Circuits That Mediate Expression of Signaled Active Avoidance Converge in the Pedunculopontine Tegmentum

Abstract

An innocuous sensory stimulus that reliably signals an upcoming aversive event can be conditioned to elicit locomotion to a safe location before the aversive outcome ensues. The neural circuits that mediate the expression of this signaled locomotor action, known as signaled active avoidance, have not been identified. While exploring sensorimotor midbrain circuits in mice of either sex, we found that excitation of GABAergic cells in the substantia nigra pars reticulata blocks signaled active avoidance by inhibiting cells in the pedunculopontine tegmental nucleus (PPT), not by inhibiting cells in the superior colliculus or thalamus. Direct inhibition of putative-glutamatergic PPT cells, excitation of GABAergic PPT cells, or excitation of GABAergic afferents in PPT, abolish signaled active avoidance. Conversely, excitation of putative-glutamatergic PPT cells, or inhibition of GABAergic PPT cells, can be tuned to drive avoidance responses. The PPT is an essential junction for the expression of signaled active avoidance gated by nigral and other synaptic afferents.SIGNIFICANCE STATEMENT When a harmful situation is signaled by a sensory stimulus (e.g., street light), subjects typically learn to respond with active or passive avoidance responses that circumvent the threat. During signaled active avoidance behavior, subjects move away to avoid a threat signaled by a preceding innocuous stimulus. We identified a part of the midbrain essential to process the signal and avoid the threat. Inhibition of neurons in this area eliminates avoidance responses to the signal but preserves escape responses caused by presentation of the threat. The results highlight an essential part of the neural circuits that mediate signaled active avoidance behavior.

Figure 1.

Avoidance and escape responses during active avoidance. A, Video tracking showing the position of an animal performing active avoidance in a shuttle box. B, Speed during active avoidance performance obtained from a group of mice (n = 36) performing ACS trials. The instantaneous speed (±SEM) is plotted versus the onset of ACS trials for avoidance responses (avoids; black) and escape responses (escapes; red). C, The instantaneous speed (±SEM) is plotted versus the occurrence of avoidance or escape responses for the data in B. D, Schematic of the circuits manipulated in this study highlighting the GABAergic pathways activated in the experiments shown in Figure 3. SC, Superior colliculus; VL, ventrolateral/ventromedial thalamus.

Figure 2.

Locations of implanted optical fibers in PPT and superior colliculus. A, Dark-field image of a parasagittal section (∼1 mm lateral from the midline) showing the trajectory of an optical fiber track coursing toward PPT. The PPT optical fibers are inserted at a 20° angle in the posterior direction. Stimulation of GABAergic nigrotegmental fibers with this optical fiber using blue light trains (66 Hz) at a power <1 mW completely abolished signaled active avoidance without affecting escape responses to the US (Fig. 3). B, 3D reconstruction of optical fiber track endings in the superior colliculus (SC; green squares) for brains cut in the coronal plane. SNr and SC are filled in semitransparent blue and orange, respectively. Scale bars, 1.0 mm. A video of the 3D reconstruction is provided (Movie 1). C, 3D reconstruction of optical fiber track endings in the PPT (yellow diamonds) for brains cut in the sagittal plane. SNr and PPT are filled in semitransparent blue and green, respectively. The bilateral optical fibers from both hemispheres are shown in the same hemisphere. Scale bars, 1.2 mm. A video of the 3D reconstruction is provided (Movie 2). D, Photomicrograph showing eYFP fluorescence in the superior colliculus of a CaMKII-SC-ChR2 mouse (coronal section). Image blends a dark-field image of the section with the green channel of the fluorescent image. The arrow points to the optical fiber tract implanted in superior colliculus. E, Photomicrograph showing eYFP fluorescence in the PPT of a CaMKII-PPT-ChR2 mouse (parasagittal section). Image blends a dark-field image of the section with the green channel of the fluorescent image. The arrow points to the optical fiber tract implanted in PPT. IC, Inferior Colliculus.

Figure 3.

