In this study, 114 male WT Crl: CD1(ICR) (RRID: IMSR_CRL:022, 5–8 weeks old, weighing 25–33 g were used). WT mice were bred and genotyped at the Medical Gene Technology Unit of the Institute of Experimental Medicine (Budapest, Hungary). Only male mice were used to eliminate the potential effects of the oestrous cycle on animal behavior. Animals were maintained on a 12:12 light–dark cycle in a temperature (23 ± 2 °C) and humidity-controlled room (60 ± 10%), with free access to food (ssniff® Souris-Elevage E, 10 mm pellet, Cat# S8189-S096; ssniff Spezialdiäten GmbH, Soest, Germany) and water. Before the experiments, 4–6 adult littermate mice were kept in standard mouse cages with corncob bedding. To enrich the environment, cardboard bedding material and tubes were placed in each cage. The experimental procedures described in this manuscript have been approved by the Semmelweis University Regional Committee (No. 116/2015) and the local Institutional Animal Care Committee of the IEM HAS (PE/EA/00513-6/2025) and followed the guidelines of the Hungarian Act of Animal Care and Experimentation guidelines (40/2013, II.14), which follows the Directive 2010/63/EU and the recommendations of the Committee on Animals in Research (FENS). Animals were treated humanely, and all effort was made to minimize animal suffering and reduce the number of animals used in experiments.

The sample size was calculated based on a pilot study of [³H] NA release after the first stimulation between control and in the presence of 1 μM RSP investigated groups. We calculated the number of animals required per group using G*Power 3.1.9.7 software (RRID: SCR_01372) (Student’s t-test: a priori: compute required sample size; tail(s): two, power: 0.7; α error probability: 0.05; effect size: 1.367; total sample size: 12).

Animal experiments were reported under ARRIVE 2.0 guidelines. The exact number of mice in each experimental group is given in the legend of the corresponding figures. The mice were randomly assigned to experimental groups, and the investigators were blinded to their experimental status.

The following chemicals were used for the release experiments: levo-[7-³H] norepinephrine (specific activity, 11.8 Ci·mmol⁻¹) and 5-hydroxytryptamine-[3H]-trifluoromethylacetate (specific activity, 80 Ci·mmol⁻¹) and [³H] choline chloride (specific activity, 80 Ci/mmol), purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO, United States). Hemicholinium-3 (Cat# H108) was obtained from Sigma-Aldrich (St. Louis, MO, United States). The irreversible VMAT-2 inhibitor RSP [(IUPAC name: 3β,16β,17α,18β,20α)-11,17-Dimethoxy-18-[(3,4,5 trimethoxybenzoyl)oxy]yohimban-16-carboxylic acid methyl ester, Cat# 2742] and the reversible VMAT-2 inhibitor TBZ (IUPAC name: 9,10-dimethoxy-3-(2-methylpropyl)-1,3,4,6,7,11b-hexahydrobenzo [a]quinolizin-2-one, Cat# 2175) were dissolved in dimethyl sulfoxide (DMSO) and were obtained from Tocris Bioscience (Minneapolis, MN, United States). The reversible VMAT-2 inhibitor VBZ (IUPAC name: (2R,3R,11bR)-3-Isobutyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-yl L-valinate, Cat# HY-16771) was purchased from MedChemExpress (Monmouth Junction, NJ, United States). Sodium channel blocker tetrodotoxin citrate (IUPAC name: (1R,5R,6R,7R,9S,11S,12S,13S,14S)-3-amino-14-(hydroxymethyl)-8,10-dioxa-2,4 diazatetracyclo[7.3.1.17,11.01,6]tetradec-3-ene-5,9,12,13,14-pentol, Cat# 1069) was obtained from Alomone labs Ltd. (Jerusalem, Israel). Trichloroacetic acid (Cat# T0669) was purchased from Merck KGaA (Darmstadt, Germany). Isoflurane (Cat# 1214) was obtained from Medicus Partner Ltd. (Biatorbágy, Hungary). Other materials used for experiments were purchased from general commercial resources and were of the highest grade.

