저자는 다음 단체를 감사 하 고 싶습니다: 국가 자연 과학 재단의 중국 (31171018, 31171355), 과학 및 기술 부의 광 (2013KJCX0054), 자연 과학 재단의 광 동성 ( 2014A030313418, 2014A030313440), 광저우 과학 및 기술 관리국 (201607010320).

동물 주택 및 모든 동물 실험 동물 관리의 국립 연구소에 의해 설립에 대 한 지침에 따라 남 중국 사범 대학에서 동물 연구에 대 한 윤리 위원회에 의해 승인 절차에 따라 실시 했다 건강.
 1. 준비의 솔루션 <ol><li>표준 인공 뇌 척추 액체의 (실제) 우리의 출판된 작업 7에서 설명 하는 표 1에 나열 된 구성 요소가 포함 된 준비 500 mL.
	신선한 실제 솔루션을 준비 하는 이온된 수로 <ol><li>사용 깨끗 한 유리 비 커. 이온된 수와 주걱을 청소 하 고 NaCl, KCl, NaH를 추가 하려면 사용 하기 전에 건조 2 포 4, NaHCO 3, MgSO 4, D-포도 당, CaCl 2 95% O 2 / 5% 솔루션 부풀어 데 후 추가 공동 2 ~ 20 분 (에 표 1)으로 500 mL 이온된 수의 혼합물. 완전히 녹아 때까지 저 어.</li>
		<li>확인은 실제는 완전 하 게 어떤 강 수 없이 취소 합니다. PH 7.2-7.4로 조정 합니다. 95% O 2와 5% CO 2 ~ 20 분에 대 한 가스 혼합물으로 거품</li>
	</ol></li> <li>슬라이스 절단 솔루션 포함 하는 표 2에 설명 된 화학 7의 250 mL를 준비.
	<ol><li>KCl, MgCl 2, NaH 2 포 4, NaHCO 3, choline·Cl, D-포도 당을 추가 하 고 1.1.1, 이온된 물 250 mL에에서 표시 된 마지막 CaCl 2를 추가 하 고 1.1.1와 1.1.2에 설명 된 대로 분해.</li>
		<li>후 버블링 95% O 2 / 5% CO 2, 배치 솔루션 15-20 분 동안 냉장고에서 절단 솔루션은 차가운 하 고 부분적으로 냉동. 
		참고: 우리가 사용 하 여이 절단 솔루션 1-2 달에서 분할 영역을 만들 때-오래 된 쥐; 수식을 마우스 2 개월 이상에 대 한 다를 수 있습니다.</li>
		<li>살 균도 무료 isotonic 식 염 수에 용 해 하 여 준비 LPS 솔루션 (0.9 %NaCl). LPS는 intraperitoneally 관리 (0.5 mg/kg) 2 h 나중 녹음 시작.</li>
	</ol></li></ol> 2. 전 두 엽 피 질 조각의 준비 <ol><li>절단 솔루션 냉동 실에서 하 고 있는 비 자금을 얻기 위해 균질. 얼음에 솔루션을 놓고 거품 지속적으로 95% O 2 / 5% CO 2.</li>
	<li>크고, 날카로운가 위를 사용 하 여 신속 하 게 설명 9 마우스를 목을 벨 고 즉시 머리 가득한 비 커에 얼음 (0-4 ° C) 절단 솔루션.</li>
	<li>좋은 위 코 꼬리 방향으로 중간 선 따라 두개골의 상단을 잘라 고 측면 두개골 부분을 제거. 얼음 처럼 차가운 절단 솔루션으로 잘 주걱와 드롭 뇌 제거. 
	주: 단계 2.2-2.3 빠르게 수행 되어야 합니다 (이상적으로 &#60; 1 분).</li>
	<li>얼음으로 채워진 9 cm 배양 접시의 뚜껑에는 두뇌를 놓습니다. 린스 차가운 절단 솔루션 두뇌. 소 뇌 및 후 각 전구는 면도날으로 잘라.</li>
