DOI: 10.5061/dryad.5qfttdz5t
title: All-trans retinoic acid induces synaptopodin-dependent metaplasticity in mouse dentate granule cells

description: Previously we showed that the vitamin A metabolite all-trans retinoic acid
 (atRA) induces synaptic plasticity in acute brain slices prepared from the
 mouse and human neocortex (Lenz et al., 2021). Depending on the brain
 region studied, distinct effects of atRA on excitatory and inhibitory
 neurotransmission have been reported. Here, we used intraperitoneal
 injections of atRA (10 mg/kg) in adult C57BL/6J mice to study the effects
 of atRA on excitatory and inhibitory neurotransmission in the mouse fascia
 dentate – a brain region implicated in memory acquisition. No major
 changes in synaptic transmission were observed in the ventral hippocampus
 while a significant increase in both sEPSC frequencies and synapse numbers
 were evident in the dorsal hippocampus 6 hours after atRA administration.
 The intrinsic properties of hippocampal dentate granule cells were not
 significantly different and hippocampal transcriptome analysis revealed no
 essential neuronal changes upon atRA treatment. In light of these
 findings, we tested for the metaplastic effects of atRA, i.e., for its
 ability to modulate synaptic plasticity expression in the absence of major
 changes in baseline synaptic strength. Indeed, in vivo long-term
 potentiation (LTP) experiments demonstrated that systemic atRA treatment
 improves the ability of dentate granule cells to express LTP. The
 plasticity-promoting effects of atRA were not observed in
 synaptopodin-deficient mice, therefore, extending our previous results
 regarding the relevance of synaptopodin in atRA-mediated synaptic
 strengthening in the mouse prefrontal cortex. Taken together, our data
 show that atRA mediates synaptopodin-dependent metaplasticity in mouse
 dentate granule cells.
description: <b>Key Resource
 Table.</b> <b>Reagent type (species) or
 resource</b> 
 <b>Designation</b> <b>Source of
 reference</b> 
 <b>Identifiers</b> <b>Additional
 information</b> Chemical compound, drug
 All-trans
 retinoic acid (atRA) 
 Sigma-Aldrich Cat#:
 R2625 Final
 concentration: 10 mg/kg; Injection vehicle: Corn oil + 5% DMSO
 
 Chemical compound, drug
 Dimethyl Sulfoxide
 (DMSO) 
 Sigma-Aldrich Cat#:
 D2650  
 
 Chemical compound, drug
 Paraformaldehyde
 (PFA) Carl
 Roth Cat#:
 0335.3 Final concentration: 4% (w/v) in
 PB or PBS Chemical compound, drug
 
 Glutardialdehyd Carl
 Roth Cat#:
 4157.2 Final concentration: 2%
 (w/v) Chemical compound, drug
 CNQX
 
 Biotrend Cat#:
 BN0153 Final concentration: 10
 µM Chemical compound, drug
 D-APV
 abcam
 Cat#:
 ab120003 Final concentration: 10
 µM Chemical compound, drug
 DAPI (1 mg/ml in
 water) Thermo
 Scientific  Cat#:
 62248 IF and post-hoc
 labelling (“1:5000”) Commercial assay, kit
 
 Monarch<sup>®</sup> Total RNA Miniprep Kit
 New England
 Biolabs 
 #T2010S  
 Genetic reagent (Mus musculus),
 male 
 B6.129-Synpo<sup>tm1Mndl</sup>/Dllr;
 Synpo<sup>-/-</sup> Vlachos et al.,
 2013 PMID: 23630268 MGI:
 6423115 Obtained from
 Deller Lab (Frankfurt) Peptide, recombinant protein
 Streptavidin,
 Alexa Fluor™ 488-Conjugate 
 Invitrogen Cat#: S32354
 RRID:AB_2315383 Post-hoc
 labelling (“1:1000”) Software, algorithm
 Prism
 
 GraphPad 
 RRID:SCR_002798  
 
 Software, algorithm
 Clampfit (pClamp
 software package) Molecular
 Devices 
 RRID:SCR_011323  
 Software, algorithm
 ImageJ
  
