Experimental design and materials
All animal procedures used in experiments were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh School of Medicine. Experiments in DCN slices were performed blind to the treatment. Experiments using ZnT3 KO and WT animals were performed blind to the genotype of the animal. All key materials used are summarized in the Supplementary Materials (table S1).
Neuronal cultures. Cortical cultures were prepared from embryonic day 16 rats. Briefly, pregnant rats (Charles River Laboratories) were sacrificed via CO2 inhalation. Embryonic cortices were dissociated with trypsin and plated at 670,000 cells per well on 12-mm glass coverslips in six-well plates. Non-neuronal cell proliferation was inhibited after 2 weeks in culture with cytosine arabinoside (1 to 2 μM). Cultures were used during the 3 and 4 weeks in vitro (20 to 27 DIV) for PLA and electrophysiology experiments.
Cell line culture and transfection. HEK (HEK-tsa201) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% GlutaMAX. Cells were plated in 35-mm petri dishes with three 15-mm glass coverslips treated with poly-d-lysine (0.1 mg/ml) and rat tail collagen (0.1 mg/ml) at a density of 1 × 105 cells per dish. Eighteen to 30 hours after plating, the cells were cotransfected using FuGENE 6 Transfection Reagent with complementary DNA (cDNA) coding for enhanced green fluorescent protein (eGFP) for identification of transfected cells and the WT rat NMDAR subunits GluN1-1a (GluN1; GenBank X63255) and GluN2A (GenBank M91561 in pcDNA1). GluN1-1a and eGFP were expressed using a specialized pCl-neo vector with cDNA encoding eGFP inserted between the cytomegalovirus (CMV) promoter and the GluN1 open reading frame to express eGFP and GluN1 as separate proteins. At the time of transfection, 200 μM DL-2-amino-5-phosphonovaleric acid (APV) was added to culture medium to prevent NMDAR-mediated cell death. For experiments testing the effect of N2AZ and scN2AZ, cells were incubated with 3 μM peptide for 3 to 6 hours before recording.
Proximity ligation assay. PLAs were performed using a Duolink PLA kit. Cortical cultures were treated overnight with either N2AZ or scN2AZ (3 μM, dissolved in water). Coverslips were fixed in ice-cold methanol for 5 min, rinsed in phosphate-buffered saline (PBS), and then permeabilized with 0.1% Triton X in PBS. Coverslips were then incubated with primary antibodies: rabbit anti-ZnT1, mouse anti-GluN2A, and chicken anti-MAP2. Coverslips were incubated with a donkey anti-chicken fluorescent secondary antibody targeting MAP2 antibodies to visualize neuron morphology. The PLA reaction was then completed according to Duolink PLA protocol. Briefly, coverslips were incubated in Duolink secondary antibodies (anti-rabbit and anti-mouse), which are conjugated with complementary oligonucleotides. Ligation solution was added to hybridize connector oligonucleotides and PLA probes, allowing the oligonucleotides to join in a closed loop when secondary antibodies were in close proximity. Next, the reaction was amplified with rolling-circle amplification (RCA) using the closed loop hybridized probes as a template. PLA probes were fluorescently labeled with oligonucleotides, which hybridized to the RCA product during amplification. Coverslips from sister cultures were treated with either scN2AZ or N2AZ, and reactions were run simultaneously using the same preparation of reagents. Coverslips were mounted on glass slides using Duolink mounting medium, and four random fields of view were imaged from each coverslip using a 60× oil objective on a Nikon A1R laser scanning confocal microscope. PLA puncta, reflecting GluN2A-ZnT1 interactions, were counted automatically with Fiji ImageJ (version 2.0) software. We used maximum intensity projection of eight sequential images in the z plane. All images were normalized to the same intensity threshold using the Yen threshold setting before automated quantification of puncta.
PLA experiments were performed four independent times, each time from a different tissue culture (different pregnant dam). The n in Fig. 2 refers to the number of coverslips analyzed per treatment (scN2AZ versus N2AZ). The average and SEM of these experiments are shown in the bar graphs of Fig. 2. Each individual data point shown in the same figure panel represents a single coverslip, each with an average of four images obtained in random areas within each coverslip.
For a given culture, cortical tissue is normally obtained from approximately eight rat brain embryos, pooled and dissociated; cultures are plated at a density of 680,000 cells per 35-mm culture dish. To plate the cells, we previously place five 12-mm coverslips in each 35-mm dish, although the area of the dish could accommodate six coverslips (that is, six coverslips would occupy nearly the entire area of the dish). This culture procedure results in approximately 20% neurons and 80% glia, mostly astrocytes (49). Thus, our best estimate is that, at most, there are ~22,500 neurons per coverslip (680,000 × 0.2/6). We transfer individual coverslips from this culture dish to a separate culture plate to perform each experiment noted.
