Ultra-sensitive fluorescent proteins for imaging neuronal activity methods
Aim. Evidence-backed execution summary for Ultra-sensitive fluorescent proteins for imaging neuronal activity methods from Ultra-sensitive fluorescent proteins for imaging neuronal activity.
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mouse
Subject model for the experiment.
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Methods
reagent used in the protocol.
- Use
- Neurons were stimulated in imaging buffer containing a drug cocktail to inhibit synaptic receptors (10 µM CNQX, 10 µM (R)-CPP, 10 µM gabazine, 1 mM (S)-MCPG, Tocris). Under these conditions, intracellular calcium increases are presumably caused by the opening of voltage sensitive calcium channels.
Methods
reagent used in the protocol.
- Use
- Hippocampi were dissected and dissociated in papain. Cells were plated at a density of 225,000 viable cells/well in 24-well glass-bottom plates (Mattek, #1.5 glass coverslips), pre-coated with Matrigel (BD Biosciences). Cells were cultured in growth medium (28 mM glucose, 2.4 mM sodium bicarbonate, 100 µg/mL tr...
Methods
reagent used in the protocol.
- Use
- Lentiviral particles were made in a biosafety level 2 laboratory by transfecting a prolentiviral construct and packaging and coat pseudotyping DNA constructs (pCAG-SIVgprre, pCAG4-RTR-SIV, pCMV-VSV-G), into HEK293T/17 cells (ATCC) in 10-cm plates. After 72 h, supernatant was collected (6 mL) and filtered. Neuronal...
Methods
reagent used in the protocol.
- Use
- Control experiments varying stimulation voltage, frequency, and pulse width insured suprathreshold stimulation of neurons. Voltage imaging using the ArchWT-GFP archaerhodopsin-based voltage sensor confirmed that individual pulses (1 ms, 40 V, 83 Hz) reliably triggered single APs. The imaging and stimulation system w...
Methods
Action potentials (APs) (83 Hz) were evoked by field stimulation with a Grass Technologies S48 stimulation unit and a custom-built 24-well cap stimulator with pairs of parallel platinum wires. The microscope was an Olympus IX81 with a 10 × (0.4 NA) air objective lens and EMCCD camera (Andor 897, 512 × 512 p...
- Use
- Action potentials (APs) (83 Hz) were evoked by field stimulation with a Grass Technologies S48 stimulation unit and a custom-built 24-well cap stimulator with pairs of parallel platinum wires. The microscope was an Olympus IX81 with a 10 × (0.4 NA) air objective lens and EMCCD camera (Andor 897, 512 × 512 p...
Methods
Control experiments varying stimulation voltage, frequency, and pulse width insured suprathreshold stimulation of neurons. Voltage imaging using the ArchWT-GFP archaerhodopsin-based voltage sensor confirmed that individual pulses (1 ms, 40 V, 83 Hz) reliably triggered single APs. The imaging and stimulation system w...
- Use
- Control experiments varying stimulation voltage, frequency, and pulse width insured suprathreshold stimulation of neurons. Voltage imaging using the ArchWT-GFP archaerhodopsin-based voltage sensor confirmed that individual pulses (1 ms, 40 V, 83 Hz) reliably triggered single APs. The imaging and stimulation system w...
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Methods
Neurons were stimulated in imaging buffer containing a drug cocktail to inhibit synaptic receptors (10 µM CNQX, 10 µM (R)-CPP, 10 µM gabazine, 1 mM (S)-MCPG, Tocris). Under these conditions, intracellular calcium increases are presumably caused by the opening of voltage sensitive calcium channels.
Introduction
Dissociated rat hippocampal neurons in 24-well plates were transduced with GCaMP variants (one per well), together with nuclear mCherry, using lentivirus-mediated gene transfer. Electrodes triggered trains of action potentials in all neurons within each well (Methods). Time-lapse images (35 Hz) of ∼800 µm fields of view containing 10-100 neurons were acquired, while delivering a series of action potential trains ( ). Fluorescence changes extracted from single neurons were used to compare the sensitivity, dynamic range, and kinetics of individual GCaMP variants and OGB1-AM ( ). We monitored the resting brightness of the sensor by measuring green fluorescence relative to red mCherry fluorescence.
