Name: whole-cell currents induced by puff application of gaba in brain slices
Uploaded: 2017-10-12T14:00:00
Description: Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

Forfatterne vil gerne takke følgende organisationer: National Natural Science Foundation of China (31171018, 31171355), videnskab og teknologi afdeling af Guangdong (2013KJCX0054), Natural Science Foundation i Guangdong provinsen ( 2014A030313418, 2014A030313440), og Guangzhou videnskab og teknologi Bureau (201607010320).

Dyr bolig og alle dyr eksperimenter blev udført efter procedurer, der er godkendt af den etiske komité til dyreforsøg på South China Normal University, efter retningslinjerne for Animal Care etableret af National Institute of Sundhed.
 1. forberedelse af løsninger <ol><li>forberede 500 mL af standard kunstige cerebrospinalvæske (ACSF) der indeholder de komponenter, der er beskrevet i vores offentliggjorte arbejde 7 som anført i tabel 1.
	<ol><li>Brug rene glas bæ...

<ol>
	<li>Neher, E., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+currents+recorded+from+membrane+of+denervated+frog+muscle+fibres.">Single-channel currents recorded from membrane of denervated frog muscle fibres.</a> Nature. 260 (5554), 799-802 (1976).</li><li>Sakmann, B., Edwards, F., Konnerth, A., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+used+for+studying+synaptic+transmission+in+slices+of+mammalian+brain.">Patch clamp techniques used for studying synaptic transmission in slices of mammalian brain.</a> Q J Exp Physiol. 74 (7), 1107-1118 (1989).</li><li>Eyo, U. B., 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=Neuronal+hyperactivity+recruits+microglial+processes+via+neuronal+NMDA+receptors+and+microglial+P2Y12+receptors+after+status+epilepticus.">Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors after status epilepticus.</a> J Neurosci. 34 (32), 10528-10540 (2014).</li><li>Dickinson, G. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+determining+the+recruitment+of+inositol+trisphosphate+receptor+channels+during+calcium+puffs.">Factors determining the recruitment of inositol trisphosphate receptor channels during calcium puffs.</a> Biophys J. 105 (11), 2474-2484 (2013).</li><li>Lee, J., 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=Columnar+distribution+of+activity+dependent+gabaergic+depolarization+in+sensorimotor+cortical+neurons.">Columnar distribution of activity dependent gabaergic depolarization in sensorimotor cortical neurons.</a> Mol Brain. 5, 33 (2012).</li><li>Glykys, J., Mody, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+main+source+of+ambient+GABA+responsible+for+tonic+inhibition+in+the+mouse+hippocampus.">The main source of ambient GABA responsible for tonic inhibition in the mouse hippocampus.</a> J Physiol. 582, 1163-1178 (2007).</li><li>Chen, M., 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=APP+modulates+KCC2+expression+and+function+in+hippocampal+GABAergic+inhibition.">APP modulates KCC2 expression and function in hippocampal GABAergic inhibition.</a> Elife. 6, (2017).</li><li>Dunn, A. J., Swiergiel, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+interleukin-1+and+endotoxin+in+the+forced+swim+and+tail+suspension+tests+in+mice.">Effects of interleukin-1 and endotoxin in the forced swim and tail suspension tests in mice.</a> Pharmacol Biochem Behav. 81 (3), 688-693 (2005).</li><li>Engel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+Patch-clamp+Recordings+from+the+Somatodendritic+Domain+of+Nigral+Dopamine+Neurons.">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J Vis Exp. (117), (2016).</li><li>Wondolowski, J., Frerking, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-dependent+postsynaptic+expression+of+kainate+receptors+on+hippocampal+interneurons+in+area+CA1.">Subunit-dependent postsynaptic expression of kainate receptors on hippocampal interneurons in area CA1.</a> J Neurosci. 29 (2), 563-574 (2009).</li><li>Yang, L., Wang, Z., Wang, B., Justice, N. J., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+regulates+Cav1.2+L-type+calcium+channel+levels+and+function+to+influence+GABAergic+short-term+plasticity.">Amyloid precursor protein regulates Cav1.2 L-type calcium channel levels and function to influence GABAergic short-term plasticity.</a> J Neurosci. 29 (50), 15660-15668 (2009).</li><li>Huo, Q., 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=Prefrontal+Cortical+GABAergic+Dysfunction+Contributes+to+Aberrant+UP-State+Duration+in+APP+Knockout+Mice.">Prefrontal Cortical GABAergic Dysfunction Contributes to Aberrant UP-State Duration in APP Knockout Mice.</a> Cereb Cortex. , (2016).</li><li>Zhou, W. L., Gao, X. B., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+Acts+through+Nicotinic+Receptors+to+Enhance+the+Firing+Rate+of+a+Subset+of+Hypocretin+Neurons+in+the+Mouse+Hypothalamus+through+Distinct+Presynaptic+and+Postsynaptic+Mechanisms.">Acetylcholine Acts through Nicotinic Receptors to Enhance the Firing Rate of a Subset of Hypocretin Neurons in the Mouse Hypothalamus through Distinct Presynaptic and Postsynaptic Mechanisms.</a> eNeuro. 2 (1), (2015).</li></ol>

Puff program er almindeligt anvendt til at evaluere postsynaptiske receptor funktion3,4,7, men kræver præcis kontrol i hvert forsøg. Vi beskriver her en procedure, der omfatter hele-celle patch fastspænding, som demonstrerer GABA-puff induceret IPSCs (dvs. eIPSCs) i præfrontal kortikale hjernen skiver. Modstanden i optagelse elektrode er omkring 5 MΩ, mens puff Mikropipetter tip diameter er omkring 2-5 µm. Puff pres er me...

