Animals
C57BL/6J mice (6–8 weeks of age) and Sprague-Dawley rats (27–29 month of age) obtained from the Yale Animal Resources Center (YARC) were housed under controlled temperature (25 ℃), humidity (50%), and lighting (12/12 light/dark cycle) conditions. Slack−/−/Slick−/− mice [30] were a gift of Dr. C.J. Lingle. Animals that underwent surgery were single housed following the procedure. All efforts were made to reduce animal suffering and the numbers used. All experiments were conducted in accordance with the guidelines of Yale University Institutional Animal Care and Use Committee (IACUC), and Public Health Service requirements for animal use as described in the Guide for the Care and Use of Laboratory Animals.
Primary Cortical Neuron Culture
Primary cortical neurons were prepared from E16-17 mouse embryos as described previously with modifications specific for this study [60]. After isolation of frontal cortex from embryonic brains, neurons were dissociated and seeded (on coverslips inside a 6 well plate: 0.2E6 cells/well) onto plates containing NB plus [Neurobasal medium supplemented with B27 (Invitrogen GIBCO Life Technologies), GlutaMAX (GIBCO), and penicillin/streptomycin (GIBCO)] and 5% FBS (GIBCO). After 2 hours incubation, primary cultures were maintained in NB plus without FBS in a 5% CO2 and 20% O2 incubator at 37 °C. Subsequently, half the medium was replaced every 2 days.
Tissue Preparation
For HCN1 and Slack channel immunolabeling, 3 male, 27 to 29-month-old Sprague-Dawley rats and 3 male 2-month-old C57BL/6J mice were used. Aged rats were used due to potentially increased HCN1 actions mediated by disinhibited cAMP-PKA signaling in the mPFC with advancing age [61]. Animals were anesthetized with Nembutal (50mg/mL, i.p.) and perfused transcardially with 4% paraformaldehyde (PFA), 0.05% glutaraldehyde in 0.1M phosphate buffer (PB; pH7.4). After perfusion, brains were removed from the skull and immersed in 4% PFA overnight at 4 °C. Coronal 60 μm-thick sections were then cut on a Vibratome (Leica V1000) and collected in 0.1 M PB.
Immunofluorescence
For immunocytochemistry of primary cortical neurons on DIV 14, coverslips were fixed in 4% paraformaldehyde for 10 min and permeabilized in blocking buffer (0.2% Triton X-100 and 3% normal goat serum in PBS) for 5 min at room temperature. For immunohistochemistry of cerebral cortex from 2-month-old mice, brain slices were permeabilized and incubated in blocking buffer for one hour at room temperature. After blocking, sections from DIV 14 cortical neuronal cultures or 2-month-old mice were overlaid with primary antibodies to Slack (1:200, Neuromab), HCN1 (1:200, Thermo Fisher Scientific) and MAP2 (1:200, Santa Cruz Biotechnology) overnight at 4 °C. Then, the corresponding Alexa Fluor 488-, 546- or 647-conjugated secondary antibodies were applied. Stained sections were mounted with DAPI-containing mounting solution and sealed with glass coverslips. Confocal imaging of primary cortical neurons and brain slices was carried out on a Leica SP5 MP. Three-dimensional z-stack images were taken and stitched into tiles to cover the entirety of the neurons of interest.
Post-embedding Immunogold Immunohistochemistry
In order to visualize the subcellular localization and interaction between HCN1 and Slack (Slo2.2, KCNT1) channels in rat mPFC layer II/III circuits, we utilized post-embedding immunogold dual-labeling techniques. Briefly, frozen brain tissue was freeze substituted in a Leica EM AFS2 unit starting from -90°C, and gradually increasing temperature to -45°C. After 3 changes of acetone, tissue was infiltrated with Lowicryl HM20 resin. The polymerization was carried out at -20°C with ultraviolet. Hardened blocks were cut using a Leica UltraCut UC7. Sixty nanometer sections were collected on formvar/carbon-coated nickel grids and stained using 2% uranyl acetate and lead citrate. For immunolabeling of resin sections, grids were placed section side down on drops of 1% hydrogen peroxide for 5 min, rinsed, and blocked for nonspecific binding with 3% bovine serum albumin in Tris-buffered saline (TBS) containing 1% Triton X-100 for 30 min. For single immunogold labeling, grids were incubated with anti-HCN1 or anti-Slack primary antibody 1:100 overnight, rinsed in TBS then incubated with 15 nm protein A gold (UMC, Utrecht, The Netherlands) for 30 min. For gold-gold dual-labeling immunohistochemistry, grids were incubated with anti-HCN1 and anti-Slack primary antibodies 1:100 overnight, rinsed in TBS then incubated with 15 nm (HCN1) and 5 nm (Slack) protein A gold (UMC, Utrecht, The Netherlands) for 30 min. The grids were rinsed in PBS, fixed using 1% glutaraldehyde for 5 min, rinsed again, dried, and heavy metal stained using 2% aqueous uranyl acetate and lead citrate. Specimen preparation for electron microscopy was carried out by the Yale CCMI Electron Microscopy facility.
