Coherent activity at three major lateral hypothalamic neural outputs controls the onset of motivated behavior responses

The lateral hypothalamus (LH) plays an important role in motivated behavior. However, it is not known how LH neural outputs dynamically signal to major downstream targets to organize behavior. We used multi-fiber photometry to show that three major LH neural outputs projecting to the dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and lateral habenula (LHb) exhibit significant coherent activity in mice engaging motivated responses, which decrease during immobility. Mice engaging active coping responses exhibit increased activity at LH axon terminals that precedes an increase in the activity of serotonin neurons and dopamine neurons, indicating that they may play a role in initiating active responses stemming from LH signal transmissions. The optogenetic activation of LH axon terminals in either the DRN, VTA, or LHb was sufficient to increase mobility but had different effects on passive avoidance and sucrose consumption, suggesting that LH outputs use complementary mechanisms to control behavioral responses. Our results support the notion that the three LH neural outputs play complementary roles in initiating motivated behaviors.


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An animal that wishes to maximize its survival must select optimal behavioral responses in order to 26 correctly adapt to its environment. Deciding to execute or refrain from a specific action depends 27 largely on the actual state of the environment that needs to be accurately integrated and then 28 translated into adaptive responses (Tye, 2018; Verharen et al., 2020). In other words, an animal 29 will engage active behavioral responses to obtain a reward, avoid a punishment, or escape from 30 an aversive context. Alternatively, it can engage passive coping behaviors to avoid being seen by a 31 predator or to avoid exhaustion. 32 The lateral hypothalamus (LH) is a heterogeneous brain region that has been associated with 33 a variety of behaviors related to motivation, reward, stress, arousal, and feeding (reviewed in Bon-  Sucrose consumption events are represented by pink shaded box in H. The magenta lines are mobility scores. Repeated measures three-way ANOVA between factors group (GCaMP6s-and eYFP-expressing mice) and within factors pathway (LH→DRN, LH→VTA, and LH→LHb), and time period (different for each test) with post hoc Dunnett's test. The p values were adjusted using the Bonferroni multiple testing correction method. * < 0.05, ** < 0.01, *** < 0.001.         To investigate how these pathways are modulated in an appetitive context, the same mice were 89 water-deprived for 24 h and were then given free access to a 2% sucrose solution for 10 min (Fig-90 ure 1G). In these sessions of sucrose consumption (SCT), the mice readily learned to drink from 91 the sucrose dispenser. Drinking events were automatically detected with a lickometer, and are 92 represented by pink boxes in the representative traces shown in Figure 1H). Aligning  Previous studies have shown that the LH→VTA and LH→LHb pathways play a role in motivating de-102 fensive behaviors. However, our results suggest that the LH neural outputs may also play a role in 103 controlling spontaneously motivated behavior. For example, the mice fled when given an aversive 104 airpuff, increased their mobility to reach a drinking spout, and stayed put during consumption.

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To directly examine the role of LH neural outputs in motivated behavior, activity was measured 106 at LH neural outputs and movements were tracked with an automated ANY-maze video tracking 107 system. The mice were free to explore either an open field (OFT) (Figure 2A-C) or were suspended 108 by the tail (tail suspension test, TST) ( Figure 2D-F). OFT  relation analysis confirmed the coherence between these pathways, which we did not observe in 116 eYFP-expressing mice (Figure 1-Figure Supplement 2 histological analysis and quantified LH neurons that were positive for one or more fluorescent 124 markers (Figure 1-Figure Supplement 5  . When the mice were tested using the same behaviors described 140 above, the calcium signals perfectly replicated the results previously obtained from the axon ter-141 minals recordings. These results indicate that the LH sends out largely independent projections, 142 that it signals to the DRN, VTA and LHb, and that this activity is coherent to mobility onset.  (Figure 2A), indicating that these pathways play a role in controlling movement.

