Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture

Oscar Maldonado; Alexandra Jenkins; Helen M Belalcazar; Helena Hernandez-Cuervo; Katelynn M Hyman; Giannina Ladaga; Lucia Padilla; Gabriel A de Erausquin

doi:10.1371/journal.pone.0223633

. 2020 Jul 23;15(7):e0223633. doi: 10.1371/journal.pone.0223633

Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture

Oscar Maldonado ^1,^#, Alexandra Jenkins ^2,^#, Helen M Belalcazar ³, Helena Hernandez-Cuervo ², Katelynn M Hyman ², Giannina Ladaga ¹, Lucia Padilla ¹, Gabriel A de Erausquin ^1,^*

Editor: Faramarz Dehghani⁴

PMCID: PMC7377472 PMID: 32701951

Abstract

Background

Small conductance, calcium-activated (SK3) potassium channels control the intrinsic excitability of dopaminergic neurons (DN) in the midbrain and modulate their susceptibility to toxic insults during development.

Methods

We evaluated the age-dependency of the neuroprotective effect of an SK3 agonist, 1-Ethyl-1,3-dihydro-2H-benzimidazol-2-one (1-EBIO), on Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) excitotoxicity to DN in ventral mesencephalon (VM) organotypic cultures.

Results

Most tyrosine hydroxylase (TH)+ neurons were also SK3+; SK3+/TH- cells (DN+) were common at each developmental stage but more prominently at day in vitro (DIV) 8. Young DN+ neurons were small bipolar and fusiform, whereas mature ones were large and multipolar. Exposure of organotypic cultures to AMPA (100 μm, 16 h) had no effect on the survival of DN+ at DIV 8, but caused significant toxicity at DIV 15 (n = 15, p = 0.005) and DIV 22 (n = 15, p<0.001). These results indicate that susceptibility of DN to AMPA excitotoxicity is developmental stage-dependent in embryonic VM organotypic cultures. Immature DN+ (small, bipolar) were increased after AMPA (100 μm, 16 h) at DIV 8, at the expense of the number of differentiated (large, multipolar) DN+ (p = 0.039). This effect was larger at DIV 15 (p<<<0.0001) and at DIV 22 (p<<<0.0001). At DIV 8, 30 μM 1-EBIO resulted in a large increase in DN+. At DIV 15, AMPA toxicity was prevented by exposure to 30 μM, but not 100 μM 1-EBIO. At DIV 22, excitotoxicity was unaffected by 30 μM 1-EBIO, and partially reduced by 100 μM 1-EBIO.

Conclusion

The effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by neuronal developmental stage.

Introduction

The intrinsic excitability and suceptiblity to toxic inuslts during development of dopaminergic neurons (DN) in the midbrain are regulated by small conductance, calcium-activated potassium (SK3) channels [1–3]. Abnormalities in SK3 channel function may contribute to the risk of schizophrenia, as mutations in the KCNN3 gene are associated with dopamine neuron dysfunction [4, 5] as well as with risk of schizophrenia in some patient samples [6–9]. Developmental processes in dopaminergic neurons (DN) have been extensively studied and characterized using primary cultures of embryonic mesencephalon [10, 11]. Indeed, the signaling pathways that define neuronal identity, maturation and differentiation of DN were described in primary cultures [12]. Furthermore, DN in cultures show mature functional phenotypes [13–17]. Our previous work in primary cultures firmly established that protracted stimulation of glutamate receptors activated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) results in phenotype-specific toxicity to a subpopulation of DN [18–21]. By contrast, we found that direct application of the glutamate receptor agonist N-Methyl-d-aspartate (NMDA) is not toxic to cultured DN [20], and fails to induce significant changes in intracellular free calcium concentrations or increase phosphorylation of c-AMP responsive element binding protein (CREB) in the same system [18]. Thus, rather than an accurate model of excitotoxicity to DN in neurodegenerative diseases such as Parkinson’s disease, AMPA-induced death of DN appears to mimic natural cell death of DN occurring during neurodevelopment [22] and the mechanism by which susceptible populations are affected in psychiatric neurodevelopmental disorders such as schizophrenia [23]. Indeed, primary cultures are plated during a critical period of the ontogeny of excitatory glutamatergic circuitry between the subthalamic nucleus and the substantia nigra pars compacta, in which AMPA receptors reach the peak of expression at the midbrain [24, 25].

In this model, commitment to die requires previous suppression of SK3 channel activity, and pharmacological enhancement of SK3 conductivity results in neuroprotection of DN [3]. We also found that a similar cell death mechanism can be triggered in developing midbrain in utero by maternal infection with neurotropic influenza virus, [18] and proposed that the same molecular pathways may act as a putative mechanism for the loss of neurons in the mesocortical dopaminergic projection in abnormal neurodevelopment leading to negative symptoms in schizophrenia [23]. However, primary cultures are limited in their viability in vitro and only allow observation of short-term effects of toxicity [26]. Organotypic cultures retain some of the 3D shape and some modicum of connections with normal anatomical targets, and most importantly, can be maintained in culture for much longer periods of time [26]. This report describes age-dependent susceptibility to excitotoxity of dopaminergic neurons in organotypic culture, as well as the impact of neuroprotection by an SK3 agonist, 1-EBIO, in organotypic cultures of different ages.

Materials and methods

Animals and dissections

Organotypic cultures were established from the ventral mesencephalon (VM) of rat embryos. Timed pregnant female Sprague-Dawley rats (n = 30; Charles River Laboratories, MA, USA) were euthanized by exposure to CO₂ on day 14 of gestation. Following laparotomy, embryos were quickly removed and placed in ice-cold Hanks Balanced Salt Solution (Sigma-Aldrich, MO, USA). Brains were dissected, VMs isolated, and meninges carefully removed [27] using a stereoscope (Zeiss, Oberkochen, Germany). VMs used in toxicological stereology experiments were carefully sliced at the midline for treatment and control sections. Animals were treated in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The University of South Florida Institutional Animal Care and Use Committee approved all procedures (protocol IS00000427).

Organotypic cultures

Dissected VMs were cultured following established procedures [28]. Briefly, VMs were transferred to Millicell-CM inserts (EMD Millipore, MA, USA) in six-well plates with 2 VM hemi-sections on each insert. Each well received 1.2 ml plating medium containing Neurobasal medium (Invitrogen, MA, USA) supplemented with 20% horse serum (Sigma-Aldrich, MO, USA), B-27® (50X, Invitrogen, MA, USA), Glutamax™ (100X, Invitrogen, MA, USA), D-(+)-glucose (1M, Sigma-Aldrich, MO, USA), penicillin-streptomycin (100X, Sigma-Aldrich, MO, USA), and basic fibroblast growth factor (25 μg/ml, Sigma-Aldrich, MO, USA). Differentiation was induced after 5 days in vitro (DIV) by introducing culture medium containing Neurobasal medium (Invitrogen, MA, USA) supplemented with 1% horse serum (Sigma-Aldrich, MO, USA), B-27® (50X, Invitrogen, MA, USA), Glutamax™ (100X, Invitrogen, MA, USA), and penicillin-streptomycin (100X, Sigma-Aldrich, MO, USA). Culture medium was renewed every 48 hours until toxicity experiments were performed on DIV 7, 14 and 21. Cultures were maintained at 37°C in an atmosphere of 5% CO₂ and 100% relative humidity.

Pharmacological treatments

All drug concentrations were chosen based on recommendations from manufacturers or previously published data by our own and other relevant groups [3, 18]. For DN survival experiments cultures were exposed to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid (AMPA) (100 μM) for 24 hours (DIV 7, 14, 21). Treatment was stopped by briefly rinsing with phosphate buffered saline (PBS) (Sigma-Aldrich, MO, USA), followed by fixation (DIV8, DIV15, DIV22). Cultures used for SK3 channel activation experiments were exposed to 1-ethyl-2-benzimidazolinone (1-EBIO) (30, 100 μM), and (AMPA) (100 μM) for 16 hours (DIV 7, 14, 21) in the following treatment groups: 1-EBIO 30 μM, 1-EBIO 100 μM, 1-EBIO 30 μM plus AMPA 100 μM, 1-EBIO 100 μM plus AMPA 100 μM, and AMPA 100 μM. Treatments were stopped by rinsing briefly with PBS, followed by fixation (DIV 8, DIV 15, DIV 22) for TH and SK3 immunofluorescence. 1-EBIO was dissolved in DMSO (Sigma-Aldrich, MO, USA) and AMPA in double distilled, deionized water as indicated in the corresponding product sheets. Both pharmacological agents were obtained from Tocris Bioscience, MO, USA.

Immunohistochemistry

Cultures were fixed in 4% formaldehyde (Fisher Scientific, MA, USA)– 4% sucrose (Sigma-Aldrich, MO, USA) in PBS at 4°C for 24 hours. Cultures were then sliced at a thickness of 50 μm using a Vibratome V1200 (Leica Biosystems, Wetzlar, Germany) and mounted on Fisherbrand™ Superfrost™ microscope slides. Mounted sections were permeabilized with 0.2% Triton (Sigma-Aldrich, MO, USA) for 15 minutes and blocked for endogenous peroxidase with 3% H₂O₂ (Sigma-Aldrich, MO, USA) for 10 minutes, followed by blocking with 10% normal goat serum (NGS) (Sigma-Aldrich, MO, USA) for 30 minutes. Sections were then incubated with the primary antibody mouse anti-TH (1:1000 EMD Millipore, MA, USA) for one hour at room temperature, followed by incubation with a goat anti-mouse IgG (H+L) poly-HRP secondary antibody (1:250 Invitrogen, MA, USA) for one hour at room temperature. For each antibody, tissue sections were incubated with primary antibody alone to test for autofluorescence, and with the secondary antibody alone to test for non-specific binding, at the same concentrations and in the same conditions used for the results; non-specific staining and autofluorescence were negligible (data not shown), For visualization, sections were then incubated with SIGMA FAST DAB (3,3’-Diaminobenzidine) per manufacturer’s instructions (Sigma-Aldrich, MO, USA), followed by a brief washing with PBS to stop the reaction. Tissue sections were then dehydrated and cleared using increasing concentrations of ethanol and xylene, followed by coverslip application using Crystal mount ™ aqueous mounting medium (Sigma-Aldrich, MO, USA). All antibody solutions were made using 1% NGS in PBS.

Immunofluorescence

Cultures were fixed in 4% formaldehyde—4% sucrose in PBS at 4°C for 24 hours. Cultures were then sliced at a thickness of 30 μm using a Vibratome V1200 (Leica Biosystems, Wetzlar, Germany) and mounted on Fisherbrand™ Superfrost™ microscope slides. Mounted sections were permeabilized in 0.15% Tween-20/0.05% Triton (Sigma-Aldrich, MO, USA) for 15 minutes and blocked in 10% normal goat serum (NGS) (Sigma-Aldrich, MO, USA) for 30 minutes, followed by blocking for endogenous avidin and biotin (avidin/biotin blocking kit, Invitrogen, MA, USA) per manufacturer’s instructions. Sections were then incubated with the primary antibodies mouse anti-TH (1:1000 EMD Millipore, MA, USA) and rabbit anti-SK3 (1:1,500 Alomone labs, Jerusalem, Israel) for 20 hours at 4°C followed by incubation with biotinylated goat anti-rabbit IgG (H+L) (Abcam, Cambridge, UK) for 15 minutes. Sections were incubated with the secondary antibodies goat anti-mouse Alexa Fluor® 568 (1:500) and Alexa Fluor® 488 conjugated to streptavidin (1:500) (Invitrogen, MA, USA) for 2 hours at room temperature. Sections were rinsed three times with PBS and cover-slipped using Prolong® Gold antifade reagent with DAPI (Invitrogen, MA, USA). All antibody solutions were made using 1% NGS/0.02% each Tween-20 and Triton in PBS.