Brain slices. Male and female mice (postpartum days 18 to 28) were anesthetized with isoflurane and sacrificed. Brains were rapidly dissected and sectioned with a vibratome (Leica, VT1000S) into 210-μm-thick coronal slices of the brainstem containing DCN. Slices were incubated in artificial cerebrospinal fluid (ACSF) containing 130 mM NaCl, 3 mM KCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 20 mM NaHCO3, 3 mM Hepes, and 10 mM glucose, saturated with 95% O2/5% CO2 (v/v), pH ∼7.3, ∼300 mOsm at 35°C for 1 hour before being moved to room temperature. During preparation, ACSF was treated with Chelex 100 resin to remove any contaminating zinc. After applying Chelex to the ACSF, high-purity calcium and magnesium salts were added (99.995% purity). All plastic and glassware were washed with 5% high-purity nitric acid.
Electrophysiology. Whole-cell voltage-clamp recordings from HEK-tsa201 cells were performed 18 to 30 hours after transfection. Pipettes were fabricated from borosilicate capillary tubing (outside diameter, 1.5 mm; inside diameter, 0.86) using a Flaming Brown P-97 electrode puller (Sutter Instruments) and fire-polished to a resistance of 2.5 to 4.5 megohms with an in-house fabricated microforge. Intracellular pipette solutions consisted of 130 mM CsCl, 10 mM Hepes, 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), and 4 mM MgATP with pH balanced to 7.2 ± 0.05 using CsOH and final osmolality of 280 ± 10 mOsm. Extracellular recording solution contained 140 mM NaCl, 2.8 mM KCl, 1 mM CaCl2, 10 mM Hepes, 10 mM tricine, and 0.1 mM glycine and was balanced to pH 7.2 ± 0.05 and osmolality 290 ± 10 mOsm with NaOH and sucrose, respectively. Glutamate (Glu) and ZnCl2 were diluted from concentrated stock solutions in extracellular solution each day of experiments. Buffered Zn2+ solutions were prepared as previously described (20) via serial dilution. Extracellular solutions were delivered to the cell using a fast perfusion system. Whole-cell currents were recorded using an AxoPatch 200A patch-clamp amplifier (Molecular Devices), low pass–filtered at 5 kHz, and sampled at 20 kHz in pClamp10.7 (Molecular Devices). In all recordings from HEK-tsa201cells, series resistance was compensated 85 to 90% and an empirically determined −6-mV liquid junction potential between the intracellular pipette solution and the extracellular recording solution was corrected.
The effect of the N2AZ on Zn2+ inhibition of GluN1/2A receptors was determined using the protocol shown in Fig. 5I. Glu (1 mM) was applied for 30 s until current reached steady state, followed by sequential applications (5 s each) of 1 mM Glu and Zn2+ at 1, 3, 10, 30, 100, and 300 nM. A final 30-s application of Glu in the absence of Zn2+ was then performed to allow recovery from inhibition. Zn2+ IC50 was estimated by fitting the following equation to data
where IZn/IGlu was calculated as the mean current over the final 1 s of Zn2+ application divided by the average of the mean steady-state currents (final 1 s) elicited by Glu before and after Zn2+ application. A (IZn/IGlu at saturating Zn2+), IC50, and nH (Hill coefficient) were free parameters during fitting. Curve fitting and statistical comparisons were performed in Prism 8. IC50s were compared by one-way ANOVA.
Whole-cell recordings from cultured cortical neurons were obtained with glass micropipettes (3 to 6 megohms) containing 140 mM CsF, 10 mM CsEGTA, 1 mM CaCl2, and 10 mM Hepes, pH 7.2, 295 mOsm. Extracellular recording solution contained 150 mM NaCl, 2.8 mM KCl, 1.0 mM CaCl2, 10 mM Hepes, and 10 μM glycine, pH ~7.2, ~300 mOsm. Using Ephus and a Multiclamp 700B amplifier (Molecular Devices), NMDAR EPSCs were recorded in voltage clamp (held at −70 mV) in the presence of tetrodotoxin (300 nM, sodium channel blocker), DNQX (20 μM, AMPA and kainate receptor antagonist), and MNI-caged glutamate (40 μM). Neurons were visualized by including 10 μM Alexa 594 in the internal solution. To evoke NMDAR EPSCs, we photolytically uncaged MNI-caged glutamate onto dendrites at four locations 0, 40, 80, and 120 μm from the cell soma using 1-ms pulses of ultraviolet laser light (355 nm, DPSS Lasers). The ZX1-mediated potentiation for each cell was calculated as the average percent increase in responses following application of the metal chelator across these four uncaging locations.