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This correlation was significantly higher with the TST, an aversive context engaging active and pas-149 sive coping responses (Commons et al., 2017). To further investigate whether the high correlation 150 between activity and the mobility score could be attributed to specific time events during the OFT 151 and TST, we performed peri-event correlation analyses at specific time points (events). Events of 152 four types were chosen: at mobility onset, at immobility onset, and at random time points during 153 mobility and immobility. For each event, a Pearson correlation was measured for each 6-seconds 154 peri-event traces of the mobility score and the calcium signal recorded at each of the LH outputs.

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Correlations with < 0.001 and > 0.6 were considered as positive correlation events, < 0.001 156 and < 0.6 as negative correlation events, and the others events as having no correlation. The Pearson correlation between 2+ signal at the LH→DRN, LH→VTA, and LH→LHb pathways, and mobility scores during the OFT or TST. One sample t-test. Three-way mixed ANOVA between factors group (GCaMP6s-and eYFP-expressing mice), and within factors test (OFT and TST) and pathways (LH→DRN, LH→VTA, and LH→LHb) with post hoc Tukey test. The p values were adjusted using the Bonferroni multiple testing correction method. (B) Schematic of the event selection. Events at the onset mobility and immobility, and random events during mobility and immobility were chosen, and the Pearson correlation at 6 seconds peri-events between the 2+ signal and the mobility score was calculated. Correlations with p < 0.001 and r > 0.6 were considered as positive, p < 0.001 and r < 0.6 as negative, and the others as uncorrelated. (C) Fraction of positive (green), negative (red), and uncorrelated events (gray) in the OFT and TST for the LH→DRN, LH→VTA, and LH→LHb pathways. Four-way MANOVA between the factors group (GCaMP6s-and eYFP-expressing mice), test (OFT and TST), and pathways (LH→DRN, LH→VTA, and LH→LHb), and within factor events (during mobility, mobility onset, during immobility, immobility onset) for positive and negative correlations with a post hoc four-way mixed ANOVA and Tukey test for multiple comparisons. The p values were adjusted using the Bonferroni multiple testing correction method. The main effect was due to differences in the number of positive correlations. There was no difference in factor output and its interactions with other factors. Asterisks on the bars show the difference between tests. (D) Cross-correlation analysis between the 2+ signal and the mobility score at mobility onset peri-events. The lines represent means ± SEM of correlations vs. lag times and means ± SEM of lag times with maximum correlation. One sample t-test. Two-way ANOVA within factors tests (OFT and TST) and pathways (LH→DRN, LH→VTA, and LH→LHb) with post hoc Tukey test. The p values were adjusted using the Bonferroni multiple testing correction method. * < 0.05, ** < 0.01, *** < 0.001.    tivity from all three LH neural outputs preceded the change in mobility at mobility onset when the 167 mice initiated an active coping response in the TST ( Figure 2D). These results indicate that there is 168 a significant correlation in activity between these three LH neural outputs, and suggest that activity 169 at these LH neural outputs controls motivated behavior, particularly to engage a coping response 170 in a stressful context. . Peri-event plots of the average 2+ signals at all mobility onsets and plots for AUC during immobility and mobility, and at mobility pre-onset and onset (right). The lines represent the means ± SEM (standard error of mean). Same convention as with B, E for the TST (C, F). the magenta lines are mobility scores. Repeated measures two-way ANOVA within factors pathways (DRN 5 and LH→DRN, or VTA and LH→VTA) and time periods (during immobility and mobility, at mobility pre-onset and onset) with post hoc Dunnett's test. The p values were adjusted using the Bonferroni multiple testing correction method. * < 0.05, ** < 0.01, *** < 0.001.     Peri-event plots of average 2+ signals to all CS events followed by escape at the LH→DRN, LH→VTA, and LH→LHb axonal terminals and plots for AUC at baseline, during CS and escape. The lines represent means ±SEM. The top plots represent the results during early training and the bottom plots represents the results during late training. Same convention as C for mobility onsets of avoidance during late training D. The magenta lines are mobility scores. Two-way repeated measures ANOVA within factors pathways (LH→DRN, LH→VTA, and LH→LHb) and time periods (different for CS and mobility onset) with post hoc Dunnett's test.
The p values were adjusted using the Bonferroni multiple testing correction method. * < 0.05, ** < 0.01, *** < 0.001.    To determine whether increased activity at LH neural outputs is sufficient to motivate behavior, the 242 LH of wild-type mice were injected with an AAV encoding the opsin Channelrhodopsine-2 (ChR2) 243 fused to the fluorescent protein eYFP (AAV-ChR2-eYFP), or with eYFP alone (AAV-eYFP) as a control.

