Basal Ganglia Output Has a Permissive Non-Driving Role in a Signaled Locomotor Action Mediated by the Midbrain

Abstract

The basal ganglia are important for movement and reinforcement learning. Using mice of either sex, we found that the main basal ganglia GABAergic output in the midbrain, the substantia nigra pars reticulata (SNr), shows movement-related neural activity during the expression of a negatively reinforced signaled locomotor action known as signaled active avoidance; this action involves mice moving away during a warning signal to avoid a threat. In particular, many SNr neurons deactivate during active avoidance responses. However, whether SNr deactivation has an essential role driving or regulating active avoidance responses is unknown. We found that optogenetic excitation of SNr or striatal GABAergic fibers that project to an area in the pedunculopontine tegmentum (PPT) within the midbrain locomotor region abolishes signaled active avoidance responses, while optogenetic inhibition of SNr cells (mimicking the SNr deactivation observed during an active avoidance behavior) serves as an effective conditioned stimulus signal to drive avoidance responses by disinhibiting PPT neurons. However, preclusion of SNr deactivation, or direct inhibition of SNr fibers in the PPT, does not impair the expression of signaled active avoidance, indicating that SNr output does not drive the expression of a signaled locomotor action mediated by the midbrain. Consistent with a permissive regulatory role, SNr output provides information about the state of the ongoing action to downstream structures that mediate the action.SIGNIFICANCE STATEMENT During signaled active avoidance behavior, subjects move away to avoid a threat when directed by an innocuous sensory stimulus. Excitation of GABAergic cells in the substantia nigra pars reticulata (SNr), the main output of the basal ganglia, blocks signaled active avoidance, while inhibition of SNr cells is an effective stimulus to drive active avoidance. Interestingly, many SNr cells inhibit their firing during active avoidance responses, suggesting that SNr inhibition could be driving avoidance responses by disinhibiting downstream areas. However, interfering with the modulation of SNr cells does not impair the behavior. Thus, SNr may regulate the active avoidance movement in downstream areas that mediate the behavior, but does not drive it.

Keywords: accumbens; avoidance; escape; movement; striatum; threat.

Figure 1.

Basic active avoidance trials. A, Schematic of the basic types of trials (ACS, ACS+LCS, LCS alone, and No CS trials) used in this study. Active avoidance trials consist of a sequence of avoidance, escape, and intertrial intervals. An effective response during the avoidance interval (e.g., shuttling in a shuttle box) avoids the occurrence of the escape interval (foot-shock and white noise [WN]). The stimulus that signals the active avoidance interval defines each trial type. In ACS trials, an 8 kHz auditory tone (ACS) signals the active avoidance interval. In LCS alone trials, optogenetic (OPTO) light (LCS) signals the active avoidance interval. In ACS+LCS trials, the ACS and LCS are delivered simultaneously during the avoidance interval to determine whether the LCS affects the ability of the ACS to drive avoidance responses. In No CS trials, the avoidance interval is not signaled to measure the incidence of spurious avoidance responses because of exploratory movement. When the optogenetic light is delivered during the avoidance interval, it continues during the escape interval until the animal escapes. The difference between AA1 and AA2 procedures is that in AA2 the intertrial crossings (ITCs) that occur during the intertrial interval (ITI) are punished (0.2 s foot-shock and WN). Thus, in AA2, the intertrial interval is a passive avoidance interval. B, Video tracking showing movement speed from a group of mice (n = 27 from this study) performing ACS trials in a shuttle box. Top, The instantaneous speed (±SEM) is plotted versus the onset of ACS trials for avoidance responses (avoids; blue) and escape responses (escapes; gray). Bottom, The instantaneous speed (±SEM) is plotted versus the occurrence of avoidance or escape responses revealing the faster speed of escapes.

Figure 2.

