Article Figures & Data
Figures
- Figure 1.
Expression of VAChT12Q increases mEPSC amplitude. A, Representative traces of mEPSCs recorded from L3 aCC/RP2 in GAL4 (shown) and UAS (not shown) controls and following expression of VAChT12Q in all cholinergic neurons (chaB19>VAChT12Q). Scale bar: 10 pA/30 ms. B, VAChT 12Q increases both mEPSC amplitude: p < 0.0001 and frequency: p = 0.0001. Whereas expression of wild-type VAChT increases mEPSC frequency p < 0.0001 but not amplitude (p = 0.1). C, Representative SRCs recorded from L3 aCC/RP2 in GAL4 (not shown) and UAS (shown) controls and chaB19>VAChT12Q. Scale bar: 400 pA/500 ms. D, Following expression of VAChT12Q, SRCs show significantly increased duration (chaB19/+: p = 0.001, UAS/+: p = 0.005) and reduced frequency (chaB19/+: p = 0.01, UAS/+: ***p = 0.0003) with no effect to amplitude (p = 0.23). All data points are mean ± SEM, n is stated in each bar. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.
- Figure 2.
Expression of VAChT12Q increases optogentically-evoked EPSC duration. A, Traces of EPSCs recorded from L3 aCC/RP2 in control (chaB19>ChR2) versus experimental (chaB19>ChR2; VAChT or chaB19>ChR2; VAChT12Q) conditions. EPSCs shown are composite averages derived from 9, 6 and 9 cells respectively. Scale bar: 50 pA/500 ms. B, Paired-pulse stimulations, at 1 Hz, show the presence of VAChT12Q enables presynaptic release to resist run-down that occurs in the control (p = 0.007). This is not seen in wild-type VAChT expression (p = 0.13). This effect is abrogated when the second stimulus is applied at 0.05 Hz (p = 0.92). C, Expression of VAChT12Q increased duration of optogenetically-evoked EPSCs (p = 0.03) but did not influence amplitude (p = 0.81). Expression of wild-type VAChT also increased EPSC duration (p = 0.02), again with no effect on amplitude (p = 0.49). All data points are mean ± SEM, n is stated in each bar. *p ≤ 0.05, **p ≤ 0.01.
- Figure 3.
VAChT polyQ manipulation alters spontaneous neurotransmission. A, Representative traces of mEPSCs recorded from embryonic late stage 17 aCC/RP2 between control, VAChT14Q, and VAChTΔQ. Scale bar: 3 pA/30 ms. B, VAChT14Q mutants display significantly reduced mEPSC amplitude (p = 0.005) and frequency (p = 0.005). However, no obvious difference in mEPSC kinetics is observed in VAChT ΔQ mutants for either amplitude (p = 0.37) or frequency (p = 0.24). All data points are mean ± SEM, n stated in each bar.
- Figure 4.
VAChT polyQ manipulation alters evoked neurotransmission. A, Representative traces of SRCs recorded from aCC/RP2 between control, VAChT14Q, and VAChTΔQ. Scale bar: 50 pA/300 ms. Data points are mean ± SEM, n stated in each bar. B, VAChT14Q mutants lack any observable SRCs. By contrast, VAChT ΔQ mutants show SRCs with no observable change in amplitude (p = 0.91). However, VAChT ΔQ mutants exhibit increased SRC duration (p = 0.003) and reduced SRC frequency (**p = 0.01). Control: C, Representative traces (from a total of four experiments) of ChR2ChETA evoked EPSCs recorded from RP2 between control (upper trace) and VAChT14Q (lower trace). Scale: 50 pA/300 ms (upper), 2 V/300 ms (lower).
Tables
- Table 1.
Primers used for creation of Drosophila VAChT UTR with modified PAM sites (5’: a, b, c, d and 3’: e, f, g, h) and modified PolyQ regions (5’: i, j, k, l and 3’: m, n, o, p)
Sequence Use ATCGGGCGCGCCGAATTCATGCTTGGGTCGACTTAAGCTC a a + b (5’PAM) ACAAAGTTCTGATGCAGTTTCTTTGG b CCAAAGAAACTGCATCAGAACTTTGT c c + d CTTAAATAGTCGGGTATAATCGGTACTA d GTACACTAGTTCGTGTTCTTTTGCACACCTCC e e + f (3’PAM) ACGTACCACTTGGCTATATGTCTATA f TATAGACATATAGCCAAGTGGTACGT g g + h GCTACTCGAGAAGTCCGCCACAATGACAACC h GTGCCTACTGGACGGGCT i i + j VAChTΔQ CAGGACCTCTGCTCTGGACGAAGGGATTGGCCACACGG j CCGTGTGGCCAATCCCTTCGTCCAGAGCAGAGGTCCTG k k + l GCTATTAATTAACATATGTAGGAGTATCTGTTCGGGGCAA l GTGCCTACTGGACGGGCT m m + n VAChT+Q CTGCTGCTGCTGCTGTTGTTGTTGCTGCTGCTGCTGCTGCTG n CAGCAGCAGCAGCAACAACAACAACAACAGCAGCAGGTCCAGAGC o o + p GCTATTAATTAACATATGTAGGAGTATCTGTTCGGGGCAA p
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