Patch-clamp-teknik, en primære værktøj til at undersøge elektriske signaler i neuroner, blev udviklet i 1970s1,2. En stor fordel ved denne teknik er, at det giver viden om hvordan specifikke behandlinger (fx farmakologiske) kan ændre neuronal funktioner eller kanaler i realtid3. Farmakologiske evaluering af neuronal funktion under hele-celle optagelse i en hjerne skive kræver anvendelse af lægemidler, såsom agonister eller antagonister af specifikke receptorer på neuroner at blive optaget. Denne metode giver mulighed for identifikation af neuronale ændringer, der opstår efter anvendelse af et bestemt stof, hvilket fører til en bedre forståelse af de fysiologiske og patologiske egenskaber af neuroner4. Selv om farmakologiske administration kan foretages via enten perfusion5 eller puff6, er sidstnævnte den overlegne teknik. Navnlig: (i) puff ansøgning hurtigt stigninger narkotika koncentration omkring indspillede neuron til et niveau, således at desensibilisering af membranreceptorer hindres; (ii) mængden af stof pustede er ekstremt lavt, derfor er der ringe effekt på bad-løsning, som derfor reducerer eventuelle uønskede virkninger af de administrerede kemikalier på hjernen udsnit; (iii) puff-protokollen kan blive indstillet og gemt, at gøre forsøget meget præcist reproducerbar; (iv) puff program repræsenterer økonomisk brug af agonister/antagonists, især hvor sådanne reagenser er dyrt eller svært at opnå.
Her vil vi fokusere på optagelsen hele-celle strømninger induceret ved puffing GABA i akut forberedte hjerne skiver, et præparat, der har fordelen, relativt velbevaret hjernens kredsløb. Hvordan vi gennemfører puff-induceret hæmmende strømninger7 vil blive beskrevet i denne artikel. Ved hjælp af cæsium (Cs+)-baseret indre løsninger, og holder neuronerne på 0 mV, vil vi introducere GABA-puff fremkaldte hæmmende postsynaptiske strømme (eIPSCs) med passende tekniske detaljer. Ved hjælp af en musemodel af depression induceret af LPS (LPS) injektion8, vi viser, at eIPSC reduceres amplitude fremkaldt af en GABA puff betydeligt i skiver af LPS-indsprøjtning mus i forhold til køretøjets forskellige betjeningsapparater. Vores hensigt er, at denne artikel bør vise, hvordan puff teknik er alment gældende for undersøgelser med henblik på evaluering af effekten af kemikalier, forbindelser eller narkotika på neuronal aktiviteter i hjernen skiver.

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whole-cell currents induced by puff application of gaba in brain slices

Farmakologiske administration er almindeligt anvendt, når der udføres hele-celle patch-klemme optagelse i hjernen skiver. En af de bedste metoder til drug application under elektrofysiologiske optagelse er puff teknik, som kan bruges til at studere effekten af farmakologisk reagenser på neuronal aktiviteter i hjernen skiver. Den største fordel af puff ansøgning er, at narkotika koncentrationen omkring optagelse site stiger hurtigt, hvilket forhindrer desensibilisering af membranreceptorer. Vellykket brug af puff anvendelse indebærer omhyggelig opmærksomhed på følgende elementer: koncentrationen af stoffet, parametrene for puff mikropipette, afstanden mellem spidsen af en puff mikropipette og neuron indspillet og varighed og pres kørsel pust (pounds per kvadrattomme, psi). I denne artikel beskrives en trinvis procedure til at optage hele-celle strømninger induceret ved puffing gamma - aminosmørsyre (GABA) på en neuron af en præfrontal kortikale skive. Især, kan den samme procedure anvendes med mindre ændringer til andre områder i hjernen som hippocampus og striatum og til forskellige præparater, såsom cellekulturer.

Puff teknik gør det muligt for forskere at studere effekten af lægemidler på bestemte receptorer på overfladen af en given neuron. I denne artikel vil fokusere vi på strømninger medieret af GABA receptorer. Figur 1 viser de puf og optagelse Mikropipetter. At fjerne nogen effekt på registrerede neuroner i det fysiske pres genereret af puff, ACSF og saccharose pust (fig. 2A) bruges som kontrol. Reaktion frem...

Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Hele-celle strømninger induceret af Puff anvendelse af GABA i hjernen skiver

Vi beskriver puff teknik, hvormed farmakologiske reagenser kan administreres under hele-celle patch-klemme optagelse, og fremhæve forskellige aspekter af de funktioner, der er afgørende for dens succes.

