November 24, 2020

Re: PONE-D-20-04655

“A longitudinal characterization of sex-specific somatosensory and spatial memory deficits in HIV Tg26 heterozygous mice”

Editor:

Responses to Reviewer #1

Reviewer #1: In this paper, the authors carried out a longitudinal study to characterize sensory and spatial memory deficits in a non-infectious model of HIV-1 (Tg26 mice). This HIV transgenic line contains an integrated transgene encoding the entire NL4-3 HIV-1 genome with a deletion of the gag/pol genes (on a C57Bl/6J background). The stability of the transgene on this background allowed the authors to test its effects across the lifespan of the animals, thus assessing the progression of HIV associated neurological disorders (HAND) with age. This is a significant advancement of previous studies that have used a cross sectional design to study HAND in either young or aged mice. In addition, the authors studied the progression of HAND in males versus female mice. They found that while both sexes did not show abnormal reflexive responses or weight loss due to viral gene expression, both males and female transgenic mice developed hyposensitivity to heat at 10 months of age. However, this hyposensitivity is evident in females even at 2.5 months of age. The authors then used 2 behavioral tasks to assess spatial memory – novel object location and the Barnes maze. The authors again saw an effect of gender, with female transgenic mice showing more severe deficits in spatial memory compared to males and manifesting at the earlier age of 3 months of age compared to 8 months in males. Finally, the authors did not find any change in tibial bone attributes due to viral gene expression. In conclusion, this study highlights important differences in the progression of HIV induced neurological deficits in male versus female mice as well as establishes the HIV-1 Tg26 mouse as a promising model of HAND.

Response: Thank you for the nice synopsis.

Major points:

1. Given that the authors use a number of subjective scoring techniques (the SHIRPA behaviors), they need to give a more detailed description of what constitutes an ‘abnormal’ response in each situation and how these were translated to a quantitative score (especially in Fig. 2F and Supplementary Fig.1).

Response: A detailed explanation of the scoring method was provided in a newly provided Table 1 and more explanation into the text of the methods.

2. The authors need to describe how the discriminative index score was calculated for the novel object location test. The authors should also indicate the chance level of novel object location exploration. With the possible exception of 2.5 mo Tg26-/- male mice, all the other groups have poor discriminative scores at the 1 week time point. Without a probabilistic measure of chance exploration, we cannot determine if these mice remember the novel location at all. Also, given that most studies using this paradigm use 24h as a measure of long term memory, why did the authors test 7 days later?

Response:

A description of the discriminative index score has been added to the methods section on page 10: “Discrimination between the objects was calculated using a discrimination ratio, calculated as the absolute difference in the time spent exploring the novel and familiar objects, divided by the total time spent exploring the objects [1-3]. This calculation takes into account the individual differences in the total amount of exploration [1-3].” This method reduces the possibility of chance exploration.

Timing of novel object location tests: We used a time choice of one week to match the Barnes timing. We may have missed a best long-term retention time by the wild type mice. The reason for the timing was added to the methods, and the potential weakness of this design was added to the discussion.

3. While the authors have used a variety of parameters to study the spatial memory of mice in the Barnes maze, they have not included some of the more standard measures, such as total errors or strategy use during the retention tests. Given the genotype differences in running speed and distance travelled seen in a previous study of these mice (Putatunda et al. 2019, Ref. 3) as well as the variability in running speed observed in Supplementary fig 3. , the above parameters may be a more accurate measure of spatial memory than latency to the target hole. Measurements of strategy use (direct search versus serial search) would especially add value to the conclusion made in page 22 line 22.

Response:

a. We appreciate the suggestion. Total errors have now been calculated and are shown below. Unfortunately, as shown by several other researchers [4-8], a calculation of total errors into the target hole was less sensitive than a measurement based on locating the escape hole using a nose to head defection (a primary error measurement) at the first encounter of the mouse with the escape hole. Also, as suggested by O’Leary in 2011 [8], mice trained with cues make fewer errors overall than mice trained without cues. Since our mice were all trained on the Barnes maze with olfactory cues, the total error measurements may be altered by the provision of cues and does not appear to accurately reflect what the mouse has learned. We added the cues part to our discussion. However, we did not add the less sensitive [in this study at least] “total error” calculation to the manuscript.

THIS FIGURE IS AVAILABLE IN THE RESPONSE ATTACHED WITH THE INDIVIDUAL FILES.

b. Distance around the Barnes maze per testing time point, and the velocity used around the Barnes maze, findings were moved from the Supplemental data section and is now presented in a new Figure 9. This data was reassessed from the raw data and then re-analyzed more stringently using the same 3 step statistical strategu as the other Barnes Maze data (all together first in a 3 way repeated measures model, and then after separating out by sex). This improved the findings and now shows that male Tg26 -/- mice moved more slowly around the maze during the acquisition Day 0 phase, similar to Putatunda et al, 2019 findings during the during the acquisition (see below).

c. To clarify the reviewer’s comments on the Putatunda et al 2019 findings versus the results of this study, in the Putatunda study, significant differences were observed only during the acquisition phases. In that study, we observed that female Tg26 +/- mice had longer distances but similar speeds during the initial acquisition phase, as female wild type mice; in contrast, male Tg26 +/- mice traveled the same distance, but at a slower speed, than the male wild type mice. This matches our Figure 9D and E results exactly. Further, no genotype differences were observed between the sexes during the retention/probe phases in Putatunda et al 2019, again matching our current findings exactly.

d. Measurements of strategy use (direct search versus serial search) would especially add value to the conclusion made in page 22 line 22.

