerve. A-G” (all images) Z projection. Scale bars: 100 μm. <i>n</i> = 4 per group.</p

Cellular proliferation is abnormal in <i>cln3</i> ATG MO morphant zebrafish.

<p>(A, A’) Proliferation, assayed at 4 dpf using anti-PH3 (a marker of proliferative cells in mitotic M phase), is observed throughout the 4 dpf WT retina, jaw and the brain. (B, B’) A marked reduction in the amount of cellular proliferation throughout the retina can be seen in the 1.6 ng <i>cln3</i> ATG MO morphant. Although not quantified, it appears that proliferation in the morphant brain (B) is increased compared to WT. Confocal images are Z-projections. Scale bar: 100 μm (A, A’, B) and 50 μm (B’). Lateral views. Anterior is to the left. Dorsal is up. (C) Quantification of these data show that the number of proliferating cells in the morphant retina is significantly reduced from 100.3 cells in WT to 50.3 cells in morphants; ***<i>p</i><0.0006 (<i>n</i> = 3 zebrafish per group). (D) Quantification demonstrating a significantly reduced mean retinal area in the morphants (0.0566 mm² for WT retinae compared to 0.0135 mm² for morphant retinae; ****<i>p</i><0.0001 (n = 10 zebrafish per group)). (C, D) Data represent mean ±SD; results were evaluated using a 2-tailed unpaired Student’s <i>t</i>-test.</p

Cellular proliferation is abnormal in <i>cln3</i> ATG MO morphant zebrafish.

<p>(A, A’) Proliferation, assayed at 4 dpf using anti-PH3 (a marker of proliferative cells in mitotic M phase), is observed throughout the 4 dpf WT retina, jaw and the brain. (B, B’) A marked reduction in the amount of cellular proliferation throughout the retina can be seen in the 1.6 ng <i>cln3</i> ATG MO morphant. Although not quantified, it appears that proliferation in the morphant brain (B) is increased compared to WT. Confocal images are Z-projections. Scale bar: 100 μm (A, A’, B) and 50 μm (B’). Lateral views. Anterior is to the left. Dorsal is up. (C) Quantification of these data show that the number of proliferating cells in the morphant retina is significantly reduced from 100.3 cells in WT to 50.3 cells in morphants; ***<i>p</i><0.0006 (<i>n</i> = 3 zebrafish per group). (D) Quantification demonstrating a significantly reduced mean retinal area in the morphants (0.0566 mm² for WT retinae compared to 0.0135 mm² for morphant retinae; ****<i>p</i><0.0001 (n = 10 zebrafish per group)). (C, D) Data represent mean ±SD; results were evaluated using a 2-tailed unpaired Student’s <i>t</i>-test.</p

Identification and morpholino targeting of the zebrafish Cln3 gene.

<p>(A) CLUSTAL alignment indicates that zebrafish Cln3 amino acid sequence (second row) is 62% similar and 49% identical to human CLN3 (first row). Black background indicates identical residues shared between human and zebrafish, grey background indicates similarity, ‘-’ indicates a gap in the alignment. (B) RT-PCR using primer pairs AF1-AR1 and AF1-AR2 (see C), followed by sequencing, confirmed that the <i>cln3</i> SPL MO affected mRNA splicing. Lanes one and two show the PCR product from cDNA made from <i>cln3</i> SPL MO injected embryos and WT embryos respectively. Arrow in lane three points to the positive control, which is the PCR product generated using plasmid containing <i>cln3</i> mRNA as a template. (C) i) the gene structure of WT <i>cln3</i> showing the relative position of the PCR primers used. ii) The WT mature mRNA. iii) Splice variants caused by injection of <i>cln3</i> SPL MO. Key: lines, introns; boxes, exons; white boxes, untranslated regions; red asterisk, stop codon.</p

Apoptotic cells and lysosomal storage were found in the <i>cln3</i> ATG MO morphant brain.

<p>Acridine orange and Lysotracker staining was carried out on live WT and <i>cln3</i> ATG MO morphant zebrafish aged 4 dpf. (A-A”) WT fish showed low levels of programmed cell death in the brain and very little bright lysosomal staining. (B-B”) Morphants injected with 1.6 ng of <i>cln3</i> ATG MO had increased levels of programmed cell death (acridine orange, bright green) in the brain, this localised particularly to the optic tectum, but only a slight increase in lysosomal staining (red). (C-C”) Morphants injected with 2.9 ng of <i>cln3</i> ATG MO showed very high levels of programmed cell death in the forebrain, midbrain, retina and yolk-body boundary; lysosomal hypertrophy was also observed. (D-D”) Morphants injected with 2.9 ng of <i>cln3</i> ATG MO exhibit abundant apoptotic bodies in the forebrain that often co-localises with lysosomes (arrows). Bright green, acridine orange in apoptotic bodies; Red, Lysotracker red in lysosomes. Abbreviations: ab, apoptotic body; lys, lysosome; ot, optic tectum. A-A", B-B", C-C” are Z projections. D-D” are the same Z slice. Lateral views. Anterior to the left. Dorsal up. The scale bars represent 200 μm and apply to all panels.</p

Survival and activity are compromised in <i>cln3</i> ATG MO morphant zebrafish.

<p>(A-B) <i>cln3</i> ATG MO morphants die prematurely. Progression of <i>cln3</i> ATG morphants demonstrated by monitoring cohorts of fish injected with 1.6 ng or 2.9 ng of <i>cln3</i> ATG MO and uninjected WT siblings. (A) The Kaplan-Meier survival curve shows that all WT larvae survive beyond 6 dpf, whereas the majority of morphants injected with 1.6 ng <i>cln3</i> ATG MO die between 3 and 6 dpf (median survival 5 dpf). Log rank (Mantel-Cox) test <i>p</i><0.0001 (<i>n</i> = 46 WT and <i>n</i> = 23 morphants). (B) The Kaplan-Meier survival curve shows that all WT larvae survive beyond 4 dpf, whereas the morphants injected with 2.9 ng <i>cln3</i> ATG MO die between 1 and 4 dpf (median survival 1 dpf). Log rank (Mantel-Cox) test <i>p</i><0.0001 (<i>n</i> = 16 WT and <i>n</i> = 31 morphants). Error bars indicate ± SE. (C-E) Increased activity was observed in <i>cln3</i> ATG MO morphants at 36 hpf. Analysis of number of spontaneous coils or tail flicks within a 3 minute time period comparing 1.6 ng <i>cln3</i> ATG MO morphants with age matched WT siblings was carried out at 24 hpf (C), 36 hpf (D) and 48 hpf (E). At 24 and 48 hpf the data show no significant difference in the number of coils or flicks. At 36 hpf the data show a significant difference in the number of flicks; <i>p</i><0.0001 (<i>n</i> = 10 zebrafish per treatment group). Data represent mean ± SD; results were evaluated using a 2-tailed unpaired Student’s <i>t</i>-test. (F-G) The <i>cln3</i> ATG MO morphant escape response is diminished. (F) The WT (control) fish aged 4 dpf respond to touch with a C-bend, a turn away from the stimulus and rapid swimming (0.03 second intervals). (G) 1.6 ng c<i>ln3</i> ATG MO morphants aged 4 dpf display a greatly attenuated escape response (0.06 second intervals).</p