A Signaled Locomotor Avoidance Action Is Fully Represented in the Neural Activity of the Midbrain Tegmentum

Abstract

Animals, including humans, readily learn to avoid harmful and threatening situations by moving in response to cues that predict the threat (e.g., fire alarm, traffic light). During a negatively reinforced sensory-guided locomotor action, known as signaled active avoidance, animals learn to avoid a harmful unconditioned stimulus (US) by moving away when signaled by a harmless conditioned stimulus (CS) that predicts the threat. CaMKII-expressing neurons in the pedunculopontine tegmentum area (PPT) of the midbrain locomotor region have been shown to play a critical role in the expression of this learned behavior, but the activity of these neurons during learned behavior is unknown. Using calcium imaging fiber photometry in freely behaving mice, we show that PPT neurons sharply activate during presentation of the auditory CS that predicts the threat before onset of avoidance movement. PPT neurons activate further during the succeeding CS-driven avoidance movement, or during the faster US-driven escape movement. PPT neuron activation was weak during slow spontaneous movements but correlated sharply with movement speed and, therefore, with the urgency of the behavior. Moreover, using optogenetics, we found that these neurons must discharge during the signaled avoidance interval for naive mice to effectively learn the active avoidance behavior. As an essential hub for signaled active avoidance, neurons in the midbrain tegmentum process the conditioned cue that predicts the threat and discharge sharply relative to the speed or apparent urgency of the avoidance (learned) and escape (innate) responses.SIGNIFICANCE STATEMENT During signaled active avoidance behavior, subjects move away to avoid a threat when directed by an innocuous sensory stimulus. Using imaging methods in freely behaving mice, we found that the activity of neurons in a part of the midbrain, known as the pedunculopontime tegmentum, increases during the presentation of the innocuous sensory stimulus that predicts the threat and also during the expression of the learned behavior as mice move away to avoid the threat. In addition, inhibiting these neurons abolishes the ability of mice to learn the behavior. Thus, neurons in this part of the midbrain code and are essential for signaled active avoidance behavior.

Keywords: avoidance; basal ganglia; escape; goal-directed behavior; midbrain; motor plan.

Figure 1.

Effect of auditory tones on F/F_o calcium signals measured from PPT neurons using fiber photometry. A, Parasagittal section showing the optical fiber tract reaching PPT and GCaMP6f fluorescence around the fiber ending expressed in CaMKII neurons. The main panel blends a dark-field image of the section with the green channel of the GCaMP6f fluorescent image. Inset, The fluorescent image alone without blending. Bottom 2 panels, Close-ups of the GCaMP6f-labeled neurons in an area imaged by the optical fiber. Ceb, Cerebellum; Thal, thalamus; SNr, substantia nigra pars reticulata. B, Same as in A, but from another animal. C, F/F_o calcium imaging signals evoked from PPT neurons by auditory tones (1 s) of different saliency. The tones vary in frequency (kHz) and SPL (dB). Bottom, Movement speed obtained by tracking a color marker on the head of the animal. The traces are mean ± SEM of the auditory mapping sessions (177 audio sessions from 7 mice). D, Area of F/F_o and speed measured during a time window (0-3 s) after tone onset. Significant Tukey test values comparing F/F_o for each tone versus pre-tone (baseline): 16 kHz, 75 dB t₍₂₅₄₄₎ = 6.1, p < 0.0001; 8 kHz, 81 dB t₍₂₅₄₄₎ = 14.0, p < 0.0001; 12 kHz, 82 dB t₍₂₅₄₄₎ = 13.9, p < 0.0001; 6 kHz, 86 dB t₍₂₅₄₄₎ = 16.0, p < 0.0001; 4 kHz, 92 dB t₍₂₅₄₄₎ = 22.0, p < 0.0001. Significant Tukey tests comparing speed for each tone versus pre-tone (baseline): 16 kHz, 75 dB t₍₂₅₄₄₎ = 4.4, p = 0.048; 8 kHz, 81 dB t₍₂₅₄₄₎ = 5.8, p = 0.0013; 12 kHz, 82 dB t₍₂₅₄₄₎ = 6.5, p = 0.0001; 6 kHz, 86 dB t₍₂₅₄₄₎ = 6.6, p < 0.0001; 4 kHz, 92 dB t₍₂₅₄₄₎ = 8.8, p < 0.0001. E, Percentage of avoidance responses and response latencies for animals performing signaled active avoidance (AA1 procedure) using six of the auditory tones tested during the auditory mapping sessions in C and D as a CS. The low saliency tone (32 kHz, 57 dB) was less effective at driving avoids compared with the other five more salient tones (9 sessions from 3 mice).

