Author response:
Reviewer #1 (Public review):
Summary:
Sun et al. are interested in how experience can shape the brain and specifically investigate the plasticity of the Toll-6 receptor-expressing dopaminergic neurons (DANs). To learn more about the role of Toll-6 in the DANs, the authors examine the expression of the Toll-6 receptor ligand, DNT-2. They show that DNT-2 expressing cells connect with DANs and that loss of function of DNT-2 in these cells reduces the number of PAM DANs, while overexpression causes alterations in dendrite complexity. Finally, the authors show that alterations in the levels of DNT-2 and Toll-6 can impact DAN-driven behaviors such as climbing, arena locomotion, and learning and long-term memory.
Strengths:
The authors methodically test which neurotransmitters are expressed by the 4 prominent DNT-2 expressing neurons and show that they are glutamatergic. They also use Trans-Tango and Bac-TRACE to examine the connectivity of the DNT-2 neurons to the dopaminergic circuit and show that DNT-2 neurons receive dopaminergic inputs and output to a variety of neurons including MB Kenyon cells, DAL neurons, and possibly DANS.
We are very pleased that Reviewer 1 found our connectivity analysis a strength.
Weaknesses:
(1) To identify the DNT-2 neurons, the authors use CRISPR to generate a new DN2-GAL4. They note that they identified at least 12 DNT-2 plus neurons. In Supplementary Figure 1A, the DNT-2-GAL4 driver was used to express a UAS-histoneYFP nuclear marker. From these figures, it looks like DNT-2-GAL4 is labeling more than 12 neurons. Is there glial expression?
Indeed, we claimed that DNT-2 is expressed in at least 12 neurons (see line 141, page 6 of original manuscript), which means more than 12 could be found. The membrane tethered reporters we used – UAS-FlyBow1.1, UASmcD8-RFP, UAS-MCFO, as well as UAS-DenMark:UASsyd-1GFP – gave a consistent and reproducible pattern. However, with DNT-2GAL4>UAS-Histone-YFP more nuclei were detected that were not revealed by the other reporters. We have found also with other GAL4 lines that the patterns produced by different reporters can vary. This could be due to the signal strength (eg His-YFP is very strong) and perdurance of the reporter (e.g. the turnover of His-YFP may be slower than that of the other fusion proteins).
We did not test for glial expression, as it was not directly related to the question addressed in this work.
(2) In Figure 2C the authors show that DNT-2 upregulation leads to an increase in TH levels using q-RT-PCR from whole heads. However, in Figure 3H they also show that DNT-2 overexpression also causes an increase in the number of TH neurons. It is unclear whether TH RNA increases due to expression/cell or the number of TH neurons in the head.
Figure 3H shows that over-expression of DNT-2 FL increased the number of Dcp1+ apoptotic cells in the brain, but not significantly (p=0.0939). The ability of full-length neurotrophins to induce apoptosis and cleaved neurotrophins promote cell survival is well documented in mammals. We had previously shown that DNT-2 is naturally cleaved, and that over-expression of DNT-2 does not induce apoptosis in the various contexts tested before (McIlroy et al 2013 Nature Neuroscience; Foldi et al 2017 J Cell Biol; Ulian-Benitez et al 2017 PLoS Genetics). Similarly, throughout this work we did not find DNT-2FL to induce apoptosis.
Instead, in Figure 3G we show that over-expression of DNT-2FL causes a mild yet statistically significant increase in the number of TH+ cells. This is an important finding that supports the plastic regulation of PAM cell number. We thank the Reviewer for highlighting this point, as we had forgotten to add the significance star in the graph. In this context, we cannot rule out the possibility that the increase in TH mRNA observed when we over-express DNT-2FL could not be due to an increase in cell number instead. Unfortunately, it is not possible for us to separate these two processes at this time. Either way, the result would still be the same: an increase in dopamine production when DNT-2 levels rise.
(3) DNT-2 is also known as Spz5 and has been shown to activate Toll-6 receptors in glia (McLaughlin et al., 2019), resulting in the phagocytosis of apoptotic neurons. In addition, the knockdown of DNT-2/Spz5 throughout development causes an increase in apoptotic debris in the brain, which can lead to neurodegeneration. Indeed Figure 3H shows that an adult-specific knockdown of DNT-2 using DNT2-GAL4 causes an increase in Dcp1 signal in many neurons and not just TH neurons.
Indeed, we did find Dcp1+ cells in TH-negative cells too (although not widely throughout the brain). This is not surprising, as DNT-2 neurons have large arborisations that can reach a wide range of targets; DNT-2 is secreted, and could reach beyond its immediate targets; Toll-6 is expressed in a vast number of cells in the brain; DNT-2 can bind promiscuously at least also Toll-7 and other Keks, which are also expressed in the adult brain (Foldi et al 2017 J Cell Biology; Ulian-Benitez et al 2017 PLoS Genetics; Li et al 2020 eLife). Together with the findings by McLaughlin et al 2019, our findings further support the notion that DNT-2 is a neuroprotective factor in the adult brain. It will be interesting to find out what other neuron types DNT-2 maintains.