Effect of activating SNr output pathways on active avoidance responses. A, Effect of blue light applied in the ventrolateral/ventromedial (VL/VM) thalamus, superior colliculus, or PPT on ACS+LCS trials for animals that express ChR2 in GABAergic fibers originating in the SNr (Vgat-SNr-ChR2). ACS+LCS trials measure the effect of optogenetic stimulation on avoidance responses driven by the ACS. Plots in all figures display mean ± SEM and asterisks denote Tukey tests. The plot also shows data for the No Opsin group of animals (open triangles), which compares the effect of all the light patterns used (combined together) versus ACS. B, Trial speed, trial velocity and intertrial speed for the data in A. C, Effect of blue light applied in the VL/VM thalamus or superior colliculus on LCS alone trials for animals that express ChR2 in GABAergic fibers originating in the SNr (Vgat-SNr-ChR2). LCS alone trials measure the ability of the optogenetic stimulation to drive avoidance responses in the absence of the ACS. Note the efficacy of continuous blue light (cont) in the superior colliculus on driving avoidance responses. The asterisks highlight nonsignificant difference versus ACS trials. The plot also shows data for the No Opsin group of animals (open triangles). D, Trial speed, trial velocity, and intertrial speed for the data in C. The asterisks highlight nonsignificant difference versus ACS trials.

Figure 4.

Effect of blue light in the PPT of Vgat-SNr-ChR2 mice on licking behavior. During the licking sessions, blue light was delivered (3 s OFF/3 s ON) in the PPT using trains of 1 ms pulses (40–66 Hz) at the same blue light power that blocked avoidance responses in each animal. The asterisks denote significant differences in licks between the light ON periods (six 0.5 s periods) and the last light OFF period immediately preceding the light ON in the sequence.

Figure 5.

Postsynaptic effects of activating GABAergic fibers with optogenetics. A, Example intracellular whole-cell responses obtained in two different neurons after activating GABAergic fibers that express ChR2 with continuous pulses or trains of 1 ms pulses of blue light. The GABAergic fibers originate in SNr and the recorded cells are in PPT (top) or superior colliculus (bottom). Each trace is an average of six trials. B, Effect of blue light on the firing of neurons recorded intracellularly for the different pathways: SNr-to-PPT and SNr-to- superior colliculus (SC). Firing was spontaneous (resting V_m) or induced with positive current injection. C, Adaptation of the hyperpolarizing responses (IPSPs) caused by activating GABAergic fibers. The area of hyperpolarization measured at the end (400–500 ms) of each type of optogenetic stimulus is plotted as a percentage of the area at the onset (0–100 ms) of the stimulus to reveal the amount of adaptation of the response as a function of the stimulus type. D, Cells (n = 12) recorded in PPT that were inhibited by SNr GABAergic afferents. E, Cells (n = 13) recorded in superior colliculus that were inhibited by SNr GABAergic afferents. F, Units recorded in the superior colliculus and PPT of urethane-anesthetized Vgat-SNr-Chr2 mice during application of blue light trains in the superior colliculus. Blue light trains delivered in superior colliculus at 40 Hz inhibit cells in PPT, presumably through antidromic activation of SNr cells that project to both superior colliculus and PPT.

Figure 6.

Effect of activating or inhibiting cells in the superior colliculus on avoidance responses driven by the ACS. A, Schematic of the superior colliculus cells inhibited in the experiments shown in B and C. B, Effect of green light applied in the superior colliculus on ACS+LCS trials for animals that express Arch in GABAergic (Vgat-SC-Arch) or putative glutamatergic (CaMKII-SC-Arch) cells in the superior colliculus. C, Trial speed, trial velocity, and intertrial speed for the data in B. D, Schematic of the superior colliculus cells activated in the experiments shown in E and F. E, Effect of blue light applied in the superior colliculus on ACS+LCS trials for animals that express ChR2 in GABAergic (Vgat-SC-ChR2) or putative glutamatergic (CaMKII-SC-ChR2) cells in the superior colliculus. F, Trial speed, trial velocit,y and intertrial speed for the data in B.

