Gjd2b-mediated gap junctions promote glutamatergic synapse formation and dendritic elaboration in Purkinje neurons

  1. Sahana Sitaraman
  2. Gnaneshwar Yadav
  3. Vandana Agarwal
  4. Shaista Jabeen
  5. Shivangi Verma
  6. Meha Jadhav
  7. Vatsala Thirumalai

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    Evaluation Summary:

    Sitaraman and colleagues address the fundamental question of whether gap junctions facilitate the formation of chemical synapses. To do so they test the function of the gap junction protein, Gjd2b, in early stages of synaptogenesis in larval Zebrafish cerebellar Purkinje neurons. They provide convincing evidence that Gjd2b is necessary for the development of glutamatergic synapses and dendritic arbor growth Purkinje neurons in vivo and that CaMKII plays a role in regulating arbor development. This study will be an important contribution to our understanding of molecular and cellular mechanisms underlying brain development.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

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Abstract

Gap junctions between neurons serve as electrical synapses, in addition to conducting metabolites and signaling molecules. During development, early-appearing gap junctions are thought to prefigure chemical synapses, which appear much later. We present evidence for this idea at a central, glutamatergic synapse and provide some mechanistic insights. Loss or reduction in the levels of the gap junction protein Gjd2b decreased the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in cerebellar Purkinje neurons (PNs) in larval zebrafish. Ultrastructural analysis in the molecular layer showed decreased synapse density. Further, mEPSCs had faster kinetics and larger amplitudes in mutant PNs, consistent with their stunted dendritic arbors. Time-lapse microscopy in wild-type and mutant PNs reveals that Gjd2b puncta promote the elongation of branches and that CaMKII may be a critical mediator of this process. These results demonstrate that Gjd2b-mediated gap junctions regulate glutamatergic synapse formation and dendritic elaboration in PNs.

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  1. Version published to 10.7554/elife.68124 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    [...] The study provides convincing evidence that gap junctions are important for glutamatergic synaptogenesis and dendritic arbor development, but how this happens is less clear. As I was reading the paper, I thought the data support the synaptotrophic hypothesis, which states that synapse formation facilitates dendritic arbor development, and that they added an essential early step that gap junctions are required for development of glutamatergic synapses. This would suggest that the sequence of local events is: gap junction formation -> synaptogenesis->branch extension, repeat. The authors suggest an alternate sequence: gap junction formation -> branch extension-> synaptogenesis, repeat. I don't think the current data allow us to choose one or the other of these options and the implied causality, but the study clearly demonstrates a role for gap junctions in the process, which was the goal of the study. Given this ambiguity, the discussion should be modified to accommodate both interpretations, or to explain why one interpretation is favored over the other.

    We agree that the sequence can be either gap junction formation→ synaptogenesis→ branch extension, repeat or gap junction formation → branch extension→ synaptogenesis, repeat. We do not have direct evidence to favour one hypothesis versus the other. However, if synaptogenesis precedes branch extension, and the stunted arbor in gjd2b mutants is a result of reduced AMPAR synapses, one might expect to see increased branch retractions as well. As demonstrated by (Haas et al., 2006), loss of AMPAR synapses leads to more dynamic dendritic branches, which are added and retracted at faster rates compared to wild type. This is not what we see. Infact, in gjd2b mutants, branch elongations were reduced and branch retractions were not affected at all. In addition, when we expressed Gjd2b in wild type PNs and imaged in 5-minute windows, branches containing Gjd2b punctum elongated more; the retractions were not affected (Figure 7C). These observations lead us to favor the Gjd2b→ branch elongation→ synaptogenesis view, but we acknowledge that without a direct assay, the two scenarios cannot be disambiguated. We have added the above to the discussion section (line 440).

    The implied mechanistic link between camk2 transcript expression and pharmacological inhibition of CaMKII enzymatic activity on dendritic arbor growth is not convincing to me. It is clear that the transcript observation is unexpected and suggests that somehow interfering with gjd2b affects camk2 transcript expression. Perhaps other synaptic proteins are affected as well. This point would be worth commenting on. But transcript level does not necessarily correlate with protein level or function, particularly for a calcium activated kinase, which is itself tightly regulated in terms of protein expression and function by multiple mechanisms. The main issue concerns causality. The authors state that the gjd2b regulates glutamatergic synaptogenesis by reducing CaMKII levels. The authors do not provide evidence for this statement of cause and effect.

    In the revised manuscript, we have removed claims of causality between observed camk2 expression levels and glutamatergic synaptogenesis. (lines 42, 327, 349, 475). We have inserted the following sentence into the discussion (Line 462):

    “Further experiments are required to verify whether this increase in expression level of CaMKII isoforms translates to increased enzymatic activity.”

