Light tunes long-term threat avoidance behavior in male mice

Domain: neuroscience
Published: 2026-02-13
Editorial outcome: major_revision

Abstract
Animals must constantly scan their environment for imminent threats to their safety. However, they must also integrate their past experiences across long timescales to assess the potential recurrence of new threats. Though visual inputs are critical for the detection of environmental danger, whether and how visual information shapes an animal’s assessment of whether a new threat is likely to reappear in a given context is unknown. In this work, we developed a behavioral assessment of long-term threat avoidance behavior where animals will avoid a familiar location where they previously experienced a single threat exposure. This avoidance behavior is highly sensitive and lasts for multiple days. Intriguingly, we find that the melanopsin-expressing, intrinsically photosensitive retinal ganglion cells tune this behavior via a perihabenula-nucleus accumbens circuit distinct from canonical visual threat detection circuits in male mice. These findings define a long-term threat avoidance behavior that is shaped by a defined retinal cell type based on prior experience.

Introduction
Detecting existing threats is key to animal survival, and organisms have evolved many mechanisms for threat detection and escape. However, animals entering each new or familiar environment must also constantly draw on past experience to calibrate their behavior based on the expectation that a new threat will appear. The ability to properly tune risk-taking versus avoidance behavior based on past experience has a clear evolutionary advantage for optimizing animal survival, foraging, and reproduction. The circuits underlying threat avoidance calculations must therefore be plastic, well-calibrated, and operate across varied contexts and timescales. Despite their importance for survival and decision making, these threat avoidance circuits are not well understood.

Visual information is a key sensory input for both threat detection and for providing contextual information about new and familiar environments to shape behavior. It is therefore likely that visual circuits tune threat avoidance behavior. In this work, to better define these key circuits, we report development of a behavioral paradigm to characterize and quantify threat avoidance based on past experience over long timescales, a behavior we have termed long-term threat avoidance (LTTA). Though escape circuits are often hardwired, we find that context-based decisions in the absence of the threat itself are tunable by visual input. Specifically, we find that avoidance is facilitated by input from the melanopsin-expressing, intrinsically photosensitive retinal ganglion cells via a retino-thalamic-limbic circuit. Thus, the LTTA paradigm has allowed us to define a previously unknown visual circuit key for shaping avoidance behavior based on past experience.

Results
A paradigm to measure long-term threat avoidance in mice
We sought to develop a paradigm to quantitatively assess threat avoidance over long timescales. To do this, we chose to test the impact of a single visual looming threat exposure on an animal’s future avoidance behavior in a previously neutral context (Fig. 1A). The visual looming stimulus has many features that make it ideal to probe the circuits and mechanisms underlying threat avoidance over long timescales because 1) a single exposure to a looming stimulus drives an innate fear response through well-characterized visual circuits, 2) it is highly ethologically relevant for the mouse, and 3) it does not rely on physically aversive stimuli, which make it difficult to distinguish visual from nociceptive contributions1–4. In this LTTA paradigm, dark-adapted male mice are placed in a square arena with a monitor overhead projecting a neutral gray background (see “Methods”). We track each animal’s exploratory behavior during this pre-exposure phase before any stimulus is presented. During Pre-exposure, we find that control animals readily explore the edges (80% of total time) and center (20% of total time) of the arena (Supplementary Fig. 1). Four minutes later, animals enter the Exposure phase, where they are exposed to a single looming stimulus in the center of the arena, which we refer to as the “threat zone” (TZ)5,6. Animals then remain in the arena for 1-min following Exposure, after which they are returned to their home cage. Two days later, we assess whether the prior exposure to that single looming stimulus drives avoidance of the TZ by placing animals back in the same arena for a 4-minute Test phase where they are allowed to freely explore the arena and no threat stimulus is presented (Fig. 1A).

We first analyzed looming detection of expanding disks varying in contrast from low (6.6% Michelson contrast) to high (36.5%, maximum achievable contrast for a black disk on gray, see “Methods”) by quantifying freezing behavior during the Exposure phase (Fig. 1B, C and Supplementary Fig. 1). Mice showed detectable freezing behavior starting at 13.1% Exposure that increased with higher contrast. Animals showed no detectable freezing behavior at 6.6% Exposure (Fig. 1B, C and Supplementary Fig. 1). These results defined a subthreshold contrast (6.6%) for freezing behavior and support previous findings that increased looming stimulus contrast enhances saliency to drive increased freezing behavior5–7.

We then tested long-term avoidance behavior by quantifying and comparing time in the TZ during the Test versus Pre-exposure phases of LTTA (Fig. 1A and see methods). Animals previously exposed to a looming stimulus of any contrast, even 6.6%, spent significantly less time in the TZ compared to 0% Exposure (i.e., no loom Exposure) controls (Fig. 1D, Supplementary Fig. 1), indicating that TZ avoidance is not simply due to habituation. We were surprised to observe robust TZ avoidance during the Test phase for this contrast, because freezing behavior was not detectable during the 6.6% looming Exposure itself (Fig. 1B–D, Supplementary Fig. 1). These findings indicate that the circuits underlying LTTA are more sensitive and likely distinct from the well-characterized, canonical, looming detection circuits5,8–11. Importantly, LTTA behavior requires visual input because animals placed in the dark during the Test phase following 6.6% Exposure fail to avoid the TZ, in stark contrast to animals in 10 lux and standard 40 lux illumination (Fig. 1E-F, Supplementary Fig. 1). Moreover, LTTA is not due to increased general anxiety because mice showed no differences in the Elevated Zero Maze 2 days post 0% (no stimulus control) or 6.6% Exposure (Fig. 1G-H). Thus, the circuits underlying LTTA are highly sensitive, long-lasting, and likely distinct from those driving anxiety or looming-dependent freezing responses.

ipRGCs tune long-term threat avoidance
We next sought to identify the retinal cell types involved in LTTA. The melanopsin-(Opn4)-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are a good candidate for tuning LTTA because melanopsin signaling within ipRGCs encodes environmental luminance over a large dynamic range and across long timescales12–14. To determine whether melanopsin signaling is required for LTTA, we compared TZ avoidance of melanopsin null (Opn4−/−) animals to littermate control animals following 6.6% or 0% Exposure. 6.6% looming contrast was chosen to isolate impacts on avoidance behaviors versus looming detection itself. Indeed, Opn4−/− and control littermates show no freezing behavior during Exposure at either contrast (Supplementary Fig. 2). However, during the Test phase, Opn4−/− animals spent significantly more time in TZ compared to controls following 6.6% Exposure, indicating that melanopsin signaling in ipRGCs is required for LTTA (Fig. 2A-C, Supplementary Fig. 2). Importantly, Opn4−/− mice begin to exhibit freezing behavior at the same contrast, 13.1%, as control animals, indicating that impacts on LTTA are not due to deficits in detecting the looming stimulus itself. Moreover, Opn4−/− mice spend similar time in TZ to controls in both the Pre-exposure and Test phases following 0% Exposure (Supplementary Fig. 2), indicating that differences in LTTA are not due to differences in baseline exploratory behavior or habituation (Fig. 2B, C, Supplementary Fig. 2).

Additionally, these differences are unlikely due to altered anxiety because Opn4−/− mice showed no differences in anxiety-like behaviors compared to control littermates (Supplementary Fig. 3).

Previous studies indicate that ipRGCs can influence brain development, which could lead to secondary impacts on LTTA15–17. If melanopsin itself is required for LTTA, then removal of melanopsin in adult animals should lead to LTTA deficits similar to those of Opn4−/− animals. To test this, we removed melanopsin in adult ipRGCs using a conditional knockout approach. Specifically, we used Opn4fl/fl littermate control (P60 intravitreal Vehicle or AAV-mCherry injection) or conditional knockout (Opn4cKO; P60 intravitreal AAV-Cre-TdTomato injection) (Fig. 2D, E). Mice were then exposed to the LTTA paradigm and the efficiency of Opn4 ablation was tested post-mortem. Opn4cKO mice injected with AAV-Cre-TdTomato, showed decreased LTTA compared to control littermates, similar to Opn4−/− animals (Fig. 2G, H, Supplementary Fig. 4). Post-mortem analysis of melanopsin expression showed a ~95% reduction in ipRGCs in Opn4cKO animals, demonstrating a high efficiency of ablation (Fig. 2F, Supplementary Fig. 4), These results indicate that it is melanopsin signaling itself that tunes LTTA, and not secondary developmental impacts on brain development in Opn4−/− animals.

If ipRGCs tune LTTA then disrupting ipRGC glutamate signaling should similarly alter LTTA. Indeed, conditional knockout of the vesicular glutamate transporter Vglut2 in ipRGCs (Vglut2cKO: Opn4Cre/+; Vglut2fl/fl)18 caused similar deficits in TZ avoidance to Opn4−/− animals despite similar baseline exploratory behavior to control littermates (Fig. 2I-K, Supplementary Fig. 4). Likewise, ablation of non-SCN-projecting Brn3b-positive ipRGCs using the Brn3bDTA line (Opn4Cre/+; Brn3bzDTA/+)19, resulted in similar TZ avoidance deficits (Fig. 2L-N, Supplementary Fig. 4). Combined, these results indicate that ipRGCs release glutamate at non-SCN targets to tune LTTA behavior.

The perihabenular nucleus is required for tuning LTTA
We next sought to identify the ipRGC-recipient target(s) required for LTTA using an unbiased cFos screen of brain regions differentially activated during the Test phase in control versus Opn4−/− animals (Fig. 3A). If a given ipRGC-recipient region is required for LTTA, then we would expect that target to show patterns in cFos activation that parallel differences in avoidance phenotypes (i.e., cFos levels in all groups are similar with the exception of 6.6% Exposed controls). Remarkably, the only ipRGC-recipient target showing the predicted differences in cFos labeling was the perihabenular nucleus (PHb) (Fig. 3B, C), suggesting an ipRGC-PHb circuit may underly LTTA. Notably, regions implicated in the canonical looming circuit such as medial superior colliculus (mSC) and the ventral lateral geniculate nucleus (vLGN)5,11,20 and ipRGC-recipient regions directly upstream of looming detection circuits, such as medial amygdala and periaqueductal gray, showed no differences in cFos expression following LTTA (Supplementary Fig. 5), again pointing to the possibility that the LTTA circuit architecture is distinct from the looming detection circuit.

ipRGCs primarily innervate GABAergic neurons in the caudal PHb (PHbGABA), which in turn make local connections with excitatory-relay PHb neurons (PHbGlu) and anterior PHbGABA neurons (Fig. 3D)21–23. If PHbGABA neurons are required for LTTA, then silencing that specific population should disrupt LTTA. To test this, we injected Cre-dependent Gi-DREADDs in the PHb of Gad2Cre mice to silence PHbGABA (Fig. 3E-F, see Supplementary Table 1) neurons. We then intraperitoneally (i.p.) injected either vehicle or the DREADD agonist clozapine-N-oxide (CNO) 30 min prior to both the Pre-exposure and Test phases of the LTTA paradigm (Fig. 3G). Importantly, i.p. CNO injection did not impact LTTA in naïve WT mice (Supplementary Fig. 6) and did not alter exploratory behavior during Pre-exposure phase compared to vehicle-injected mice, indicating that i.p. CNO itself does not alter baseline exploratory behavior (Fig. 3H). Silencing of PHbGABA (Fig. 3E–J), but not PHbGlu neurons (Supplementary Fig. 6), disrupted LTTA, causing animals to spend significantly more time in the TZ, confirming that the GABAergic neurons of the ipRGC-recipient PHb are required for LTTA.

