This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.
Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.
The encoding and storage of information in the brain still remains the most significant and keenly pursued challenge in neuroscience. Over the years, long-term potentiation (LTP) and long-term depression (LTD) have emerged as the leading cellular correlates of memory1,2. These activity dependent changes, which exhibit input specificity and associativity, result in the stabilization of memory traces in the neuronal networks 1,3,4. The maintenance of the two forms of synaptic plasticity requires the synthesis of plasticity-related products (PRPs)5-10. Synapse specificity that involves the interaction of newly synthesized protein only with specific activated synapses expressing LTP or LTD, is critical to memory. This specificity is explained by the concept of ‘Synaptic Tagging and Capture’ (STC), where the PRPs interact with recently active, ‘tagged’ synapses11,12. The STC process offers a framework for associative properties of memories at the cellular level. It provides us with a conceptual basis of how short-term forms of plasticity are transformed into long-lasting forms of plasticity in an associative and time-dependent manner13.
During the process of STC, a strong tetanization in one input that leads to protein synthesis dependent late-LTP, results in the reinforcement of a protein synthesis independent early-LTP induced in another independent input on to the same population of neurons into a persistent one13. The setting of a local synaptic tag by a transient neural activity and the synthesis of the diffusible PRPs by the strong neural activity are the two key events during STC13,14. The capture of the PRPs by the recently potentiated ‘tagged’ synapses is fundamental to the maintenance of long-term potentiation. Many studies have been done to confirm the existence of STC phenomenon15-17 and identify the candidate ‘tags’18 and ‘PRPs’19. Calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase1/2 (ERK1/2); CaMKIV, Protein Kinase M (PKM) and brain-derived neurotrophic factor (BDNF) are some of the candidate molecules for ‘tag’ and ‘PRP’ respectively19-21. The synaptic tagging model has further been expanded to include the positive associative interactions between LTP and LTD – the “synaptic cross-tagging”22. In synaptic cross-tagging, a late LTP/ LTD in one synaptic input transforms the opposite protein synthesis-independent early-LTD/LTP in an independent input into its long-lasting form or vice versa22.
The hippocampal slice preparation is the most widely used model in the studies of long-term synaptic plasticity23,24. Much of the understanding about the concepts of synaptic tagging and cross- tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the labs are still unable to study these processes. Experimental conditions for the preparation of rat hippocampal slices and the recording of stable late-LTP/LTD for extended hours are extremely important to study synaptic tagging/cross-tagging23,25,26. This article describes the detailed experimental procedures for studying long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.
Akut hippocampale udsnit er en fremragende model til undersøgelse af LTP og andre funktionelle plasticitetsegenskaber processer såsom STC og cross-capture. Det bevarer en stor del af det laminare strukturelle netværk af hippocampus kredsløb, giver præcise Elektrodeplaceringerne og tilbyder ved siden af, en åben platform til hurtig neurofarmakologiske manipulation uden blod-hjerne-barrieren.
Denne artikel beskriver den metode for udarbejdelse af levedygtige akutte hippocampusskiver fra unge voksne rotter og bruge dem til at undersøge STC og cross-tagging. Tidligere forskning har understreget, at køn og alder af de dyr, er vigtige faktorer at overveje til brug i elektrofysiologi undersøgelser. 27,28 Derfor unge voksne dyr med fuldt udtrykt voksne receptor funktioner (Wistar hanrotter i alderen 5-7 uger) anvendes. 23 Asymmetries i forbindelserne mellem venstre og højre hippocampus er blevet bemærket i gnavere 29 ogDer er rapporteret om store forskelle i NMDA-receptor-ekspression samt 34. Vi har anvendt den rigtige hippocampus for at være i overensstemmelse med vores tidligere undersøgelser LTP. 23,32 imidlertid en af hippocampusen kan anvendes, så længe sammenhæng opretholdes.
Som i enhver protokol, er det meget vigtigt at udføre isolering og udskæring procedurer hurtigt, men pas på, at vævet ikke er strakt, beskadiget, gjort tør eller hypoksisk. Variationerne i pH, temperatur og ioniske sammensætning af løsninger kan have dybtgående virkning på levedygtigheden af skiverne og resultaterne. Derfor bør undgås sådanne variationer. Det er blevet observeret, at glutamat receptor-afhængig frigivelse calcium forekommer under fremstillingstrinnene irreversibelt kan påvirke proteinsyntese i nervevæv 35,36, 37. Brug af manuel væv skæremaskiner kan bidrage til at minimere dette ved at tillade, at processen kan gennemføres meget hurtigt sammenlignet med VIbraslicers. Men mange laboratorier også effektivt at bruge vibraslicers med de nødvendige forholdsregler for at bevare skive levedygtighed. En anden vigtig faktor at overveje, er den lange inkubationstid, før du starter forsøgene. Det er blevet bemærket at være virkelig afgørende at opnå stabilitet i metaboliske statslige og kinaseaktivering niveauer i skiver efter forstyrrelse forårsaget under forberedelse 23. Sådan stabilitet er nødvendig for konsistens i de lange optagelser. Vi igen understrege på denne iagttagelse og foreslå den lange inkubation timer på omkring 3 timer.
