Peer review process
Revised: This Reviewed Preprint has been revised by the authors in response to the previous round of peer review; the eLife assessment and the public reviews have been updated where necessary by the editors and peer reviewers.
Read more about eLife’s peer review process.Editors
- Reviewing EditorHelen ScharfmanNathan Kline Institute, Orangeburg, United States of America
- Senior EditorJohn HuguenardStanford University School of Medicine, Stanford, United States of America
Reviewer #1 (Public review):
Summary:
In this study, authors explored how galanin affects whole-brain activity in larval zebrafish using wide-field Ca2+ imaging, genetic modifications, and drugs that increase brain activity. The authors conclude that galanin has a sedative effect on the brain under normal conditions and during seizures, mainly through the galanin receptor 1a (galr1a). However, acute "stressors(?)" like pentylenetetrazole (PTZ) reduce galanin's effects, leading to increased brain activity and more seizures. Authors claim that galanin can reduce seizure severity while increasing seizure occurrence, speculated to occur through different receptor subtypes. This study confirms galanin's complex role in brain activity, supporting its potential impact on epilepsy.
Strengths:
The overall strength of the study lies primarily in its methodological approach using whole-brain Calcium imaging facilitated by the transparency of zebrafish larvae. Additionally, the use of transgenic zebrafish models is an advantage, as it enables genetic manipulations to investigate specific aspects of galanin signaling. This combination of advanced imaging and genetic tools allows for addressing galanin's role in regulating brain activity.
Weaknesses:
The weaknesses of the study also stem from the methodological approach, particularly the use of whole-brain Calcium imaging as a measure of brain activity. While epilepsy and seizures involve network interactions, they typically do not originate across the entire brain simultaneously. Seizures often begin in specific regions or even within specific populations of neurons within those regions. Therefore, a whole-brain approach, especially with Calcium imaging with inherited limitations, may not fully capture the localized nature of seizure initiation and propagation, potentially limiting the understanding of Galanin's role in epilepsy.
Furthermore, Galanin's effects may vary across different brain areas, likely influenced by the predominant receptor types expressed in those regions. Additionally, the use of PTZ as a "stressor" is questionable since PTZ induces seizures rather than conventional stress. Referring to seizures induced by PTZ as "stress" might be a misinterpretation intended to fit the proposed model of stress regulation by receptors other than Galanin receptor 1 (GalR1).
The description of the EAAT2 mutants is missing crucial details. EAAT2 plays a significant role in the uptake of glutamate from the synaptic cleft, thereby regulating excitatory neurotransmission and preventing excitotoxicity. Authors suggest that in EAAT2 knockout (KO) mice galanin expression is upregulated 15-fold compared to wild-type (WT) mice, which could be interpreted as galanin playing a role in the hypoactivity observed in these animals.
However, the study does not explore the misregulation of other genes that could be contributing to the observed phenotype. For instance, if AMPA receptors are significantly downregulated, or if there are alterations in other genes critical for brain activity, these changes could be more important than the upregulation of galanin. The lack of wider gene expression analysis leaves open the possibility that the observed hypoactivity could be due to factors other than, or in addition to, galanin upregulation.
Moreover, the observation that in double KO mice for both EAAT2 and galanin there was little difference in seizure susceptibility compared to EAAT2 KO mice alone further supports the idea that galanin upregulation might not be the reason to the observed phenotype. This indicates that other regulatory mechanisms or gene expressions might be playing a more pivotal role in the manifestation of hypoactivity in EAAT2 mutants.
These methodological shortcomings and conceptual inconsistencies undermine the perceived strengths of the study, and hinders understanding of Galanin's role in epilepsy and stress regulation.
Comments on revisions:
The revised manuscript and the answers of the authors is appreciated. However, the criticisms were addressed only partially and main weaknesses of the manuscript are still remaining.
Reviewer #2 (Public review):
This revised study is an investigation of galanin and galanin receptor signaling on whole-brain activity in the context of recurrent seizure activity or under homeostatic basal conditions. The authors primarily use calcium imaging to observe whole-brain neuronal activity accompanied by galanin qPCR to determine how manipulations of galanin or the galr1a receptor affect the activity of the whole-brain under non-ictal conditions or when seizure activity occurs. The authors use their eaat2a-/- model (introduced in their Glia 2022 paper, PMID 34716961) that shows recurrent seizure activity as well as suppression of neuronal activity and locomotion interictally. It is compared to the well-known pentylenetetrazole (PTZ) pharmacological model of seizures in zebrafish. Given the literature cited in their Introduction, the authors hypothesize that galanin will exert a net inhibitory effect on brain activity in models of seizures/epilepsy. They were surprised to find that this hypothesis was only moderately supported in their eaat2a-/- model. In contrast, after PTZ, fish with galanin overexpression showed increased seizure number and reduced duration while fish with galanin KO showed reduced seizure number and increased duration.