Stereology and assessment of DN survival

Every other section of VM was stained with TH for unbiased stereological cell counting using the optical fractionator method [29]. The total number of TH+ neurons were counted in VM’s (DIV 8, DIV 15, DIV 22) (n = 14–15 toxicity experiments per DIV) by individuals blind to treatment conditions. Counts of TH+ neurons within a counting frame (50% screen height²) were made at regular predetermined intervals (frame spacing = 150 μm) by means of a grid program (Stereologer 2000, Stereology Resource Center, Inc., FL, USA). Each VM was viewed at low magnification (Nikon Apo 4x dry objective) and the entire section was outlined as the region of interest (ROI). Then, at a systematically determined starting point, TH+ neurons were counted at high magnification (Nikon Apo 60x oil objective). Cells were counted as TH+ if they exhibited immunoreactivity in the soma and the dendrites, and if the nucleus of the cell was within the focus between the top and bottom boundaries of the counting frame without touching the exclusion lines. Neurons were differentiated from non-neuronal cells based on size, shape and lack of TH staining. After counting the total number of TH+ neurons in each section, the program generated an estimate of the total number of TH+ neurons using the sample sectioning fraction (SSF), the area sampling fraction (ASF), and the mean thickness.

Effects of 1-EBIO on DN survival

One in three sections of control and 1-EBIO/AMPA treated VM were stained with TH and SK3, and were used for unbiased stereological cell counting (Stereologer, 2000, Stereology Resource Center, Inc., FL, USA). The total number of TH+/SK3+ co-stained neurons were counted in VM’s (DIV 8, DIV 15, DIV 22) (n = 3 toxicity experiments per DIV) using the optical fractionator probe in a blind-treated manner. Counts of TH+/SK3+ neurons within a counting frame (50% screen height²) were made at regular predetermined intervals (frame spacing = 100 μm) by means of a grid program (Stereologer 2000, Stereology Resource Center, Inc., FL, USA). Stereological estimates for total number of TH+ neurons were calculated as described above. Estimates of total number of SK3+ neurons were calculated manually using the fractionator equation as described by Peter Mouton [29]. All stereological counting was carried out using a Nikon Diaphot 300 inverted microscope (Nikon, Tokyo, Japan) equipped with a stereologer system (Stereologer 2000, Stereology Resource Center, Inc., FL, USA), 4x dry and 60x oil immersion objectives, 75 W xenon lamp, rhodamine and fluorescein filters, motorized XYZ stage (Stereology Resource Center, Inc., FL, USA), and Panasonic industrial CDD Gp-KR22 digital camera (Panasonic, Kadoma, Japan).

Measurement of soma size

Digital images of microscope fields containing at least one DN were captured, and DN soma diameter was measured at the widest point using ImageJ (NIH, MD, USA). Soma diameter was measured in pixels and converted to μm using a known measurement to set the scale within ImageJ.

Statistical analysis

Data were analyzed using SPSS (version 22 for Macintosh, IBM, USA). Results are reported as mean ± standard error. For DN survival at least 14 AMPA toxicity replicates were performed for each DIV. Statistical analysis was performed by two-way ANOVA with DIV and treatment as fixed factors, followed by protected Post-hoc Test for multiple comparisons. For 1-EBIO experiments at least 3 replicates were performed for each treatment group. Statistical analysis was performed by one-way ANOVA on each DIV, with treatment as the fixed factor, followed (in significant ANOVAS) by simple contrasts to compare each treatment group to the control. The alpha level for significance was set at P ≤ 0.05. Prior to analysis all data was checked for normality using the Shapiro-Wilk normality test, and checked for outliers using the outlier-labeling rule [30].

Results

SK3 channels are expressed in DN organotypic cultures

Virtually all TH+ neurons observed were also SK3+. Only occasionally did we observe neurons that were TH+/SK3-, most frequently at DIV 8. SK3 staining was mainly localized to the soma and proximal dendrites, with staining being more intense closer to the cell body (Fig 1, panel A). SK3+/TH- cells were common at each developmental stage but more prominently at DIV8, which contrasts with our results using primary cultures. TH+/SK3+ neurons exhibited two distinctive morphologies: small to medium bipolar fusiform and large multipolar. Most TH+/SK3+ neurons at DIV 8 were small, spherical, and bipolar, whereas at DIV 22 TH+/SK3+ they were frequently large and multipolar (Fig 1, panels A, C).

Fig 1 — Images show examples of stained neurons at DIV 8 (panel A), DIV 15 (panel B) and DIV 22 (panel C) merged color images show high co-localization for anti-TH (red) and anti-SK3 (green) antibodies. At each DIV there are also a number of cells that are positive for anti-SK3, but negative for anti-TH. At DIV 8 DNs were typically bipolar with small fusiform shaped somas and displayed little branching. While small bipolar neurons were still present at DIV 15, DN morphology began shifting towards multipolar cells with larger somas and increased branching. Multipolar cells with large somas was the predominant DN morphology at DIV 22, with small bipolar cells being rare. Scale bar = 20 μm.

DNs in organotypic cultures are differentially susceptible to AMPA-receptor mediated excitotoxicity

AMPA-receptor mediated excitotoxicity was assessed in organotypic VM cultures at DIV 8, DIV 15, and DIV 22. After 16 hours of exposure to AMPA (100 μm), DIV 8 treated tissue did not exhibit a detectable loss of TH+ cells compared to control tissue (CT) (n = 14, F = 0.124; p = 0.127, Fig 2, panel A and top row of panel B). At DIV 15, however, AMPA treatment (100 μm, 16 hr) showed a significant decrease in the number of TH+ cells compared to CT tissue (n = 15, F = 10.826; p<0.005, Fig 2 panel A and middle row of panel B). The most dramatic effect of AMPA treatment (100 μm, 16 hr) on DN survival was observed at DIV 22, where the frequency of TH+ cells was diminished by more than half (n = 15, F = 21.651, p<0.0001, Fig 2, panel A and bottom row of panel B). These results indicate that susceptibility of DN to AMPA excitotoxicity is developmental stage-dependent in embryonic VM organotypic cultures.

Fig 2 — (A) VM were incubated with AMPA (100 μM) for 16 hr at DIV 8 (n = 14), DIV 15 (n = 15), and DIV 22 (n = 15). Survival is expressed as total number of TH+ neurons in untreated VM (CT) compared to VM treated with AMPA (100 μM) for each DIV. Data are expressed as mean ± SEM. **p < 0.01, ***p < 0.001. (B) Representative photomicrographs of DN at DIV 8, DIV 15 and DIV 22 comparing untreated (CT) VM and VM treated with AMPA (100 μM) for 16 hr. Scale bar = 20 μm.

Since the most notable change in the appearance of DN with time in vitro was their soma size, we also measured the soma diameter of individual DNs in AMPA-treated and CT tissue at each DIV to establish the impact of differentiation on susceptibility. Interestingly, although we did not detect an effect of AMPA treatment on overall DN survival at DIV 8, the number of large DN was significantly lower at DIV 8 compared to CT tissue (n_CT = 163, n_AMPA = 126, p = 0.039, Fig 3, panel B). This pattern was most dramatic at DIV 15 (n_CT = 151, n_AMPA = 146, p = 2.0 x 10⁻²⁴, Fig 3, panel B) and continued to be significant at DIV 22 (n_CT = 133, n_AMPA = 111, p = 1.7 x 10⁻¹⁰, Fig 3, panel B). These results indicate that AMPA treatment has a differential effect on more differentiated DN in embryonic VM organotypic culture.

Fig 3 — (A) Histogram representing the frequency of DN soma size in untreated (CT) VM and VM treated with AMPA (100 μM) for 16 hr at DIV 8 (n_CT = 126, n_AMPA = 163), DIV 15 (n_CT = 151, n_AMPA = 146) and DIV 22 (n_CT = 133, n_AMPA = 111). (B) Mean DN soma size following 16 hr AMPA treatment in DIV 8, DIV 15, and DIV 22. Soma size is expressed as mean DN diameter (μm) ± SEM for all DIV.

Protection conferred by SK3 channel activation is developmental stage-dependent in organotypic cultures of embryonic VM

The effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by developmental age (Fig 4, two-way ANOVA F = 36.242, p < 0.00001). At DIV 8 there was no significant toxicity of AMPA, but treatment with 30 μM resulted in a large increase in double stained neurons (Fig 4, panel A). At DIV 15, AMPA toxicity was prevented by exposure to 30 μM, but not 100 μM 1-EBIO (Fig 4, panel B). Lastly, at DIV 22, the profound excitotoxic effect of AMPA was unaffected by 30 μM 1-EBIO, and only partially reduced by exposure to 100 μM 1-EBIO (Fig 4, panel C).

Fig 4 — Bars represent the number of TH+/SK3+ neurons (expressed as % of the control condition) at DIV 8 (panel A), DIV 15 (panel B), and DIV 22 (panel C). Organotypic cultures were treated with conditioned media (control condition, black bars) or with AMPA (100 μM, gray bars) for 16 h. The y axes represent increasing concentrations of 1-EBIO (0, 30 or 100μM). DN survival is expressed as percentage of TH+/SK3+ neurons in each treatment group relative to the control. Data are expressed as mean ± SEM, and are normalized with respect to controls.

Discussion

Dopamine neuron susceptibility to excitotoxic injury is potentially relevant to a number of pathological conditions, and therefore so is the development of potential neuroprotective strategies, including the use of pharmacological modulators of SK3 channel function [31–34]. We previously showed that 1-EBIO effectively protects DN in primary cultures of ventral mesencephalon against AMPA-mediated toxicity by sustaining SK3 channel conductance [3]. Specifically, we found that the inactivation of SK3 channels by apamin or NS8593 promotes specific death of DN sharing a convergent pathway with AMPA-induced excitotoxicity [3]. By contrast, the SK3 channel agonists 1-EBIO and CyPPA increased SK3 currents and promoted both survival and differentiation of cultured DN, as well as increased neurogenesis [3, 35]. The overall expression level of SK3 channel increases with the synaptic maturation [36]. The trophic effects of SK3 agonists are likely explained by the rescue of DN that degenerate as a result of culture-related neuronal attrition [3]. However, primary cultures can be studied over a very brief period of time, and lack much of the natural geometry of the brain tissue including important interactions with glial cells and other neurons. Thus, we studied organotypic cultures of the ventral mesencephalon in the same species and were harvested at the same age, but were maintained in vitro long enough to allow evaluation of DN susceptibility at different developmental stages up to 22 days in vitro.

Perhaps not surprisingly, we found that in organotypic cultures, susceptibility of DN to AMPA-mediated excitotoxicity increases with age and differentiation status, such that the more complex, larger DN that become more common as the tissue matures, are much more susceptible (Fig 3, panel A) than the embryonic-appearing, small and round neurons more common earlier in development (Fig 3, panel B). This is consistent with observations where age plays a determining role in the increased susceptibility of DN to chronic rotenone exposure that is accompanied by severe locomotor deficits and decreased lifespan [37]. Overall, AMPA did not induce cell death in DN at 8 days in vitro, but toxicity became apparent at 15 DIV and most intense at 22 DIV. These findings are consistent with knowledge about the expression of AMPA receptors in DN in VM or rodents [3]. Activity-dependent changes in synaptic expression of AMPA receptors are tightly regulated [3]. During development in rodents, the four subunits of AMPA receptors (GlurA to GlurD) are first detected in VM neurons at E13, and thereafter their expression increases until roughly E17 [24].

The molecular pathway of AMPA-mediated excitotoxicity in embryonic DN requires activation of voltage-dependent calcium channels, destabilization of calcium homeostasis, activation of a mitochondrial transition pore and nuclear translocation of nuclear factor kappa b, and phosphorylation of p53, this then leads to the activation of a programmed cell death process [18, 19, 38]. However, the key step prior to commitment to die is the suppression of the SK3 current in DN, such that the entire process can be mimicked by the SK3 antagonist apamin, and prevented by SK3 activation by either 1-EBIO or CyPPA (N-cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine) [3]. Thus, the natural molecular target for neuroprotection in a more intact system is the SK3 channel. Our data is also consistent with the neuroprotective effect of SK channel activation in cultured human postmitotic dopaminergic neurons in vitro following rotenone treatment; indeed, sub-lethal concentrations of rotenone are significantly more toxic to DN and cause more motor impairment in older when compared to younger drosophila flies [37]. Additionally susceptibility of differentiated DN from the human neuroblastoma cell line SH-SY5Y is also increased by time in vitro [37].