If PHb-ipRGCs are required for LTTA, then activation of PHb-ipRGCs in Opn4−/− animals should rescue the observed deficits in LTTA. Light activates melanopsin in ipRGCs, triggering a Gq-mediated phototransduction cascade that ultimately depolarizes the cells and drives glutamate release onto downstream targets24. We have previously shown that Gq activation with Gq-DREADDs in ipRGCs ex vivo restores melanopsin-like photoresponses in Opn4−/− ipRGCs25. We therefore adapted this approach to our in vivo LTTA paradigm by bilaterally injecting retrograde (rg) AAV into the PHb of melanopsin null (Opn4Cre/Cre) animals to drive Cre-dependent expression of Gq-DREADDs (rgAAV-hSyn-DIO-hM3Gq-mCherry) selectively in PHb-ipRGCs (Fig. 4A, see Supplementary Table 1). We then intraperitoneally injected either vehicle or CNO 30 min prior to both the Pre-Exposure and Test phases (Fig. 4A). CNO-injected Opn4−/− animals avoided the TZ, spending significantly less time in the TZ than vehicle-injected Opn4−/− littermates. This indicates that Gq-DREADD activation of PHb-ipRGCs restores LTTA in Opn4−/− mice (Fig. 4B-D). CNO administration did not alter baseline exploratory behavior during the Pre-exposure phase compared to vehicle-injected mice (Supplementary Fig. 7), again suggesting that CNO does not affect exploratory behavior. These effects are not through collateral outputs of PHb-ipRGCs to the vLGN, a collateral target of PHb-ipRGCs (Supplementary Fig. 8, see Supplementary Table 1) previously implicated in looming-response behavior5,11, because chemogenetic silencing of the vLGN itself did not affect LTTA in WT mice (Supplementary Fig. 9, Supplementary Table 1 and see methods). Collectively, these findings indicate that PHb-ipRGCs are required for LTTA.

ipRGCs tune PHb neuron activity to facilitate recall of the threat-context association
To determine whether PHb-ipRGCs are important for LTTA during the formation of association between threat and context (i.e., at Pre-exposure/Exposure phases) or whether they facilitate the recall of this association upon re-exposure (i.e., at Test phase), we again bilaterally injected retrograde (rg) AAV-Gq-DREADDs into the PHb of melanopsin null (Opn4Cre/Cre) animals, but this time administered a single dose of i.p. CNO to Opn4−/− mice prior to either Pre-exposure (CNO/Veh group) or Test (Veh/CNO group) phases (Fig. 4A-D, see Supplementary Table 1). A third group received two vehicle injections (Veh/Veh group) as control. Opn4−/− animals that received CNO prior to the Test phase (Veh/CNO), but not those that received CNO prior to the Pre-exposure phase (CNO/Veh), showed rescued LTTA and decreased time in the TZ (Fig. 4D). These results were identical to those where animals received CNO in both the Pre-exposure and Test phases (Fig. 4B-D), indicating that PHb-ipRGC activity is required for LTTA only during the Test phase to facilitate the recall of the association between the threat and context.

To further test whether the PHb-ipRGC circuit is required for the recall of the threat–context association, we selectively, locally inhibited PHb-projecting retinal axon terminals only during the Test phase. We intravitreally injected inhibitory Gi-DREADDs (AAV2-Syn-hM4Di-mCherry) into WT mice and implanted bilateral cannulas targeting the PHb for local infusion of Vehicle or CNO (Fig. 4E-G). After 4 weeks, vehicle was infused prior to the Exposure phase and either vehicle or CNO prior to the Test phase of the LTTA paradigm. CNO-infused mice spent significantly more time in the TZ compared to Vehicle-infused mice (Fig. 2H-I and Supplementary Fig. 7). These results demonstrate that inhibition of PHb-projecting ipRGC terminals during recall disrupts LTTA, confirming that PHb-ipRGC activity facilitates retrieval of the threat–context association. Moreover, this terminal-specific manipulation rules out confounding effects mediated by collateral ipRGC projections to the LGN (Supplementary Fig. 8).

Our data indicate that silencing PHbGlu neurons does not impact LTTA, in agreement with ipRGC synaptic input to these cells21. However, if PHbGlu neurons are downstream of PHbGABA neurons (ipRGC-PHbGABA-PHbGlu), it is possible that exciting, rather than inhibiting them, would drive deficits in LTTA. To test this, we next assessed the contribution of excitatory-relay PHbGlu neurons to the LTTA behavior during the Test phase. To do this, we bilaterally injected Cre-dependent Gq DREADDs in the PHb of Vglut2Cre mice to specifically excite PHbGlu neurons (Fig. 3K–M and Supplementary Fig. 6). We then intraperitoneally injected vehicle prior to Pre-exposure either vehicle or CNO prior to the Test phase of the LTTA paradigm (Fig. 3L and Supplementary Fig. 6). Chemogenetic excitation of PHbGlu neurons disrupted LTTA (Fig. 3N-P), indicating that PHbGlu neurons are likely inhibited by PHbGABA neurons during LTTA.

To understand PHb signaling dynamics and the impact of melanopsin signaling during LTTA, we next used fiber photometry (FP) to measure bulk Ca2+ activity in the PHb in vivo in Opn4−/− and control littermates at different phases of the LTTA paradigm. To do this, we injected an AAV containing GCamp6s (AAV1-CAG-GCaMP6s-WPRE-SV40) and implanted a FP canula unilaterally in the PHb of control and Opn4−/− littermates (Fig. 5A-B, see Supplementary Table 1 and methods). We then compared overall activity in the Pre-exposure versus Test phases of the LTTA paradigm using a 6.6% contrast looming stimulus. In agreement with cFos expression analysis (Fig. 3C), we found decreased PHb activity in Opn4−/− mice compared to control littermates during the Test, but not during the Pre-exposure phase of the LTTA paradigm (Fig. 5C-D). This indicates that ipRGC signaling during the Test phase serves to increase baseline PHb activity.

We next examined how PHb neuron activity varied as animals exhibited certain behaviors during the LTTA paradigm. To identify key parameters for further analysis, we performed a principal component analysis (PCA) of bulk Ca2+ activity in the PHb and behavioral parameters data (Supplementary Fig. 10). We observed that the calcium signal z-score in the PHb was highly correlated with behavioral parameters when mice remained in the edges (i.e., increased avoidance) compared to those behavioral parameters when mice were in the TZ (Supplementary Fig. 10). To better understand this, we analyzed the calcium signal z-score in the PHb from control or Opn4−/− animals at different phases of the LTTA paradigm. We did not observe changes in calcium signal z-score in the PHb during the Exposure phase (Fig. 5E-H). Then, we analyzed the activity in the PHb while mice occasionally transitioned from the edge to the TZ during the Pre-exposure and the Test phases (Fig. 5I). In control mice, we found a significant decrease in the calcium signal z-score in the PHb when control mice transitioned from the edge to the TZ, but no significant change during transitions of Opn4−/− mice, indicating that the PHb may be particularly important for signaling entry into the TZ (Fig. 5M-O). These changes were not observed during the Pre-exposure phase (Fig. 5I-L), suggesting again that ipRGC to PHb signaling is required to facilitate the recall of the threat/context association. We did not observe changes when mice were transitioning from the TZ to the edges in the Test phase (Supplementary Fig. 10), indicating that the PHb nucleus is key for encoding environmental information while mice are entering the TZ and that this process requires melanopsin signaling within ipRGCs.

A PHb to nucleus accumbens (NAc) circuit is required for LTTA
The PHb nucleus is part of a thalamo-frontocortico-striatal loop projecting to the ventromedial prefrontal cortex (vmPFC) and the nucleus accumbens (NAc) (Fig. 6A-C)21–23. We therefore next tested whether PHb-NAc or PHb-vmPFC neurons are required for LTTA. If PHb-NAc or PHb-vmPFC neurons are required for LTTA, then activating them with Gq-DREADDs in Opn4−/− animals should rescue the LTTA deficits in these mice. To test this, we expressed excitatory Gq-DREADDs in vmPFC-, or NAc-projecting PHb neurons into the PHb of Opn4−/− mice (Fig. 6D, Supplementary Fig. 11, Supplementary Table 1 and see methods) Chemogenetic excitation of PHb-NAc circuit rescued LTTA deficits evidenced by a decreased time in the TZ (i.e., increased avoidance) in i.p. CNO-injected Opn4−/− mice compared to vehicle-injected Opn4−/− littermates (Fig. 6D-I). However, we did not observe differences in LTTA when PHb-vmPFC was chemogenetically excited in Opn4−/− mice (Supplementary Fig. 11). These results suggest that the PHb-NAc circuit, and not PHb-vmPFC, is required for LTTA.

To further test whether the PHb-NAc circuit is required for the recall of the threat–context association, we selectively, locally inhibited NAc-projecting PHb axon terminals only during the Test phase. If PHb-NAc circuit is required for LTTA, then silencing PHb neuron terminals in the NAc in WT mice should cause LTTA deficits in these mice. To test this, we bilaterally injected inhibitory Gi-DREADDs (AAV2-Syn-hM4Di-mCherry) into the PHb of WT mice and implanted bilateral cannulas targeting the NAc for local infusion of Vehicle or CNO (Fig. 6J, K and Supplementary Fig. 11). After 4 weeks, vehicle was infused prior to the Exposure phase and either vehicle or CNO prior to the Test phase of the LTTA paradigm. CNO-infused mice spent significantly more time in the TZ compared to Vehicle-infused mice (Fig. 6L, N and Supplementary Fig. 11). These results demonstrate that inhibition of NAc-projecting PHb terminals during recall disrupts LTTA, confirming that ipRGC-PHb-NAc neurons are required. Moreover, this terminal-specific manipulation rules out any other potential outcome effects mediated by the PHb nucleus22,23.

