Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics

Abstract

Single-cell genetic manipulation is expected to substantially advance the field of systems neuroscience. However, existing gene delivery techniques do not allow researchers to electrophysiologically characterize cells and to thereby establish an experimental link between physiology and genetics for understanding neuronal function. In the mouse brain in vivo, we found that neurons remained intact after 'blind' whole-cell recording, that DNA vectors could be delivered through the patch-pipette during such recordings and that these vectors drove protein expression in recorded cells for at least 7 d. To illustrate the utility of this approach, we recorded visually evoked synaptic responses in primary visual cortical cells while delivering DNA plasmids that allowed retrograde, monosynaptic tracing of each neuron's presynaptic inputs. By providing a biophysical profile of a cell before its specific genetic perturbation, this combinatorial method captures the synaptic and anatomical receptive field of a neuron.

Figure 1. Recording methodology and biocytin recovery rates

a, Seal test current trace used to monitor pipette resistance. b, Contact with cell membrane while stepping the electrode indicated by rhythmic changes in pipette resistance associate with heartbeat-related movement. c, Traces showing seal formation and d, whole-cell access. e, IV relationship (current injections 50 pA steps for 600 ms) recorded from the cell shown in i. f, Current traces in response to voltage steps immediately prior and g, during pipette retraction whereby the outside-out patch configuration is achieved h. Inset trace: detail from h showing single channel currents. i, Example morphology of a biocytin-labeled layer 2/3 pyramidal cell (animal sacrificed 29 hours after recording). j, Success rates of biocytin-filled cell recovery plotted against series resistance of recording (determined within 30 seconds after break-in). Circles represent, on a cell-by-cell basis, recovery success or failure. Grey-filled circles indicate those recordings that did not meet retraction and/or series resistance criteria. Large open red circles represent the mean success for each bin (range: 20–40, 41–50, 53–70, 71–200 MΩ). All error bars are SD. Numbers indicate the n for the respective bin. Dashed lines are linear fits to the individual data points. k, Success rates of cell recovery plotted against duration of recording (bin ranges: 1–4, 5–6, 7–9 and 10–13 mins) and l, the interval between recording and sacrificing the animal (bin ranges: 23–24, 25–29, 31–45, 47–51 hours).

Figure 2. Physiological characterization and genetic manipulation

a, The firing profile and IV relationship recorded from the layer 5 neuron shown in panels d1 and d2. Voltage traces shown are in response to 50 pA current steps of 600 ms duration. b, Membrane voltage traces showing spontaneous synaptic activity and the kinetics of a resultant action potential. c1, Synaptic responses (average of 10 sweeps) to drifting gratings moving in the direction indicated by the arrows (left). Arrow (top) indicates onset of grating drift. c2, Polar plot showing integral of the membrane voltage traces (for 1400ms from stimulus onset). d, Layer 5 pyramidal cell in primary visual cortex 26 hours after recording showing GFP expression (d1) and biocytin-labeling (d2). Note the weak, non-specific biocytin signal along the electrode track due to the plume of biocytin expelled while approaching the recorded cell. e, GFP signal in dendritic spines and axonal arborizations in the same cell. f, Success rates of GFP-labeled cell recovery plotted against series resistance of recording (determined within 30 seconds after break-in). Circles represent, on a cell-by-cell basis, recovery success or failure. Grey-filled circles are cases that did not meet retraction and/or series resistance criteria. Open green circles represent the mean success for each bin (range 20–40, 41–50, 53–70, 71–200 MΩ. All error bars are SD. Numbers indicate the n for the respective bin. Dashed lines are linear fits to the individual data points. g, Success rates of cell recovery plotted against duration of recording (bin ranges: 1–4, 5–6, 7–8, 9–13 mins) and h, the interval between recording and sacrificing the animal (bin ranges: 23–25, 26–28, 29–33, 40–47, 48–51 hours).

Figure 3. Multiple gene delivery

a, Native fluorescence images of a layer 5 neuron patched in motor cortex 3 days after patching with pCAGGS-tdTomato (top right) and pCAGGS-Cerulean (50 ng/μl each bottom right), scale bar: 1 mm and 20 μm. b, Gallery of native fluorescence images for a layer 5 cell in primary somatosensory cortex 5 days after recording with pCAGGS-DsRed2, pCAGGS-Venus and pCAGGS-ChR2-Cerulean. Scale bar: 20 μm.

Figure 4. Synaptic receptive mapping and connectivity of a visual cortical neuron

a, The firing profile and IV relationship recorded from the GFP-expressing cell shown in panel d. Voltage traces shown are in response to 50 pA current steps of 600 ms duration. b, Synaptic responses (average of 10 sweeps) to drifting gratings moving in the direction indicated by the arrows (left). Arrow (top) indicates onset of drift. Current injection (−200 pA) was delivered to isolate evoked synaptic responses from AP activity. c, Polar plot showing integral of the voltage traces (red) recorded during the duration of the drifting grating. The integral of synaptic responses (1400 ms from stim onset) are normalized to largest response. Below are 5 example traces recorded for the least and most preferred orientation. For the spiking receptive field, holding current was removed and the stimulus sequence (b) was repeated five times. d, Coronal slice (100 μm thick) showing anti-GFP (green) and anti-RFP (red) fluorescence. The recorded layer 5 neuron (green-yellow) in primary visual cortex (V1) and mCherry-labeled cells (red) are clearly identifiable. In this slice approximately 200 presynaptic cells were observed. Scale bar = 500 μm. Abbreviations: V2ML: secondary visual cortex mediolateral, V2L: secondary visual cortex lateral, CA1–3: hippocampus fields CA1–3, DLG: dorsolateral geniculate nucleus. e, High magnification image showing GFP and mCherry labeled cells in V1. Scale bar = 100 μm. f. In this slice 8 mCerry-labeled cells were observed in the DLG. Scale bar = 50 μm. g. Five maximum intensity projections for consecutive 100 μm thick slices. The middle panel contains the host cell (green; scale bar: 200 μm). This mouse was sacrificed 7 and 9 days after virus injection and recording respectively.