Mice were sacrificed in a 2-step manner according to the American Veterinary Medical Association guidelines for the Euthanasia of Animals. First, the animals were anaesthetized with a relatively high dose of isoflurane for rapid loss of consciousness (>1.5 v/v %) in a sufficiently large container to prevent hypoxia. Then, mice were decapitated, and the brain was immediately removed and placed into ice-cold Krebs solution [composition (in mM): 113 NaCl, 4.7 KCl, 1.2 MgSO₄, 2.5 CaCl₂, 25 NaHCO₃, 1.2 KH₂PO₄, 115 glucose, 0.3 Na₂EDTA, and 0.03 ascorbic acid]. After removal, the hippocampus was isolated and sliced into 400 μm thick slices (Milusheva et al., 1994) using a tissue chopper and incubated for 45 min in 1 mL of Krebs buffer (see above) containing radioactive [³H] NA or [³H] 5-HT (5 μCi·mmol⁻¹). After this incubation period, the incubation medium was replaced, allowing complete washout of any residual volatile anesthetic. Although we did not directly measure the residual isoflurane concentration, similar incubation and washout protocols have been applied in our previous studies (Csölle et al., 2013; Tod et al., 2024; Gölöncsér et al., 2024), where animals were anesthetized using the same procedure and no confounding effects on neurotransmitter uptake or release were observed. Therefore, it is unlikely that isoflurane exposure affected the present results. Possible residual effects of isoflurane anesthesia were controlled by the incubation and washout procedures described in the Discussion (see Discussion, Limitations, Methodological considerations). Experiments were conducted at 37 °C in a modified Krebs solution, continuously saturated with carbogen gas (95% O₂ and 5% CO₂). To investigate the effects of RSP, TBZ, and VBZ, three different treatment protocols were applied (Figure 1). (i) In some experiments, mice received intraperitoneal injections of the corresponding drug (RSP 1 mg·kg⁻¹, TBZ 2.5 mg·kg⁻¹, VBZ 2.5 mg·kg⁻¹) 24, 48, or 72 h before hippocampal slices were prepared for release measurements (Figures 4, 5, 8). (ii) In other experiments, the drugs were added only during a 45-min incubation period in 1 mL Krebs solution, after which they were washed out and were no longer present during the measurement (Figure 7). (iii) In a third set of experiments, the drugs were continuously present in the Krebs perfusate until the end of the measurement (Figures 2, 3, 6). Following the incubation, the preparations were washed three times with 10 mL of ice-cold, oxygenated Krebs solution, and three to four slices were transferred to a thermoregulated four-channel (internal volume, 100 μL) microperfusion system (Hennings et al., 1999) and maintained at 37 °C. The release experiments were performed as previously described (Milusheva et al., 2008); the preparation was superfused with Krebs solution at a rate of 0.5 mL·min⁻¹ for 60 min before each measurement. After 60 min of preperfusion, the outflow was collected in 1.5 mL·min⁻¹ fractions (3 min each) for 60 min (19 fractions). During the third (S₁) and 13^th (S₂) collection periods, the preparations were field-stimulated electrically with the following parameters: [³H] NA: 20 V, 2 Hz, with a 2 ms impulse duration for 1 min (120 pulses) and [³H] 5-HT: 40 V, 2 Hz, with a 2 ms impulse duration for 1 min (120 pulses) using a Grass S88 stimulator. The vesicular release of radioactivity evoked by electric field stimulation was sensitive to tetrodotoxin, which was applied at a concentration of 1 μM. The supernatant (500 μL) from each fraction was added to 2 mL of scintillation mixture (Ultima Gold; Packard, Canberra, Australia). At the end of the perfusion period, the tissue was removed from the chamber and the residual radioactively labeled neurotransmitters were extracted with 5 mL of 10% trichloroacetic acid for 30 min; 100 μL of the supernatant was added to 2 mL of scintillation mixture, and the radioactivity was measured using a Packard 1900 Tricarb and 5110 TR liquid scintillation counter (Packard). The samples’ radioactivity was expressed in disintegrations per gram of wet tissue weight (Becquerels per gram, Bq·g⁻¹).

The tissue’s [³H] NA and [³H] 5-HT uptake by the tissue slices was quantified as the amount of radioactivity present in the tissue at the beginning of the perfusion period (CB), estimated using Equation (1):

where FRi corresponds to the radioactivity detected in the i-th fraction, and CE refers to the remaining radioactivity in the tissue measured at the end of the experiment.

