Bilateral equalization of synaptic output in olfactory glomeruli of Xenopus tadpoles
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eLife Assessment
This manuscript investigates inter-hemispheric interactions in the olfactory system of Xenopus tadpoles. Using a combination of electrophysiology, pharmacology, imaging, and uncaging, the transection of the contralateral nerve is shown to lead to larger odor responses in the unmanipulated hemisphere, and implicates dopamine signaling in this process. The study uses a rich and sophisticated array of tools to investigate olfactory coding and uncovers valuable mechanisms of signaling. However, the data is incomplete, with a few of the conclusions not being well-supported by the data; the interpretation should be adjusted with some caveats, or additional experiments should be done to support these conclusions.
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Abstract
Odorants stimulate olfactory sensory neurons (OSNs) to create a bilateral sensory map defined by a set of glomeruli present in the left and right olfactory bulbs. Using Xenopus tropicalis tadpoles we challenged the notion that glomerular activation is exclusively determined ipsilaterally. Glomerular responses evoked by unilateral stimulation were potentiated following transection of the contralateral olfactory nerve. The gain of function was observed as early as 2 hours after injury and faded away with a time constant of 4 days. Potentiation was mediated by the presence of larger and faster calcium transients driving glutamate release from OSN axon terminals. The cause was the reduction of the tonic presynaptic inhibition exerted by dopamine D2 receptors. Inflammatory mediators generated by injury were not involved. These findings reveal the presence of a bilateral modulation of glomerular output driven by dopamine that compensates for imbalances in the number of operative OSNs present in the two olfactory epithelia. Considering that the constant turnover of OSNs is an evolutionary conserved feature of the olfactory system and determines the innervation of glomeruli, the compensatory mechanism here described may represent a general property of the vertebrate olfactory system to establish an odor map.
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eLife Assessment
This manuscript investigates inter-hemispheric interactions in the olfactory system of Xenopus tadpoles. Using a combination of electrophysiology, pharmacology, imaging, and uncaging, the transection of the contralateral nerve is shown to lead to larger odor responses in the unmanipulated hemisphere, and implicates dopamine signaling in this process. The study uses a rich and sophisticated array of tools to investigate olfactory coding and uncovers valuable mechanisms of signaling. However, the data is incomplete, with a few of the conclusions not being well-supported by the data; the interpretation should be adjusted with some caveats, or additional experiments should be done to support these conclusions.
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Reviewer #1 (Public review):
In this study, the authors investigate LFP responses to methionine in the olfactory system of the Xenopus tadpole. They show that this response is local to the glomerular layer, arises ipsilaterally, and is blocked by pharmacological blockade of AMPA and NMDA receptors, with little modulation during blockade of GABA-A receptors. They then show that this response is translently enlarged following transection of the contralateral olfactory nerve, but not the optic lobe nerve. Measurement of ROS- a marker of inflammation- was not affected by contralateral nerve transection, and LFP expansion was not affected by pharmacological blockade of ROS production. Imaging biased towards presynaptic terminals suggests that the enlargement of the LFP has a presynaptic component. A D2 antagonist increases the LFP size and …
Reviewer #1 (Public review):
In this study, the authors investigate LFP responses to methionine in the olfactory system of the Xenopus tadpole. They show that this response is local to the glomerular layer, arises ipsilaterally, and is blocked by pharmacological blockade of AMPA and NMDA receptors, with little modulation during blockade of GABA-A receptors. They then show that this response is translently enlarged following transection of the contralateral olfactory nerve, but not the optic lobe nerve. Measurement of ROS- a marker of inflammation- was not affected by contralateral nerve transection, and LFP expansion was not affected by pharmacological blockade of ROS production. Imaging biased towards presynaptic terminals suggests that the enlargement of the LFP has a presynaptic component. A D2 antagonist increases the LFP size and variability in intact tadpoles, while a GABA-B antagonist does not. On this basis, the authors conclude that the increase driven by contralateral nerve transection is due to DA signaling.