We found that the effects of 1-EBIO on DN survival following excitotoxicity were complex, but largely consistent with the expected neuroprotection. The contrasting results between our current experiments and those reported by us previously in primary cultures of VM, may be due to differences in culture systems, including the presence of SK3-expressing microglia, which is particularly abundant in the midbrain at this developmental stage [3, 23, 39–41]. Interestingly, SK3 activation in primary cultures led to an increase in TH-expressing neurons, in the absence of any susceptibility to AMPA-induced toxicity [3]; this suggests that SK3 channel expression precedes the ability of DN to trigger the programmed cell death induced by AMPA [19, 21]. At DIV 8, exposure to increasing concentrations of 1-EBIO yielded an inverted U-shaped curve, with an optimum effect at 30 μM and no change in the number of TH+/SK3+ neurons after exposure to 100 μM (Fig 4, panels A-C). The lack of effect at the higher concentration was also seen at DIV15, where 30 μM was clearly neuroprotective but 100 μM had no effect.

On the other hand, at DIV22 exposure of the cultures to 30 μM 1-EBIO did not afford any protection, whereas 100 μM did, suggesting a shift to the right in the concentration-response curve. Again, this may be explained at least in part by the maturational stage of the DN, and possibly also by the indirect effects of 1-EBIO on SK3 expressing microglia or non-dopaminergic neurons, both of which are much more infrequent at DIV22 (Fig 1). Furthermore, SK3 mRNA in the rat brain was found exclusively in areas that also contained large numbers of DA neurons including the substantia nigra (SN) and the ventral tegmental area (VTA), and younger animals express higher levels and less regional variation in SK3 transcripts [42, 43]. As we found, fully differentiated DN continue to express high levels of SK3 channels, which are heavily expressed in the soma and, to a lesser extent, throughout the dendritic arbor [38, 44]. Somatodendritic expression of SK3 channels is consistent with their involvement in pace making activity in DNs, which is also modulated during early development but stable after a week after birth in the rat [45].

A note is needed regarding expression of SK3 immunoreactivity in cells other than DN, which again was most apparent at earlier developmental stages (Fig 1). Since the focus of this publication is the effect of 1-EBIO on the susceptibility of DN we did not attempt to characterize the SK3 expressing cells, but such transient expression has been reported in mesencephalic microglia [39, 46]. Indeed, SK3 channels can modulate inflammatory responses in microglial cells, and SK3 channel blockade inhibited microglial activation and reduced their ability to kill neurons [39, 46]. Thus, it is possible that the presence of microglia in large numbers may alter the overall impact of SK3 activation on excitotoxicity, and that the effect may disappear when microglial numbers are smaller.

Our observations are consistent with findings of the effects of SK3 channel agonists in intact animals [31, 38, 47, 48]. In conditionally SK3-deficient mice, baseline levels of striatal extracellular dopamine are increased [31] while both CyPPA spontaneous firing rate and dopamine release are decreased [49]. As a result, the duration of the apamin-sensitivity afterhyperpolarization is increased and causes an activity-dependent inhibition of current-evoked action potentials in DN [47]. After bilateral lesions with 6-hydroxydopamine into the striatum, SK3 channel expression is reduced in the SN, and motor impairment is reversed by administration of CyPPA [48]. Likewise, inhibition of mitochondrial complex I with rotenone disrupts the dendritic network of human DN and induces neuronal death, and SK3 channel activation preserves the dendritic network, cell viability and ATP levels after rotenone challenge [38].

KCNN3, the gene that encodes SK3, is mutated in some families in association with chronic psychosis [4]. Consistent with the results using AMPA excitotoxicity, transgenic expression of the human disease-related SK3 mutation (hSK3Δ) in DN of mice suppresses endogenous SK3 currents, reducing coupling between SK channels and NMDA receptors (NMDARs) and resulting in increased burst firing [4]. Also, hSK3Δ increased excitability of DN through increased evoked calcium signals and potentiated evoked dopamine release [4]. Notably, in mice transgenic for the causal gene for human Huntington Disease, DN excitability was increased due to functional loss and abnormal distribution of SK3 channels. Progressive expansion in the CAG repeat number and nuclear localization of mutant protein within the SN pars compacta resulted in an increase in dopamine release related to a loss of SK3 channel function [50].

Conclusion

We found that in organotypic cultures of VM, dopaminergic neurons are increasingly susceptible to excitotoxicity with time in vitro, and in direct proportion to their soma size and neurite complexity. Just as we had shown in primary cultures of VM, in orgaontypic cultures treatment with an agonist of SK3 channels results in neuroprotection against excitotoxicity. Notably, we found that the protective effect of SK3 agonists is modulated by age in vitro as well, such that it is most apparent in more mature DN. Our data suggest that SK3 agonists may be a potentially useful target for neuroprotection of developing DN.

Abbreviations

VM: ventral mesencephalon
1-EBIO: 1-Ethyl-1,3-dihydro-2H-benzimidazol-2-one; 1-Ethylbenzimidazolinone
CyPPA: Cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine
PBS: phosphate buffered saline
AMPA: Amino-3-hydroxy-5-methylisoxazole-4-propionic acid
NMDAR: N-methyl-D-Aspartate type glutamate receptor
TH: tyrosine hydroxylase
SK: small conductance calcium-activated potassium channel
SK3: small conductance calcium-activated potassium channel 3 also known as K_Ca2.3
DIV: days in vitro
DN: dopaminergic neurons
KCNN3: potassium calcium-activated channel subfamily N member 3 (human) gene
DMSO: Dimethyl Sulfoxide
NGS: normal goat serum
DAB: 3,3′-Diaminobenzidine
HRP: horseradish peroxidase
DAPI: 4′,6-diamidino-2-phenylindole
ROI: region of interest

Data Availability

All relevant data are included within the manuscript.