The release evoked by electrical field stimulation (S₁ and S₂) was calculated as the total radioactivity released above the baseline (resting) levels, represented by R1 and R2, respectively. R1 was defined as the average radioactivity measured in the first and second fractions, while R2 corresponded to the average radioactivity in the 14th and 15th fractions. A custom-made equation was used, and the radioactivity measured in the sample was calculated as the fractional release (FR) Equation 2:

The release in response to electric field stimulation was evaluated in fractional release (FR) and as the ratio of the area under the curve of the total radioactivity release to the resting in response to supramaximal electrical field stimulation, applied during the third (S₁) and 13^th (S₂) fractions, in the absence (FRS1) and presence (FRS2) of the drug. The FRS2/FRS1 ratio was then calculated to evaluate the drug’s effect, with the S1-evoked release serving as the internal standard. Similarly, the impact on resting release was assessed using the FRR2/FRR1 ratio, where FRR1, defined as the average radioactivity from the first and second fractions, served as the internal reference, unless stated otherwise. Previous studies reported that 88% of the stimulation-induced increase in [³H] release under identical experimental conditions was due to the increased release of [³H] NA (Vizi and Burnstock, 1988), as well as [³H] 5-HT.

There is several evidence that the cholinergic system is also involved in the pathophysiology of depression through nAChR alpha7 activation (Pahlavani, 2024); therefore, we investigated the effect of RSP on the stimulation-evoked release of acetylcholine. For the detection of [³H] ACh release, hippocampal slices were obtained as described for [³H] NA. First, we conducted a 45-min-long incubation with [³H]-choline (5 μCi/mL) in a 1 mL organ bath containing 37 °C Krebs solution. At the end of the incubation period, the slices were washed five times with Krebs solution and transferred to the four-channel microperfusion system and superfused with Krebs solution at a rate of 0.5 mL/min for 60 min. To selectively prevent the [³H] choline reuptake, high-affinity choline transporter inhibitor hemicholinium-3 (10 μM) was added throughout the experiments, which did not affect ACh’s resting and axonal stimulation-induced release. After 60 min of preperfusion, nineteen 3-min samples were collected, and the tissues were stimulated twice, at the third fraction (S₁) and the 13th fraction (S₂). Supramaximal electrical train stimulation was performed using a Grass S88 stimulator, with the following parameters: 40 V, 2 Hz, 2 ms, 1 min. The total radioactivity released from the tissue and collected in the perfusion fluid was considered to represent the amount of vesicular [³H] ACh released following hydrolysis rather than that of [³H] choline released directly from nerve terminals (Milusheva et al., 1994). At the end of radioactivity collection, supernatant (500 μL) from each fraction was added to 2 mL of scintillation mixture (Ultima Gold; Packard). After sample collection, the tissues were removed from the chambers, and the residual [³H] ACh was extracted using 5 mL of 10% trichloroacetic acid for 30 min. Subsequently, 100 μL of supernatant was added to 2 mL of scintillation mixture; the radioactivity was determined using a liquid scintillation counter (Packard 1900 Tricarb and 5110 TR) and expressed in disintegrations per gram of wet tissue weight (Bq·g⁻¹).

The release of [³H] ACh at rest and in response to axonal stimulation was calculated in FR and Bq·g⁻¹ as described for NA and 5-HT. The effect of axonal stimulation or drugs on the release was evaluated as the ratio of the area under the curve of the total radioactivity release to the resting release in response to the first and second stimulation (FRS2/FRS1 or S₂/S₁ in Bq·g⁻¹). A similar calculation was performed for the resting release (FRR2/FRR1 or S₂/S₁ in Bq·g⁻¹).

