Laminar specificity and coverage of viral-mediated gene expression restricted to GABAergic interneurons and their parvalbumin subclass in marmoset primary visual cortex

Reviewer #1 (Public Review):

Summary:

Federer et al. tested AAVs designed to target GABAergic cells and parvalbumin-expressing cells in marmoset V1. Several new results were obtained. First, AAV-h56D targeted GABAergic cells with >90% specificity, and this varied with serotype and layer. Second, AAV-PHP.eB.S5E2 targeted parvalbumin-expressing neurons with up to 98% specificity. Third, the immunohistochemical detection of GABA and PV was attenuated near viral injection sites.

Strengths:

Vormstein-Schneider et al. (2020) tested their AAV-S5E2 vector in marmosets by intravenous injection. The data presented in this manuscript are valuable in part because they show the transduction pattern produced by intraparenchymal injections, which are more conventional and efficient.

Weaknesses:

The conclusions regarding the effects of serotype are based on data from single injection tracks in a single animal. I understand that ethical and financial constraints preclude high throughput testing, but these limitations do not change what can be inferred from the measurements. The text asserts that "...serotype 9 is a better choice when high specificity and coverage across all layers are required". The data presented are consistent with this idea but do not make a strong case for it.

A related criticism extends to the analysis of injection volume on viral specificity. Some replication was performed here, but reliability across injections was not reported. My understanding is that individual ROIs were treated as independent observations. These are not biological replicates (arguably, neither are multiple injection tracks in a single animal, but they are certainly closer). Idiosyncrasies between animals or injections (e.g. if one injection happened to hit one layer more than another) could have substantial impacts on the measurements. It remains unclear which results regarding injection volume or serotype would hold up had a large number of injections been made into a large number of marmosets.

Reviewer #2 (Public Review):

This is a straightforward manuscript assessing the specificity and efficiency of transgene expression in marmoset primary visual cortex (V1), for 4 different AAV vectors known to target transgene expression to either inhibitory cortical neurons (3 serotypes of AAV-h56D-tdTomato) or parvalbumin (PV)+ inhibitory cortical neurons in mice. Vectors are injected into the marmoset cortex and then postmortem tissue is analyzed following antibody labeling against GABA and PV. It is reported that: "in marmoset V1 AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 80% efficiency, depending on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency."

These claims are largely supported but slightly exaggerated relative to the actual values in the results presented. In particular, the overall efficiency for the best h56D vectors described in the results is: "Overall, across all layers, AAV9 and AAV1 showed significantly higher coverage (66.1{plus minus}3.9 and 64.9%{plus minus}3.7)". The highest coverage observed is just in middle layers and is also less than 80%: "(AAV9: 78.5%{plus minus}9.1; AAV1: 76.9%{plus minus}7.4)". For the AAV-PHP.eB-S5E2 the efficiency reported in the abstract ("86-90%) is also slightly exaggerated relative to the results: "Overall, across all layers coverage ranged from 78%{plus minus}1.9 for injection volumes >300nl to 81.6%{plus minus}1.8 for injection volumes of 100nl."

These data will be useful to others who might be interested in targeting transgene expression in these cell types in monkeys. Suggestions for improvement are to include more details about the vectors injected and to delete some comments about results that are not documented based on vectors that are not described (see below).

Major comments:

Details provided about the AAV vectors used with the h56D enhancer are not sufficient to allow assessment of their potential utility relative to the results presented. All that is provided is: "The fourth animal received 3 injections, each of a different AAV serotype (1, 7, and 9) of the AAV-h56D-tdTomato (Mehta et al., 2019), obtained from the Zemelman laboratory (UT Austin)." At a minimum, it is necessary to provide the titers of each of the vectors. It would also be helpful to provide more information about viral preparation for both these vectors and the AAVPHP.eB-S5E2.tdTomato. Notably, what purification methods were used, and what specific methods were used to measure the titers?