Figure 2.

3D reconstruction showing the PPT areas imaged by fiber photometry. The PPT region is the volume in semitransparent green. The small bright volumes (red, yellow, blue, pink, cyan) around the dorsal and middle portions of PPT represent the areas imaged per animal. This was traced by identifying the location of the optical fiber endings in parasagittal histologic sections and traced the estimated imaging area protruding from the cannula ending. Semitransparent pink represents the superior colliculus (dorsal to PPT). Semitransparent blue represents the SNr (rostral to PPT). Semitransparent orange represents the zona incerta (rostral to SNr). Figure 2 is a placeholder for Movie 1.

Figure 3.

Effect of signaled active avoidance on F/F_o calcium signals measured from PPT neurons. A, F/F_o calcium and speed traces (mean ± SEM) from signaled active avoidance (AA1 procedure) sessions (35 sessions from 5 mice). At time 0, traces are aligned from the CS onset (left panels) or from the response occurrence (right panels). The traces include all trials averaged together per session (dashed black) or separated according to the animal's response in the trial: avoids (blue) and escapes (dashed red). Also shown are the intertrial crossings (ITCs; produced during the ITI) aligned from response occurrence (green). The CS and US terminate when the mice avoid or escape, respectively; the mean ± SEM avoid and escape response latencies (from CS or US onset, respectively) are noted. B, Close-up of the traces in A. Peak 1 in the F/F_o calcium signal evoked by the CS for both avoids and escapes occurs before the onset of response movement (speed). Peak 2 in the F/F_o calcium signal is associated with a rise in speed during avoidance responses and is fully revealed when the trace is aligned from the avoidance response occurrence (right panels). This F/F_o Peak 2 is virtually inexistent during ITCs, which involve the same movement as avoidance responses but performed more slowly. C, Area of F/F_o and speed measured in 1 s windows from CS onset (left) and from response occurrence (right) for avoids, escapes, and ITCs. Filled symbols represent a significant difference (Tukey test p < 0.01) compared with a pretrial window shown on the left (Pre). D, The left 3 panels compare F/F_o peak amplitude, time to peak, and area between avoids and escapes for 3 windows from CS onset (0-1, 2-7, and 7-10 s). Right, Comparison of F/F_o area between avoids, escapes, and ITCs for a window around response occurrence (−1 to 1 s). Significant Tukey test values comparing F/F_o (from CS onset) between avoids versus escapes: Peak Amplitude (7-10 s), t₍₂₀₎ = 57.24, p < 0.0001; Time to Peak (0-1 s), t₍₂₀₎ = 4.46, p = 0.0049; Time to Peak (2-7 s), t₍₂₀₎ = 9.38, p < 0.0001; Area (2-7 s), t₍₂₀₎ = 3.75, p = 0.015; Area (7-10 s), t₍₂₀₎ = 35.17, p < 0.0001. Significant Tukey test values comparing F/F_o area (from response): t₍₃₈₎ = 28.33, p < 0.0001 (avoids vs escapes); t₍₃₈₎ = 10.20, p < 0.0001 (avoids vs ITCs); t₍₃₈₎ = 38.54, p < 0.0001 (escapes vs ITCs).

Figure 4.

