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, 11 (1), 262

Control of Locomotor Speed, Arousal, and Hippocampal Theta Rhythms by the Nucleus Incertus

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Control of Locomotor Speed, Arousal, and Hippocampal Theta Rhythms by the Nucleus Incertus

Lihui Lu et al. Nat Commun.

Abstract

Navigation requires not only the execution of locomotor programs but also high arousal and real-time retrieval of spatial memory that is often associated with hippocampal theta oscillations. However, the neural circuits for coordinately controlling these important processes remain to be fully dissected. Here we show that the activity of the neuromedin B (NMB) neurons in the nucleus incertus (NI) is tightly correlated with mouse locomotor speed, arousal level, and hippocampal theta power. These processes are reversibly suppressed by optogenetic inhibition and rapidly promoted by optogenetic stimulation of NI NMB neurons. These neurons form reciprocal connections with several subcortical areas associated with arousal, theta oscillation, and premotor processing. Their projections to multiple downstream stations regulate locomotion and hippocampal theta, with the projection to the medial septum being particularly important for promoting arousal. Therefore, NI NMB neurons functionally impact the neural circuit for navigation control according to particular brains states.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The activity of NI NMB neurons is correlated with locomotion, arousal, and theta power.
a Up, images showing the colocalization of NMB mRNA (green), mCherry (red), and NeuN (blue) in the NI of an NMB-Cre mouse (98.4% NMB+ neurons expressed mCherry, n = 2284/2322 neurons; 89.0% mCherry+ neurons expressed NMB, n = 2284/2565 neurons, 24 sections from 4 mice), scale bars = 100 µm. NI NMB neurons were labeled with mCherry by injecting AAV-DIO-mCherry vectors into the NI of an NMB-Cre mouse. Bottom, the zoom-in view of the dashed rectangular area, scale bars = 50 µm. b Expressing GCaMP6m in NI NMB neurons. Scale bars = 200 µm. c The method of simultaneously monitoring GCaMP signals, animal locomotion, arousal, and hippocampal local field potentials (LFP) from a head-fixed mouse running on a wheel treadmill. DM, dichroic mirror; PMT, photomultiplier tube. An infrared camera was used to measure pupil diameter as a proxy of arousal. d Behavior paradigm and example data from one experimental trial. LFP signals are shown as a raw bandpassed signals (0.1–200 Hz), together with the 1–20 Hz spectrogram. Video frame images of the mouse’s eye (1–4) are shown where acquired at the times indicated in the pupil recording trace. Pupil diameter was extracted posthoc via a fitted ellipse (red). The left dashed line indicates the onset of cue and the right dashed line indicates the onset of liquid reward. e The group data of GCaMP signals, the locomotor speed, the normalized pupil diameter, lick signal, and theta rhythm spectrogram (n = 6 mice). Red segments indicate statistically significant increase from the baseline (P < 0.01; multivariate permutation test). Shaded areas indicate SEM. f Cross-correlation analysis of GCaMP signal, locomotor speed (locom), normalized pupil diameter, theta power change, and lick rates. Color scale to the right indicates correlation values.
Fig. 2
Fig. 2. Inhibiting NI NMB neurons suppresses locomotion, arousal, and theta power.
a Experimental schematic for inhibiting NI NMB neurons and recording hippocampal LFP in head-fixed mice. b A blue laser pulse (5 s) abolished action potential firing elicited by current injection into a GtACR1-expressing neuron in the NI (n = 7 cells tested). c Raw traces showing the effect of optogenetic inhibition (blue bar) of NI NMB neurons on locomotion, arousal, and theta rhythms. d Locomotor speed aligned to laser onset. e Bar plot showing the inhibition effects on locomotor speed (n = 6 mice for all groups). f, g The effect of optogenetic inhibition on the pupil diameter across time (f) and summary data (g). h, i Grand mean of hippocampal LFP spectrograms aligned to laser onset (h) and summary data of optogenetic inhibition on theta power normalized to the sum of 0.1–12 Hz power (i). j, k Schematic (j) and time line (k) illustrating the food-chasing task, in which a mouse chased after a moving food tray to retrieve food pellets. In the third behavioral session we optogenetically inhibited NI neurons (k). l, m Optogenetic inhibition of NI neurons suppressed the chasing speed (l) and aborted the success rate of reaching the moving food tray (m). Shaded areas (d, f) and error bars (e, g, i, l, m) indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired t test; see Supplementary Table 1 for detailed statistical analysis. