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. 2023 Feb 2;186(3):543-559.e19.
doi: 10.1016/j.cell.2022.12.035. Epub 2023 Jan 19.

All-optical physiology resolves a synaptic basis for behavioral timescale plasticity

Linlin Z Fan  1 , Doo Kyung Kim  1 , Joshua H Jennings  1 , He Tian  2 , Peter Y Wang  1 , Charu Ramakrishnan  1 , Sawyer Randles  1 , Yanjun Sun  3 , Elina Thadhani  1 , Yoon Seok Kim  1 , Sean Quirin  1 , Lisa Giocomo  3 , Adam E Cohen  4 , Karl Deisseroth  5
Affiliations

All-optical physiology resolves a synaptic basis for behavioral timescale plasticity

Linlin Z Fan et al. Cell. .

Abstract

Learning has been associated with modifications of synaptic and circuit properties, but the precise changes storing information in mammals have remained largely unclear. We combined genetically targeted voltage imaging with targeted optogenetic activation and silencing of pre- and post-synaptic neurons to study the mechanisms underlying hippocampal behavioral timescale plasticity. In mice navigating a virtual-reality environment, targeted optogenetic activation of individual CA1 cells at specific places induced stable representations of these places in the targeted cells. Optical elicitation, recording, and modulation of synaptic transmission in behaving mice revealed that activity in presynaptic CA2/3 cells was required for the induction of plasticity in CA1 and, furthermore, that during induction of these place fields in single CA1 cells, synaptic input from CA2/3 onto these same cells was potentiated. These results reveal synaptic implementation of hippocampal behavioral timescale plasticity and define a methodology to resolve synaptic plasticity during learning and memory in behaving mammals.

Keywords: all-optical electrophysiology; excitability; hippocampal behavioral timescale plasticity; imaging; optogenetics; place cells; synaptic plasticity.

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Conflict of interest statement

Declaration of interests A.E.C. is a founder of Q-State Biosciences. K.D. is a member of the Cell advisory board.

