Data (Information)

Plateau potentials underlie calcium indicator signals in vivo

Alejandre Garcia, Maria Mildred Tzitzitlini

Calcium imaging is widely used to measure the activity of neuronal populations, as calcium accumulations are interpreted as proxies for action-potential (APs) firing. However, by using simultaneous whole-cell and two-photon calcium imaging, we recorded spontaneous activity of pyramidal neurons in mouse layer 2/3 visual cortex in vivo and find that somatic calcium transients correlate better with plateau potentials than with AP trains. Our results demonstrate that somatic calcium signals in vivo primarily reflect plateau potentials, which should be explicitly considered when interpreting calcium imaging data and calibrating spike-inference models.

Keywords: Cortex, Imaging, Pyramidal neuron, Two-photon

Subjects

Files

  • thumbnail for Tigre_GCamp6sLinearR_1.mat Tigre_GCamp6sLinearR_1.mat application/x-matlab-data 131 MB
  • thumbnail for Tigre_GCamp6sLinearR_4.mat Tigre_GCamp6sLinearR_4.mat application/x-matlab-data 118 MB
  • thumbnail for Tigre_GCamp6sLinearR_2.mat Tigre_GCamp6sLinearR_2.mat application/x-matlab-data 123 MB
  • thumbnail for GCamp6sLinearR_1.mat GCamp6sLinearR_1.mat application/x-matlab-data 107 MB
  • thumbnail for GCamp8mLinearR_3.mat GCamp8mLinearR_3.mat application/x-matlab-data 69.6 MB
  • thumbnail for GCamp8mLinearR_1.mat GCamp8mLinearR_1.mat application/x-matlab-data 68.3 MB
  • thumbnail for GCamp6sLinearR_3.mat GCamp6sLinearR_3.mat application/x-matlab-data 136 MB
  • thumbnail for GCamp8mLinearR_4.mat GCamp8mLinearR_4.mat application/x-matlab-data 81.9 MB
  • thumbnail for GCamp6sLinearR_2.mat GCamp6sLinearR_2.mat application/x-matlab-data 101 MB
  • thumbnail for Tigre_GCamp6sLinearR_3.mat Tigre_GCamp6sLinearR_3.mat application/x-matlab-data 89.8 MB
  • thumbnail for GCamp8mLinearR_2.mat GCamp8mLinearR_2.mat application/x-matlab-data 83.8 MB

More About This Work

Academic Units
Biological Sciences
Published Here
January 29, 2026

Notes

We investigated the biophysical origin of calcium transients by combining two-photon calcium imaging with whole-cell recordings from layer 2/3 pyramidal neurons in mouse visual cortex in vivo. We measured two-photon fluorescence from two genetically encoded indicators: GCaMP8m, expressed in Vglut1-Cre transgenic mice with viral injection of AAV-FLEX-jGCaMP8m, and GCaMP6s, expressed by crossing Vglut1-Cre mice with TIGRE2.0 Ai162 reporter mice. We inspected movies and selected neurons with clear somatic fluorescent transients for targeted whole-cell recordings.

We evaluated each neuron’s membrane-voltage distribution using raw voltage recorded during 5 minutes of spontaneous activity. Calcium imaging experiments were performed on a two-photon microscope (Ultima IV, Bruker), equipped with a Ti:sapphire laser (Chameleon Ultra II, Coherent) tuned to λ = 920 nm. Laser power at the sample (20-30 mW) was regulated with a Pockels cell (Conoptics 350-105), using 2 PMTs filtered for green (510/20 nm) and red (607/45). Scanning used combined resonant and galvanometric mirrors. Fluorescence was collected through a 25x/1.05 N.A. water immersion objective (XLPlan N, Olympus).

Recordings focused on pyramidal-neuron soma in a single imaging plane. The field of view (FOV) was 512 x 512 pixels scanned at ~33 Hz (230 x 230 μm). Patch-clamp voltage signals were synchronously with calcium imaging using Prairie View (Bruker).

Recordings were analyzed with custom routines in MATLAB. The data was read and storage as '.mat' files per recorded neuron.

FILES:
GCaMP6:
Tigre_GCamp6sLinearR_1.mat
Tigre_GCamp6sLinearR_2.mat
Tigre_GCamp6sLinearR_3.mat
Tigre_GCamp6sLinearR_4.mat
GCamp6sLinearR_1.mat
GCamp6sLinearR_2.mat
GCamp6sLinearR_3.mat
GCaMP8:
GCamp8mLinearR_1.mat
GCamp8mLinearR_2.mat
GCamp8mLinearR_3.mat
GCamp8mLinearR_4.mat

Variable naming:
“Cell_caTransient” Correspond to normalized calcium transient of the recorded neuron.
“recording_method”: Correspond to voltage membrane (mV) of the recorded neuron.
“Par_Ca” (1x1 struct) “frame_period”: Calcium imaging, frame recording.
“frame_relativeTime”: Calcium imaging, total recording duration.
“AP_inference”: number of AP per calcium imaging frame
“Par_VR” (1x1 struct) “Voltage”: voltage membrane (mV)
“Current”: Current membrane (pA)
“Spike_time”: Spike events time
“APs_per_frame”: AP per voltage recording frame
“FIT” (1x1 struct) • “x”: voltage membrane suprathreshold duration per calcium transient.
• “y”: calcium transient amplitude.
“APs”:
• “x”: number of APs per calcium transient.
• “y”: calcium transient amplitude.
“ins_freq”:
• “x”: APs averaged frequency per calcium transient.
• “y”: calcium transient amplitude.