Funding Statement

This work was supported in part by the Zachry Foundation Chair to Gde, and the Center for Brain Health grant from the Valley Baptist Legacy Foundation to GdE. In addition, the authors received funding from Roskamp Chair at USF Morsani College of Medicine and Fundacion FULTRA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Ji H., Hougaard C., Herrik K.F., Strøbaek D., Christophersen P., Shepard P.D., 2009. Tuning the excitability of midbrain dopamine neurons by modulating the Ca2+ sensitivity of SK channels. Eur. J. Neurosci. 29, 1883–1895. 10.1111/j.1460-9568.2009.06735.x [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Wolfart J., Neuhoff H., Franz O., Roeper J., 2001. Differential expression of the small-conductance, calcium-activated potassium channel SK3 is critical for pacemaker control in dopaminergic midbrain neurons. J. Neurosci. 21, 3443–3456. 10.1523/JNEUROSCI.21-10-03443.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Benítez B.A., Belálcazar H.M., Anastasía A., Mamah D.T., Zorumski C.F., Mascó D.H., et al. , 2011. Functional reduction of SK3-mediated currents precedes AMPA-receptor mediated excitotoxicity in dopaminergic neurons. Neuropharmacology 60, 1176–1186. 10.1016/j.neuropharm.2010.10.024 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Soden M.E., Jones G.L., Sanford C.A., Chung A.S., Güler A.D., Chavkin C., et al. , 2013. Disruption of dopamine neuron activity pattern regulation through selective expression of a human KCNN3 mutation. Neuron 80, 997–1009. 10.1016/j.neuron.2013.07.044 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Tomita H., Shakkottai V.G., Gutman G.A., Sun G., Bunney W.E., Cahalan M.D., et al. , 2003. Novel truncated isoform of SK3 potassium channel is a potent dominant-negative regulator of SK currents: implications in schizophrenia. Mol. Psychiatry 8, 524–535, 460. 10.1038/sj.mp.4001271 [DOI] [PubMed] [Google Scholar]
6.Grube S., Gerchen M.F., Adamcio B., Pardo L.A., Martin S., Malzahn D., et al. , 2011. A CAG repeat polymorphism of KCNN3 predicts SK3 channel function and cognitive performance in schizophrenia. EMBO Mol Med 3, 309–319. 10.1002/emmm.201100135 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Ivković M., Ranković V., Tarasjev A., Orolicki S., Damjanović A., Paunović V.R., et al. , 2006. Schizophrenia and polymorphic CAG repeats array of calcium-activated potassium channel (KCNN3) gene in Serbian population. Int. J. Neurosci. 116, 157–164. 10.1080/00207450341514 [DOI] [PubMed] [Google Scholar]
8.Ritsner M., Amir S., Koronyo-Hamaoui M., Gak E., Ziv H., Halperin T., et al. , 2003. Association study of CAG repeats in the KCNN3 gene in Israeli patients with major psychosis. Psychiatr. Genet. 13, 143–150. 10.1097/00041444-200309000-00002 [DOI] [PubMed] [Google Scholar]
9.Ritsner M., Modai I., Ziv H., Amir S., Halperin T., Weizman A., et al. , 2002. An association of CAG repeats at the KCNN3 locus with symptom dimensions of schizophrenia. Biol. Psychiatry 51, 788–794. 10.1016/s0006-3223(01)01348-8 [DOI] [PubMed] [Google Scholar]
10.Branton RL, Clarke DJ. Apoptosis in primary cultures of E14 rat ventral mesencephala: time course of dopaminergic cell death and implications for neural transplantation. Exp Neurol. 1999. November;160(1):88–98. 10.1006/exnr.1999.7207 [DOI] [PubMed] [Google Scholar]
11.Falkenburger BH, Schulz JB. Limitations of cellular models in Parkinson’s disease research. J Neural Transm Suppl. 2006;(70):261–8. 10.1007/978-3-211-45295-0_40 [DOI] [PubMed] [Google Scholar]
12.Alavian KN, Scholz C, Simon HH. Transcriptional regulation of mesencephalic dopaminergic neurons: the full circle of life and death. Mov Disord. 2008. February 15;23(3):319–28. 10.1002/mds.21640 [DOI] [PubMed] [Google Scholar]
13.Greco D, Volpicelli F, Di Lieto A, Leo D, Perrone-Capano C, Auvinen P, et al. Comparison of gene expression profile in embryonic mesencephalon and neuronal primary cultures. PLoS ONE. 2009;4(3):e4977 10.1371/journal.pone.0004977 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Chiodo LA, Kapatos G. Membrane properties of identified mesencephalic dopamine neurons in primary dissociated cell culture. Synapse. 1992. August;11(4):294–309. 10.1002/syn.890110405 [DOI] [PubMed] [Google Scholar]
15.Liss B, Franz O, Sewing S, Bruns R, Neuhoff H, Roeper J. Tuning pacemaker frequency of individual dopaminergic neurons by Kv4.3L and KChip3.1 transcription. EMBO J. 2001. October 15;20(20):5715–24. 10.1093/emboj/20.20.5715 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Neuhoff H, Neu A, Liss B, Roeper J. I(h) channels contribute to the different functional properties of identified dopaminergic subpopulations in the midbrain. J Neurosci. 2002. February 15;22(4):1290–302. 10.1523/JNEUROSCI.22-04-01290.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Seutin V, Massotte L, Renette MF, Dresse A. Evidence for a modulatory role of Ih on the firing of a subgroup of midbrain dopamine neurons. Neuroreport. 2001. February 12;12(2):255–8. 10.1097/00001756-200102120-00015 [DOI] [PubMed] [Google Scholar]
18.de Erausquin G., Brooker G., Costa E., Hanbauer I., 1994. Persistent AMPA receptor stimulation alters [Ca2+]i homeostasis in cultures of embryonic dopaminergic neurons. Brain Res. Mol. Brain Res. 21, 303–311. 10.1016/0169-328x(94)90261-5 [DOI] [PubMed] [Google Scholar]
19.de Erausquin G.A., Hyrc K., Dorsey D.A., Mamah D., Dokucu M., Mascó D.H., et al. , 2003. Nuclear translocation of nuclear transcription factor-kappa B by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors leads to transcription of p53 and cell death in dopaminergic neurons. Mol. Pharmacol. 63, 784–790. 10.1124/mol.63.4.784 [DOI] [PubMed] [Google Scholar]
20.Isaacs KR, de Erausquin G, Strauss KI, Jacobowitz DM, Hanbauer I. Differential effects of excitatory amino acids on mesencephalic neurons expressing either calretinin or tyrosine hydroxylase in primary cultures. Brain Res Mol Brain Res. 1996. February;36(1):114–26. 10.1016/0169-328x(95)00252-n [DOI] [PubMed] [Google Scholar]
21.Dorsey D.A., Mascó D.H., Dikranian K., Hyrc K., Masciotra L., Faddis B., et al. , 2006. Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons. Apoptosis 11, 535–544. 10.1007/s10495-006-5268-y [DOI] [PubMed] [Google Scholar]
22.Burke RE. Ontogenic cell death in the nigrostriatal system. Cell Tissue Res. 2004. October;318(1):63–72. 10.1007/s00441-004-0908-4 [DOI] [PubMed] [Google Scholar]
23.de Erausquin G.A., Hanbauer I. (1995) AMPA Receptor-Induced Dopaminergic Cell Death: A Model for the Pathogenesis of Hypofrontality and Negative Symptoms in Schizophrenia In: Mednick S.A., Hollister J.M. (eds) Neural Development and Schizophrenia. NATO ASI Series (Series A: Life Sciences), vol 275 Springer, Boston, MA [Google Scholar]
24.Lilliu V, Pernas-Alonso R, Trelles RD, di Porzio U, Zuddas A, Perrone-Capano C. Ontogeny of AMPA receptor gene expression in the developing rat midbrain and striatum. Brain Res Mol Brain Res. 2001. November 30;96(1–2):133–41. 10.1016/s0169-328x(01)00280-7 [DOI] [PubMed] [Google Scholar]
25.Marani E, Heida T, Lakke EAJF, Usunoff KG. The subthalamic nucleus. Part I: development, cytology, topography and connections. Adv Anat Embryol Cell Biol. 2008;198:1–113, vii. [PubMed] [Google Scholar]
26.Lopes FM, Bristot IJ, da Motta LL, Parsons RB, Klamt F. Mimicking Parkinson's Disease in a Dish: Merits and Pitfalls of the Most Commonly used Dopaminergic In Vitro Models. Neuromolecular Med. 2017. September;19(2–3):241–255. 10.1007/s12017-017-8454-x [DOI] [PubMed] [Google Scholar]
27.Costantini LC, Lin L, Isacson O. Medial fetal ventral mesencephalon: a preferred source for dopamine neuron grafts. Neuroreport. 1997. July 7;8(9–10):2253–7 10.1097/00001756-199707070-00032 [DOI] [PubMed] [Google Scholar]
28.Jaeger C, Gonzalo Ruiz A, Llinás R. Organotypic slice cultures of dopaminergic neurons of substantia nigra. Brain Res Bull. 1989. June;22(6):981–91 10.1016/0361-9230(89)90010-5 [DOI] [PubMed] [Google Scholar]
29.Mouton PR, Phoulady HA, Goldgof D, Hall LO, Gordon M, Morgan D. Unbiased estimation of cell number using the automatic optical fractionator. J Chem Neuroanat. 2017. March;80:A1–A8. 10.1016/j.jchemneu.2016.12.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Hoaglin D. C., and Iglewicz B. (1987), “Fine Tuning Some Resistant Rules for Outlier Labeling”, Journal of American Statistical Association., 82, 1147–1149. [Google Scholar]
31.Noh W., Pak S., Choi G., Yang Sungchil, Yang,et al. , 2019. Transient Potassium Channels: Therapeutic Targets for Brain Disorders. Front Cell Neurosci 13, 265 10.3389/fncel.2019.00265 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Hipólito L, Fakira AK, Cabañero D, Blandón R, Carlton SM, Morón JA, et al. In vivo activation of the SK channel in the spinal cord reduces the NMDA receptor antagonist dose needed to produce antinociception in an inflammatory pain model. Pain. 2015. May;156(5):849–58. 10.1097/j.pain.0000000000000124 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Hopf FW, Bowers MS, Chang S-J, Chen BT, Martin M, Seif T, et al. Reduced nucleus accumbens SK channel activity enhances alcohol seeking during abstinence. Neuron. 2010. March 11;65(5):682–94. 10.1016/j.neuron.2010.02.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Blank T, Nijholt I, Kye M-J, Spiess J. Small conductance Ca2+-activated K+ channels as targets of CNS drug development. Curr Drug Targets CNS Neurol Disord. 2004. June;3(3):161–7. 10.2174/1568007043337472 [DOI] [PubMed] [Google Scholar]
35.Liebau S, Vaida B, Proepper C, Grissmer S, Storch A, Boeckers TM, et al. Formation of cellular projections in neural progenitor cells depends on SK3 channel activity. J Neurochem. 2007. June;101(5):1338–50. 10.1111/j.1471-4159.2006.04437.x [DOI] [PubMed] [Google Scholar]
36.Obermair GJ, Kaufmann WA, Knaus H-G, Flucher BE. The small conductance Ca2+-activated K+ channel SK3 is localized in nerve terminals of excitatory synapses of cultured mouse hippocampal neurons. Eur J Neurosci. 2003. February;17(4):721–31. 10.1046/j.1460-9568.2003.02488.x [DOI] [PubMed] [Google Scholar]
37.Sur M., Dey P., Sarkar A., Bar S., Banerjee D., Bhat S., et al. , 2018. Sarm1 induction and accompanying inflammatory response mediates age-dependent susceptibility to rotenone-induced neurotoxicity. Cell Death Discov 4, 114 10.1038/s41420-018-0119-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Tessner T.G., Rock C.O., Kalmar G.B., Cornell R.B., Jackowski S., 1991. Colony-stimulating factor 1 regulates CTP: phosphocholine cytidylyltransferase mRNA levels. J. Biol. Chem. 266, 16261–16264. [PubMed] [Google Scholar]
39.Dolga A.M., Letsche T., Gold M., Doti N., Bacher M., Chiamvimonvat N., et al. , 2012. Activation of KCNN3/SK3./K(Ca)2.3 channels attenuates enhanced calcium influx and inflammatory cytokine production in activated microglia. Glia 60, 2050–2064. 10.1002/glia.22419 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Schlichter L.C., Kaushal V., Moxon-Emre I., Sivagnanam V., Vincent C., 2010. The Ca2+ activated SK3. channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro. J Neuroinflammation 7, 4 10.1186/1742-2094-7-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Thion M.S., Ginhoux F., Garel S., 2018. Microglia and early brain development: An intimate journey. Science 362, 185–189. 10.1126/science.aat0474 [DOI] [PubMed] [Google Scholar]
42.Tacconi S., Carletti R., Bunnemann B., Plumpton C., Merlo Pich E., Terstappen G.C., 2001. Distribution of the messenger RNA for the small conductance calcium-activated potassium channel SK3. in the adult rat brain and correlation with immunoreactivity. Neuroscience 102, 209–215. 10.1016/s0306-4522(00)00486-3 [DOI] [PubMed] [Google Scholar]
43.Sarpal D., Koenig J.I., Adelman J.P., Brady D., Prendeville L.C., Shepard P.D., 2004. Regional distribution of SK3. mRNA-containing neurons in the adult and adolescent rat ventral midbrain and their relationship to dopamine-containing cells. Synapse 53, 104–113. 10.1002/syn.20042 [DOI] [PubMed] [Google Scholar]
44.Deignan J., Luján R., Bond C., Riegel A., Watanabe M., Williams J.T., et al. , 2012. SK2 and SK3. expression differentially affect firing frequency and precision in dopamine neurons. Neuroscience 217, 67–76. 10.1016/j.neuroscience.2012.04.053 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Dufour M.A., Woodhouse A., Goaillard J.-M., 2014. Somatodendritic ion channel expression in substantia nigra pars compacta dopaminergic neurons across postnatal development. J. Neurosci. Res. 92, 981–999. 10.1002/jnr.23382 [DOI] [PubMed] [Google Scholar]
46.Dolga A.M., de Andrade A., Meissner L., Knaus H.-G., Höllerhage M., Christophersen P., et al. , 2014. Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons. Cell Death Dis 5, e999 10.1038/cddis.2013.530 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Herrik K.F., Redrobe J.P., Holst D., Hougaard C., Sandager-Nielsen K., Nielsen A.N., et al. , 2012. CyPPA, a Positive SK3./SK2 Modulator, Reduces Activity of Dopaminergic Neurons, Inhibits Dopamine Release, and Counteracts Hyperdopaminergic Behaviors Induced by Methylphenidate. Front Pharmacol 3, 11 10.3389/fphar.2012.00011 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Mourre C., Manrique C., Camon J., Aidi-Knani S., Deltheil T., Turle-Lorenzo N., et al. , 2017. Changes in SK channel expression in the basal ganglia after partial nigrostriatal dopamine lesions in rats: Functional consequences. Neuropharmacology 113, 519–532. 10.1016/j.neuropharm.2016.11.003 [DOI] [PubMed] [Google Scholar]
49.Jacobsen J.P.R., Weikop P., Hansen H.H., Mikkelsen J.D., Redrobe J.P., Holst D., et al. , 2008. SK3. K+ channel-deficient mice have enhanced dopamine and serotonin release and altered emotional behaviors. Genes Brain Behav. 7, 836–848. 10.1111/j.1601-183X.2008.00416.x [DOI] [PubMed] [Google Scholar]
50.Dallérac G.M., Levasseur G., Vatsavayai S.C., Milnerwood A.J., Cummings D.M., Kraev I., et al. , 2015. Dysfunctional Dopaminergic Neurones in Mouse Models of Huntington’s Disease: A Role for SK3. Channels. Neurodegener Dis 15, 93–108. 10.1159/000375126 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0223633.r001

Decision Letter 0

Faramarz Dehghani

3 Dec 2019

PONE-D-19-26792

Age-dependent neuroprotective effect of an SK3. channel agonist on excitotoxity

to dopaminergic neurons in organotypic culture

PLOS ONE

Dear Dr de Erausquin,

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Reviewer #1: In the present study the authors examined the age-dependency of the neuroprotective effect of an small-conductance calcium activated potassium channel 3 (SK3) agonist, 1-EBIO, on AMPA excitoxicity to dopaminergic neurons (DN) in organotypic VM cultures.

These results indicate that susceptibility of DN to AMPA excitotoxicity is dependent on developmental stag in embryonic VM organotypic cultures, as well as the effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by neuronal developmental stage (DIVs). In all, the data suggest that activation of SK3 channels in DN may be a potentially useful target for neuroprotection.

General impression: this manuscript is interesting and definitely provides the new insight nto the age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture.

However, these manuscript need to be substantial improve and I have numerous remarks that have to be considered:

General points:

The layout of the manuscript is absolutely not according to PLOS ONE. Please do it according to PLOS ONE.

Please say in the whole manuscript “SK3” (without point) or “SK3.” Or is there an interlectual reason to use two versions?

Please change/transfer all references/citations in the manuscript to numbers according to References-List at the end of the manuscript.

Please change the References-List at the end of the manuscript in accordance to PLOS ONE.

Please check the MS for obviously missing word - some sentences do no really give sense as they stand now, eg. last sentence on page 3, first sentence on page 4, last sentence of page 11, ...

Do not say cell culture - you studied organotypic VM cultures.

At page 5 you clearly defined the groups tested. Please stick to these definitions throughout the text - otherwise it is confusing.

Special points:

Abbreviation: Please add all abbreviation used in the manuscript into “Abbreviations” (page 1). Please add also to abbreviations: VM; KCNN3; SK; NMDAR and so on..

Abstract

*Line/row 2: please correct to “calcium-activated”; please write out: 1-EBIO and AMPA.

Line 3: “TH” – please write out.

Line 8 and 12: please use usual and typical mathematic symbols when expressing levels of significance. Please check and correct the similar symbols in the whole manuscript.

Line 9: “VM” – please write out.

Keywords:

Please also add to keywords: “neuroprotective effect”.

Introduction:

Line/row 5: please write “KCNN3” in “italic” for genes.

Line 10: … (AMPA)-mediated excitotoxicity (?); - please add references after these conversation.

Line 20: However, primary cultures are limited in their viability in vitro and only allow observation of short term effects of toxicity (.???.): please add references.

Line 23: Organotypic cultures that retain some of the 3D shape and some modicum of connections with normal anatomical targets, and most importantly, can be maintained in culture for much longer periods of time (.???.) – please add references.

The Introduction should be improve!

Please show more exactly the reason and justify, why you chosen this study design. Please add more description and information about the similar studies in the literature up to date? Please add whole this information into the introduction before: .. .Line 7: Thus, we have focused on understanding the determinants of DN susceptibility to excitotoxic challenges during development.

Materials and Methods:

Please delete “-“at the beginning of the each parts of the Materials and methods.

Pharmacological treatmens

Line 14: .. and AMPA in water as indicated. Please add: which kind of water and more information about this indication (???.).

