We wish to thank Yvonne Ducho and Kathrin Pohlmann for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 779) and by the State Saxony-Anhalt / European Regional Development Fund (ERDF) via the Center for Behavioral Brain Sciences (CBBS).

<ol>
	<li>Lamprecht, R., LeDoux, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+plasticity+and+memory.">Structural plasticity and memory.</a> Nat Rev Neurosci. 5 (1), 45-54 (2004).</li><li>Bingol, B., Schuman, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+protein+degradation+by+the+ubiquitin+proteasome+system.">Synaptic protein degradation by the ubiquitin proteasome system.</a> Curr Opin Neurobiol. 15 (5), 536-541 (2005).</li><li>Richter, J. D., Klann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+synaptic+plasticity+and+memory+last:+mechanisms+of+translational+regulation.">Making synaptic plasticity and memory last: mechanisms of translational regulation.</a> Genes Dev. 23 (1), 1-11 (2009).</li><li>Rosenberg, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+protein+expression+in+synaptic+plasticity+and+memory+consolidation.">The roles of protein expression in synaptic plasticity and memory consolidation.</a> Front Mol Neurosci. 7, 86 (2014).</li><li>Scheich, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+semantics+of+learning+and+crossmodal+processing+in+auditory+cortex:+the+semantic+processor+concept.">Behavioral semantics of learning and crossmodal processing in auditory cortex: the semantic processor concept.</a> Hear Res. 271 (1-2), 3-15 (2011).</li><li>K&#228;hne, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+proteome+changes+in+mouse+brain+regions+upon+auditory+discrimination+learning.">Synaptic proteome changes in mouse brain regions upon auditory discrimination learning.</a> Proteomics. 12 (15-16), 2433-2444 (2012).</li><li>Reichenbach, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+dopamine+signalling+on+long-term+memory+formation+and+consolidation+in+rodent+brain.">Differential effects of dopamine signalling on long-term memory formation and consolidation in rodent brain.</a> Proteome Sci. 13, 13 (2015).</li><li>K&#228;hne, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+rearrangements+after+auditory+learning:+high-resolution+profiling+of+synapse-enriched+protein+fractions+from+mouse+brain.">Proteome rearrangements after auditory learning: high-resolution profiling of synapse-enriched protein fractions from mouse brain.</a> J Neurochem. , (2016).</li><li>Li, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+proteomics+of+rat+synaptic+proteins:+correlation-profiling+by+isotope-coded+affinity+tagging+in+conjunction+with+liquid+chromatography-tandem+mass+spectrometry+to+reveal+post-synaptic+density+specific+proteins.">Organelle proteomics of rat synaptic proteins: correlation-profiling by isotope-coded affinity tagging in conjunction with liquid chromatography-tandem mass spectrometry to reveal post-synaptic density specific proteins.</a> J Proteome Res. 4 (3), 725-733 (2005).</li><li>Carlin, R. K., Grab, D. J., Cohen, R. S., Siekevitz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+postsynaptic+densities+from+various+brain+regions:+enrichment+of+different+types+of+postsynaptic+densities.">Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.</a> J Cell Biol. 86 (3), 831-845 (1980).</li><li>Smalla, K. H., Klemmer, P., Wyneken, U., Dermietzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Cytoskeleton - Imaging, Isolation, and Interaction. , 265-282 (2012).</li><li>Smalla, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synaptic+glycoprotein+neuroplastin+is+involved+in+long-term+potentiation+at+hippocampal+CA1+synapses.">The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses.</a> Proc Natl Acad Sci U S A. 97 (8), 4327-4332 (2000).</li><li>Shapiro, A. L., Vinuela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Biophys Res Commun. 28 (5), 815-820 (1967).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Shevchenko, A., Wilm, M., Vorm, O., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+sequencing+of+proteins+silver-stained+polyacrylamide+gels.">Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.</a> Anal Chem. 68 (5), 850-858 (1996).</li><li>Thingholm, T. E., Larsen, M. R., Ingrell, C. R., Kassem, M., Jensen, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TiO(2)-based+phosphoproteomic+analysis+of+the+plasma+membrane+and+the+effects+of+phosphatase+inhibitor+treatment.">TiO(2)-based phosphoproteomic analysis of the plasma membrane and the effects of phosphatase inhibitor treatment.</a> J Proteome Res. 7 (8), 3304-3313 (2008).</li><li>Carmona-Saez, P., Chagoyen, M., Tirado, F., Carazo, J. M., Pascual-Montano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GENECODIS:+a+web-based+tool+for+finding+significant+concurrent+annotations+in+gene+lists.">GENECODIS: a web-based tool for finding significant concurrent annotations in gene lists.</a> Genome Biol. 8 (1), 3 (2007).</li><li>Bonn, S., Seeburg, P. H., Schwarz, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+expression+of+alpha-+and+gamma-protocadherins+alters+their+presenilin-dependent+processing.">Combinatorial expression of alpha- and gamma-protocadherins alters their presenilin-dependent processing.</a> Mol Cell Biol. 27 (11), 4121-4132 (2007).</li><li>Tweedie-Cullen, R. Y., Reck, J. M., Mansuy, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+mapping+of+post-translational+modifications+on+synaptic,+nuclear,+and+histone+proteins+in+the+adult+mouse+brain.">Comprehensive mapping of post-translational modifications on synaptic, nuclear, and histone proteins in the adult mouse brain.</a> J Proteome Res. 8 (11), 4966-4982 (2009).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differential+hippocampal+phosphoproteome+of+Apodemus+sylvaticus+paralleling+spatial+memory+retrieval+in+the+Barnes+maze.">The differential hippocampal phosphoproteome of Apodemus sylvaticus paralleling spatial memory retrieval in the Barnes maze.</a> Behav Brain Res. 264, 126-134 (2014).</li></ol>