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Activating NI NMB neurons promotes locomotion, arousal, and theta power.
a Experimental schematic for viral expression of ChR2, stimulating NI NMB neurons, and recording hippocampal LFP. b A coronal section shows ChR2-mCherry expression in NI NMB neurons. Scale bar = 200 µm. c Recording from brain slices confirmed that brief blue laser pulses at 5, 10, 20 and 50 Hz (5 ms width, 5 mW) reliably elicited the firing of action potentials in a ChR2-expressing NI neuron. d A representative example showing the effect of optogenetic activation of NI NMB neurons (blue bar) on inducing locomotion, arousal, and theta oscillations. e Locomotor speed aligned to different frequency laser onset. f Summary of the stimulation effects on locomotor speed. g, h The effect of optogenetic activation of NI NMB neurons on the pupil diameter across time (g) and summary data (h). i, j Grand average of LFP spectrograms for the entire test group (i) and summary data on the theta power (j). Shaded areas (e, g) and error bars (f, h, j) indicate SEM. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001; ns, not significant; Tukey’s multiple comparisons test; see Supplementary Table 1 for detailed statistical analysis. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. The effect of NI NMB neurons on locomotion, arousal, and theta power are decomposable.
a Schematics show the method of optogenetic stimulation of NI NMB neurons (left), time line of behavior test (middle), and stimulation protocol (right). b The effect of optogenetic stimulation on average locomotor speed (left), pupil diameter change (middle), and grand average of LFP spectrograms (right) for NMB-NIChR2 mice with saline control injection (ip; n = 7 mice). c The effect of applying the adrenergic alpha-2 receptor agonist clonidine (0.1 mg kg−1, ip) of NI NMB neurons (n = 7 mice). d The effect of applying the muscle relaxant pancuronium (0.18 mg kg−1, ip; n = 4 mice). eg Summary data of showing the effect of clonidine and pancuronium on locomotor speed change, pupil diameter change, and theta power change in response to optogenetic stimulation of NI NMB neurons. Shaded areas (bd) and error bars (eg) indicate SEM. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001; ns, not significant; Paired t test and Mann–Whitney test; see Supplement Table 1 for detailed statistical analysis. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. NI NMB neurons receive inputs from brain areas associated with arousal and locomotion.
a The strategy for monosynaptic retrograde tracing of NI NMB neurons. b The expression pattern of TVA-mCherry (red) and RV-GFP (green) at the injection site within the NI. Dual labeled cells indicate starter cells competent for retrograde transsynaptic traversal. c Ratio of total retrogradely-labeled neurons in various upstream stations of NI NMB neurons (n = 4 mice). d Schematic showing the inputs of NI NMB neurons. e Schematic diagram showing terminal photostimulation of the upstream neurons and whole-cell patch recording of the NI NMB+ cells. f Presynaptic RV+ cells in the MS. g Postsynaptic responses from a NMB+ neuron in the NI following photostimulation of ChR2+ MS neuron axonal terminals under the conditions of control (latency, 8.1 ± 0.8 ms), drug applications, and wash. Postsynaptic current was measured holding at −65 mV. The right panel shows the summary effect of DNQX and the addition of Gabazine (n = 6 NMB+ cells from 2 mice). h, i Presynaptic RV+ cells in the LH (h) and the physiological effect of photostimulating ChR2-expressing LH axonal terminals on NI NMB neurons (i; latency, 6.6 ± 0.6 ms). Right panel in (i) shows the drug effects (n = 9 NMB+ cells from 2 mice). j, k Presynaptic RV+ cells in the LHb (j) and the physiological effect of activating LHb axonal terminals on NI NMB neurons (k; latency, 8.5 ± 1.0 ms; n = 6 NMB+ cells from 2 mice). l, m Presynaptic RV+ cells in the IPN (l) and the physiological effect of activating IPN axonal terminals on NI NMB neurons (m; latency, 6.2 ± 1.2 ms; n = 6 NMB+ cells from 2 mice). Error bars (c, g, i, k, m) indicate SEM. Scale bars = 200 µm (b, f, h, j, and l). Source data are provided as a Source Data file.