Stereology and assessment of DN survival

Every other section of VM were stained with TH, and used for unbiased stereological cell counting using the optical fractionator method – please add references to this method.

Counts of TH+ neurons within a counting frame (50% screen height 2) were made at regular predetermined intervals (frame spacing=150 μm) by means of a grid program (Stereologer 2000) - please add the exactly information about this program.

Statistical analysis

.. “by protected post-hoc test for”: please correct to “Post-hoc-Test.

Results:

Don’t use the numbers“1,2,3” before each part of the Results.

2. DNs in organotypic cultures are differentially susceptible to AMPA-receptor mediated excitotoxicity: Please indicate every time very exactly after the Fig. 2 and Fig. 3, wheater it is A or B!! The reader is not a detective for finding the right subfigure.

Please add and correct:

p=0.127, Fig.2????).

to CT tissue (n=15, p=0.005, Fig.2???).

more than half (n=15, p=0.0000000, Fig.2???).

CT tissue (nCT=163,nAMPA=126, p=0.039, Fig. 3 ???).

This pattern was most dramatic at DIV 15 (nCT=151, nAMPA=146, p=2.0 x 10-24, Fig. 3???) and continued to be significant at DIV 22 (nCT=133, nAMPA=111, p=1.7 x10-10, Fig. 3????).

It would be a good idea to write down the exact number and not only the result of the comparative statistics. From the figures the exact values and the SDs or SEMs cannot be seen.

3. Protection conferred by SK3. channel activation is developmental stage-dependent in organotypic cultures of embryonic VM:

The effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by developmental age (Fig. 4, ??? Which panel do you mean??). It would be a good idea to write down the exact number and not only the result of the comparative statistics. From the figures the exact values and the SDs or SEMs cannot be seen. Also say exactly to the previously defined groups.

Discussion:

Please use sometimes instead of “indeed".

Please correct: Perhaps not surprisingly, we found that in organotypic cultures, susceptibility of DN to AMPA-mediated excitotoxicity increases with age and differentiation status, such that the more complex, larger DN that become more common as the tissue matures, are much more susceptible than the embryonic-appearing, small and round neurons more common earlier in development (Fig. 3??? ). A or B?? Please add exactly.

Please add the references after these sentence: We found that the effects of 1-EBIO on DN survival following excitotoxicity were complex, but largely consistent with the expected neuroprotection. The contrasting results between our current and previous experiments (…?? please add the references). What are the previous ones ?

Did you mentioned these results also in the Results part of the manuscript? Interestingly, SK3 activation in younger cultures led to an increase in TH expressing neurons, in the absence of any susceptibility to AMPA-induced toxicity; this suggests that SK3 channel expression precedes the ability of DN to trigger the programmed cell death induced by AMPA (de Erausquin et al., 2003; Dorsey et al., 2006). Indeed, at DIV exposure to increasing concentrations of 1-EBIO yielded an inverted U-shaped curve, with an optimum effect at 30 μM and no change in the number of TH+/SK3+ neurons after exposure to 100 μM. The lack of effect at the higher concentration was also seen at DIV15, where 30 μM was clearly neuroprotective but 100 μM had no effect. On the other hand, at DIV22 exposure of the cultures to 30 μM 1-EBIO did not afford any protection, whereas 100 μM did, suggesting a shift to the right in the concentration response curve.

KCNN3, the gene that encodes SK3. is mutated in some families in association with chronic psychosis (Soden et al., 2013): Please write “KCNN3” in “italic” for gene.

Please come up with a conclusio at the ned.

Figure Legends:

Please write in the Fig. 2 and Fig. 3: (A), (B) instead of A, B.

Please use in all Figures: ± SEM instead of +/- SEM.

Figure 4: The Legend should be rewritten!! It is not understandable and it is not possible to see in the Fig. 4 the different incubation conditions: Cultures were incubated with 1-EBIO alone (30-100μM), AMPA alone (100 μM), or 1- EBIO (30-100μM) plus AMPA (100 μM) for 16 hr at DIV 8, DIV 15, and DIV 22. Please include all these conditions into layout of the Fig. 4.

Supplementary Fig. 1: Please describe the legend in full, not refering to <fig 4=""></fig>

Reviewer #2: The manuscript describes the protective effects of a SK3 enhancer in an organotypic culture model of AMPA toxicity.

The experimental model is very artifactual. If it can be used to study basic, molecular properties of neurons that can endure dissection and long-term culturing, it is questionable whether this in vitro system is able to model normal neurodevelopment and associated pathological conditions.

Materials and methods are sufficiently clear and detailed.

Results are very limited and sometimes contradictory.

One example: why 1-EBIO is protective at 30 and not 100 uM at one stage and the other way at later stage?

Also, the paper suffers from a number of limits that make it unsuitable for publication at this stage.

1) the quality of language, both in terms of english form and logic presentation of concepts is very poor.

2) the abstract lacks an introductory section which is normally required for the reader to understand the aim of the study.

3) It is unclear the rationale linkfing AMPA toxicity and neurological disorder such as schizophrenia as, according to the current comprehension of the disease, there is no neurodevelopmental neuronal loss linked to the disease.

4) the quality of images in immunostaining figures is not acceptable.

5) the SK3 staining shows very high background. I find it hard to believe that the signal is specific.

The discussion is too long for such a scarce amount of data. Finally, it contains some useful background information that should be moved forward into the intro.

Reviewer #3: In this paper, Authors aimed to investigate age-dependent neuroprotective effects of a conductance calcium activated potassium channel 3 (SK3) agonist, 1-EBIO, on AMPA excitoxicity to dopaminergic neurons (DN) in organotypic cultures. They claimed that exposure of organotypic cultures to AMPA (100 μm, 16 h) had no effect on the survival of DN+ at DIV 8, but caused significant toxicity at DIV 15. Moreover, immature DN+ (small, bipolar) were increased after AMPA (100 μm, 16 h) at DIV 8. This effect was more significant at DIV 15 and at DIV 22. Administration of 30 μM 1-EBIO resulted in a large increase in DN+ at DIV 8 and in AMPA toxicity prevention at DIV 15, while no effects on excitotoxicity were observed at DIV 22. Different results were observed with administarion of 100 um of the same compound, therefore suggesting that its effects were concentration-dependent and influenced by neuronal developmental stage.

I found this paper interesting. However, there are some major concerns that Authors should address before it can be reconsidered for publication:

1. In the Introduction section, the general and specific aims of the study should be better presented.

2. References should be provided for the dissection procedure (Materials and Methods section).

3. The same as above for the description of cell culture (Materials and Methods section).

4. References should be also provided for the doses of the pharmacological treatment used in this study.

5. For section Immunohistochemistry and Immunofluorence, Authors should detail the validation process of the antibody they used, especially with respect to the use of negative controls.

6. References should be provided for section Stereology and assessment of DN survival.

7. Authors should include the F values obtained for each performed ANOVA (in the description of results or in the figure legends).

8. The graphical quality of Images include in Fig.1 should be improved.

9. The quality of Figure 3 should be improved, especially for panel B.

10. I found the discussion quite fragmented and, in some of its part, a repetition of the obtained results. Therefore, Authors should revise this section, better discussing their results, also in the light of the existing literature in the field, better elaborating on the novelty of their findings.</fig>

<fig 4="">**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Andreas Wree

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

</fig>

PLoS One. 2020 Jul 23;15(7):e0223633. doi: 10.1371/journal.pone.0223633.r002

Author response to Decision Letter 0

3 Apr 2020

Please find our replies to the comments in the attached file.

Attachment

Submitted filename: rebuttal fo reviewers.docx

Click here for additional data file.^{(25.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0223633.r003

Decision Letter 1

Faramarz Dehghani

17 Apr 2020

PONE-D-19-26792R1

Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture

PLOS ONE

Dear Dr de Erausquin,

We would appreciate receiving your revised manuscript by Jun 01 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Faramarz Dehghani

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Dear Dr. de Erausquin,

I have now received the comments from the reviewers on the revised version of your manuscript. Since reviewer 3 was satisfied with the changes made both other reviewers still have substantial concerns.

You will find the detailed comments below. I share the points of criticism but encourage you to address all points accordingly and thoroughly revise the manuscript.

Best regards,

Faramarz Dehghani

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: This manuscript was substantively improved after revision.

The main massage of the study became substabtionally clearer.

Unfortunately, authors provided not all considerable corrections and therefore, this manuscript should be further improved and corrected before publishing.

There are still a lot of many "minors":

- Minor spell check required.

- Please indicate all references according to PlosOne.

- Please say “SK3” and check and correct once again in whole manuscript.

One example in Abstract: “The effects of the SK3. channel agonist 1-EBIO on the survival of SK3.- expressing dopaminergic neurons were concentration-dependent and influenced by neuronal developmental stage”. one more: in Results section: “SK3. channels are expressed in DN organotypic cultures”.

- Abstract: Please structure your Abstract section according to PlosOne: The Abstract section should include a separate parts: Background; Methods; Results; Conclusion. Already before, it was a proposal from me to design your manuscript according to PlosOne.

- Introduction: The Introduction section is still too short to describe the situation of the appropriate literature and the results of the previous publications. The Introduction section should be once again improve. Please describe more exactly why you selected an AMPA-model for your study?

- Please add more exactly the results of the Reference 3. Please describe more exactly and add to Introduction the description and the results of the References 10, 14,15.

- Pharmacological treatments: You said: “Cultures used for SK3 channel activation experiments were exposed to 1-ethyl-2-benzimidazolinone (1-EBIO) (30, 100 μM), and (AMPA) (100 μM) for 16 hours (DIV 7, 14, 21) in the following treatment groups: 1-EBIO 30 μM, 1-EBIO 100 μM, 1-EBIO 30 μM plus AMPA 100 μM, 1-EBIO 100 μM plus AMPA 100 μM, and AMPA 100 μM”.

Please add, why you used this appropriate doses and volumes?

- Protection conferred by SK3 channel activation is developmental stage-dependent in organotypic cultures of embryonic VM: Please say: (Fig. 4, panel A); (Fig. 4, panel B); (Fig. 4, panel C) like as above. Please use the same abbreviations style in whole text, all Figures and Legends.

- Discussion: Please add more references at the end of this sentence:” Dopamine neuron susceptibility to excitotoxic injury is potentially relevant to a number of pathological conditions, and therefore so is the development of potential neuroprotective strategies, including the use of pharmacological modulators of SK3 channel function”.

- You said: “This is consistent with observations in other models, including susceptibility to rotenone-induced toxicity (21). Please add more exactly description of the study and results of the Reference 21.

- You said: “Overall, AMPA did not induce cell death in DN at 8 days in vitro, but toxicity became apparent at 15 DIV and most intense at 22 DIV”. Please add to discussion, what you think about that. This - I think - is very essential in the message of the study.

- You said: “We previously showed that 1-EBIO effectively protects DN in primary cultures of ventral mesencephalon against AMPA-mediated toxicity by sustaining SK3 channel conductance (3). Please add more exactly description of the study and results of the Reference 3.

- You said:” Our data are also consistent with the neuroprotective effect of SK channel activation in cultured human postmitotic dopaminergic neurons in vitro following rotenone treatment (23). Please add more exactly description of the study and results of the Reference 23.

- You said: “The contrasting results between our current experiments and those reported by us previously in primary cultures of VM (3)”. Please add more exactly description of the study and results of the Reference 3.

- Please add more references at the end of this sentence: “Our observations are consistent with findings of the effects of SK3 channel agonists in intact animals“.

- Conclusion

The Conclusion section is too short. Please add the meaning of your method and your findings for experimental research and clinical practice. Please add also to Conclusion section the future perspectives.

- Figures: The quality of Figures 1 and 3 is still not acceptable for publishing. They should be substantial once more improved.

- Figure 1: Please indicate the panels A, B and C directly in your Figure 1.

Please indicate the DIV8, DIV15 and DIV22 directly in your Figure 1, like as Figures 2 and 3.

- Figure 4: Please indicate the panels A, B and C directly in your Figure 4.

Figure Legends:

Fig. 1:

Please indicate the panels instead of a, b, c like as A, B, C, the same with text and images and in other Legends.

Reviewer #2: The manuscript entitled "Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture" was, in the initial version, unsuited for publication due to, primarily, an unacceptable number of typos, imprecisions and omissions in the text and general lack of accuracy.

