Figure 2. Basolateral amygdala (BLA) ACh signaling aligns with salient events during reward learning.

(A) Diagram and example of injection and fiber placement sites in the BLA for recording from mice expressing a fluorescent acetylcholine sensor (ACh3.0). Left: Diagram of BLA ACh3.0 injection and fiber tip placement. Right: Representative coronal brain slice with fiber tip and ACh3.0 expression. Blue: DAPI, Green: ACh3.0. White dashed line: BLA outline. Gray dashed rectangle: fiber track. Scale = 500 µm. Individual fiber placements are shown in Figure 2—figure supplement 1A. (B) Behavioral responses of mice expressing ACh3.0 in BLA. Individual mice acquired the task at different rates as measured by rewards earned. Horizontal white line: acquisition threshold, when a mouse began to earn 20 rewards consistently in Training. Incorrect nose pokes shown in Figure 2—figure supplement 2A. Pre-Training (PT): blue shaded area, Training: pink shaded area, Extinction (Ext): orange shaded area. (C) Fluorescence traces from BLA of ACh3.0-expressing mouse. A significant increase in fluorescence representing BLA ACh release consistently coincided with correct (green line) but not incorrect (gray line) nose pokes on the last day of PT (data are shown from Mouse 1). The mean Z-scored precent ΔF/F0 (Z%ΔF/F0) overlaid on bootstrapped 99% confidence intervals (99% bCIs). Shaded significance bars under traces represent time points where 99% bCIs do not contain 0 for at least 0.5 s. Correct: n = 24; downsampled incorrect: n = 24 of 58. Traces of signal and reference channels (%ΔF/F0) during nose pokes are shown in Figure 2—figure supplement 1B–C. Incorrect nose pokes on the last day of PT versus Training Day 1 shown in Figure 2—figure supplement 2B. (D) Heatmap of BLA ACh signaling in Mouse 1 across all training phases, aligned to tone onset (Tone), correct nose poke (NP), and receptacle entry (Rec). Each row is the average of rewarded trials across a training session. White dashed horizontal line: first Training day earning 10 rewards. Horizontal white line: acquisition threshold, when a mouse began to earn 20 rewards consistently in Training. Black horizontal lines: divisions between training phases. Black vertical lines: divisions between breaks in time to allow for variable latencies in tone onset, correct nose poke, and receptacle entry (reward retrieval). The bCI plot for Mouse 1 is in Figure 2—figure supplement 1G. Individual heatmaps for mice 2–4 in Figure 2—figure supplement 1D–F. Incorrect nose pokes heatmaps for individual mice shown in Figure 2—figure supplement 2C–F. (E) Heatmap of BLA ACh signaling averaged across mice. Signal aligned as in (D) with a selection of data from key days in the behavioral paradigm shown. From bottom to top: PT Day 1, PT Day 5, Early Training Day, First Training day earning 10 rewards (white dashed horizontal line), Mid Training Day, Acquisition Day (white horizontal line), Last Training Day, Last Extinction Day. The bCI plot for cohort averaged data is in Figure 2—figure supplement 1H. Incorrect nose poke heatmap and bCI plot averaged across mice shown in Figure 2—figure supplement 2G–H. (F) Diagram and example of Nucleus Basalis of Mynert (NBM)-BLA terminal fiber recordings. Left: DIO-GCaMP7s was injected in the NBM of ChAT-IRES-Cre mice, individual injection sites are shown in Figure 2—figure supplement 5A. Representative coronal brain slice showing GCaMP7s expression. White dashed lines: internal capsule and globus pallidus outlines. Blue: DAPI, Green: GCaMP7s, Red: ChAT. Scale = 500 µm; separate channels shown in Figure 2—figure supplement 5B. Right: An optical fiber was implanted above the ipsilateral BLA, individual fiber placements are shown in Figure 2—figure supplement 5A. Representative coronal brain slice showing GCaMP7 expression and fiber tip placement. White dashed line: BLA outline. Gray dashed rectangle: fiber tract. Blue: DAPI, Green: GCaMP7s, Red: ChAT. Scale = 500 µm; separate channels shown in Figure 2—figure supplement 5C. (G) Behavioral responses of mice during NBM-BLA terminal fiber recordings. White horizontal line: acquisition threshold, when a mouse began to earn 20 rewards consistently in Training. Incorrect nose pokes shown in Figure 2—figure supplement 6A. (H) NBM-BLA terminal fiber activity is similar to ACh3.0 recordings. NBM-BLA terminal fiber activity significantly increased with correct (green line) but not incorrect (gray line) nose pokes on the last day of PT (data shown for Mouse 1). Mean Z%ΔF/F0 overlaid on bootstrapped 99% confidence intervals (99% bCIs). Shaded significance bars under traces represent time points where 99% bCIs do not contain 0 for at least 0.5 s. Correct: n = 42; downsampled incorrect: n = 42 of 101. Signal and reference channels (%ΔF/F0) during nose pokes are shown in Figure 2—figure supplement 5D–E. Incorrect nose pokes on the last day of PT versus Training Day 1 shown in Figure 2—figure supplement 6B. See Figure 2—figure supplement 9A–H for simultaneous ACh3.0 and NBM-BLA terminal fiber recordings. (I) Heatmap of NBM-BLA terminal fiber activity in Mouse 1 across all training phases, as in (D-E). Blanks in the heatmaps indicate time bins added for alignment. bCI plot for Mouse 1 in Figure 2—figure supplement 7F. Mouse 2 individual heatmap shown in Figure 2—figure supplement 5F. Incorrect nose pokes heatmaps for individual mice shown in Figure 2—figure supplement 6C–D. (J) Heatmap of NBM-BLA terminal fiber activity averaged across mice. Signal aligned as in (D-E, I) with a selection of key days shown, from bottom to top: PT Day 1, PT Day 4, Early Training, Acquisition Day (white horizontal line), Last Training Day, Last Extinction Day. The bCI plot for cohort averaged data in Figure 2—figure supplement 7G. Incorrect nose poke heatmap and bCI plot averaged across mice shown in Figure 2—figure supplement 6E + Figure 2—figure supplement 8E.

