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Molecular mechanisms that stabilize short term synaptic plasticity during presynaptic homeostatic plasticity

  1. Jennifer M Ortega
  2. Özgür Genç
  3. Graeme W Davis  Is a corresponding author
  1. Kavli Institute for Fundamental Neuroscience, University of California San Francisco, California
Research Article
Cite this article as: eLife 2018;7:e40385 doi: 10.7554/eLife.40385
9 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Preservation of Release Dynamics During Presynaptic Homeostatic Plasticity.

(A) Example traces at the indicated external calcium concentration in the presence or absence of PhTx and PdBu. (B) Paired-pulse ratio (EPSC4/EPSC1) versus initial EPSC amplitude (EPSC1) at two external calcium concentrations as indicated. (C) Data from (B) re-plotted (gray) with the addition of data recorded in the presence of PhTx (light and dark red) for indicated external calcium concentrations. (D) Data from (B) re-plotted (gray) with the addition of data recorded in the presence of PdBu (light and dark blue) for indicated external calcium concentrations. (E) Data for mEPSP, EPSC and quantal content for control (ctrl), control in the presence of PdBu (PdBu), control in the presence of PhTx (PhTx) and control synapses incubated in PhTx followed by PdBU (PhTx +PdBu). At right, percent change is calculated as quantal content recorded in the presence of PdBu versus control in the absence of PdBu. (F) EPSC amplitude in the presence (red) and absence (black) of PdBu at the indicated extracellular calcium concentrations. (G) Schematic highlighting the homeostatic doubling of the pool of docked primed vesicles during presynaptic homeostatic plasticity (PHP) in the presence of PhTx. The ratio of primed (light red) to super-primed (dark red) vesicles is held constant, thereby preserving presynaptic release dynamics. ns, not significant; *p<0.05, **p<0.01, ***p<0.001; Data represent mean ±SEM. Student’s t-test, two tailed.

https://doi.org/10.7554/eLife.40385.002
Figure 1—figure supplement 1
PdBu-dependent potentiation converges to wild type steady state during a prolonged stimulus train.

Average peak amplitudes of 60 Hz trains in the absence (black) and presence (red) of PdBu (n = 6 for control, n = 4 for PdBu, data are mean ±SEM). The average of the last three data points at the end of the stimulus trains are statistically similar (p>0.1; Student’s t-test, two tails).

https://doi.org/10.7554/eLife.40385.003
Identification of Rop as a SNARE-Associated Molecule Involved in the Rapid Induction of Presynaptic Homeostasis.

(A) Schematic of the Drosophila Rop gene locus (top) and protein (bottom). Coding exon is shown in dark purple and non-coding DNA is in gray. Protein is shown in light purple. Point mutations in Rop mutant alleles (RopG27 and RopG11) are indicated by yellow stars. Deficiency Df(3L)BSC735 uncovers the Rop gene locus as indicated. Syntaxin-binding domains (SBD) of the Rop protein are shown (pink). (B) Average data for mEPSP amplitude in the absence (baseline) and presence (PhTx) of PhTx for WT and heterozygous deficiency chromosome Df(3L)BSC735 (DfRop/+). PhTx application reduces amplitudes in all genotypes (p<0.01). Data represent mean ± SEM. (C) Average data for EPSP amplitude as in (B); sample sizes for data in (B–D) are shown on bar graph; ns, not significant; *p<0.05; Student’s t-test. (D) Average mEPSP amplitude and quantal content are normalized to values in the absence of PhTx for each genotype. ***p<0.001. (E) Sample traces showing EPSP and mEPSP amplitudes ± PhTx for indicated genotypes. (F–H) Average mEPSP (F) EPSP (G) and Quantal Content (H) for indicated genotypes; ns, not significant; ****p<0.0001; **p<0.01. (I) Average percent change in mEPSP amplitude and quantal content in PhTx compared to baseline for indicated genotypes; **p<0.01. Data are mean ±SEM for all figures. Student’s t-test, two tailed.

https://doi.org/10.7554/eLife.40385.004
Rop is Required Neuronally During Presynaptic Homeostasis.

(A) Sample traces showing EPSP and mEPSP amplitudes ± PhTx for indicated genotypes. (B) Average data for mEPSP when UAS-Rop-RNAi is expressed pan-neuronally (c155-GAL4)±Phtx as indicated. PhTx reduces amplitudes in all genotypes; p<0.01. (C) Average data for EPSP as in B; ns, not significant; **p<0.01. (D) Average data for Quantal Content as in B; **p<0.01. (E) Average percent change in mEPSP amplitude and quantal content in PhTx compared to baseline for indicated genotypes; ***p<0.001. (F) Sample traces showing mEPSPs for indicated genotypes (left) and average mEPSP frequencies (Hz) (right). Student’s t-test, two tailed.

https://doi.org/10.7554/eLife.40385.005
Suppression of Presynaptic Homeostasis is maintained with increased [Ca2+]e in Rop mutants.