The modest improvements made in this resubmission are by all means insufficient. In fact, the manuscript is still flawed by many of the serious shortcomings that all reviewers pointed out in the previous version.

If such generalized untidiness is regrettable in a first submission, I find it intolerable in a resubmission.

A) Major issues

Abstract: line 1, “an small conductance (..a small…)

Introduction:

1) Line 2, a bracket is missing in the citation.

2) Line 5, “…risk of schizophrenia in some samples…”. Maybe the author meant “examples”?

3) Line 6, “To further understanding of the determinants” (…understand the…).

4) Line 16, a number is missing in the citation in brackets.

5) Line 18, “Organotypic cultures that…”. “That” should be removed.

Results

1) Title: “SK3. channels…”. The abbreviation SK3 is still “SK3.” here and in many spots throughout the text. Again, this was incomprehensible in the first version, it is unacceptable after this issue was explicitly raised by reviewers.

2) 1st paragraph, line 4, “panel A”. There is no panel A, B or C in figure 1, only three pictures in a left to right row.

3) Page 11, line 3: AMPA-receptor-mediated. There is one too many hyphens.

4) Page 11, line 4, “DIV8 treated…”. Treated at DIV8 would be more correct.

5) Page 13, line 16, “…leading activation…” (…leading to the activation…)

6) Page 14, line 9, “At DIV…”. 8? 15? 22? Please specify.

7) Page 16, Conclusion. The section was clearly added simply to meet the journal required formta, but a 2-line period is not acceptable.

8) Acknowledgements: if, as I understand, no acknowledgements are to be made, please insert at least one sentence stating this.

References: SK3. is abundantly present throughout.

Legends and relative figures

1) Figure 1. There are no A, B and C letters in the figure. The resolution of the pictures is very low, so that individual, large pixels can easily be distinguished in paper and on screen. What is the vertical “background” writing on the right? Last, but not least, there is no scale bar in the photographs, although it is mentioned at the end of the legend.

2) Figure 3, the resolution is insufficient, the appearance of the text is very blurred in both panels A and B

3) Figure 4. There are no A, B or C letters in the panels of figure 4. Line 4 of legend, …the y axes…(They are actually the x axes).

B) Minor issues: beyond form, that makes it unacceptable, the MS still suffers from many of the scientific and conceptual shortcomings raised by reviewers.

1) Abstract. The abstract was minimally revised. In fact, it still lacks an overview of the field and the scientific question the MS aims to address. This is indispensable in the abstract of a scientific publication. Instead, the 2nd sentence reports an unnecessary characterization of the model system.

2) Discussion. Page 13, second paragraph; Stage-dependent susceptibility to AMPA toxicity, the underpinnings of which are not discussed in any detail here, is likely due to progressive maturation of excitatory synapses and/or intracellular AMPA signals. Susceptibility to rotenone, a mitochondrial poison, does not seem a useful example of similarity. Rather, I find it confounding.

3) Page 13-14. Neuroprotection afforded by 1-EBIO (only hypothetically via activation of SK3 channels as there is no direct demonstration of K current enhancement in the model) is indeed complex, not to say hardly intelligible. One result stands out that has been inadequately discussed and that conflicts with the authors interpretation of the link between AMPA toxicity and 1-EBIO-dependent neuroprotection: 30 uM 1-EBIO increases the number of TH+/SK3+ neurons at DIV 8 in the absence of toxic AMPA effects (fig 4, top). This suggests a neurotrophic action of 1-EBIO treatment, at least at this stage. Consistently, the neuroprotection afforded 1-EBIO in other conditions may be, at least in part, explained by this effect and, thus, not solely a counteraction of AMPA activation/signaling.

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

PLoS One. 2020 Jul 23;15(7):e0223633. doi: 10.1371/journal.pone.0223633.r004

Author response to Decision Letter 1

3 May 2020

Comment: “The main massage of the study became substantially clearer.”

Reply: We appreciate the encouragement.

Comment: Unfortunately, authors provided not all considerable corrections and therefore, this manuscript should be further improved and corrected before publishing. There are still a lot of many "minors"

Reply: We will address them individually.

Comment: Minor spell check required.

Reply: We thoroughly spell checked the manuscript.

Comment: Please indicate all references according to PlosOne.

Reply: References were individually and manually edited according to PlOS One guidelines.

Comment: Please say “SK3” and check and correct once again in whole manuscript.

Reply: We have corrected the abbreviation where needed.

Comment: Abstract: Please structure your Abstract section according to PlosOne: The Abstract section should include a separate parts: Background; Methods; Results; Conclusion. Already before, it was a proposal from me to design your manuscript according to PlosOne.

Reply: We apologize about missing the comment the first time. We have formatted the Abstract as required.

Comment: Introduction: The Introduction section is still too short to describe the situation of the appropriate literature and the results of the previous publications. The Introduction section should be once again improved. Please describe more exactly why you selected an AMPA-model for your study?

Reply: As requested, we have expanded the rationale for the use of this model. Of note, the laboratory of the corresponding author established this model two decades ago, and has contributed significantly to the literature on the topic.

Comment: Please add more exactly the results of the Reference 3. Please describe more exactly and add to Introduction the description and the results of the References 10, 14,15.

Reply: We respectfully disagree. The present manuscript is not a review. The requested results are already sufficiently described in great detail in the referenced manuscripts by our own group, and any reader can satisfy their questions by seeking out those references. Further repetition here would detract from the main purpose of this original publication.

Comment: Pharmacological treatments: You said: “Cultures used for SK3 channel activation experiments were exposed to 1-ethyl-2-benzimidazolinone (1-EBIO) (30, 100 μM), and (AMPA) (100 μM) for 16 hours (DIV 7, 14, 21) in the following treatment groups: 1-EBIO 30 μM, 1-EBIO 100 μM, 1-EBIO 30 μM plus AMPA 100 μM, 1-EBIO 100 μM plus AMPA 100 μM, and AMPA 100 μM”.

Please add, why you used this appropriate doses and volumes?

Reply: The doses were chosen based on recommendations from manufacturers or previously published data by our own and other relevant groups. We have added this sentence to the manuscript.

Comment: Protection conferred by SK3 channel activation is developmental stage-dependent in organotypic cultures of embryonic VM: Please say: (Fig. 4, panel A); (Fig. 4, panel B); (Fig. 4, panel C) like as above. Please use the same abbreviations style in whole text, all Figures and Legends.

Reply: We corrected the requested format.

Comment: Discussion: Please add more references at the end of this sentence:” Dopamine neuron susceptibility to excitotoxic injury is potentially relevant to a number of pathological conditions, and therefore so is the development of potential neuroprotective strategies, including the use of pharmacological modulators of SK3 channel function”.

Reply: We added three references as requested.

Comment: You said: “This is consistent with observations in other models, including susceptibility to rotenone-induced toxicity (21). Please add more exactly description of the study and results of the Reference 21.

Reply: The relevant findings described in reference 21 have been added to the discussion text.

Comment: You said: “Overall, AMPA did not induce cell death in DN at 8 days in vitro, but toxicity became apparent at 15 DIV and most intense at 22 DIV”. Please add to discussion, what you think about that. This - I think - is very essential in the message of the study.

Reply: We have added two paragraphs to the discussion to address this comment.

Comment: You said: “We previously showed that 1-EBIO effectively protects DN in primary cultures of ventral mesencephalon against AMPA-mediated toxicity by sustaining SK3 channel conductance (3). Please add more exactly description of the study and results of the Reference 3.

Reply: We have added several paragraphs expounding the findings of our previous publication (reference 3).

Comment: You said:” Our data are also consistent with the neuroprotective effect of SK channel activation in cultured human postmitotic dopaminergic neurons in vitro following rotenone treatment (23). Please add more exactly description of the study and results of the Reference 23.

Reply: We have added a paragraph with the description of the study referenced.

Comment: You said: “The contrasting results between our current experiments and those reported by us previously in primary cultures of VM (3)”. Please add more exactly description of the study and results of the Reference 3.

Reply: The results from our previous publication are now extensively discussed in previous paragraphs.

Comment: Please add more references at the end of this sentence: “Our observations are consistent with findings of the effects of SK3 channel agonists in intact animals“.

Reply: All of the available studies are referenced. We regret to say that no other data are available, but this is why our paper is relevant…

Comment: The Conclusion section is too short. Please add the meaning of your method and your findings for experimental research and clinical practice. Please add also to Conclusion section the future perspectives.

Reply: We have expanded the conclusion as requested.

Comment: Figures: The quality of Figures 1 and 3 is still not acceptable for publishing. They should be substantial once more improved.

Reply: The figures are very high quality and passed the quality tests by PlOS One. We wonder if the reviewer is looking at the uploaded tiff files or instead referencing the jpg transformations inside the manuscript?

Comments: Figure 1: Please indicate the panels A, B and C directly in your Figure 1. Please indicate the DIV8, DIV15 and DIV22 directly in your Figure 1, like as Figures 2 and 3. Figure 4: Please indicate the panels A, B and C directly in your Figure 4. Figure Legends: Fig. 1: Please indicate the panels instead of a, b, c like as A, B, C, the same with text and images and in other Legends.

Reply: We have corrected the figures and legends as suggested.

Comment: Reviewer #2: The manuscript entitled "Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture" was, in the initial version, unsuited for publication due to, primarily, an unacceptable number of typos, imprecisions and omissions in the text and general lack of accuracy. The modest improvements made in this resubmission are by all means insufficient. In fact, the manuscript is still flawed by many of the serious shortcomings that all reviewers pointed out in the previous version. If such generalized untidiness is regrettable in a first submission, I find it intolerable in a resubmission.

Reply: Dear Editor, the corresponding author for this manuscript is a PlOS One Academic Editor and reviews frequently for other highly ranked journals. I find the tone and content of this comment unprofessional, inaccurate and offensive. To state that a typo (“an” for “a”) in an abstract, or the lack of a bracket in a citation are “major issues” is flatly outrageous and not in keeping with review guidelines or policy by PlOS One. In spite of this problem, and for the sake of facilitating publication and editorial review, we have corrected each and everyone of the very minor issues raised by this reviewer, and reply to each in the following paragraphs.

Comment: Abstract: line 1, “an small conductance (..a small…)

Reply: The full sentence read: “an small conductance … agonist” and was grammatically correct. Nonetheless, we have changed it to read “an SK3 agonist”, to make it better sounding.

Comment: 1) Line 2, a bracket is missing in the citation.

Reply: We added the bracket.

Comment: 2) Line 5, “…risk of schizophrenia in some samples…”. Maybe the author meant “examples”?.

Reply: No, we meant samples of patients with schizophrenia. We corrected it to “patient samples” to clarify the meaning of the sentence.

Comment: 3) Line 6, “To further understanding of the determinants” (…understand the…).

Reply: The sentence is grammatically correct and uses the gerund “understanding” as a form that is derived from a verb but that functions as a noun.

Comment: 4) Line 16, a number is missing in the citation in brackets.

Reply: We added the missing bracket.

Comment: 5) Line 18, “Organotypic cultures that…”. “That” should be removed.

Reply: The reviewer is correct, and we removed the word “That” from the sentence.

Comment: 1) Title: “SK3. channels…”. The abbreviation SK3 is still “SK3.” here and in many spots throughout the text. Again, this was incomprehensible in the first version, it is unacceptable after this issue was explicitly raised by reviewers.

Reply: We have corrected the usage of SK3 throughout the manuscript.

Comment: 2) 1st paragraph, line 4, “panel A”. There is no panel A, B or C in figure 1, only three pictures in a left to right row.

Reply: We have corrected the figures as requested by reviewer #1.

Comment: 3) Page 11, line 3: AMPA-receptor-mediated. There is one too many hyphens.

Reply: We removed the second hyphen.

Comment: 4) Page 11, line 4, “DIV8 treated…”. The sentence is no longer in the text of the manuscript.

Comment: 5) Page 13, line 16, “…leading activation…” (…leading to the activation…)

Reply: The reviewer is correct and the sentence was changed accordingly.

Comment: 6) Page 14, line 9, “At DIV…”. 8? 15? 22? Please specify.

Reply: It should have read At DIV8. The typo was corrected (thank you).

Comment: 7) Page 16, Conclusion. The section was clearly added simply to meet the journal required formta, but a 2-line period is not acceptable.

Reply: As requested by reviewer #1 also, we have expanded the conclusion.

