1. Volume 74, Issue 5, Pages 830-844 (June 2012)
Use-Dependent Inhibition of Synaptic Transmission by the Secretion of Intravesicularly Accumulated Antipsychotic Drugs 
Carsten H. Tischbirek, Eva M. Wenzel, Fang Zheng, Tobias Huth, Davide Amato, Stefan Trapp, Annette Denker, Oliver Welzel, Katharina Lueke, Alexei Svetlitchny, Manfred Rauh, Janina Deusser, Annemarie Schwab, Silvio O. Rizzoli, Andreas W. Henkel, Christian P. Müller, Christian Alzheimer, Johannes Kornhuber, Teja W. Groemer 
Neuron 
Volume 74, Issue 5, Pages (June 2012)
DOI: /j.neuron
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 APDs Accumulate in Synaptic Vesicles
(A) Representative images of living hippocampal neurons. Left panel shows functional synapses that are fluorescently labeled with anti-synaptotagmin1-cypHer5 (αSyt1-cypHer5). Middle panel illustrates staining of the same region with 50 nM LTR. Right panel presents overlay of αSyt1-cypHer5 and LTR staining. LTR exhibits synaptic and extrasynaptic fluorescence.
(B) Photoconversion electron microscopy image of a hippocampal bouton labeled with LTR. Synaptic vesicles (SV) and large extrasynaptic vesicle-like structures (asterisk) are visible. Scale bar, 200 nm.
(C) Experimental procedure for (D) and (E). LTR fluorescence intensity was determined before and after the administration of APDs (“Drug”) to viable LTR- and αSyt1-cypHer5-stained hippocampal neurons.
(D) Representative images of hippocampal neurons stained with 50 nM LTR or FM4-64 before (Pre) and after (Post) the administration of the drug or a vehicle (DMSO).
(E) Quantification of normalized mean synaptic LTR fluorescence intensity (n = 3 experiments each).
(F) Quantification of normalized (Norm.) fluorescence intensity of 1 μM LTR in saline and of FM4-64-stained synapses after the addition of the indicated substances (n = 3 experiments each). The tested APDs did not quench but instead displaced LTR from the synaptic terminals. Ctrl, control.
Error bars represent SEM. ∗p < 0.05, ∗∗p < 0.01, t tests. Scale bars in (A) and (D), 5 μm.
Please see Figure S1.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2
Acidic Intravesicular pH Is Needed for the Accumulation of APDs and Not Changed by APD Treatment
(A) Experimental procedure. Rat hippocampal neurons were stained with LTR in the presence or absence of the vATPase inhibitor folimycin (Foli, 80 nM) with physiological (2.5 mM) or elevated (10 mM) K+ levels. To simultaneously visualize synapses and their acidification, the neurons were coincubated with anti-Syt1-Oy488 (Oyster 488) and an antibody with pH-dependent dye αSyt1-cypHer5.
(B) Representative images of hippocampal neurons stained as indicated in (A). Scale bar, 5 μm.
(C) Quantification of normalized (Norm.) mean synaptic fluorescence intensity in the four different staining paradigms (n = 3). Error bars represent SEM.
(D) Normalized calculated LTR concentrations in synaptic vesicles and whole synapses for folimycin and K+ administration. The vesicle diameter, number of vesicles, and synapse volume used to calculate volume fractions were taken from Schikorski and Stevens (1997).
(E) Upper panel shows average spH fluorescence intensity profiles for the administration of ammonium chloride (NH4Cl) (2.5 mM, n = 4, and 5.0 mM, n = 5) and control (n = 4). Constant perfusion with standard saline was switched to the vehicle or the NH4Cl containing saline during minutes 4.5 and 9.0. Stim., electrical stimulation (50 AP, 10 Hz). Lower panel presents average spH fluorescence intensity profile. Cells were incubated with 80 nM folimycin to dissipate the intravesicular pH gradient of recycling vesicles. Stim., electrical stimulation (1,200 AP).
(F) pH changes by administration of the different APDs are limited to <0.1 pH. Baseline fluorescence levels and corresponding intravesicular pH before (Pre) and during (Drug) administration of substances. Indicated are DMSO (1:500), 5 μM HAL, 5 μM CPZ, 10 μM CLO, 15 μM RSP, 2.5 mM and 5.0 mM NH4Cl (corresponding to a 0.4 and 0.7 pH change, respectively) and 80 nM folimycin (complete deacidification of the recycling pool of vesicles to a pH of 7.5).