Figure 2—figure supplement 1. BLA ACh3.0 recording.

(A) Squares indicate optical fiber tips for individual mice. 1 (red), 2 (blue), 3 (teal), 4 (purple). (B) Increase in fluorescence (%ΔF/F0) following correct nose pokes is specific to the signal (465 nm, green) channel and is not observed in the reference channel (405 nm, tan). Data from Mouse 1 PT Day 5 as in Figure 2C. Mean ± SEM, n = 24. (C) Minimal increase in fluorescence (%ΔF/F0) following incorrect nose pokes. Signal (465 nm, gray) channel, reference channel (405 nm, tan). Data from Mouse 1 PT Day 5 as in Figure 2C. Downsampled mean ± SEM, n = 24 of 58. (D–F) Individual mouse data for mice 2–4 as shown in Figure 2D. Dashed white horizontal line: first Training day earning 10 rewards (10 Rew.). White horizontal line: acquisition threshold (Acq.). (G) Mouse 1 combined action bCI plot for subset of days used in cohort averaged heatmap Figure 2E (from bottom to top: PT Day 1, PT Day 5, Early Training Day, First Training day earning 10 rewards, Mid Training Day, Acquisition Day, Last Training Day, Last Extinction Day). Mean overlaid on 99% bCIs for tone onset, correct nose poke, and receptacle entry. Pink and blue significance bars under traces denote time points where 99% bCIs are above or below 0 for at least 0.5 s, respectively. Horizontal scale = 1 s. Vertical scale = 5 Z%ΔF/F0. (H) Cohort averaged combined action bCI plot for subset of days used in (G) and cohort averaged heatmap Figure 2E. Trial level mean overlaid on 99% bCIs for tone onset, correct nose poke, and receptacle entry. Pink and blue significance bars under traces denote time points where 99% bCIs are above or below 0 for at least 0.5 s, respectively. Horizontal scale = 1 s. Vertical scale = 5 Z%ΔF/F0.

Figure 2—figure supplement 2. BLA ACh3.0 recording: incorrect nose pokes.