Calcium transients in dendritic spines
Pyramidal neuron output is shaped by thousands of excitatory synapses distributed across the dendritic arbor. Activation of single excitatory synapses causes calcium accumulations in individual dendritic spines, mediated by NMDA-Rs,, which can be imaged to measure the tuning of single synapses in vivo,. We used GCaMP6s to image synaptic calcium signals within dendritic spines over chronic timescales. In V1 with sparsely labeled L2/3 pyramidal neurons (Methods) small dendritic branches were imaged at high magnification during visual stimulation ( ). We first focused on neurons that did not fire visually evoked action potentials (∼40% of neurons) to avoid calcium changes caused by action potentials back-propagating into dendrites. Individual spines showed large fluorescence transients, often independent of their parent dendrites ( and ). Spine responses were orientation-tuned...
Methods
Hippocampi were dissected and dissociated in papain. Cells were plated at a density of 225,000 viable cells/well in 24-well glass-bottom plates (Mattek, #1.5 glass coverslips), pre-coated with Matrigel (BD Biosciences). Cells were cultured in growth medium (28 mM glucose, 2.4 mM sodium bicarbonate, 100 µg/mL transferrin, B-27 supplement (1X, Invitrogen), 500 µM L-glutamine, 50 units/mL penicillin, 50 mg/mL streptomycin, 5% fetal bovine serum in MEM).
Methods
Lentiviral particles were made in a biosafety level 2 laboratory by transfecting a prolentiviral construct and packaging and coat pseudotyping DNA constructs (pCAG-SIVgprre, pCAG4-RTR-SIV, pCMV-VSV-G), into HEK293T/17 cells (ATCC) in 10-cm plates. After 72 h, supernatant was collected (6 mL) and filtered. Neuronal cultures were infected at 3 days in vitro. Each well of a 24-well plate was incubated overnight with 0.5 mL of lentivirus in conditioned growth medium. The growth medium was supplemented with 4 µM AraC to inhibit glial proliferation. In some experiments, OGB1-AM was loaded into cells by incubating neurons in 1 mL of 2 µM OGB1-AM (Invitrogen) for 30 min and rinsing 3 times with imaging buffer (145 mM NaCl, 2.5 mM KCl, 10 mM glucose, 10 mM HEPES pH 7.4, 2 mM CaCl 2, 1 mM MgCl 2 ).
Methods
Action potentials (APs) (83 Hz) were evoked by field stimulation with a Grass Technologies S48 stimulation unit and a custom-built 24-well cap stimulator with pairs of parallel platinum wires. The microscope was an Olympus IX81 with a 10 × (0.4 NA) air objective lens and EMCCD camera (Andor 897, 512 × 512 pixels, 35 frames/s), Cairn OptoLED illumination system, and GFP (Excitation: 450-490 nm; Dichroic: 495 nm long-pass; Emission: 500-550 nm) and TxRed (Excitation: 540-580 nm; Dichroic: 585 nm long-pass; Emission: 593-668 nm) filter sets. The field of view was 800 µm × 800 µm. Images were background subtracted (mean of 5% lowest pixel values). Responses were quantified for each cell as change in fluorescence divided by baseline fluorescence measured one second prior to stimulation. Signal-to-noise ratio (SNR) was quantified as peak ΔF/F 0 response over the...
Methods
Control experiments varying stimulation voltage, frequency, and pulse width insured suprathreshold stimulation of neurons. Voltage imaging using the ArchWT-GFP archaerhodopsin-based voltage sensor confirmed that individual pulses (1 ms, 40 V, 83 Hz) reliably triggered single APs. The imaging and stimulation system was controlled by custom scripts written in MetaMorph software (version 7.7.5, Molecular Devices) and Ephus software (ephus.org). Detailed neuronal culture screening methods will be described elsewhere (T.J.W., T.W.C., E.R.S., R.A.K., V.J., L.L.L., K.S., and D.S.K., manuscript in preparation).
Image analysis
Occasional BAP related calcium signals that invaded the imaged spines were removed using a subtraction method, implemented in three steps ( ). First, a region covering the entire parent dendritic shaft (∼30 micrometers of dendritic length; excluding all spines) was drawn for each recorded dendritic segment to estimate BAP related global dendritic signal, ΔF/F 0_dendrite. Because of the much larger volume of the dendritic shaft compared to tuned dendritic spines (100-fold), spines above and below the focal plane were expected to contribute negligible signal to ΔF/F 0_dendrite. This was verified using principle component analysis (data not shown). Plotting ΔF/F 0_spine against ΔF/F 0_dendrite reveals two components of spine signals, a BAP-related component and a spine-specific component. Second, the BAP-related component was removed from the spine signals by...