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

The overall goal of this puff technique is to conduct the pharmacological administration in whole-cell patch-clamp recording. This method can help answer key questions in the electrophysiology field, such as pharmacological administration. The main advantage of this technique is that the drug concentration around the recording site increases rapidly, thus preventing the membrane receptors from desensitization.Generally, individual new to this method will struggle because the preparation of acute prefrontal cortex slice and the position of puff micropipette are relatively difficult. To begin this procedure, crush the frozen cutting solution to obtain a slush. Keep the slushy solution on ice, and bubble it with 95%oxygen, 5%carbon dioxide.Next, place the dissected head into a beaker filled with ice-cold cutting solution, and take it out after a few seconds. Cut the skull along the midline in a caudal-rostral direction with fine scissors, and remove the lateral skull portions. Then, remove the brain with a fine spatula, and drop it into the ice-cold cutting solution.Afterward, place the brain on the lid of a nine-centimeter Petri dish filled with ice. Rinse the brain with ice-cold cutting solution. Then, cut off the cerebellum and olfactory bulb with a razor blade.Subsequently, apply cyanoacrylate glue to the Vibratome specimen holder. Carefully place the brain block on the drop of glue, rostral side up, and immediately immerse it in the ice-cold cutting solution. Adjust the blade holder to an angle of 15 degrees with reference to the horizontal plane.Section the brain tissues into 350 micron coronal slices. Then, transfer the slides into the recovery chamber filled with ACSF, and incubate them for one hour with constant bubbling of 95%oxygen, 5%carbon dioxide. In our experiment, the preparation of acute prefrontal cortex slice is also critical in the procedure.A healthy brain slice means active neurons that can be patched tightly. In this procedure, make glass borosilicate micropipettes for use as recording electrodes or puff micropipettes. For recording electrodes, tip resistances are in the range of four to six megaohm, while puff micropipettes have tip diameters in the range of two to five micrometers.Add sodion channel blocker to ACSF to block fast sodion channel and thus action potentials, and maintain a constant flow rate of this solution of two to three milliliter per minute in the recording chamber. Bubble the ACSF with 95%oxygen, 5%carbon dioxide constantly to ensure the viability of the slices. Transfer a brain slice to the recording chamber, using a Pasteur pipette with its fine tip cut to fit the size of the slice.Next, place a platinum slice anchor above the slice to hold it on the platform. After that, fill the recording micropipettes with internal solution, and fill the puff micropipettes with GABA at 10 micromolar, 50 micromolar, and 100 micromolar, or ACSF as vehicle control. To prevent blockage with debris, apply slight positive pressure using a one-milliliter syringe before immersing the recording electrode in the ACSF.Under a microscope, position the recording electrode and puff micropipette so that the tips appear in the center of the monitor. Then, identify a target neuron. Adjust the microscope focus while gradually lowering the puff micropipette, and place it above the recording neuron at an angle of 45 degrees.Keep the distance between the tip of the puff micropipette and the target neuron in the range of 20 to 40 micrometers. Slowly and carefully approach the neuron with the recording electrode, and then release the positive pressure. Apply a weak and brief suction through the tubing connected to the electrode holder to create a gigaohm seal.Maintain the voltage at zero millivolts. After the formation of the gigaohm seal, compensate the fast and slow capacitance manually or automatically. Then, apply a brief and strong suction through the tubing to break into the whole-cell mode.Subsequently, record evoked IPSC currents in voltage clamp mode. Deliver single or paired GABA puffs through the puff micropipette controlled by a Master-8 voltage-step generator. Change the puff duration to obtain the best evoked IPSC currents results, and measure the evoked IPSC currents in the LPS-induced depression model.Shown here, are the sample IPSCs induced by 10, 50, and 100 micromolar GABA. And here are the puff duration response curves. The repetitive currents evoked by GABA at 50 micromolar puffs at interpuff intervals of one, two, or three seconds are shown here.One mastered, this technique can be done in two hours if it is performed properly. While attempting this procedure, it's important to remember to obtain healthy brain slices. After its development, this technique paved the way for researchers in the field of pharmacological administration to explore the effect of drug in electrophysiology.After watching this video, you should have a good understanding of how to conduct the pharmacological administration in whole-cell patch-clamp recording. Don't forget that working with TTX can be extremely hazardous and precautions, such as wearing gloves, should always be taken while performing this procedure.

Research

JoVE Journal

Neuroscience

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

Hurtig Mikro-iontophoresis af glutamat og GABA: et nyttigt redskab til Undersøge Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Forbundne Helcelle optagelser i Organotypiske hippocampusskiver

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-celle Patch-clamp Optagelser i Brain Skiver

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Anvendelse af automatiseret billedstyret patchklemme til undersøgelse af neuroner i hjerneskiver

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

Evaluering af Synapse tæthed i hippocampus gnaver hjernen skiver

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Optagelse og graduering af Epileptiform aktivitet i gnavere hjernen skiver koblet til mikroelektrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Forberedelse af akut rygmarven skiver til hele-celle Patch-klemme optagelse i Substantia Gelatinosa neuroner

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Vurdering af langvarig depression induktion i voksne cerebellar skiver

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...