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Optical fibers were implanted as described for the fiber photometry recordings ( Figure 6A) (Figure 6C). The stimulation had no effect on the mobility of mice expressing eYFP 252 only ( Figure 6D). The same results were also obtained in the OFT (Figure 6-Figure Supplement 1). 253 These results show that increased activity at individual LH neural outputs is sufficient to promote 254 motivated behavior.           The LH is a central nucleus that connects many brain regions that orchestrates vital behaviours 276 (Bonnavion et al., 2016; Fakhoury et al., 2020). It is the central brain region of an interconnected  send coherent signals to downstream brain regions, and (2) they support the validity of recording 312 activity at axon terminals to study signal transmission along specific neural pathways.

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The LHb receives a net excitatory input from the LH (Lazaridis et al., 2019; Trusel et al., 2019). 314 Recent work has shown that aversive stimuli are sufficient to activate the LH→LHb pathway and 315 promote escape behavior (Lecca et al., 2017). Moreover, this pathway encodes negative valences 316 and rapidly develops prediction signals for negative events and aversive cues, making this pathway 317 a critical node for value processing and avoidance learning (Lazaridis et al., 2019; Trusel et al.,   318   2019). The results we obtained in our active avoidance task are consistent with these findings.

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The VTA receives both excitatory and inhibitory inputs from the LH, both of which play roles 320 in motivated behaviors (Nieh et al., 2015, 2016; Barbano et al., 2016, 2020; de Jong et al., 2019). 321 Glutamatergic transmission at the LH→VTA pathway (LH →VTA) plays a role in motivating innate 322 escape responses, signaling unexpected aversive outcomes, and signaling cues predicting aversive . 340 Here we show that the LH→DRN pathway is activated when mice are presented with an aversive 341 airpuff or a mild foot shock. This pathway is also prominently engaged before the mice reach the 342 reward spout, as we observed with the LH→VTA pathway and at mobility onset, which indicates 343 that there is a significant correlation with mobility in the TST.  (Celada et al., 2002). However, it remains to be deter-354 mined whether this increase in serotoninergic activity is mediated by direct activation of 5-HT DRN 355 neurons or by indirect disinhibition through local GABAergic neurons. 356 We also show that all three LH neural outputs are activated prior to the onset of movement in 357 the TST, an energetically-demanding and aversive context (Proulx et al., 2014; Warden et al., 2012). 358 Recent miniscopic in vivo calcium imaging of LHb neurons in mice responding in looming experi-359 ments has revealed that specific LHb neuron ensembles are active before the mice start running 360 away from an aversive threat (Lecca et al., 2020). Our results suggest that some of the excitatory 361 inputs driving these neuronal ensembles may be provided by the LH. Our results are also remi-362 niscent of recent studies that directly measured the activity of orexin-expressing LH neurons and 363 shown that this cell population is activated by aversive stimuli and stressors and is inhibited dur-364 ing food consumption, which is independent of food taste and texture (González et al., 2016a,b). 365 Moreover, the same group recently showed that a large fraction of orexin-expressing LH is acti-366 vated at the onset of the initiation of movement (Karnani et al., 2020). We propose that signal 367 transmission from this genetically-defined population in the LH play an important role in motivat-

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ing the initiation of movement by processing reward and sensory information in the DRN, VTA, and 369 LHb (Flanigan et al., 2020; Harris et al., 2005). Since most orexin-expressing neurons in the LH co- Open field test.

