PSD-95 drives binocular vision maturation critical for predation

Reviewer #1 (Public review):

Summary:

PSD95 has long been studied in detail to understand molecular mechanisms of synaptic plasticity as related to specific cell types (excitatory), circuits (visual cortex) and circuit development and function (ocular dominance plasticity ). While much was known about the molecular and cellular details of its function, it remained unclear whether and how it might contribute to the development of specific aspects of visual perception. While overall vision is preserved in PSD95 KO (Knockout) mice, studying natural, visually-guided prey capture behavior revealed robust, yet specific, perturbations to binocular processing during the behavior.

Strengths:

A major strength of the paper is being able to quantify precise measures of the visual aspects versus the motor aspects of prey pursuit. Comparing changes in behavior due to monocular occlusion was particularly revealing that mice indeed employ binocular summation to extract visual cues useful for prey pursuit. This result further suggested that in cases with poor binocular vision, monocular input can improve perceptual and behavioral processes as it does in human subjects with comparable challenges.

The study not only provided a useful finding regarding the function of PSD95, but also outlined a useful general approach toward identifying and quantifying specific deficits in binocular summation. This is likely to broadly impact studies of visual system development, behavior, and neural circuit function. The careful attention to details, observations, and openness about subject variance will also be helpful to those studying specific visual pursuit and natural prey capture behavior in the mouse.

Weaknesses:

Lack of eye movement monitoring and detailed head movement analysis preclude total certainty for the interpretation of observed behaviors.

Reviewer #2 (Public review):

Summary:

This manuscript studies the impacts of knocking out a protein known to be involved in synapse maturation in mice, measuring their ability to hunt prey items (and to discriminate simple visual patterns) under binocular and monocular viewing conditions. The main results are that the mice with this protein knocked out are impaired when performing visual tasks with binocular viewing, but are actually better when they perform monocularly. The interpretation is that the knocked-out protein has affected binocular visual integration.

Strengths:

Overall, the attempt to connect a protein to behavior/perception, via known mechanistic effects on synapse development and visual critical periods, is admirable.

The use of multiple visual conditions and behavioral paradigms (binocular/monocular, cricket hunting/orientation discrimination, light/dark) strengthens and enriches the results.

Weaknesses:

The primary interpretation - that binocular integration is affected in the PSD-95 knockouts- is not supported by the behavioral evidence. Using behavior to isolate a particular stage in visual processing (and further, to distinguish it from elements of generating the behavioral response and/or acquiring the visual information in the first place) is notoriously difficult. Such attempts are, of course, the domain of psychophysics. In fact, the most classical and loveliest success is in the domain of binocular integration- Bela Julesz's "psychoanatomy" that used random dot stereograms to isolate stereoscopic computations.

I mention this example because it is, in fact, directly relevant to my primary concern about the evidence used as support for the favored interpretation here. Julesz's stimuli were extremely clever in isolating binocular mechanisms (i.e., binocular mechanisms MUST be used to perform the task), and any perceptual/behavioral reports are very straightforward to interpret (i.e., a stereoscopically-defined shape can be identified, or not).

Now compare this to the work described in this manuscript. KO (knockout) mice are worse than wild types at chasing prey items or at moving towards a rewarded orientation, but they get better when performing this task monocularly. No argument that that is an interesting bit of scientific phenomenology to characterize. However, the behaviors do not require binocular integration, the freely-moving paradigms involve a variety of gaze and body-movement strategies, and the metrics used to quantify performance are similarly high-dimensional. Bottom line, it is not possible to glean whether the KO's intriguing binocular-vs-monocular differences are due to binocular integration per se, or something better thought of as fundamentally sensorimotor in origin. The tasks do not isolate visual from sensorimotor processing, and the behaviors and associated metrics cannot definitely adjudicate between a multitude of possible specific interpretations.

More specifically, the KO mice may have abnormal patterns of binocular coordination. Eye movements were not tracked in these studies, despite the availability of such instrumentation and their successful application in many preceding studies of mouse prey capture. If the KO mice do not coordinate their eye movements (in task-specific/task-relevant ways), they might receive binocular input that is abnormal. Under monocular conditions, that mismatched or inappropriately coordinated binocular input is absent, which would relieve them of the confusing visual information. That is rather different than having an impairment of binocular integration, as it is basically a question of whether the visual system is impaired, or whether the inputs to the visual system are abnormal due to differences in binocular coordination.

It is also possible that the binocular deficit, as measured in behavior,r occurs in a distinct part of the sensorimotor loop. Even if the binocular eye movements are normal, and binocular visual integration is normal, PSD-95 KO mice may be confused or distracted by the larger visual field that comes from binocular viewing (quite profound in species with mostly lateralized eyes). Such a "post-sensory" interpretation related to target selection (from what could be a totally normal visual representation) is difficult to rule out as well.

In summary, this reviewer appreciates the value of trying to connect this molecular mechanism to sensory processing and behavior. The use of naturalistic tasks and freely-moving paradigms is also something to commend. However, the sorts of visual stimuli and behavioral paradigms used here are not well-suited to supporting the rather specific interpretation that has been put forth in this manuscript.

Reviewer #3 (Public review):

Summary:

Bhattacharya et al. describe significant differences in prey capture behaviour in PSD-95 KO (Knockout) and wild-type (WT) mice. This work develops logically from their previous findings that KO of PSD-95 inhibits the maturation in the primary visual cortex. However, their previous work revealed that the visual deficits in the KO mice were relatively modest. Here, by employing an ethologically-relevant behavioural task, they show that several aspects of prey capture are impaired in the KO. Importantly, the deficits in predatory behavior in the KO mouse improved with monocular deprivation, consistent with deficits in binocular vision.