The first paragraph of the results includes brief anecdotal claims without any data to support them and without any details about the relevant vectors that would allow any data that might have been collected to be critically assessed. These statements should be deleted. Specifically, delete: "as well as 3 different kinds of PV-specific AAVs, specifically a mixture of AAV1-PaqR4-Flp and AAV1-h56D-mCherry-FRT (Mehta et al., 2019), an AAV1-PV1-ChR2-eYFP (donated by G. Horwitz, University of Washington)," and delete "Here we report results only from those vectors that were deemed to be most promising for use in primate cortex, based on infectivity and specificity. These were the 3 serotypes of the GABA-specific pAAV-h56D-tdTomato, and the PV-specific AAVPHP.eB-S5E2.tdTomato." These tools might in fact be just as useful or even better than what is actually tested and reported here, but maybe the viral titer was too low to expect any expression.

Based on the description in the Methods it seems that no antibody labeling against TdTomato was used to amplify the detection of the transgenes expressed from the AAV vectors. It should be verified that this is the case - a statement could be added to the Methods.

Reviewer #3 (Public Review):

Summary:

Federer et al. describe the laminar profiles of GABA+ and of PV+ neurons in marmoset V1. They also report on the selectivity and efficiency of expression of a PV-selective enhancer (S5E2). Three further viruses were tested, with a view to characterizing the expression profiles of a GABA-selective enhancer (h56d), but these results are preliminary.

Strengths:

The derivation of cell-type specific enhancers is key for translating the types of circuit analyses that can be performed in mice - which rely on germline modifications for access to cell-type specific manipulation - in higher-order mammals. Federer et al. further validate the utility of S5E2 as a PV-selective enhancer in NHPs.

Additionally, the authors characterize the laminar distribution pattern of GABA+ and PV+ cells in V1. This survey may prove valuable to researchers seeking to understand and manipulate the microcircuitry mediating the excitation-inhibition balance in this region of the marmoset brain.

Weaknesses:

Enhancer/promoter specificity and efficiency cannot be directly compared, because they were packaged in different serotypes of AAV.

The three different serotypes of AAV expressing reporter under the h56D promoter were only tested once each, and all in the same animal. There are many variables that can contribute to the success (or failure) of a viral injection, so observations with an n=1 cannot be considered reliable.

The language used throughout conflates the cell-type specificity conferred by the regulatory elements with that conferred by the serotype of the virus.

Author response:

eLife assessment

Unlocking the potential of molecular genetic tools (optogenetics, chemogenetics, sensors, etc.) for the study of systems neuroscience in nonhuman primates requires the development of effective regulatory elements for cell-type specific expression to facilitate circuit dissection. This study provides a valuable building block, by carefully characterizing the laminar expression profile of two viral vectors, one designed for general GABA+ergic neurons and the second for parvalbumin+ cell-type selective expression in the marmoset primary visual cortex. The authors provide solid evidence for the first enhancer S5E2 and incomplete evidence for the second one, h56D. This study contributes to our understanding of these tools but is limited by the understandably small number of animals used.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Federer et al. tested AAVs designed to target GABAergic cells and parvalbumin-expressing cells in marmoset V1. Several new results were obtained. First, AAV-h56D targeted GABAergic cells with >90% specificity, and this varied with serotype and layer. Second, AAV-PHP.eB.S5E2 targeted parvalbumin-expressing neurons with up to 98% specificity. Third, the immunohistochemical detection of GABA and PV was attenuated near viral injection sites.

Strengths:

Vormstein-Schneider et al. (2020) tested their AAV-S5E2 vector in marmosets by intravenous injection. The data presented in this manuscript are valuable in part because they show the transduction pattern produced by intraparenchymal injections, which are more conventional and efficient.

Our manuscript additionally provides detailed information on the laminar specificity and coverage of these viral vectors, which was not investigated in the original studies.

Weaknesses:

The conclusions regarding the effects of serotype are based on data from single injection tracks in a single animal. I understand that ethical and financial constraints preclude high throughput testing, but these limitations do not change what can be inferred from the measurements. The text asserts that "...serotype 9 is a better choice when high specificity and coverage across all layers are required". The data presented are consistent with this idea but do not make a strong case for it.

We are aware of the limitations of our results on the AAV-h56D. We agree with the Reviewer that a single injection per serotype does not allow us to make strong statements about differences between the 3 serotypes. Therefore, in the revised version of the manuscript we will temper our claims about such differences and use more caution in the interpretation of these data. Despite this weakness, we feel that these data still demonstrate high efficiency and specificity across cortical layers of transgene expression in GABA cells using the h56D promoter, at least with two of the 3 AAV serotypes we tested. We feel that in itself this is sufficiently useful information for the primate community, worthy of being reported. Due to cost, time and ethical considerations related to the use of primates, we chose not to perform additional experiments to determine precise differences among serotypes. Thus, for example, while it is possible that had we replicated these experiments, serotype 7 would have proven equally efficient and specific as the other two serotypes, we felt answering this question did not warrant additional experiments in this precious species.