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The mice were placed in an open field (50 cm x 50 cm) and movement was tracked using a camera 488 and ANY-maze video-tracking software as with to the tail-suspension test.

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Avoidance Test.  (Mathis et al., 2018). 499 Optogenetic manipulations 500 For the optogenetic experiments, the mice were connected to a 450-nm laser (Doric Lenses) through 501 an optical fiber and a rotary joint. Pulses of blue light were controlled by the ANY-maze software.

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The stimulation protocol was 20-Hz trains and 5-ms pulses for 1 s every 4 s. The light intensity was 503 adjusted to provide 10 mW at the tips of the implanted optical fiber cannulae. During a trial, the 504 18 of 26 mice were connected to a single optical fiber cannula implanted above the DRN, VTA, or LHb. For 505 each test, the mice were tested on consecutive days using a Latin square experimental design.

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Real-time place preference test.

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The mice were placed in a chamber with two compartments connected by a small corridor. After 508 1 min of habituation, one of the compartments was paired with an optogenetic stimulation. A 509 mouse received a photostimulation every time it entered the paired chamber (randomly assigned).

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To maximize novelty and exploratory behavior on consecutive testing days, the RTPP apparatus 511 was used as follows: day 1 with a plain floor, day 2 with bedding covering the floor, and day three 512 with finely ground food pellet on the floor. Stimulation chambers were randomly assigned on each 513 of the three days of testing. The location of the mouse (chamber 1, chamber 2, or corridor) was 514 tracked, and laser activation was controlled using the ANY-maze video-tracking system.

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Tail suspension test and open field test.

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The mice were suspended by their tail for 20 min. The photostimulation was alternated between 517 a 2-min periods without stimulation and with a 2-min period with photostimulation trains (20 Hz, 518 1 second, 5-ms pulse duration, every 4 seconds).

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The same protocol used for the fiber photometry recordings was used for the optogenetic ma- Fiber photometry data.

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To store, process, analyze, and visualize the fiber photometry data, a Python package of objects 590 and functions was created, which is available at Fiber Photometry Data Analysis GitHub repository. ). To account for different tracking setups and experimental setups, 606 all the speeds were transformed to a standardized range [0, 1] (mobility score).

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A mobility score was used to define mobility and immobility onsets. An immobility bout was 608 defined as a period where the values were < 0.1 and lasted at least 2 s.

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∕ was aligned and averaged around the specific events such as airpuff, consumption onset,

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For the RTPP, ANY-maze data of laser activity over time was exported from ANY-maze and the 618 preference score was calculated using the following formula: = − 619 as detailed before (Proulx et al., 2018). 620 For the OFT and TST, the speeds tracked by ANY-maze were transformed into a standardized Events at the onset of mobility and immobility and random events during mobility and immobility were chosen, and the Pearson correlation at 6-seconds peri-events between the 2+ signal and the mobility score was calculated. Correlations with p < 0.001 and r > 0.6 were considered as positive, p < 0.001 and r < 0.6 -negative, the others -not correlated. (C) Fraction of positive (green), negative (red), and uncorrelated events (gray) in the OFT and TST for the LH→DRN, LH→VTA, and LH→LHb pathways. Statistical analysis was performed along with the data from the mice expressing GCaMP6s.  The preference scores measured in the RTPP during optostimuation of the LH→DRN, LH→VTA, or LH→LHb pathways in mice expressing ChR2-eYFP or eYFP. Two-way ANOVA between factor of group (ChR2-eYFP-and eYFP-expressing mice) and within factor pathway (LH→DRN, LH→VTA, or LHA→LHb) with post hoc Tukey test. The p values were adjusted using the Bonferroni multiple testing correction method. # < 0.1,* < 0.05, ** < 0.01, *** < 0.001, ns (not significant) -> 0.2.