Strengths:

Overall, the data presented are convincing and valuable, and support the idea that PSD-95 expression is important for the maturation of visual responses.

Weaknesses:

The manuscript could be strengthened by consideration of the following points:

(1) The deficits in predatory behavior are interpreted to reveal several possible visual defects, including the absence of binocularity, binocular summation, or binocular mismatch in V1 neurons. Yet this is done with insufficient detail about each possible mechanism and without direct neuronal evidence.

(2) The observation that binocular visual field bias is intact in the PSD-95 KO mice is interesting but appears to contradict other data suggesting the absence of binocularity in the KO visual system, and this is not discussed in sufficient detail.

(3) No consideration of previous work using constitutive PSD-95 KOs that documented a learning deficit.

(4) Throughout the manuscript, including the first paragraph of the discussion, the authors state that "This study explored whether the maturation of CP closure, inhibited by PSD-95 influences binocular visual behaviour". However, if this were the case, the current experiments would have compared cricket capture behavior at two ages across the two genotypes: pre- and post-CP closure in WTs and at matching chronological ages in KOs.

(5) Freeman and others have shown that the influence of binocular summation on orientation discrimination is highest at low stimulus contrast and short duration stimuli. How does this impact the interpretation of predatory behavior and discrimination in the VWT?

Author response:

We thank the reviewers for their thorough and constructive evaluation of our manuscript titled “PSD-95 drives binocular vision maturation critical for predation”. The reviewers raised several important conceptual and technical points. Here, we address and provide additional context on the major themes and outline our planned revisions.

We acknowledge that the current prey capture task cannot directly adjudicate between PSD-95 binocular vision impairments or sensorimotor processing deficits. However, we did not observe any major impairment supporting a sensorimotor processing deficit, in contrast to a major impairment in line with binocular vision impairment. Evidence from Huang et al. (2015) [1], Favaro et al. (2018) [2] and our data with the visual water task (VWT) — thus requiring identical sensorimotor but differential visual processing—clearly demonstrated intact visual acuity but impaired orientation discrimination in PSD-95 KO mice. Therefore, we believe that a binocular integration deficit is the most likely explanation of PSD-95 KO binocular impairments. In line with this, it is unlikely that aberrations in binocular eye movements account for the observations. We appreciate that alternative explanations remain possible and merit explicit discussion. Accordingly, we intend to expand the discussion of these alternatives.

Importantly, we will provide additional experimental data demonstrating that knock-down of PSD-95 in V1 but not in superior colliculus, significantly decreases orientation discrimination analyzed with the VWT, as we had shown for PSD-95 KO mice (while control knock-down does not have this effect). We believe that this new evidence better delineates the potential neuroanatomical locus of the PSD-95-associated deficits.

Furthermore, we will provide additional head movement analyses, as suggested by Reviewer 1. Specifically, we will investigate the head angle in relation to the cricket (azimuth) in time (±1 second) around prey contact under light and dark conditions.

We will also address the potential impact of PSD-95 KO learning deficits. We agree that there are more impairments in the PSD-95 KO brain, as has been published previously. But strikingly, the binocular impairment was dominating the sensory processing. This cannot be convincingly explained by learning deficits. In fact, we have observed improved learning of PSD-95 KO mice with some tasks (e.g. cocaine conditioned place preference) [3], but no significant differences in the VWT [1,2]. Learning differences were described for another PSD-95 mouse line, expressing the N-terminus with two PDZ domains [4]. To avoid potential learning dependent confounds, we have chosen salient stimuli, like water aversion, and prey capture to reduce impacts of potential learning defects.

We agree on the strength of the random dot stereograms to isolate stereoscopic computations. However, it requires special filters in front of either eye, which renders it unsuitable for the VWT. The lengthy training with less silent stimuli of water reward, could potentially add additional confounds of PSD-95 KO deficits. Thus, we think that this would be something for future experiments to allow for integration of different visual inputs. However, the combined improved performance of WT mice with binocular vision for prey capture (depth percept) and orientation discrimination (summation) is already supporting the importance of binocular vision in mice and the dominant defect in PSD-95 KO mice.

Finally, we will address the other points raised by the reviewers through clearer exposition and reorganization of the manuscript.

Once again, we would like to thank the reviewers for their thoughtful and constructive feedback, which we believe will substantially strengthen the manuscript.

(1) Huang, X., Stodieck, S. K., Goetze, B., Cui, L., Wong, M. H., Wenzel, C., Hosang, L., Dong, Y., Löwel, S., and Schlüter, O. M. (2015). Progressive maturation of silent synapses governs the duration of a critical period. Proc. Natl. Acad. Sci. 112, E3131–E3140. https://doi.org/10.1073/pnas.1506488112.

(2) Favaro, P.D., Huang, X., Hosang, L., Stodieck, S., Cui, L., Liu, Y., Engelhardt, K.-A., Schmitz, F., Dong, Y., Löwel, S., et al. (2018). An opposing function of paralogs in balancing developmental synapse maturation. PLOS Biol. 16, e2006838. https://doi.org/10.1371/journal.pbio.2006838.

(3) Shukla, A., Beroun, A., Panopoulou, M., Neumann, P.A., Grant, S.G., Olive, M.F., Dong, Y., and Schlüter, O.M. (2017). Calcium‐permeable AMPA receptors and silent synapses in cocaine‐conditioned place preference. EMBO J. 36, 458–474. https://doi.org/10.15252/embj.201695465.

(4) Migaud, M., Charlesworth, P., Dempster, M., Webster, L.C., Watabe, A.M., Makhinson, M., He, Y., Ramsay, M.F., Morris, R.G.M., Morrison, J.H., et al. (1998). Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396, 433–439. https://doi.org/10.1038/24790.