    Reviewer #2 (Public Review):

    [...] Strengths:

    1. The sheer amount of work that has gone into this paper is impressive. Each technique is tedious, time consuming and labor intensive so it is quite impressive that the authors have a substantial number of Ns for their experiments. All the experiments and analysis have been performed carefully and the data is of high quality.
    1. The authors have done a thorough job of generating and characterizing Gjd2b mutant fish which will be useful for the entire zebrafish neuroscience community.*

    We thank the reviewer for these very nice comments about our work.

    *Weaknesses:

    1. Overall, while the experiments and data are clearly presented, several experimental results have significant technical limitations, which in turn open them up to alternative explanations which cannot be easily ruled out based on current data.*

    We agree that there are technical limitations and have discussed these in the “Supplementary File 4” document.

    1. Knocking out gap-junctions will affect spontaneous activity in early development which is propagated via gap junctions. Given that spontaneous activity is likely dampened in Gjd2b knockout fish, a substantial concern is that effects that the authors attribute to the absence of gap junction mediated activity could equally likely be a consequence of homeostatic changes in synaptic input. One possible way to alleviate this issue is to perform transplant experiments from mutant fish to wild type fish which ensures that the rest of the circuit is unaffected.

    The rescue experiment we performed is akin to transplant experiments, in the sense that we expressed Gjd2b in single Purkinje neurons in the mutant background. This way, the rest of the circuit is unaffected and remains Gjd2b null while only the neuron expressing Gjd2b is rescued.

    1. Rescuing Gjd2b is an interesting experiment, but its unclear how functional electrical synapses form by expressing the functional protein in only one neuron.

    We speculate that this can be due to heterotypic gap junctions formed between Gjd2b and other connexins expressed on the coupled neuron. Such heterotypic gap junctions have been documented in the Mauthner and CoLo neurons in larval zebrafish (Miller et al., 2017). (Line 300).

    1. The authors suggest that dendritic growth is reduced because of the absence of gap junctions, but its unclear whether the reduced dendritic growth is simply a consequence of fewer excitatory synapses, and thus a downstream consequence of the absence of gap junctions rather than specific information being transmitted through gap junctions.

    This point was raised by Reviewer #1 as well. Please see response above.

  3. eLife

    Evaluation Summary:

    Sitaraman and colleagues address the fundamental question of whether gap junctions facilitate the formation of chemical synapses. To do so they test the function of the gap junction protein, Gjd2b, in early stages of synaptogenesis in larval Zebrafish cerebellar Purkinje neurons. They provide convincing evidence that Gjd2b is necessary for the development of glutamatergic synapses and dendritic arbor growth Purkinje neurons in vivo and that CaMKII plays a role in regulating arbor development. This study will be an important contribution to our understanding of molecular and cellular mechanisms underlying brain development.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This study tests the function of the gap junction protein, Gjd2b, in early stages of synaptogenesis in larval Zebrafish cerebellar Purkinje neurons. It uses morpholino-mediated knockdown of Gjd2b and talen-mediated knockout of Gjd2b, both of which resulted in decreased mEPSP frequency and mEPSPs with faster decay kinetics compared to controls. The decreased mEPSP frequency suggests there are fewer synapses. In EM analyses, comparing synapse density and maturation in the cerebellar molecular layer in WT and Gjd2b KO animals, the authors find the KO animals have decreased synaptic density, but that the maturation index is unaffected, suggesting that Gjd2b-/- animals form fewer synapses, but once synaptogenesis is initiated, maturation occurs normally.

    In vivo time-lapse imaging of PC dendritic arbors was used to analyze effects of Gjd2b on arbor structure. This beautiful series of experiments demonstrates that Gjd2b-/- decreases dendritic arbor growth by modifying branch extensions, but not branch retractions. Further analysis shows that dendritic branches with Gjd2b puncta extend more than branches without puncta. This level of cellular mechanistic resolution provides considerable insight into the processes by which electrical junctions influence synapse formation and dendritic arbor growth. The authors attempt to identify cells that are coupled to PCs using dye labeling, but relatively few cells are dye labeled. Most dye-labeled cells are not PCs and can't be identified. Nevertheless, PC expression of functional Gjd2b is sufficient to rescue dendritic arbor growth defects seen in gjd2b-/- animals, suggesting that heterotypic gap junctions between PC and non-PCs regulate dendritic arbor growth, and furthermore that Gjd2b has cell autonomous effects on arbor development. Finally, it appears that gjd2b-/- animals have increased levels of camk2 transcripts, and that inhibiting CaMKII activity rescues the effect of gjd2b knockdown on dendritic arbor development.