(A) Sample EPSC traces in the absence (baseline) and presence (PhTx) of PhTx for WT and heterozygous RopG27 mutant at 1.5 mM extracellular calcium [Ca2+]e. (B) Average data for mEPSP amplitude ± PhTx for indicated genotypes. PhTx application reduces amplitude in all genotypes (p<0.01). Data represent mean ± SEM. (C) Average data for EPSC amplitude as in (B); ns, not significant; **p<0.01, *p<0.05; Student’s t test, two tailed. Sample sizes indicated on bar graph. (D–E) Average mEPSP (D) EPSC (E) ± PhTx for each genotype at 3.0 mM extracellular calcium [Ca2+]e; ns, not significant; **p<0.01; Data represent mean ±SEM. Student’s t-test, two tailed. (F) Relationship between mean quantal content and [Ca2+]e (left axis) and relationship between quantal content normalized to values in the absence of PhTx and [Ca2+]e (right axis) for WT and RopG27 heterozygous mutants.

https://doi.org/10.7554/eLife.40385.006
Decreased Release Probability in Rop Mutants.

(A) Sample EPSC traces (top) and cumulative EPSC amplitudes (bottom) ±PhTx for indicated genotypes. Experiment used 60 Hz stimulation (30 stimuli) in 1.5 mM [Ca2+]e. Red line is fit to cumulative EPSC data and back extrapolated to time zero. (B) Average cumulative EPSC amplitudes ± PhTx for indicated genotypes; ns, not significant. Student’s t-test, two tailed. (C) Average Ptrain ±PhTx for indicated genotypes. Ptrain = 1st EPSC/cumEPSC; ns, not significant; ***p<0.001. (D) Average EPSC amplitudes normalized to the first pulse are plotted against stimulus number for indicated genotypes.

https://doi.org/10.7554/eLife.40385.007
Rop Interacts with rim during Synaptic Homeostasis.

(A) Average data for mEPSP amplitude ±PhTx for WT, heterozygous RopG27/+ mutant heterozygous rim103/+ mutant, and transheterozygous RopG27/rim103 mutant at 0.3 mM [Ca2+]e. PhTx application reduces amplitude in all genotypes (p<0.01). Data represent mean ±SEM. Student’s t test. (B) Average data for EPSP amplitude ±PhTx for indicated genotypes; statistics as in (A); *p<0.05; ***p<0.001; ****p<0.0001, Student’s t-test, two tailed. (C) Average data for Quantal Content ±PhTx for indicated genotypes as in (A); ns, not significant; **p<0.01; ****p<0.0001; Student’s t-test, two tailed. (D) Average data for mEPSP and quantal content normalized to values in the absence of PhTx for indicated genotypes. Statistical comparisons are made within each genotype to baseline in the absence of PhTx. Student’s t-test, two tailed. (E) Each point represents average data from an individual NMJ recording. For WT, recordings in the absence of PhTx are dark gray, those with PhTx are light gray. For RopG27/+, recordings in the absence of PhTx are dark purple, those with PhTx are light purple. For rim103/+, recordings in the absence of PhTx are dark blue, those with PhTx are light blue. For RopG27/rim103, recordings in the absence of PhTx are dark red, those with PhTx are light red. The black line in the WT graph is a curve fit to this control data. The same wild type curve-fit is overlaid on all other genotypes for purposes of comparison. Dotted black lines encompass 95% of wild type data points. These same lines from wild type are superimposed on the graphs for indicated genotypes. (F–H) Average percent change in mEPSP amplitude and quantal content in PhTx compared to baseline for trans heterozygous combinations: RopG27/rbpSTOP1 (F), rim103/+; dunc13P84200/+ (G), rim103/rbpSTOP1 (H); ns, not significant; *p<0.05; Student’s t-test, two tailed.

https://doi.org/10.7554/eLife.40385.008
Enhanced action of PdBu at synapses depleted of both Rop and RIM.

(A) Paired-pulse ratio is plotted against initial EPSC amplitude for the indicated genotypes and conditions (in the absence and presence of PdBu). (B) Representative traces for the indicated genotypes in the absence (baseline) and presence of PdBu (PdBu, red). (C) The effect of PdBu application is plotted for wild type controls, the RopG27/rim103 double heterozygous condition and each heterozygous mutation alone. Each genotype is expressed as a percent change in the presence compared to absence of PdBu. Calculations are made on the first EPSC of the stimulus train (EPSC1). The average percent change is statistically significant only in the double heterozygous condition (p<0.01; ANOVA, One Way with Tukey Multiple Comparisons). (D) Data as in (C) plotting the ratio of EPSC4/EPSC1 as a percent change in the presence compared to absence of PdBu. The average percent change is statistically significant only in the double heterozygous condition (p<0.01; ANOVA, One Way with Tukey Multiple Comparisons).

https://doi.org/10.7554/eLife.40385.009
Syx rescues PHP during Synaptic Homeostasis.

