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  1. Author Response:

    Reviewer #2:

    Major comments:

    1. Despite the strong ERG phenotype, some 50% of the TADR mutant flies still show behavioral responses in the phototaxis axis, strongly arguing for a pathway acting in parallel to TADR. Comparison to a known blind mutant, such as HDC could clarify this issue.

    As an essential amino acid, each cell can only use extracellular histidine. Although TADR is a specific histidine transporter, its expression pattern suggests that it is not the only histidine transporter. Supporting this, null tadr mutants are viable and have no growth phenotype. As photoreceptor cells may uptake histidine from other histidine transporters, they have a small histidine pool and synthesize some histamine via Hdc. Supporting this, histamine levels in tadr mutants are higher than in Hdc mutants, although histamine levels are greatly reduced in both mutants (Figure 5D). Consistent with reduced histamine levels, tadr^2 mutants exhibit weak phototactic behavior, indicating the presence of another histidine transporter in Drosophila photoreceptor cells. However, given that tadr^2 mutants displayed a complete loss of ON and OFF transients, greatly reduced histamine levels, and much less phototactic behavior, we speculate that TADR is the major histidine transporter, responsible for maintaining the histidine pool and keeping visual transmission at high frequencies. We added this part to the discussion section.

    1. Such a second pathway is also consistent with the level of histamine still present in TADR flies. Although curiously this issue is not specifically addressed by the authors, the level appears to be significantly higher than in HDC mutants (Figure 5D). This should be addressed.

    We thank the reviewer for this concern. Indeed, we found that levels of histamine in compound eyes of tadr^2 mutant flies were slightly higher than in eyes from Hdc^P217 mutants. As you mentioned, we cannot not rule out the possibility that another pathway acts in parallel to TADR. We briefly explain this in the results section (highlighted). Moreover, histamine levels in tadr^2 mutant flies were higher than in Hdc^P217 mutants, suggesting that a small fraction of histidine could be supplied by other transporter systems.

    1. Beyond referring to a "complete disruption of the tadr locus", the molecular details of the mutant should be better explained: Does the mutant result in an in-frame or an out-of-frame fusion of exon3 and 5? What parts of the protein are deleted?

    We thank the reviewer for this suggestion. We verified the genomic details of the tadr^2 mutant, and indeed the tadr^2 mutation generates a truncated tadr mRNA with a frame-shift (deletion of 244 nt) at the truncated site. We added the molecular details of the mutant to the manuscript: “PCR amplification and sequencing of the tadr locus from genomic DNA isolated from wild-type and tadr^2 flies revealed a truncated tadr locus in mutant animals, resulting in an out-of-frame fusion of exon 3 and 5 (Figure 2-figure supplement 2B and 2C).”. We also modified Figure 2-figure supplement 2C showing the cDNA sequence and corresponding amino acids around the truncated stie.

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  2. Evaluation Summary:

    Han et al. report the discovery of an amino acid transporter that is required locally at axon terminals of fly photoreceptors neurons for the uptake of histidine, the precursor of the neurotransmitter histamine. This function is required for transmitter synthesis locally and neurotransmission. The work exemplifies a specialized model for local monoamine transmitter synthesis at synapses in the nervous system, the generality of which for other monoamine systems remains to be tested.

    (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 #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Han et al. present a straight-forward discovery and description of an amino acid transporter that is locally required for neurotransmitter synthesis in Drosophila photoreceptor axon terminals. The question is somewhat specific: where does the precursor histidine come from to synthesize histamine? But as the authors argue, a specific amino acid transporter that locally generates the required precursor for local monoamine transmitter synthesis has not been shown for any other system.

    Strengths/novelty include: (1) this is a local phenomenon, providing precedence for other systems that uptake of a precursor and synthesis of a monoamine transmitter is a local business and may locally be exploited at synapses to regulate transmission. (2) a previous paper from 2008 in J Neurosci (Ni et al.) published a mutation in the TADR gene that leads to neurodegeneration. The authors now made a clean CRISPR null and it has no degenerative phenotype. They convincingly show a specific defect in histamine-dependent transmission. They further provide a strong biochemical characterization of the histidine uptake role for TADR and in vivo rescue with other histidine transporters.

    Weakness: there is no claim to generality, and little discussion of how similar the system may be to other, better known monoamine transmitter systems. As such the scope of the work may be limited. On the other hand, the entire synthesis and recycling pathway for histamine as a monoamine transmitter in an in vivo system may prove valuable to other systems in principle.

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  4. Reviewer #2 (Public Review):

    The manuscript by Han et al. describes the identification of TADR as an axonally localized transporter for Histidine, the direct precursor of the Histamine neurotransmitter used by photoreceptor cells in Drosophila. A role of TADR in vision was first identified in an RNAi screen and confirmed by a CRISPR mutant and rescue experiments. These findings, as well as the experiments supporting a role of TADR as histidine transporter and its localization to photoreceptor axons, convincingly support the main conclusions of the authors.

    The critical role of this axonal histidine transporter in vision are conceptually new and will be of interest to many neuroscientists. Somewhat unfortunately the authors are not able to resolve the conflicts with previously published results about a different TADR allele with structural phenotypes in photoreceptors (PMID: 19074021). However, given the convincingly documented and quantified experiments in the current manuscript, I believe it is warranted to just let this difference stand at this time.

    Major comments:

    1. Despite the strong ERG phenotype, some 50% of the TADR mutant flies still show behavioral responses in the phototaxis axis, strongly arguing for a pathway acting in parallel to TADR. Comparison to a known blind mutant, such as HDC could clarify this issue.

    2. Such a second pathway is also consistent with the level of histamine still present in TADR flies. Although curiously this issue is not specifically addressed by the authors, the level appears to be significantly higher than in HDC mutants (Figure 5D). This should be addressed.

    3. Beyond referring to a "complete disruption of the tadr locus", the molecular details of the mutant should be better explained: Does the mutant result in an in-frame or an out-of-frame fusion of exon3 and 5? What parts of the protein are deleted?

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  5. Reviewer #3 (Public Review):

    The authors present a well-executed series of experiments and a convincing set of data supporting the idea that tadr is a histidine transporter essential for the function of photoreceptor cells. The manuscript nicely complements other papers from this group and others on transporters required for histaminergic signaling at photoreceptor cells. Indeed, this is an important paper for understanding synaptic signaling in the fly visual system. This is an important paper for understanding neuronal activity in the fly visual system anwill be of great interest to people in this field.

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