 
 RRID:SCR_003070  
 
 Software, algorithm
 
 Photoshop Adobe
 
 RRID:SCR_014199  
 Strain, strain background (Mus
 musculus) C57BL/6J;
 Synpo<sup>+/+</sup> Jackson
 Laboratory RRID:
 IMSR_JAX:000664  
 <b>Ethics
 statement.</b> All experiments were performed according to German
 animal welfare legislation and after positive evaluation by the local
 authorities (University of Freiburg, AZ G-19/152; Faculty of Medicine at
 the University of Frankfurt, AZ FU/1131). Animals were kept in a 12-hour
 light/12-hour dark cycle with access to food and water <i>ad
 libitum</i>. Every effort was made to minimize pain or distress of
 animals. <b>Pharmacological treatment.</b>
 All-trans retinoic acid (atRA; Sigma-Aldrich) was dissolved in DMSO and
 stored at -20°C until further use. The injection solution was prepared
 immediately before injection by adding corn-oil to prediluted stocks to
 achieve a final concentration of 5% DMSO (v/v). Before use, the solution
 was vortexed briefly. The solution was intraperitoneally injected in adult
 (C57BL/6J; 6–10 weeks old) male mice at an atRA concentration of 10 mg/kg.
 Control animals were injected with a vehicle-only solution (5% DMSO in
 corn oil) but otherwise treated equally. After injection, no overt
 behavioral changes were observed. Experiments were performed 3–6 hours
 after intraperitoneal injections. <b>Preparation
 of acute mouse hippocampal slices.</b> Adult mice were anesthetized
 with ketamine/xylazine (100 mg/kg ketamine and 20 mg/kg xylazine) and
 rapidly decapitated. Brains were removed and further dissected for the
 preparation of acute slices of the ventral hippocampus as previously
 described [30]. For the preparation of acute slices of the dorsal
 hippocampus, the rostral and caudal parts of the brains were removed to
 ensure stable coronal sectioning. Brains were immediately transferred to a
 cooled oxygenated extracellular solution (5°C; 5%
 CO<sub>2</sub> / 95% O<sub>2</sub>) containing (in
 mM): 92 NMDG, 2.5 KCl, 1.25
 NaH<sub>2</sub>PO<sub>4</sub>, 30
 NaHCO<sub>3</sub>, 20 HEPES, 25 glucose, 2 thiourea, 5
 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl<sub>2</sub>, and 10
 MgSO<sub>4</sub>; pH = 7.3 to 7.4 at ~7°C (NMDG-aCSF; [31]).
 300 µm tissue sections were cut with a Leica VT1200S vibratome. Slices
 were transferred to cell strainers with 40 µm pore size placed in
 NMDG-aCSF at 34°C, and the sodium levels were gradually increased
 following a protocol as described before [31]. After recovery, slices were
 maintained for further experimental assessment at room temperature in
 extracellular solution containing (in mM): 92 NaCl, 2.5 KCl, 1.25
 NaH<sub>2</sub>PO<sub>4</sub>, 30
 NaHCO<sub>3</sub>, 20 HEPES, 25 glucose, 2 thiourea, 5
 Na-ascorbate, 3 Na-pyruvate, 2 CaCl<sub>2</sub>, and 2
 MgSO<sub>4</sub>. <b>Whole-cell
 patch-clamp recordings. </b>Dentate granule cells in the
 suprapyramidal blade of the dentate gyrus were recorded in a bath solution
 (35°C) containing (in mM): 92 NaCl, 2.5 KCl, 1.25
 NaH<sub>2</sub>PO<sub>4</sub>, 30
 NaHCO<sub>3</sub>, 20 HEPES, 25 glucose, 2 thiourea, 5
 Na-ascorbate, 3 Na-pyruvate, 2 CaCl<sub>2</sub>, and 2
 MgSO<sub>4</sub>. Granule cell somata close to the molecular
 layer of the dentate gyrus were visually identified using an LN-Scope
 (Luigs & Neumann, Ratingen, Germany) equipped with an infrared
 dot-contrast and a 40x water immersion objective (Olympus, NA 0.8).
 Recorded signals were amplified using a Multiclamp 700B amplifier,
 digitized with a Digidata 1550B digitizer and visualized with the pClamp
 11 software package. For recordings of spontaneous excitatory postsynaptic
 currents (sEPSC) and intrinsic cellular properties, patch pipettes with a
 tip resistance of 3-5 MΩ were used, containing (in mM): 126 K-Gluconate, 4
 KCl, 10 HEPES, 4 MgATP, 0.3 Na<sub>2</sub>GTP, 10 PO-Creatine,
 0.3% (w/v) Biocytin (pH = 7.25 with KOH, 285 mOsm/kg). For sEPSC
 recordings, dentate granule cells were held at -80 mV in voltage-clamp
 mode. Intrinsic cellular properties were recorded in current-clamp mode. A