(A) Incorrect nose poking of individual mice throughout training. (B) Incorrect nose pokes that yield timeouts (downsampled Training Day 1, pink line, n = 24 of 66) result in a modest increase in BLA ACh signaling but incorrect nose pokes before timeouts are introduced (downsampled PT Day 5, blue line, n = 24 of 58) do not. Data from Mouse 1 as in Figure 2C, Mean Z%ΔF/F0 overlaid on bootstrapped 99% confidence intervals (99% bCIs). Shaded significance bars under traces represent time points where 99% bCIs do not contain 0 for at least 0.5 s. (C–F) Individual mouse heatmaps of BLA ACh signaling across all training phases, aligned to incorrect nose poke. Each row is the average of incorrect nose pokes that led to (or would have led to for PT) a timeout across a session. White dashed horizontal line: first Training day earning 10 rewards. Horizontal white line: acquisition threshold, when a mouse began to earn ~20 rewards consistently in Training. Black horizontal lines: divisions between training phases. (G) Heatmap of BLA ACh signaling during incorrect nose poke averaged across mice. Signal aligned as in (C-F) with a selection of data from key days in the behavioral paradigm shown. From bottom to top: PT Day 1, PT Day 5, Early Training Day, First Training day earning 10 rewards (white dashed horizontal line), Mid Training Day, Acquisition Day (white horizontal line), Last Training Day, Last Extinction Day. Black horizontal lines: divisions between training phases. (H) Cohort averaged bCI plot for subset of days used in (G). Trial level mean (downsampled to 20) overlaid on 99% bCIs for incorrect nose poke. Pink and blue significance bars under traces denote time points where 99% bCIs are above or below 0 for at least 0.5 s, respectively. It is unclear how to interpret the time points significantly below 0 before and after incorrect nose pokes pulled out by bootstrapping. This may be an artifact of the small signal across incorrect trials, although this remains to be investigated. Horizontal scale = 1 s. Vertical scale = 5 Z%ΔF/F0.

Figure 2—figure supplement 3. BLA ACh3.0 recording replicate.

(A) Squares indicate optical fiber tips for individual mice. 5 (orange), 6 (cyan), 7 (brown), 8 (navy). (B) Behavioral responses of mice during BLA ACh3.0 recordings. Individual mice acquired the task at different rates as measured by rewards earned. White horizontal line: acquisition threshold, when a mouse began to earn 20 rewards consistently in Training. Incorrect nose pokes shown in Figure 2—figure supplement 4A. (C–F) Heatmaps of BLA ACh signaling in mice 5–8 across all training phases, aligned to tone onset (Tone), correct nose poke (NP), and receptacle entry (Rec). Each row is the average of rewarded trials across a training session. Horizontal white line: acquisition threshold, when a mouse began to earn 20 rewards consistently in Training. Black horizontal lines: divisions between training phases. Black vertical lines: divisions between breaks in time to allow for variable latencies in tone onset, correct nose poke, and receptacle entry (reward retrieval). Blanks in the heatmaps indicate time bins added for alignment. (G) Heatmap of BLA ACh signaling averaged across mice 5–8. Signal aligned as in (C-F) with a selection of key days shown, from bottom to top: PT Day 1, PT Day 4, Early Training, Acquisition Day (white horizontal line), Last Training Day, Last Extinction Day. (H) Mouse 5 combined action bCI plot for subset of days used in (G). Mean overlaid on 99% bCIs for tone onset, correct nose poke, and receptacle entry. Pink and blue significance bars under traces denote time points where 99% bCIs are above or below 0 for at least 0.5 s, respectively. Horizontal scale = 1 s. Vertical scale = 5 Z%ΔF/F0. (I) Cohort (mice 5–8) averaged combined action bCI plot for subset of days used in (G-H). Trial level mean overlaid on 99% bCIs for tone onset, correct nose poke, and receptacle entry. Pink and blue significance bars under traces denote time points where 99% bCIs are above or below 0 for at least 0.5 s, respectively. Horizontal scale = 1 s. Vertical scale = 5 Z%ΔF/F0.

Figure 2—figure supplement 4. BLA ACh3.0 recording replicate: incorrect nose pokes.

(A) Incorrect nose poking of individual mice throughout training. (B–E) Individual mouse heatmaps of BLA ACh signaling across all training phases, aligned to incorrect nose poke. Each row is the average of incorrect nose pokes that led to (or would have led to for PT) a timeout across a session. Horizontal white line: acquisition threshold, when a mouse began to earn 20 rewards consistently in Training. Black horizontal lines: divisions between training phases. (F) Heatmap of BLA ACh signaling during incorrect nose poke averaged across mice. Signal aligned as in (B-E) with a selection of data from key days in the behavioral paradigm shown. From bottom to top: PT Day 1, PT Day 4, Early Training Day, Acquisition Day (white horizontal line), Last Training Day, Last Extinction Day. Black horizontal lines: divisions between training phases. (G) Cohort averaged bCI plot for subset of days used in (F). Downsampled trial level mean overlaid on 99% bCIs for incorrect nose poke. Pink and blue significance bars under traces denote time points where 99% bCIs are above or below 0 for at least 0.5 s, respectively. Horizontal scale = 1 s. Vertical scale = 5 Z%ΔF/F0.

Figure 2—figure supplement 5. NBM-BLA GCaMP7s recording in cholinergic terminal fibers.