The authors have nothing to disclose.

The study presents a methodological workflow optimized for an accurate quantitative profiling of synaptic protein expression changes during learning and memory consolidation in different brain areas of mice. The setup provides the opportunity to study the protein expression on the level of a single animal despite of the required application of at least three technical replicates per sample for mass spectrometric analysis.
The methodology takes into account the particular protein composition of the pre- and postsynapse consisting of high molecular weight scaffold proteins but also of important mediator proteins bearing medium or lower molecular weights. The in-solution digests of synaptosomal preparations result in an efficient generation and, hence, an over-representation of scaffold-derived peptides. This, in turn, may suppress the analysis of smaller or lower abundant proteins. The suggested preparation of SDS-PAGE fractions from an aliquot of each sample combined with an in-gel digestion procedure in parallel facilitates the analysis of medium and low abundance proteins and represents a highly recommended complementary method. After separate mass spectrometric application of all fractions derived from a sample (e.g. in-solution digest, in-gel digest, combined phospho-enriched fractions) the corresponding MS/MS data sets can be combined and further calculated for protein identification and quantification by PEAKS software or alternative popular software packages.
Alternatively, the individual application of in-gel-digestion-derived fractions of a sample (separately processed gel-areas of a sample lane) and fractions generated of the in-solution digested sample (e.g. by ion exchange chromatography) to mass spectrometry can increase the analytical depth. However, this extended workflow dramatically increases the required time for LS-MS/MS data acquisition. For generation of a detailed molecular sequence of synaptic protein rearrangements during learning and memory formation a specified time course of the proteomic profiling is required. This time course may start immediately after or even during the first training session and covers a close-meshed time frame until the animals&#39; performance reached the asymptotic level of the learning curve after approx. 8 - 10 days of training (see Figure 2 for details).
The analysis of phosphorylation changes of synaptic proteins requires a particular focus on the selected time frames during FMTD learning. On the one hand signaling cascades initiating synaptic protein rearrangements known to be triggered by protein phosphorylations and dephosphorylations are expected at very early stages of animal training. On the other hand, there are long lasting modifications of multiple phosphorylated synaptic proteins known which regulate the connectivity and assembly within the synaptic architecture19, 20. Those posttranslational modifications are expected even at later time points of memory consolidation.
The complex datasets generated by this proteomic workflow require bioinformatic processing to identify participating molecular pathways and key molecules. Meta-analysis shows significant overrepresented pathways, which play a role in learning and memory processes.

