Visualization of plasticity in fear-evoked calcium signals in midbrain dopamine neurons

Abstract

Dopamine is broadly implicated in fear-related processes, yet we know very little about signaling dynamics in these neurons during active fear conditioning. We describe the direct imaging of calcium signals of dopamine neurons during Pavlovian fear conditioning using fiber-optic confocal microscopy coupled with the genetically encoded calcium indicator GCaMP3. We observed calcium transients in a subset of dopamine neurons to an unconditioned fear stimulus on the first day of Pavlovian fear conditioning. On the second day, calcium transients occurred in response to conditioned and unconditioned stimuli. These results demonstrate plasticity in dopamine neuron calcium signals and the occurrence of activity-dependent processes in these neurons during fear conditioning.

Figure 1.

Expression of GCaMP3 in dopamine neurons. (A) Schematic of AAV-FLEX-GCaMP3. (B) Schematic of viral injection to the midbrain. (C) Immunohistochemistry illustrating GCaMP3 colocalization with the dopamine neuron marker tyrosine hydroxylase (TH). Scale = 100 μm. Note: GCaMP3 expression is largely restricted to the medial aspect (VTA) of the ventral midbrain. (D) Average action potential waveforms for VTA neurons transduced (green) or untransduced (blue) with AAV-FLEX-GCaMP3 (scale, 2 msec, 20 mV). (E) Average waveform properties of untransduced (n = 6) and transduced (n = 7) cells in D. (F) The average firing rate of transduced and untransduced neurons in the VTA. Data are presented as mean ± SEM.

Figure 2.

Characterization of activity-dependent GCaMP3 fluorescence in brain slice. (A) Illustration of individual fiber bundles comprising the field of view (scale 40 µm). (Inset) Higher magnification demonstrating individual fiber-optic arrangements within the probe (scale 10 µm). Characterization as a single cell requires detection by a minimum of four fibers, or an average diameter of 6 µm. (B) Jet pseudocolor fiber-optic image of dopamine neurons in an acute slice following NMDA application. White circles highlight individual cells. (C) High magnification view of cell in (B) indicated by arrow during baseline (top), muscimol (middle), and NMDA (bottom). White dots illustrate the center of individual fibers and circles represent ROI used for analysis (scale = 10 µm). (D) The change in fluorescence observed following bath application of muscimol and NMDA (blue, WF = wide-field, n = 7; green FO = fiber optic, n = 19). Arrows indicate the time of application. Data are presented as mean ± SEM. (E) WF fluorescence image of GCaMP3 expressing dopamine neuron illustrating patch electrode placement (dashed lines) to record action potential generation during imaging (scale 10 µm). (F) Average traces of stimulated action potentials recorded during imaging (three trials). Scale = 20 mV, 250 msec. (G) Change in GCaMP3 fluorescence observed following action potential stimulation using WF (top, n = 7 for 10 spikes and n = 19 for both 5 spikes and burst) or FO microscopy (bottom, n = 34 for 10 spikes, and n = 28 for both 5 spikes and burst). Scale = 1%DF/F, 2 sec. (H) Average latency to onset of fluorescence intensity change following action potential stimulation measured using FO or WF fluorescence microscopy. (I) Average duration of fluorescence change following action potential stimulation. (J) Average peak fluorescence intensity change. Data are presented as mean ± SEM.

Figure 3.

Characterization of calcium signals in dopamine neurons during fear conditioning. (A) Image of fiber-optic probe (left) and cannula (right). Scale (5 mm). (B) Image of mouse in conditioning chamber showing fiber-optic/cannula assembly. (C) Image of GCaMP3 fluorescence in VTA obtained by the fiber optic in vivo on second day of fear conditioning (scale 20 µm), (left) four cells (colored circles correspond to graph in (D); (right) fluorescence of a single cell during baseline (top), immediately after CS+ (middle), and immediately after shock (bottom). (D) Fluorescence signal acquired from four cells in (C) during a single trial on the second day of conditioning; red line is the cell in the right panels in (C). (E) Percent time mobile on Day 1 of paired trials (Bonferroni post hoc [**] P < 0.01, [***] P < 0.001). (F) Percent time mobile on Day 2 of paired trials. (Bonferroni post hoc [***] P < 0.001, [****] P < 0.0001.) (G) Percent time mobile on Day 1 of unpaired trials. (H) Percent time mobile on Day 2 of unpaired trials. (I) Average change in fluorescence observed during Day 1 of conditioning in CS+ (red) and CS− (blue) trials. Insets are average CuSums of cells responding to the US during CS presentation (left, red CS+ trials, blue CS− trials) or US presentation (right, red CS+ trials, blue CS− trials); x-axis: time (sec), 2 sec prior and 2 sec following stimulus; y-axis: cumulative summation of fluorescence signal. (J) Average change in fluorescence observed during Day 2 of conditioning in CS+ (red) and CS− (blue) trials. Insets are average CuSums of cells responding to the US during CS presentation (left, red CS+ trials, blue CS− trials) or US presentation (right, red CS+ trials, blue CS− trials). (K) Average change in fluorescence observed during Day 1 of conditioning with unpaired CS+ (red) and CS− (blue). Insets are average CuSums of all cells. (L) Average change in fluorescence observed during Day 2 of conditioning with unpaired CS+ (red) and CS− (blue). Insets are average CuSums of all cells. (M) Average normalized percent change in fluorescence during CS+ (red) and CS− (blue) trials on Day 1. (N) Average normalized percent change in fluorescence during US (red), or corresponding time in CS− (blue) trials on Day 1 (Bonferroni post hoc [*] P < 0.05; time: 0.833–2.417 sec). (O) Average normalized percent change in fluorescence during CS+ (red) and CS− (blue) trials on Day 2 (Bonferroni post hoc [*] P < 0.05; time: 1.083–1.833 sec). (P) Average normalized percent change in fluorescence during US (red), or corresponding time in CS− (blue) trials on Day 2 (Bonferroni post hoc [**] P < 0.01; time: 1.417–3.833 sec).