Effect of unsignaled US presentations on F/F_o calcium signals measured from PPT neurons. A, F/F_o calcium and speed traces (mean ± SEM) from all unsignaled US sessions (32 sessions from 7 mice). At time 0, traces are aligned from US onset. Shown are trials evoked by the US (red) or the US without white noise (US-WN, dashed blue). Also shown for comparison is the trace evoked by the CS in signaled active avoidance (avoid) trials from Figure 3A (for the same animals). B, F/F_o and speed peak amplitude, time to peak, and area measured during a 0-4 s window from US onset. The measures compare the effect of the US with (red) and without (cyan) white noise. Also shown is the latency of the escape response (bottom right). Significant Tukey test values comparing F/F_o between US versus US-WN: Peak Amplitude, t₍₂₇₎ = 9.7, p < 0.0001; Time to Peak (seconds), t₍₂₇₎ = 5.3, p = 0.0008; Significant Tukey test values comparing speed between US versus US-WN: Peak Speed, t₍₂₇₎ = 14.1, p < 0.0001; Latency, t₍₂₇₎ = 14.0, p < 0.0001. *significant at the levels indicated in the legend. n.s., not significant.

Figure 5.

Cross-correlations between speed and F/F_o calcium signals of PPT neurons during signaled active avoidance behavior and unsignaled US-evoked escape behavior. A, Cross-correlation analysis between speed and F/F_o calcium for signaled active avoidance (AA1 procedure, left) and unsignaled US sessions (right). In the signaled active avoidance sessions, the trial period (0-12 s from CS onset) was compared with the same amount of time during the ITI period (−12 to 0 s preceding each trial period). Moreover, the cross-correlations during the trial period were computed separately for trials leading to avoids and escapes. For unsignaled US sessions, the same analysis was done comparing US presentations with or without white noise. All unsignaled US trials produce escape responses. B, Peak amplitude and time to peak for the cross-correlations (xCorr) shown in C. The time to peak denotes the lag between the speed and F/F_o calcium signal. Significant Tukey test values for xCorr Peak amplitude in signaled active avoidance: t₍₅₈₎ = 23.3, p < 0.0001 (Avoids vs Escapes); t₍₅₈₎ = 22.3148, p < 0.0001 (Avoids vs ITI); t₍₅₈₎ = 45.61, p < 0.0001 (Escapes vs ITI). Significant Tukey test values for xCorr Time to Peak in signaled active avoidance: t₍₅₄₎ = 6.1, p = 0.0002 (Avoids vs ITI); t₍₅₄₎ = 6.54, p < 0.0001 (Escapes vs ITI). Significant Tukey test values for xCorr Peak Amplitude in Unsignaled US: t₍₅₆₎ = 5.83, p = 0.0003 (US vs US-WN); t₍₅₆₎ = 36.52, p < 0.0001 (US vs ITI); t₍₅₆₎ = 42.36, p < 0.0001 (US-WN vs ITI). Significant Tukey test values for xCorr Time to Peak in Unsignaled US: t₍₅₆₎ = 4.65, p = 0.0048 (US vs US-WN); t₍₅₆₎ = 14.19, p < 0.0001 (US vs ITI); t₍₅₆₎ = 9.53, p < 0.0001 (US-WN vs ITI). C, Regression analysis between speed and F/F_o during signaled active avoidance for the periods noted in A. Plotted is the r² for the ITI, avoid, and escape periods of each session. Right, The same analysis after shuffling the F/F_o signal. All r² values for the 3 periods in the left panel were statistically significant (p < 0.01), indicating a significant linear relation between speed and F/F_o (none in the right panel was significant). This relation increases sharply during avoidance and, especially, escape movement compared with spontaneous ITI movement. *significant at the levels indicated in the legend. n.s., not significant.

Figure 6.