Learning is based on the formation of memory traces and their maintenance. It is widely accepted that one underlying mechanism may represent an activity-dependent formation of new and/or rearrangement of existing synaptic contacts between neurons. On the molecular level, various protein modifications, subcellular relocalizations and changes in the turnover of synaptic proteins have been described1-4(Lamprecht, 2004 #8). However, most studies so far focused on selected proteins rather than on the global but complex synaptic proteome composition. The present approach allows an unbiased screening for synaptic proteome changes in mouse brain regions after a learning experiment. It is suitable to render time-point dependent molecular snapshots of the learning-induced reorganization of the synaptic architecture. The described workflow requires a particular teamwork of different specialists in animal behavior, protein biochemistry, mass spectrometry and bioinformatics.
The chosen learning paradigm, i.e. frequency-modulated tone discrimination (FMTD), is a well-characterized auditory discrimination task in rodents5. Learning and long-term memory formation in this shuttle box Go/No-Go-task involves mechanisms depending on increased cortical dopamine signaling and protein synthesis. Accordingly, recent proteomic studies on gerbils and mice revealed dopamine- and learning-induced plastic rearrangements of synaptic components in cortical, but also in more basal brain regions that supposedly interact during FMTD learning and memory6-8. This illustrates that memory formation involves a complex interplay of various brain regions and thus, might be differentially regulated within these regions on the proteome level. Therefore, dissection of selected cortical and subcortical mouse brain regions is included in the workflow.
Furthermore, the reliable characterization even of weak changes in synaptic protein composition requires an enrichment of pre- and postsynaptic compartments rather than the analysis of homogenates or crude membrane fractions9. Therefore, the preparation of synaptosomes utilizing established protocols prior to proteomic analysis is described in order to increase the detection level and the dynamic range for synapse-specific proteins10,11.
An essential prerequisite to use label-free high-resolution mass spectrometry for quantitative purposes is a high degree of similarity of protein samples. As rather minor changes in synaptic protein composition are expected to occur after learning, a label-free approach will be appropriate to compare corresponding protein samples obtained from trained and na&#239;ve mice. Alternatively, condition-specific label strategies of proteins/peptides using stable isotopes (e.g. TMT, iTRAQ , ICPL and SILAC) as well as MS2-based label free quantification (SWATH) are useful, but they are more expensive than the chosen label-free approach or need special mass spectrometric hardware.
Since proteomic screenings often yield complex data sets, bioinformatic processing is recommended for appropriate data interpretation. Additional meta-analyses may support a better understanding of potential molecular mechanisms underlying paradigm-related changes and the identification of involved key cellular processes and signaling pathways. Appropriate methodologies are also described below.

