automated whole-cell patch-clamp electrophysiology of neurons in
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1. O course of pipette resistance during autopatcher operation top with zoomed in 2 ae D view of the neuron hunting phase bottom i The first of the series of 8 85 T e A s sa O resistance measurements that indicate neuron detection ii the last of the D e series iii when positive pressure is released iv when suction is applied v when holding potential starts to ramp from 30 mV to 65 mV vi when it f 2m hits 65 mV vii break in b Raw traces showing patch pipette currents at E a ae A E oe s A the events in a i vii while a square voltage wave 10 Hz 10 mV is applied TA i nA o Aa E c f Quality of recordings from cortical and hippocampal neurons obtained with gt qol o i a 5 xe autopatching vs manual whole cell patch clamping c Plot of access resistances E iS IL obtained versus pipette depth left and bar graph summary of access resistances mean s d right for the final autopatcher whole cell patch validation test set black symbols n 23 the test set in which the autopatcher concludes in the gigaseal state open symbols n 24 data acquired after manual break in and the test set acquired via manual whole cell patch clamp grayed symbols n 15 d Holding current versus pipette depth left and summary data right plotted as in c e Resting potential versus pipette depth left and summary right plotted as in c f Holding times versus pip2. 2012 Nature America Inc All rights reserved Pa BRIEF COMMUNICATIONS Automated whole cell patch clamp electrophysiology of neurons in vivo Suhasa B Kodandaramaiah Giovanni Talei Franzesi Brian Y Chow Edward S Boyden amp Craig R Forest Whole cell patch clamp electrophysiology of neurons is a gold standard technique for high fidelity analysis of the biophysical mechanisms of neural computation and pathology but it requires great skill to perform We have developed a robot that automatically performs patch clamping in vivo algorithmically detecting cells by analyzing the temporal sequence of electrode impedance changes We demonstrate good yield throughput and quality of automated intracellular recording in mouse cortex and hippocampus Whole cell patch clamp recording of the electrical activity of neurons in vivo utilizes glass micropipettes to establish electrical and molecular access to the insides of neurons in intact tissue This methodology has fidelity sufficient to report the synaptic and ion channel mediated changes in subthreshold membrane potential that enable neurons to compute information and that are affected in brain disorders or by drug treatment In addi tion molecular access to the cell allows the infusion of dyes for morphological visualization as well as extraction of cell contents for transcriptomic single cell analysis which together enable the Figure 1 The autopatcher a
3. cated by increases in resistance If the pipette resistance remains constant and has an acceptable value the autopatcher program is started The program records the resistance of the pipette outside the brain and automatically lowers the pipette to a pre specified target region within the brain the stage labeled regional pipette doi 10 1038 nmeth 1993 localization in Fig la after which a second critical examination of the pipette resistance is carried out for quality control This check is followed by an iterative process of lowering the pipette by small increments while looking for a pipette resistance change that indicates proximity to a suitable neuron the neuron hunting stage The robot uses a specific sequence of resistance changes to detect proximal neurons and avoid false positives that would waste time and decrease cell yield After detecting this signature the robot halts movement and begins to actuate suction and pipette voltage changes so as to form a high quality seal con necting the pipette electrically to the outside of the cell mem brane the gigaseal formation stage thus resulting in a gigaseal cell attached recording If whole cell access is desired the robot can then be used to perform controlled application of suction in combination with brief electrical pulses to break into the cell the break in stage Supplementary Fig 3 Alternatively break in can be achieved using pulses of suction Supplementary Fig