For brain slice recordings, whole-cell recordings of NMDAR EPSCs of DCN cartwheel cells were obtained with micropipettes (3 to 6 megohms) containing 128 mM Cs(CH3O3S), 4 mM MgCl2•6H2O, 4 mM Na2ATP, 10 mM Hepes, 0.3 mM tris-GTP (guanosine triphosphate), 10 mM tris-phosphocreatine, 1 mM CsEGTA, 1 mM QX-314, and 3 mM sodium ascorbate, pH ~7.2, 300 mOsm in chelexed ACSF with the following composition: 130 mM NaCl, 3 mM KCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 20 mM NaHCO3, 3 mM Hepes, and 10 mM glucose, saturated with 95% O2/5% CO2 (v/v), pH ∼7.3, ∼300 mOsm. Cartwheel cells were identified by the presence of complex spikes (50) in cell-attached configuration before break-in or in response to current injections in current-clamp mode immediately after break-in. NMDAR EPSCs were recorded in voltage-clamp mode at a holding potential of +40 mV in the presence of DNQX (20 μM), SR95531 (20 μM, GABAAR antagonist), and strychnine (1 μM, GlyR antagonist). ZX1 (100 μM) was included in the pipette in experiments where noted. Whole-cell recordings of AMPAR EPSCs were obtained with micropipettes containing 113 mM K-gluconate, 4.5 mM MgCl2•6H2O, 14 mM tris-phosphocreatine, 9 mM Hepes, 0.1 mM EGTA, 4 mM Na2ATP, 0.3 mM tris-GTP, and 10 mM sucrose, pH 7.3, 295 mOsm. AMPAR EPSCs were recorded in voltage-clamp mode at a holding potential of −70 mV in the presence of SR95531 (20 μM) and strychnine (1 μM). Both NMDAR and AMPAR EPSCs were evoked using an Isoflex stimulator (A.M.P.I., 0.1-ms pulses) stimulating parallel fibers with voltage pulses through a theta glass electrode. For paired-pulse experiments, interstimulus interval was 50 ms. Once a stable response was established, ZX1 (100 μM) was added to the recording solution to measure the effect of zinc chelation on EPSCs. The series resistance was not compensated because the currents measured were relatively small; therefore, there was minimum voltage-clamp error. The cell parameters were monitored during the recording by delivering −5-mV voltage steps for 50 ms at each sweep. The peak current value (ΔIpeak) generated immediately after the step in the command potential was used to calculate series resistance (Rseries) using the following formula: Rseries = −5 mV/ΔIpeak. The difference between baseline and steady-state current (ΔIss) was used to calculate input resistance (RI) using the following formula: RI = −5 mV/ΔI − Rseries. Recordings were excluded from further analysis if the series resistance or input resistance changed by more than 20% compared to the baseline period. Data were low pass–filtered at 4 kHz and sampled at 10 kHz. NMDAR EPSC peak values were averaged over a 20-ms time window using custom MATLAB 2012a software. All values reported are animal-based values; in cases where multiple cells were recorded from the same animal preparation, the average of cells is presented. All recordings were performed at room temperature.
Quantitative real-time PCR. For quantitative PCR (qPCR) analysis of rat cortical cultures, cells were harvested at 5, 12, 19, and 26 DIV and RNA was isolated using the Invitrogen PureLink RNA Mini Kit. cDNA was synthesized from RNA transcripts using the iScript Select cDNA Synthesis Kit using Eppendorf Thermocycler. Quantitative real-time PCRs (qRT-PCRs) were performed on a Bio-Rad CFX qRT-PCR machine using iTaq Universal SYBR Green Supermix. Relative expression was calculated using β-actin as a reference gene. Custom primers against rat β-actin (forward: TTCAACACCCCAGCCATGT; reverse: GCATACAGGGACAACACAGCC; Invitrogen) and rat ZnT1 (forward: TGGGCGCTGACGCTTACT; reverse: GTCAGCCGTGGAGTCAATAGC; Invitrogen) were designed using the National Center for Biotechnology Information Primer-BLAST.
