Automated whole-cell patch-clamp electrophysiology of neurons in
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1. 1 2 mm diameter by thinning the skull until 100 pm 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 occurred 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 1100 um depth It is critical to ensure that bleeding is minimal and the craniotomy is clean as this allows good visualization of the pipette and minimizes 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 consisting of 126 mM NaCl 3 mM KCI 1 25 mM NaH PO 2 mM CaClo 2 mM MgSOxq 24 mM NaHCOs and 10 mM glucose to keep the brain moist until the moment of pipette insertion 17 mice were used to derive the autopatching algorithm Supplementary Fig 2 16 mice were used to validate the robot for the primary test set Fig 2 Supplementary Fig 3a and Supplementary Fig 3b For the manual experiments Fig 2c f and Supplementary Fig 3c we used 4 mice For the development of the suction based autopatching variant Supplementary Fig 5 6 we used 5 mice Out of the 5 mice used for suction based autopatching 3 mic2. YIVULIQVVM 1XO YIVULIOIVMY 1XO YIVULIOIVME Kodandaramaiah et al Page 2 electrode impedance changes gigaseal formation in which the pipette is hyperpolarized and suction applied to create the gigaseal and break in in which a brief voltage pulse zap is applied to the cell to establish the whole cell state We constructed a simple automated robot to perform this algorithm Fig 1b which actuates a set of motors and valves rapidly upon recognition of specific temporal sequences of microelectrode impedance changes achieving in vivo patch clamp recordings in a total period of 3 7 minutes of robot operation The robot is relatively inexpensive and can easily be appended to an existing patch rig We demonstrate the utility of this autopatching robot in obtaining high quality recordings which could be held for an hour or longer in the cortex and hippocampus of anesthetized mouse brain The robot Fig 1b monitors pipette resistance as the pipette is lowered into the brain and automatically moves the pipette in incremental steps via a linear actuator In principle the pipette resistance monitoring can be performed by a traditional patch amplifier and digitizer and the 3 axis linear actuator typically used for in vivo patching can be used as the robotic actuator we here for flexibility added an additional computer interface board to support pipette resistance monitoring and an additional linear actuator for pipette movem
3. 4 g Time min ii 5 ALA D g g a 0 0 4 0 8 1 2 1 6 2 0 a Time min b i ii iii vii Whole cell state romney DAA 1nA 500 ms Quality of obtained recordings cortex hippocampus algorithm ends in whole cell state e a algorithm ends in gigaseal state manual break in A manual whole cell patch clamping 4 d 120 o 200 T o shohod 100 270 100 ro e o 60 b yrs E e e o 50 80 ee o o 250 5 3 o a 100 e gt b fS L 100o Pwr 2 0 Aa aa E 60 00 8 A ta 5 3 150 e00 A AA 7 30 200 A O 200 40 SoSo 9 A D D e D o Po A AA 300 CLP Bao o A x a 20 5 250 20 ee 2 10 5 400 a 300 0 f 1 1 1 1 1 o O _ I _500 400 600 800 1000 1200 1400 1600 1800 lt 400 600 800 1000 1200 1400 1600 1800 Depth um f Depth um 45 i S oat oaoa 1000 z 50 Ei E 100 55 0 A o jee a o o ISA A a 2 Ziol egee 4 a4 ga e o A a 0 30 5 e 8 e A Ay 5 a og ro e eo A A 60 59 Pe k A 2 40 D os o AA A D 70 Q A Q o a a 40 75 oe 00 O50 xe 10 e s A D 60 T o O 20 80 e e D I I 85 we 0 400 600 800 1000 1200 1400 1600 1800 1 ea0ACA 400 600 800 1000 1200 1400 1600 1800 Depth um Depth um Figure 2 Autopatcher operation and performance a Representative timecourse of pipette resistance during autopatcher operation fop with zoomed in view of the neuron hunting phase bottom Roman numerals i the first of the series of resistance meas
4. recordings terminated spontaneously Nat Methods Author manuscript available in PMC 2012 December 01
5. 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 Alternately break in can also be achieved using pulses of suction Supplementary Fig 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 algorithm attempts to make decisions such as whether to advance to the next stage or to restart a Nat Methods Author manuscript available in PMC 2012 December 01 1XO YIVULIQVVM 1XO YIVULIOIVMY 1XO YIVULIOIVMY Kodandaramaiah et al Page 7 stage or to halt the process entirely on the temporal trajectory taken by the pipette resistance during the experiment The performance of the robot is enabled by two critical abilities of the robot its ability to monitor the pipette resistance quantitatively over time and its ability to execute actions in a temporally precise fashion upon the measured pipette resistance reaching quantitative m