Methods
Animals
All procedures were approved by the Animal Care and Use Committee at Northwestern University (Protocol number: IS00003845). Animals were housed in vivarium under 12:12 light/dark cycle conditions with ad libitum access to food and water. Temperature ranged from 21 to 23 °C and humidity ranged from 30% to 70%. P60-P120 male mice were used for this study. For circuit tracing we used WT and Opn4Cre (RRID:IMSR_JAX:035925) mice. For behavior, chemogenetic and histological studies, we used WT, Opn4Cre, Opn4fl (C57BL/6JCya-Opn4em1flox/Cya, ID: S-CKO-21264), Opn4Cre/+; Vglut2fl/fl (MGI:4879031), Brn3bDTA (RRID:IMSR_JAX:038952), Gad2Cre (IMSR_JAX:028867) and Vglut2Cre (RRID:IMSR_JAX:016963) male mice. For fiber photometry experiments we used Opn4Cre male mice. Opn4fl mouse was generated by mating “knockout-first” allele Opn4tm1a(KOMP)Wtsi (UCDAVIS KOMP Repository) to FLPO line (C57BL/6N-Tg(CAG-Flpo)1Afst/Mmucd, MMRRC.

Long-term threat anticipation paradigm
The experimental arena consisted of a 40 × 40 × 40 cm3 opaque white foamboard Plexiglas box with a screen embedded in the ceiling. Throughout the experimental sessions, the monitor displayed a dim gray background (40 lux), on which a dark expanding disk was presented as the looming stimulus during the Exposure phase (Fig. 1A). We used Adobe Flash (RRID:SCR_017258) to design the looming stimuli and VLC Media Player (RRID:SCR_003070) to trigger the stimuli. Each stimulus consisted of ten expansions of a dark disk projected onto the gray background of the monitor. A single disc expansion consisted of two phases: 1) the disk expanded over a duration of 0.5 s, 2) the disk then remained static at its maximum expansion for 0.5 s before disappearing. An interval of 0.5-s was left before the next disk expansion; thus, each stimulus presentation lasted 10 s. Each mouse was exposed once, to one unique condition: 0, 6.6, 13.1, 27.5, or 36.5% Michelson contrast. Michelson contrast was calculated based in arena illumination levels measured using a XL-500 BLE Spectroradiometer (nanoLambda Korea Co., Ltd).

Mice were dark-adapted for 20–30 min and then placed in the LTTA arena for 4 min (Pre-exposure, Fig. 1A). A single contrast of looming stimulus of contrasts ranging from 0 to 36.5% Michelson contrast and then recorded in the arena for 1 min following the Exposure phase. After a retention period of 2 days in the home cage, 30-min dark-adapted animals were then placed in the same arena for 4 min with no stimulus triggered (Test phase, Fig. 1A) and behavior was recorded using two cameras (C920 x HD, Logitech and ELP Wide Angle Infrared Webcam).

For chemogenetic experiments, 4 weeks after viral infections (see below), mice were dark-adapted and 30 min before the Pre-exposure and Test phases of the LTTA paradigm, animals were i.p. injected with saline (Vehicle, 0.9% NaCl) or Clonazepine N-Oxide (1 mg/kg) (CNO, Tocris Bioscience Cat# 6329). After i.p. injections behavioral assay was performed as described above. For local axon-terminal manipulations, 4 weeks after viral infections and cannula implantations (see below), mice were dark adapted and 20 min before the Pre-exposure and Test phases of the LTTA paradigm, animals were infused with saline (Vehicle, 0.9% NaCl) or CNO (0.3 mg/kg). For Vehicle or CNO cannula infusions, bulk catheter (Cat#: BC-22, RWD Life Science Inc.) were attached to a Hamilton Syringe (Cat# 88500) to infuse a total volume of 0.3 μL at a rate of 0.1 μL/min. After infusions, behavioral assay was performed as described above. Mice with mistargeted viral expression or cannula implantation, confirmed by post hoc histology, were excluded from all analyses.

Behavior analysis
For behavior analysis, we used eZtrack (RRID:SCR_021496) automated tracking software27 to analyze IR videos and quantify freezing behavior during Exposure phase of the LTTA paradigm at different looming stimuli contrasts. Freezing behavior was computed when mice were tracked immobile for at least 1 second. To quantify avoidance behavior, we used eZtrack software to analyze IR and regular light videos and quantify the time spent in the center of the arena during the Test phase (TZtest) of the LTTA paradigm. This parameter was normalized to the time spent in the center of the arena in the Pre-exposure phase for each mouse (TZpre-exp).

Viral infection and guide cannula implantations
Mice were anesthetized with isoflurane (Kent Scientific VetFlo system) and then unilaterally or bilaterally injected with 150 nl of AAVs (see Supplementary Table 1) in the perihabenular nucleus (PHb) (AP: −1.60, ML: ±0.55, DV: 2.65 mm), ventral lateral geniculated nucleus (vLGN) (AP: −2.15, ML: ±2.65, DV: 3.60 mm), nucleus accumbens (NAc) (AP: 1.42, ML: ±0.80, DV: 4.00 mm) or ventromedial prefrontal cortex (vmPFC) (AP: 1.94, ML: ±0.40, DV: 2.82 mm) using a stereotaxic injector (Neurostar) controlled by the software Stereodrive at an injection rate of 30 nl/min. For intravitreal injections, mice were anesthetized by intraperitoneal (IP) injection of Avertin (2,2,2-Tribromoethanol) and a 30-gauge needle was used to open a hole in the ora serrata. Each eye was intravitreally injected with 1 μL of AAVs (see Supplementary Table 1) using a custom Hamilton syringe (Borghuis Instruments) with a 33-gauge needle (Hamilton). After the surgery procedures, mice were subcutaneously injected with 20 mg/kg of meloxicam (Covetrus). Efficiency of the viral injections was tested postmortem.

In some cases, bilateral guide cannulas in the PHb or NAc were implanted followed by viral infection. For the PHb, Series I bilateral cannula (RWD Life Science Inc.) were implanted with a center-to-center (C.C) distance of 1.2 mm, and a length of 3.65 mm (DV-PHb coordinate +1 mm, Model No. 62025; O.D. 0.48 mm − 26G/C.C 1.2/B 7.8/M 3.5) guide cannula with an internal cannula of 0.5 mm projection length (O.D. 0.30 mm − 30G/C.C 1.2/Mates with M 3.5) and a dummy cannula of 0 mm projection (O.D. 0.30 mm/C.C 1.2/Mates with M 3.5). For the NAc, Series I bilateral cannula (RWD Life Science Inc.) were implanted with a C.C distance of 1.6 mm, and a length of 5.00 mm (DV-NAc coordinate +1 mm, Model No. 62029; O.D. 0.48 mm − 26G/C.C 1.6/B 7.8/M 3.5) guide cannula with an internal cannula of 0.5 mm projection length (O.D. 0.30 mm − 30G/C.C 1.6/Mates with M 3.5) and a dummy cannula of 0 mm projection (O.D. 0.30 mm/C.C 1.6/Mates with M 3.5). For both cannula sets, dust caps (O.D. 5.5 mm/L 7.0 mm/M 3.5) were attached until the day of behavioral test.

Immunohistochemical procedures
For immunological studies in brain sections, mice were anesthetized by IP injection of Avertin and intracardially perfused with saline solution (PBS) followed by PFA 4% in PBS. Brains were dissected and post-fixated in PFA 4% in PBS overnight at 4 °C. For cryoprotection, brains were incubated in sucrose 30% for 3 days, embedded in OCT and then sectioned at 40 μm using a Leica CM1950 cryostat. For immunolabeling studies in retinas, animals were anesthetized by IP injection of Avertin and sacrificed by cervical dislocation. Eyecups were enucleated and retinas were dissected and then fixed in PFA 4% in PBS for 30–60 min at RT. Brain slices and retinas were washed and then blocked at 4 °C overnight in 6% normal goat serum in 0.3% Triton PBS prior to incubating in primary antibody solution for 2–3 nights at 4 °C. Then, tissue was washed and incubated in secondary antibody solution for 2 hours at RT (Supplementary Table 2). The tissue was finally mounted using Fluoromount (Sigma) and imaged using a Leica SP5 confocal microscope. Primary and secondary antibody solutions were made in 3% normal goat serum in 0.3% Triton PBS. For melanopsin and PHb-ipRGC mCherry expression analysis, high magnification images (pixel size of 0.36 μm) with a z-stack size of 1 μm were taken. For viral expression in brain regions, high-magnification images (pixel size of 0.72 μm) with a z-stack size of 2 μm were taken. All images were processed and analyzed using ImageJ.

Fiber photometry
Mice were anaesthetized with isoflurane (Kent Scientific VetFlo system) and then unilaterally injected with 150 nl of AAV1-CAG-GCaMP6s-WPRE-SV40 (~8 × 1012 GC/mL, see Supplementary Table 1) in the PHb using a stereotaxic injector (Neurostar) controlled by the software Stereodrive at an injection rate of 30 nl/min. A FP cannula (MFC_200/230-0.48_3mm_MF1.25_FLT, DORIC) was then implanted 150 μm above the site of injection. After surgery procedures, mice were subcutaneously injected with 2 mg/kg of meloxicam (Covetrus). Efficiency of the brain injection and fiber implantation was tested postmortem.

Behavioral assay and FP recordings were made 4 weeks post-surgery. For that, dark-adapted mice were exposed to the LTTA paradigm as described above. For FP recording, an optic fiber (MFP_400/430/1100-0.57_1m_FC-ZF1.25(F)_LAF, DORIC) was attached to the head of the animals and then were placed to the LTTA arena. FP signal was collected using an assisted electrical rotary system (AERJ_24_FMC, DORIC), Amplifier (Doric Fluorescence Detector, DORIC), LED driver (Laser Diode Fiber Light Source, DORIC) and processor (RZ5P Base Processor, Tucker-Davis Technologies). FP data were extracted using pMAT (RRID:SCR_022570) software28. All FP data processing and behavior correlation analyses were made using eZtrack, Microsoft Excel (RRID:SCR_016137) and Graph Pad Prism 10.1.2 (RRID:SCR_002798).