    The study provides convincing evidence that gap junctions are important for glutamatergic synaptogenesis and dendritic arbor development, but how this happens is less clear. As I was reading the paper, I thought the data support the synaptotrophic hypothesis, which states that synapse formation facilitates dendritic arbor development, and that they added an essential early step that gap junctions are required for development of glutamatergic synapses. This would suggest that the sequence of local events is: gap junction formation -> synaptogenesis->branch extension, repeat. The authors suggest an alternate sequence: gap junction formation -> branch extension-> synaptogenesis, repeat. I don't think the current data allow us to choose one or the other of these options and the implied causality, but the study clearly demonstrates a role for gap junctions in the process, which was the goal of the study. Given this ambiguity, the discussion should be modified to accommodate both interpretations, or to explain why one interpretation is favored over the other.

    The implied mechanistic link between camk2 transcript expression and pharmacological inhibition of CaMKII enzymatic activity on dendritic arbor growth is not convincing to me. It is clear that the transcript observation is unexpected and suggests that somehow interfering with gjd2b affects camk2 transcript expression. Perhaps other synaptic proteins are affected as well. This point would be worth commenting on. But transcript level does not necessarily correlate with protein level or function, particularly for a calcium activated kinase, which is itself tightly regulated in terms of protein expression and function by multiple mechanisms. The main issue concerns causality. The authors state that the gjd2b regulates glutamatergic synaptogenesis by reducing CaMKII levels. The authors do not provide evidence for this statement of cause and effect.

  5. eLife

    Reviewer #2 (Public Review):

    Summary:

    The paper by Seetharaman, Yadav et al. establishes zebrafish as a new model system to investigate the role of gap junctions as mediators of glutamatergic synapse formation and dendrite elaboration in Purkinje neurons. After performing an initial set of experiments with morpholinos to knockdown the gap junction protein Gjd2b, the authors invest a substantial amount of effort to establish a mutant line where the gap junction protein Gjd2b is knocked out. Using electrophysiology and electron microscopy in mutant fish, the authors find reduced numbers of AMPAR synapses though only the electrophysiology data are unequivocal in attributing the reduced synapse number to Purkinje neurons. The authors then investigate dendritic development of Purkinje neurons in the mutant fish and find that mutant Purkinje neurons have significantly shorter dendrites likely because of the reduced elongation rate of dendrites. The authors then find that the dendritic structure of mutant neurons can be rescued by expressing functional Gjd2b in individual neurons in knockout fish. Finally, the authors find that CaMKII levels are elevated in mutant neurons and blocking CaMKII activity restores dendritic arbors.

    Strengths:

    1. The sheer amount of work that has gone into this paper is impressive. Each technique is tedious, time consuming and labor intensive so it is quite impressive that the authors have a substantial number of Ns for their experiments. All the experiments and analysis have been performed carefully and the data is of high quality.

    2. The authors have done a thorough job of generating and characterizing Gjd2b mutant fish which will be useful for the entire zebrafish neuroscience community.

    Weaknesses:

    1. Overall, while the experiments and data are clearly presented, several experimental results have significant technical limitations, which in turn open them up to alternative explanations which cannot be easily ruled out based on current data.

    2. Knocking out gap-junctions will affect spontaneous activity in early development which is propagated via gap junctions. Given that spontaneous activity is likely dampened in Gjd2b knockout fish, a substantial concern is that effects that the authors attribute to the absence of gap junction mediated activity could equally likely be a consequence of homeostatic changes in synaptic input. One possible way to alleviate this issue is to perform transplant experiments from mutant fish to wild type fish which ensures that the rest of the circuit is unaffected.

    3. Rescuing Gjd2b is an interesting experiment, but its unclear how functional electrical synapses form by expressing the functional protein in only one neuron.

    4. The authors suggest that dendritic growth is reduced because of the absence of gap junctions, but its unclear whether the reduced dendritic growth is simply a consequence of fewer excitatory synapses, and thus a downstream consequence of the absence of gap junctions rather than specific information being transmitted through gap junctions.

  6. eLife

    Reviewer #3 (Public Review):

    The authors tried to address the question of whether the gap-junction protein Gjd2b was involved in the development of cerebellar Purkinje cells. Using a number of complementary approaches, including electrophysiology, EM, and morphological analysis, they show that excitatory synapses are affected when it is reduced in two different ways, and that dendritic structure is affected, thus supporting their hypothesis.

    The strength of this manuscript is its thoroughness, and that key findings are shown with more than one method, for example, by the use of both morpholinos and knock-out fish. Combining electrophysiology, EM analysis, and dendritic structure analysis is excellent, and gives weight to the conclusions of the paper.

    The results are convincing, and support a role for Gjd2b in Purkinje cell maturation. The mechanism by which this acts is also addressed, although not fully elucidated. I think this makes the paper interesting and important to a number of different groups, including developmental neurobiologists, those interested in gap junction function and signaling, and cerebellar researchers.

  7. Version published to 10.1101/2020.01.31.928242v1 on bioRxiv