Zinc efflux assay. HEK cells were grown in DMEM containing penicillin (100 U/ml), streptomycin (0.1 mg/ml), 2 mM glutamine, and 10% (v/v) fetal calf serum in a 5% CO2 humidified atmosphere at 37°C. To express ZnT1, HEK293 cells were transfected with ZnT1 or empty plasmid (control) using CaPO4 precipitation. Briefly,1 μg of mouse ZnT1 (pCMV6, ZnT1; GenBank, Q60738) or empty vector plasmid (pCMV6, OriGene) was incubated with 2 M calcium chloride in Hepes-buffered solution containing 1.5 mM Na2HPO4 to generate a coprecipitate; this solution was then dispersed onto cultured cells for 6 hours. Twenty-four hours later, cells were treated overnight with N2AZ or scN2AZ (3 μM). To visualize intracellular zinc, cells were loaded with the fluorescent zinc indicator FluoZin-3 (2 μM) for 25 min at room temperature before imaging. Cells were imaged using a 480-nm excitation filter and an emission 525-nm long-pass filter on a Zeiss Axiovert 100 inverted microscope with a Polychrome IV monochromator (T.I.L.L. Photonics) and a cooled charge-coupled device camera (PCO). To measure zinc efflux, cells were superfused with Ringer’s solution (composition: 120 mM NaCl, 0.8 mM MgCl2, 5.4 mM KCl, 1.8 mM CaCl2, 20 mM Hepes, and 15 mM glucose), and 1 μM Zn2+ with 5 μM pyrithione was added for 150 s. The FluoZin-3 signal was normalized to 10-s baseline in each experiment. Rates of initial decrease of the fluorescent signal following removal of Zn2+ pyrithione were determined during a 100-s period. For each experiment, at least 30 cells were imaged per coverslip and rates were averaged for three to five coverslips performed as three independent experiments. Fluorescence imaging measurements were acquired using Axon Imaging Workbench 5.2 (INDEC BioSystems) and analyzed using Excel and Prism GraphPad.
Peptide hspot array and far-Western assay. Far-Western protein-binding affinity assays were performed as previously described (25). Peptide spot arrays (15-mers) spanning the proximal C-terminal residues 1390 to 1464 of mouse GluN2A (UniProt no.: P35436) in overlapping one-residue steps were constructed using the Spots-synthesis method. Standard 9-fluorenylmethoxy carbonyl (Fmoc) chemistry was used to synthesize the peptides and spot them onto nitrocellulose membranes, which were prederivatized with a polyethylene glycerol spacer (Intavis). Fmoc-protected and Fmoc-activated amino acids were spotted in 20 to 30 arrays on 150 mm–by–100 mm membranes using an Intavis MultiPep robot. The nitrocellulose membrane containing the immobilized peptides was soaked in N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer [10 mM CAPS (pH 11.0) with 20% (v/v) methanol] for 30 min, washed once with tris-buffered 0.1% Tween 20 (TBST), blocked for 1 hour at room temperature with gentle shaking in TBST containing 5% (w/v) nonfat milk, and then incubated with enriched Flag-tagged ZnT1 (SLC30a1) cell lysate (obtained from previously transfected HEK cells) overnight at 4°C with gentle shaking. Next, the membrane was incubated in primary antibody for Flag for 1 hour at room temperature with gentle shaking, followed by washing with TBST. Last, the membrane was incubated in secondary antibody for 45 min, washed three times for 5 min in TBST, and then visualized by infrared fluorescence (Li-Cor). Four independent peptide spot arrays were used in this study. A second set of membranes (n = 4) was treated as above but also in the presence of 100 μM N2AZ or scN2AZ and compared to 0.1% DMSO. For each experiment, an additional peptide array was done with omission of Flag-tagged ZnT1 (SLC30a1) protein to measure and correct for the background due to the primary and secondary antibodies.
Statistical analyses. Slice electrophysiology experiments using N2AZ and scN2AZ were completed blind to the identity of the peptide. Experiments in ZnT3 KO and WT animals were completed blind to the genotype. Electrophysiology recordings in cortical cultures and DCN slices were obtained using Ephus software run in MATLAB 2012a (MathWorks). Cell parameters and response peaks were calculated using custom MATLAB scripts. For neuronal culture electrophysiology, ZX1 potentiation was measured as the percent increase in NMDAR amplitude 5 min after the application of ZX1. In slice experiments, ZX1 potentiation was calculated as the average percent increase over baseline of NMDAR or AMPAR EPSCs 10 to 15 min after the addition of ZX1 (Figs. 3, 4, 5, and 6, D to F) or 15 to 20 min after the addition of ZX1 (Fig. 6, A to C). Unpaired t tests and ANOVAs were used to compare between treatments and genotypes. To determine whether ZX1 significantly potentiated responses, paired t tests were used to compare amplitude of peak responses before and after addition of ZX1. Statistical analysis was completed in Prism 8 (GraphPad).