(A) Circles indicate NBM DIO-GCaMP7s injection sites for individual mice, 1 (red), 2 (blue). Triangles indicate estimated optical fiber tips based on adjacent slices for individual mice. 1 (red), 2 (blue). (B) Representative injection site coronal slice from Figure 2F with channels separated. Scale = 500 µm. (C) Representative fiber tip site coronal slice from Figure 2F with channels separated. Scale = 500 µm. (D) Increase in fluorescence (%ΔF/F0) following correct nose pokes is specific to the signal (465 nm, green) channel and is not observed in the reference channel (405 nm, tan). Data from Mouse 1 PT Day 4 as in Figure 2H. Mean ± SEM, n = 42. (E) Minimal increase in fluorescence (%ΔF/F0) following incorrect nose pokes. Signal (465 nm, gray) channel, reference channel (405 nm, tan). Data from Mouse 1 PT Day 4 as in Figure 2H. Downsampled mean ± SEM, n = 42 of 101. (F) Individual data for Mouse 2 as shown in Figure 2I. White horizontal line: acquisition threshold. (G) Mouse 1 combined action bCI plot for subset of days used in cohort averaged heatmap Figure 2J (from bottom to top: PT Day 1, PT Day 4, Early Training Day, Acquisition Day, Last Training Day, Last Extinction Day). Mean overlaid on 99% bCIs for tone onset, correct nose poke, and receptacle entry. Pink and blue significance bars under traces denote time points where 99% bCIs are above or below 0 for at least 0.5 s, respectively. Horizontal scale = 1 s. Vertical scale = 5 Z%ΔF/F0.

Figure 2—figure supplement 6. NBM-BLA GCaMP7s recording in cholinergic terminal fibers: incorrect nose pokes.

(A) Incorrect nose poking of individual mice throughout training. (B) Incorrect nose pokes that yield timeouts (downsampled Training Day 1, pink line, n = 42 of 105) result in a significant increase in NBM-BLA terminal fiber activity, but incorrect nose pokes before timeouts are introduced (downsampled PT Day 4, blue line, n = 42 of 101) do not. Data from Mouse 1 as in Figure 2H, mean Z%ΔF/F0 overlaid on bootstrapped 99% confidence intervals (99% bCIs). Shaded significance bars under traces represent time points where 99% bCIs do not contain 0 for at least 0.5 s. (C–D) Individual mouse heatmaps of NBM-BLA terminal fiber activity across all training phases, aligned to incorrect nose poke. Each row is the average of incorrect nose pokes that led to (or would have led to for PT) a timeout across a session. Horizontal white line: acquisition threshold, when a mouse began to earn 20 rewards consistently in Training. Black horizontal lines: divisions between training phases. (E) Heatmap of NBM-BLA terminal fiber activity during incorrect nose poke averaged across mice. Signal aligned as in (C-D) with a selection of data from key days in the behavioral paradigm shown. From bottom to top: PT Day 1, PT Day 4, Early Training Day, Acquisition Day (white horizontal line), Last Training Day, Last Extinction Day. Black horizontal lines: divisions between training phases.

Figure 2—figure supplement 7. NBM-BLA GCaMP7s recording in cholinergic terminal fibers replicate.

(A) Circles indicate NBM DIO-GCaMP7s injection sites for individual mice, 3 (teal), 4 (purple). Squares indicate optical fiber tips for individual mice, 3 (teal), 4 (purple). (B) Behavioral responses of mice during NBM-BLA recordings. Individual mice acquired the task at different rates as measured by rewards earned. White horizontal line: acquisition threshold, when a mouse began to earn 20 rewards consistently in Training. Incorrect nose pokes shown in Figure 2—figure supplement 8A. (C–D) Heatmaps of NBM-BLA terminal fiber activity in mice 3–4 across all training phases, aligned to tone onset (Tone), correct nose poke (NP), and receptacle entry (Rec). Each row is the average of rewarded trials across a training session. Horizontal white line: acquisition threshold, when a mouse began to earn 20 rewards consistently in Training. Black horizontal lines: divisions between training phases. Black vertical lines: divisions between breaks in time to allow for variable latencies in tone onset, correct nose poke, and receptacle entry (reward retrieval). Blanks in the heatmaps indicate time bins added for alignment. (E) Heatmap of NBM-BLA terminal fiber activity averaged across mice 3–4. Signal aligned as in (C-D) with a selection of key days shown, from bottom to top: PT Day 1, PT Day 4, Early Training, Acquisition Day (white horizontal line), Last Training Day, Last Extinction Day. Black horizontal lines: divisions between training phases. Black vertical lines: divisions between breaks in time to allow for variable latencies in tone onset, correct nose poke, and receptacle entry (reward retrieval). (F) Mouse 3 combined action bCI plot for subset of days used in cohort averaged heatmap (E). Mean overlaid on 99% bCIs for tone onset, correct nose poke, and receptacle entry. Pink and blue significance bars under traces denote time points where 99% bCIs are above or below 0 for at least 0.5 s, respectively. Horizontal scale = 1 s. Vertical scale = 5 Z%ΔF/F0. (G) Cohort (mice 1–4) averaged combined action bCI plot for subset of days used in (E-F). Trial level mean overlaid on 99% bCIs for tone onset, correct nose poke, and receptacle entry. Pink and blue significance bars under traces denote time points where 99% bCIs are above or below 0 for at least 0.5 s, respectively. Horizontal scale = 1 s. Vertical scale = 5 Z%ΔF/F0.