We would like to thank Reviewer 1 for their positive comments on our work and their interesting and valuable feedback.
Reviewer #2 (Public review):
This paper examines how structural plasticity in neural circuits, particularly in dopaminergic systems, is regulated by Drosophila neurotrophin-2 (DNT-2) and its receptors, Toll-6 and Kek-6. The authors show that these molecules are critical for modulating circuit structure and dopaminergic neuron survival, synaptogenesis, and connectivity. They show that loss of DNT-2 or Toll-6 function leads to loss of dopaminergic neurons, dendritic arborization, and synaptic impairment, whereas overexpression of DNT-2 increases dendritic complexity and synaptogenesis. In addition, DNT-2 and Toll-6 modulate dopamine-dependent behaviors, including locomotion and long-term memory, suggesting a link between DNT-2 signaling, structural plasticity, and behavior.
A major strength of this study is the impressive cellular resolution achieved. By focusing on specific dopaminergic neurons, such as the PAM and PPL1 clusters, and using a range of molecular markers, the authors were able to clearly visualize intricate details of synapse formation, dendritic complexity, and axonal targeting within defined circuits. Given the critical role of dopaminergic pathways in learning and memory, this approach provides a good opportunity to explore the role of DNT-2, Toll-6, and Kek-6 in experience-dependent structural plasticity. However, despite the promise in the abstract and introduction of the paper, the study falls short of establishing a direct causal link between neurotrophin signaling and experience-induced plasticity.
Simply put, this study does not provide strong evidence that experience-induced structural plasticity requires DNT-2 signaling. To support this idea, it would be necessary to observe experience-induced structural changes and demonstrate that downregulation of DNT-2 signaling prevents these changes. The closest attempt to address this in this study was the artificial activation of DNT-2 neurons using TrpA1, which resulted in overgrowth of axonal arbors and an increase in synaptic sites in both DNT-2 and PAM neurons. However, this activation method is quite artificial, and the authors did not test whether the observed structural changes were dependent on DNT-2 signaling. Although they also showed that overexpression of DNT-2FL in DNT-2 neurons promotes synaptogenesis, this phenotype was not fully consistent with the TrpA1 activation results (Figures 5C and D).
In conclusion, this study demonstrates that DNT-2 and its receptors play a role in regulating the structure of dopaminergic circuits in the adult fly brain. However, it does not provide convincing evidence for a causal link between DNT-2 signaling and experience-dependent structural plasticity within these circuits.
We would like to thank Reviewer 2 for their very positive assessment of our approach to investigate structural circuit plasticity. We are delighted that this Reviewer found our cellular resolution impressive. We are also very pleased that Reviewer 2 found that our work demonstrates that DNT-2 and its receptors regulate the structure of dopaminergic circuits in the adult fly brain. This is already a very important finding that contributes to demonstrating that, rather than being hardwired, the adult fly brain is plastic, like the mammalian brain.
We are very pleased that this Reviewer acknowledges that this work provides a good opportunity to explore the role of DNT-2, Toll-6, and Kek-6 in experience-dependent structural plasticity. We provide a molecular mechanism and proof of principle, and we demonstrate a direct link between the function of DNT-2 and its receptors in circuit plasticity, and a suggestive link to neuronal activity. Finding out the direct link to lived experience is a big task, beyond the scope of this manuscript, and we will be testing this with future projects. Nevertheless, it is important to place our findings within this context, as it opens opportunities for discovery by the neuroscience community.
We would like to thank Reviewer 2 for the positive and thoughtful evaluation of our work, and for their feedback.
Reviewer #3 (Public review):
Summary:
The authors used the model organism Drosophila melanogaster to show that the neurotrophin Toll-6 and its ligands, DNT-2 and kek-6, play a role in maintaining the number of dopaminergic neurons and modulating their synaptic connectivity. This supports previous findings on the structural plasticity of dopaminergic neurons and suggests a molecular mechanism underlying this plasticity.
Strengths:
The experiments are overall very well designed and conclusive. Methods are in general state-of-the-art, the sample sizes are sufficient, the statistical analyses are sound, and all necessary controls are in place. The data interpretation is straightforward, and the relevant literature is taken into consideration. Overall, the manuscript is solid and presents novel, interesting, and important findings.
We are delighted that Reviewer 3 found our work solid, novel, interesting and with important findings. We are also very pleased that this Reviewer found that all necessary controls have been carried out.
Weaknesses:
There are three technical weaknesses that could perhaps be improved.
