PREreview of Phosphorylation of pyruvate dehydrogenase marks the inhibition ofin vivoneuronal activity

This review arose out of a course for graduate students in the life sciences at UCSF, “Peer Review in the Life Sciences,” which aims to introduce junior scientists to peer review in a critical yet constructive way. The students selected preprints to review, led discussions of them, drafted reviews, and revised them based on feedback from peers and instructors.

Summary:

Molecular markers for neural activation have been widely used for decades, but there does not yet exist a molecular marker for neural inhibition. This study aimed to identify ones by conducting a systematic and unbiased screen combining optogenetics and tandem mass tag (TMT) proteomics. The authors identified phosphorylated-pyruvate dehydrogenase (pPDH) as a novel molecular marker for the decrease in neural activity. They subsequently performed various validations both in vitro and in vivo. The compelling validations consistently showed there is an inverse correlation between the expression level of pPDH and neural activity and the change in pPDH expression quickly follows the change in neural activity. The authors also performed fiber photometry in AgRP neurons in a fast-refeed paradigm to compare pPDH expression with existing methods measuring neural inhibition.

This is a nice study that created a valuable new molecular marker that can be applied to a wide range of research topics in the field of neuroscience. While immediate early genes such as c-fos have long been used as a proxy for neural activation and to create various tools including recombinant virus and mouse lines (e.g. Fos-TRAP mice) have been developed to widen its application, there is no equivalent marker for neural inhibition, which is equally important when studying the function of specific neuronal populations and the nervous system in general. This new marker identified in this study possesses great potential to elucidate the mechanism of neural inhibition. Neuroscientists of various subfields can apply this technique to identify previously undefined neural populations in various contexts including sensory perception, behaviors, and/or disease.

Major comments:

1. The introduction could be strengthened if previous attempts (if any) made to find a molecular marker for neural inhibition were discussed. Are there any good review papers outlining previous work that could be referenced?

2. Page 3 Line 12: “An ideal marker should be temporally linked to the decreased firing of action potentials yet result in molecular changes that are amenable to stable and trackable detection. The slower kinetics of de novo gene expression events makes them less suitable for this application.” Based on this text, we are unable to fully appreciate the argument that due to de novo gene expression being slow, it is less a suitable target for the screen. If a de novo gene expression event is triggered upon the inhibition of neurons, then presumably this newly synthesized molecule could serve as a suitable marker for neural inhibition - like c-fos is a suitable marker for activation. On the other hand, if a molecule is synthesized during activation or tonic firing states while having a long half-life, then this molecule would not be an ideal proxy for neural inhibition as the concentration of the molecule will not drop significantly long after neurons are inhibited.

3. Fig 1 and Page 3 Line 36. We believe it could be useful to include a statement explaining why inhibitory opsins weren’t used. Although the authors mentioned “ChR2 is the most used channelrhodopsin”, we believe it would be helpful for readers to understand the rationale with some additional justification (e.g. “inhibitory opsins are less reliable than ChR2”).

4. Fig 1F. We are curious how the authors chose p-PDH S293 and S300 from the phosphoproteomic screen. There appears to be many other hits (with better enrichment) in this plot that could have been selected. What were the rationale and criteria behind choosing pPDH versus other candidates?

Minor comments:

1. Fig 1B. We found it unclear what the difference between the 2 ms, 5 ms, and 10 ms data were in this panel. Does this describe the time length of each pulse? 

2. Fig 1C. How was the fidelity score calculated for this panel? Our understanding is that a fidelity score represents whether a certain frequency and duration of stimulus can reliably evoke the same response across different neurons, but this was not mentioned in the text.

3. Page 3 Line 6:  There is a run-on sentence. Change to: “IEG-based methods robustly report neuronal activation; however, ….”

4. Fig 4A. There is a small text “pPDH” that is unclear which panel it belongs to and what it refers to.

5. In the discussion section, we believe it could be helpful if the authors could speculate how this technology could be used more broadly. Consider perhaps including a statement to encourage those working on parts of the nervous system other than the brain (e.g. spinal cord and PNS) to apply this new technique.

6. Page 3 line 38: Should be 470 nm instead of 488 nm.

Competing interests

The author declares that they have no competing interests.