	<li>적용 cyanoacrylate 접착제는 견본에는 vibratome의 홀더. 조심 스럽게 뇌 블록에 접착제, rostral 쪽, 방울 놓고 즉시 얼음 절단 솔루션에 몰입할.</li>
	<li>수평 비행기에 관하여 15 °의 각도를 vibratome의 블레이드 홀더를 조정 하 고 준비 350 µ m 코로나 뇌 조각 (블레이드 진동 주파수 = 85 Hz, 속도 = 0.1 m m/s).</li>
	<li>95% O 2 / 5% 상수 버블링 (RT)에 1 시간에 대 한 품 어 전송 복구 챔버로 조각을 가득 실제, 한 피 펫을 사용 하 여, CO 2.</li>
</ol> 3. 전체 셀 패치 녹음 <ol><li>확인 유리 붕 규 산 micropipettes에 대 한 기록 전극으로 사용 하거나 끌어당기는 작동 설명서에는 특정 지침에 따라 micropipettes 퍼프. 전극, 기록에 대 한 팁 저항 범위 4-6 M에에서는 &#8486;, 퍼프 micropipettes 있는 팁 직경 범위 2-5 µ m.</li>
	<li>블록 빠른 Na + 채널을 따라서 실제를 활동 전위 나 + 채널 차단 (TTX, 1 µ M)를 추가 하 고 챔버로 연동 펌프에 의해 녹음 실에서 2-3 mL/min이 솔루션의 일정 유량을 유지 그리고 그것의 포부입니다. 거품 실제 95% O 2 / 5% CO 2 조각의 생존 능력을 보장 하기 위해 지속적으로.</li>
	<li>그 좋은 팁 조각 크기에 맞게 잘라 했다 수정 된 파스퇴르 피 펫을 사용 하 여 녹음 실로 두뇌 슬라이스를 전송 합니다. 백 금 조각 앵커와 슬라이스 병렬 나일론 스레드 간격 커버 &#8805; 플랫폼에는 슬라이스를 2mm 떨어져.</li>
	<li>내부 솔루션 7 (표 3), 녹음 micropipette 작성 및 10 µ M, 50 µ M, 100 µ M (실제에 녹아 있는), 또는 차량 제어로 실제에서 GABA와 퍼프 micropipettes를 작성.</li>
	<li>파편과 막힘을 방지 하기 위해 가벼운 긍정적인 압력에는 실제 기록 전극 immersing 전에 1 mL 주사기를 사용 하 여 적용 됩니다. 팁 ( 그림 1 A) 모니터에 비디오 이미지의 중심에 표시 되도록 기록 전극과 퍼프 micropipette 위치 micromanipulator (4 x 렌즈), 현미경을 사용 하 여.</li>
	<li>퍼프 micropipette 점차 줄이면서 현미경 초점 (40 x 렌즈)를 조정 하 고 45 °의 각도에서 녹음 신경 위에 그것을 배치. 퍼프 micropipette의 끝 및 20-40 µ m ( 그림 1 B) 기록 하는 뉴런 사이의 거리를 유지. 천천히, 그리고 신중 하 게 접근 기록 전극과 신경 하 고 긍정적인 압력 다음 놓습니다. 전극 홀더 기가 옴 씰을 만들에 연결 하는 튜브를 통해 약하고 짧은 흡입 적용.</li>
	<li>유지 0 전압 mV입니다. 기가 옴 씰의 형성, 후 빠른 보상 하 고 수동 또는 자동으로 커패시턴스를 속도. 그런 다음 전체 셀 모드에 들어가 위에서 언급 한 튜브를 통해 짧고 강한 흡입을 적용.</li>
	<li>전압 클램프에 기록 eIPSC 모드입니다. 퍼프 micropipette 퍼프 ( 그림 2) 단일 또는 짝 GABA를 제공 하는 마스터 8 전압 단계 발전기에 의해 제어 하는 Picospritzer를 사용 하 여 낮은 가스 압력 (질소, 4-6 psi) 적용.</li>
	<li>최고의 eIPSC 결과 ( 그림 3) 우울증 LPS 유발 모델 ( 그림 4)에서 eIPSC를 측정 하는 퍼프 기간 변경.</li>
</ol>