Statistical comparisons
All graphs and statistical analyses were performed using Graph Pad Prism 10.1.2. Normal distribution of data was tested using Shapiro-Wilk test. Data points were excluded only if identified as statistical outliers using the ROUT method (Q = 1%), which was pre-established prior to data analysis. No other data were excluded from the analyses. Two-sided Student’s t or Mann Whitney U tests were used to compare two experimental groups. For multiple statistical comparisons, we performed one-way ANOVA followed by Tukey’s and Dunn’s tests. Significance was concluded when P < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The study by Aranda et al. introduces a novel behavioral paradigm termed long-term threat avoidance (LTTA) to quantify context-dependent avoidance behavior shaped by past threat experiences over extended timescales. While the hypothesis that melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) drive LTTA via a retino-perihabenulo-accumbens circuit (ipRGC→pHb→NAc) is conceptually innovative, the physiological and mechanistic evidence are not sufficient to support the central claims.\nMajor Concerns:\n1.The study introduces LTTA as a novel behavior, but its mechanistic overlap with classical contextual fear conditioning1 raises questions about its conceptual uniqueness. The authors should clarify how LTTA fundamentally differs from classical contextual fear conditioning. Without such distinctions, LTTA risks being interpreted as a procedural adaptation of existing paradigms rather than a fundamentally novel behavior.\n2.The authors compared the behavioral difference mainly between Opn4-/- and wild-type mice (in Figures 2-4). However, previous studies have indicated that ipRGCs can influence brain development2-4. Therefore, the observed differences cannot be ruled out to be caused by developmental-induced emotional and cognitive changes. The appropriate approach would be to compare adult mice with melanopsin rescue or wild-type mice in which the Opn4 gene expression is knocked out during adulthood.\n3.The LTTA must requires high-resolution visual context discrimination. Although ipRGCs have been implicated in image-processing circuits5-11, their physiological properties – characterized by broad receptive fields and low spatial resolution – appear fundamentally inadequate for encoding fine-grained scene features. In fact, rod/cone pathways — not ipRGCs — are evolutionarily specialized for high-acuity image processing. This dichotomy raises a pivotal mechanistic question left unresolved in the current study: Does pHb receive (1) direct context identity signals from ipRGCs (despite their poor spatial encoding fidelity), or (2) rod/cone-processed context information that is gated/modulated by ipRGC-conveyed ambient irradiance cues through parallel circuits? This critical distinction can be addressed by metameric illumination paradigms employing melanopsin-isolating stimuli with equiluminant cone-controlled backgrounds to dissociate ipRGC-driven inputs from classical photoreceptor pathways8.\n4.The chemogenetic activation of PHb→NAcc neurons via hM3Dq in Figure 5 (comparing Opn4-/- mice with/without activation) may confound results due to the emotional roles of PHb and NAcc, failing to isolate vision-specific mechanisms. To directly validate the ipRGC-PHb-NAcc pathway's causality in LTTA, the authors should use hM4Di-based inhibition in wild-type mice (as in Figure 3E-J) combined with axon-terminal-specific CNO delivery, which would abolish ipRGC-dependent effects while excluding mood-related confounds and ensuring pathway-selective manipulation.\n5.The chemogenetic activation protocol in Figure 4 (CNO-induced excitation of pHb-targeting ipRGCs) introduces significant interpretational ambiguity. Crucially, Extended Data Figure 10 demonstrates that these pHb-targeting ipRGCs exhibit collateral projections to visual processing nuclei such as the dorsal lateral geniculate nucleus (dLGN)—a central hub for high-acuity visual coding. This dual-circuit architecture creates confusion: the behavioral effects attributed to pHb modulation could equally arise from dLGN-mediated alterations in visual processing. To resolve this circuit specificity, CNO should be delivered directly into the PHb to selectively modulate axon terminals of ipRGCs projecting to the PHb (rather than their cell bodies), thereby avoiding confounding effects from their collateral projections to the dLGN. Similar to point 4, this approach should employ hM4Di-based inhibition in wild-type mice to isolate causality within the ipRGC-PHb pathway while excluding indirect visual or emotional confounds.\n6.The 0 lux control in Figure 1E invalidates the experimental logic. Complete darkness inherently prevents all visual scene recognition including rods, cones and ipRGCs, making the observed LTTA abolition uninterpretable for isolating ipRGC-specific mechanisms. Instead, the authors should test LTTA across an illuminance gradient to demonstrate parametric modulation of avoidance strength by ambient light levels. A dose-dependent relationship would better support ipRGC involvement in contextual threat association rather than binary light/dark comparisons.\n7.The absence of significant LTTA differences between Opn4−/− and control groups at higher contrast levels (27.5%, 36.5% in Extended Data Figure 4C) warrants further investigation, as it appears inconsistent with the proposed ipRGC-driven contrast dependency. A complete impairment of ipRGC signaling would predict reduced avoidance across all contrast conditions if these cells solely mediate threat-context associations.\n8. The temporal specificity of ipRGC involvement in LTTA remains unresolved due to the concurrent use of CNO during both threat exposure and test phases (Figures 3–5). This dual administration conflates the circuit’s potential roles in memory encoding (during exposure) versus contextual recall (during testing). To disentangle these mechanisms, the authors must perform time-selective manipulations: silencing or activating the ipRGC→pHb→NAc circuit either during the exposure phase (to assess memory formation) or during the test phase (to evaluate context recognition). Such experiments would clarify whether ipRGCs primarily regulate the consolidation of threat-associated visual scenes or the retrieval of these memories during avoidance behavior, another key mechanism of ipRGC-mediated LTTA.\nMinor Concerns:\n1.Preliminary data exploring ipRGC interactions with sex hormones, while intriguing, are tangential to the core narrative of this study. These findings lack direct relevance to the proposed LTTA mechanism or the retino-perihabenular-accumbens circuit. Including them risks diluting the manuscript’s focus and should be removed unless expanded into a dedicated mechanistic exploration in subsequent work.\n2.In Figure 5C, the use of dual-color labeling to distinguish left/right pHb→NAc projections introduces unnecessary complexity. The bilateral projection pattern could be unambiguously demonstrated using a single fluorophore, as hemispheric laterality adds no conceptual value to the circuit mapping. Simplifying this figure to show unilateral projections would enhance clarity without sacrificing scientific rigor.\n3.Gene names should be italicized.\nReference:\n1. Curzon P, Rustay NR, Browman KE. Cued and Contextual Fear Conditioning for Rodents. In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2009. Chapter 2.\n2. Chew, K. S. et al. A subset of ipRGCs regulates both maturation of the circadian clock and segregation of retinogeniculate projections in mice. Elife 6, e22861 (2017).\n3. Lazzerini Ospri, L. et al. Light affects the prefrontal cortex via intrinsically photosensitive retinal ganglion cells. Sci. Adv. 10, eadh9251 (2024).\n4. Hu, J. et al. Melanopsin retinal ganglion cells mediate light-promoted brain development. Cell 185, 3124-3137.e15 (2022).\n5. Ecker JL. et al.. Melanopsin-expressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision. Neuron. 2010 Jul 15;67(1):49-60.\n6. Schmidt TM. et al. A role for melanopsin in alpha retinal ganglion cells and contrast detection. Neuron. 2014 May 21;82(4):781-8.\n7. Sonoda T, Lee SK, Birnbaumer L, Schmidt TM. Melanopsin Phototransduction Is Repurposed by ipRGC Subtypes to Shape the Function of Distinct Visual Circuits. Neuron. 2018 Aug 22;99(4):754-767.e4.\n8. Allen AE. et al.. Form vision from melanopsin in humans. Nat Commun. 2019 May 22;10(1):2274.\n9. Allen AE. et al.. Melanopsin Contributions to the Representation of Images in the Early Visual System. Curr Biol. 2017 Jun 5;27(11):1623-1632.e4.\n10. Mouland JW. et al. Responses to Spatial Contrast in the Mouse Suprachiasmatic Nuclei. Curr Biol. 2017 Jun 5;27(11):1633-1640.e3.\n11. Shi Y. et al.. Non-image-forming photoreceptors improve visual orientation selectivity and image perception. Neuron. 2025 Feb 5;113(3):486-500.e13.", "reviewer_2": "This manuscript by Aranda et al. explores how looming stimuli can drive the development of long-term threat avoidance (LTTA) behavior. The authors present a novel behavioral paradigm where mice exposed to a looming stimulus in a high-contrast open field subsequently avoid that threat zone in future sessions. They further demonstrate that melanopsin-expressing retinal neurons (Opn4+) are necessary for this behavior, as Opn4 knockout mice fail to exhibit LTTA. The study then examines the role of the perihabenular nucleus (PHb), showing that looming exposure induces c-Fos activation in PHb neurons and that DREADD-mediated inhibition of GABAergic neurons in this region prevents LTTA. Fiber photometry recordings indicate larger calcium transients in wild-type animals relative to Opn4 knockouts, and the authors propose that PHb GABAergic neurons project to the nucleus accumbens (NAc) and prefrontal cortex (PFC), with inhibition of this pathway reducing LTTA. The study is well-written, with a clear presentation of behavioral, anatomical, and functional data. While the findings are interesting and provide new insight into long-term threat processing, several aspects especially of the latter half of the paper—particularly the fiber photometry data and the proposed PHb-to-NAc circuit—require further clarification and additional experiments to fully support their conclusions.\nMajor comments:\n1. There is not strong evidence in the literature for projections from PHb to NAc. To better examine this, axonal tracing of these neurons and their projections to the NAc is needed. Additionally, the paraventricular nucleus of the thalamus (PVT), a neighboring region, projects heavily to the NAc. The authors need to unequivocally demonstrate that they are not inadvertently targeting PVT neurons with their preparation.\n2. The specificity of the GAD-Cre mouse for GABAergic neurons in PHb is not well established. While it may be selective in some brain regions, this has not been demonstrated for PHb. The authors' own images show clear labeling of some cells in the lateral habenula, which is almost entirely glutamatergic, implying that their virus and mouse combination may not be exclusively targeting GABAergic neurons.\n3. The fiber photometry results do not fully support the conclusions drawn. There are concerns regarding data interpretation. What does PHb activity in wild-type and Opn4 KO mice look like aligned to loom onset during the exposure phase? This would help clarify whether PHb neurons are responding to the looming stimulus itself. The number of edge-to-threat zone (TZ) transitions during the test phase appears low (n=4 across 4 mice per group, Fig. 4J), which limits statistical power. Given this, the photometry data for transitions should be interpreted cautiously. Figure 4I suggests that PHb activity in control mice is inhibited by the edge-to-TZ transition. This is unexpected given that ipRGC input to PHb is presumed excitatory. The authors should clarify how this result fits with the overall increase in PHb activity in control mice during the test phase (Fig. 4G).