Effect of behavioral task contingency changes on avoidance responses and on F/F_o calcium signals of PPT neuron. A, F/F_o calcium and speed traces (mean ± SEM) comparing avoidance responses from the same animals performing AA1 and AA2 signaled active avoidance sessions (35 sessions from 5 mice). In AA2, animals must passively avoid during the ITI. This task contingency change leads to longer latency active avoidance responses. The behavioral data for these sessions are shown in Figure 7. B, Top panels, Comparison of F/F_o peak amplitude and time to peak for 2 windows from CS onset (0-1 and 1-7 s) and 1 window around avoidance response occurrence (−1 to 1 s) for avoidance responses during the AA1 and AA2 procedures. Bottom panels, Comparison of peak speed and time to peak speed for the same windows in the top panels. Significant Tukey test values comparing F/F_o peak amplitude between AA1 versus AA2: t₍₃₀₎ = 7.48, p < 0.0001 (0-1 s); t₍₃₀₎ = 4.56, p = 0.003 (1-7 s); t₍₃₀₎ = 11.82, p < 0.0001 (−1 to 1 s). Significant Tukey test values comparing F/F_o time to peak between AA1 versus AA2: t₍₃₀₎ = 9.16, p < 0.0001 (1-7 s). Significant Tukey test values comparing peak speed between AA1 versus AA2: t₍₃₀₎ = 6.82, p < 0.0001 (1-7 s); t₍₃₀₎ = 12.19, p < 0.0001 (−1 to 1 s). Significant Tukey test values comparing time to peak speed between AA1 versus AA2: t₍₃₀₎ = 6.56, p < 0.0001 (1-7 s). C, F/F_o calcium and speed traces (mean ± SEM) comparing signaled active avoidance responses to CS1 and signaled passive avoidance responses to CS2 for animals performing AA3 (48 sessions from 6 mice). In AA3, mice actively avoid during CS1 and passively avoid during CS2. The behavioral data for these sessions are shown in Figure 7. D, Top panels, Comparison of F/F_o peak amplitude and time to peak for 2 windows from CS onset (0-1 and 1-7 s) for AA3 procedure active avoidance responses during CS1 and passive avoidance responses during CS2. Bottom panels, Comparison of peak speed and time to peak speed for the same windows as the top panels. Significant Tukey test values comparing F/F_o Peak Amplitude between CS1 versus CS2: t₍₄₂₎ = 24.59, p < 0.0001 (0-1 s); t₍₄₂₎ = 34.73, p < 0.0001 (1-7 s). Significant Tukey test values comparing peak speed between CS1 versus CS2: t₍₄₂₎ = 10.41, p < 0.0001 (0-1 s); t₍₄₂₎ = 32.99, p < 0.0001 (1-7 s). E, F/F_o calcium and speed traces (mean ± SEM) comparing avoidance responses to CS1, CS2, and CS3 for animals performing AA4 (25 sessions from 5 mice). In AA4, the 3 distinct CS tones (8, 10, and 12 kHz at ∼80 dB) signal different active avoidance interval durations of 4, 7, and 15 s. Mice adjust their active avoidance response latency according to the duration of the signaled active avoidance interval. The behavioral data for these sessions is shown in Figure 7. F, Top panels, Comparison of F/F_o peak amplitude and time to peak for 2 windows from CS onset (0-1 and 2-7 s) and 1 window around response occurrence (−1 to 1 s) active avoidance responses during CS1, CS2, and CS3 (AA4 procedure). Bottom panels, Comparison of peak speed and time to peak speed for the same windows as the top panels. Significant Tukey test values for F/F_o peak amplitude (0-1 s): t₍₃₈₎ = 6.74, p < 0.0001 (CS1 vs CS3); t₍₃₈₎ = 5.55, p = 0.001 (CS2 vs CS3). Significant Tukey test values for F/F_o peak amplitude (1-15 s): t₍₃₈₎ = 5.84, p = 0.0005 (CS1 vs CS2); t₍₃₈₎ = 8.69, p < 0.0001 (CS1 vs CS3). Significant Tukey test values for F/F_o peak amplitude (−1 to 1 s): t₍₃₈₎ = 4.11, p = 0.0164 (CS1 vs CS3). Significant Tukey test values for F/F_o time to peak (1-15 s): t₍₃₈₎ = 4.05, p = 0.0179 (CS1 vs CS3); t₍₃₈₎ = 3.61, p = 0.0381 (CS2 vs CS3). Significant Tukey test values for peak speed (1-15 s): t₍₃₈₎ = 8.29, p < 0.0001 (CS1 vs CS2); t₍₃₈₎ = 11.48, p < 0.0001 (CS1 vs CS3). Significant Tukey test values for time to peak speed (1-15 s): t₍₃₈₎ = 4.51, p = 0.0078 (CS1 vs CS3). *significant at the levels indicated in the legend. n.s., not significant.