Measurement outputs
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Pyramidal neuron output is shaped by thousands of excitatory synapses distributed across the dendritic arbor. Activation of single excitatory synapses causes calcium accumulatio...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
GCaMP variants were made in a modified SIV-based lentiviral construct, pGP- syn -GCaMP-nls-mCherry-WPRE, derived from pCL20cSLFR MSCV-GFP. The prolentiviral vector included a 4...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Occasional BAP related calcium signals that invaded the imaged spines were removed using a subtraction method, implemented in three steps ( ). First, a region covering the entir...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
We next confirmed the effectiveness of the BAP removal algorithm. First, BAP removed spine signals showed sharp orientation tuning (OSI = 0.84 ± 0.14, mean ± s.d., n=1...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Pyramidal neuron output is shaped by thousands of excitatory synapses distributed across the dendritic arbor.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Pyramidal neuron output is shaped by thousands of excitatory synapses distributed across the dendritic arbor. Activation of single excitatory synapses causes calcium accumulatio...; GCaMP variants were made in a modified SIV-based lentiviral construct, pGP- syn -GCaMP-nls-mCherry-WPRE, derived from pCL20cSLFR MSCV-GFP. The prolentiviral vector included a 4...; Occasional BAP related calcium signals that invaded the imaged spines were removed using a subtraction method, implemented in three steps ( ). First, a region covering the entir...; We next confirmed the effectiveness of the BAP removal algorithm. First, BAP removed spine signals showed sharp orientation tuning (OSI = 0.84 ± 0.14, mean ± s.d., n=1....
from paperStatistical comparison
Pyramidal neuron output is shaped by thousands of excitatory synapses distributed across the dendritic arbor. Activation of single excitatory synapses causes calcium accumulatio...; How orientation-tuned neurons connect to other orientation-tuned neurons remains controversial,. Some measurements suggest that individual neurons sample heterogeneous input,...; We next confirmed the effectiveness of the BAP removal algorithm. First, BAP removed spine signals showed sharp orientation tuning (OSI = 0.84 ± 0.14, mean ± s.d., n=1...
from paperReporting output
Report representative outputs alongside summary comparisons for Pyramidal neuron output is shaped by thousands of excitatory synapses distributed across the dendritic arbor. Activation of single excitatory synapses causes calcium accumulatio..., GCaMP variants were made in a modified SIV-based lentiviral construct, pGP- syn -GCaMP-nls-mCherry-WPRE, derived from pCL20cSLFR MSCV-GFP. The prolentiviral vector included a 4..., Occasional BAP related calcium signals that invaded the imaged spines were removed using a subtraction method, implemented in three steps ( ). First, a region covering the entir..., We next confirmed the effectiveness of the BAP removal algorithm. First, BAP removed spine signals showed sharp orientation tuning (OSI = 0.84 ± 0.14, mean ± s.d., n=1....
inferred from protocolStructured statistical methods
Pyramidal neuron output is shaped by thousands of excitatory synapses distributed across the dendritic arbor. Activation of single excitatory synapses causes calcium accumulatio...; How orientation-tuned neurons connect to other orientation-tuned neurons remains controversial,. Some measurements suggest that individual neurons sample heterogeneous input,...; We next confirmed the effectiveness of the BAP removal algorithm. First, BAP removed spine signals showed sharp orientation tuning (OSI = 0.84 ± 0.14, mean ± s.d., n=1...
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Evidence quotes (8)
Neurons were stimulated in imaging buffer containing a drug cocktail to inhibit synaptic receptors (10 µM CNQX, 10 µM (R)-CPP, 10 µM gabazine, 1 mM (S)-MCPG, Tocris). Under these conditions, intracellular calcium increases are presumably caused by the opening of voltage sensitive calcium channels.
Dissociated rat hippocampal neurons in 24-well plates were transduced with GCaMP variants (one per well), together with nuclear mCherry, using lentivirus-mediated gene transfer. Electrodes triggered trains of action potentials in all neurons within each well (Methods). Time-lapse images (35 Hz) of ∼800 µm fields of view containing 10-100 neurons were acquired, while delivering a series of action potential trains ( ). Fluorescence changes extracted from single neurons were used to compare the sensitivity, dynamic range, and kinetics of individual GCaMP variants and OGB1-AM ( ). We monitored the resting brightness of the sensor by measuring green fluorescence relative to red mCherry fluorescence.