6. will be made available online at http autopatcher org Details of Autopatcher Program Execution The autopatcher evaluates the pipette electrical resistance is evaluated outside the brain e g between 3 9 MQ is typical for 30 60 seconds to see if AgCl pellets or other particulates internally clog the pipette indicated by increases in resistance If the pipette resistance remains constant and is of acceptable resistance 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 localization in Fig la followed by a second critical examination of the pipette resistance 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 indicative of proximity to a neuron suitable for recording the neuron hunting stage During this stage the robot looks for a specific sequence of resistance changes that indicates that a neuron is proximal attempting to 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 connecting the pipette electrically to the outside of the cell membrane the gigaseal formation stage thus
7. 075421 and other NIH Grants 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 the McGovern Institute Neurotechnology Award Program MIT Media Lab NARSAD Paul Allen Distinguished Investigator Award Alfred P Sloan Foundation and the Wallace H Coulter Foundation C R F acknowledges funding by the NSF CISE 1110947 EHR 0965945 as well as American Heart Association LOGRNT4430029 the Georgia Economic Development Association the Wallace H Coulter Foundation Center for Disease Control NSF National Nanotechnology Infrastructure Network NNIN and from Georgia Tech Institute for BioEngineering and BioSciences Junior Faculty Award Technology Fee Fund Invention Studio and the George W Woodruff School of Mechanical Engineering Nat Methods Author manuscript available in PMC 2012 December 01 1XO YIVULIQVVM 1XO YIVULIOIVMY 1XO YIVULIOIVME Kodandaramaiah et al Page 8 REFERENCES FOR THE ONLINE METHODS SECTION 1 Hamill OP Marty A Neher E Sakmann B Sigworth FJ Improved patch clamp techniques for high resolution current recording from cells and cell free membrane patches Pflugers Arch 1981 391 85 100 PubMed 6270629 Margrie TW Brecht M Sakmann B In vivo low resistance whole cell recordings from neurons in the anaesthetized and awake mammalian brain Pflugers Arch 2002 444 49
8. 1 498 PubMed 12136268 Eberwine J et al Analysis of gene expression in single live neurons Proc Natl Acad Sci U S A 1992 89 3010 3014 PubMed 1557406 Lee AK Epsztein J Brecht M Head anchored whole cell recordings in freely moving rats Nat Protocols 2009 4 385 392 Kitamura K Judkewitz B Kano M Denk W Hausser M Targeted patch clamp recordings and single cell electroporation of unlabeled neurons in vivo Nat Meth 2008 5 61 67 Trachtenberg MC Pollen DA Neuroglia Biophysical Properties and Physiologic Function Science 1970 167 1248 1252 PubMed 5411911 Harvey CD Collman F Dombeck DA Tank DW Intracellular dynamics of hippocampal place cells during virtual navigation Nature 2009 461 941 946 PubMed 19829374 DeWeese MR Zador AM Non Gaussian membrane potential dynamics imply sparse synchronous activity in auditory cortex J Neurosci 2006 26 12206 12218 PubMed 17122045 DeWeese MR Whole cell recording in vivo Curr Protoc Neurosci 2007 Chapter 6 Unit 6 22 PubMed 18428661 10 Boyden ES A history of optogenetics the development of tools for controlling brain circuits with light F1000 Biology Reports 2011 3 11 Pak N Dergance MJ Emerick MT Gagnon EB Forest CR An Instrument for Controlled Automated Production of Micrometer Scale Fused Silica Pipettes Journal of Mechanical Design 2011 133 061006 12 Boyden ES Raymond JL Active reversal of
9. Fo IT M AHIO Open Access Articles Automated whole cell patch clamp electrophysiology of neurons in vivo The MIT Faculty has made this article openly available Please share how this access benefits you Your story matters As Published Publisher Version Accessed Citable Link Terms of Use DACE MUO l il MASSACHUSETTS INSTITUTE OF TECHNOLOGY Kodandaramaiah Suhasa B Giovanni Talei Franzesi Brian Y Chow Edward S Boyden and Craig R Forest Automated Whole cell Patch clamp Electrophysiology of Neurons in Vivo Nature Methods 9 no 6 May 6 2012 585 587 http dx doi org 10 1038 nmeth 1993 Nature Publishing Group Author s final manuscript Fri Jun 05 03 25 18 EDT 2015 http hdl handle net 1721 1 79604 Article is made available in accordance with the publisher s policy and may be subject to US copyright law Please refer to the publisher s site for terms of use DSpace MIT 1XO YILULIOIVMY 1XO YIVULIOVE ME 1XO YIVULIQVM Va NIH Public Access 8 Author Manuscript Crews Me on Published in final edited form as Nat Methods 2012 June 9 6 585 587 doi 10 1038 nmeth 1993 Automated whole cell patch clamp electrophysiology of neurons in vivo Suhasa B Kodandaramaiah Giovanni Talei Franzesi Brian Y Chow Edward S Boyden and Craig R Forest MIT Media Lab McGovern Institute Dept of Biological Engineering and Dept of Brain and Co