(A) Sample traces showing EPSP and mEPSP amplitudes in the absence (baseline) and presence (PhTx) of PhTx for syx1A heterozygous null allele (syx1AΔ229/+) and heterozygous syx1AΔ229 placed in trans with RopG27 mutant (RopG27/syx1AΔ229). (B) Average data for mEPSP amplitude ±PhTx for indicated genotypes. PhTx application reduces amplitude in all genotypes (p<0.01). Data represent mean ±SEM. Student’s t test. (C–E) Average data for EPSP amplitude (C) Quantal Content (D) and mEPSP and quantal content normalized to values in the absence of PhTx (E) for indicated genotypes. Statistical comparisons are made to wild type for each genotype in (E). **p<0.01; ****p<0.0001, Student’s t-test, two tailed. (F) Average mEPSP frequencies (Hz) (left) and sample traces showing mEPSPs for indicated genotypes (right). Student’s t test, two tailed.

https://doi.org/10.7554/eLife.40385.010
RopG11 abolishes the biochemical interaction between Rop and syx1A and rescues homeostasis defect in Rop.

(A) Schematic of the Drosophila Rop protein. Point mutation RopG11 is indicated by red star at syntaxin-binding domain (SBD) of the Rop protein shown in pink. RopG11 converts Aspartic Acid (D45) to Asparagine (N). This site is conserved in mammalian Rop (munc18-1). (B) Coomassie stains of in vitro binding assays. (left) MBP-Rop (110 kDa) coprecipitated with bead-bound GST fusions of syx1A (GST-syx1AΔC) (60 kDa). MBP-Rop does not bind to GST-syx1AΔC in the presence of single point mutation at the N-terminal of Rop (MBP-RopG11). (right) Free MBP-Rop in the absence of GST-syx1A. (C) Binding curves quantify dissociation constant (Kd) for MBP-Rop and MBP-RopG11 binding to GST-syx1AΔC; x-axis is concentration of MBP recombinant protein used (μM); y-axis is the fraction of protein bound; n = 2 (D) Sample traces showing EPSC amplitudes ± PhTx for RopG11 heterozygous null allele (RopG11/+), heterozygous RopG11 placed in trans with RopG27 mutant (RopG27/RopGG11), and heterozygous RopG11 placed in trans with DfRop (DfRop/RopGG11). (E) Average data for mEPSP amplitude ±PhTx for indicated genotypes. PhTx application reduces amplitude in all genotypes (p<0.01). Data represent mean ±SEM. Student’s t test. two tailed. (F) Average data for EPSC amplitude as in (B); ns, not significant; Student’s t test, two tailed.