\nMinor comments:\n1. The introduction should provide more background on the ipRGC signaling cascade (light → melanopsin → Gq cascade → glutamate signaling) to better justify the focus on this pathway. This would strengthen the rationale for the DREADD experiments targeting PHb GABAergic neurons.\n2. The discussion should include a clearer model of the proposed neural circuit. The claim that chemogenetic excitation of PHb neurons reduces LTTA is intriguing, but it would be more convincing if excitation were restricted to only NAc-projecting PHb GABAergic neurons. Does activating PHb neurons indiscriminately have other behavioral effects?\n3. A simpler photometry analysis may improve interpretability: Consider analyzing transitions (edge→TZ and TZ→edge) only when not followed by another transition within a window where fluorescence could decay to baseline. Compare average activity in the edge versus TZ across different phases (pre-exposure, exposure, and test). This could reveal circuit-consistent activity patterns. Including a figure showing behavioral events overlaid with fluorescence traces could help contextualize neural activity changes.", "reviewer_3": "In this study, Aranda and colleagues introduces a conceptually interesting behavioral paradigm (LTTA) and explores underexamined neural substrates. However, major methodological and interpretive flaws limit its impact. The central hypothesis that ipRGCs drive LTTA via PHb-NAc projections remains inadequately substantiated due to insufficient circuit-level evidence and incomplete experimental validation. While the work holds promise, I recommend rejecting the manuscript in its current form. Substantial revisions are needed to bolster mechanistic claims and clarify the study's novelty. Specifically, the authors must rigorously validate the proposed pathway and better contextualize their findings within the broader literature on threat avoidance circuits.\nMajor:\n1. Inadequate analysis of sex-specific effects: The reported estrous cycle-dependent variation in LTTA behavior in female mice remains superficially analyzed. Given the critical importance of sex-specific effects in behavioral studies, the authors should expand their analysis of female cohorts across all estrous stages, including hormonal profiling (e.g., estrogen/progesterone levels). They should investigate whether sex hormones modulate ipRGCs-PHb-NAc pathway activity. If feasible, key experiments should be repeated in female-specific cohorts to validate generalizability.\n2. Unresolved local circuit interactions: While the authors demonstrate that silencing PHb GABA neurons (but not PHb Glu neurons) disrupts LTTA (Fig. 3E-J), the experimental design does not fully address potential local inhibitory interactions. Specifically, PHb GABA neurons may tonically suppress PHb Glu neurons under baseline conditions. Silencing PHb GABA neurons could thus result in disinhibition of PHb Glu neurons, potentially confounding the interpretation that PHb Glu neurons are functionally irrelevant to LTTA. To establish direct causality, the authors should selectively activate PHb Glu neurons while monitoring LTTA behavior and quantify whether artificial activation significantly alters time spent in the TZ.\n3. Insufficient validation of chemogenetic approaches: The validation experiments for assessing chemogenetic efficiency require substantial supplementation. Critical methodological controls, including both hM4Di and hM3Dq validation approaches, are conspicuously absent. Specifically, the key experimental evidence supporting chemogenetic efficiency claims in Figure 3E and Figure 4B lacks essential validation data.\n4. Weak mechanistic validation: The proposed link between ipRGCs and the PHb-NAc pathway in regulating LTTA lacks robust validation. Current evidence, relying primarily on behavioral and limited electrophysiological data, fails to establish a conclusive causal relationship at molecular or cellular levels. The authors should incorporate molecular/cellular validation (e.g., conditional knockout models, pharmacological interventions, or optogenetic manipulation of the ipRGCs-PHb-NAc pathway) and include recordings of NAc neuronal activity during LTTA behavior to strengthen the proposed mechanism.\n5. Unclear rationale for experimental timeline: The justification for selecting a 2-day post-stimulus interval in the LTTA experimental design requires clarification. The authors should provide direct empirical evidence supporting the temporal specificity of ipRGC and PHb neuronal plasticity measurements following looming stimulation. This could include either:\n•Citations to established temporal patterns of threat-conditioned neural plasticity in this specific pathway, or\n•Time-course validation data demonstrating that 48 hours represents the optimal window for detecting stimulus-induced changes in the measured parameters.\nThe manuscript would be strengthened by addressing whether published literature demonstrates that neuroplastic changes (e.g., dendritic remodeling, synaptic protein expression, or electrophysiological alterations) in ipRGC and PHb neurons exhibit peak manifestation precisely at 48 hours post-stimulation, or if pilot studies showed differential outcomes between 2-day and longer-term (e.g., 5-day) intervals."}, "author_response": "We are resubmitting a revised version of our work entitled “Light tunes a novel long-term threat avoidance\nbehavior” for consideration by Nature Communications. We thank the reviewers for their thoughtful and in-depth\nreviews of the initial submission of this manuscript. In this study, we identify a novel visual circuit that tunes long-\nterm threat avoidance (LTTA) behavior based on past experience.\nOur resubmission includes multiple new approaches to define the circuits by which light tunes LTTA via an\nipRGC-PHb-NAc pathway. Here, we provide a list of the new experiments added to the resubmission.\n- We provided information on the spectral illumination in the LTTA assay (Suppl. Fig. 1).\n- We exposed Control mice to 10 lux illumination during the Test phase of the LTTA assay to assess the\nimpact of light on the behavior (Fig. 1E).\n- Opn4 gene knocked out in adult mice to mitigate concerns of developmental impacts on brain circuits.\nFor that we injected AAV-Cre (AAV-hSyn-Cre-P2A-dTomato) in Opn4fl/fl mice followed by LTTA assay\n(Fig. 2D-H and Suppl. Fig. 4).\n- Additional Control and Opn4-/- mice exposed to higher looming contrasts to increase experimental\nnumbers (27.5 and 36.5% Michelson contrast) in the LTTA assay (Suppl. Fig. 2).\n- WT naïve mice injected with Vehicle or CNO, to assess potential off target effects of CNO on LTTA\n(Suppl. Fig. 6).\n- Additional chemogenetic manipulations to test functional role of PHb-ipRGCs in LTTA. We combined\nintravitreal injections of Gi-DREADDs with axon-terminal-specific Vehicle or CNO delivery using guide\ncannulas in PHb followed by exposure to the LTTA assay (Fig. 4E-I and Suppl. Fig. 7).\n- Additional chemogenetic manipulations to test functional role of PHbGlu neurons in LTTA. We injected Gq\nDREADDs in PHb of Vglut2Cre mice followed by exposure to the LTTA assay (Fig. 3K-P).\n- Additional chemogenetic manipulations to test functional role of PHb-NAc neurons in LTTA. We\ncombined injections of Gi-DREADDs in PHb with axon-terminal-specific Vehicle or CNO delivery using\nguide cannulas in NAc followed by exposure to the LTTA assay (Fig. 6J-N).\n- Additional fiber photometry recordings from the PHb of Control and Opn4-/- littermates and deeper data\nanalysis:\nAdditional animal transitions from edge to Threat zone (TZ) in Pre-exposure (Fig. 5I-L) and Test\no\nphases (Fig. 5M-O) of the LTTA paradigm.\nCalcium signal during the Exposure phase of the LTTA paradigm (Fig. 5E-H).\no\nAnimal transitions from TZ to edges in the Test phase of the LTTA paradigm (Suppl. Fig. 10).\no\nCorrelations of calcium signal and behavioral parameters during the Test phase of the LTTA\no\nparadigm (Suppl. Fig. 10).\nWe are excited to report that these new results provide additional converging lines of evidence that further\nsupport our original conclusions and have greatly strengthened the manuscript. We hope that the reviewers will\nappreciate the multiple additional supporting experiments and controls in this updated version. Our specific\nresponses are detailed below.\nReviewer #1\nThe study by Aranda et al. introduces a novel behavioral paradigm termed long-term threat avoidance (LTTA) to\nquantify context-dependent avoidance behavior shaped by past threat experiences over extended timescales.\nWhile the hypothesis that melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) drive\nLTTA via a retino-perihabenulo-accumbens circuit (ipRGC→pHb→NAc) is conceptually innovative, the\nphysiological and mechanistic evidence are not sufficient to support the central claims.\nMajor Concerns:\n1.The study introduces LTTA as a novel behavior, but its mechanistic overlap with classical contextual fear\nconditioning1 raises questions about its conceptual uniqueness. The authors should clarify how LTTA\nfundamentally differs from classical contextual fear conditioning. Without such distinctions, LTTA risks being\ninterpreted as a procedural adaptation of existing paradigms rather than a fundamentally novel behavior.\nWhile both LTTA and classical contextual fear conditioning involve a learned association between a context and\nan aversive event, LTTA fundamentally differs from classical contextual fear conditioning paradigms in multiple\nimportant ways:\n1. The conceptual uniqueness of the LTTA paradigm lies in the response to a purely visual, ethologically\nrelevant, stimulus without any physical discomfort or harm to the animal.\n2. A key benefit of LTTA is that during the Test phase we are able to observe and quantify how the mouse\ndecides to explore the arena relative to the location of the prior threat presentation (i.e. the “Threat Zone”).\nIn contrast, in a contextual fear conditioning paradigm the mouse cannot escape the floor bars that\ndelivered the shock and has no opportunity to decide whether or not to revisit the location of the threat.\n3. The purely visual nature of the looming stimulus in LTTA allows us to present a “threat” without physically\ninducing pain, harm, or injury to the animal. This allows us to isolate response to a perceived threat rather\nthan recall of a physically uncomfortable experience. This removes confounds of pain thresholds and\nnegates the need for actual physical discomfort.\n4. Our data implicate an ipRGC-PHb-NAc circuit unrelated to those linked to contextual fear conditioning.\nThough this is not part of our initial rationale for using LTTA, this newly identified circuit alone further\nseparates the LTTA paradigm from classical contextual fear conditioning. The single, 10 second\nexposure to the looming stimulus during the Exposure phase also highlights the high sensitivity and\nadaptability of these ethologically important circuits, marking the LTTA circuit as an important target for\nfuture research into threat assessment, risk-based decision making, and whether it modulates other yet-\nunidentified behaviors.\nWe now highlight these key differences in the Discussion to better highlight the distinctions for future readers.\n2.The authors compared the behavioral difference mainly between Opn4-/- and wild-type mice (in Fig.s 2-4).\nHowever, previous studies have indicated that ipRGCs can influence brain development2-4. Therefore, the\nobserved differences cannot be ruled out to be caused by developmental-induced emotional and cognitive\nchanges. The appropriate approach would be to compare adult mice with melanopsin rescue or wild-type mice\nin which the Opn4 gene expression is knocked out during adulthood.