<table><tbody><tr><td>3 M Empore Solid Phase Extraction- Filter</td><td>3M Bioanalytical Technologies</td><td>4245SD</td><td>7 mm/3 ml</td></tr><tr><td>Acclaim PepMap 100</td><td>Dionex/Thermo Scientific</td><td>164564</td><td>100 &amp;micro;m x 2 cm, C18</td></tr><tr><td>Acclaim PepMap 100</td><td>Dionex/Thermo Scientific</td><td>164569</td><td>75 &amp;micro;m x 25 cm, C18</td></tr><tr><td>Acetic acid</td><td>Carl Roth GmbH</td><td>3738.1</td><td></td></tr><tr><td>Acetonitrile (ACN)</td><td>Carl Roth GmbH</td><td>AE70.2</td><td></td></tr><tr><td>Acrylamide (30%)</td><td>AppliChem</td><td>A0951</td><td></td></tr><tr><td>Ammonium hydrogen carbonate&amp;nbsp;&amp;nbsp;</td><td>Fluka</td><td>9830</td><td></td></tr><tr><td>Ammonium hydroxide&amp;nbsp;</td><td>Fluka&amp;nbsp;</td><td>44273</td><td></td></tr><tr><td>Ammonium persulfate&amp;nbsp; (APS)</td><td>AppliChem</td><td>A2941</td><td></td></tr><tr><td>Biofuge &lt;em&gt;pico&lt;/em&gt;</td><td>Heraeus GmbH</td><td>75003280</td><td></td></tr><tr><td>Blue R-250&amp;nbsp;</td><td>SERVA Electrophoresis GmbH</td><td>17525</td><td></td></tr><tr><td>Bromophenol Blue</td><td>Pharmacia Biotech</td><td>17132901</td><td></td></tr><tr><td>C57BL/6J mice</td><td>Charles River&amp;nbsp;</td><td></td><td></td></tr><tr><td>Cantharidin</td><td>Carl Roth GmbH</td><td>3322.1</td><td></td></tr><tr><td>Centrifuge tubes for MLS-50</td><td>Beckman Coulter</td><td>344057</td><td></td></tr><tr><td>Centrifuge tubes for TLA 100.1 rotor</td><td>Beckman Coulter</td><td>343776</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>AppliChem</td><td>A1101</td><td></td></tr><tr><td>Eppendorf 5417R centrifuge</td><td>VWR</td><td>22636138</td><td></td></tr><tr><td>Eppendorf A-8-11 rotor</td><td>VWR</td><td>5407000317</td><td></td></tr><tr><td>Formic acid</td><td>Fluka</td><td>14265</td><td></td></tr><tr><td>GeneCodis</td><td>http://genecodis.cnb.csic.es/</td><td></td><td></td></tr><tr><td>Gephi</td><td>https://gephi.org/</td><td></td><td></td></tr><tr><td>Glycerol</td><td>AppliChem</td><td>A1123</td><td></td></tr><tr><td>Glycine</td><td>AppliChem</td><td>A1067</td><td></td></tr><tr><td>HALT Phosphatase Inhibitor Cocktail&amp;nbsp;</td><td>Pierce /Thermo Scientific</td><td>78420</td><td></td></tr><tr><td>HEPES Buffer solution&amp;nbsp;</td><td>PAA Laboratories GmbH</td><td>S11-001</td><td></td></tr><tr><td>Homogenization vessel 2 ml</td><td>Sartorius AG</td><td>854 2252</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>Ingenuity Pathway Analysis</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Iodoacetamide (IAA)</td><td>Sigma-Aldrich</td><td>I1149</td><td></td></tr><tr><td>Laboratory drilling drive &amp;nbsp;K-ControlTLC 4957&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Kaltenbach &amp;amp; Vogt GmbH</td><td>182997</td><td></td></tr><tr><td>LTQ Tune Plus 2.7.0.1112 SP2</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>LTQ Orbitrap Velos Pro</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Macs-mix tube rotator</td><td>Miltenyi Biotech</td><td>130-090-753</td><td></td></tr><tr><td>Magic Scan 4.71</td><td>UMAX</td><td></td><td></td></tr><tr><td>Methanol</td><td>Carl Roth GmbH</td><td>AE71.2</td><td></td></tr><tr><td>MLS-50 rotor</td><td>Beckman