Figure 7.

Behavioral performance during the different signaled active avoidance procedures. Percentage of active avoidance responses (top), response latency (middle), and intertrial crossings (bottom) for the different signaled active avoidance procedures shown in Figure 6 (AA1, AA2, AA3, and AA4). In AA3, the correct response to CS2 is a passive avoidance (top, shown in gray). The middle panel also shows in gray the avoidance latencies (i.e. response latency excluding escape latency). Significant Tukey test values for Active Avoids: t₍₄₂₎ = 54.25, p < 0.0001 (CS1 vs CS2 in AA3); t₍₃₈₎ = 5.27, p = 0.0018 (CS1 vs CS2 in AA4); t₍₃₈₎ = 5.41, p = 0.0013 (CS1 vs CS3 in AA4). Significant Tukey test values for latency: t₍₃₈₎ = 9.54, p < 0.0001 (CS1 vs CS3 in AA4); t₍₃₈₎ = 8.63, p < 0.0001 (CS2 vs CS3 in AA4). Significant Tukey test values for avoid latency: t₍₂₃₎ = 9.63, p < 0.0001 (AA1 vs AA2); t₍₃₈₎ = 10.98 p < 0.0001 (CS1 vs CS3 in AA4); t₍₃₈₎ = 9.21, p < 0.0001 (CS2 vs CS3 in AA4). Significant Tukey test values for intertrial crossings: t₍₂₃₎ = 10.42, p < 0.0001 (AA1 vs AA2).

Figure 8.

Effect of PPT neuron inactivation during the avoidance interval on signaled active avoidance learning. A, Parasagittal section showing the optical fiber tract reaching PPT and eArchT3.0 expression around the fiber ending expressed in CaMKII neurons (CaMKII-PPT-Arch mice). The main panel blends a bright-field image of the section with the green channel of the eYFP fluorescent image. Inset, The fluorescent image alone without blending. Cx, Cortex; Ceb, cerebellum; Hippo, hippocampus; Thal, thalamus; Str, striatum. B, Percentage of avoidance responses, latency, and intertrial crossings for mice that express eArchT3.0 in PPT neurons (CaMKII-PPT-Arch; 5 mice). Plots represent 6 blocks of sessions. In the initial 2 blocks (5 sessions per block), naive mice perform ALCS trials during which optogenetic green light is delivered bilaterally to inhibit PPT neurons together with the CS during the avoidance interval (AA1 Procedure). The third block (Test 1) tests the ability of the mice to avoid during presentation of the CS without optogenetic light (the escape interval and US are not presented if the animal does not avoid). No CS trials are presented randomly for comparison (solid, red squares). In No CS trials, the CS is omitted during the avoidance interval to check for spurious avoids because of normal movement. The fourth and fifth blocks are identical to the first 2 blocks but without optogenetic light. The final block is a repeat of the test done in the third block. Gray filled circles in the first block represent a group of No Opsin mice (that do not express eArchT3.0; n = 4) implanted with dual cannulas in PPT and subjected to the same optogenetic light protocol as the experimental mice. These mice learned the task in the first block indicating that the impairment in the experimental mice was caused by inhibiting PPT neurons, not by the light itself. Significant Tukey test values comparing CS trials (without US) and No CS trials in the two test sessions: t₍₄₎ = 5.6, p = 0.009 (Test 2). C, Trial speed, trial velocity, and intertrial speed for the data shown in B. D, Overlay of speed for escape responses triggered by presentation of the US during the absence (control) or presence of green light in the PPT of CaMKII-PPT-Arch mice. Escape response speed is similar in both conditions.