\nIf brain development effects are driving Opn4-/- deficits in LTTA, then these deficits should disappear if the Opn4\ngene is knocked out in adult stages. To test this, we now include a conditional knockout approach using Opn4fl/fl\nanimals to remove Opn4 in adult mice. We intravitreally injected an AAV carrying the Cre recombinase gene\n(AAV2-hSyn-Cre-P2A-TdTomato) into Opn4fl/fl mice and then compared LTTA behavior to vehicle injected\ncontrols. Opn4fl/fl mice injected with AAV-Cre, showed deficits in LTTA behavior (Fig. 2D-H and Suppl. Fig. 4)\nsimilar to those observed in germline Opn4-/- animals compared to control mice, indicating that this phenotype is\ndue to loss of melanopsin from ipRGCs, not off-target developmental impacts in the germline melanopsin\nknockout. Following behavioral testing of LTTA, we confirmed that conditional knockout animals showed a ~95%\nreduction in ipRGC numbers (Suppl. Fig. 4).\nIt's important to note that there are other lines of evidence in the manuscript beyond the Opn4fl/fl animals that\nalso argue against developmental effects in Opn4-/- animals driving LTTA deficits:\n1. Exploratory behavior is similar in Opn4-/- mice and control in both Pre-exposure and Test phases followed\nby a 0% Michelson contrast, suggesting that exploratory is similar in Opn4-/- and control littermates.\n2. General anxiety levels are similar in Control and Opn4-/- littermates following either a 0 or 6.6% Michelson\ncontrast exposure in the LTTA paradigm, arguing that anxiety is not the source of differences in LTTA\n3. The specific chemogenetic activation of PHb-ipRGCs in adult Opn4-/- animals rescued the observed\ndeficits in LTTA, demonstrating that adult activation of ipRGCs is sufficient to rescue LTTA, arguing\nagainst a fundamental change in circuit function and for LTTA deficits arising simply due to absence of\nipRGC signaling.\nCombined, our data indicate that LTTA deficits in Opn4-/- animals arise from the absence of melanopsin signaling\nin ipRGCs, rather than from developmental rewiring of visual circuits.\n3.The LTTA must requires high-resolution visual context discrimination. Although ipRGCs have been implicated\nin image-processing circuits5-11, their physiological properties – characterized by broad receptive fields and low\nspatial resolution – appear fundamentally inadequate for encoding fine-grained scene features. In fact, rod/cone\npathways — not ipRGCs — are evolutionarily specialized for high-acuity image processing. This dichotomy\nraises a pivotal mechanistic question left unresolved in the current study: Does pHb receive (1) direct context\nidentity signals from ipRGCs (despite their poor spatial encoding fidelity), or (2) rod/cone-processed context\ninformation that is gated/modulated by ipRGC-conveyed ambient irradiance cues through parallel circuits? This\ncritical distinction can be addressed by metameric illumination paradigms employing melanopsin-isolating stimuli\nwith equiluminant cone-controlled backgrounds to dissociate ipRGC-driven inputs from classical photoreceptor\npathways8.\nThe low PHb activity during the Pre-exposure phase in our fiber photometry experiments suggests that ipRGCs\ndo not passively convey spatial or contextual information about the arena because PHb activity only emerges\nonce the environment becomes associated with the threatening (looming) stimulus (Fig.5). This observation\nargues against the notion that ipRGCs provide fine-grained scene features and instead supports a role for the\nPHb in relaying information about environmental luminance. In support of this idea (see Point 6 below), we find\nthat LTTA is tuned by luminance, with control animals showing more TZ avoidance in the Test phase at higher\nlight, less in lower light, and none in darkness.\nWe have opted against using metameric stimuli in this case. Isolating melanopsin-driven responses under cone-\nequiluminant conditions only produces subtle variations in what is already a fairly high melanopic irradiance.\nThus, this approach limits the range of detectable behavioral or physiological outcomes compared to the multiple\ngenetic and chemogenetic manipulations. Here it is worth noting that only Opn4fl/fl mice with an ipRGC loss of\ngreater than 80% showed detectable changes in LTTA behavior, indicating that even sub-saturating melanopsin\nactivation is sufficient for normal LTTA (Reviewer Fig. 1). Thus, reductions in melanopsin activation of 10-15%\nusing metameric stimuli1 are unlikely to drive differences in LTTA behavior, making the building and careful\ncalibration of such a metameric stimulus delivery system unlikely to be time- or cost-effective.\nIt is important to note that Opn4-/- animals will still allow for rod/cone signals to be relayed to the brain, and retinal\nrecordings from Opn4-/- ipRGCs show detectable rod/cone responses that arise and terminate on much faster\ntimescales than the sustained melanopsin-based response. Our newly added experiments in conditional\nmelanopsin knockout animals further supports a likely cell autonomous role for melanopsin.\nWhether melanopsin null deficits arise from how rod/cone signals combine within ipRGCs or instead arise due\nto an absence of melanopsin signaling itself is not something we could determine even with the help of metameric\nstimuli.\nIn order to provide a better characterization of the illumination set up in the LTTA paradigm, in the new version\nof the manuscript we now include specifics of the spectral power illumination at all looming contrast during the\nExposure phase and at different light intensities in the Test phase (Suppl. Fig. 1).\n4.The chemogenetic activation of PHb→NAcc neurons via hM3Dq in Fig. 5 (comparing Opn4-/- mice with/without\nactivation) may confound results due to the emotional roles of PHb and NAcc, failing to isolate vision-specific\nmechanisms. To directly validate the ipRGC-PHb-NAcc pathway's causality in LTTA, the authors should use\nhM4Di-based inhibition in wild-type mice (as in Fig. 3E-J) combined with axon-terminal-specific CNO delivery,\nwhich would abolish ipRGC-dependent effects while excluding mood-related confounds and ensuring pathway-\nselective manipulation.\nWe thank the reviewer for this suggestion. To better isolate vision-specific effects on LTTA, we injected Gi\nDREADDs (AAV2-Syn-hM4Gi-mCherry) in the PHb and co-implanted bilateral guide cannulas in the NAc of WT\nmice to allow for axon-terminal-specific manipulations. 4 weeks post-surgery, Vehicle or CNO was infused in the\nguide cannulas 20-30 min before each phase of the LTTA paradigm. After histological examination of 9 mice,\nwe excluded 2 mice where the PHb or NAc were mistargeted (Reviewer Fig. 2). We observed that CNO-infused\nmice showed increased time in TZ compared to Vehicle-infused WT mice (Fig. 6J-N), further supporting a role\nfor ipRGC input in tuning LTTA.\nIt's also important to note that in our initial version, we did not find difference in the baseline exploratory behavior\nof either Vehicle or CNO injected Opn4-/- littermates during the Pre-exposure phase, suggesting that CNO itself\ndoes not impact exploratory behavior in the absence of a prior threat exposure.\n5.The chemogenetic activation protocol in Fig. 4 (CNO-induced excitation of pHb-targeting ipRGCs) introduces\nsignificant interpretational ambiguity. Crucially, Extended Data Fig. 10 demonstrates that these pHb-targeting\nipRGCs exhibit collateral projections to visual processing nuclei such as the dorsal lateral geniculate nucleus\n(dLGN)—a central hub for high-acuity visual coding. This dual-circuit architecture creates confusion: the\nbehavioral effects attributed to PHb modulation could equally arise from dLGN-mediated alterations in visual\nprocessing. To resolve this circuit specificity, CNO should be delivered directly into the PHb to selectively\nmodulate axon terminals of ipRGCs projecting to the PHb (rather than their cell bodies), thereby avoiding\nconfounding effects from their collateral projections to the dLGN. Similar to point 4, this approach should employ\nhM4Di-based inhibition in wild-type mice to isolate causality within the ipRGC-PHb pathway while excluding\nindirect visual or emotional confounds.\nAs described above, we find that Gi-DREADD mediated inhibition of ipRGC terminals via CNO delivery to the\nPHb drives deficits in LTTA that match those seen in Opn4-/- animals (Fig. 4E-I). These results are further\nsupportive of our findings that inhibition of vLGN, a collateral target of ipRGCs, does not alter LTTA (Suppl. Fig.\n9). Moreover, with the addition of new animals to our fiber photometry recordings, we continue to observe altered\nPHb signaling in Opn4-/- animals (Fig. 5). Combined, our data provide strong support for a role of ipRGC signaling\ntuning PHb neuron activity during LTTA.\n6.The 0 lux control in Fig. 1E invalidates the experimental logic. Complete darkness inherently prevents all visual\nscene recognition including rods, cones and ipRGCs, making the observed LTTA abolition uninterpretable for\nisolating ipRGC-specific mechanisms. Instead, the authors should test LTTA across an illuminance gradient to\ndemonstrate parametric modulation of avoidance strength by ambient light levels. A dose-dependent relationship\nwould better support ipRGC involvement in contextual threat association rather than binary light/dark\ncomparisons.\nWe appreciate the reviewer’s excellent suggestion. In the new version we added a new experimental group\nwhere animals were exposed to 10 lux during the Test phase of the LTTA paradigm and compared it to animals\nexposed to 0 lux or 40 lux during the Test phase. Excitingly, we observed a dose-dependent relationship between\navoidance behavior and ambient light levels, suggesting that luminance levels tune avoidance strength (Fig. 1E).\n7.The absence of significant LTTA differences between Opn4−/− and control groups at higher contrast levels\n(27.5%, 36.5% in Extended Data Fig. 4C) warrants further investigation, as it appears inconsistent with the\nproposed ipRGC-driven contrast dependency. A complete impairment of ipRGC signaling would predict reduced\navoidance across all contrast conditions if these cells solely mediate threat-context associations.\nWe have added additional animals at higher contrasts. With the higher sample size, differences between control\nand Opn4-/- littermates now reach statistical significance at these higher contrasts (Suppl. Fig. 2). Thus, ipRGCs\ndo tune threat avoidance across the full range of contrasts, including those below threshold (6.6%) for a\ndetectable looming response and those that reliably drive detectable looming responses (36.5%) in all animals.\n8. The temporal specificity of ipRGC involvement in LTTA remains unresolved due to the concurrent use of CNO\nduring both threat exposure and test phases (Fig.s 3–5). This dual administration conflates the circuit’s potential\nroles in memory encoding (during exposure) versus contextual recall (during testing). To disentangle these\nmechanisms, the authors must perform time-selective manipulations: silencing or activating the\nipRGC→pHb→NAc circuit either during the exposure phase (to assess memory formation) or during the test\nphase (to evaluate context recognition). Such experiments would clarify whether ipRGCs primarily regulate the\nconsolidation of threat-associated visual scenes or the retrieval of these memories during avoidance behavior,\nanother key mechanism of ipRGC-mediated LTTA.\nIn our initial version, we did in fact test CNO delivery during only the Pre-Exposure or only during the Test phase\n(now Fig. 4A-D) in Opn4-/- animals where only PHb ipRGCs expressed Gq-DREADDs. We found that LTTA in\nOpn4-/- mice was only restored in animals receiving CNO during the Test phase, but not for those receiving CNO\nonly during the Pre-Exposure phase, indicating that ipRGCs are required only during the Test phase, when the\nanimal must recall the threat-context association. This was further bolstered by fiber photometry recordings in\ncontrol mice that indicate PHb signaling differences only during edge-TZ transitions of the Test phase, but not\nthe Pre-Exposure phase (Fig. 5). Moreover, the differences between control and Opn4-/- PHb signaling in fiber\nphotometry experiments were only observed during the Test phase.