Coulter</td><td>367280</td><td></td></tr><tr><td>Optima MAX Ultracentrifuge</td><td>Beckman Coulter</td><td>364300</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder</td><td>Thermo Scientific</td><td>26616</td><td></td></tr><tr><td>PEAKS 7.5</td><td>Bioinformatic Solutions&amp;nbsp;</td><td></td><td></td></tr><tr><td>Phosphatase Inhibitor Cocktail 3</td><td>Sigma-Aldrich</td><td>P0044</td><td></td></tr><tr><td>PhosphoRS 3.1</td><td>IMP/IMBA/GMI</td><td></td><td></td></tr><tr><td>PhosSTOP</td><td>Roche</td><td>4906845001</td><td></td></tr><tr><td>Plunger/pestle made of PTFE</td><td>Sartorius AG</td><td>854 2651</td><td></td></tr><tr><td>PotterS homogenizer</td><td>Sartorius AG</td><td>853 3024</td><td></td></tr><tr><td>Protease Inhibitor complete mini</td><td>Roche</td><td>4693159001</td><td></td></tr><tr><td>Quantity One 4.5.1</td><td>BioRad</td><td></td><td></td></tr><tr><td>RapiGest</td><td>Waters</td><td>186002122</td><td></td></tr><tr><td>Shuttle box</td><td>Coulbourne Instruments</td><td></td><td></td></tr><tr><td>Sodium dodecylsulfate (SDS)</td><td>AppliChem</td><td>A1112</td><td></td></tr><tr><td>Sodium molybdate</td><td>Carl Roth GmbH</td><td>274.2</td><td></td></tr><tr><td>Sodium tartrate dihydrate</td><td>Sigma-Aldrich</td><td>228729</td><td></td></tr><tr><td>SONOREX RK 156 Ultrasonic Bath</td><td>BANDELIN electronic GmbH &amp;amp; Co. KG</td><td>305</td><td></td></tr><tr><td>Soundproof chamber</td><td>Industrial Acoustics Company</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Carl Roth GmbH</td><td>4621.2</td><td></td></tr><tr><td>Tetramethyl ethylene -1,2-diamine (TEMED)</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Thermomixer&lt;em&gt; basic&lt;/em&gt;</td><td>CallMedia</td><td>111000</td><td></td></tr><tr><td>&lt;a name=&quot;RANGE!A60&quot;&gt;Titansphere TiO 5&amp;micro;m&lt;/a&gt;</td><td>GL Sciences Inc. Japan</td><td>502075000</td><td></td></tr><tr><td>TLA 100.1 rotor</td><td>Beckman Coulter</td><td>343840</td><td></td></tr><tr><td>Trifluoro acetic acid&amp;nbsp; (TFA)</td><td>Sigma-Aldrich</td><td>T6508</td><td></td></tr><tr><td>Tris ( hydroxymethyl) aminomethane (TRIS)</td><td>AppliChem</td><td>A1086</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8532</td><td></td></tr><tr><td>Trypsin Gold</td><td>Promega</td><td>V5280</td><td></td></tr><tr><td>Ultimate 3000 Ultra HPLC&amp;nbsp;</td><td>Dionex/Thermo Scientific</td><td></td><td></td></tr><tr><td>Ultracentrifuge tube</td><td>Beckman Coulter</td><td>343776</td><td></td></tr><tr><td>Unijet II Refrigerated Aspirator</td><td>Uniequip Laborger&amp;auml;te- und Vertriebs GmbH</td><td></td><td></td></tr><tr><td>UNIVAPO 100 H Concentrator Centrifuge&amp;nbsp;</td><td>Uniequip Laborger&amp;auml;te- und Vertriebs GmbH</td><td></td><td></td></tr><tr><td>Urea</td><td>AppliChem</td><td>A1049</td><td></td></tr><tr><td>Water (high quality purifed)</td><td></td><td></td><td>Resistivity: &gt; 18.2 M&amp;Omega;*cm at 25 &amp;deg;C&amp;nbsp; Pyrogens: &amp;lt; 0.02 EU/ml&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TOC: &amp;lt; 10 ppb</td></tr><tr><td>Xcalibur 3.0.63</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>ZipTip&lt;sub&gt;C18&lt;/sub&gt; Pipette Tips</td><td>MILLIPORE</td><td>ZTC18S960</td><td></td></tr></tbody></table>