Electron Microscopy and Data Analysis
Tissue blocks containing prelimbic mPFC layer II/III were analyzed under a JEM1010 (Jeol, Tokyo, Japan) transmission electron microscope at 80 kV. Several blocks of each brain were examined and structures were digitally captured at x25,000-x100,000 magnification with Bioscan camera (Gatan, Pleasanton, CA, United States of America) and individual panels were adjusted for brightness and contrast using Adobe Photoshop and Illustrator CC.2017.01 image editing software (Adobe Systems Inc., San Jose, CA, United States of America). Approximately, 400 micrographs of selected areas of neuropil with immunopositive profiles were used for analyses. For profile identification, we adopted the criteria summarized in [62].
Co-immunoprecipitation (Co-IP) and Western Blotting
Two-month-old mice frontal cortex were prepared using Pierce IP Lysis Buffer (Thermo Scientific) supplemented with cOmplete EDTA-free Protease Inhibitor Cocktail (Millipore Sigma) according to manufacturer’s protocol. Protein quantification was performed using Pierce BCA Protein Assay Kit (Thermo Scientific).
For Co-IP experiments, frontal cortex lysates were incubated with 5 μg anti-Slack IgY (AvesLabs) antibody [24] or IgY control (AvesLabs) overnight at 4°C. 100 μL Anti-IgY PrecipHen beads (AvesLabs) was added to sample and allowed to incubate for 2 hours, followed by wash and collection of beads. Beads were transferred to 2x Laemelli-Buffer with 5% beta- mercaptoethanol and incubated at room temperature for 30 minutes prior to Western blotting [43].
For all immunoblotting experiments, protein samples were electrophoretically separated on an SDS-PAGE gel (4%–15% gradient gel, Bio-Rad) and transferred onto PVDF membranes (0.2 μm pores, Bio-Rad, USA). Blots were blocked in 5% nonfat milk in Tris-buffered saline and Tween 20 (TBST) for 1 h at room temperature and probed with the primary antibodies to Slack (1:3000, AvesLabs) and HCN1 (1:1000, Abcam) overnight at 4°C. After overnight incubation, the blots were washed three times in TBST for 30min, followed by incubation with corresponding horseradish peroxidase-conjugated secondary antibodies (1:1000; Abcam) at room temperature for 1 h. Protein bands were visualized via enhanced chemiluminescence and quantified with analyzed with ImageJ (NIH) software.
Kuhl-H Cells
HEK293 and HEK293 Slack-positive cells were cultured in DMEM, 10% FBS, and 1% Penicillin (100U/mL). For experiments, the cells were plated in 35mm plastic plates at 2ml per well (~3x104 cell/mL). To introduce components into the cells using baculovirus, HCN2 and the photoactivated cyclase, bPAC were packaged in BacMam (Montana Molecular, Bozeman, MT). The virus titers were bPAC (2 x 1010 VG/mL) and HCN2 (1.57 x 1010 VG/mL). The additional Ca2+ biosensor in baculovirus was obtained from Montana Molecular: R-GECO (2 x 1010 VG/mL). The cells were transduced following the manufacturer's recommended protocol, plated in 35 mm plastic plates and transduced with the appropriate mix of viruses and HDAC inhibitor sodium butyrate (2mM final concentration). Two days later, the media was exchanged with PBS before imaging. Cells were imaged using post brief illumination from a blue LED (488nm) for rapid stimulation of bPAC. The yellow illumination was provided with a LED (560nm).
For data analysis, image data was stored in a Z-stack tiff file and loaded into the FIJI distribution of the IMAGEJ software. The cells were selected using a freehand ROI surrounding the cell of interest. The average total intensity pixel value within the ROI for each frame was collected using the time series analyzer plugin and saved as a .csv file. The raw fluorescence data was then put into PRISM and ORIGIN for further analysis. The fluorescence traces were rescaled from 0 - 100% and normalized to zero using PRISM software.
