Orexin population activity precisely reflects net body movement across behavioral and metabolic states

Reviewer #1 (Public review):

Summary:

This manuscript by Tesmer and colleagues uses fiber photometry recordings, sophisticated analysis of movement, and deep learning algorithms to provide compelling evidence that activity in hypothalamic hypocretin/orexin neurons (HONs) correlates with net body movement over multiple behaviors. By examining projection targets, the authors show that hypocretin/orexin release differs in projection targets to the locus coeruleus and substantia nigra, pars compacta. Ablation of HONs does not cause differences in the power spectra of movements. The movement-tracking ability of HONs is independent of HON activity that correlates with blood glucose levels. Finally, the authors show that body movement is not encoded to the same extent in other neural populations.

Strengths:

The major strengths of the study are the combination of fiber photometry recordings, analysis of movement in head-fixed mice, and sophisticated classification of movement using deep learning algorithms. The experiments seem to be well performed, and the data are well presented, visually. The data support the main conclusions of the manuscript.

Weaknesses:

The weaknesses are minor, mostly consisting of writing and data visualization throughout the manuscript. To some degree, it is already known that hypocretin/orexin neurons correlate with movement and arousal, although this manuscript studies this correlation with unprecedented sophistication and scale. It is also unfortunate that most of the experiments throughout the study were only performed in male mice.

Taken together, this study is likely to be impactful to the field and our understanding of HONs across behavioral states.

Reviewer #2 (Public review):

Summary:

Despite several methodological strengths, the major and highly significant drawback is the confound of arousal with movement. This confound is not resolved, so the results could be explained by previously established relationships between orexin and arousal/wakefulness.

Strengths:

The authors show that orexin neuron activity is associated with body movement and that this information is conveyed irrespective of the fasted state. They also report differences in different orexin target brain regions for orexin release during movement.

This paper contains an impressive array of cutting-edge techniques to examine a very important brain system, the orexin-hypocretin system. The authors offer an original perspective on the function of this system. The authors showed that orexin neuron activity scales to some degree with the magnitude of body movement change; this is unaffected by a fasted state and seems to be somewhat unique to orexin neurons.

The investigation of other genetically-defined subcortical neuron populations to determine the specificity of findings is also a strength, as is the ability to quantify movement and use deep learning to classify specific behaviors adds sophistication to analysis. The authors also show heterogeneity in orexin projections to specific target nuclei, which is interesting.

The authors "speculate that narcolepsy-cataplexy, caused by HON loss-of-function, is perhaps explained by oscillations into unwanted sleep-states and motor programs due to impaired control loops for wakefulness and movement". This is quite an interesting aspect of their work, and deserving of further study.

Weaknesses:

Despite the strengths, there are several major and minor weaknesses that detract significantly from the study.

Weaknesses - Major

My main concern with this work is the confound of arousal with movement so that correlations with one might reflect a relationship instead with the other. The orexin system is well known to play an important role in arousal, with elevated activity of orexin neurons reported for waking and high arousal. Orexin signaling has also been strongly associated with motivation, which also is associated with arousal and movement. The authors offer no compelling evidence that the relationships they describe between different movements and orexin signaling do not simply reflect the known relationship between arousal and motivation.

The authors could address this concern by including classical arousal measurements, eg, cortical EEG recorded simultaneously with movements. Often, EEG arousal occurs independently of movement, so this could provide one approach to disentangling this confound. The idea that orexin signaling plays a role in arousal rather than movement is supported by their finding that orexin lesions using the orexin-DTR mouse model did not impact movements. In contrast, prior lesion and pharmacologic studies have found that decreased orexin signaling significantly decreases arousal and waking.

Another way they could test their idea would be to paralyze and respirate animals so that orexin activity could be recorded without movement. Alternatively, animals could be trained to remain motionless to receive a reward. Thus, there are several ways to test the overall hypothesis of this work that have not been examined here.

The authors propose that "a simple interpretation of their results is that, via HON movement tracking, the brain creates a "wake up" signal in proportion to movement". This seems to argue for the role of the orexin system in arousal and motivation rather than in movement per se.

There are several studies that have examined the effect of orexin antagonist treatment in rodents on locomotor and other motor activities. These studies have largely found no consistent effect of antagonizing orexin signaling, especially at the OxR1 receptor, on simple motor activity. These studies are not referenced here but should be taken into account in the authors' conclusions.

Figure 3, panel F: I understand HON-DTR is a validated model but a picture of HONs ablation is necessary, including pictures of HONs outputs ablation within the SNc and LC.

The discussion lacks a more extensive paragraph on the distinct signal and role of Ox->SNc and Ox-LC projections.

Reviewer #3 (Public review):

Summary

The study presents an investigation into how hypothalamic orexin neurons (HONs) track body movement with high precision. Using techniques including fiber photometry, video-based movement metrics, and empirical mode decomposition (EMD), the authors demonstrate that HONs encode net body movement consistently across a range of behaviors and metabolic states. They test the ability of HONs to track body movement to that of other subcortical neural populations, from which they distinguish HONs activity from other subcortical neural populations.

Strengths:

The study characterizes HONs activity as key indicators of movement and arousal, and this method may have potential implications for understanding sleep disorders, energy regulation, and brain-body coordination. Overall, I think this is a very interesting story, with novel findings and implications about sensorimotor systems in animals. The manuscript is clearly written and the evidence presented is rigorous. The conclusions are well supported by experimental data with clear statistical analyses.

Weaknesses/suggestions:

There are a couple of issues I think the authors could address to make the paper better and more complete:

(1) The study primarily focuses on steady-state behaviors. It would be interesting if the authors' current dataset allows analyses of HON dynamics during transitions between behavioral states (e.g., resting to running or grooming to sniffing). This could provide additional insights into how HONs adapt to rapid changes in body movement.

(2) Given the established role of HONs in arousal and wakefulness, the study could further investigate how movement-related HON dynamics interact with arousal states. For example, does HON encoding of movement differ during sleep versus wakefulness?

(3) Although HON ablation experiments suggest that HONs do not shape movement frequency profiles. It would be more compelling if the authors could investigate whether HONs contribute to specific types of movements (e.g., fine motor vs. gross motor movements) or modulate movement initiation thresholds.

(4) The heterogeneous movement-related orexin dynamics observed in the LC and SNc raise intriguing questions about the circuit-level mechanisms underlying these differences. Optogenetic or chemogenetic manipulation of these projections could validate the functional implications of these dynamics.