high resolution quantitative synaptic proteome profiling of mouse brain regions after auditory discrimination learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

Figure 1 summarizes the complete workflow of quantitative synaptic proteome profiling of mouse brain regions after auditory discrimination learning. It starts with the animal training in a shuttle box. In the example shown in Figure 2, mice started to show significant FM tone discrimination in the 4th training session, indicating efficient learning. Animals are sacrificed at selected time points for brain area dissection. The required enrichment of synapses can either be achieved by the preparation of synaptosomes or alternatively by the preparation of a PSD-enriched fraction, both described in detail in Figure 3. The PSD-enrichment method has been developed for low tissue amounts, e.g. 1 - 2 hippocampal slices from rat brain12, 18. It requires small tubes, PTFE pestles fitting to these tubes, and a laboratory drilling drive for powering the pestle.
Due to the particular protein composition of synaptosomes, it is strongly recommend to perform the sample preparation in two different but complementary ways. Scaffolds of the PSDs are often very high molecular weight proteins occurring in high stoichiometry. In-solution digest is the best way to extract them efficiently but may lead to an oversampling of the generated peptide mixture. The in-gel digest performed of the same sample in parallel can exclude those high molecular weight proteins and favor the analysis of proteins with medium and lower molecular weight. For a comprehensive analysis both types of proteolytic digests are recommended.
The different amounts of tissues of the brain areas investigated require an adjustment of the applied material for better comparison. Within the four investigated brain areas the auditory cortex is generally the limiting factor. The material of all other brain areas should carefully be adjusted to the amount of the auditory cortex after preparation of synaptosomes or PSD-enriched fractions (see 3.1.1.). Typical weights of freshly prepared brain areas from mice are as following: auditory cortex (AC): ~ 50 mg; hippocampus (HIP): ~ 90 mg; striatum (STR): ~ 120 mg and frontal cortex (FC): ~ 100 mg.
The PSD-enrichment method described in section 2.3 allowed the identification of approximately 1500 different proteins and approximately 250 different phospho-peptides per brain region on the level of a single animal (Table 1). Proteomic analysis 24 h after the first training session revealed that 7.3% of the identified proteins and 5.8% of the phospho-peptides showed significant (p&#60; 0.05) quantitative changes in their synaptic expression compared to na&#239;ve controls (Table 1). A conspicuous tendency for down regulation of synaptic scaffolds may point to a pronounced rearrangement of the synaptic architecture during early stages of FMTD learning. The vast majority of the regulated proteins were altered in a brain region-specific manner, whereas only 22% were found to be regulated in two or more brain areas. Six selected examples are shown in Figure 4.
Meta-analysis of the complex results by IPA provides evidence for the particular participation/manipulation of the following canonical pathways: &#34;Clathrin-mediated Endocytosis Signaling&#34;, &#34;Axonal Guidance Signaling&#34;, &#34;Calcium Signaling&#34;, &#34;RhoA Signaling&#34;, &#34;Notch Signaling&#34;, &#34;Remodeling of Epithelial Adherens Junctions&#34;, &#34;Glutamate Receptor Signaling&#34;, &#34;GABA Receptor Signaling&#34;, &#34;Dopamine Receptor Signaling&#34; and &#34;Synaptic Long-Term Potentiation&#34;.
Single enrichment analysis revealed significant overrepresented biological processes in the frontal cortex concerning protein transport, cell adhesion, phosphorylation, endocytosis, vesicle-mediated transport, forebrain development and axonogenesis (Figure 5). In the auditory cortex biological processes including ion transport, translation, mRNA transport, protein transport and learning were noticeable. The analysis of the protein fraction of the hippocampus detects significantly enriched processes related to ion transport, cell cycle, translation, phosphorylation and nervous system development. In the striatum, overrepresented biological processes including mRNA transport, vesicle-mediated transport, axonogenesis, proteolysis, protein transport and endocytosis were found.
<img alt="Figure 1" src="/files/ftp_upload/54992/54992fig1.jpg" /> 
Figure 1: Systematic Workflow of the Methodological Approach. This figure schematically summarizes the workflow of high resolution quantitative profiling of brain area specific synaptic protein composition. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54992/54992fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54992/54992fig2.jpg" /> 
Figure 2: Example of the Performance of Mice in the FM Tone Discrimination Task. Animals show an increasing rate of hits (blue curve) and a decreasing rate of false alarms (black curve) in the course of training sessions. Significant discrimination occurs from the fourth session. Error bars are provided as SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54992/54992fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54992/54992fig3.jpg" /> 
