Aging-Related Changes in Expression and Function of Glutamate Transporters in Rat Spinal Cord Astrocytes

Round 1

Reviewer 1 Report

Authors Sharan et al., investigated differences in the morphological and electrophysiological properties of astrocytes surrounding the dorsal and ventral horn motor neurons among three distinct age groups to address changes associated with normal aging. Authors hypothesized that the properties of astrocytes greatly depend upon the environment surrounding and ageing resulting into a differential response to the glutamate excitotoxicity. Authors report evidence of impaired glutamate regulation by astrocytes with aging and differential morphological and physiological properties of DH and VH spinal astrocytes.

Overall, the manuscript is well written and has potential to contribute to the literature. Authors have cited recent and appropriate references throughout the manuscript.

I do not have any major concern. However, I do have few minor corrections and suggestions to recommend.

1. While the introduction is well-structured, the extensive literature review can be somewhat overwhelming. I suggest either providing a more concise summary or transferring some of this content to the discussion section. Additionally, consider expanding on the last two paragraphs of the introduction, focusing on the significance and function of astrocytes in the context of VH and DH to enhance the study's objectives. This could contribute to further strengthen the goal of the manuscript.

2. The data in Figure 1A doesn't effectively convey the information presented in Figure 1D. This discrepancy might be due to the utilization of low-magnification images, where the astrocytic processes' signals seem comparable between juvenile and old groups for both DH and VH, in contrast to the young adult group. It would be beneficial to consider acquiring high-magnification images at 40-60x, as they could offer a more accurate representation of the estimated area data. Please comment and if feasible, incorporating corresponding high-magnification images would be valuable.

3. Multiple bold and non-bold comparison lines in the Fig 1B-D, 2E and 4B are confusing. To enhance clarity, it is advisable to eliminate non-significant comparisons from the histograms.

4. In Figure 2C and 3C, the EAAT1 immunostaining doesn't reveal discernible astrocytic cell bodies. It raises questions about the validation and titration of the antibodies, including the secondary antibodies used. To enhance the clarity of the images, it would have been beneficial to include high magnification inset images displaying both cell bodies and processes.

5. A varying font size of the label ‘Glu’ in Fig 4A can be misleading to some readers.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript, Sharan et al. examined the effect of aging on the cellular function of spinal cord astrocytes by measuring the expression level of glutamate transporters EAAT1 and EAAT2, astrocytes density and morphology, as well as glutamate uptake function in the dorsal horn and ventral horn of the spinal cord. Using a combination of western blotting, immunostaining followed by quantitative image analysis, and whole cell patch clam electrophysiology, the authors discovered inherent differences in astrocyte density, EAAT1/2 expression, and glutamate update capability in differently aged mice. Further differences were revealed between the astrocytes located in the dorsal horn and the ventral horn. Given the important homeostatic role along with many others possessed by astrocytes, these results in which dorsal horn and ventral horn astrocytes experience differential changes with aging, provide an explanation for the vulnerability observed for the motor neurons residing in the ventral horn compared to the sensory neurons in the dorsal horn.

Overall the manuscript is clearly written with an emphasis on the differential changes observed for spinal cord astrocytes in the DH and VH with aging. This study is important because it used an in vivo model to examine the functions of astrocytes. The current version of the manuscript falls short of the publication criteria and I have listed my major comments below:

1.       The introduction is too long and all over the place.

(1)    Not sure the importance of having this paragraph (line 79-84).

(2)    GFAP level was mentioned several times, could you either combine or remove some redundancy? Further, none of your results was related to GFAP level, despite the fact that an antibody against GFAP was listed as one of the reagents used in the method section.

(3)    Too many short paragraphs between line 75 -line 99. What is the key message?

(4)    It’s very easy to get lost going through your introduction in its current way of presentation. I suggest you cut the length in half to make it read more smoothly.

2.       Description of results not consistent with the data shown in figures.

(1)    Take Fig 1B for example, the Y axis values are completely off. On line 168, the value was mentioned as 131 while on the graph it’s around 250. The sample applies to figures 1C, 1D, 1E.

(2)    I was confused with your quantification of S100B-labeled astrocyte density in young adults between VH and DH (as shown in Figure 1C), because with the images provided, it’s not clear to me that VH had a lower density than the DH.

(3)    To better illustrate the morphological complexities of the processes of the astrocytes (line 189-193), could you provide some close up images of the cells?

(4)    How is glial coverage defined on line 198?

(5)    Line 201-202, the fold change of 48 is clearly wrong based on the values provided in the text, from 37 to 456.

(6)    Line 256-258, the text reads “EAAT1 expression significantly decreased in the DH, while VH remained unchanged” when referring to Fig2B. I can not tell if in DH there was really that much decrease, but I can clearly see that VH had a significant increase in EAAT1 based on the western bot you provided. Please also confirm if your quantification was normalized using beta-actin control.

(7)    Quantification not consistent with the images shown in  Fig 2C and 2F (line 266-268)

(8)    line 286, the text reads “the VH of the young adult and old groups exhibited higher EAAT2 expression”. However, it should be LOWER.

(9)    Line 288-294. The description of the distribution pattern of EAAT1 and EAAT2 does not seem to align properly with the images shown in figure 2 and 3. For example, line 290, it says “EAAT1 immunoreactivity was predominantly observed in lamina II…”, but on the image layer II was basically devoid of signal. Is the sectioning not accurate on the image? Same for the description of EAAT2.

3.       The last sentence of your discussion was not complete. Please elaborate more on “it will be interesting to elucidate”.

English language does not have too much problem except for some places the choice of words was odd. However, the introduction section must be improved from its current state (explained in the previous section)

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

 the authors have mostly addressed my comments.