Previous concerns about sex or developmental biological variables were addressed, as their model's seizure phenotype emerges rapidly and long prior to the establishment of zebrafish sexual maturity. However, in the course of re-review, some additional concerns (below) were detected that, if addressed, could further improve the manuscript. These concerns relate to how seizures were defined from the measurement of fluorescent calcium imaging data. Overall, this study is important and convincing, and carries clear value for understanding the multifaceted functions that neuronal galanin can perform under homeostatic and disease conditions.
Additional Concerns:
- The authors have validated their ability to measure behavioral seizures quantitatively in their 2022 Glia paper but the information provided on defining behavioral seizures was limited. The definition of behavioral seizure activity is not expanded upon in this paper, but could provide detail about how the behavioral seizures relate to a seizure detected via calcium imaging.
- Related to the previous point, for the calcium imaging, the difference between an increase in fluorescence that the authors think reflects increased neuronal activity and the fluorescence that corresponds to seizures is not very clear. This detail is necessary because exactly when the term "seizure" describes a degree of increased activity can be difficult to distinguish objectively.
- The supplementary movies that were added were very useful, but raised some questions. For example, what brain regions were pulsating? What areas seemed to constantly exhibit strong fluorescence and was this an artifact? It seemed that sometimes there was background fluorescence in the body. Perhaps an anatomical diagram could be provided for the readers. In addition, there were some movies with much greater fluorescence changes - are these the seizures? These are some reasons for our request for clarified definitions of the term "seizure".
Reviewer #3 (Public review):
Summary:
The neuropeptide galanin is primarily expressed in the hypothalamus and has been shown to play critical roles in homeostatic functions such as arousal, sleep, stress, and brain disorders such as epilepsy. Previous work in rodents using galanin analogs and receptor-specific knockout have provided convincing evidence for anti-convulsant effects of galanin.
In the present study, the authors sought to determine the relationship between galanin expression and whole-brain activity. The authors took advantage of the transparent nature of larval zebrafish to perform whole-brain neural activity measurements via widefield calcium imaging. Two models of seizures were used (eaat2a-/- and pentylenetetrazol; PTZ). In the eaat2a-/- model, spontaneous seizures occur and the authors found that galanin transcript levels were significantly increased and associated with reduced frequency of calcium events. Similarly, two hours after PTZ galanin transcript levels roughly doubled and the frequency and amplitude of calcium events were reduced.
The authors also used a heat shock protein line (hsp70I:gal) where galanin transcripts levels are induced by activation of heat shock protein, but this line also shows higher basal transcript levels of galanin. Due to problems with whole-brain activity in wild-type larvae, the authors used the line without heat shock. They found higher level of galanin in hsp70I:gal larval zebrafish resulted in a reduction of calcium events and a reduction in amplitude of events. In contrast, galanin knockout (gal-/-) increased calcium activity, indicated by an increased number of calcium events, but a reduction in amplitude and duration. New data in the supplementary figure 2 used antibody staining to confirm the absence of galanin expression in gal-/- knockouts. Knockout of the galanin receptor subtype galr1a via crispants also increased the frequency of calcium events. New data in the revised manuscript reports that galr1aKO did not cause an upregulation of galanin, thereby ruling out genetic compensation effects.
In subsequent experiments in eaat2a-/- mutants were crossed with hsp70I:gal or gal-/- to increase or decrease galanin expression, respectively. These experiments showed modest effects, with eaat2a-/- x gal-/- knockouts showing an increased normalized area under the curve and seizure amplitude.
Lastly, the authors attempted to study the relationship between galanin and brain activity during a PTZ challenge. The hsp70I:gal larva showed increased number of seizures and reduced seizure duration during PTZ. In contrast, gal-/- mutants showed increased normalized area under the curve and a stark reduction in number of detected seizures, a reduction in seizure amplitude, but an increase in seizure duration. The authors then ruled out the role of Galr1a in modulating this effect during PTZ, since the number of seizures was unaffected, whereas the amplitude and duration of seizures was increased.
Strengths:
(1) The gain- and loss-of function galanin manipulations provided convincing evidence that galanin influences brain activity (via calcium imaging) during interictal and/or seizure-free periods. In particular, the relationship between galanin transcript levels and brain activity in figures 1 & 2 was convincing. New antibody staining confirms the absence of galanin in gal-/- mutants. New data also shows galanin transcript levels were unchanged in galr1ako brains.
(2) The authors use two models of epilepsy (eaat2a-/- and PTZ).
(3) Focus on the galanin receptor subtype galr1a provided good evidence for an important role of this receptor in controlling brain activity during interictal and/or seizure-free periods.
(4) The authors have added supplementary video files for calcium imaging to support their observations.
Weaknesses:
(1) Although the relationship between galanin and brain activity during interictal or seizure-free periods was clear, the revised manuscript still lacks mechanistic insight in the role of galanin during seizure-like activity induced by PTZ.
(2) The revised manuscript continues to heavily rely on calcium imaging of different mutant lines. Confirmation of knockouts has been provided with immunostaining in a new supplementary figure. Additional methods could strengthen the data, translational relevance, and interpretation (e.g., acute pharmacology using galanin agonists or antagonists, brain or cell recordings, biochemistry, etc).