We calculated the animal numbers required for group sizes using G*Power 3.1.9.7 software, as shown above. Values in the paper were expressed as the mean ± SEM (error bars). Six animals were used in each experimental group. The normality of the experimental data distribution was tested using the Shapiro–Wilk normality test. Depending on the datasets, statistical analyses were performed with unpaired Student’s t-test or one-way ANOVA with Tukey’s post hoc test if the data were normally distributed; if not, Kruskal-Wallis test with multiple comparisons with Dunn’s multiple comparisons post hoc test using the STATISTICA version 14.0.1 software (TIBCO Software Inc., Palo Alto, CA, United States). Post hoc tests were only performed when F in ANOVA achieved p < 0.05. p-values of less than 0.05 were considered statistically significant throughout the study.

After loading the tissue with [³H] NA and washout, we measured the resting and electric field stimulation-evoked vesicular release of [³H] NA from hippocampus slice preparations. Figure 2 shows the release of [³H] NA during resting and in response to electric stimulation. In Figure 2A, the radioactivity content of each perfusate sample was expressed as a percentage of the tissue content calculated at the time of sampling (FR%). Figure 2B represents the release calculated in Bq·g⁻¹. The resting release in the first 3-min collection period was 12.66 ± 2.63 kBq·g⁻¹, and it was relatively constant from one collection period to the next throughout the experiments. Radioactivity released by the first stimulation (S₁) was 18.20 ± 2.94 kBq·g⁻¹. The release of radioactivity in two consecutive stimulation periods (FRS2/FRS1) was 0.98 ± 0.04 in control experiments (Figure 2A), indicating that the amount of fractional release in response to consecutive electric field stimulations remained relatively constant. The tissue’s [³H] NA content was 420.0 ± 17.1 kBq·g⁻¹. The release during resting was also constant: FRR2 ± FRR1 = 0.93 ± 0.03. In the presence of tetrodotoxin (1 μM), there was no significant increase in [³H] NA release in response to electric stimulation, indicating that the evoked release of tritium is of neuronal origin and depends on the propagation of action potentials along the neuronal membrane. This is a well-established basic finding that has been demonstrated previously by others (Raiteri et al., 1990; Westphalen et al., 2009) and also in our earlier work (Török et al., 2004; Tod et al., 2024). The effects of RSP, a competitive inhibitor of the VMAT-2 enzyme (Wu et al., 2024), were measured on electric field stimulation-evoked vesicular release (Figure 3A) and storage capacity (Figure 3B) of ex vivo hippocampal slices. To demonstrate that VMAT-2 is responsible for transporting transmitters into vesicles at axon terminals and that its inhibition results in a reduction of quantal, exocytotic transmitter release evoked by axonal stimulation, the effects of TBZ and VBZ, non-competitive selective VMAT-2 inhibitors (Eiden et al., 2004) were also studied and compared to RSP loaded and control groups in these preparations (Figure 3).

RSP and the other two antagonists were administered at a 10 μM concentration during the tissue loading and kept in the solution. RSP and TBZ significantly reduced the electric field stimulation-evoked [³H] NA vesicular release throughout the experiment (RSP: S₁: 4.0 ± 0.4 kBq·g⁻¹; p = 0.0115; S₂: 1.1 ± 0.3 kBq·g⁻¹; p < 0.0001; TBZ: S₁: 4.2 ± 0.5 kBq·g⁻¹; p = 0.0132; S₂: 2.1 ± 0.6 kBq·g⁻¹; p = 0.0001). The presence of VBZ decreased the release significantly, exclusively following the second stimulation (S₁: 5.3 ± 1.1 kBq·g⁻¹; p = 0.0766; S₂: 2.8 ± 1.2 kBq·g⁻¹; p < 0.0001), compared to the control group (S₁: 18.2 ± 2.9 kBq·g⁻¹; S₂: 13.7 ± 2.4 kBq·g⁻¹) (Figures 3A,B). As far as the storage capacity is concerned, 10 μM RSP (88.9 ± 6.0 kBq·g⁻1: p = 0.0065) and TBZ (77.1 ± 11.2 kBq·g⁻¹: p = 0.0009) significantly reduced the tissue’s [³H] NA content, compared to the control group (420.0 ± 17.2 kBq·g⁻¹) (Figure 3C). VBZ (121.0 + 9.4 kBq·g⁻¹) did not alter the tissue’s [³H] NA uptake. Compared to the RSP-loaded group, there was no significant difference neither at the electric field stimulation-evoked [³H] NA vesicular release nor at the tissue’s [³H] NA uptake regarding the TBZ and VBZ loaded groups.