Figures

Figure 1.
Figure 1.. Voltage imaging of hippocampal dynamics during virtual reality (VR) behavior
(A) Optical system and VR setup. Holographic structured-illumination voltage imaging (red), micromirror-patterned optogenetic stimulation (blue), and VR rig (cyan and gray). Details in STAR Methods. (B) Visual wall cues of VR environment. Red box: reward delivered at end of track on 80% of VR trials. (C) Voltage imaging of CA1 cells during VR behavior. Red: single-trial unfiltered fluorescence traces of somQuasAr6a recorded at 1 kHz. Gray shaded zones: simultaneously recorded cells. Black: mouse running velocity. Dark red line with ticks at bottom: reward delivery. Cyan: detected licking periods (reward retrieval). Middle: magnified views of boxed region at left. Right: corresponding cells showing GEVI fluorescence. Scale bars, 20 μm. (D) Spike-triggered autocorrelogram showing (left) refractory period and (right) theta oscillation (n = 117 cells, 9 mice). (E and F) (E) Spike-triggered average waveform, and (F) power spectra showing theta oscillation peaking at ~7 Hz. (G) Two representative place cells: red: GEVI fluorescence traces as a function of time for selected VR trials. Dark red: ramp-like subthreshold membrane potential (spikes removed and data low-pass filtered <3 Hz). Blue: theta oscillation (spikes removed and data bandpass filtered 6–10 Hz). Insets on the right: magnified views of the boxed regions at left. Place field size: left: 67.5 cm, right: 103.5 cm. (H) Top: firing rate of all VR trials across virtual space for the cells shown in (G). Mean firing rate maps (black), average subthreshold membrane potential changes (red), and mean theta-rhythm amplitude (blue) for the cells shown in (G). (I) Normalized firing rates of all place cells (n = 30 cells) ordered by virtual space. (J–M) (J) Mean complex spike rate, (K) mean firing rate, (L) mean subthreshold membrane potential, and (M) mean amplitude of theta-rhythm inside and outside place-field. All error bars and shading: SEM.
Figure 2.
Figure 2.. All-optical induction and recording of hippocampal BTSP
(A) Closed-loop targeted optogenetic stimulation of single cells at specific locations. (B) Two example cells: fluorescence traces as a function of time for VR trials pre-, during, and post-optogenetic stimulation. Blue shaded boxes: 300 ms targeted optogenetic stimulation at 90 cm. Insets on the right: magnified views of the black dashed regions at left. Note that voltage imaging was performed by sampling through the Pre, Stim, and Post epochs (VR trial numbers, left: 7–8-14–15-23–24-26–27-39–40-41–49-50–51-56–57-58–62-63–64-69–70-71–77-78–84-85–86; right: 9–10-11–12-41–42-43–44-62–63-64–65-73–74-75–76-87–88-89–90-106–107-108–109-255–256-257–258). (C) Firing rate of all VR trials (pre-, during, and post-optogenetic stimulation) across virtual space for the two cells shown in (B). White: 300-ms targeted optogenetic stimulation at the 90 cm location. Bottom: average firing rate maps pre, during, and post-optogenetic stimulation for the cells shown in (B). The cell on the right was silent in the recorded VR trials before optogenetic stimulation. (D) Firing rate of all VR trials for two example cells stimulated at 60 cm (n = 5 cells) or 120 cm (n = 7 cells). (E and F) (E) Normalized firing rates, and (F) average firing rate maps of all optogenetically stimulated cells pre- and post-optogenetic stimulation (n = 32 cells). Data from different optogenetic stimulation locations aligned at 0. Bottom (F): quantification of firing rate at 10–30 cm before the optically stimulated location for Pre, Stim, and Post epochs. (G and H) (G) Normalized firing rates, and (H) average firing rate maps, of optogenetically stimulated cells pre-, post-, and 24 h (day 2) post-optogenetic stimulation (n = 10 cells). Bottom (H): firing rate at the optically stimulated location significantly increased both immediately and 24 h after optogenetic stimulation compared with pre-optogenetic stimulation. (I) For n = 25 cells passing the significance test for place cells, optogenetic stimulation did not affect out-of-field firing rate but significantly increased in-field firing rate. Optogenetically induced place cells had similar in-field and out-of-field firing rates as the natural place cells (same dataset as in Figure 1). All error bars and shading: SEM.
Figure 3.
Figure 3.. Induced BTSP exhibited enhanced subthreshold potentials and voltage correlations among simultaneously associated neurons
(A) Mean subthreshold membrane potential changes (spikes removed, low-pass filtered <3 Hz) for cells shown in Figure 2B. Black: pre-optogenetic stimulation. Red: post-optogenetic stimulation. (B) Mean subthreshold membrane potential (for n = 32 cells aligned at 0 cm). (C) Optogenetic stimulation did not affect out-of-field membrane potential but did increase in-field membrane potential (n = 25 cells). Optogenetically induced place cells had similar relative in-field and out-of-field membrane potential dynamics as natural place cells; values for natural place cells (orange bars, right) from the dataset in Figure 1. (D) Example of simultaneously optically stimulated cells forming stable representations at the same location (n = 18 pairs). Red: GEVI fluorescence traces as a function of time for selected VR trials. Blue: simultaneous 300 ms targeted optogenetic stimulation at 120 cm. Gray shaded zones indicate simultaneously recorded cells. Asterisks indicate concurrent spiking failures inside the place field. Middle: Firing rates from all VR trials (pre-, during, and post-optogenetic stimulation) across virtual space. Bottom: mean firing rate maps. (E) Same as (D) but control condition wherein the left cell is simultaneously imaged but not optogenetically stimulated. (F) Firing rate across virtual space for simultaneously optically stimulated and associated cells (n = 5 cells). (G) Pearson’s correlation coefficients of the firing rate over virtual space between simultaneously stimulated cells and between simultaneously imaged cells under control conditions. All error bars and shading: SEM.
Figure 4.
Figure 4.. Stability in optically assessed single-cell excitability parameters before vs. after induction of place-field plasticity