En række stimuleringsparametre vides at inducere LTP, men de molekylære mekanismer fremkaldt i hvert fald ikke være den samme (for oversigt se 38). Dette kan påvirke holdbarheden og andre egenskaber ved LTP, som til gengæld kan påvirke resultaterne af synaptiske tagging og capture eksperimenter. Derfor er det vigtigt at validere stimulation paradigmer og karakteristikaaf den fremkaldte LTP under betingelserne for den udøvende laboratoriet og vedligeholde konsistens.
Vi generelt anser ikke eksperimenter med meget store præsynaptiske fiber flugtninger og med maksimal fEPSPs mindre end 0,5 mV og forsøg med væsentlige ændringer i fiberen volley under optagelserne også afvises. Endvidere, mens de udfører to-pathway eller tre-pathway eksperimenter, er det vigtigt at sikre pathway uafhængighed. Dette kan udføres med en parret-puls lettelse protokol 28.
En ulempe af grænsefladen registreringssystemer er dannelsen af kondens dråber på elektroderne i de lange optagelse timer på grund af den temperatur og luftfugtighed forskelle mellem kammeret og omgivelserne. Disse dråber skal nøje blottet fra tid til anden. Ellers kan dråberne dryppe ned på skiver og forårsage forstyrrelser eller endda tab af signaler. Vi plejer tackle dette by dygtigt blotting dråberne styret under mikroskop under anvendelse af en slank filtrerpapir væge, uden at røre elektroderne. Imidlertid ville den bedste løsning være at bruge en centraliseret varmesystem, såsom ETC system udviklet af University of Edinburgh forskere.
På en afsluttende bemærkning, en række metoder eksisterer i de laboratorier over hele verden, der anvendes til fremstilling af hippocampusskiver til forskellige eksperimentelle formål. Hver af proceduren giver nogle fordele i forhold til den anden. Man skal omhyggeligt optimere minut detaljer af protokollen, der passer til formålet med forsøget. Vi håber, at denne artikel hjælper med at forbedre nogle aspekter af metoden til at studere sent associative processer såsom STC og cross-capture.
The authors have nothing to disclose.
This video article is sponsored by Cerebos Pacific Limited. This work is supported by National Medical Research Council Collaborative Research Grant (NMRC-CBRG-0041/2013) and Ministry of Education Academic Research Funding (MOE AcRF- Tier 1 – T1-2012 Oct -02).
I. Dissection Tools | |||
1. Bandage scissors | KLS Martin, Germany | 21-195-23-07 | |
B-Braun/Aesculap, Germany | LX553R | ||
2. Iris scissors | B-Braun/Aesculap, Germany | BC140R, | |
BC100R | |||
3. Bone rongeur | World Precision Instruments (WPI), Germany | 14089-G | |
4. Scalpel | World Precision Instruments (WPI), Germany; | 500236-G | |
B-Braun/Aesculap, Germany | |||
BB73 | |||
5. Scalpel blade#11 | B-Braun/Aesculap, Germany | BB511 | |
6. Sickle scaler | KLS Martin, Germany | 38-685-00 | |
7. Angled forceps | B-Braun/Aesculap, Germany | BD321R | |
8. Anesthetizing/Induction chamber | MIP Anesthesia Technologies (Now, Patterson Scientific), Oregon | AS-01-0530-LG | |
II. ACSF component chemicals | |||
1. Sodium chloride (NaCl) | Sigma-Aldrich | S5886 | |
2. Potassium chloride (KCl) | Sigma-Aldrich | P9541 | |
3. Magnesium sulphate heptahydrate (MgSO4.7H20) | Sigma-Aldrich | M1880 | |
4. Calcium chloride dihydrate (CaCl2.2H2O) | Sigma-Aldrich | C3881 | |
5. Potassium phosphate monobasic (KH2PO4) | Sigma-Aldrich | P9791 | |
6. Sodium bicarbonate (NaHCO3) | Sigma-Aldrich | S5761 | |
7. D-Glucose anhydrous (C6H12O6) | Sigma-Aldrich | G7021 | |
III. Electrophysiology Instruments | |||
1. Microscope | Olympus, Japan | Model SZ61 | |
2. Temperature Controller | Scientific Systems Design Inc. Canada | PTC03 | |
3. Differential AC Amplifier | AM Systems, USA | Model 1700 | |
4. Isolated Pulse Stimulator | AM Systems, USA | Model 2100 | |
5. Oscilloscope | Rhode & Schwarz | HM0722 | |
6. Digital-Analog Converter | Cambridge Electronic Design Ltd. Cambridge, UK | CED-Power 1401-3 | |
7. Interface Brain Slice Chamber | Scientific Systems Design Inc. Canada | BSC01 | |
8. Tubing Pump | Ismatec, Idex Health & Science, Germany | REGLO-Analog | |
9. Carbogen Flowmeter | Cole-Parmer | 03220-44 | |
10. Fiber Light Illuminator | Dolan-Jenner Industries | Fiber Lite MI-150 | |
11. Micromanipulators | Marzhauser Wetzlar, Germany | 00-42-101-0000 (MM-33) | |
00-42-102-0000 (MM-32) | |||
12. Electrodes | AM Systems, USA | 571000 (Stainless steel; 0.010, 5MΩ, 8 degree) |