\nGiven our previously observed differences in the Test phase only, we opted to perform multiple, new\nchemogenetic approaches using cannulated CNO delivery in either the PHb or NAc of control animals (Fig. 4E-\nI and Fig. 6J-N) only at the Test phase. We find that inhibition of PHb-ipRGCs or NAc-PHb neurons during the\nTest phase leads to deficits in LTTA. Thus, these newly added experiments confirm what we found in our rescue\nand CNO experiments (Fig.s 4A-D and Fig. 6D-I). suggesting that an ipRGC-PHb-NAc circuit tunes LTTA by\nimpacting recall/retrieval of the threat-context association.\nMinor Concerns:\n1.Preliminary data exploring ipRGC interactions with sex hormones, while intriguing, are tangential to the core\nnarrative of this study. These findings lack direct relevance to the proposed LTTA mechanism or the retino-\nperihabenular-accumbens circuit. Including them risks diluting the manuscript’s focus and should be removed\nunless expanded into a dedicated mechanistic exploration in subsequent work.\nWe thank the reviewer for this suggestion and have consequently decided to limit the current study to male mice.\nAs suggested by the reviewer, we will expand the estrous-cycle dependent effect of female mice on LTTA in a\nsubsequent work.\n2.In Fig. 5C, the use of dual-color labeling to distinguish left/right pHb→NAc projections introduces unnecessary\ncomplexity. The bilateral projection pattern could be unambiguously demonstrated using a single fluorophore,\nas hemispheric laterality adds no conceptual value to the circuit mapping. Simplifying this Fig. to show unilateral\nprojections would enhance clarity without sacrificing scientific rigor.\nWe now include unilateral projections in Fig. 6C.\n3.Gene names should be italicized.\nWe changed this formatting in the new version.\nReference:\n1. Curzon P, Rustay NR, Browman KE. Cued and Contextual Fear Conditioning for Rodents. In: Buccafusco JJ,\neditor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press/Taylor &\nFrancis; 2009. Chapter 2.\n2. Chew, K. S. et al. A subset of ipRGCs regulates both maturation of the circadian clock and segregation of\nretinogeniculate projections in mice. Elife 6, e22861 (2017).\n3. Lazzerini Ospri, L. et al. Light affects the prefrontal cortex via intrinsically photosensitive retinal ganglion cells.\nSci. Adv. 10, eadh9251 (2024).\n4. Hu, J. et al. Melanopsin retinal ganglion cells mediate light-promoted brain development. Cell 185, 3124-\n3137.e15 (2022).\n5. Ecker JL. et al.. Melanopsin-expressing retinal ganglion-cell photoreceptors: cellular diversity and role in\npattern vision. Neuron. 2010 Jul 15;67(1):49-60.\n6. Schmidt TM. et al. A role for melanopsin in alpha retinal ganglion cells and contrast detection. Neuron. 2014\nMay 21;82(4):781-8.\n7. Sonoda T, Lee SK, Birnbaumer L, Schmidt TM. Melanopsin Phototransduction Is Repurposed by ipRGC\nSubtypes to Shape the Function of Distinct Visual Circuits. Neuron. 2018 Aug 22;99(4):754-767.e4.\n8. Allen AE. et al.. Form vision from melanopsin in humans. Nat Commun. 2019 May 22;10(1):2274.\n9. Allen AE. et al.. Melanopsin Contributions to the Representation of Images in the Early Visual System. Curr\nBiol. 2017 Jun 5;27(11):1623-1632.e4.\n10. Mouland JW. et al. Responses to Spatial Contrast in the Mouse Suprachiasmatic Nuclei. Curr Biol. 2017 Jun\n5;27(11):1633-1640.e3.\n11. Shi Y. et al.. Non-image-forming photoreceptors improve visual orientation selectivity and image perception.\nNeuron. 2025 Feb 5;113(3):486-500.e13.\nReviewer #2\nThis manuscript by Aranda et al. explores how looming stimuli can drive the development of long-term threat\navoidance (LTTA) behavior. The authors present a novel behavioral paradigm where mice exposed to a looming\nstimulus in a high-contrast open field subsequently avoid that threat zone in future sessions. They further\ndemonstrate that melanopsin-expressing retinal neurons (Opn4+) are necessary for this behavior, as Opn4\nknockout mice fail to exhibit LTTA. The study then examines the role of the perihabenular nucleus (PHb),\nshowing that looming exposure induces c-Fos activation in PHb neurons and that DREADD-mediated inhibition\nof GABAergic neurons in this region prevents LTTA. Fiber photometry recordings indicate larger calcium\ntransients in wild-type animals relative to Opn4 knockouts, and the authors propose that PHb GABAergic\nneurons project to the nucleus accumbens (NAc) and prefrontal cortex (PFC), with inhibition of this pathway\nreducing LTTA. The study is well-written, with a clear presentation of behavioral, anatomical, and functional data.\nWhile the findings are interesting and provide new insight into long-term threat processing, several aspects\nespecially of the latter half of the paper—particularly the fiber photometry data and the proposed PHb-to-NAc\ncircuit—require further clarification and additional experiments to fully support their conclusions.\nMajor comments:\n1. There is not strong evidence in the literature for projections from PHb to NAc. To better examine this, axonal\ntracing of these neurons and their projections to the NAc is needed. Additionally, the paraventricular nucleus of\nthe thalamus (PVT), a neighboring region, projects heavily to the NAc. The authors need to unequivocally\ndemonstrate that they are not inadvertently targeting PVT neurons with their preparation.\nIn addition to projections shown in the current manuscript (Fig. 6), the projections from the PHb to the NAc have\nbeen well-described in the literature in the mouse2-4 and in the tree shrew5. These references are included in the\nmanuscript. In all our chemogenetic approaches, we corroborate efficiency and leakage of viral expression and\nmistargeted mice were excluded from the analyses.\nIt’s important to note that any animal exhibiting spread of the 150 nl viral injection volume outside of the PHb\nwas excluded from the study. This information is included in the methods. We note that we did not observe viral\nspread that extended medially enough to reach the PVT, and we did not observe infection of PVT neurons in\nany included animals.\n2. The specificity of the GAD-Cre mouse for GABAergic neurons in PHb is not well established. While it may be\nselective in some brain regions, this has not been demonstrated for PHb. The authors' own images show clear\nlabeling of some cells in the lateral habenula, which is almost entirely glutamatergic, implying that their virus and\nmouse combination may not be exclusively targeting GABAergic neurons.\nAlthough the specificity of GAD-Cre lines can vary across brain regions, previous work has shown that the PHb\ncontains a well-defined population of GABAergic neurons. These neurons have been reported to be labeled by\nthe Gad2Cre reporter line3 and characterized by expression of GABA4 and co-expression of Gad2, Gad1 and Vgat\nmRNA3. This provides a biological basis for using GAD-Cre–dependent approaches in the PHb.\nRegarding the reviewer’s concern about labeling in the lateral habenula (LHb), we do observe occasional\nreporter-positive cells at the border with LHb. We interpret this to likely be labeling of perhaps a sparse\nGABAergic population at the border of the two brain regions. This represents only a small fraction of the neurons\nlabeled in the PHb. Notably, we include multiple other manipulations supporting a role for the PHb in LTTA\nincluding viral approaches that manipulate only PHb neurons projecting to the NAc (Fig. 3K-P) or only manipulate\nipRGC terminals in the PHb (Fig. 4E-I).\nThus, these multiple lines of evidence suggest a role for the PHb, not the LHb, in tuning LTTA behavior.\n3. The fiber photometry results do not fully support the conclusions drawn. There are concerns regarding data\ninterpretation. What does PHb activity in wild-type and Opn4 KO mice look like aligned to loom onset during the\nexposure phase? This would help clarify whether PHb neurons are responding to the looming stimulus itself.\nThe number of edge-to-threat zone (TZ) transitions during the test phase appears low (n=4 across 4 mice per\ngroup, Fig. 4J), which limits statistical power. Given this, the photometry data for transitions should be interpreted\ncautiously. Fig. 4I suggests that PHb activity in control mice is inhibited by the edge-to-TZ transition. This is\nunexpected given that ipRGC input to PHb is presumed excitatory. The authors should clarify how this result fits\nwith the overall increase in PHb activity in control mice during the test phase (Fig. 4G).\nWe have increased the sample size for fiber photometry recordings in the PHb (Now Fig. 5 and Suppl. Fig. 10).\nHigher sample sizes allowed us to expand our analyses, and we now include 1) transitions from edge to TZ in\nPre-exposure and Test phases of the LTTA paradigm; 2) calcium signal aligned to looming onset during the\nExposure phase of the LTTA paradigm; 3) animal transitions from TZ to edges in the Test phase of the LTTA\nparadigm, and 4) correlations of calcium signal and behavioral parameters during the Test phase of the LTTA\nparadigm.\nWith the new analysis, we observed that the PHb activity increased when mice move within the edges in control\nmice during the Test phase. Because control mice spend more time exploring the edges during the Test phase\n(i.e. TZ avoidance), it follows that they would exhibit an overall increase in this parameter in this phase compared\nto Opn4-/- mice where this phenomenon is not evident.\nIt is in fact difficult to predict PHb calcium responses during LTTA because we are measuring bulk calcium\nsignals under consistent ambient lighting conditions, not necessarily measuring light-evoked responses per se\nin the PHb.\nMinor comments:\n1. The introduction should provide more background on the ipRGC signaling cascade (light → melanopsin →\nGq cascade → glutamate signaling) to better justify the focus on this pathway.\nWe now include more background on the ipRGC signaling cascade in the introduction of the new version of the\nmanuscript.\n2. The discussion should include a clearer model of the proposed neural circuit. The claim that chemogenetic\nexcitation of PHb neurons reduces LTTA is intriguing, but it would be more convincing if excitation were restricted\nto only NAc-projecting PHb GABAergic neurons. Does activating PHb neurons indiscriminately have other\nbehavioral effects?\nIn our initial submission, we showed that inhibition (not excitation) of PHbGABA neurons causes deficits in LTTA.\nIn the resubmitted manuscript, we more fully examine the circuits within the PHb involved in LTTA (see also\nresponse to Point 3 from Reviewer 3 below). To do this we injected Cre-dependent Gq DREADDs in the PHb of\nVglut2Cre to specifically excite PHbGlu neurons. We then intraperitoneally injected either vehicle or CNO prior to\nboth the Pre-Exposure and Test phases of the LTTA paradigm. Chemogenetic excitation of PHbGlu neurons\ndisrupted LTTA (Fig. 3K-P), indicating that PHbGlu neurons are likely inhibited by PHbGABA neurons during LTTA.\nWe now include a working model of the proposed circuit in the new version of the manuscript (Suppl. Fig. 12).\n3. A simpler photometry analysis may improve interpretability: Consider analyzing transitions (edge→TZ and\nTZ→edge) only when not followed by another transition within a window where fluorescence could decay to\nbaseline. Compare average activity in the edge versus TZ across different phases (pre-exposure, exposure, and\ntest). This could reveal circuit-consistent activity patterns. Including a Fig. showing behavioral events overlaid\nwith fluorescence traces could help contextualize neural activity changes.