Figure 3: Preparation of the Synaptosome and the PSD-enriched Fraction. A: Synaptosome preparation. B: PSD-enriched fraction preparation. Both figures explain the detailed workflow of preparation of synaptosomes or alternatively PSD-enriched fractions from brain tissues. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54992/54992fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54992/54992fig4.jpg" /> 
Figure 4: Selected Quantitative Proteomic Results. The relative synaptic abundances of selected proteins are compared between mice trained on the FMTD task (AV, n= 6) and na&#239;ve control mice (NV, n= 6) 24 hr after the first training session. The abundance values were calculated as median of the peak areas of the three most intense peptides of a protein. Proteins with significant abundance changes (AV/NV; t-test) are marked within the plots: * p&#60; 0.05, ** p&#60; 0.01, *** p&#60; 0.005. Error bars are provided as SD. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54992/54992fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54992/54992fig5.jpg" /> 
Figure 5: Visualization of Biological Pathways for Frontal Cortex by GeneCodis/Gephi. Only significant terms of the Gene Ontology (GO) database (http://geneontology.org) related to &#8220;Biological process&#8221; with a minimum protein number of three are shown here. Nodes represent GO terms, the size of the node, the line width and number of connections of a certain node depict the number of proteins, which share this GO term with other nodes. Due to the &#8220;Force Atlas&#8221; method of Gephi, related nodes are clustering closely together.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54992/54992fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brain region</td>
			<td>AC</td>
			<td>FC</td>
			<td>HIP</td>
			<td>STR</td>
			<td>&#8721;</td>
		</tr>
		<tr>
			<td>identified&#160; proteins</td>
			<td>1435</td>
			<td>1758</td>
			<td>1572</td>
			<td>1507</td>
			<td>6272</td>
		</tr>
		<tr>
			<td>regulated proteins (p&#60;0.05)</td>
			<td>59</td>
			<td>130</td>
			<td>162</td>
			<td>108</td>
			<td>459</td>
		</tr>
		<tr>
			<td>&#8593; AV/NV</td>
			<td>8</td>
			<td>4</td>
			<td>76</td>
			<td>35</td>
			<td>123</td>
		</tr>
		<tr>
			<td>&#8595; AV/NV</td>
			<td>51</td>
			<td>126</td>
			<td>86</td>
			<td>73</td>
			<td>336</td>
		</tr>
		<tr>
			<td>identified phosphomotifs</td>
			<td>197</td>
			<td>361</td>
			<td>273</td>
			<td>278</td>
			<td>1109</td>
		</tr>
		<tr>
			<td>regulated phosphomotifs (p&#60;0.05)</td>
			<td>8</td>
			<td>22</td>
			<td>21</td>
			<td>14</td>
			<td>65</td>
		</tr>
		<tr>
			<td>&#8593; AV/NV</td>
			<td>4</td>
			<td>17</td>
			<td>5</td>
			<td>9</td>
			<td>35</td>
		</tr>
		<tr>
			<td>&#8595; AV/NV</td>
			<td>4</td>
			<td>5</td>
			<td>16</td>
			<td>5</td>
			<td>30</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of a Proteomic Result. This table summarizes a representative proteomic experiment of trained mice (AV, n= 6) 24 hr after the first training session compared to their na&#239;ve controls (NV, n= 6). The sum of 459 regulated proteins includes overlapping regulations. 283 different regulations were determined as brain specific. In detail, 57 proteins are regulated in two brain regions, 18 protein regulations were detected in three brain regions and only 2 proteins are regulated in all four investigated brain areas.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Error tolerances</td>
		</tr>
		<tr>
			<td>precursor mass (fourier transformation mass spectrometry)</td>
			<td>10 ppm</td>
		</tr>
		<tr>
			<td>fragment ion mass (linear ion trap)</td>
			<td>0.6 Da</td>
		</tr>
		<tr>
			<td>Maximum missed cleavages per peptide</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Fixed modifications</td>
		</tr>
		<tr>
			<td>for in-gel-digested samples</td>
			<td>Carbamidomethylation of Cysteine</td>
		</tr>
		<tr>
			<td>for in-solution-digested samples</td>
			<td>Methylthiolation of Cysteine</td>
		</tr>
		<tr>
			<td>Variable modifications</td>
			<td>Oxidation of Methionine</td>
		</tr>
		<tr>
			<td></td>
			<td>Deamidations of Asparagin and/or Glutamine&#160;</td>
		</tr>
		<tr>
			<td>Database</td>
			<td>Uniprot/Sprot&#160;</td>
		</tr>
		<tr>
			<td>Taxonomy</td>
			<td>mouse</td>
		</tr>
		<tr>
			<td>Statistical identification-acceptance settings</td>
		</tr>
		<tr>
			<td>de novo average local confidence (ALC)</td>
			<td>&#160;&#62; 50%</td>
		</tr>
		<tr>
			<td>Peptide-false discovery rate (FDR, based on est. decoy-fusion)</td>
			<td>&#160;&#60; 1%&#160;</td>
		</tr>
		<tr>
			<td>Protein significance (-10logP, based on modified T-test)</td>
			<td>&#62; 20&#160;</td>
		</tr>
		<tr>
			<td>unique peptides / protein</td>
			<td>&#160;&#8805; 1&#160;</td>
		</tr>
		<tr>
			<td>Quantification settings:</td>
		</tr>
		<tr>
			<td>Peptides used for quantification if:</td>
		</tr>
		<tr>
			<td>Peptide significance (-10logP)</td>
			<td>&#62; 30</td>
		</tr>
		<tr>
			<td>Peptide identification in&#160;</td>
			<td>&#160;&#8805; 50% of samples</td>
		</tr>
		<tr>
			<td>Peptide signal quality</td>
			<td>&#62;1</td>
		</tr>
		<tr>
			<td>Peptide average area</td>
			<td>&#160;&#62; 1E5</td>
		</tr>
		<tr>
			<td>Peptide retention time tolerance</td>
			<td>&#160;&#60; 5 min</td>
		</tr>
		<tr>
			<td>Normalization&#160;</td>
			<td>by total ion current (TIC)</td>
		</tr>
	</tbody>
</table>
Table 2: Settings for Protein Identification (step 4.2.2).