10. VULIOIVMG 1XO YIVULIOIVME Kodandaramaiah et al Page 4 within a single brain region may open up new strategies for understanding how different cell types function in the living milieu Online Methods Surgical procedures All animal procedures were approved by the Massachusetts Institute of Technology MIT Committee on Animal Care Adult male C57BL 6 mice 8 12 weeks old were purchased from Taconic Upon arrival 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 for the period before the experiment On the day of the experiment they were anesthetized using ketamine xylazine initially at 100 mg kg and 10 mg kg and redosed at 30 45 minute intervals with 10 15 of the initial ketamine 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 headplate affixed using dental acrylic as previously described Once set 20 minutes the mice were removed from the stereotactic aparatus and placed in a custom built low profile holder A dental drill was used to open up one or more craniotomies
11. direct access to these measurements this can be omitted c Current clamp traces during current injection top 2 s long pulses of 60 0 and 80 pA current injection and at rest bottom note compressed Nat Methods Author manuscript available in PMC 2012 December 01 1XO YIVULIQVVM 1XO YIVULIOIVMY 1XO YIVULIOIVME Kodandaramaiah et al Page 10 timescale relative to the top trace for an autopatched cortical neuron Access resistance 44 MQ input resistance 41 MQ depth of cell 832 um below brain surface d Current clamp traces during current injection top 2 s long pulses of 60 0 and 40 pA current injection and at rest bottom for an autopatched hippocampal neuron 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 Nat Methods Author manuscript available in PMC 2012 December 01 1XO YIVULIQVVME 1XO YIVULIOIV MY 1XO YIVULIOIVME Kodandaramaiah Cc Access resistance MQ Resting potential mV et al Page 11 a Timecourse of autopatching algorithm 1 1 Gigaseal Formation AT r i g 2500 lt Neuron Hunting and Breakla Wi g 2000 i S f w 1500 F f Gigaseal Whole 3 Iv state i e cell o 1000 f for cell state oy iv attached a 500 f iii A patch 0 0 5 1 1 5 2 2 5 3 3 5
12. e were used for the throughput estimations Supplementary Note 6 For biocytin filling experiments Fig 1f and Supplementary Fig 4 and validation of heartbeat modulation as a method for confirming neuronal detection Supplementary Note 1 we used 6 additional mice At the end of the patch clamp recording mice were euthanized while still fully anesthetized via cervical dislocation unless biocytin filling was attempted In the case of biocytin filling the mice were isoflurane anesthetized then transcardially perfused in 4 ice cold through the left cardiac ventricle with 40 mL of ice cold 4 paraformaldehyde in phosphate buffered saline PBS see Histology and Imaging section for more details Electrophysiology Borosilicate glass pipettes Warner were pulled using a filament micropipette puller Flaming Brown P97 model Sutter Instruments within a few hours before beginning the Nat Methods Author manuscript available in PMC 2012 December 01 1XO YIVULIQVVM 1XO YIVULIOIVMG 1XO YIVULIOIVME Kodandaramaiah et al Page 5 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 solution consisted of in mM 125 potassium gluconate with more added empirically at the end to bring osmolarity up to 290 mOsm 0 1 CaCly 0 6 MgClo 1 EGTA 10 HEPES 4 Mg ATP 0 4 Na GTP 8 NaCl pH 7 23 osmolarity 289 mOsm similar as to w
13. ed voltage pulses from which the pipette resistances were derived are shown in Fig 2b Note the small visual appearance of the change in pipette currents observed when a neuron is detected Fig 2b event ii See Online methods for details of the autopatcher timecourse and execution The quality of cells recorded by the autopatcher was comparable to those in published studies conducted by skilled human investigators 7 9 and to our own fully manually patched cells Fig 2c f Supplementary Fig 5 These comparisons showed no statistically significant difference between n 23 auto whole cell patched and n 15 fully manually patched neurons 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 is easy to use it to derive alternative or specialized algorithms e g if a specialized cell type is the target or if image guided or other styles of patching is desired or if the technology is desired to be combined with other technologies such as optogenetics for cell type identification As an example we derived a variant of the algorithm that uses pulses of suction to break in to cells rather than zap Supplementary Fig 6 the yields cell qualities and cell properties obtained by the suction pulse variation of the autopatch algorithm were c