4. while still fully anesthetized via cervical dislocation unless bio cytin filling was attempted In the case of biocytin filling the mice were anesthetized with isoflurane then transcardially perfused with 4 paraformaldehyde in phosphate buffered saline PBS see Histology and imaging Electrophysiology Borosilicate glass pipettes Warner were pulled using a filament micropipette puller Flaming Brown P97 model Sutter Instruments within a few hours before the NATURE METHODS beginning of the experiment and stored in a closed Petri dish to reduce dust contamination We pulled glass pipettes with resistances between 3 9 MQ The intracellular pipette solu tion consisted of in mM 125 potassium gluconate with more added empirically at the end to bring osmolarity up to 290 mOsm 0 1 CaCl 0 6 MgCl 1 EGTA 10 HEPES 4 MgATP 0 4 Na GTP 8 NaCl pH 7 23 osmolarity 289 mOsm similar as to what has been used in the past For experiments with biocytin 0 5 biocytin weight volume was added to the solu tion before the final gluconate based osmolarity adjustment and osmolarity then adjusted to 292 mOsm with potassium gluconate We performed manual patch clamping using previously described protocols with some modifications and iterations as explained in the text in order to prototype algorithm steps and to test them Robot construction We assembled the autopatcher Fig 1b and Supplementary Fig 1 through modif
5. 6 Throughout the process the robot applies a voltage square wave to the pipette 10 Hz 10 mV alternating with 0 mV relative to pipette holding voltage and the current is measured in order to calculate the resistance of the pipette at a given depth or stage of the process Throughout the entire process of robot operation this pipette resistance is the chief indicator of pipette quality cell presence seal quality and recording quality and the algo rithm attempts to make decisions such as whether to advance to the next stage restart a stage or halt the process entirely on the basis of the temporal trajectory taken by the pipette resist ance during the experiment Robot performance is enabled by two critical abilities its ability to monitor the pipette resistance quantitatively over time and its ability to execute actions in a temporally precise fashion once the measured pipette resistance reaches quantitative milestones Focusing on the data for the n 47 neurons in the main vali dation test set the neuron hunting stage took on average 2 5 1 7 min The time to find a target that later led to successful as compared to an unsuccessful gigaseal did not differ significantly P 0 8114 t test n 58 unsuccessful gigaseal formation trials that is failed trials did not take longer than successful ones The gigaseal formation took 2 6 1 0 min including the few seconds required for break in for the whole cell autopatched case fai
6. algo rithm that uses pulses of suction for break in rather than a zap Supplementary Fig 6 the yields cell qualities and cell properties obtained by the suction pulse variation of the autopatch algorithm were comparable to those obtained by the original algorithm Supplementary Fig 7 The inherent data logging of the robot allows quantitative analyses of the patch process for example revealing that the probability of success of autopatching starts at 50 70 in the first hour and then drops to 20 50 over the next few hours presumably due to cellular displacement intrinsic to the in vivo patching process Supplementary Fig 7d We have developed a robot that automatically performs patch clamping in vivo and demonstrated its use in the cortex and hippocampus of live mice We anticipate that other applications of robotics to the automation of in vivo neuroscience experiments and to other in vivo assays in bioengineering and medicine will be possible The ability to automatically make micropipettes in a high throughput fashion and to install them automatically might eliminate some of the few remaining steps requiring human intervention The use of automated respiratory and temperature monitoring could enable a single human operator to control many rigs at once further increasing throughput see Supplementary Note 5 for discussion of throughput METHODS Methods and any associated references are available in the online version of t