Watch this Scientific Journal Video about High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning at JoVE.com

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.5K Views.  Leibniz Institute for Neurobiology (LIN). The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

Video: High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

Behavior

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

LFP Recording in Mouse Brain for Cognitive Research and Drug Analysis

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal populations. It serves as a crucial tool in cognitive research, particularly in brain regions like the hippocampus and prefrontal cortex. Dual LFP recordings between these areas are of particular interest as they allow the exploration of interregional signal communication. However, methods for performing these recordings are rarely described, and most commercial recording devices are either expensive or lack adaptability to accommodate specific experimental designs. This study presents a comprehensive protocol for performing dual-electrode LFP recordings in the mouse hippocampus and the prefrontal cortex to investigate the effects of antipsychotic drugs and potassium channel modulators on LFP properties in these areas. The technique enables the measurement of LFP properties, including power spectra within each brain region and coherence between the two. Additionally, a low-cost, custom-designed recording device has been developed for these experiments. In summary, this protocol provides a means to record signals with high signal-to-noise ratios in different brain regions, facilitating the investigation of interregional information communication within the brain.

Author Spotlight: Studying Drug Impacts on Brain Signals Using Dual LFP Recording Protocol in Mice

This protocol outlines the use of a custom-designed recording device and electrodes to record local field potentials and investigate information flow in the mouse hippocampus and prefrontal cortex.

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal ...

Quantifying the Distribution of a Presynaptic Protein in the Mouse Brain Using Immunofluorescence

Source: Wallrafen, R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58940">Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence.</a> J. Vis. Exp. (2019).
This video demonstrates the immunofluorescence staining of a mouse brain slice to quantify the distribution of a specific presynaptic protein. The process involves blocking nonspecific sites, applying primary antibodies for the target protein and a reference marker, followed by secondary antibody labeling and nuclear counterstaining. The reference marker ensures accurate quantification by providing a consistent baseline, and the distribution of the presynaptic protein is analyzed across brain regions using confocal microscopy.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunofluorescence

	...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

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