Pyramidal neuron output is shaped by thousands of excitatory synapses distributed across the dendritic arbor. Activation of single excitatory synapses causes calcium accumulations in individual dendritic spines, mediated by NMDA-Rs,, which can be imaged to measure the tuning of single synapses in vivo,. We used GCaMP6s to image synaptic calcium signals within dendritic spines over chronic timescales. In V1 with sparsely labeled L2/3 pyramidal neurons (Methods) small dendritic branches were imaged at high magnification during visual stimulation ( ). We first focused on neurons that did not fire visually evoked action potentials (∼40% of neurons) to avoid calcium changes caused by action potentials back-propagating into dendrites. Individual spines showed large fluorescence transients, often independent of their parent dendrites ( and ). Spine responses were orientation-tuned ( ), as expected from the fact that most input to V1 neurons originates from (presumably orientation-tuned) V1 neurons. Neighboring spines were often tuned with different preferred orientations. The correlation between orientation tuning and distance between spines was weak (R=0.08; p>0.05). Overa...
Hippocampi were dissected and dissociated in papain. Cells were plated at a density of 225,000 viable cells/well in 24-well glass-bottom plates (Mattek, #1.5 glass coverslips), pre-coated with Matrigel (BD Biosciences). Cells were cultured in growth medium (28 mM glucose, 2.4 mM sodium bicarbonate, 100 µg/mL transferrin, B-27 supplement (1X, Invitrogen), 500 µM L-glutamine, 50 units/mL penicillin, 50 mg/mL streptomycin, 5% fetal bovine serum in MEM).
Lentiviral particles were made in a biosafety level 2 laboratory by transfecting a prolentiviral construct and packaging and coat pseudotyping DNA constructs (pCAG-SIVgprre, pCAG4-RTR-SIV, pCMV-VSV-G), into HEK293T/17 cells (ATCC) in 10-cm plates. After 72 h, supernatant was collected (6 mL) and filtered. Neuronal cultures were infected at 3 days in vitro. Each well of a 24-well plate was incubated overnight with 0.5 mL of lentivirus in conditioned growth medium. The growth medium was supplemented with 4 µM AraC to inhibit glial proliferation. In some experiments, OGB1-AM was loaded into cells by incubating neurons in 1 mL of 2 µM OGB1-AM (Invitrogen) for 30 min and rinsing 3 times with imaging buffer (145 mM NaCl, 2.5 mM KCl, 10 mM glucose, 10 mM HEPES pH 7.4, 2 mM CaCl 2, 1 mM MgCl 2 ).
Action potentials (APs) (83 Hz) were evoked by field stimulation with a Grass Technologies S48 stimulation unit and a custom-built 24-well cap stimulator with pairs of parallel platinum wires. The microscope was an Olympus IX81 with a 10 × (0.4 NA) air objective lens and EMCCD camera (Andor 897, 512 × 512 pixels, 35 frames/s), Cairn OptoLED illumination system, and GFP (Excitation: 450-490 nm; Dichroic: 495 nm long-pass; Emission: 500-550 nm) and TxRed (Excitation: 540-580 nm; Dichroic: 585 nm long-pass; Emission: 593-668 nm) filter sets. The field of view was 800 µm × 800 µm. Images were background subtracted (mean of 5% lowest pixel values). Responses were quantified for each cell as change in fluorescence divided by baseline fluorescence measured one second prior to stimulation. Signal-to-noise ratio (SNR) was quantified as peak ΔF/F 0 response over the standard deviation of the signal during a one second period prior to stimulation.
Control experiments varying stimulation voltage, frequency, and pulse width insured suprathreshold stimulation of neurons. Voltage imaging using the ArchWT-GFP archaerhodopsin-based voltage sensor confirmed that individual pulses (1 ms, 40 V, 83 Hz) reliably triggered single APs. The imaging and stimulation system was controlled by custom scripts written in MetaMorph software (version 7.7.5, Molecular Devices) and Ephus software (ephus.org). Detailed neuronal culture screening methods will be described elsewhere (T.J.W., T.W.C., E.R.S., R.A.K., V.J., L.L.L., K.S., and D.S.K., manuscript in preparation).
Occasional BAP related calcium signals that invaded the imaged spines were removed using a subtraction method, implemented in three steps ( ). First, a region covering the entire parent dendritic shaft (∼30 micrometers of dendritic length; excluding all spines) was drawn for each recorded dendritic segment to estimate BAP related global dendritic signal, ΔF/F 0_dendrite. Because of the much larger volume of the dendritic shaft compared to tuned dendritic spines (100-fold), spines above and below the focal plane were expected to contribute negligible signal to ΔF/F 0_dendrite. This was verified using principle component analysis (data not shown). Plotting ΔF/F 0_spine against ΔF/F 0_dendrite reveals two components of spine signals, a BAP-related component and a spine-specific component. Second, the BAP-related component was removed from the spine signals by subtracting a scaled version of the dendritic shaft signal, ΔF/F 0_spine_specific =ΔF/F 0_spine - α ·ΔF/F 0_dendrite. α was determined using robust regression (MATLAB function 'robustfit.m') of ΔF/F 0_spine vs. ΔF/F 0_dendrite (the slope of the fi...