Error bars represent the SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. not significant, t tests.
Please see Figures S2 and S3.
Neuron  , DOI: ( /j.neuron )
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4. Figure 3 APDs Are Secreted from Presynaptic Terminals during Activity
(A) Scheme of the experimental procedure. The LTR fluorescence intensity was determined before, during, and after the electrical stimulation of LTR- and αSyt1-cypHer5-stained hippocampal neurons.
(B) Normalized LTR fluorescence intensity at synaptic sites over time. Electrical stimulation was performed in the presence (200 AP, 10 Hz and 600 AP, 30 Hz) or absence (0 mM Ca2+, 600 AP, 30 Hz) of calcium ions. LTR is released by electrical stimulation in a calcium-dependent manner.
(C) Pie plot of the LTR fluorescence fractions at synaptic sites. A 20% or 25% dye loss would correspond to the release of 30% or 37% of the vesicular fluorescence, respectively.
(D) Representative images of LTR-stained organelles that colocalize (arrowheads) or do not colocalize (triangles) with synaptic sites, as defined by αSyt1-cypHer5 before (F0) and after (F600AP) electrical stimulation. Synaptic (αSyt1-positive) and nonsynaptic (αSyt1-negative) LTR fluorescence intensities are indicated over time. LTR is released from αSyt1-cypHer5-positive sites.
(E) In vivo microdialysis experiments in freely moving rats that were chronically treated with HAL (0.5 mg/kg/d) by osmotic minipumps were performed in various brain regions: light blue indicates prefrontal cortex (PFC), red shows nucleus accumbens (Nac), and dark blue represents dorsal striatum (DStr).
(F) Extracellular HAL concentration (mean + SEM) after a local 100 mM K+ challenge administered by reverse dialysis (gray bar) in the PFC and DStr.
(G) Extracellular concentration of DA and 5-HT in the NAc (n = 5 experiments).
Error bars represent the SD. ∗p < 0.05, t tests versus last baseline sample.
Neuron  , DOI: ( /j.neuron )
Copyright © 2012 Elsevier Inc. Terms and Conditions

5. Figure 4
CPZ, HAL, CLO, and RSP Reversibly Reduce Synaptic Vesicle Exocytosis in a Dose-Dependent Manner, as Measured with spH
(A) Image details of rat hippocampal neurons transfected with spH in a representative experiment. The intensity (in arbitrary units) is indicated by color maps. (a) Initial fluorescence intensity (F0). Scale bar, 5 μm. (b) Fluorescence intensity reached after electrical stimulation with 50 action potentials (AP) at 10 Hz (F50AP). (c) Difference image of (a) and (b) (dF). (d) Automatically detected regions of interest (ROI) marked by white circles. (e) spH time course upon electrical stimulation with a scheme of the calculated parameters. The relative spH amplitudes are normalized to the dF values.
(B) Fluorescence time course in representative experiments. Average fluorescence intensity profiles for the administration of the vehicle (DMSO, 1:1,000, n = 5) and of 5 μM HAL (n = 5). Constant perfusion with standard saline was switched to the vehicle or the drug containing saline during min 6–18. Stimulation, electrical stimulation (50 AP, 10 Hz).
(C) Time courses showing the mean normalized evoked spH fluorescence increase with the maximal concentration-dependent amplitude reduction relative to the vehicle control (column plots) (n = 5 or 6, n > 120). ANOVA: CPZ, F4,103 = , p < 0.001; HAL, F5,126 = , p < 0.001; CLO, F2,65 = 20.67, p < 0.001; RSP, F3,80 = 49.81, p < Error bars represent SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, t tests.
See Figure S4.
Neuron  , DOI: ( /j.neuron )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 5
APD-Induced Inhibition of Exocytosis Correlates with a Reduction in Stimulation-Dependent Ca2+ Entry
(A) Image details of fluo-4- and FM5-95-stained rat hippocampal neurons in a representative experiment. Scale bar, 5 μm. Intensity scales are indicated for every image. (a) Initial fluorescence intensity of fluo-4-stained cells. Scale bar, 5 μm. (b) Electrical stimulation with 50 action potentials (AP) at 10 Hz provokes a Ca2+ influx that leads to an increase in fluo-4 fluorescence. (c) Staining with FM5-95. (d) FM5-95 is unloaded by electrical stimulation with 600 AP at 30 Hz. (e) The difference image between (c) and (d) enables the localization of the synaptic boutons. Automatically detected regions of interest (ROIs) are marked by white circles. (f) The ROIs defined in (e) were used for the fluo-4 analysis.