To explore the length of the inhibitory effect of RSP (1 mg·kg⁻¹, i.p.) on the vesicular release of [³H] NA and storage capacity in hippocampal slices, we studied its action (Figure 4), 24, 48, and 72 h after RSP i.p. treatment. The slices were loaded with [³H] NA and maintained throughout the experiments.

RSP administrations’ effect on vesicular release and storage capacity of [³H] NA proved to be irreversible (Figure 4). The administration of RSP inhibited the electric field stimulation-evoked release of [³H] NA 24, and even after 48 h after treatment (24 h: S₁: 8.1 ± 10.7 kBq·g⁻¹; p = 0.018; S₂: 4.2 ± 0.7 kBq·g⁻¹; p < 0.0001; 48 h: S₁: 9.1 ± 1.7 kBq·g⁻¹; p = 0.0037; S₂: 5.2 ± 0.6 kBq·g⁻¹; p = 0.0001) compared to the control group (S₁: 21.8 ± 3.4 kBq·g⁻¹; S₂: 15.5 ± 2.4 kBq·g⁻¹). However, after 72 h, a decreased [³H] NA efflux was seen exclusively after the S₂, while after S₁ there was no difference (S₁: 14.0 ± 1.0 kBq·g⁻¹; p = 0.1020; S₂: 9.3 ± 0.5 kBq·g⁻¹; p = 0.0139), compared to the control group (Figures 4A,B). Twenty-four hours after RSP treatment, the tissue’s [³H] NA content was reduced significantly (93.0 ± 7.2 kBq·g⁻¹; p = 0.0001), compared to the control group (612.0 ± 28.3 kBq·g⁻¹), indicating that 84.9% of NA is not stored in vesicles. Forty-eight hours and seventy-two hours after RSP administration, the radioactivity measured in the tissue (138 ± 12.6and 148 ± 8.4 kBq·g⁻¹; p = 0.0766 and p = 0.2474) was not significantly reduced compared to the control group (Figure 4C).

We studied the effects of TBZ, VBZ, and RSP treatments 24 h after i.p. administration. We measured the axonal stimulation-evoked release of radioactivity from slice preparations loaded with [³H] NA in response to RSP (1 mg·kg⁻¹), TBZ (2.5 mg·kg⁻¹), and VBZ (2.5 mg·kg⁻¹) administration, expressed in Bq·g⁻¹ (Figure 5A). The pre-administration of RSP inhibited the S₁ and S₂ induced release of [³H] NA from the hippocampus (S₁: 8.1 ± 10.7 kBq·g⁻¹; p = 0.0116; S₂: 4.2 ± 0.7 kBq·g⁻¹; p = 0.0029) compared to the control group (S₁: 21.8 ± 3.4 kBq·g⁻¹; S₂: 15.5 ± 2.4 kBq·g⁻¹), indicating that its effect is long-lasting and irreversible. The pre-administration of TBZ (S₁: 17.2 ± 2.8 kBq· g⁻¹; p = 0.6463 and S₂: 12.7 ± 2.1 kBq·g⁻¹; p = 0.7389) and VBZ (S₁: 19.1 ± 2.6 kBq·g⁻¹; p = 0.8964; S₂: 16.1 ± 2.2 kBq·g⁻¹; p = 0.9953) failed to reduce the electric field stimulation-evoked release of [³H] NA, compared to the non-pretreated control group (Figures 5A,B). RSP pretreatment significantly reduced the tissue’s [³H] NA content (93.0 ± 7.2 kBq·g⁻¹; p = 0.0004), while the administration of VBZ and TBZ did not alter it (341.0 ± 32.5 and 574.0 ± 32.4 kBq·g⁻¹; p = 0.0858 and p > 0.9999), compared to the control group (612.0 ± 28.3 kBq·g⁻¹) (Figure 5C). The administration of the RSP dose was very efficient and reduced both vesicular release of [³H] NA and the tissue’s [³H] NA uptake by 62.9 and 84.9%, respectively. TBZ and VBZ had no reducing effect (Figure 5), indicating that their action was short-lasting and reversible.