First, the model of reciprocal, inhibitory feedback loops (Figure 2F) is speculative. On the one hand, glutamate can act in flies as an excitatory or inhibitory transmitter (line 157), and either situation can be the case here. On the other hand, it is not clear how an increase or decrease in cAMP level translates into transmitter release. One can only conclude that two types of neurons potentially influence each other.
Thank you for pointing out that glutamate can be inhibitory. In mammals, the neurotrophin BDNF has an important function in glutamatergic synapses, thus we were intrigued by a potential evolutionary conservation. Our evidence that DNT-2A neurons could be excitatory is indirect, yet supportive: exciting DNT-2 neurons with optogenetics resulted in an increase in GCaMP in PAMs (data not shown); over-expression of DNT-2 in DNT-2 neurons increased TH mRNA levels; optogenetic activation of DNT-2 neurons results in the Dop2R-dependent downregulation of cAMP levels in DNT-2 neurons. Dop2R signals in response to dopamine, which would be released only if dopaminergic neurons had been excited. Accordingly, glutamate released from DNT-2 neurons would have been rather unlikely to inhibit DANs.
cAMP is a second messenger that enables the activation of PKA. PKA phosphorylates many target proteins, amongst which are various channels. This includes the voltage gated calcium channels located at the synapse, whose phosphorylation increases their opening probability. Thus, a rise in cAMP could facilitate neurotransmitter release, and a downregulation would have the opposite effect. Other targets of PKA include CREB, leading to changes in gene expression. Conceivably, a decrease in PKA activity could result in the downregulation of DNT-2 expression in DNT-2 neurons. This negative feedback loop would restore the homeostatic relationship between DNT-2 and dopamine levels.
Our data indeed demonstrate that DNT-2 and PAM neurons influence each other, not potentially, but really. We have provided data that: DNT-2 and PAMs are connected through circuitry; that the DNT-2 receptors Toll-6 and kek-6 are expressed in DANs, including in PAMs; that alterations in the levels of DNT-2 (both loss and gain of function) and loss of function for the DNT-2 receptors Toll-6 and Kek-6 alter PAM cell number, alter PAM dendritic complexity and alter synaptogenesis in PAMs; alterations in the levels of DNT-2, Toll-6 and kek-6 in adult flies alters dopamine dependent behaviours of climbing, locomotion in an arena and learning and long-term memory. These data firmly demonstrate that the two neuron types DNT-2 and PAMs influence each other.
We have also shown that over-expression of DNT-2 in DNT-2 neurons increases TH mRNA levels, whereas activation of DNT-2 neurons decreases cAMP levels in DNT-2 neurons in a dopamine/Dop2R-dependent manner. These data show a functional interaction between DNT-2 and PAM neurons.
Second, the quantification of bouton volumes (no y-axis label in Figure 5 C and D!) and dendrite complexity are not convincingly laid out. Here, the reader expects fine-grained anatomical characterizations of the structures under investigation, and a method to precisely quantify the lengths and branching patterns of individual dendritic arborizations as well as the volume of individual axonal boutons.
Figure 5C, D do contain Y-axis labels, all our graphs in main manuscript and in supplementary files contain Y-axis labels.
In fact, we did use a method to precisely quantify the lengths and branching patterns of individual dendritic arborisations, volume of individual boutons and bouton counting. These analyses were carried out using Imaris software. For dendritic branching patterns, the “Filament Autodetect” function was used. Here, dendrites were analysed by tracing semi-automatically each dendrite branch (ie manual correction of segmentation errors) to reconstruct the segmented dendrite in volume. From this segmented dendrite, Imaris provides measurements of total dendrite volume, number and length of dendrite branches, terminal points, etc. For bouton size and number, we used the Imaris “Spot” function. Here, a threshold is set to exclude small dots (eg of background) that do not correspond to synapses/boutons. All samples and genotypes are treated with the same threshold, thus the analysis is objective and large sample sizes can be analysed effectively. We have already provided a description of the use of Imaris in the methods section.
Third, Figure 1C shows two neurons with the goal of demonstrating between-neuron variability. It is not convincingly demonstrated that the two neurons are actually of the very same type of neuron in different flies or two completely different neurons.
We thank Reviewer 3 for raising this interesting point. It is not possible to prove which of the four DNT-2A neurons per hemibrain, which we visualised with DNT-2>MCFO, were the same neurons in every individual brain we looked at. This is because in every brain we have looked at, the soma of the neurons were not located in exactly the same location. Furthermore, the arborisation patterns are also different and unique, for each individual brain. Thus, there is natural variability in the position of the soma and in the arborisation patterns. Such variability presumably results from the combination of developmental and activity-dependent plasticity.
We would like to thank Reviewer 3 for the very positive evaluation of our work and the interesting and valuable feedback.