A related criticism extends to the analysis of injection volume on viral specificity. Some replication was performed here, but reliability across injections was not reported. My understanding is that individual ROIs were treated as independent observations. These are not biological replicates (arguably, neither are multiple injection tracks in a single animal, but they are certainly closer). Idiosyncrasies between animals or injections (e.g. if one injection happened to hit one layer more than another) could have substantial impacts on the measurements. It remains unclear which results regarding injection volume or serotype would hold up had a large number of injections been made into a large number of marmosets.

For the AAV-S5E2, we made a total of 7 injections (at least 2 at the same volume), all of which, irrespective of volume, resulted in high specificity and efficiency for PV interneurons. Our conclusion is that larger volumes are slightly less specific, but the differences are minimal and do not warrant additional injections. Additionally, all of our injections involved all cortical layers, and the ROIs we selected for counts encompassed reporter protein expression across all layers. To provide a better sense of the reliability of the results across injections, in the revised version of the manuscript we will provide a supplementary table with results for each injection case separately.

Reviewer #2 (Public Review):

This is a straightforward manuscript assessing the specificity and efficiency of transgene expression in marmoset primary visual cortex (V1), for 4 different AAV vectors known to target transgene expression to either inhibitory cortical neurons (3 serotypes of AAV-h56D-tdTomato) or parvalbumin (PV)+ inhibitory cortical neurons in mice. Vectors are injected into the marmoset cortex and then postmortem tissue is analyzed following antibody labeling against GABA and PV. It is reported that: "in marmoset V1 AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 80% efficiency, depending on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency."

These claims are largely supported but slightly exaggerated relative to the actual values in the results presented. In particular, the overall efficiency for the best h56D vectors described in the results is: "Overall, across all layers, AAV9 and AAV1 showed significantly higher coverage (66.1{plus minus}3.9 and 64.9%{plus minus}3.7)". The highest coverage observed is just in middle layers and is also less than 80%: "(AAV9: 78.5%{plus minus}9.1; AAV1: 76.9%{plus minus}7.4)".

In the abstract, we indeed summarize the overall data and round up the decimals, and state that these parentages are upper bound and that they vary by serotype and layer, while in the Results we report the detailed counts with decimals. To clarify this, in the revised version of the Abstract we will change 80% to 79% and emphasize even more clearly the dependence on serotype and layer. We will amend this sentence of the Abstract as follows: “We show that in marmoset V1 AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer.”

For the AAV-PHP.eB-S5E2 the efficiency reported in the abstract ("86-90%) is also slightly exaggerated relative to the results: "Overall, across all layers coverage ranged from 78%{plus minus}1.9 for injection volumes >300nl to 81.6%{plus minus}1.8 for injection volumes of 100nl."

Indeed, the numbers in the Abstract are upper bounds, for example efficiency in L4A/B with S5E2 reaches 90%. To further clarify this important point, in the revised abstract we will state ”AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency, depending on layer”.

These data will be useful to others who might be interested in targeting transgene expression in these cell types in monkeys. Suggestions for improvement are to include more details about the vectors injected and to delete some comments about results that are not documented based on vectors that are not described (see below).

Major comments:

Details provided about the AAV vectors used with the h56D enhancer are not sufficient to allow assessment of their potential utility relative to the results presented. All that is provided is: "The fourth animal received 3 injections, each of a different AAV serotype (1, 7, and 9) of the AAV-h56D-tdTomato (Mehta et al., 2019), obtained from the Zemelman laboratory (UT Austin)." At a minimum, it is necessary to provide the titers of each of the vectors. It would also be helpful to provide more information about viral preparation for both these vectors and the AAVPHP.eB-S5E2.tdTomato. Notably, what purification methods were used, and what specific methods were used to measure the titers?

We thank the Reviewer for this comment. In the revised version of the manuscript, we will provide a Table with titers of each viral vector injected as well as more information regarding viral preparation methods. In fact, the methods for viral preparation and purification are detailed in the original publications so we feel it may be sufficient to cite the original papers?