In further experiments, we studied the stimulation-evoked vesicular release of radioactivity from slices loaded with [³H] 5-HT in the presence of 0.1, 0.3, 1.0, and 10 μM RSP (Figure 6). All applied doses significantly reduced the electric field stimulation-evoked vesicular release in the hippocampus, compared with the control group at S₁ and S₂ (control: S₁: 10.5 ± 1.2 kBq·g⁻¹; S₂: 4.5 ± 0.64 kBq·g⁻¹; 0.1 μM: S₁: 4.4 ± 0.5 kBq·g⁻¹; p = 0.0002; S₂: 1.9 ± 0.16 kBq·g⁻¹: p = 0.0007; 10 μM: S₁: 1.29 ± 0.24 kBq·g⁻¹: p < 0.0001; S₂: 0.83 ± 0.5 kBq·g⁻¹: p < 0.0001) (Figures 6A,B). The S₁ and S₂-induced release of [³H] 5-HT was inhibited, indicating that the maximum vesicular axonal activity-induced release was reduced by 87.6%. The tissue’s [³H] 5-HT content was decreased in a concentration-dependent manner, as by loading the tissue at 1.0 μM concentration, the storage capacity was inhibited to 91.7 ± 5.84 kBq·g⁻¹: p > 0.9999, respectively, and by loading the tissue at 10 μM concentration, the storage capacity was significantly altered by 72.4 ± 3.9 kBq·g⁻¹: p < 0.0001, compared to the control group (381 ± 18.9 kBq·g⁻¹) (Figure 6C).

In further experiments, to investigate which antagonist binds permanently to the transporters at a concentration of 1 μM, we studied the electric stimulation-evoked vesicular release of radioactivity from hippocampal slices loaded with [³H] 5-HT for 45 min. RSP, TBZ, and VBZ (1 μM) were present only during the incubation phase. After 60-min washout periods, RSP significantly reduced the electric field stimulation-evoked vesicular release in the hippocampus throughout the experiment (S₁: 2.8 ± 0.8 kBq·g⁻¹: p = 0.0261; S₂: 1.9 ± 0.4 kBq·g⁻¹: p = 0.0401), while VBZ increased it (S₁: 17.1 ± 2.7 kBq·g⁻¹: p = 0.0335; S₂: 12.4 ± 1.7 kBq·g⁻¹: p = 0.0038), compared to the control group (S₁: 10.1 ± 1.3 kBq·g⁻¹; S₂: 6.3 ± 1.2 kBq·g⁻¹). TBZ failed to change the vesicular release (Figures 7A,B). The tissue’s [³H] 5-HT content was reduced in the hippocampus by loading the tissue with RSP to 102.0 ± 23.1 kBq·g⁻¹: p = 0.0009, respectively, compared to the control group (401.5 ± 52.0 kBq·g⁻¹). TBZ and VBZ did not alter the tissue’s [³H] 5-HT storage capacity (287.0 ± 47.0; 523.0 ± 55.4 kBq·g⁻¹: p = 0.3223 and p = 0.2725) (Figure 7C).

VAChT (vesicular ACh transporter) and VMAT-2 are members of the SCL18 family of vesicular transporters, and VAChT shares 40% amino acid identity with VMAT-2; therefore, it seemed interesting to study the effect on the vesicular release of ACh. We measured the [³H] ACh release from hippocampal slices, from control, and 24 h before the experiment, pretreated (with 1 mg kg⁻¹ RSP i.p.) mice. RSP treatment failed to reduce the electric field stimulation-evoked release of [³H] ACh (S₁: 9.08 ± 1.2 kBq·g⁻¹:p = 0.9197 and S₂: 7.8 ± 0.72 kBq·g⁻¹:p = 0.2525) (Figures 8A,B) and the tissue’s [³H] ACh content (251.0 ± 19.7 kBq·g⁻¹p = 0.1462) compared to the control group (S₁: 8.92 ± 1.02 kBq·g⁻¹ and S₂: 6.5 ± 0.83 kBq·g⁻¹; tissue [³H] ACh content: 214.0 ± 12.9 kBq·g⁻¹) (Figure 8C). This observation is consistent with the biochemical studies proving that RSP does not affect this transporter (Arvidsson et al., 1997).