Active avoidance task in head-fixed mice on a spherical treadmill. A, Head-fixed mouse on the spherical treadmill used to record SNr cells during ACS trials. The mice are subjected to ACS trials detailed in Figure 1A, except that the US during the escape interval is a train of air puffs (delivered to the back of the animal) and white noise (WN); movement during the intertrial interval is not punished. B, Parasagittal section showing the location of an electrode marking lesion in the dorsal border of SNr, at the point where the electrode enters the nucleus and cell sampling begins. CP, Cerebral peduncle. C, Speed tracking during active avoidance performance obtained from a group of mice (n = 8) performing ACS trials on the spherical treadmill. Left, The instantaneous speed (±SEM) is plotted versus the onset of ACS trials for avoidance responses (avoids; blue) and escape responses (escapes; gray). Right, The instantaneous speed (±SEM) is plotted versus the occurrence of avoidance or escape responses. D, Peristimulus time histograms (PSTHs) of SNr cells single-unit firing recorded during active avoidance performance on the spherical treadmill. Blue bars represent PSTHs where time 0 is the occurrence of the avoidance response. Gray bars represent PTSHs for the same data where time 0 is the onset of the ACS. The overlaid red lines plot the speed from the occurrence of the avoidance responses for the same trials. The plots represent two groups of cells based on their activity during active avoidance. Left, SNr cells (n = 19) that inhibited their firing (deactivated) during the occurrence of avoidance responses (note that speed is inverted for comparison). Right, SNr cells (n = 10) that enhanced their firing (activated) during the occurrence of avoidance responses. E, Cross-correlations between movement speed and neuronal firing for the cells that deactivated and activated during the avoidance interval. The cross-correlations were calculated during the avoidance interval and during an equivalent period of the intertrial interval per every trial (0.1 s bin; −3 to 3 s). For the shuffled cross-correlations (insets), one of the signals was randomly reordered before calculating the cross-correlation.

Figure 3.

Location of AAV injections in SNr and optical fiber endings. A, Example parasagittal section showing YFP fluorescence after an AAV injection in SNr to express Arch3.0 in a Vgat-SNr-Arch mouse. The main image blends a bright-field image of the section with the green channel of the eYFP fluorescence image. Insets, Bright-field (left) and the fluorescence image (right) alone. B, Closeup of YFP fluorescence in SNr cell bodies from another animal. C, Example coronal section showing the bilateral tracks of optical fibers coursing to SNr. D, Locations of implanted optical fibers in SNr and PPT. 3D reconstruction of optical fiber track endings in SNr (red circles) and PPT (yellow circles) for brains cut in the sagittal plane. SNr is filled in semitransparent blue and PPT in semitransparent green. Scale bars, 1.0 mm. The mean ± SEM distance from the end of optical fiber tips to the targeted SNr border was 3.8 ± 40 µm (n = 38 tips), which means that, on average, the optical fibers targeting SNr were placed right at the border of SNr. The image is a placeholder for Movie 1. Cx, Cortex; Hippo, hippocampus; SC, superior colliculus; Thal, thalamus; IC, inferior colliculus; Ceb, cerebellum; MG, medial geniculate; PAG, periaqueductal gray.

Figure 4.