Figure 2—figure supplement 8. NBM-BLA GCaMP7s recording in cholinergic terminal fibers replicate: incorrect nose pokes.

(A) Incorrect nose poking of individual mice throughout training. (B–C) Individual mouse heatmaps of NBM-BLA terminal fiber activity across all training phases, aligned to incorrect nose poke. Each row is the average of incorrect nose pokes that led to (or would have led to for PT) a timeout across a session. Horizontal white line: acquisition threshold, when a mouse began to earn 20 rewards consistently in Training. Black horizontal lines: divisions between training phases. (D) Heatmap of NBM-BLA terminal fiber activity during incorrect nose poke averaged across mice 3–4. Signal aligned as in (B-C) with a selection of data from key days in the behavioral paradigm shown. From bottom to top: PT Day 1, PT Day 4, Early Training Day, Acquisition Day (white horizontal line), Last Training Day, Last Extinction Day. Black horizontal lines: divisions between training phases. (E) Cohort (mice 1–4) averaged bCI plot for subset of days used in (D). Trial level mean (downsampled to 20) overlaid on 99% bCIs for incorrect nose poke. Pink and blue significance bars under traces denote time points where 99% bCIs are above or below 0 for at least 0.5 s, respectively. As for other experiments measuring signal before and after incorrect nosepokes, it is unclear how to interpret the time points significantly below 0 pulled out by bootstrapping. As mentioned previously, this may be an artifact of the small signal across incorrect trials, although this remains to be investigated. Horizontal scale = 1 s. Vertical scale = 5 Z%ΔF/F0.

Figure 2—figure supplement 9. Simultaneous BLA ACh3.0 + GCaMP7s recording in NBM-BLA cholinergic terminal fibers.

(A) Left: DIO-jRCaMP1b was injected in the NBM of ChAT-IRES-Cre mice. Representative coronal brain slice showing jRCaMP1b expression. Yellow dashed lines: internal capsule and globus pallidus outlines. Scale = 500 µm. (White box: higher magnification area shown in B.) (B) Higher magnification of injection site. Scale = 100 µm. (C) Circle indicates NBM DIO-jRCaMP1b injection site for Mouse 1. (D) ACh3.0 was injected into the ipsilateral BLA and an optical fiber was implanted above the BLA. White dashed line: BLA outline. Scale = 500 µm. (E) Squares indicate optical fiber tips for individual mice. ACh3.0 + RCaMP (red), ACh3.0 + RCaMP sham (gray), (F) A substantial increase in both fluorescence representing BLA ACh release (green line) and NBM-BLA cholinergic terminal activity (magenta line) coincided with correct nose pokes on the last day of PT. Mean Z%ΔF/F0 overlaid on bootstrapped 99% confidence intervals (99% bCIs). Shaded significance bars under traces represent time points where 99% bCIs do not contain 0 for at least 0.5 s. n = 42. (G) Modest increase in fluorescence in both channels following incorrect nose pokes on the last day of PT. Mean Z%ΔF/F0 overlaid on bootstrapped 99% confidence intervals (99% bCIs). Shaded significance bars under traces represent time points where 99% bCIs do not contain 0 for at least 0.5 s. Downsampled n = 42 of 94. (H) jRCaMP1b signal is not simply crosstalk from ACh3.0 channel. A substantial increase in fluorescence representing BLA ACh release (green line) following correct nose pokes did not necessitate signal in RCaMP sham red channel (gray line). Last day of PT. Mean Z%ΔF/F0 overlaid on bootstrapped 99% confidence intervals (99% bCIs). Shaded significance bars under traces represent time points where 99% bCIs do not contain 0 for at least 0.5 s. n = 44. (I) Incorrect nose pokes on the last day of PT, as in G, for ACh3.0 + RCaMP sham mouse. Downsampled n = 44 of 135.