 pipette capacitance of 2.0 pF was corrected and series resistance was
 compensated using the automated bridge balance tool of the Multiclamp
 commander. IV-curves were generated by injecting 1 s square pulse currents
 starting at -100 pA and increasing in 10 pA steps until +500 pA injection
 was reached (sweep duration: 2 s). Spontaneous inhibitory postsynaptic
 currents (sIPSCs) were recorded in the same extracellular solution by
 adding the AMPA receptor inhibitor CNQX (10 µM, Biotrend) and the NMDA
 receptor inhibitor APV (10 µM, Abcam). Patch pipettes for sIPSC recordings
 contained (in mM): 40 CsCl, 90 K-gluconate, 1.8 NaCl, 1.7
 MgCl<sub>2</sub>, 3.5 KCl, 0.05 EGTA, 2 MgATP, 0.4
 Na<sub>2</sub>GTP, 10 PO-Creatine, 10 HEPES (pH = 7.25 with
 KOH, 290 mOsm) and granule cells were held at -70 mV during the
 recordings. Series resistance was monitored and recordings were discarded
 if series resistance reached &gt; 30 MΩ.
 <b>Post-hoc labeling of patched dentate granule cells.
 </b>Acute slice preparations were fixed in 4% PFA/ 4% sucrose (w/v,
 phosphate buffered saline, PBS) at room temperature and stored at 4°C
 overnight in the same solution. After fixation, slices were washed in PBS
 and consecutively incubated for 1 hour with 10% (v/v) normal goat serum
 (NGS) in 0.5% (v/v) Triton X-100 containing PBS to reduce unspecific
 staining. For <i>post hoc</i> visualization of patched dentate
 granule cells, sections were incubated for 3 hours with streptavidin-Alexa
 Fluor 488 (Invitrogen, #S32354; 1:1000 dilution in 10% (v/v) NGS, 0.1%
 (v/v) Triton X-100 containing PBS) at room temperature. Sections were
 washed in PBS and incubated with DAPI for 10 minutes (Thermo Scientific,
 #62248; 1:5000 dilution in PBS) to visualize the cytoarchitecture. After
 washing, sections were transferred onto glass slides and mounted with
 fluorescence anti-fading mounting medium (DAKO Fluoromount). Confocal
 images were acquired using a Leica SP8 laser-scanning microscope equipped
 with a 20x multi-immersion (NA 0.75; Leica) and a 40x oil-immersion (NA
 1.30; Leica) objective. Image stacks were acquired in tile scanning mode
 with the automated stitching function of the LasX software
 package. <b>Electron microscopy. </b>Adult
 mice of both sexes were anesthetized using intraperitoneal injection of
 ketamine (100 mg/kg) and xylazine (20 mg/kg).<b> </b>Deeply
 anesthetized mice were transcardially perfused using 2% (w/v; 0.1 M
 phosphate buffer, PB) glutaraldehyde and 4% (w/v; 0.1 M phosphate buffer,
 PB) paraformaldehyde. Post-hoc fixation of the brains were continued
 overnight in the same fixation solution. After fixation, frontal sections
 containing the dorsal hippocampus were generated using a Leica VT1000S
 vibratome. Isolated dorsal hippocampal slices were washed for 4 hours in
 0.1 M PB (phosphate buffer). Subsequently, slices were incubated with 1%
 osmium tetroxide for 60 min, washed in graded ethanol (up to 50% (v/v))
 for 5 min each, and incubated overnight with uranyl acetate (1% (w/v) in
 70% (v/v) ethanol) overnight. Slices were then dehydrated in graded
 ethanol (80%, 90%, 98% for 5 min each, 2 times 100% for 10 min each).
 Subsequently, two washing steps were performed in propylene oxide for 10
 min each prior to incubation with durcupan/propylene oxide (1:1 for 1
 hour) and transferred to durcupan (overnight at room temperature). Slices
 were embedded in durcupan, and ultra-thin sectioning (55 nm) was performed
 using a Leica UC6 Ultracut. Sections were mounted onto copper grids
 (Plano), at which point an additional Pb-citrate contrasting step was
 performed (3 min). Electron microscopy was performed with a LEO 906E
 microscope (Carl Zeiss) at 4646× magnification. For each sample, 12 images
 from the outer two thirds of the molecular layer were acquired and further
 analyzed.  <b>RNA isolation and transcriptome
 analysis. </b>Hippocampi were isolated from the brain of adult mice
 and immediately transferred into RNA protection buffer (New England