The first paragraph of the results includes brief anecdotal claims without any data to support them and without any details about the relevant vectors that would allow any data that might have been collected to be critically assessed. These statements should be deleted. Specifically, delete: "as well as 3 different kinds of PV-specific AAVs, specifically a mixture of AAV1-PaqR4-Flp and AAV1-h56D-mCherry-FRT (Mehta et al., 2019), an AAV1-PV1-ChR2-eYFP (donated by G. Horwitz, University of Washington)," and delete "Here we report results only from those vectors that were deemed to be most promising for use in primate cortex, based on infectivity and specificity. These were the 3 serotypes of the GABA-specific pAAV-h56D-tdTomato, and the PV-specific AAVPHP.eB-S5E2.tdTomato." These tools might in fact be just as useful or even better than what is actually tested and reported here, but maybe the viral titer was too low to expect any expression.

This data is indeed anecdotal, and while we could delete it from the manuscript, as suggested by the Reviewer, we feel it could be useful information for the scientific community. It could prevent other labs from wasting resources, animals and time, particularly, as some of these vectors have been reported to be selective and efficient in the primate cortex, which we have not been able to confirm. We made several injections in several animals of those vectors that failed either to infect a sufficient number of cells or turned out to be poorly specific. Therefore, the negative results have been consistent. But we agree with the Reviewer that our negative results could have depended on factors such as titer. In the revised version of the manuscript, we will provide a supplementary Methods section in which we will report the specifics of the vectors that failed in our hands (i.e. number of injections made in how many animals, volumes, survival time, and titers).

Based on the description in the Methods it seems that no antibody labeling against TdTomato was used to amplify the detection of the transgenes expressed from the AAV vectors. It should be verified that this is the case - a statement could be added to the Methods.

That is indeed the case. We used no immunohistochemistry to enhance the reporter proteins as this was unnecessary. The native / non-emplified tdT signal was strong.

Reviewer #3 (Public Review):

Summary:

Federer et al. describe the laminar profiles of GABA+ and of PV+ neurons in marmoset V1. They also report on the selectivity and efficiency of expression of a PV-selective enhancer (S5E2). Three further viruses were tested, with a view to characterizing the expression profiles of a GABA-selective enhancer (h56d), but these results are preliminary.

Strengths:

The derivation of cell-type specific enhancers is key for translating the types of circuit analyses that can be performed in mice - which rely on germline modifications for access to cell-type specific manipulation - in higher-order mammals. Federer et al. further validate the utility of S5E2 as a PV-selective enhancer in NHPs.

Additionally, the authors characterize the laminar distribution pattern of GABA+ and PV+ cells in V1. This survey may prove valuable to researchers seeking to understand and manipulate the microcircuitry mediating the excitation-inhibition balance in this region of the marmoset brain.

Weaknesses:

Enhancer/promoter specificity and efficiency cannot be directly compared, because they were packaged in different serotypes of AAV.

The three different serotypes of AAV expressing reporter under the h56D promoter were only tested once each, and all in the same animal. There are many variables that can contribute to the success (or failure) of a viral injection, so observations with an n=1 cannot be considered reliable.

This is an important point that was also brought up by the Reviewer 1, which we thoroughly addressed in our comments. For clarity and convenience, we copied our response to Reviewer 1 below:.

We are aware of the limitations of our results on the AAV-h56D. We agree with the Reviewer that a single injection per serotype does not allow us to make strong statements about differences between the 3 serotypes. Therefore, in the revised version of the manuscript we will temper our claims about such differences and use more caution in the interpretation of these data. Despite this weakness, we feel that these data still demonstrate high efficiency and specificity across cortical layers of transgene expression in GABA cells using the h56D promoter, at least with two of the 3 AAV serotypes we tested. We feel that in itself this is sufficiently useful information for the primate community, worthy of being reported. Due to cost, time and ethical considerations related to the use of primates, we chose not to perform additional experiments to determine precise differences among serotypes. Thus, for example, while it is possible that had we replicated these experiments, serotype 7 would have proven equally efficient and specific as the other two serotypes, we felt answering this question did not warrant additional experiments in this precious species.

The language used throughout conflates the cell-type specificity conferred by the regulatory elements with that conferred by the serotype of the virus.

In the revised version of the manuscript we will correct ambiguous language.