Optogenetic inhibition of SNr GABAergic cells. A, Left, Coronal section showing YFP fluorescence in a Vgat-Arch slice expressing ArchT completely filling the SNr. The main image blends a bright-field image of the slice with the green channel of the eYFP fluorescence image. Insets, The bright-field (top) and the fluorescence image (bottom) alone. Right, Reconstruction of SNr cells that were whole-cell recorded in slices to test the effects of green light in Vgat-Arch and Vgat-SNr-Arch mice. B, Left, Example showing the effect of green light on the firing of a whole-cell recorded SNr cell from a Vgat-SNr-Arch mouse slice (in vitro). The overlaid traces (6 per condition) show spontaneous firing (gray) and the effect of a continuous pulse of green light (red). Note the robust inhibition of cell firing. Right, Population data obtained from a group of SNr cells inhibited by green light in Vgat-SNr-Arch mice. C, Left, Example of the effect of green light (1 s pulses) on SNr unit firing tested in an isoflurane-anesthetized Vgat-SNr-Arch mouse in vivo. Plot represents overlaid raw traces (10 trials) and a PSTH (30 trials). Note the robust inhibition of SNr cell firing during the continuous green light pulse. Right, Population data obtained from a group of SNr units (n = 56 of 102 recording sites surrounding 6 implanted optical fibers in 3 mice) inhibited by continuous green light (25-35 mW) in Vgat-SNr-Arch mice. D, Effect of longer duration green light pulses on SNr unit firing inhibition in isoflurane-anesthetized Vgat-SNr-Arch mice in vivo. Top, Population PSTH of SNr units (n = 12) inhibited by a 5 s continuous pulse of green light. Bottom, Comparison between baseline firing and the first or last second of a 5 s green light pulse. The inhibition persists during the long duration pulses; the amount of inhibition does not differ between the first and last second of the 5 s pulse. E, Top, Example of the effect of green light applied in the SNr of an isoflurane-anesthetized Vgat-SNr-Arch mouse in vivo on the firing of a cell in PPT. Bottom, Population data obtained from PPT units (n = 58) excited when SNr GABAergic cells are inhibited with green light applied in the SNr of Vgat-SNr-Arch mice. F, Vgat-SNr-Arch mice (n = 5) that trained to avoid the US signaled by the ACS learn rapidly to avoid the US signaled by SNr inhibition caused by the LCS (continuous green light in SNr). Plots represent the percentage of avoidance responses (top), response latency from trial onset (middle), and the number of intertrial crossings per trial (bottom) for blocks of five sessions (AA1). During the first block of sessions (1-5), animals were trained with ACS trials to avoid the US signaled by the ACS. During the next three blocks of sessions (6-20), the animals received ACS and LCS alone trials (half of each). In these sessions, there were no significant differences between the ACS and LCS alone trials in avoidance rates or other measures. During the last 2 blocks, No CS trials were also presented (13-20). In No CS trials, performance was significantly impaired compared with ACS trials. The No Opsin column represents a group of control animals that do not express Arch (No Opsin, n = 4) but were subjected to the same LCS and ACS trials during an equivalent number of sessions. The LCS was ineffective at driving avoidance responses in these mice; responding levels were similar to No CS trials. Thus, in these mice, avoidance rates were much higher during ACS trials than during LCS alone trials.

Figure 5.

SNr inhibition is an effective CS to drive active and passive avoidance responses. A, Schematic of the AA3 procedure used to test the ability of SNr inhibition to drive passive avoidance responses. In AA3, ACS and LCS trials are presented randomly, but LCS trials require mice to passively avoid by not shuttling during the presentation of the LCS. If animals shuttle during the LCS-signaled passive avoidance interval, they are punished (0.5 s foot-shock and white noise [WN]). B, Plots (details in Fig. 4F) show three blocks of sessions for the same mice. In the first block (No US), naive (untrained) mice (n = 6) were presented with ACS and LCS alone trials that did not include a US (escape interval). The naive animals did not learn to shuttle during presentation of the ACS or the LCS in the absence of a US and the shuttling did not differ between these trials. During the next block of sessions (Include US), the animals received ACS and LCS alone trials that included a US (AA1); No CS trials were also presented. The animals learned to avoid the US when the ACS and LCS began to predict the US. There was no difference in avoidance rates or other measures between ACS and LCS alone trials. During the last block of sessions (AA3), the AA3 procedure was applied, which requires passive avoidance in LCS trials (instead of the active avoidance required in the previous sessions). Mice rapidly learned passive avoidance signaled by the LCS, while continuing active avoidance signaled by the ACS. C, Trial speed, trial velocity, and intertrial speed for the data in B. Trial velocity is not available for LCS trials in AA3 since there is no effective direction in passive avoidance. In AA3, trial speed was lower in LCS trials compared with ACS trials, since the mice learned not to move in response to the LCS.

Figure 6.

3D reconstructions showing typical results of AAV injections to express ChR2 in GABAergic cells of the dorsal striatum (A) and ventral striatum (B). In these experiments, cannulas were implanted in SNr. All sites showing some level of eYFP expression are labeled (green), which reflects both the cells labeled at the injection site and all the projecting fibers. The striatum and SNr are filled in semitransparent red and blue, respectively. The images are placeholders for Movies 2 and 3, respectively. C, D, Example parasagittal sections from two different mice showing eYFP fluorescence after injection in dorsal (C) or ventral (D) striatum. The images blend a dark-field image of the section with the green channel of the eYFP fluorescent image. Note the fluorescence in striatum (injection site) and in SNr (projection site). E, The schematic highlights the pathways targeted by optogenetics in the experiments shown in Figure 7. Two different GABAergic striatonigral pathways (black) originating in the dorsal or ventral striatum were targeted. During active avoidance, striatonigral fibers were excited by activating ChR2 with blue light applied in the SNr through optical fibers (blue arrow). Semitransparent structures refer to other pathways/areas manipulated in this or in our previous work.