 Biolabs) and RNA was consecutively isolated using a column based RNA
 isolation kit according to the manufacturer’s instructions
 (Monarch<sup>®</sup> Total RNA Miniprep Kit; #T2010S New
 England Biolabs). Strand specific cDNA library preparation from polyA
 enriched RNA (150 bp mean read length) and RNA sequencing was performed by
 Eurofins Genomics (Eurofins Genomics Europe Sequencing GmbH, Konstanz,
 Germany). RNA sequencing was performed using the genome sequencer Illumina
 HiSeq technology in NovaSeq 6000 S4 PE150 XP sequencing mode. For further
 analysis .fastq-files were provided. All files contained more than 45 M
 high-quality reads having at least a phred quality of 30 ( &gt; 90% of
 total reads). <b>In vivo perforant path long-term
 potentiation.</b> 3-month-old C57BL/6J
 (Synpo<sup>+/+</sup>) or synaptopodin-deficient male animals
 (Synpo<sup>-/-</sup>; with C57BL/6J genetic background) were
 kept in a 12-hour light/12-hour dark cycle (Scantainer) with access to
 food and water <i>ad libitum</i>. To achieve stable
 anesthesia, an initial dose of urethane (1.25 g/kg, in sodium chloride
 solution) was injected subcutaneously (s.c.); a supplemental dose of 0.1
 g/kg was given as needed. After stable anesthesia was reached, atRA (10
 mg/kg in 5% DMSO) or vehicle-only was intraperitoneally injected (blind to
 experimenter). The surgery and electrode placement were performed as
 previously described [32; 33]. Briefly, the mouse was placed in a
 stereotactic frame (David Kopf Instruments) and local anesthesia with
 prilocaine (Xylonest 1%, Astra Zeneca, s.c. to the scalp) was applied.
 Cranial access to the brain was established according to coordinates from
 the mouse brain atlas (Franklin and Paxinos; stimulation electrode: 2.5 mm
 lateral to the midline, 3.8 mm posterior to bregma; recording electrode:
 1.2 mm lateral to the midline, 1.7 mm posterior to bregma). The ground
 electrode was placed in the neck musculature. Electrophysiological signals
 were amplified using a Grass P55 A.C. pre-amplifier (Astro-Med) and
 digitized at a 10 kHz sampling rate (Digidata 1440A, Molecular Devices).
 Extracellular stimulation was performed using a STG1004 stimulator
 (Multichannel Systems). A bipolar stimulation electrode (NE-200, 0.5 mm
 tip separation, Rhodes Medical Instruments) was lowered 1.5-2.2 mm below
 the surface of the brain to target the angular bundle of the perforant
 path. Then a tungsten recording
 electrode (TM33B01KT, World Precision Instruments) was lowered in 0.1 mm
 increments while monitoring the waveform of the field excitatory
 postsynaptic potential (fEPSP) in response to 500 µA test pulses until the
 granule cell layer in the dorsal part of the hippocampus was reached
 (1.7-2.2 mm below the surface). The correct
 placement of the stimulation electrode in the medial portion of the
 perforant path was verified electrophysiologically by the latency of the
 population spike (approximately 4 ms), although the activation of some
 lateral perforant path fibers could not be excluded. Recordings started a
 minimum of 3 hours after experimental treatment with atRA or vehicle-only.
 An input-output curve was generated by 30-800 µA current pulses, repeated
 three times at each intensity, 0.1 ms pulse duration, 60 pulses total at
 0.1 Hz. Perforant path-dentate gyrus (PP/DG)-long-term potentiation (LTP)
 was recorded by applying stimuli with a current intensity set to elicit a
 1-2 mV population spike (0.1 Hz, 0.1 ms pulse duration). PP/DG-LTP was
 induced using a weak theta-burst stimulation (TBS) protocol [34] composed
 of three series of six trains with six 400 Hz current pulses at double the
 baseline intensity and pulse duration (with 200 ms interval between trains
 and 20 s interval between series). Following LTP induction, evoked fEPSPs
 were recorded for 1 hour using the baseline stimulation
 parameters. <b>Quantification and
 statistics.</b> RNA sequencing data were uploaded to the galaxy web
 platform (public server: usegalaxy.eu; [35; 36; 37]) and transcriptome