7. direct access to these measurements this can be omitted During autopatcher operation the USB 6259 board sent commands to the patch amplifier after acquisition of cell attached or whole cell patched neurons the patch amplifier would instead receive commands from the Digidata we used a software controlled TTL co axial BNC relay CX230 Tohtsu for driving signal switching between the USB 6259 BNC and the Digidata so that only one would be empowered to command the patch amplifier at any time The patch amplifier streamed its data to the analog input ports of both the USB DAQ and the Digidata throughout and after autopatching For pneumatic control of pipette pressure we used a set of three solenoid valves two input one output LHDA0533215H A Lee Company They were arranged and operated in the configuration shown in Supplementary Figure 1 The autopatcher program was coded in and run by Labview 8 6 National Instruments Detailed instructions for robot construc tion are described in the Supplementary Software Autopatcher User Manual doi 10 1038 nmeth 1993 2012 Nature America Inc All rights reserved Pa The USB6259 DAQ sampled the patch amplifier at 30 KHz and with unity gain applied and it then filtered the signal using a moving average smoothening filter half width six samples with triangular envelope The amplitude of the current pulses was measured using the peak to peak measurement function of Labview During t
8. USA gt McGovern Institute Massachusetts Institute of Technology Cambridge Massachusetts USA Correspondence should be addressed to C R F cforest gatech edu or E S B esb media mit edu RECEIVED 31 AUGUST 2011 ACCEPTED 30 MARCH 2012 PUBLISHED ONLINE 6 MAY 2012 DOI 10 1038 NMETH 1993 NATURE METHODS VOL 9 NO 6 JUNE 2012 585 2012 Nature America Inc All rights reserved Pa BRIEF COMMUNICATIONS set of motors and valves rapidly upon recognition of specific tem poral sequences of microelectrode impedance changes achiev ing in vivo patch clamp recordings in a total period of 3 7 min of robot operation The robot is relatively inexpensive and can easily be appended to an existing patch rig The robot Fig 1b monitors pipette resistance as the pipette is lowered into the brain and it automatically moves the pipette in incremental steps via a linear actuator The robot also contains a set of valves connected to pressure reservoirs to provide positive pressure during pipette insertion into the brain and negative pressure for gigaseal formation and attainment of the whole cell state Supplementary Fig 1 After the regional pipette localization stage pipettes that undergo increases of resistance of gt 300 kQ after this descent to depth are rejected which greatly increases the yield of later steps Supplementary Note 1 During neuron hunting when the pipette is lowered into the brain in a stepwise fashion the ke
9. d at stereotaxic coordinates anteroposterior 2 mm relative to bregma mediolateral 0 75 1 25 mm left or right of the midline neuron hunting began at 1 100 um depth It is critical to ensure that bleeding is minimal and the craniotomy is clean to allow good visualization of the pipette and minimize the number of pipettes blocked after insertion into the brain The dura was removed using a pair of fine forceps The craniotomy was superfused with artificial cerebrospinal fluid ACSF con sisting of 126 mM NaCl 3 mM KCl 1 25 mM NaH PO 2 mM CaCl 2 mM MgSO 24 mM NaHCO and 10 mM glucose to keep the brain moist until the moment of pipette insertion Seventeen mice were used to derive the autopatching algorithm Supplementary Fig 3 Sixteen mice were used to validate the robot for the primary test set Fig 2 Supplementary Fig 5a and Supplementary Fig 5b For the manual experiments Fig 2c f and Supplementary Fig 5c we used four mice For the develop ment of the suction based autopatching variant Supplementary Figs 6 7 we used five mice Out of the five mice used for suction based autopatching three were used for the throughput estima tions Supplementary Note 5 For biocytin filling experiments Fig le and Supplementary Fig 4 and validation of heartbeat modulation as a method for confirming neuronal detection Supplementary Note 1 we used six additional mice At the end of the patch clamp recording mice were euthanized
10. d of a stereomicroscope Nikon The pipette was lowered until it just touched the brain surface indicated by dimpling of the surface and retracted back by 20 30 um The autopatcher software then denoted this position just above the brain surface as z 0 for the purposes of executing the algo rithm Supplementary Fig 3 and acquired the baseline value R O of the pipette resistance The z axis is the vertical axis perpendicular to the earth s surface with greater values going downward The pipette voltage offset was automatically nullified by the pipette offset function in the Multiclamp Commander Molecular Devices We ensured that the electrode wire in the pipette was sufficiently coated with silver chloride