<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>

저자는 공개 없다.

퍼프 응용 프로그램은 널리 postsynaptic 수용 체 기능3,,47, 평가 하는 데 사용 됩니다 하지만 각 실험에 정확한 컨트롤을 요구 한다. 여기에 나온 전 두 엽 피 질 뇌 조각에서 Ipsc (즉, eIPSCs)을 유도 하는 GABA 퍼프 전체 셀 패치 클램핑 관련 절차를 설명 합니다. 기록 전극의 저항은 약 5 m ω, 퍼프 micropipettes의 팁 직경에 대해 2-5 µ m. ?...

패치 클램프 기술, 뉴런의 전기 신호를 조사 하기 위한 기본 도구는 1970 년대1,2에서 개발 되었다. 이 기술의 주요 장점은 어떻게 특정 치료에 지식을 제공 한다는 것입니다 (예를 들어, 약리) 신경 기능 또는 실시간으로3채널을 변경할 수 있습니다. 전체 셀 두뇌 조각에서 녹음 하는 동안 신경 기능 약리학 평가 기록 되 고 신경에 촉진제 또는 특정 수용 체의 길 항 제와 같은 약물의 응용 프로그램을 필요 합니다. 이 메서드는4신경 생리 적 및 병 적인 속성을 더 잘 이해 하는 특정 약물의 응용 프로그램에 따라 발생 하는 신경 변경 식별 수 있습니다. 약리학 관리 중 관류5 또는 퍼프6을 통해 수행할 수 있습니다, 비록 후자의 우수한 기술입니다. 특히: (i) 응용 프로그램을 빠르게 퍼프 증가 약물 농도 수준으로 기록 된 신경 주위를 막 수용 체의 탈 감 작 예방; (ii) 부풀어 약물의 볼륨이 매우 낮은, 따라서 뇌 조각;에 관리 화학 물질의 어떤 바람직하지 않은 효과 감소 시키는 목욕 솔루션에는 효과가 거의 되도록 (iii) 퍼프 프로토콜 수 설정 하 고 저장, 실험을 매우 정확 하 게 재현할 수; 만들기 (iv) 퍼프 응용 촉진제/길 항 근, 특히 어디 이러한 시 약 인지 비싼 얻기 어려운의 경제적인 사용을 나타냅니다.
여기, 우리는 심하게 준비 뇌 조각, 상대적으로 잘 보존 된 두뇌 회로의 장점이 준비에에서 GABA를 피에 의해 유도 된 전체 셀 전류 기록에 집중할 것 이다. 우리가 퍼프 유발 억제 전류7 을 수행 하는 방법이 문서에서 설명 합니다. 세 슘 (Cs+)를 사용 하 여-기반 내부 솔루션, 그리고 0에서 신경 세포를 들고 mV, GABA-퍼프 갖는 소개 합니다 적절 한 기술적 세부 사항 가진 금지 postsynaptic 전류 (eIPSCs). 우리는 eIPSC 보여 lipopolysaccharide (LPS) 주입8에 의해 유도 된 우울증의 마우스 모델을 사용 하 여 GABA 퍼프에 의해 갖는 진폭 차량 컨트롤에 비해 LPS-주입 마우스의 조각에 상당히 절감 됩니다. 우리의 의도이 문서가 어떻게 퍼프 기술 두뇌 조각에서 신경 활동에 어떤 화학 물질, 화합물 또는 약의 효과 평가 하기 위한 연구에 널리 적용 되는 표시 한다.

<table><tbody><tr><td>Glass Borosilicate micropipettes</td><td>Shutter Instruments</td><td>BF150-86-10</td><td>1.50 mm outer diameter; 0.86 mm inner diameter</td></tr><tr><td>Micropipette Puller</td><td>Shutter Instruments</td><td>MODEL P-97&amp;nbsp;</td><td>Flaming/Brow Micropipette Puller</td></tr><tr><td>Micromanipulators</td><td>Shutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Computer controlled Amplifier&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Digital Acquisition system&amp;nbsp;</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Imaging Camera</td><td>Nikon&amp;nbsp;</td><td>2115001</td><td>Inspection equipment</td></tr><tr><td>Microscopy&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td>Eclipse FN1&amp;nbsp;</td><td></td></tr><tr><td>Master 8</td><td>A.M.P.I.</td><td>Master-8 Pulse stimulator</td><td></td></tr><tr><td>Vibratome Slicer</td><td>Leica</td><td>VT 1000S</td><td></td></tr><tr><td>Razor blade</td><td>Gillette</td><td>74-S</td><td>FLYING EAGLE</td></tr><tr><td>Video monitor</td><td>Panasonic</td><td>WV-BM 1410</td><td></td></tr><tr><td>502 Glue</td><td>Deli</td><td>7146</td><td>Cyanoacrylate Glue</td></tr><tr><td>Peristaltic pump</td><td>Shanghai JIA PENG Corporation</td><td>BT100-1F</td><td></td></tr><tr><td>Video Camera</td><td>Olympus America Medical</td><td>OLY-150</td><td></td></tr><tr><td>Transfer Pipets</td><td>Biologix</td><td>30-0138A1</td><td></td></tr></tbody></table>