 analysis was performed using the Galaxy platform in accordance with the
 reference-based RNA-seq data analysis tutorial [38]. Adapter sequences,
 low quality and short reads were removed via the CUTADAPT tool (Galaxy
 version 1.16.5). Reads were mapped using RNA STAR (Galaxy version 2.7.6a)
 with the mm10 Full reference genome (Mus Musculus). For initial assessment
 of gene expression, unstranded FEATURECOUNT (Galaxy version 2.0.1)
 analysis was performed from RNA STAR output. Statistical evaluation was
 performed using DESeq2 (Galaxy version 2.11.40.6+galaxy1) with treatment
 as the primary factor that might affect gene expression. Genes were
 considered as differentially expressed if the adjusted p-value was
 &lt; 0.05. Heat maps were generated based on z-scores of the
 normalized count table. Single-cell recordings were
 analyzed offline using Clampfit 11 of the pClamp11 software package
 (Molecular Devices). sEPSC and sIPSC properties were analyzed using the
 automated template search tool for event detection [12]. Input resistance
 was calculated for the injection of -100 pA current at a time frame of 200
 ms with a maximum distance to the initial hyperpolarization. Resting
 membrane potential was calculated as the mean baseline value. Action
 potential (AP) detection was performed using the input-output curve
 threshold search event detection tool, and the AP frequency was assessed
 by the number of APs detected during the respective current injection
 time. One individual cell (control group, ventral hippocampus) was
 excluded from the analysis of intrinsic membrane properties, since the
 membrane patch lost its integrity during the recordings. The action
 potential plots in Figures 2 and 4 depict cellular responses until 300 pA
 current injection. Beyond 300 pA, subsets of granule cells in both groups
 failed to maintain regular action potential firing. The treatment did not
 significantly affect action potential frequency beyond 300 pA current
 injection. <i>In vivo</i> perforant path LTP was analyzed
 using Clampfit 10.2 and custom MATLAB (Mathworks) scripts. In these
 experiments, one Synpo<sup>-/-</sup> animal in the
 vehicle-only group was excluded from further analysis, since an
 insufficient response to increasing stimulus intensities was detected in
 the input-output curve. Electron microscopy images were
 analyzed and cross-checked by five investigators blind to experimental
 conditions. Image analysis was performed using the ImageJ software package
 (available at http://imagej.nih.gov/ij/). For post-synaptic density (PSD) assessment, all visible PSDs in one image were counted and normalized to the image area. Subsequently, PSD length and presynaptic vesicle abundance was manually assessed. In case of perforated synapses, each PSD was analyzed individually. For statistical evaluation, each individual synapse was considered a biological replicate. Data were statistically evaluated using GraphPad Prism 7 (GraphPad software, USA). Statistical comparisons were made using the non-parametric Mann-Whitney test. For statistical comparison of XY-plots in whole-cell patch-clamp recordings and fEPSP input-output curves, we used an RM two-way ANOVA test (repeated measurements/analysis) with Sidak’s multiple comparisons. Statistical analysis of fEPSP slope data was performed using the Mann-Whitney test for the three terminal data points. p-values smaller 0.05 were considered a significant difference. In the text and figures, values represent mean ± standard error of the mean (s.e.m.). Statistical significance in XY-plots is indicated in the figure panel. U-values were provided for significant results only. *, p &lt; 0.05; ***, p &lt; 0.001; ‘ns’, non-significant differences. <b>Digital Illustrations.</b> Figures were prepared using Photoshop graphics software (Adobe, San Jose, CA, USA). Image brightness and contrast were adjusted.
description: RNA sequencing data
 are accessible from the galaxy web platform via the following link: https://usegalaxy.eu/u/maximilian.lenz/h/transcriptome-analysisatra-6h-vs-controlhippocampus.