(B) Average fluorescence intensity profiles for the vehicle (DMSO, 1:1,000) and HAL (10 μM). Synaptic regions were identified by FM5-95 (Ac–Af, Ba, Bb). Subsequent fluo-4 imaging allowed for repetitive measurements of evoked synaptic Ca2+ transients (Aa, Ab, Af, Bb, Bc). Constant perfusion with standard saline was switched to either the vehicle or the drug containing saline between min 4.5 and 9. Stim., electrical stimulation (600 AP, 30 Hz for FM5-95 and 50 AP, 10 Hz for fluo-4).
(C) Normalized average fluorescence intensity profiles of spH and fluo-4. Vehicle (VEH, 1:500), CPZ (10 μM), HAL (10 μM), CLO (20 μM), RSP (30 μM). Scale, 1.0 versus 1 min (n = 5, n > 200 for spH; n = 5 or 6, n > 900 for fluo-4).
(D) Mean spH and fluo-4 amplitude reductions relative to the vehicle control during the perfusion of the indicated drug (“Drug”) or after the perfusion of the drug (“Post drug”). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
(E) Correlation of the relative fluorescence increase by spH exocytosis and fluo-4-indicated Ca2+ influx in the APD and control experiments. The inhibition of Ca2+ entry correlates with the inhibition of synaptic vesicle exocytosis (n = 5, R = 0.86; p < 0.001). Values are taken from the experiments depicted in (C). Dotted line shows linear regression (y = ax). p Value is for the hypothesis a = 0. Error bars represent SEM.
Neuron  , DOI: ( /j.neuron )
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7. Figure 6
APDs Inhibit Sodium Channels in a State- and Use-Dependent Fashion
(A) Electrical stimulation requires signal propagation along the axon to achieve synaptic transmission and is inhibited by the application of TTX. Chemical stimulation with 40 mM K+, 20 s (comparable spH amplitude as evoked by 200 AP, 10 Hz) under 1 μM TTX was used to selectively stimulate presynaptic terminals independent of sodium channel action.
(B) Synaptic vesicle exocytosis was reported by spH and monitored before and after drug administration. One micromolar TTX completely blocked vesicular release by electrical stimulation (200 AP 10 Hz). The chemically induced exocytosis was insensitive to TTX and HAL application. ∗∗∗p < 0.001; n.s., not significant; t tests.
(C) Dose-response curves for HAL block of endogenous (Nav1.1/Nav1.2) and Nav1.6r current in N1E115 neuroblastoma cells were determined from steady-state recordings using the activation protocol depicted as inset (Vh −90mV).
(D) Steady-state activation curves were constructed from the activation protocol of (A) and the inactivation protocol (depicted as an inset) before and during the application of HAL (25 μM). Data points were fitted with the use of a Boltzmann equation. Half-activation potential Vmid was −13.8 ± 1.4mV (Nav1.6r), −13.6 ± 1.0mV (Nav1.6r plus HAL), −8.9 ± 1.1mV (Nav1.1/Nav1.2), and −10.7 ± 1.0mV (Nav1.1/Nav1.2 plus HAL), with p = for control versus 25 μM HAL in Nav1.1/Nav1.2. Half-inactivation potential Vmid was −61.4 ± 1.1mV (Nav1.6r), −79.3 ± 1.4mV (Nav1.6r plus HAL), −61.9 ± 1.7mV (Nav1.1/Nav1.2), and −72.6 ± 2.2mV (Nav1.1/Nav1.2 plus HAL), with p = 8.9E-6 for control versus 25 μM HAL for Nav1.6r and p = 1.0E-5 for control versus 25 μM HAL for Nav1.1/Nav1.2. There was p = for a HAL-induced voltage shift of Nav1.6r versus Nav1.1/Nav1.2 (n = 8 for Nav1.6r, n = 7 for Nav1.1/Nav1.2).
(E) A gradual recovery from inactivation in a double-pulse protocol with an increasing interpulse interval (inset) is shown here for the endogenous current under the control conditions. The test pulse duration was 5 ms. The intervals between test pulses (Δt) are denoted above the peak of the second current response.