Comment: 8) Acknowledgements: if, as I understand, no acknowledgements are to be made, please insert at least one sentence stating this.

Reply: We added the requested sentence.

Comment: References: SK3. is abundantly present throughout.

Reply. The usage has been corrected throughout.

Comment: 1) Figure 1. There are no A, B and C letters in the figure. The resolution of the pictures is very low, so that individual, large pixels can easily be distinguished in paper and on screen. What is the vertical “background” writing on the right? Last, but not least, there is no scale bar in the photographs, although it is mentioned at the end of the legend. 2) Figure 3, the resolution is insufficient, the appearance of the text is very blurred in both panels A and B. 3) Figure 4. There are no A, B or C letters in the panels of figure 4. Line 4 of legend, …the y axes… (They are actually the x axes).

Reply: As in the comment from reviewer #1, I wonder if the reviewers are looking at the pdf file instead of at the uploaded tiff individual files? The figures are highest quality and passed the PlOS One quality test. We fixed the other issues raised by the comment.

Comment: 1) Abstract. The abstract was minimally revised. In fact, it still lacks an overview of the field and the scientific question the MS aims to address. This is indispensable in the abstract of a scientific publication. Instead, the 2nd sentence reports an unnecessary characterization of the model system.

Reply: The abstract has been thoroughly re-written.

Comment: 2) Discussion. Page 13, second paragraph; Stage-dependent susceptibility to AMPA toxicity, the underpinnings of which are not discussed in any detail here, is likely due to progressive maturation of excitatory synapses and/or intracellular AMPA signals. Susceptibility to rotenone, a mitochondrial poison, does not seem a useful example of similarity. Rather, I find it confounding.

Reply: This aspect of the discussion has been expanded following suggestions from reviewer #1 as well. We agree with the reviewer on the likely molecular mechanism of the age-dependent effects.

Comment: 3) Page 13-14. Neuroprotection afforded by 1-EBIO (only hypothetically via activation of SK3 channels as there is no direct demonstration of K current enhancement in the model) is indeed complex, not to say hardly intelligible. One result stands out that has been inadequately discussed and that conflicts with the authors interpretation of the link between AMPA toxicity and 1-EBIO-dependent neuroprotection: 30 uM 1-EBIO increases the number of TH+/SK3+ neurons at DIV 8 in the absence of toxic AMPA effects (fig 4, top). This suggests a neurotrophic action of 1-EBIO treatment, at least at this stage. Consistently, the neuroprotection afforded 1-EBIO in other conditions may be, at least in part, explained by this effect and, thus, not solely a counteraction of AMPA activation/signaling.

Reply: The direct effects of AMPA current modulation on dopaminergic neuron survival and on the mechanism of action of SK3 agonists was extensively demonstrated in our previous work (Benitez et al). We have included those results in greater detail in the current version of the discussion.

Attachment

Submitted filename: PlOS reviewers comments and rebuttal(1).docx

Click here for additional data file.^{(9.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0223633.r005

Decision Letter 2

Faramarz Dehghani

2 Jun 2020

PONE-D-19-26792R2

Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture

PLOS ONE

Dear Dr. de Erausquin,

Please submit your revised manuscript by Jul 17 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Faramarz Dehghani

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: This manuscript was very substantively improved after both major revisions.

Unfortunately, the authors provided once again not all considerable corrections and furthermore, some problems emerged acutely during the last revision by authors.

Therefore, this manuscript need once more a bit mprovement and corrections before publishing. I hope, then eberything will fine.

Correction points:

Abstract

Authors said: Background: Small conductance, calcium-activated (SK3) potassium channels control the intrinsic excitability of dopaminergic neurons (DN) in the midbrain and modulate their susceptibility to toxic insults during development.

Please use another sentence, this sentence is absolutely the same with the first sentence in your Introduction section.

Materials and Methods

Animals and Dissections

Authors said: Timed pregnant female Sprague-Dawley rats (Charles River Laboratories, MA, USA) were euthanized by exposure to CO2 on day 14 of gestation.

Please add the total number of pregnant female Sprague-Dawley rats used, (n=?).

Pharmacological treatment

Authors said: All drug concentrations were chosen based on recommendations from manufacturers or previously published data by our own and other relevant groups.

Please add the corresponding references for appropriate previously published data by yourself and others.

Discussion

Authors said: These findings are consistent with knowledge about the expression of AMPA receptors in DN in VM or rodents.

Please add references at the end of this sentence.

Authors said: Additionaly susceptibility of differentiated DN from the human cell line SH-SY5Y is also increased by time in vitro (37).

Please write out: SH-SY5Y. By the way, please correct “Additionaly” to “Additionally”.

Authors said: Our observations are consistent with findings of the effects of SK3 channel agonists in intact animals.

Please add references at the end of this sentence.

Figure 1

Please indicate the DIV8, DIV15 and DIV22 directly in your Figure 1, like as Figures 2 and 3.

Figure 4

Please indicate the panels A, B and C directly in your Figure 4.

Figure Legends

Fig. 1:

Please indicate the panels instead of a, b, c like as A, B, C, the same with text and images and in other Legends.

Please correct two time “anti-SK3.” to “anti-SK3”

Please see both sentences: Images show examples of stained neurons at DIV 8 (panel a), DIV 15 (panel b) and DIV 22 (panel c) merged color images show high co-localization for anti-TH (red) and anti-SK3. (green) antibodies. At each DIV there are also a number of cells that are positive for anti-SK3., but negative for anti-TH. At DIV 8 DNs were typically bipolar with small fusiform shaped somas and displayed little branching.

Fig. 3:

Please delete from the legends: *p < 0.05, **p < 0.01, ***p <0.001. These p values and asterisks were not used in the Figure 3.

**********

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PLoS One. 2020 Jul 23;15(7):e0223633. doi: 10.1371/journal.pone.0223633.r006

Author response to Decision Letter 2

5 Jul 2020

Reply to Reviewer’s comments

Comment: “Please use another sentence, this sentence is absolutely the same with the first sentence in your Introduction section”.

Reply: Thank you for pointing this out. We re-wrote the introduction.

Comment: Please add the total number of pregnant female Sprague-Dawley rats used, (n=?).

Reply: we added the requested number

Comment: Please add the corresponding references for appropriate previously published data by yourself and others.

Reply: references numbers were added as requested

Comment:Please add references at the end of this sentence.

Reply: reference number was added as requested

Comment: Please write out: SH-SY5Y.

Reply: The code proided is the only name used for that cell line, not an abbreviation. There are multiple references (included the one we provide), but see for instance https://www.sigmaaldrich.com/catalog/product/sigma/cb_94030304?lang=en&region=US&gclid=EAIaIQobChMIiJizttmx6gIVEL7ACh10qAQdEAAYASAAEgIbWvD_BwE

We clarified that it is a human neuroblastoma cell line.

Comment: By the way, please correct “Additionaly” to “Additionally”.

Reply: Corrected as noted. Thank you.

Comment: Please add references at the end of this sentence.

Reply: Added the reference as requested.

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PLoS One. doi: 10.1371/journal.pone.0223633.r007

Decision Letter 3

Faramarz Dehghani

8 Jul 2020

Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture

PONE-D-19-26792R3

Dear Dr. de Erausquin,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

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6. Review Comments to the Author

Reviewer #1: (No Response)

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PLoS One. doi: 10.1371/journal.pone.0223633.r008

Acceptance letter

Faramarz Dehghani

10 Jul 2020

PONE-D-19-26792R3

Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture

Dear Dr. de Erausquin:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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PLOS ONE Editorial Office Staff

on behalf of

Dr. Faramarz Dehghani

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PLOS ONE

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[pone.0223633.ref001] 1.Ji H., Hougaard C., Herrik K.F., Strøbaek D., Christophersen P., Shepard P.D., 2009. Tuning the excitability of midbrain dopamine neurons by modulating the Ca2+ sensitivity of SK channels. Eur. J. Neurosci. 29, 1883–1895. 10.1111/j.1460-9568.2009.06735.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref002] 2.Wolfart J., Neuhoff H., Franz O., Roeper J., 2001. Differential expression of the small-conductance, calcium-activated potassium channel SK3 is critical for pacemaker control in dopaminergic midbrain neurons. J. Neurosci. 21, 3443–3456. 10.1523/JNEUROSCI.21-10-03443.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref003] 3.Benítez B.A., Belálcazar H.M., Anastasía A., Mamah D.T., Zorumski C.F., Mascó D.H., et al. , 2011. Functional reduction of SK3-mediated currents precedes AMPA-receptor mediated excitotoxicity in dopaminergic neurons. Neuropharmacology 60, 1176–1186. 10.1016/j.neuropharm.2010.10.024 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref004] 4.Soden M.E., Jones G.L., Sanford C.A., Chung A.S., Güler A.D., Chavkin C., et al. , 2013. Disruption of dopamine neuron activity pattern regulation through selective expression of a human KCNN3 mutation. Neuron 80, 997–1009. 10.1016/j.neuron.2013.07.044 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref005] 5.Tomita H., Shakkottai V.G., Gutman G.A., Sun G., Bunney W.E., Cahalan M.D., et al. , 2003. Novel truncated isoform of SK3 potassium channel is a potent dominant-negative regulator of SK currents: implications in schizophrenia. Mol. Psychiatry 8, 524–535, 460. 10.1038/sj.mp.4001271 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref006] 6.Grube S., Gerchen M.F., Adamcio B., Pardo L.A., Martin S., Malzahn D., et al. , 2011. A CAG repeat polymorphism of KCNN3 predicts SK3 channel function and cognitive performance in schizophrenia. EMBO Mol Med 3, 309–319. 10.1002/emmm.201100135 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref007] 7.Ivković M., Ranković V., Tarasjev A., Orolicki S., Damjanović A., Paunović V.R., et al. , 2006. Schizophrenia and polymorphic CAG repeats array of calcium-activated potassium channel (KCNN3) gene in Serbian population. Int. J. Neurosci. 116, 157–164. 10.1080/00207450341514 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref008] 8.Ritsner M., Amir S., Koronyo-Hamaoui M., Gak E., Ziv H., Halperin T., et al. , 2003. Association study of CAG repeats in the KCNN3 gene in Israeli patients with major psychosis. Psychiatr. Genet. 13, 143–150. 10.1097/00041444-200309000-00002 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref009] 9.Ritsner M., Modai I., Ziv H., Amir S., Halperin T., Weizman A., et al. , 2002. An association of CAG repeats at the KCNN3 locus with symptom dimensions of schizophrenia. Biol. Psychiatry 51, 788–794. 10.1016/s0006-3223(01)01348-8 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref010] 10.Branton RL, Clarke DJ. Apoptosis in primary cultures of E14 rat ventral mesencephala: time course of dopaminergic cell death and implications for neural transplantation. Exp Neurol. 1999. November;160(1):88–98. 10.1006/exnr.1999.7207 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref011] 11.Falkenburger BH, Schulz JB. Limitations of cellular models in Parkinson’s disease research. J Neural Transm Suppl. 2006;(70):261–8. 10.1007/978-3-211-45295-0_40 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref012] 12.Alavian KN, Scholz C, Simon HH. Transcriptional regulation of mesencephalic dopaminergic neurons: the full circle of life and death. Mov Disord. 2008. February 15;23(3):319–28. 10.1002/mds.21640 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref013] 13.Greco D, Volpicelli F, Di Lieto A, Leo D, Perrone-Capano C, Auvinen P, et al. Comparison of gene expression profile in embryonic mesencephalon and neuronal primary cultures. PLoS ONE. 2009;4(3):e4977 10.1371/journal.pone.0004977 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref014] 14.Chiodo LA, Kapatos G. Membrane properties of identified mesencephalic dopamine neurons in primary dissociated cell culture. Synapse. 1992. August;11(4):294–309. 10.1002/syn.890110405 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref015] 15.Liss B, Franz O, Sewing S, Bruns R, Neuhoff H, Roeper J. Tuning pacemaker frequency of individual dopaminergic neurons by Kv4.3L and KChip3.1 transcription. EMBO J. 2001. October 15;20(20):5715–24. 10.1093/emboj/20.20.5715 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref016] 16.Neuhoff H, Neu A, Liss B, Roeper J. I(h) channels contribute to the different functional properties of identified dopaminergic subpopulations in the midbrain. J Neurosci. 2002. February 15;22(4):1290–302. 10.1523/JNEUROSCI.22-04-01290.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref017] 17.Seutin V, Massotte L, Renette MF, Dresse A. Evidence for a modulatory role of Ih on the firing of a subgroup of midbrain dopamine neurons. Neuroreport. 2001. February 12;12(2):255–8. 10.1097/00001756-200102120-00015 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref018] 18.de Erausquin G., Brooker G., Costa E., Hanbauer I., 1994. Persistent AMPA receptor stimulation alters [Ca2+]i homeostasis in cultures of embryonic dopaminergic neurons. Brain Res. Mol. Brain Res. 21, 303–311. 10.1016/0169-328x(94)90261-5 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref019] 19.de Erausquin G.A., Hyrc K., Dorsey D.A., Mamah D., Dokucu M., Mascó D.H., et al. , 2003. Nuclear translocation of nuclear transcription factor-kappa B by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors leads to transcription of p53 and cell death in dopaminergic neurons. Mol. Pharmacol. 63, 784–790. 10.1124/mol.63.4.784 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref020] 20.Isaacs KR, de Erausquin G, Strauss KI, Jacobowitz DM, Hanbauer I. Differential effects of excitatory amino acids on mesencephalic neurons expressing either calretinin or tyrosine hydroxylase in primary cultures. Brain Res Mol Brain Res. 1996. February;36(1):114–26. 10.1016/0169-328x(95)00252-n [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref021] 21.Dorsey D.A., Mascó D.H., Dikranian K., Hyrc K., Masciotra L., Faddis B., et al. , 2006. Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons. Apoptosis 11, 535–544. 10.1007/s10495-006-5268-y [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref022] 22.Burke RE. Ontogenic cell death in the nigrostriatal system. Cell Tissue Res. 2004. October;318(1):63–72. 10.1007/s00441-004-0908-4 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref023] 23.de Erausquin G.A., Hanbauer I. (1995) AMPA Receptor-Induced Dopaminergic Cell Death: A Model for the Pathogenesis of Hypofrontality and Negative Symptoms in Schizophrenia In: Mednick S.A., Hollister J.M. (eds) Neural Development and Schizophrenia. NATO ASI Series (Series A: Life Sciences), vol 275 Springer, Boston, MA [Google Scholar]