14. embedding matrix to preserve tissue integrity and 40 um thick slices were cut at 20 C using a cryostat Leica The brain slices were mounted on charged glass slides e g SuperFrost and incubated at room temperature for 4 hours in PBS containing 0 5 Triton X vol vol and 2 goat serum vol vol This was followed by 12 14 hours 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 slices were thoroughly washed in PBS containing 100 mM glycine and 0 5 Triton X vol vol followed by washing in PBS with 100 mM glycine Slices were then mounted in Vectashield with DAPI Vector Labs covered using a coverslip and sealed using nail polish 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 needed to reconstruct full neuron morphology multiple such maximum intensity projection images were auto leveled then montaged using Photoshop CS5 software Supplementary Material Refer to Web version on PubMed Central for supplementary material Acknowledgments We would like to acknowledge electronic switch design by G Holst at Georgia Tech E S B acknowledges funding by the National Institute of Health NIH Director s New Innovator Award DP20D002002 and the NIH EUREKA Award program 1R0O1NS
15. ent The robot also contains a set of valves connected to pressure reservoirs to provide positive pressure during pipette insertion into the brain as well as negative pressure as necessary to result in gigaseal formation and attainment of the whole cell state see Supplementary Fig for details The algorithm derivation took place in the cortex and the validation of the algorithm then took place in both cortex and hippocampus to confirm generality After the regional pipette localization stage pipettes that undergo increases of resistance of greater than 300 kQ after this descent to depth are rejected which greatly increases the yield of later steps Supplementary Note 1 During neuron hunting the key indicator of neuron presence is that as the pipette is lowered into the brain in a stepwise fashion there is a monotonic increase in pipette resistance across several consecutive steps e g a 200 250 KQ increase in pipette resistance across three 2 um steps Successfully detected neurons also exhibited an increase in heartbeat modulation of the pipette current Supplementary Fig 2 as has been noted before although we did not utilize this in our current version of the algorithm due to the variability in the shape and frequency of the heartbeat from cell to cell Supplementary Note 1 Gigaseal formation was implemented as a simple feedback loop introducing negative pressure and hyperpolarization of the pipette as needed to fo
16. g 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 3 axis linear actuator Siskiyou to manually position the pipette tip over the craniotomy using a control joystick with the aid of a stereomicroscope Nikon The pipette was lowered until it just touched the brain surface indicated by dimpling of surface and retracted back by 20 30 micrometers The autopatcher software then denotes this position just above the brain surface as z 0 for the purposes of executing the algorithm Supplementary Fig 2 acquiring the baseline value R O of the pipette resistance at this time the z axis is the vertical axis perpendicular to the earth s surface with greater values going downwards The pipette voltage offset was automatically nullified by the pipette offset function in the Multiclamp Commander Molecular Devices We ensured that electrode wire in the pipette was chlorided enough so as to minimize pipette current drift which can affect the detection of the small resistance measurements that occur during autopatcher operation 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
17. gnitive Sciences MIT Cambridge MA George W Woodruff School of Mechanical Engineering Georgia Institute Of Technology Atlanta GA Abstract 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 recordings 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 exhibits signal quality and temporal fidelity sufficient to report the synaptic and ion channel mediated subthreshold membrane potential changes that enable neurons to compute information and that are affected in brain disorders or by drug treatment In addition molecular access to the cell enables infusion of dyes for ee visualization as well as extraction of cell contents for transcriptomic single cell analysis which together enable the integrative analysis of molecular anatomical and electrophysiological properties of single cells in the intact brain However in vivo patching