to minimize pipette current drift which can affect the detection of the small resistance measurements that occur during autopatcher opera tion The brain surface was then superfused with ACSF and the autopatcher program was started See included Supplementary Software Autopatcher User Manual for detailed description of running the Labview program for autopatching Updated versions of the software and user manual will be made available online at http autopatcher org Details of autopatcher program execution The autopatcher evaluates the pipette electrical resistance outside the brain for example between 3 9 MQ is typical for 30 60 s to check whether AgCI pellets or other particulates internally clog the pipette indi
11. e 8 12 weeks old were purchased from Taconic During the period before the experiment the mice were housed in standard cages in the MIT animal facility with ad libitum food and water in a controlled light dark cycle environment with standard monitoring by veterinary staff On the day of the experiment they were anesthetized using ketamine and xylazine initially at 100 mg kg and 10 mg kg respectively and redosed at 30 45 min intervals with 10 15 of the initial keta mine dose as needed using toe pinch reflex as a standard metric of anesthesia depth The scalp was shaved and the mouse placed in a custom stereotax with ophthalmic ointment applied to the eyes and with Betadine and 70 ethanol used to sterilize the surgical area Three self tapping screws FOOOCE094 Morris Precision Screws and Parts were attached to the skull and a plastic head plate was affixed using dental acrylic as previously described Once set 20 min the mice were removed from the stereotactic apparatus and placed in a custom built low profile holder A dental drill was used to open up one or more craniotomies 1 2 mm diameter by thinning the skull until 100 um thick and then a small aperture was opened up with a 30 gauge needle tip Cortical craniotomies occurred at stereotaxic coordinates anteroposterior 0 mm relative to bregma mediolateral 0 1 mm left or right of the midline neuron hunting began at 400 um depth Hippocampal craniotomies occurre
12. ette depth left and summary right plotted as in c including recordings that were either deliberately or spontaneously terminated 4 SO 2 oO co Seo Oe Oe oS Depth um 586 VOL 9 NO 6 JUNE 2012 NATURE METHODS BRIEF COMMUNICATIONS 2012 Nature America Inc All rights reserved Pa be visualized histologically Fig 1e and Supplementary Fig 4 Focusing on the robot s performance after regional pipette locali zation that is leaving out losses due to pipette blockage during the descent to depth the autopatcher was successful at whole cell patch clamping 43 6 of the time Supplementary Table 1 n 24 out of 55 attempts starting with the neuron hunting stage and at gigaseal cell attached patch clamping 45 8 of the time n 27 out of 59 attempts Of the successful recordings approximately 10 were putative glia as reflected by their capacitance and lack of spiking 4 out of 51 successful autopatched recordings 2 out of 17 successful fully manual recordings For simplicity we analyzed only the neurons in the rest of the paper their various firing patterns are described in Supplementary Note 2 From the beginning of the neuron hunting stage to acquisition of successful whole cell or gigaseal cell attached recordings took 5 2 min for the robot to perform Supplementary Table 1 not significantly different from the duration of fully manual patching 543 min P 0 7539 t test n 47 autopatched neuro
13. he entire procedure a square wave of voltage was applied 10 mV in amplitude at 10 Hz to the patch amplifier pipette output via the USB6259 DAQ analog output Resistance values were then computed by dividing applied voltage by the peak to peak current observed for five consecutive voltage pulses and then these five values were averaged Once the autopatch process was complete neurons were recorded using Clampex software Molecular Devices Signals were acquired at standard rates for example 30 50 KHz and low pass filtered Bessel filter 10 KHz cutoff All data was analyzed using Clampfit software Molecular Devices and MATLAB MathWorks Robot operation At the beginning of the experiment we installed a pipette after filling it with pipette solution using a thin polyimide fused silica needle Microfil attached to a syringe 1 mL with syringe filter 0 2 um We removed excess ACSF to improve visualization of the brain surface in the pipette lowering stage and then applied positive pressure 800 1 000 mbar low positive pressure 25 30 mbar and suction pressure 15 to 20 mbar at the designated ports Fig 1 Supplementary Fig 1 and clamped the tubing to the input ports with butterfly clips the initial state of high positive pressure was present at this time with all valves electrically off We used the three axis linear actuator to manu ally position the pipette tip over the craniotomy using a control joystick with the ai