\nAt the reviewer’s suggestion, we added new analyses and plots to the resubmission (Fig. 5 and Suppl. Fig. 10)\nincluding: 1) transitions from edge to TZ in Pre-exposure and Test phases of the LTTA paradigm; 2) calcium\nsignal aligned to the loom onset during the Exposure phase of the LTTA paradigm; 3) animal transitions from TZ\nto edges in the Test phase of the LTTA paradigm, and 4) correlations of calcium signal and behavioral\nparameters during the Test phase of the LTTA paradigm. Additionally, we added a correlation analysis involving\ndifferent behavioral and FP data parameters. We found that the z-score has a strong positive correlation with\nthe distance travelled in the edges in control mice. This suggests that PHb neuronal activity increases when mice\nare exploring the edges, compared to the TZ.\nReviewer #3\nIn this study, Aranda and colleagues introduces a conceptually interesting behavioral paradigm (LTTA) and\nexplores underexamined neural substrates. However, major methodological and interpretive flaws limit its\nimpact. The central hypothesis that ipRGCs drive LTTA via PHb-NAc projections remains inadequately\nsubstantiated due to insufficient circuit-level evidence and incomplete experimental validation. While the work\nholds promise, I recommend rejecting the manuscript in its current form. Substantial revisions are needed to\nbolster mechanistic claims and clarify the study's novelty. Specifically, the authors must rigorously validate the\nproposed pathway and better contextualize their findings within the broader literature on threat avoidance\ncircuits.\nMajor:\n1. Inadequate analysis of sex-specific effects: The reported estrous cycle-dependent variation in LTTA behavior\nin female mice remains superficially analyzed. Given the critical importance of sex-specific effects in behavioral\nstudies, the authors should expand their analysis of female cohorts across all estrous stages, including hormonal\nprofiling (e.g., estrogen/progesterone levels). They should investigate whether sex hormones modulate ipRGCs-\nPHb-NAc pathway activity. If feasible, key experiments should be repeated in female-specific cohorts to validate\ngeneralizability.\nWe take the reviewers point about the need for an expanded analysis of females in the LTTA paradigm if females\nare included in the manuscript. We have opted to follow Reviewer 1’s suggestion to remove the female data\nbecause it would more than triple the volume of data in the current manuscript, which is already substantial in its\ndata content. We will expand on the circuit architecture and hormone-dependent effects on LTTA in females in\na future manuscript.\n2. Unresolved local circuit interactions: While the authors demonstrate that silencing PHb GABA neurons (but\nnot PHb Glu neurons) disrupts LTTA (Fig. 3E-J), the experimental design does not fully address potential local\ninhibitory interactions. Specifically, PHb GABA neurons may tonically suppress PHb Glu neurons under baseline\nconditions. Silencing PHb GABA neurons could thus result in disinhibition of PHb Glu neurons, potentially\nconfounding the interpretation that PHb Glu neurons are functionally irrelevant to LTTA. To establish direct\ncausality, the authors should selectively activate PHb Glu neurons while monitoring LTTA behavior and quantify\nwhether artificial activation significantly alters time spent in the TZ.\nAs suggested, we expressed excitatory DREADDs in PHbGlu neurons and then injected CNO to activate PHbGlu\nneurons (Fig. 3K-P). As the reviewer predicted, i.p. injection of CNO led to disruption of LTTA, indicating that\nPHbGlu neurons are also involved in the local PHb circuit. We thank the reviewer for this insightful comment. We\nhave updated our results and discussion section to incorporate this new information into the manuscript and\nmodel.\n3. Insufficient validation of chemogenetic approaches: The validation experiments for assessing chemogenetic\nefficiency require substantial supplementation. Critical methodological controls, including both hM4Di and\nhM3Dq validation approaches, are conspicuously absent. Specifically, the key experimental evidence supporting\nchemogenetic efficiency claims in Fig. 3E and Fig. 4B lacks essential validation data.\nWe thank the reviewer for raising this important point. The manuscript now includes addition and/or clarification\nof multiple key points that support functionality of Gq and Gi DREADDs and rule out mislocalization or off-target\nCNO effects. These include:\n1. WT naïve mice were injected with Vehicle or CNO, to assess potential effects of i.p. CNO per se in LTTA\n(Supplementary Fig. 6A). There was no change in LTTA in the presence of CNO versus Vehicle, arguing\nagainst off-target CNO impacts on LTTA.\n2. We performed additional chemogenetic manipulations to more specifically test the functional role of PHb-\nipRGCs in LTTA. We combined intravitreal injections of Gi-DREADDs with axon-terminal-specific Vehicle\nor CNO delivery using guide cannulas in PHb followed by exposure to the LTTA assay (Fig. 4E-I,\nSupplementary Fig. 7C and Reviewer Fig. 2). Inhibition of ipRGC terminals resulted in LTTA deficits,\nfurther supporting a role for PHb-ipRGCs in LTTA.\n3. We performed additional chemogenetic manipulations to test functional role of PHbGlu neurons in LTTA.\nWe injected Gq DREADDs in PHb of Vglut2Cre mice followed by exposure to the LTTA assay (Fig. 3K-P).\nExcitation of PHbGlu neurons caused deficits in LTTA, suggesting these neurons also play a role in this\nbehavior.\n4. We performed additional chemogenetic manipulations to test functional role of PHb-NAc neurons in\nLTTA. We combined injections of Gi-DREADDs in PHb with axon-terminal-specific Vehicle or CNO\ndelivery using guide cannulas in NAc followed by exposure to the LTTA assay (Fig. 6J-N,\nSupplementary Fig. 11-G-H and Reviewer Fig. 2). Inhibition of NAc-PHb neurons caused deficits in LTTA, further\nsupporting the conclusion that NAc-projecting PHb neurons are required for LTTA.\n5. We have performed anatomical validation of properly localized viral expression and/or guide cannula\nplacement for all animals and clearly state this in the methods.\nNotably, cannula-delivered CNO results replicate those found with i.p. CNO injection, providing a second,\nconverging line of evidence supporting our original interpretations. Inhibitory and excitatory DREADD\nmanipulations throughout the manuscript drive the expected, opposing phenotypes on LTTA behavior, providing\nsupport for the fact that the Gi and Gq DREADDs manipulations are working as expected and in opposing ways.\nMoreover, CNO injection in WT naïve mice does not impact LTTA compared to Vehicle, allowing us to rule out\noff target, surgical, or viral-induced impacts on this behavior. Our anatomical validation across multiple sets of\nexperiments support a role for the PHb and NAc in the LTTA circuit. Combined, these results and controls\nstrengthen support for our interpretation of the various DREADD manipulations. We have expanded and clarified\ndiscussion of existing control experiments and combined it with discussion of newly added data.\n4. Weak mechanistic validation: The proposed link between ipRGCs and the PHb-NAc pathway in regulating\nLTTA lacks robust validation. Current evidence, relying primarily on behavioral and limited electrophysiological\ndata, fails to establish a conclusive causal relationship at molecular or cellular levels. The authors should\nincorporate molecular/cellular validation (e.g., conditional knockout models, pharmacological interventions, or\noptogenetic manipulation of the ipRGCs-PHb-NAc pathway) and include recordings of NAc neuronal activity\nduring LTTA behavior to strengthen the proposed mechanism.\nWe have now added experiments to show necessity and sufficiency of ipRGCs, PHbGlu, PHbGABA, and PHb-NAc\nneurons. Below we outline the manipulations that support involvement at each node of the circuit. We hope the\nreviewers will agree that there is extensive evidence to support each node. While we agree that additional\nmolecular or mechanistic evidence will be key points for future research, additional work at the molecular and\ncellular level is beyond the scope of the current, already extensive, study.\nEvidence for ipRGC involvement in LTTA\n• Opn4-/- mice show disrupted LTTA (Fig. 2A-C and Suppl. Fig. 2)\n• Opn4fl/fl mice show disrupted LTTA following adult removal of the Opn4 gene (Fig. 2D-H and Suppl. Fig.\n4)\n• Brn3bDTA mice lacking Brn3b positive ipRGCs show disrupted LTTA (Fig. 2L-N and Suppl. Fig. 4)\n• Vglut2cKO animals lacking Vglut2 in ipRGCs show disrupted LTTA (Fig. 2I-K and Suppl. Fig. 4)\n• Gq DREADD activation of PHb-ipRGCs in Opn4-/- animals show rescue of disrupted LTTA (Fig. 4A-D\nand Suppl. Fig. 7)\n• Gi DREADD inhibition of PHb-ipRGC terminals in control animals show disrupted LTTA (Fig. 4E-I and\nSuppl. Fig. 7)\n• Opn4-/- mice show altered PHb signaling during LTTA (Fig. 5)\nEvidence for PHb involvement in LTTA\n• cFos induction during Test phase in exposed control animals but not unexposed control or Opn4-/- animals\n(Fig. 3A-C)\n• Gi DREADD inhibition of PHbGABA neurons causes disrupted LTTA (Fig. 3E-J)\n• Gq DREADD activation of PHbGlu neurons causes disrupted LTTA (Fig. 3K-P)\n• Gq DREADD activation of NAc-PHb neurons in Opn4-/- animals show rescue of disrupted LTTA (Fig. 6D-\nI)\n• Gq DREADD activation of PHb-ipRGCs in Opn4-/- animals show rescue of disrupted LTTA (Fig. 4A-D\nand Suppl. Fig. 7)\n• Gi DREADD inhibition of PHb-ipRGC terminals via cannula delivery of CNO in control animals show\ndisrupted LTTA (Fig. 4E-I and Suppl. Fig. 7)\n• Opn4-/- mice show altered PHb signaling during LTTA (Fig. 5)\nEvidence for NAc involvement in LTTA\n• Gq DREADD activation of NAc-PHb neurons in Opn4-/- animals show rescue of disrupted LTTA (Fig. 6D-\nI)\n• Gi DREADD inhibition of NAc-PHb terminals via cannula delivery of CNO in control animals show\ndisrupted LTTA (Fig. 6J-N and Suppl. Fig. 10)\n5. Unclear rationale for experimental timeline: The justification for selecting a 2-day post-stimulus interval in the\nLTTA experimental design requires clarification. The authors should provide direct empirical evidence supporting\nthe temporal specificity of ipRGC and PHb neuronal plasticity measurements following looming stimulation. This\ncould include either:\n•Citations to established temporal patterns of threat-conditioned neural plasticity in this specific pathway, or\n•Time-course validation data demonstrating that 48 hours represents the optimal window for detecting stimulus-\ninduced changes in the measured parameters.\nThe manuscript would be strengthened by addressing whether published literature demonstrates that\nneuroplastic changes (e.g., dendritic remodeling, synaptic protein expression, or electrophysiological alterations)\nin ipRGC and PHb neurons exhibit peak manifestation precisely at 48 hours post-stimulation, or if pilot studies\nshowed differential outcomes between 2-day and longer-term (e.g., 5-day) intervals.\nWe thank the reviewer for this comment. In pilot studies (Reviewer Fig. 3), we tested 2 and 4 days as different\nretention periods for the Test phase followed by 6.6 % Michelson contrast in the Exposure phase. While mice\nexposed to the Test phase 2 days after the Exposure showed robust LTTA behavior, mice exposed to the Test\nphase after 4 days of the Exposure phase did not present TZ avoidance, comparable to LTTA levels of the\nstandard 0% Exposure mice (2 days of retention period). Therefore, we decided to use 2-day post-stimulus in\nthe LTTA paradigm."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Based on the author's responses, which have addressed the vast majority of my concerns, I believe the manuscript in its current form is now suitable for publication in Nature Communications. Congratulations！", "reviewer_2": "The authors have sufficiently address all of my comments. I have no further concerns and find the paper suitable for publication.", "reviewer_3": "The authors have addressed my concerns. This reviewer has no further comments."}, "author_response": ""}]

Decision letter