https://doi.org/10.7554/eLife.40385.011

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene
(Drosophila
melanogaster)
RopNAFLYB:
FBgn0004574
Gene
(D. melanogaster)
RimNAFLYB:
FBgn0053547
Gene
(D. melanogaster)
RbpNAFLYB:
FBgn0262483
Gene
(D. melanogaster)
unc-13NAFLYB:
FBgn0025726
Gene
(D. melanogaster)
Syx1ANAFLYB:
FBgn0013343
Strain -
strain
background
WT - w1118NAw1118
Genetic
reagent
(D. melanogaster)
RopG27Bloomington
Drosophila
Stock Center
BDSC: 4381
FLYB FBst0
004381
RRID: DGGR_107715
Flybase symbol: bw(Aravamudan et al., 1999); Rop[G27] st(Aravamudan et al., 1999)/TM6B, Tb[+]
Genetic
reagent
(D. melanogaster)
DfRop (Df(3L)
BSC735)
Bloomington
Drosophila
Stock Center
BDSC:
26833 FLYB
FBst0026833
RRID:
BDSC_26833
Flybase symbol: w[1118]; Df(3L)
BSC735/TM6C, Sb
(Aravamudan et al., 1999)
cu(Aravamudan et al., 1999)
Genetic
reagent
(D. melanogaster)
elavC155-
GAL4
Bloomington
Drosophila
Stock Center
BDSC:
458 FLYB
FBst0000458
RRID:
BDSC_458
Flybase symbol: P{w[+mW.hs]=GawB}elav[C155]
Genetic
reagent (D. melanogaster)
UAS-Rop RNAiVienna Drosophila
RNAi Center
VDRC:
19696
FLYB FBst0
453580
RRID: FlyBase_FBst0453580
Flybase symbol: w[1118]; P{GD1523}v19696/TM3
Genetic
reagent (D. melanogaster)
rim103; rim(Müller et al., 2012b) PMID: 23175813
Genetic
reagent (D. melanogaster)
rbpSTOP1(Liu et al., 2011) PMID: 22174254gift from
Stephan Sigrist
Genetic
reagent (D. melanogaster)
dunc-13P84200Kyoto Stock
Center
KSC: 101911
RRID: DGGR_101911
Flybase symbol: ry[506]; P{ry11}l(4)ry16
(Aravamudan et al., 1999)
/ci[D]
Genetic
reagent (D. melanogaster)
syx1AΔ229Kyoto Stock
Center
KSC: 107713
RRID: DGGR_107713
Flybase symbol: Syx1A[Delta229] ry[506]/TM3, ry[RK] Sb(Aravamudan et al., 1999)
Ser(Aravamudan et al., 1999)
Genetic
reagent
(D. melanogaster)
RopG11(Harrison et al., 1994) PMID: 7917291gift from
Hugo Bellen
Recombinant DNA reagentPMAL-
c5E (vector)
New England
Biolabs
NEB: N8110
Recombinant
DNA
reagent
PGEX-4T1 (vector)Addgene27-4580-01
Recombinant
DNA
reagent
Rop (cDNA)Drosophila
Genomics
Resource
Center
DGRC:
SD04216
Recombinant
DNA reagent
Syx1A (cDNA)Drosophila
Genomics
Resource
Center
DGRC:
LD43943
Recombinant
DNA reagent
PMAL-RopWT
(plasmid)
This paperPrimers
CGCGGATCCATGGCCTTGAAAGTGCTGGTGG and CC
GGAATTCTTAGTCCTCC
TTCGAGAGACTGC were used to amplify Rop, which was then cloned into PMAL-5ce vector
Recombinant
DNA reagent
PMAL-RopG11
(plasmid)
This paperGenerated using
site-directed
mutageneis with
primer
GGCGGGTGCTGGTGGTGAACAAGCTGGGTATGCGC
Recombinant
DNA reagent
PGEX-Syx1AΔC
(plasmid)
This paperPrimers
CGCGGATCCA
TGACTAAAGA
CAGATTAGCCG
and TCCCCCGGG
TTACATGAAATAAC
TGCTAACAT were
used to
amplify Syx1A,
which was
then cloned
into PGEX-4T1, site-
directed mutagenesis
with primer
GTAAAGCCCGA
CGAAAG
TAGATCATGAT
ACTGATC was used to remove the
C-terminal tail
Peptide,
recombinant
protein
MBP-RopWT
/MBP-RopG11
This paperRecombinant
MBP-Rop was
expressed from
PMAL-Rop in
RosettaTM cells,
purified using
amylose resin,
and eluted with
maltose
Peptide,
recombinant
protein
GST-Syx1AΔCThis paperRecombinant
GST-Syx1AΔC
was expressed
from PGEX-
Syx1AΔC
in RosettaTM,
purified using
GST resin,
and eluted
with glutathione
Commercial
assay or kit
QuikChange
Lightning Site-
Directed
Mutagenesis
Kit
Agilent210518
Commercial
assay or kit
Coomassie
Blue R-250
Solution
TekNovaC1050
Chemical
 compound,
drug
Phorbol
12-myristate
13-acetate
Phorbol Ester
(PdBU)
Sigma-AldrichSigma-
Aldrich CAS:
16561-29-8
Stock
concentration:
10 mM Final
Concentration
(in HL3 saline):
1 μM
Chemical
compound,
drug
Philanthotoxin-
433 (PhTX)
Sigma-Aldrich
(disc.) Santa
Cruz Biotech.
Sigma Aldrich
CAS: 276684-27-6
Santa Cruz
Biotech. sc-255421
Stock
concentration:
5 mM Final
Concentration
(in HL3 saline):
10–20 μM
Software,
algorithm
Sharp-
electrode
recordings
Molecular DevicesClampex (10.3.1.5)
Software,
algorithm
EPSP analysisMolecular DevicesClampfit (10.3.1.5)
Software,
algorithm
EPSC and
Pr analysis
Wave-MetricsIgor Pro (6.3.4.1)
RRID: SCR_000325
custom script
Software,
algorithm
RRP,
train analysis
(Müller et al., 2015)
Software,
algorithm
mEPSP analysisSynaptosoftMini Analysis 6.0.7
RRID: SCR_002184
Software,
algorithm
GraphPad Prism (7.0 c)GraphPadRRID: SCR_002798
Software,
algorithm
FijiNIHRRID: SCR_002285
OtherAmylose ResinNew England BiolabsNEB: E8021used to purify
MBP recombi
nant protein
OtherGST Bind ResinNovagen70541used to purify GST
recombinant protein
and for pull-down of
recombinant protein

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