14. he paper Note Supplementary information is available in the online version of the paper ACKNOWLEDGMENTS We would like to acknowledge electronic switch design by G Holst at Georgia Tech E S B acknowledges funding by the US National Institutes of Health NIH Director s New Innovator Award DP20D002002 and the NIH EUREKA Award program 1RO1NS075421 and other NIH grants the New York Stem Cell Foundation Robertson Neuroscience Award the National Science Foundation NSF CAREER award CBET 1053233 and other NSF grants Jerry and Marge Burnett Google Human Frontiers Science Program MIT McGovern Institute and McGovern Institute Neurotechnology Award Program MIT Media Lab NARSAD Paul Allen Distinguished Investigator Award Alfred P Sloan Foundation and Wallace H Coulter Foundation C R F acknowledges funding by the NSF CISE 1110947 EHR 0965945 as well as the American Heart Association 10GRNT4430029 Georgia Economic Development Association Wallace H Coulter Foundation Center for Disease Control and NSF National Nanotechnology Infrastructure Network NNIN and from the Georgia Tech Institute for BioEngineering and BioSciences Junior Faculty Award Technology Fee Fund Invention Studio and George W Woodruff School of Mechanical Engineering AUTHOR CONTRIBUTIONS S B K G T F B Y C E S B and C R F designed devices and experiments and wrote the paper S B K conducted experiments G T F assisted with experiments and aut
15. ication of a standard in vivo patch clamping system The standard system comprised a three axis linear actuator MC1000e Siskiyou Inc for hold ing the patch headstage and a patch amplifier Multiclamp 700B Molecular Devices that connects its patch headstage to a com puter through an analog digital interface board Digidata 1440A Molecular Devices For programmable actuation of the pipette in the vertical direction we mounted a programmable linear motor PZC12 Newport onto the three axis linear actuator Note that if the vertical axis of the three axis linear actuator is computer controlled this can be omitted For experiments where we attempted biocytin filling we mounted the programmable linear motor at a 45 angle to the vertical axis to reduce the amount of background staining in the coronal plane that we did histological sectioning along The headstage was in turn mounted on the pro grammable linear motor through a custom mounting plate The programmable linear motor was controlled using a motor controller PZC200 Newport Inc that was connected to the computer through a serial COM port An additional data acquisition DAQ board USB 6259 BNC National Instruments Inc was connected to the computer via a USB port and to the patch amplifier through BNC cables for control of patch pipette voltage commands and acquisition of pipette current data during the execution of the autopatcher algorithm If the patch amplifier provides
16. ld at 65 mV for at least 5 min n 24 out of 73 attempts and successful gigaseal cell attached patch clamp recording 36 of the time defined as a stable seal of gt 1 GQ resistance n 27 out of 75 attempts success rates that are simi lar to or exceed those of a trained investigator manually perform ing blind whole cell patch clamping in vivo for us 28 8 success at whole cell patching n 17 out of 59 fully manual attempts see also refs 2 4 5 Example traces from neurons autopatched in cor tex and hippocampus are shown in Figure 1c d When biocytin was included in the pipette solution morphologies of cells could Cortex Hippocampus A a _ 2 900 p lt Neuron hunting mrs and break in yi Algorithm ends in whole cell state g Algorithm ends in gigaseal state manual break in O A lt 2 000 Manual whole cell patch clamping A O PE 1200 Gigaseal Whole cell c amp a 2 state i e state 1 000 for cell 9 8 attached g D 500 patch a D a T p F 0 0 5 1 0 15 2 0 2 5 3 0 3 5 D D 0 O Time min 2 H DOD OD CO QO CQ PO LO lt Q Soe Ce oc Oo 5 w Depth um N e AOAOA d lt aa F 0 0 4 0 8 1 2 1 6 z Time min 5 5 b EER ii i vii Whole cell state ES E Tale h N RET a Pr Oe Cec a MM a rth JAE FF FIPPHESs 1nA e ote Depth um 500 ms E 50 z w 55 E 60 2 g g _65 T Figure 2 Autopatcher operation and performance a Representative time 2 70 s
