Development of a platform for automated, two-photon targeted quad patch-clamping of neurons
File(s)
Author(s)
Vera Gonzalez, Gema
Type
Thesis
Abstract
Whole-cell patch-clamp allows for the study of ion channel biophysics, membrane
properties, and synaptic responses in intact brains. Multi-patching
promises to elucidate the functional and connectivity properties of neuronal
microcircuits, but the high skill and labour required (and low throughput)
hinders its widespread dissemination. Automated multi-patching may solve
this problem, but current ‘blind’ autopatchers are impractical for targeting
non-primary cell types or perform connectivity studies. We have developed
an automated two-photon targeted quad-channel patch-clamping technology
platform for ex vivo electrophysiology, extending the system of Annecchino
et al. (Neuron 2017, 95:1048-55), as a promising step towards an in vivo
targeted, quad-patching setup.
The platform comprises a two-photon microscope, four micromanipulators
(Sensapex/Scientifica) fitted with mechanical stability clamps (to improve
pipette resistance against vibration, tissue deformation and pressure changes),
custom-developed electronically-controlled pressure regulator, patch-clamp
amplifier and DAQs. Control is via a custom-developed LabView program
which acquires frames directly from the microscope. After each trial, pipettes
are automatically cleaned and returned to their previous positions, allowing
for their reuse. A ‘follow’ function is available to allow the user to move a
single pipette and have the rest follow, both in the z and x/y coordinates, to
facilitate patching in new, undisturbed brain regions. Temperature control
for the perfusion system is also possible within the software. The output
and cell information of each channel is automatically recorded for each trial.
Basic injection and connectivity protocols are also available to run within
the program, making the software a complete set for the basic needs of any
experimenter, regardless of their skill set.
Our targeted quad patch-clamp system allows scalable and reproducible electrophysiology
studies to be conducted across a variety of laboratory settings,
offering for the first time robotically automated recording of subthreshold
signals from multiple genetically and optically targeted cells simultaneously.
properties, and synaptic responses in intact brains. Multi-patching
promises to elucidate the functional and connectivity properties of neuronal
microcircuits, but the high skill and labour required (and low throughput)
hinders its widespread dissemination. Automated multi-patching may solve
this problem, but current ‘blind’ autopatchers are impractical for targeting
non-primary cell types or perform connectivity studies. We have developed
an automated two-photon targeted quad-channel patch-clamping technology
platform for ex vivo electrophysiology, extending the system of Annecchino
et al. (Neuron 2017, 95:1048-55), as a promising step towards an in vivo
targeted, quad-patching setup.
The platform comprises a two-photon microscope, four micromanipulators
(Sensapex/Scientifica) fitted with mechanical stability clamps (to improve
pipette resistance against vibration, tissue deformation and pressure changes),
custom-developed electronically-controlled pressure regulator, patch-clamp
amplifier and DAQs. Control is via a custom-developed LabView program
which acquires frames directly from the microscope. After each trial, pipettes
are automatically cleaned and returned to their previous positions, allowing
for their reuse. A ‘follow’ function is available to allow the user to move a
single pipette and have the rest follow, both in the z and x/y coordinates, to
facilitate patching in new, undisturbed brain regions. Temperature control
for the perfusion system is also possible within the software. The output
and cell information of each channel is automatically recorded for each trial.
Basic injection and connectivity protocols are also available to run within
the program, making the software a complete set for the basic needs of any
experimenter, regardless of their skill set.
Our targeted quad patch-clamp system allows scalable and reproducible electrophysiology
studies to be conducted across a variety of laboratory settings,
offering for the first time robotically automated recording of subthreshold
signals from multiple genetically and optically targeted cells simultaneously.
Version
Open Access
Date Issued
2023-01
Date Awarded
2023-04
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Schultz, Simon
Brickley, Stephen
Publisher Department
Biomedical Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)