Figure 7.

Effect of striatonigral pathway activation in SNr on signaled active avoidance responses. A, LCS alone trial sessions to see effects of blue light applied in the SNr on animals that express ChR2 in striatal GABAergic fibers originating in ventral (Vgat-StrV-ChR2), dorsal (Vgat-StrD-ChR2), or both (Vgat-StrDV-ChR2) portions of the striatum (33-55 sessions in 4 mice/group). The percentage of avoidance responses (top), response latency from trial onset (middle), and the number of intertrial crossings per trial (bottom) are shown. No CS trials (red) were also presented and are averaged for all sessions and shown in the first, ACS column. In LCS alone sessions: ^#Nonsignificant differences versus ACS trials to denote equivalency in the ability of the optogenetic light to drive avoidance responses. Also shown are the results from a group of No Opsin mice subjected to the same procedures (open triangles combine the different optogenetic stimulation patterns since they did not differ). B, Trial speed, trial velocity, and intertrial speed for the data in A. C, Effect of blue light applied in the SNr on ACS+LCS trial sessions for the groups in A (26-29 sessions in 4 mice). In ACS+LCS sessions: *Significant differences versus ACS trials to denote the effect of the optogenetic light on the ability of the ACS to drive avoidance responses. Also shown are the results from a group of No Opsin mice subjected to the same procedures (open triangles combine the different optogenetic stimulation patterns since they did not differ). D, Trial speed, trial velocity, and intertrial speed for the data in C.

Figure 8.

Location of AAV injections in NAc to express ChR2 in GABAergic neurons. A, B, Example parasagittal sections from 2 animals with typical ChR2 expression in NAc and corresponding projections to SNr and PPT after an AAV injection in Vgat-Acb-ChR2 mice. Top right insets, An image of the maximal intensity projection of the fluorescence. Bottom right insets, Closeup of the top inset around SNr and PPT. Note the eYFP fluorescent fibers coursing via SNr into PPT, which fills PPT. The faint fluorescence in the cortex rostral to the accumbens, visible in Animal A, but not in Animal B, appears as dotted puncta at high magnification, which could be projection fibers from accumbens or faint expression in local GABAergic interneurons. C, 3D reconstructions showing the results of AAV injections to express ChR2 in GABAergic cells of NAc. The image is a placeholder for Movie 4. D, Example parasagittal section showing the tracks of an optical fiber implanted in PPT.

Figure 9.

Effect of NAc pathway activation in SNr or PPT on signaled active avoidance responses. A, Example IPSPs evoked in an SNr cell (whole-cell recording) from a Vgat-Acb-ChR2 mouse slice by exciting GABAergic fibers originating in NAc cells with blue light. Each trace is the average of 6 stimulus trials and reflects different patterns of blue light stimulation, including trains of 1 ms pulses (at 5, 20, 40, and 66 Hz) and continuous pulses delivered for 1 s. B, Population data showing the effect of exciting GABAergic fibers of NAc cells in SNr with blue light on the firing of SNr units (n = 26) recorded in isoflurane-anesthetized Vgat-Acb-ChR2 mice. Plot represents the spontaneous firing of SNr cells compared with different patterns of blue light simulation, including trains of 1 ms pulses (10, 40, and 66 Hz) and continuous pulses delivered for 1 s. C, Blue light applied in SNr of Vgat-Acb-ChR2 mice inhibits cells in PPT, but there is much less inhibition when continuous light is used. Top, Example PPT unit activity showing the effect of a train (40 Hz) and a continuous pulse (1 s) of blue light. Note the robust inhibition in response to the train but not the continuous pulse. Bottom, Population data comparing spontaneous PPT cell (n = 16) firing versus different patterns of blue light simulation applied in SNr, including trains of 1 ms pulses (10, 40, and 66 Hz) and continuous pulses delivered for 1 s. D, Schematic of the pathways activated in these experiments, which includes striatonigral and striatotegmental fibers that originate in the NAc. E, Effect of blue light applied in the SNr (blue closed circles) or the PPT (red open squares) on ACS+LCS trials for animals that express ChR2 in GABAergic fibers that originate in the NAc (Vgat-Acb-ChR2; 26-28 sessions in 4 mice/group). Note the strong inhibition of avoidance responses for blue light trains at 40-66 Hz delivered in either SNr or PPT, indicating that excitation of GABAergic striatotegmental fibers that originate in the NAc and course through SNr block signaled active avoidance. Right, Trial speed, trial velocity, and intertrial speed for the data in the left panel.