17. led attempts to form gigaseals were truncated at the end of the ramp down procedure and thus took 85 s These durations are simi lar to those obtained by trained human investigators practicing published protocols Histology and imaging For experiments with biocytin filling of cells mice were perfused through the left cardiac ventricle with 40 mL of ice cold 4 paraformaldehyde in PBS while anes thetized with isoflurane Perfused brains were then removed from the skull and postfixed overnight in the same solution at 4 C The fixed brains were incubated in 30 sucrose solu tion for 2 d until cryoprotected that is until the brains sank The brains were flash frozen in isopentane cooled using dry ice at temperatures between 30 C and 40 C The flash frozen brains were mounted on mounting plates using OCT as base and covered with tissue embedding matrix to preserve tissue integrity Slices 40 um thick were cut at 20 C using a cryostat Leica The brain slices were mounted on charged glass slides for example SuperFrost and incubated at room temperature NATURE METHODS 2012 Nature America Inc All rights reserved for 4 h in PBS containing 0 5 Triton X vol vol and 2 goat serum vol vol This was followed by 12 14 h of incubation at 4 C in PBS containing 0 5 Triton X vol vol 2 goat serum vol vol and Alexa 594 conjugated with streptavidin Life Technologies diluted 1 200 After incubation the
18. ns 15 fully manually patched neurons A representative autopatcher run plotting the pipette resistance versus time is shown in Figure 2a with key events indicated by roman numerals raw current traces resulting from the continu ously applied voltage pulses from which the pipette resistances were derived are shown in Figure 2b Note the change in pipette currents observed when a neuron is detected Fig 2b event ii See Online Methods for details of the autopatcher time course and execution The quality of cells recorded by the autopatcher was comparable to those in published studies conducted by skilled human investigators 7 and to our own fully manually patched cells Fig 2c f and Supplementary Fig 5 These comparisons showed no statistically significant difference between n 23 whole cell autopatched and n 15 fully manually patched neu rons for access resistance holding current resting membrane potential holding time gigaseal resistance cell membrane capacitance or cell membrane resistance detailed statistics in Supplementary Notes 3 and 4 Once the robot has been assembled it can easily be used to derive alternative or specialized algorithms for example if a specialized cell type is the target if image guided or other styles of patching are desired or if it is desirable to combine the auto patcher with other technologies such as optogenetics for cell type identification As an example we derived a variant of the
19. opatcher pilot testing COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests Published online at http www nature com doifinder 10 1038 nmeth 1993 Reprints and permissions information is available online at http www nature com reprints index html 1 Hamill 0 P Marty A Neher E Sakmann B amp Sigworth F J Pflugers Arch 391 85 100 1981 Margrie T W Brecht M amp Sakmann B Pflugers Arch 444 491 498 2002 Eberwine J et al Proc Natl Acad Sci USA 89 3010 3014 1992 Lee A K Epsztein J amp Brecht M Nat Protoc 4 385 392 2009 Kitamura K Judkewitz B Kano M Denk W amp Hausser M Nat Methods 5 61 67 2008 Trachtenberg M C amp Pollen D A Science 167 1248 1252 1970 Harvey C D Collman F Dombeck D A amp Tank D W Nature 461 941 946 2009 8 DeWeese M R amp Zador A M J Neurosci 26 12206 12218 2006 9 DeWeese M R Curr Protoc Neurosci 38 6 22 1 6 22 15 2007 10 Boyden E S F1000 Biol Rep 3 11 2011 11 Pak N Dergance M J Emerick M T Gagnon E B amp Forest C R J Mech Des 133 061006 2011 Or BR W PO N O NATURE METHODS VOL 9 NO 6 JUNE 2012 587 2012 Nature America Inc All rights reserved Pa ONLINE METHODS Surgical procedures All animal procedures were approved by the Massachusetts Institute of Technology MIT Committee on Animal Care Adult male C57BL 6 mic