Response:

We did present the strategy used during the retention tests in the first submission. It was presented as “path efficiency”. It is a quantitative AnyMaze software measurement of the direct versus serial search. The original “path efficiency” as measured by AnyMaze is the same method that we used in Putatunda et al, 2019 for reporting the search strategy and is necessary to keep for continuity between the studies. In this revision, we kept our original “path efficiency” as measured by AnyMaze in what is now Figure 8A , and page 24. We also added a manual method of strategy search and results (see below).

We have now defined path efficiency” in the methods page 12 and below in response # 9. It is defined as: “Path efficiency is a measure performed by the AnyMaze software and is an index of the efficiency of the path taken by the animal to get from the first position in the test (i.e., the center site of the maze where they are placed initially) to the last position (i.e., the target). For this, the software divides the straight line distance between the first position in the test and the last position by the total distance travelled by the animal during the test. A value of 1 indicates perfect efficiency - the animal moved in a straight line indicative of a “direct search” - values less than 1 indicate decreasing efficiency. The lowest value would be indicative of the most inefficient “serial search”.

Yet, to meet the reviewer’s concerns, the proportion of animals per group exhibiting a direct, serial versus random strategy for reaching the target hole was also manually scored from the AnyMaze plot tracts. These new results are shown in Figure 8B, and described on pages 12 and 24.

e. Also, to meet the reviewer’s concerns, we also added a summary of this finding to the conclusion.

4. The rationale behind analyzing tibial bones in the context of this study is unclear. The HIV Tg26 mouse line has been reported to show a range of symptoms including lymphomas, cardiomyopathy and inflammation as the authors have noted in their introduction. The authors’ decision to examine osteoclastogenesis as part of a study that focuses on the cognitive deficits associated with HIV needs to be further explained.

Response: We did not study osteoclastogenesis (no histology was performed). Instead, we novelly forwarded the characterization of bone morphology in Tg26 mice (similar to kidney, cardiac and bone morphology in past studies of Tg26 rats).

The rationale was expanded in the Introduction page 5, “As a consequence, mice heterozygous for the transgene develop many clinical features of HIV infection and AIDS including a disease similar to HIV-associated nephropathy (HIVAN), as well as changes in other tissues and organs, including, inflammation, B cell lymphomas, skin lesions consistent with cutaneous papillomas, cardiomyopathy, and muscle wasting between 3 and 6 months of age on the FVB/N background [9-12]. Interestingly, degenerative changes in bone, another key organ, have been observed in mature HIV-1 Tg heterozygous rats (NL4-3�gag/pol Fisher344 rats), compared to mature wildtype rats [13, 14]. However, possible bone degeneration that may occur as a result of altered general physical characteristics or neurological issues has yet to be examined in HIV mouse models.”

We also added rationale to the results section on page 29: “Degenerative changes in bone have been observed in mature HIV-1 Tg heterozygous rats (NL4-3�gag/pol Fisher344 rats), compared to mature wildtype rats [13, 14]. Thus, on study completion, tibial bones were collected at 11 months of age and the proximal ends assayed using ex vivo microCT to determine if there were bone degenerative changes in mature HIV Tg mice as a result of the general physical characteristics or the observed neurological issues.”

We would also like to direct the reviewer to page 34 of the Discussion where more is discussed on bone and other organ, and body weight differences between HIV Tg mice versus rats. We expanded this paragraph some as well.

Minor points:

1. The authors bred the Tg26 line by back crossing the original strain from the FVB/N background to a C57Bl/6J background. Did they analyze the genome of the new strain for SNP variations to confirm the C57Bl/6J background?

Response: The reviewer has a good suggestion for the future. Unfortunately, we did not examine the back-crossed animals for SNP variations. But we did perform traditional backcrosses for a minimum of 6 generations in order to establish the C57Bl/6J background, as mentioned in the text on page 6, line 9 -11. We also cited our past paper using the same back crossing method [15].

2. Page 7, Line 10-13 of the Methods section states that the Irwin Observation Test battery was used to measure indices of neuropsychiatric function. Page 7, Line 2 (legend for Fig. 1) then introduces the term SHIRPA, which is a behavioral screen developed in 1997 based on the Irwin battery. If the authors are using the SHIRPA screen (i.e. modified from the original Irwin battery) for their study, they should state this clearly in their methods, name the test in full and cite the original paper.

Response: We have re-written this section of the methods to be more clear (General health and neurological testing) and added a new Table 1 showing the exact tests used and their scoring methods. We are unsure which author(s) specifically the reviewer wants us to cite, as several authors modified the original Irwin screening method [16] in parallel in 1997 [17, 18]. Also, that 1997 modified SHIRPA version has been further modified and expanded by many researchers across the years in order to enhance the effectiveness of these screenings. Therefore, we also cited those papers of expansion and/or explanation as well (also needed since of the few of the earlier papers were not as clear regarding the methods as some of the latter) [19-25].