18. hat has been used in the past For experiments with biocytin 0 5 biocytin weight volume was added to the solution 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 c through modification of a standard in vivo patch clamping system The standard system comprised a 3 axis linear actuator MC1000e Siskiyou Inc for holding the patch headstage and a patch amplifier Multiclamp 700B Molecular Devices that connects its patch headstage to a computer through a 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 3 axis linear actuator 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 programmable linear motor through a custom mounting plate The programmable linear motor was controlled using a motor controller PZC200 Newport Inc that was connected
19. ilestones Focusing on the data for the n 47 neurons in the main validation test set the neuron hunting stage took on average 2 5 1 7 minutes 7 47 with the time to find a target that later led to successful gigaseal not differing significantly from the time to find a target that does not lead to a gigaseal 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 minutes including for the whole cell autopatched case the few seconds required for break in failed attempts to form gigaseals were truncated at the end of the ramp down procedure and thus took 85 seconds These durations are similar 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 phosphate buffered saline PBS while anesthetized with isoflurane Perfused brains were then removed from the skull then postfixed overnight in the same solution at 4 C The fixed brains were incubated in 30 sucrose solution for 2 days until cryoprotected i e the brains sank The brains were flash frozen in isopentane cooled using dry ice at temperatures between 30 C to 40 C The flash frozen brains were mounted on mounting plates using OCT as base and covered with tissue
20. motor memories reveals rules governing memory encoding Neuron 2003 39 1031 1042 PubMed 12971901 13 Chow BY et al High performance genetically targetable optical neural silencing by light driven proton pumps Nature 2010 463 98 102 PubMed 20054397 Nat Methods Author manuscript available in PMC 2012 December 01 1XO YIVULIQVVM 1XO YIVULIOIVMY 1XO YIVULIOIVME Kodandaramaiah et al Page 9 a pipette gt brain ae aaa a Regional Gigaseal Break In Pipette f a Hunting Formation Localization 3 axis linear actuator computer programmable linear motor patch digital board secondary digital board high positive pressure low positive pressure atmospheric pressure suction pressure headstage pipette holder valves C d gt E lt 1s 62 mV 60 mV gt E Jo 10s Figure 1 The autopatcher a 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 note that if the vertical axis of the 3 axis linear actuator is computer controlled this can be omitted a controllable bank of pneumatic valves for pressure control and a secondary computer interface board if the patch amplifier provides
21. n Nat Methods Author manuscript available in PMC 2012 December 01 1XO YIVULIQVVM 1XO YIVULIOIVMY 1XO YIVULIOIVME Kodandaramaiah et al Page 3 stage i e 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 2 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 described in the previous paragraph 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 just the neurons in the rest of the paper their various firing patterns are described in the 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 minutes for the robot to perform Supplementary Table 1 not significantly different from the duration of fully manual patching 5 3 minutes p 0 7539 n 47 autopatched neurons 15 fully manually patched neurons A representative autopatcher run plotting the pipette resistance versus time is shown in Fig 2a with key events indicated by Roman numerals raw current traces resulting from the continuously appli
22. omparable to those obtained by the original algorithm Supplementary Fig 7 The inherent data logging of the robot allows fine scale 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 algorithmically detecting cells by analyzing the temporal sequence of electrode impedance changes and demonstrated it 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 increasing throughput further see Supplementary Note 5 for discussion of throughput As a final example the ability to control many pipettes within a single brain and to perform parallel recordings of neurons Nat Methods Author manuscript available in PMC 2012 December 01 1XO YIVULIQVVM 1XO YI