Fig. 6
Fig. 6. NI NMB neurons project to multiple brain areas.
a The presence of VGAT (slc32a1) mRNA (green) in mCherry-expressing NI NMB neurons (n = 937 VAGT+/1234 mCherry+ neurons; 937 mCherry+ /2089 VGAT+ neurons; 20 sections from 4 mice). Arrows, dual labeled cells; Arrowheads, mCherry + cells that clearly lacked VGAT mRNA expression. b Schematic shows that expressing synaptophysin-EGFP in NI NMB neurons labels terminals in several subcortical structures. c The normalized density of innervations (total EGFP+ pixels divided by the area of each nucleus; n = 4 mice). d Schematic diagram showing the method of optogenetic stimulation and recordings from the MS, the LH or the IPN in brain slices. e, f Terminal expression of synaptophysin-EGFP in the MS (e) and the effect of activating ChR2-expressing terminals on evoking IPSCs from MS neurons (f). The left panel in (f) shows representative traces of light-evoked IPSCs from an MS neuron before (red; latency, 4.8 ± 1.1 ms), during (black), and after (gray) Gabazine application. The right panel shows the group data on the effect of Gabazine on blocking the light-evoked IPSCs (n = 9 cells from 4 mice). Postsynaptic current was measured holding at −10 mV. g, h Terminal projection pattern of NI NMB neurons in the LH (g) and the effects of activating NI NMB neurons on evoking IPSCs from LH neurons (h; latency, 3.9 ± 1.8 ms; n = 5 cells in 5 mice). i, j Distribution of NI NMB axonal terminals in the IPN (i) and the effects of activating the terminals on the Gabazine-sensitive IPSCs of IPN neurons (j; latency, 6.1 ± 0.5 ms; n = 8 cells from 5 mice). Error bars (c, f, h, j) indicate SEM. Scale bars = 50 µm (a), 200 µm (e, g, i). Source data are provided as a Source Data file.
Fig. 7
Fig. 7. NI-MS projections differentially modulates locomotion, arousal, and theta power.
a Experimental schematic for viral expression of ChR2 and stimulation of NI terminals at different target regions. b, c Average traces (b) and summary data (c) showing the locomotor speed change evoked by terminal stimulation in the MS (red; n = 5 mice), the LH (blue; n = 7 mice), the IPN (cyan; n = 5 mice), and the IO (magenta; n = 6 mice) as well as stimulation of soma area in the NI (n = 7 mice). Dashed blue line marks the duration of laser stimulation. d, e Average traces (d) and summary data (e) showing the change in normalized pupil diameter elicited by stimulating different projections and NI somata. f, g Grand average of LFP spectrograms (f) and summary data of theta power change (g) elicited by terminal stimulations in different target regions and the NI soma area (theta power is normalized to its base in each group). h Experimental schematic for viral expression of GtACR1 and optogenetic inhibition of NI terminals in various target regions. i, j Average normalized locomotor speed traces (i) and summary data showing the effect of optogenetically inhibiting axonal terminals in the MS (red; n = 4 mice), LH (blue; n = 5 mice), and IPN (cyan; n = 4 mice). Blue horizontal line and dashed vertical lines marks the duration of light inhibition. k, l Average traces (k) and summary data showing the effect of terminal inhibition and soma inhibition on pupil diameter. m, n Grand average of LFP spectrograms and summary of theta power change within different target regions and the NI soma area. The data for soma activation and inhibition in this Figure are identical to those presented in Figs. 2 and 3 for comparison purpose. Error bars (c, e, g, j, l, n) indicate SEM. *P< 0.05, **P< 0.01, ***P < 0.001, ****P< 0.0001; ns, not significant; Dunnett’s multiple comparisons test; see Supplemental Table 1 for detailed statistical analysis. Source data are provided as a Source Data file.

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