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. 2016 Apr;11(4):634-54.
doi: 10.1038/nprot.2016.007. Epub 2016 Mar 3.

Assembly and Operation of the Autopatcher for Automated Intracellular Neural Recording in Vivo

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Free PMC article

Assembly and Operation of the Autopatcher for Automated Intracellular Neural Recording in Vivo

Suhasa B Kodandaramaiah et al. Nat Protoc. .
Free PMC article

Abstract

Whole-cell patch clamping in vivo is an important neuroscience technique that uniquely provides access to both suprathreshold spiking and subthreshold synaptic events of single neurons in the brain. This article describes how to set up and use the autopatcher, which is a robot for automatically obtaining high-yield and high-quality whole-cell patch clamp recordings in vivo. By following this protocol, a functional experimental rig for automated whole-cell patch clamping can be set up in 1 week. High-quality surgical preparation of mice takes ∼1 h, and each autopatching experiment can be carried out over periods lasting several hours. Autopatching should enable in vivo intracellular investigations to be accessible by a substantial number of neuroscience laboratories, and it enables labs that are already doing in vivo patch clamping to scale up their efforts by reducing training time for new lab members and increasing experimental durations by handling mentally intensive tasks automatically.

Conflict of interest statement

CONFLICT OF INTEREST

ACS, GTF, MLM, CRF and ESB declare no competing interests. IRW, SBK and GLH received financial remuneration from Neuromatic Devices Inc. for technical consulting services provided in 2012, 2013 and 2012–2015 respectively.