Patch-clamp Recordings
Whole-cell patch-clamp recordings were performed with patch-clamp amplifiers (MultiClamp 700B; Molecular Devices) under the control of pClamp 11 software (Molecular Devices). Data were recorded with a sampling rate at 20 kHz and filtered at 6 kHz. Rs compensation of 70% was used. Primary cortical neurons at DIV 13-14 or mPFC pyramidal neurons in brain slices from 2-month-old mice or HEK cells were recorded at physiological temperature (37°C). Recording electrodes were pulled from filamented borosilicate glass pipettes (Sutter Instrument, CA), and had tip resistances between 4 and 6 MΩ when filled with the following internal solution (in mM): 124 K-gluconate, 2 MgCl2, 13.2 NaCl, 1 EGTA, 10 HEPES, 4 Mg-ATP, and 0.3 Na-GTP (pH 7.3, 290-300 mOsm). The extracellular medium contained the following (in mM): either 140 NaCl, 5.4 KCl, 10 HEPES, 10 glucose, 1 MgCl2, and 1 CaCl2 (pH 7.4, 310 mOsm). For voltage-clamp recording on primary cortical neurons, neurons were held at -80 mV and given 200 ms voltage pulses in 10 mV steps over a range of -90 to +50 mV. For voltage-clamp recording on mPFC pyramidal neurons, neurons were held at -60 mV and given 500 ms voltage pulses in 20 mV steps over a range of -120 to +120 mV. For voltage-clamp recording on HEK cells, cells were held at -80 mV and given 300 ms voltage pulses in 20 mV steps over a range of -100 to +80 mV. ZD 7288 was purchased from MedChemExpress (Cat#: HY-101346) and was bath applied.
SLK-1 Methods
SLK-01 was prepared and its structure confirmed by the Yale Center for Molecular Discovery. at Yale). Electrophysiological recordings were carried out on an HEK cell line stably expressing the human Slack channel. The cells were cultured in a modified low sodium DMEM medium supplemented with 10% fetal bovine serum and penicillin-streptomycin (Invitrogen Inc, Carlsbad, CA). Whole-cell patch recordings were obtained at room temperature (21-23 oC) using electrodes of 3-5 MW resistance. These were pulled from TW150F-6 micropipettes (World Precision Instruments Inc., Sarasota, FL) on a horizontal Flaming/Brown micropipette puller (Model P-87, Sutter Instrument Co., Novato, CA). During recordings, the cells were bathed in a solution containing (in mM): 140 NaCl, 1 CaCl2, 5 KCl, 29 glucose, and 25 HEPES, pH 7.4. The pipette solution contained (in mM): 100 K-gluconate, 30 KCl, 5 Na-gluconate, 29 glucose, 5 EGTA, 2 Na2ATP, 0.2 GTP, and 10 HEPES, pH 7.3. In general, currents elicited by voltage steps between -100 and +60 mV from a holding potential of -80 mV. SLK-01 stock solutions were prepared at 10 mM in DMSO and were diluted to the final working concentration in recording solution before use.
Delayed Alternation Task
Rats were trained on the delayed alternation test of spatial working memory in a T-shaped maze. They were first adapted to handling and to eating treats (highly palatable miniature chocolate chips) on the maze prior to cognitive training. In this task, the rat was placed in the start box at the bottom of the ‘T’. When the gate was lifted, the rat proceeded down the stem of the maze to the choice point. On the first trial, rats were rewarded for entering either arm, but for each subsequent trial, were rewarded only if they chose the arm that they had not visited in the previous trial. Between trials, they were picked up and returned to the start box for a prescribed delay period. The choice point was cleaned with alcohol between each trial to remove olfactory clues (scent trails) often used by rodents to mark their previous locations. Successful performance of this task requires many operations carried out by the PFC: the rats must update and maintain the spatial information over the delay period for each trial, resist the distraction of being picked up and carried to the start box, and use response inhibition to overcome the tendency to repeat a rewarded action. The delay period was adjusted for each rat, such that they were performing at a stable baseline of 60–80% before drug treatment, thus leaving room for either impairment or improvement in performance. Rats were tested by experimenters who were highly familiar with the baseline behaviors of each individual animal, but blind to drug treatment conditions. Rats were observed for any potential differences in normative behavior (e.g. grooming, distracted sniffing), physical appearance, or physiological functioning (e.g. defecation/urination).
Surgery and Drug Infusions into mPFC
Following training on the delayed alternation task, rats underwent aseptic stereotaxic surgery, under ketamine+xylazine anesthesia with metacam analgesic pretreatment, to implant cannula aimed just above the prelimbic mPFC (AP: +3.2 mm; ML: ±0.75 mm; DV: -4.2 mm). Details of the surgical procedure and post-surgical treatment can be found in [12]. After rats had fully recovered from the surgery, they were adapted to the infusion procedure to minimize stress. Once stable baseline performance was re-established, rats received intra-PFC infusions of vehicle vs. drug (0.5μl per side) over 5 minutes; performance was assessed 10 min after the infusion by a researcher unaware of the drug treatment conditions. There was at least a one-week washout between drug infusions.
Statistical Analysis
GraphPad Prism Version 8 was used for statistical analysis. Two-tailed Student’s t test (parametric) or unpaired two-tailed Kolmogorov-Smirnov-test (non-parametric) was used for single comparisons between two groups. Other data were analyzed using two-way ANOVA with Bonferroni correction (parametric). All data were expressed as mean ± SEM, with statistical significance determined at p values < 0.05. In details, *Indicates p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 in all figures.