23. oving average smoothening filter half width 6 samples with triangular envelope and the amplitude of the current pulses was measured using the peak to peak measurement function of Labview During the entire procedure a square wave of voltage was applied 10 mV in amplitude at 10 Hz to the pipette via the USB6259 DAQ analog output Resistance values were then computed by dividing applied voltage by the peak to peak current observed for 5 consecutive voltage pulses and then these 5 values were averaged Once the autopatch process was complete neurons were recorded using Clampex software Molecular Devices Signals were acquired at standard rates e g 30 50 KHz and low pass filtered Bessel filter 10 KHz cutoff All data was analyzed using Clampfit software Molecular Devices and MATLAB Mathworks Nat Methods Author manuscript available in PMC 2012 December 01 1XO YIVULIQVVME 1XO YIVULIOIVMY 1XO YIVULIOIVME Kodandaramaiah et al Page 6 Robot Operation At the beginning of the experiment we installed a pipette and filled it with pipette solution using a thin polyimide fused silica needle Microfil attached to a syringe filter 0 2 um attached to a syringe 1 mL We removed excess ACSF to improve visualization of the brain surface in the pipette lowering stage and then applied positive pressure 800 1000 mBar low positive pressure 25 30 mBar and suction pressure 15 to 20 mBar at the designated ports Fi
24. requires skill being something of an art to perform and is laborious This has posed a challenge for its broad adoption in neuroscience and biology and precluded systematic or scalable in vivo experiments We have discovered that unbiased non image guided n vivo whole cell patching blind patch clamping of neurons Fig 1a 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 regional pipette localization in which the pipette is rapidly lowered to a desired depth under positive pressure 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 Correspondence to Craig R Forest Georgia Institute of Technology 813 Ferst Dr Room 411 Atlanta GA 30332 cforest gatech edu Office phone 404 385 7645 and Edward S Boyden Massachusetts Institute of Technology E15 421 20 Ames St Cambridge MA 02139 esb media mit edu Office phone 650 468 5625 Authors Contributions S B K 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 autopatcher pilot testing Competing financial interests The authors declare no competing financial interests 1XO
25. rm the seal Finally break in was implemented through the application of suction and the application of a zap voltage pulse to enable the whole cell state Information about the algorithm are indicated in Online Methods Supplementary Fig 3 and Supplementary Note 1 Detailed instructions for robot construction are described in Supplementary Software Autopatcher User Manual We validated the algorithm and robot on targets within the cortex and hippocampus of anesthetized mice The robot running the algorithm Fig la b 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 held at 65 mV for at least 5 minutes 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 similar to or exceed those of a trained investigator manually performing 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 4 5 Example traces from neurons autopatched in cortex and hippocampus are shown in Fig 1c d When biocytin was included in the pipette solution morphologies of cells could be visualized Fig le and Supplementary Fig 4 histologically Focusing on the robot s performance after the regional pipette localizatio
26. 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 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 2 input 1 output LHDA0533215H A Lee Company They were arranged and operated in the configuration shown in Supplementary Fig 1 The autopatcher program was coded in and run by Labview 8 6 National Instruments Detailed instructions for robot construction are described in the Supplementary Software Autopatcher User Manual The USB6259 DAQ sampled the patch amplifier at 30 KHz and with unity gain applied and then filtered the signal using a m
27. urements that indicate neuron detection ii the last of the 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 hits 65 mV vii break in b Raw traces showing patch pipette currents while a square voltage wave 10 Hz 10 mV is applied at the events flagged by Roman numerals in Fig 2a c f Quality of recordings obtained with the autopatcher vs by manual whole cell patch clamping c e t Plot of Nat Methods Author manuscript available in PMC 2012 December 01 1XO YIVULIQVVM 1XO YIVULIOIVMY 1XO YIVULIOIVME Kodandaramaiah et al Page 12 access resistances obtained versus pipette depth and right bar graph summary of access resistances mean s d for the final autopatcher whole cell patch validation test set closed 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 for cortical circles and hippocampal triangles neurons d eft Resting potential versus pipette depth and right summary data plotted as in c e eft Holding current versus pipette depth and right summary plotted as in c f eft Holding times versus pipette depth and right summary plotted as in c including recordings that were deliberately terminated as well as
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