Machine-readable layer
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"text": "Dissociated rat hippocampal neurons in 24-well plates were transduced with GCaMP variants (one per well), together with nuclear mCherry, using lentivirus-mediated gene transfer. Electrodes triggered trains of action potentials in all neurons within each well (Methods). Time-lapse images (35 Hz) of ∼800 µm fields of view containing 10-100 neurons were acquired, while delivering a series of action potential trains ( ). Fluorescence changes extracted from single neurons were used to compare the sensitivity, dynamic range, and kinetics of individual GCaMP variants and OGB1-AM ( ). We monitored the resting brightness of the sensor by measuring green fluorescence relative to red mCherry fluorescence."
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"text": "Pyramidal neuron output is shaped by thousands of excitatory synapses distributed across the dendritic arbor. Activation of single excitatory synapses causes calcium accumulations in individual dendritic spines, mediated by NMDA-Rs,, which can be imaged to measure the tuning of single synapses in vivo,. We used GCaMP6s to image synaptic calcium signals within dendritic spines over chronic timescales. In V1 with sparsely labeled L2/3 pyramidal neurons (Methods) small dendritic branches were imaged at high magnification during visual stimulation ( ). We first focused on neurons that did not fire visually evoked action potentials (∼40% of neurons) to avoid calcium changes caused by action potentials back-propagating into dendrites. Individual spines showed large fluorescence transients, often independent of their parent dendrites ( and ). Spine responses were orientation-tuned..."
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"text": "Hippocampi were dissected and dissociated in papain. Cells were plated at a density of 225,000 viable cells/well in 24-well glass-bottom plates (Mattek, #1.5 glass coverslips), pre-coated with Matrigel (BD Biosciences). Cells were cultured in growth medium (28 mM glucose, 2.4 mM sodium bicarbonate, 100 µg/mL transferrin, B-27 supplement (1X, Invitrogen), 500 µM L-glutamine, 50 units/mL penicillin, 50 mg/mL streptomycin, 5% fetal bovine serum in MEM)."
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"text": "Action potentials (APs) (83 Hz) were evoked by field stimulation with a Grass Technologies S48 stimulation unit and a custom-built 24-well cap stimulator with pairs of parallel platinum wires. The microscope was an Olympus IX81 with a 10 × (0.4 NA) air objective lens and EMCCD camera (Andor 897, 512 × 512 pixels, 35 frames/s), Cairn OptoLED illumination system, and GFP (Excitation: 450-490 nm; Dichroic: 495 nm long-pass; Emission: 500-550 nm) and TxRed (Excitation: 540-580 nm; Dichroic: 585 nm long-pass; Emission: 593-668 nm) filter sets. The field of view was 800 µm × 800 µm. Images were background subtracted (mean of 5% lowest pixel values). Responses were quantified for each cell as change in fluorescence divided by baseline fluorescence measured one second prior to stimulation. Signal-to-noise ratio (SNR) was quantified as peak ΔF/F 0 response over the..."
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"text": "Control experiments varying stimulation voltage, frequency, and pulse width insured suprathreshold stimulation of neurons. Voltage imaging using the ArchWT-GFP archaerhodopsin-based voltage sensor confirmed that individual pulses (1 ms, 40 V, 83 Hz) reliably triggered single APs. The imaging and stimulation system was controlled by custom scripts written in MetaMorph software (version 7.7.5, Molecular Devices) and Ephus software (ephus.org). Detailed neuronal culture screening methods will be described elsewhere (T.J.W., T.W.C., E.R.S., R.A.K., V.J., L.L.L., K.S., and D.S.K., manuscript in preparation)."
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"text": "Occasional BAP related calcium signals that invaded the imaged spines were removed using a subtraction method, implemented in three steps ( ). First, a region covering the entire parent dendritic shaft (∼30 micrometers of dendritic length; excluding all spines) was drawn for each recorded dendritic segment to estimate BAP related global dendritic signal, ΔF/F 0_dendrite. Because of the much larger volume of the dendritic shaft compared to tuned dendritic spines (100-fold), spines above and below the focal plane were expected to contribute negligible signal to ΔF/F 0_dendrite. This was verified using principle component analysis (data not shown). Plotting ΔF/F 0_spine against ΔF/F 0_dendrite reveals two components of spine signals, a BAP-related component and a spine-specific component. Second, the BAP-related component was removed from the spine signals by..."
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