We provided a new Table 1 indicated exactly which tests were performed and the scoring system used. We also expanded in the methods on the subtest that we had to modify (the touch escape) in order to address the habituation of the mice to either the tester or the test across time.

3. Page 8, Line 20. The authors should elaborate on what constitutes ‘spontaneous behaviors’ and how these were quantitatively scored.

Response: A very detailed explanation of the scoring method was provided in a newly provided Table 1, and the text of the methods.

4. Page 9, Line 16-17. The authors need to elaborate on how the ‘magnitude of reaction’ was quantified and translated into the discriminative indices shown in Fig. 6.

Response: A discriminative index score was used. Its description has been added to the methods section, as were references: “Discrimination between the objects was calculated using a discrimination ratio, calculated as the absolute different in the time spent exploring the novel and familiar objects, divided by the total time spent exploring the objects [1-3]. This calculation takes into account the individual differences in the total amount of exploration [1-3].”

5. The results in Fig. 5, the authors have described differences in the discriminative index of female Tg26-/- mice. Were there any differences in the total time these mice interacted with all the objects? This could be another measure of anxiety as well as confound the interpretation of their discriminative ability.

Response: There was no significant difference in total time the mice interacted with all of the objects, between the groups in this longitudinal study. The touch escape results also indicate that there was no anxiety. The longitudinal nature of this study likely reduced anxiety in the mice as they were exposed to the same tester and testing room from 1 months of age for another 10 months.

We added this to the discussion: “In this current study, the 10 month long period of handling and testing gave ample opportunity for the mice to habituate to the tester (R.L. in each case), the room and the testing apparati, giving an underlying reason for a lack of anxiety in these Tg26 +/- mice. This lack of anxiety is further supported by the touch escape results, in which the mice began crawling into the tester’s hands rather than escaping during the 7 month testing point.”

6. Fig. 5C – The legend shows male Tg26 -/- mice are indicated with blue squares. However, the figure uses blue circles to represent this group.

Response: Thank you. Fixed.

7. Fig. 6E – The figure legend does not describe the * shown in this figure. What comparison does this signify?

Response: Thank you. This has been added to that figure legend: “*:p<0.05 and **: p<0.01, compared to wild type -/- mice of same sex.”

8. Page 21, Line 18-19. “Results are organized by time of detection of significant differences.” This statement is unclear.

Response: We removed this statement altogether as it was not really needed.

9. Fig. 7 – The authors need to elaborate on how path efficiency was calculated.

Response: The following was added to the methods: “Path efficiency is a measure performed by the AnyMaze software and is an index of the efficiency of the path taken by the animal to get from the first position in the test (i.e., the center site of the maze where they are placed initially) to the last position (i.e., the target). For this, the software divides the straight line distance between the first position in the test and the last position by the total distance travelled by the animal during the test. A value of 1 indicates perfect efficiency - the animal moved in a straight line indicative of a “direct search” - values less than 1 indicate decreasing efficiency. The lowest value would be indicative of the most inefficient “serial search”.

10. Page 24-25, Lines 21-5. At 8 months of age, wild type and transgenic mice of both sexes seem unable to distinguish the target hole versus any other hole in the Barnes maze (Fig. 8D). The non-target holes should not have any spatial valence for the mice and hence entry into these should be entirely a matter of chance. Given the above, I disagree with the authors that differences in entries into non-target holes at this time point (8 mo, 1 week) is indicative of any spatial memory deficits. The differences seen by the authors could be due to unforeseen underlying preferences or an artefact of a small sample size. As mentioned before, a calculation of errors would be a better indicator of spatial memory than the heading errors used in this study.

a. Response: We appreciate the suggestion. Total errors have now been calculated and are shown above. Unfortunately, as shown by several other researchers [4-8], a calculation of total errors into the target hole was less sensitive than a measurement based on locating the escape hole using a nose to head defection (a primary error measurement) at the first encounter of the mouse with the escape hole. Also, as suggested by O’Leary in 2011 [8], mice trained with cues make fewer errors overall than mice trained without cues. Since our mice were all trained on the Barnes maze with olfactory cues, the total error measurements may be altered by the provision of cues and does not appear to accurately reflect what the mouse has learned. Thus, we did not add the less sensitive [in this study at least] “total error” calculation to the manuscript.

We also added the same explanation for the test used, as we had placed in the Putatunda et al 2019 paper. We apologize for not including it in the first submission of this paper: “To assess whether Tg26(+/-) mice recognized the target hole in relation to the other holes on the maze, the number of times the mice entered each hole was measured during the short term (24 hours) and long term (14 days) probe sessions. This reference error metric is a robust and reliable method for assessment of hippocampal-dependent spatial memory [4, 26]. This same method was also used in a recent paper from the lab using a different cohort of Tg26 -/- and +/- mice [15], thus, allowing for comparison to that prior study.”

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