Figure 7.

Effect of DREADDs in superior colliculus or PPT on active avoidance. Comparison of the effects of saline and CNO on ACS trials in mice that express hM4D(Gi) or hM3D(Gq) in GABAergic neurons (black circles) or non-selectively in neurons (hSyn promoter; red squares) of superior colliculus (A, B) or PPT (C, D).

Figure 8.

Effect of activating or inhibiting cells in the superior colliculus on driving avoidance responses in the absence of the ACS. A, Schematic of the superior colliculus cells inhibited in the experiments shown in B and C. B, Effect of green light applied in the superior colliculus on LCS alone trials for animals that express Arch in GABAergic (Vgat-SC-Arch) or putative glutamatergic (CaMKII-SC-Arch) cells in the superior colliculus. The asterisks highlight nonsignificant difference versus ACS trials. C, Trial speed, trial velocity, and intertrial speed for the data in B. The asterisks highlight nonsignificant difference versus ACS trials. D, Schematic of the superior colliculus cells activated in the experiments shown in E and F. E, Effect of blue light applied in the superior colliculus on LCS alone trials for animals that express ChR2 in GABAergic (Vgat-SC-ChR2) or putative glutamatergic (CaMKII-SC-ChR2) cells in the superior colliculus. The asterisks highlight nonsignificant difference versus ACS trials. F, Trial speed, trial velocity, and intertrial speed for the data in E. The asterisks highlight nonsignificant difference versus ACS trials.

Figure 9.

Effect of activating or inhibiting cells in the PPT on avoidance responses driven by the ACS. A, Schematic of the PPT cells inhibited in the experiments shown in B and C. B, Effect of green light applied in the PPT on ACS+LCS trials for animals that express Arch in GABAergic (Vgat-PPT-Arch) or putative glutamatergic (CaMKII-PPT-Arch) cells in PPT. C, Trial speed, trial velocity, and intertrial speed for the data in B. D, Schematic of the PPT cells activated in the experiments shown in E and F. E, Effect of blue light applied in the PPT on ACS+LCS trials for animals that express ChR2 in GABAergic (Vgat-PPT-ChR2) or putative glutamatergic (CaMKII-PPT-ChR2) cells in the PPT. F, Trial speed, trial velocity, and intertrial speed for the data in E.

Figure 10.

Effect of activating or inhibiting cells in the PPT on driving avoidance responses in the absence of the ACS. A, Schematic of the PPT cells inhibited in the experiments shown in B and C. B, Effect of green light applied in the PPT on LCS alone trials for animals that express Arch in GABAergic (Vgat-PPT-Arch) cells in the PPT. The asterisks highlight nonsignificant difference versus ACS trials. C, Trial speed, trial velocity, and intertrial speed for the data in B. D, Schematic of the PPT cells activated in the experiments shown in E and F. E, Effect of blue light applied in the PPT on LCS alone trials for animals that express ChR2 in GABAergic (Vgat-PPT-ChR2) or putative glutamatergic (CaMKII-PPT-ChR2) cells in the PPT. The asterisks highlight nonsignificant difference versus ACS trials. F, Trial speed, trial velocity, and intertrial speed for the data in E.

Figure 11.

Effect of different light powers on avoidance responses driven by the ACS in Vgat-PPT-Arch and CaMKII-PPT-ChR2 mice. A, Effect of green light applied in the PPT at different powers on ACS+LCS trials for animals that express Arch in putative glutamatergic PPT cells (CaMKII-PPT-Arch). B, Effect of blue light applied in the PPT at different powers on ACS+LCS trials for animals that express ChR2 in GABAergic PPT cells (Vgat-PPT-ChR2).

Figure 12.

Effect of exciting cholinergic cells in PPT on active avoidance responses. The panels show the effect of high-power blue light (6 mW) applied in the PPT on ACS+LCS (closed black circles) and LCS alone (open green circles) trials for animals that express ChR2 in cholinergic PPT cells (Chat-PPT-ChR2).