Figures

FIGURE 1
FIGURE 1. The autopatcher – a robot for automated whole cell patch clamp recordings in vivo: overview of algorithm and schematic
(a) The algorithm for autopatching (adapted from Kodandaramaiah et al 2012): The six stages of obtaining whole cell patch clamp recordings in vivo include: (i) all the manual steps (Steps 12–31: installing a pipette, software initialization, etc.) that need to be performed after which the autopatcher will programmatically perform Steps 24–31 (ii) an initial assessment of the resistance of the patch pipette to eliminate unsuitable pipettes (BOX 2, Autopatching Step 1), (iii) lowering of the patch pipette to the region of interest followed by a second assessment of pipette tip fidelity (BOX 2, Autopatching Step 2), (iv) the neuron hunting stage during which the autopatcher scans for neurons (BOX 2, Autopatching Step 3), (v) attempting gigasealing by modulating the pressure inside the pipette and pipette voltage after contact with a cell has been established (BOX 2, Autopatching Step 4–5), and (vi) the break-in stage during which pulses of high negative pressure are applied to achieve the whole cell patch clamp state (Step 36). Some fraction of the autopatcher trials result in end points other than acquisition of whole cell patched or cell attached recordings. Such instances are highlighted with the red arrows and explained in the corresponding steps in the protocol. (b) Schematic of the autopatcher system capable of performing the autopatching algorithm (adapted from Kodandaramaiah et al 2012): The system consists of a conventional in vivo patch setup (i.e., pipette, headstage, 3 axis linear actuator, patch amplifier and computer), equipped with a few additional modules: a programmable linear motor and a custom control box for data acquisition to enable closed-loop control of the motor based upon a series of pipette resistance measurements. The control box also performs closed-loop pneumatic pressure control of the patch pipette. (a) and (b) adapted from Kodandaramaiah et al 2012.
FIGURE 2
FIGURE 2. The autopatcher: equipment photographs
(a) Photograph of the autopatcher showing the general layout of major equipment. (b) Photograph focusing on the autopatcher control box and its interface with the patch amplifier and external digitizer. (c) Photograph focusing on the pipette actuator assembly. (d) Schematic of the autopatcher control box. A central digitizer board equipped with analog inputs, as well as analog and digital outputs, in the autopatcher control box sends command voltage signals to the patch amplifier and reads the patch measurements from the amplifier output. Digital outputs on the same board are sent to a bank of pneumatic valves (described in ref and in the assembly manual ‘Autopatcher control box assembly manual.pdf’ in Supplementary Data 4) to switch between different pressure states during autopatcher operation. The four pressures are generated by downregulating a compressed air source of ~2580 mBar using manual and electronic pressure regulators whose outputs can be controlled using knobs on the front panel of the control box (potentiometers in the lower left hand corner). Vacuum pressures are generated using Venturi tube vacuum generators also installed inside the autopatcher control box.
FIGURE 3
FIGURE 3. Optimum pipettes used for autopatching
(a) Photomicrographs of an ideal patch pipette pulled using a Flaming Brown pipette puller focusing on the pipette tip with 0.9 μm tip diameter (6.2 MΩ resistance) visualized with a 40× magnification objective (left) and 100× water immersion objective (right) in comparison to (b) a patch pipette with 1.5 μm tip diameter (3.3 MΩ resistance) visualized with a 40× magnification objective (left) and 100× water immersion objective (right). (c) Comparison of a convex tapered pipette which is ideal for autopatching (left) vs. concave tapered pipettes (right) and (d) illustration of an ideal patch pipette exhibiting broad cone angle. Larger tip angles, as measured in the image at the very tip of the pipette, are ideal for rapid gigasealing, stable recordings, and easier break-in attempts.
FIGURE 4
FIGURE 4. Surgical procedure for headplate implantation
(a) top (left) and side (right) views of the Delrin headplate to be affixed to the skull for head stabilization during autopatching. Scale bar indicates 5 mm. (b–f) Preparation and surgery of the anesthetized mouse after administering approved anesthetic, followed by shaving and sterilizing the scalp. First perform longitudinal incision of the scalp (b, c) to expose the skull (d). Clear the skull further around the desired recording region, (d; red circle). Using a burr drill bit, drill three anchor holes (~500 μm diameter, three small red circles; e) and implant skull screws. Finally implant the headplate by applying freshly mixed dental acrylic cement around the skull screws and around the periphery of the headplate window (f). (g) Photographs of the mouse skull after implantation of the skull screws (top) and after implantation of the headplate (bottom). Scale bars indicate ~2mm (h–k) Illustration of the craniotomy procedure. Identify the desired craniotomy location (h), thin down a 1–2 mm wide pit at the desired craniotomy location until the remaining bone is ~100 μm thick (i), carefully dislodge the flaky bone tissue with a tip of a needle (j), and lift off the bone tissue and clear any remaining bone fragments (k). CAUTION: All animal use should comply with institutional and federal regulations.
FIGURE 5
FIGURE 5. Autopatcher software graphical user interface (GUI)
Red dotted lines outline different panels of the GUI. Panel (1): Pipette status indicators that display the instantaneous pipette position (in μm) from surface of the brain, pressure applied to the pipette (in kPa), and the holding voltage (in mV) applied to the pipette during autopatcher operation. Panel (2): Text indicators that display the current status of the autopatcher trial, and a log of all autopatching trials attempted during an experiment, as well as an entry box where the experimenter can log comments. Panel (3): Includes control elements that allow the experimenter to control the programmable motor. This displays the absolute position of the pipette in motor coordinates. Panel (4): The interactive elements displayed in this panel depend on the stage of autopatching, and include methods for setting the beginning and ending depth ranges within which the autopatcher will scan for neurons, amongst other things (see Figs. 6 and 7 and Supplementary Video 1 to see how this box changes throughout the protocol).