Overall, I found the array of techniques and approaches applied in this study to be creatively and effectively employed. However, several of the conclusions made in the Discussion are too strong, given the evidence presented. For example, the authors state that "The observed potentiation was not related to inflammatory mediators associated to inury, because it was caused by a release of the inhibition made by D2 dopamine receptor present in OSN axon terminals." This statement is too strong - the authors have shown that D2 receptors are sufficient to cause an increase in LFP, but not that they are required for the potentiation evoked by nerve transection. The right experiment here would be to get rid of the D2 receptors prior to transection and show that the potentiation is now abolished. In addition, the authors have not shown any data localizing D2 receptors to OSN axon terminals.
Similarly, the authors state, "the onset of LFP changes detected in glomeruli is determined by glutamate release from OSNs." Again, the authors have shown that blockade of AMPA/NMDA receptors decreases the LFP, and that uncaging of glutamate can evoke small negative deflections, but not that the intact signal arises from glutamate release from OSNs. The conclusions about the in vivo contribution of this contralateral pathway are also rather speculative. Acute silencing of one hemisphere would likely provide more insight into the moment-to-moment contributions of bilateral signals to those recorded in one hemisphere.
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Author response:
Thank you for your time and for considering our manuscript as a Reviewed Preprint. We also would like to thank Reviewer 1 for their evaluation of our manuscript.
Here, we present a provisional response to reviewer comments and following their suggestions we will make an effort to: i) increase evidence for the role of dopamine in olfactory glomeruli and ii) delineate the circuit involved mediating the observed potentiation. Next, we briefly describe the set of experiments that are in progress or will be performed to improve our paper.
We will carry out immunostainings for tyrosine hydroxylase to certify that dopamine can be released on the genetically labelled glomerulus. There is a lack of good commercial antibodies for Xenopus (we already tried one and did not work, PA1-4679, Thermofisher scientific), but we will look …
Author response:
Thank you for your time and for considering our manuscript as a Reviewed Preprint. We also would like to thank Reviewer 1 for their evaluation of our manuscript.
Here, we present a provisional response to reviewer comments and following their suggestions we will make an effort to: i) increase evidence for the role of dopamine in olfactory glomeruli and ii) delineate the circuit involved mediating the observed potentiation. Next, we briefly describe the set of experiments that are in progress or will be performed to improve our paper.
We will carry out immunostainings for tyrosine hydroxylase to certify that dopamine can be released on the genetically labelled glomerulus. There is a lack of good commercial antibodies for Xenopus (we already tried one and did not work, PA1-4679, Thermofisher scientific), but we will look for alternatives. In a previous set of experiments, we attempted to measure dopamine release in the glomerular layer by electroporating olfactory sensory neurons or olfactory bulb neurons with the dopamine sensors dLight1.1 (Addgene #111053) or dLight1.3 (Addgene # 111056). In our hands, fluorescence signals were extremely weak, barely undetectable. Similar results were obtained after electroporating the tectum or the rhombencephalon. We propose to repeat experiments using a more sensitive sensor such as GRAB_DA2m. Other approaches, such as performing single cell transcriptomics of olfactory sensory neurons might be considered to confirm the expression of D2 receptors.
We agree with the reviewer that we should obtain more lines of evidence in support for a presynaptic inhibition mediated by D2 receptors.To gain insight on the bilateral circuit mediating the observed potentiation of glomerular responses we are currently investigating the role of dorsolateral pallium neurons. In Xenopus tadpoles the lateral pallium plays an analogous role to the olfactory cortex in amniotes. Preliminary observations show that neurons located in this pallial region respond to ipsilateral stimulation of the olfactory epithelium and if damaged, a contralateral potentiation of glomerular output occurs. We aim to conclude this set of experiments and include it in the paper as we believe it clarifies the circuitry involved.
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