20. robot for in vivo patch clamping a The four stages of the automated in vivo patch algorithm detailed in Supplementary Fig 3 b Schematic of a simple robotic system capable of performing the autopatching algorithm consisting of a conventional in vivo patch setup equipped with a programmable linear motor a controllable bank of pneumatic valves for pressure control and a secondary computer interface board c Current clamp traces for an autopatched cortical neuron during current injection top 2 s long pulses of 60 0 and 80 pA current injection and at rest bottom note compressed timescale relative to the top trace Access resistance 44 MQ input resistance 41 MQ depth of cell 832 um below brain surface d Current clamp traces for an autopatched hippocampal neuron during current injection top 2 s long pulses of 60 0 and 40 pA current injection and at rest bottom Access resistance 55 MQ input resistance 51 MQ depth of cell 1 320 um e Biocytin fill of a representative autopatched cortical pyramidal neuron Scale bar 50 um integrative analysis of molecular anatomical and electrophysio logical properties of single cells in the intact brain However the skill and labor required for in vivo patching have posed a challenge for its broad adoption in neuroscience and biology and precluded systematic integrative experiments We have discovered that unbiased non image guided in vivo whole cell patching blind pa
21. slices were thoroughly washed in PBS containing 100 mM glycine and 0 5 Triton X vol vol followed by PBS with 100 mM glycine Slices were then mounted in Vectashield with DAPI Vector Labs covered using a coverslip and sealed using nail polish NATURE METHODS Image stacks were obtained using a confocal microscope Zeiss with 20x objective lens Maximum intensity projections of the image stacks were taken using ImageJ software If full neuron morphology needed to be reconstructed multiple such maxi mum intensity projection images were auto leveled and then montaged using Photoshop CS5 software 12 Boyden E S amp Raymond J L Neuron 39 1031 1042 2003 13 Chow B Y et al Nature 463 98 102 2010 doi 10 1038 nmeth 1993
22. tch clamping of neurons in which micropipettes are lowered until a cell is detected and then an opening in the cell membrane created for intracellular recording can be reduced to a reliable algorithm The patch algorithm takes place in four stages Fig la i regional pipette localization in which the pipette is rapidly lowered to a desired depth under positive pressure ii neuron hunting in which the pipette is advanced more slowly at lower pressure until a neuron is detected as reflected by a specific temporal sequence of electrode imped ance changes iii gigaseal formation in which the pipette is hyperpolarized and suction applied to create the gigaseal and iv break in in which a brief voltage pulse zap is applied to the cell to establish the whole cell state We constructed a simple auto mated robot to perform this algorithm Fig 1b which actuates a a Ww Pipette Brain SEAS Regional l pipette Neuron Gigaseal Break in localization hunting formation Three axis linear actuator Programmable linear motor Patch digital Headstage board E Pipette holder Pipette High positive pressure Low positive pressure Atmospheric pressure Suction pressure 62 mV l PARTEN 2 10s Media Lab Massachusetts Institute of Technology Cambridge Massachusetts USA George W Woodruff School of Mechanical Engineering Georgia Institute of Technology Atlanta Georgia
23. y indicator of neuron presence is a monotonic increase in pipette resistance across several consecutive steps for example a 200 to 250 kQ increase in pipette resistance across three 2 um steps In our experiments successfully detected neurons also exhib ited an increase in heartbeat modulation of the pipette current Supplementary Fig 2 as has been noted before although we omitted this in our current version of the algorithm because Gigaseal formation of the variability in the shape and frequency of the heartbeat from cell to cell Supplementary Note 1 Gigaseal formation is implemented as a simple feedback loop introducing negative pressure and hyperpolarization of the pipette as needed to form the seal Finally break in is implemented through the application of suction and a zap Information about the algorithm is given in Online Methods Supplementary Fig 3 and Supplementary Note 1 Detailed instructions for robot construction are described in Supplementary Software Autopatcher User Manual The above algorithm whose derivation took place in the cortex was then validated in both the cortex and hippocampus to con firm generality The robot running the algorithm Fig 1a b and Supplementary Fig 3 obtained successful whole cell patch recordings 32 9 of the time Supplementary Table 1 defined as lt 500 pA of current when he
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