(A) Excitability was assessed out of the VR environment, before vs. after optogenetically induced plasticity. (B and C) Paired recordings of the same cells before and after optically induced plasticity. Blue: patterned optogenetic stimulation with steps of blue light (500 ms duration, 0–20 mW/mm2). Black: example fluorescence of somQuasAr6a during optogenetic stimulation. Red: fluorescence recording of the same cell after optically induced plasticity. Bottom: spike raster before and after optically induced plasticity; n = 14 cells. (D) Mean firing rate as a function of optogenetic stimulus strength (F-I curve) pre- (black) and post-(red) optical plasticity induction. (E) Excitability before and after optical plasticity, summarized for each cell as the slope of the F-I curve from 0–17 mW/mm2. Slope of the F-I curve did not change (pre: 1.9 ± 0.2, post: 1.8 ± 0.2, p = 0.67, two-sided paired-sample t test; n = 14 cells; control: pre: 1.9 ± 0.2, post: 1.8 ± 0.2, p = 0.43, two-sided paired-sample t test; n = 21 cells; pre vs. control-pre: p = 0.91, two-tailed t test; post vs. control-post: p = 0.92, two-tailed t test). Error bars/shading: SEM.
Figure 5.
Figure 5.. All-optical physiology of CA2/3-to-CA1 synaptic transmission in behaving mice
(A) Optical assay of synaptic function between hippocampal CA2/3 and CA1. CAV2-Cre, Flpo and Flp-on-somQuasAr6a(GEVI)-P2A-sombC1C2TG(opsin) were injected into CA1, and Cre-on-ChRmine-oScarlet-Kv2.1 was injected into contralateral CA2/3. (B) Confocal images of fixed brain slices showing fiber positioning and expression of ChRmine (red) in CA2/3 and somQuasAr6a (cyan) in CA1. Scale bars, 1,000 mm (representative data; similar results n = 5 preparations). (C) Representative confocal image of fixed brain slices expressing ChRmine (red) stained with the vesicular GABA transporter (VGAT) (cyan). 103/103 neurons expressing oScarlet were VGAT-negative (n = 5 mice). Bottom: magnified view of the boxed region at top. (D) All-optical assessment of EPSPs. Orange: optical stimulation of CA2/3 neurons through fiber (594 nm, 20 ms duration, repeated at 1 Hz). Left top: example fluorescence traces from CA1 neurons. Bottom: spike raster (n = 8 neurons). Right: expanded fluorescence waveforms from boxed region at left. (E) Distribution of delays between CA2/3 stimulation onset and peak of evoked spike (27 spiking events from 96 trials; latency: 17 ± 6 ms, mean ± SD, n = 8 cells). (F) Spike-triggered average waveform of spontaneous (left) and CA2/3 stimulus-evoked action potentials (right, n = 8 neurons). (G) CA2/3 stimulation-triggered mean fluorescence to stimuli that failed to evoke spikes corresponding to (D). (H) As in (D) except no CA2/3 stimulation-evoked spikes were detected. Instead, decreased fluorescence signal resulted (n = 6 cells). (I) Mean spike rate during CA2/3 stimulation corresponding to (H). (J) CA2/3 stimulation-triggered mean fluorescence corresponding to (H). Error bars/shading: SEM.
Figure 6.
Figure 6.. Potentiation of CA2/3-to-CA1 synaptic inputs by optogenetically induced plasticity
(A and B) Synaptic function assessed out of VR environment before vs. after optogenetically induced plasticity. (C–E) Example of CA1 plasticity induction by optogenetic stimulation of CA1 cells as in Figure 2, but in animals configured as in (B) with soma-localized ChRmine expression and fiber implantation in CA2/3. (F) Top: examples of paired measurements of fluorescence signals from the same cells in response to CA2/3 stimulation, pre- vs. post- CA1-induced plasticity. Bottom: corresponding spike raster, pre- vs. post-CA1-induced plasticity. Right: magnified views of the boxed regions at left. (G) Mean spike rate during CA2/3 stimulation pre- and post-opto-plasticity. (H) Quantification of CA2/3 stimulation effect, pre- vs. post-CA1-induced plasticity. CA2/3 stimulation evoked a substantial increase in spike rate post CA1-induced plasticity, measured over 1–20 ms window following CA2/3 test-pulse onset (n = 17 cells). Importantly, the spontaneous firing rate defined by the 200 ms time window before the CA2/3 test-pulses did not change. Error bars/shading: SEM.
Figure 7.
Figure 7.. Optogenetic silencing of CA2/3 reveals role of CA2/3 activity for full plasticity induction in CA1
(A) Optogenetic inhibition of CA2/3 during CA1-targeted optogenetic stimulation. Flpo and Flp-on-somQuasAr6a(GEVI)-P2A-sombC1C2TG(excitatory opsin) were injected into CA1, and eHcKCR1–3.0(inhibitory opsin)-oScarlet-Kv2.1 into contralateral CA2/3. (B) Confocal images of fixed brain slices showing fiber positioning and expression of eHcKCR1–3.0 (red) in CA2/3. Scale bars, 200 μm (representative data; similar results n = 3 preparations). (C) Closed-loop CA1-targeted optogenetic stimulation at specific locations with or without optogenetic silencing of CA2/3 neurons. (D) Two example cells: fluorescence traces as a function of time for VR trials during Pre, Stim with CA2/3 inhibition, Post I, Stim without CA2/3 inhibition, and Post II epochs. Insets on the right: magnified views of the black dashed regions at left. (E) Firing rate of all VR trials (Pre, Stim with CA2/3 inhibition, Post I, Stim without CA2/3 inhibition, and Post II epochs) across virtual space for the two cells shown in (D). Orange and white boxes: 300 ms targeted optogenetic stimulation at 90 cm location with (orange box) and without (white box) CA2/3 inhibition. Bottom: average firing rate maps for the cells shown in (D). Black: Pre. Orange: Post I. Red: Post II. (F) Normalized firing rates of all cells (n = 14 cells). Optogenetic stimulation locations were at aligned at 0 cm. (G) Top: average firing rate maps of n = 14 cells aligned at 0 cm. Bottom: quantification of firing rate at 10–30 cm before the optically stimulated location for Pre, Post I, and Post II epochs. (H) Top: mean subthreshold membrane potential of n = 14 cells aligned at 0 cm. Bottom: subthreshold membrane potential at 10–30 cm before the optically stimulated location. Subthreshold potential was significantly higher in both Post I and Post II epochs compared with the Pre epoch. Error bars/shading: SEM.
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