(F) Plot summarizing the effect of HAL (25 μM) on the recovery from inactivation. The peak current elicited by the second pulse was normalized to the peak current of the first pulse. The data points were fitted using a biexponential function and were plotted against the interpulse interval. Here, n = 3 for Nav1.6r and n = 3 for Nav1.1/Nav1.2, at 20 ms. Additionally, p = for control versus 25 μM HAL for Nav1.1/Nav1.2, and p = 0.04 for control versus 25 μM HAL for Nav1.6r.
Neuron  , DOI: ( /j.neuron )
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8. Figure 7
The Inhibition of Synaptic Transmission by APDs Is Use Dependent
(A) Experimental procedure for the optical measurement of use-dependent inhibition. spH-reported electrically induced exocytosis was monitored before and after drug administration.
(B) The ratio of spH fluorescence increases before and after the administration of HAL (0.5 or 5 μM), which was evoked with either 60 or 180 action potentials (AP) at 40 Hz (n = 3 to 6, n > 120). Error bars represent SEM. ∗p < 0.05, ∗∗p < 0.01, n.s. not significant, t tests.
(C and D) The incubation of hippocampal slices with HAL (0.5 and 5 μM) for 2–5 hr dramatically enhanced the use-dependent reduction of EPSC amplitudes (vehicle, n = 5; HAL 0.5 μM, n = 6; HAL 5 μM, n = 5) during 100 stimuli at 25 Hz. This effect was mimicked by low-dose TTX (25 nM, 2–4 hr incubation, n = 5). EPSCs were normalized to their amplitudes before the train. Illustrations in (C) are typical traces from different experiments. Insets depict superimposed EPSCs before and 2 s after the start of a 25 Hz train with and without HAL incubation.
(E) Incubation of hippocampal slices (for 2–5 hr) with HAL (5 μM, n = 7), CPZ (30 μM, n = 5), RSP (30 μM, n = 5), and TTX (25 nM, n = 5) reduced CA1 axonal activity during a 25 Hz train (vehicle n = 11). CA1 FVs were normalized to their amplitudes before the train. Insets depict superimposed FVs before and 2 s after the start of a 25 Hz train.
(F and G) Incubation of slices containing NAc with HAL (5 μM) for 2–6 hr dramatically enhanced the use-dependent inhibition of EPSCs (vehicle, n = 8; HAL 5 μM, n = 7) during 100 stimuli at 25 Hz. Insets depict superimposed EPSCs before and 1 s after the start of a 25 Hz train with and without HAL incubation.
(H) Incubation of NAc slices with HAL (5 μM) for 2–6 hr reduced axonal activity (vehicle, n = 6; HAL 5 μM, n = 6) during 100 stimuli at 25 Hz. Insets depict superimposed FVs before and 1 s after the start of a 25 Hz train.
Neuron  , DOI: ( /j.neuron )
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9. Figure 8
Folimycin Prevents the Efficient Inhibition of Synaptic Vesicle Exocytosis and Calcium Influx by HAL
(A) Experimental procedure for (B) and (C). The destaining of FM4-64-loaded synaptic terminals by 600 AP at 30 Hz was monitored in the presence of either the vehicle (DMSO), HAL (5 μM) or HAL and folimycin (Foli+HAL, 80 nM folimycin and 5 μM HAL).
(B) Normalized fluorescence intensities of FM4-64-stained hippocampal boutons as a function of time.
(C) Normalized ΔF before and after electrical stimulation (n = 4 experiments, n > 150 synapses). Error bars represent SEM. ∗p < 0.05, ∗∗∗p < 0.001, t tests.
(D) Image details of fluo-4-stained rat hippocampal neurons from representative experiments. Scale bars, 5 μm. Upper panel illustrates that the ΔF (pre) of fluo4 induced by electrical stimulation with 50 AP at 10 Hz was reduced in the presence of 10 μM HAL (ΔF drug). Lower panel shows that in the presence of 80 nM folimycin, the HAL-induced inhibition of calcium influx was less pronounced.
(E) Normalized average fluorescence intensity profiles of fluo-4 experiments. Vehicle (DMSO, 1:500), folimycin (Foli, 80 nM), HAL (10 μM), and folimycin plus HAL (Foli+HAL, 80 nM folimycin and 10 μM HAL).
(F) Normalized average Δf values of fluo-4 amplitudes relative to the DMSO (n = 2 experiments) control (n = 3–5 experiments, n > 500 synapses). Error bars represent SEM. ∗∗∗p < 0.001, t tests. n. s., not significant.
See Figure S5.
Neuron  , DOI: ( /j.neuron )
Copyright © 2012 Elsevier Inc. Terms and Conditions