[pone.0223633.ref024] 24.Lilliu V, Pernas-Alonso R, Trelles RD, di Porzio U, Zuddas A, Perrone-Capano C. Ontogeny of AMPA receptor gene expression in the developing rat midbrain and striatum. Brain Res Mol Brain Res. 2001. November 30;96(1–2):133–41. 10.1016/s0169-328x(01)00280-7 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref025] 25.Marani E, Heida T, Lakke EAJF, Usunoff KG. The subthalamic nucleus. Part I: development, cytology, topography and connections. Adv Anat Embryol Cell Biol. 2008;198:1–113, vii. [PubMed] [Google Scholar]

[pone.0223633.ref026] 26.Lopes FM, Bristot IJ, da Motta LL, Parsons RB, Klamt F. Mimicking Parkinson's Disease in a Dish: Merits and Pitfalls of the Most Commonly used Dopaminergic In Vitro Models. Neuromolecular Med. 2017. September;19(2–3):241–255. 10.1007/s12017-017-8454-x [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref027] 27.Costantini LC, Lin L, Isacson O. Medial fetal ventral mesencephalon: a preferred source for dopamine neuron grafts. Neuroreport. 1997. July 7;8(9–10):2253–7 10.1097/00001756-199707070-00032 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref028] 28.Jaeger C, Gonzalo Ruiz A, Llinás R. Organotypic slice cultures of dopaminergic neurons of substantia nigra. Brain Res Bull. 1989. June;22(6):981–91 10.1016/0361-9230(89)90010-5 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref029] 29.Mouton PR, Phoulady HA, Goldgof D, Hall LO, Gordon M, Morgan D. Unbiased estimation of cell number using the automatic optical fractionator. J Chem Neuroanat. 2017. March;80:A1–A8. 10.1016/j.jchemneu.2016.12.002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref030] 30.Hoaglin D. C., and Iglewicz B. (1987), “Fine Tuning Some Resistant Rules for Outlier Labeling”, Journal of American Statistical Association., 82, 1147–1149. [Google Scholar]

[pone.0223633.ref031] 31.Noh W., Pak S., Choi G., Yang Sungchil, Yang,et al. , 2019. Transient Potassium Channels: Therapeutic Targets for Brain Disorders. Front Cell Neurosci 13, 265 10.3389/fncel.2019.00265 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref032] 32.Hipólito L, Fakira AK, Cabañero D, Blandón R, Carlton SM, Morón JA, et al. In vivo activation of the SK channel in the spinal cord reduces the NMDA receptor antagonist dose needed to produce antinociception in an inflammatory pain model. Pain. 2015. May;156(5):849–58. 10.1097/j.pain.0000000000000124 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref033] 33.Hopf FW, Bowers MS, Chang S-J, Chen BT, Martin M, Seif T, et al. Reduced nucleus accumbens SK channel activity enhances alcohol seeking during abstinence. Neuron. 2010. March 11;65(5):682–94. 10.1016/j.neuron.2010.02.015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref034] 34.Blank T, Nijholt I, Kye M-J, Spiess J. Small conductance Ca2+-activated K+ channels as targets of CNS drug development. Curr Drug Targets CNS Neurol Disord. 2004. June;3(3):161–7. 10.2174/1568007043337472 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref035] 35.Liebau S, Vaida B, Proepper C, Grissmer S, Storch A, Boeckers TM, et al. Formation of cellular projections in neural progenitor cells depends on SK3 channel activity. J Neurochem. 2007. June;101(5):1338–50. 10.1111/j.1471-4159.2006.04437.x [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref036] 36.Obermair GJ, Kaufmann WA, Knaus H-G, Flucher BE. The small conductance Ca2+-activated K+ channel SK3 is localized in nerve terminals of excitatory synapses of cultured mouse hippocampal neurons. Eur J Neurosci. 2003. February;17(4):721–31. 10.1046/j.1460-9568.2003.02488.x [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref037] 37.Sur M., Dey P., Sarkar A., Bar S., Banerjee D., Bhat S., et al. , 2018. Sarm1 induction and accompanying inflammatory response mediates age-dependent susceptibility to rotenone-induced neurotoxicity. Cell Death Discov 4, 114 10.1038/s41420-018-0119-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref038] 38.Tessner T.G., Rock C.O., Kalmar G.B., Cornell R.B., Jackowski S., 1991. Colony-stimulating factor 1 regulates CTP: phosphocholine cytidylyltransferase mRNA levels. J. Biol. Chem. 266, 16261–16264. [PubMed] [Google Scholar]

[pone.0223633.ref039] 39.Dolga A.M., Letsche T., Gold M., Doti N., Bacher M., Chiamvimonvat N., et al. , 2012. Activation of KCNN3/SK3./K(Ca)2.3 channels attenuates enhanced calcium influx and inflammatory cytokine production in activated microglia. Glia 60, 2050–2064. 10.1002/glia.22419 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref040] 40.Schlichter L.C., Kaushal V., Moxon-Emre I., Sivagnanam V., Vincent C., 2010. The Ca2+ activated SK3. channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro. J Neuroinflammation 7, 4 10.1186/1742-2094-7-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref041] 41.Thion M.S., Ginhoux F., Garel S., 2018. Microglia and early brain development: An intimate journey. Science 362, 185–189. 10.1126/science.aat0474 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref042] 42.Tacconi S., Carletti R., Bunnemann B., Plumpton C., Merlo Pich E., Terstappen G.C., 2001. Distribution of the messenger RNA for the small conductance calcium-activated potassium channel SK3. in the adult rat brain and correlation with immunoreactivity. Neuroscience 102, 209–215. 10.1016/s0306-4522(00)00486-3 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref043] 43.Sarpal D., Koenig J.I., Adelman J.P., Brady D., Prendeville L.C., Shepard P.D., 2004. Regional distribution of SK3. mRNA-containing neurons in the adult and adolescent rat ventral midbrain and their relationship to dopamine-containing cells. Synapse 53, 104–113. 10.1002/syn.20042 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref044] 44.Deignan J., Luján R., Bond C., Riegel A., Watanabe M., Williams J.T., et al. , 2012. SK2 and SK3. expression differentially affect firing frequency and precision in dopamine neurons. Neuroscience 217, 67–76. 10.1016/j.neuroscience.2012.04.053 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref045] 45.Dufour M.A., Woodhouse A., Goaillard J.-M., 2014. Somatodendritic ion channel expression in substantia nigra pars compacta dopaminergic neurons across postnatal development. J. Neurosci. Res. 92, 981–999. 10.1002/jnr.23382 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref046] 46.Dolga A.M., de Andrade A., Meissner L., Knaus H.-G., Höllerhage M., Christophersen P., et al. , 2014. Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons. Cell Death Dis 5, e999 10.1038/cddis.2013.530 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref047] 47.Herrik K.F., Redrobe J.P., Holst D., Hougaard C., Sandager-Nielsen K., Nielsen A.N., et al. , 2012. CyPPA, a Positive SK3./SK2 Modulator, Reduces Activity of Dopaminergic Neurons, Inhibits Dopamine Release, and Counteracts Hyperdopaminergic Behaviors Induced by Methylphenidate. Front Pharmacol 3, 11 10.3389/fphar.2012.00011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0223633.ref048] 48.Mourre C., Manrique C., Camon J., Aidi-Knani S., Deltheil T., Turle-Lorenzo N., et al. , 2017. Changes in SK channel expression in the basal ganglia after partial nigrostriatal dopamine lesions in rats: Functional consequences. Neuropharmacology 113, 519–532. 10.1016/j.neuropharm.2016.11.003 [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref049] 49.Jacobsen J.P.R., Weikop P., Hansen H.H., Mikkelsen J.D., Redrobe J.P., Holst D., et al. , 2008. SK3. K+ channel-deficient mice have enhanced dopamine and serotonin release and altered emotional behaviors. Genes Brain Behav. 7, 836–848. 10.1111/j.1601-183X.2008.00416.x [DOI] [PubMed] [Google Scholar]

[pone.0223633.ref050] 50.Dallérac G.M., Levasseur G., Vatsavayai S.C., Milnerwood A.J., Cummings D.M., Kraev I., et al. , 2015. Dysfunctional Dopaminergic Neurones in Mouse Models of Huntington’s Disease: A Role for SK3. Channels. Neurodegener Dis 15, 93–108. 10.1159/000375126 [DOI] [PubMed] [Google Scholar]

PERMALINK

Age-dependent neuroprotective effect of an SK3 channel agonist on excitotoxity to dopaminergic neurons in organotypic culture

Oscar Maldonado

Alexandra Jenkins

Helen M Belalcazar

Helena Hernandez-Cuervo

Katelynn M Hyman

Giannina Ladaga

Lucia Padilla

Gabriel A de Erausquin

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Materials and methods

Animals and dissections

Organotypic cultures

Pharmacological treatments

Immunohistochemistry

Immunofluorescence

Stereology and assessment of DN survival

Effects of 1-EBIO on DN survival

Measurement of soma size

Statistical analysis

Results

SK3 channels are expressed in DN organotypic cultures

Fig 1. Double immunofluorescence labeling of embryonic rat VM organotypic cultures with antibodies against TH and SK3.

DNs in organotypic cultures are differentially susceptible to AMPA-receptor mediated excitotoxicity

Fig 2. Susceptibility to AMPA-induced excitotoxicity is developmental stage dependent in organotypic cultures of embryonic VM.

Fig 3. AMPA treatment preferentially targets DN with larger soma size in embryonic VM organotypic cultures.

Protection conferred by SK3 channel activation is developmental stage-dependent in organotypic cultures of embryonic VM

Fig 4. Concentration-response curves for 1-EBIO effect on DN survival in organotypic cultures.

Discussion

Conclusion

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Faramarz Dehghani

Roles

Author response to Decision Letter 0

Decision Letter 1

Faramarz Dehghani

Roles

Author response to Decision Letter 1

Decision Letter 2

Faramarz Dehghani

Roles

Author response to Decision Letter 2

Decision Letter 3

Faramarz Dehghani

Roles

Acceptance letter

Faramarz Dehghani

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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