Figure 10.

Effects of precluding SNr deactivation on signaled active avoidance. A, Schematic detailing LS and LS-ACS trials. LS trials are No CS trials in which optogenetic light begins randomly during the preceding intertrial interval and continues during the unsignaled avoidance interval. LS-ACS trials are ACS trials in which optogenetic light begins randomly during the preceding intertrial interval and continues during the avoidance and escape intervals (until the animal avoids or escapes). The optogenetic light applied in the SNr of Vgat-SNr-Arch (green light) and of Vgat-SNr-IC++ (blue light) mice is used to inhibit cells during the intertrial interval to preclude the SNr deactivation that occurs when animals actively avoid during the avoidance interval. Shuttling during the intertrial interval (with or without optogenetic light) are intertrial crossings, which in AA2 procedures are punished, requiring passive avoidance during the intertrial interval. B, Effect of precluding SNr deactivation in GABAergic SNr cells on signaled active avoidance. The results from Vgat-SNr-Arch and Vgat-SNr-IC++ animals were combined into a single group (n = 8) since the effects did not differ significantly. Right panels, Trial speed, trial velocity, and intertrial speed for the data in the left panels. No Opsin animals (open triangles) subjected to the same procedures are also shown (n = 6). Precluding SNr deactivation did not impair signaled active avoidance performance; the percentage of avoids was normal in LS-ACS trials and much higher than in LS trials for both AA1 and AA2 procedures. C, Effect of LS-ACS trials in naive mice to determine whether precluding SNr deactivation impairs learning of signaled active avoidance (AA2 procedure). Naive Vgat-SNr-Arch and Vgat-SNr-IC++ were trained using only LS-ACS trials (n = 6), and an additional group of mice (n = 6) trained only with ACS trials (No light). Precluding SNr deactivation did not impair learning of signaled active avoidance.

Figure 11.

Effects of modulating (exciting or inhibiting) striatal, NAc, or SNr cells with DREADDs on signaled active avoidance. A, Example coronal sections showing mCherry fluorescence after an AAV injection in the SNr of a Vgat-SNr-hM4Di mouse. Top, Centered on SNr. Bottom, The zona incerta located more rostral, which is devoid of fluorescence. The main images blend a dark-field image of the section with the red channel of the mCherry fluorescence image. Insets, The dark-field (top) and the fluorescence image (bottom) alone. CP, Cerebral peduncle. B–D, Comparison of the effects of saline (Sal) and CNO on ACS trials in mice that express hM4D(Gi) or hM3D(Gq) to inhibit or excite, respectively, GABAergic neurons in broad areas of striatum, including dorsal and ventral striatum (B), NAc (C), and SNr (D). All animals are subjected to AA1 procedures, and subsequently to AA2 procedures if the number of intertrial crossings was increased by CNO during AA1. Excitation of GABAergic cells in the NAc (not in dorsal or ventral striatum), or inhibition of GABAergic cells in SNr, increased the number of intertrial crossings during AA1 procedures. In AA2, these manipulations produced modest failures in passive avoidance with negligible effects on signaled active avoidance (n = 6/group). *p < 0.01, Sal versus CNO (Tukey).

Figure 12.

Effects of inhibiting PPT GABAergic afferents that originate in SNr and zona incerta on signaled active avoidance. A, Effect of green light applied in the PPT on ACS+LCS trials for animals that express Arch in GABAergic fibers originating in SNr and zona incerta (Vgat-SNZI-Arch; n = 6). This did not impair active avoidance. The same procedures were applied to a group of No Opsin mice (B6-No Opsin; n = 6). B, Trial speed, trial velocity, and intertrial speed for the data in A. Inhibiting GABAergic afferents in PPT suppressed trial speed (at the higher green light powers) only in the Vgat-SNZI-Arch mice.