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This review reflects comments and contributions from Femi Arogundade, Prithviraj Rajebhosale & Ryan Cubero. Review synthesized by Ryan Cubero

In this preprint, Hong and colleagues delve into the significance of calcium-permeable AMPA receptors (CP-AMPARs) in shaping the low feature selectivity of parvalbumin-positive (PV) interneurons in the brain. By lowering CP-AMPAR levels in PV interneurons, the study demonstrates an increased feature selectivity without significantly changing connection rates. Their findings highlight the role of CP-AMPARs in influencing the sensory representation of PV interneurons and shed light on the computational distinctions between inhibitory and excitatory neurons. The study raises questions about the broader implications of CP-AMPARs in neural circuits and their potential impact on intelligent behavior.

Overall, we believe that the paper’s strengths enumerated below make their findings compelling and highly valuable to the scientific community:

• The study provides strong insights into the role of CP-AMPARs in shaping the selectivity of PV interneurons. It highlights the previously underappreciated influence of these receptors on the sensory representation of inhibitory neurons, expanding our understanding of how neural circuits process information.

• The study encompasses a diverse array of animal models, encompassing ferrets, rodents, marmosets, and even humans. This broad exploration across species enhances the strength of the results and implies a shared (and potentially conserved) molecular mechanism that influences both inhibitory and excitatory neurons.

• The study demonstrates a direct cause-and-effect connection between the presence of CP-AMPARs and the limited feature selectivity observed in PV interneurons. Through targeted alterations in the expression of these receptors, the researchers modified the selectivity of PV interneurons, providing compelling evidence of the crucial role these receptors play in shaping the way neural responses are generated.

• The study offers valuable understanding regarding the consequences of changes in AMPAR composition for neurological disorders. It delves into the significance of these findings for conditions like intellectual disability and behaviors associated with AMPAR mutations in individuals with autism, shedding light on their potential implications.

• The study combines a range of methodologies, such as electrophysiology, transcriptomics, and computational modeling. This interdisciplinary strategy enriches the depth of the results and reinforces the credibility of the conclusions drawn from the study.

• The authors offer details regarding the accessibility of data and code utilized in the research. This transparent approach promotes reproducibility and empowers the scientific community to validate and expand upon the study's findings.

Moreover, we do believe that the following aspects need to be addressed to strengthen the observations, interpretation and implications made in the study. We have organized them as either major or minor aspects to be addressed. We also note some avenues where the authors can improve on reporting, and outlined ideas for future experiments.

Major aspects that needs to be addressed:

• The authors remove CP-AMPARs from PV interneurons by overexpressing GluA2 and demonstrate that this results in gain of direction and orientation selectivity. Next, they make the point that increasing presence of CP-AMPARs by GluA2 knockout in pyramidal neurons results in a loss of direction and orientation selectivity. While they do see a reduction in OSI and DSI indices (Fig. 4), the representative traces that the authors show in Fig. 4b and the plot in Fig. 4e show that the major effect is an overall increase in the amplitude of responses whereas the selectivity is largely maintained compared to effects seen in PV interneurons. The authors provide detailed verification of alterations in AMPA subunit expression, and rectification upon manipulating GluA2 expression in PV interneurons. Similar verification detailing possible changes to subunit composition and verification of a change in rectification is lacking for the pyramidal neuron experiments. Providing this could help support a discussion regarding the cell-type specific effects.

• Furthermore, how is the selectivity of excitatory neurons affected by the increased selectivity of PV interneurons with more GluA2? Will one expect a shift in the distribution of direction/orientation selectivity of excitatory neurons when lowering CP-AMPAR levels?

• The authors make a convincing argument that PV interneurons’ lack of selectivity is due to presence of CP-AMPARs and not solely because they receive dense excitatory input; that CP-AMPARs likely mask the selectivity. On the other hand, it is possible that the contribution of pyramidal neuron connectivity being sparse is a major driver for selectivity within this cell type and increasing CP-AMPARs is insufficient to drown this effect. If the authors can demonstrate a measurable effect on behavior on a perceptual decision making task in both PV-Cre;lsl-eGFP-GluA2 and GluA2 knockout mice, they could convince readers that while the effect on selectivity is mild, it is meaningful. 

• It is known that inhibitory neurons form connections among each other. Furthermore, it has been shown in Pfeffer et al., 2013 (Nature Neuroscience) that PV interneurons strongly inhibit each other. Could the increased feature selectivity also arise from these types of inhibitory interactions, i.e., increased inhibition in offsets from preferred stimuli? Moreover, how will connections between PV interneurons and other interneuron types be affected by a decrease in AMPAR calcium permeability?

• It is very interesting to note that the authors only see a difference in GluA2 transcriptomic expression between the two conditions, while seeing excitatory-like responses. Have the authors found any other differentially expressed gene in their dataset? Do these differentially expressed genes correspond to those genes that are differentially expressed between Pvalb cells vs glutamatergic neurons from the Allen Brain transcriptomic atlas?

Minor aspects:

• The discussion could benefit from adding a section on what is known regarding selectivity in SST and VIP interneurons. Recent reports suggest that SST interneurons have low selectivity like PV interneurons, whereas VIP interneurons display more selectivity on some measures - See Milman JD., et al. 2020 (eLife) and Guy J., et al. 2023 (Cell Reports). These findings might help strengthen the arguments made by the authors.

• The authors have chosen mice at P32-P62 and P69-P77 in their experiments. What was the rationale behind using these specific age ranges?

• Were there any challenges or potential biases in identifying PV interneurons based on eGFP or SEP-GluA2 expression?

• Are there any modifications or improvements that could be made to the FICSRseq workflow for even greater accuracy or efficiency?

• Transcriptomically, Pvalb and Sst are tightly linked clusters and exhibit a continuous phenotype. Does the R:Q ratio also exhibit a continuum, going from high to low as one traverses from Pvalb cells towards Sst cells, or is this stable across the cluster and only change close to their overlap?

Comments on reporting:

• Providing Fig 2f, 3b, 4b as mean +/- SEM would aid in transparency.

• In Fig 2 and 3, statistical comparisons have been done on technical replicates using a KW nonparametric ANOVA. This test assumes independence of data points within and between groups. The authors should perform statistics on biological replicates to check if the findings are robust to statistical testing. Same applies to the other comparisons authors are making in this manuscript using Mann-Whitney and t-tests. 

Suggestions for future studies:

• Extend the analysis to encompass various species and different regions of the brain. This approach will help determine whether the influence of CP-AMPARs on modulating neuronal selectivity remains consistent throughout evolutionary history and whether there are adaptations specific to each species.

• Furthermore, the authors can extend the analysis to consider cases of multiplexed neural codes. In this case, will lowering CP-AMPAR levels increase multiple modalities or is there a preference to certain features? 

• Examine whether disruptions in the expression or functioning of CP-AMPARs are connected to neurological or psychiatric disorders. Investigate the possibility of using CP-AMPAR manipulation as a therapeutic approach for conditions characterized by changes in inhibitory neural circuits.

We commend the authors for this extraordinary work and we wish them all the best in their future scientific pursuits!

Competing interests

The author declares that they have no competing interests.