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

약리학 관리 전체 셀 패치 클램프 두뇌 조각에서 녹화를 할 때 주로 사용 됩니다. Electrophysiological 녹음 중 약물 응용 프로그램의 가장 좋은 방법 중 하나는 두뇌 조각에서 신경 활동에 약리 시 약의 효과 연구 하는 데 사용할 수 있습니다 퍼프 기술입니다. 퍼프 응용 프로그램의 가장 큰 장점은 그 녹음 사이트 약물 농도 증가, 따라서 방지 막 수용 체의 둔감. 퍼프 응용 프로그램의 성공적인 사용 주의 다음과 같은 요소를 포함: 약물의 농도, 퍼프 micropipette, 퍼프 micropipette 기록, 신경의 끝 및 기간 사이의 거리의 매개 변수 및 퍼프는 운전 압력 (평방 인치 당 파운드 psi). 이 문서에서는 피 감마-aminobutyric 산 (GABA) 전 두 엽 피 질 조각의 뉴런에 의해 유도 된 전체 셀 전류를 기록 하기 위한 단계별 절차를 설명 합니다. 특히, 마가, 등 다른 두뇌 지역 그리고 다른 준비, 세포 배양 등 사소한 수정 같은 절차를 적용할 수 있습니다.

퍼프 기술은 특정된 뉴런의 표면에 특정 수용 체에 약물의 효과 연구 하는 연구자를 수 있습니다. 이 문서에서는, 우리는 GABA 수용 체에 의해 중재 전류에 초점. 그림 1 에서는 퍼프와 녹음 micropipettes. 퍼프, 실제에 의해 생성 된 실제 압력의 기록 된 신경 세포에 어떤 영향을 제거 하 고 자당 퍼프 (그림 2A) 컨트롤로 ?...

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

전체 셀 전류 두뇌 분할 영역에서 GABA의 퍼프 응용 프로그램에 의해 유도 된

우리는 약리 시 약 전체 셀 패치 클램프 기록 하는 동안 관리 될 수 및 그것의 성공에 대 한 중요 한 기능의 다양 한 측면을 강조 퍼프 기술을 설명 합니다.

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

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Research

JoVE Journal

Neuroscience

9.3K 조회수.  South China Normal University. 우리는 약리 시 약 전체 셀 패치 클램프 기록 하는 동안 관리 될 수 및 그것의 성공에 대 한 중요 한 기능의 다양 한 측면을 강조 퍼프 기술을 설명 합니다.

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

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, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

랫 뇌 조각의 GABA 활성화된 단일 채널 및 토닉 해류

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, ...

연구

신경과학

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- 및 Neurochemically 확인 된 해마의 interneurons에서 전체 세포 패치 클램프 녹음

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

슬라이스 뜨거운 것이 : 생리적 온도에서 급성 성인 뇌 슬라이스

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

뇌 조각에서 전체 셀 패치 클램프 녹음

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

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

두뇌 조각에서 신경 모 수석에서 2 광자 칼슘 이미지

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

설치류와 인간의 조직에 급성 Ictal 활동의 생성 및 요청 시 개시

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

뇌 조각에서 구 심성 경로의 광유전 적 활성화 및 휘발성 마취제에 의한 반응의 조절

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

마우스 뇌의 수평 해마 조각

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

Source: Feng, Y. et al., <a href="https://app.jove.com/v/56387">Whole-cell Currents Induced by Puff Application of GABA in Brain Slices.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the recording of whole-cell currents induced by gamma-aminobutyric acid, or GABA, in a mouse prefrontal cortical brain slice. Controlled delivery of GABA opens chloride channels in a postsynaptic neuron, generating inhibitory postsynaptic currents, or IPSCs. These IPSCs increase with higher GABA concentrations, reflecting GABA&#39;s inhibitory activity on synaptic transmission.

mouse brain slice에서 GABA Puffing을 사용한 전체 세포 전류 기록

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

	...