FIGURE 6
FIGURE 6. Autopatcher software graphical user interface: neuron hunting
(a) The autopatcher software GUI displayed during the neuron hunting stage (BOX 2, Autopatching Step 3 in the protocol) of autopatching: red dotted lines highlight the indicators and user controls in Panel 4 of Figure 5 that can be accessed during this stage. The measured resistances after each step taken during neuron hunting are plotted on the “NEURON HUNTING RESISTANCE MONITOR” graph. The ‘SKIP TO GIGASEALING’ button allows the experimenter to override the neuron detection algorithm of the autopatcher and proceed to the gigasealing stage. The resistance threshold the algorithm uses to switch from neuron hunting to gigasealing mode can be changed as needed in the ‘Neuron detection threshold (M-Ohms)’ numerical entry box (default, 0.25 MΩ). (b) Representative screen shot of the “NEURON HUNTING RESISTANCE MONITOR” graph displaying the resistance measurements logged during a successful trial resulting in a whole cell patch recording. The autopatcher initially lowered the pipette to a depth of 650 μm and scanned to a depth of 720 μm before encountering a neuron as indicated by the monotonic increase in pipette resistance in the last data points.
FIGURE 7
FIGURE 7. Autopatcher software graphical user interface: gigasealing and break-in
(a) Panel 4 in Figure 5, now showing the autopatcher software GUI displayed during the gigasealing and break-in stages of autopatching (BOX 2, Autopatching Steps 4 and 5, Step 34 and 35). The ‘GIGASEALING RESISTANCE MONITOR’ graph displays the recorded seal resistances during a gigasealing attempt. The ‘RETURN TO NEURON HUNT’ button allows the experimenter to override the autopatcher operation and return to the neuron hunting stage of autopatching. The ‘MANUAL APPLICATION OF SUCTION’ button allows the experimenter to override the autopatcher algorithm’s negative pressure application to manually apply additional negative pressure at any time during gigasealing and allows exploration of alternate strategies for gigasealing used in in vivo and in vitro slice patching, . Once a successful gigaseal is formed, break-in can be attempted by using the ‘ATTEMPT BREAK IN’ button, which causes the autopatcher to apply pulses of negative pressure of selected time duration. Alternatively, break-in can be attempted by applying voltage pulses using the ‘ZAP!’ button. The ‘WHOLE CELL CURRENTS MONITOR’ graph displays the currents in response to injected voltage square waves after a break-in attempt so the user can assess whether it has achieved the whole cell configuration. (b) Screen capture of seal resistance measurements displayed in the ‘GIGASEALING RESISTANCES MONITOR’ graph in the autopatcher software GUI during a successful gigasealing attempt. A GΩ seal was obtained at time point (i). The ‘ATTEMPT BREAK IN’ button was utilized by the experimenter at t = 105 s to break into the cell. Whole cell configuration was obtained at time point (ii). (c) Illustration of currents measured and displayed in the ‘WHOLE CELL CURRENTS MONITOR’ graph in the autopatcher software GUI after a successful break-in attempt resulting in a whole cell patched neuron.
FIGURE 8
FIGURE 8. Autopatcher software graphical user interface: recording
(a) Panel 4 in Figure 5, now showing the autopatcher software GUI displayed during the recording stage after autopatching (Step 37–39). A toggle switch allows the user to use either the built-in data acquisition feature under ‘Autopatcher Control’ or to use an external data acquisition software under ‘External Control’. If the ‘Autopatcher Control’ option is used, a second toggle switch allows the user to switch between voltage clamp and current clamp mode. A numerical entry box allows the user to set the holding voltage (in mV, if recording in voltage clamp mode) or holding current (in pA, if recording in current clamp mode). When the ‘RECORD’ button is pressured, the measured currents (if recording in voltage clamp mode), or measured voltage (if recording in current clamp mode), are displayed in the graph indicator. The ‘REPEAT’ button can be used to acquire data continuously, and the ‘SAVE TO FILE’ button saves the file to disk. Pressing the ‘START OVER’ button ends the recording and the autopatcher will retract the patch pipette back to the surface in one quick step lasting ~500 ms for pipette retrieval and replacement to start a new trial. To recover the morphology of the recorded cell, biocytin filling is attempted by pressing the ‘SLOWLY RETRACT PIPETTE’ button, which will result in the autopatcher withdrawing the patch pipette back to the surface of the brain at a rate of 3 μm/s.
FIGURE 9
FIGURE 9. Example data acquired by the autopatcher
(a) Current clamp recording from autopatched cortical neuron during current injection (2s long pulses of −30, 0, +30, +60, +90, +120, +150 and +180 pA current injection). Access resistance, 44 MΩ; input resistance, 66 MΩ; depth of cell 442 μm below surface of the brain. (b–d) Voltage clamp recordings from an autopatched cortical neuron clamped at −80 mV (b) showing spontaneous excitatory post-synaptic potentials (EPSCs), (c) zooming in on single synaptic event indicated by black arrow in (b), and (d) zooming in synaptic barrage event indicated by black bar in (b). Access resistance 23 MΩ; input resistance 124 MΩ; depth of cell 544 μm from surface of the brain. (e) Biocytin fill of the autopatched cortical pyramidal neuron recorded in (a). (f) Autopatching in awake headfixed mice. Current clamp recording from a layer-4 cortical neuron in barrel cortex of an awake headfixed mouse showing persistent depolarization of membrane potential during active whisking (periods of whisker movements indicated by black bar). Access resistance 37 MΩ; input resistance, 88 MΩ; depth of cell 468 μm below surface of the brain. (g–h) Simultaneous whole cell recording and optogenetic stimulation in vivo. (g) Jaws-expressing neuron in cortex showing hyperpolarization at onset of red light delivery. Adapted from ref . (h) Channelrhodopsin-2 expressing cortical neuron in a Thy1-ChR2 mouse, showing evoked spiking in response to 20ms blue light pulses. All animal use complied with institutional and federal regulations. (g) adapted from Chuong et al Nat Neuroscience 2014.

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