Toxoplasma gondii injected neurons localize to the cortex and striatum and have altered firing
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This study uses a new approach to map all neurons in the brain that have been infected with Toxoplasma gondii or injected with parasite proteins. The authors show that Toxoplasma injected neurons are heterogeneously distributed in murine brain tissues, that excitatory neurons are the primary targets, and that injection of parasite proteins leads to neuronal death. This work provides new insights into Toxoplasma-neuron interactions that underlie the pathology and potential changes in behaviour of infected individuals. The manuscript will be of interest to those working in neuroscience and/or parasitic infections.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Microbiology and Infectious Disease (eLife)
Abstract
Toxoplasma gondii is an intracellular parasite that causes a long-term latent infection of neurons. Using a custom MATLAB-based mapping program in combination with a mouse model that allows us to permanently mark neurons injected with parasite proteins, we found that Toxoplasma -injected neurons (TINs) are heterogeneously distributed in the brain, primarily localizing to the cortex followed by the striatum. Using immunofluorescence co-localization assays, we determined that cortical TINs are commonly (>50%) excitatory neurons (FoxP2 + ) and that striatal TINs are often (>65%) medium spiny neurons (MSNs) (FoxP2 + ). As MSNs have highly characterized electrophysiology, we used ex vivo slices from infected mice to perform single neuron patch-clamping on striatal TINs and neighboring uninfected MSNs (bystander MSNs). These studies demonstrated that TINs have highly abnormal electrophysiology, while the electrophysiology of bystander MSNs was akin to that of MSNs from uninfected mice. Collectively, these data offer new neuroanatomic and electrophysiologic insights into CNS toxoplasmosis.
Article activity feed
-
Author Response:
Reviewer #1 (Public Review):
The primary strength of this paper is the attempt to characterize the neurons injected by Toxoplasma and the electrophysiological changes that ensue. Three major problems are however noted.
- Figure 1 attempts to identify regions of the brain more profoundly impacted by Toxoplasma and does so by normalizing the numbers of injected neurons to the size of the region. But since the reporter system used requires the parasite injected protein to interact with a neuron's nucleus, The authors claims can only be valid after normalizing not to size but to density of nuclei in a region. This is especially important in the cortex where different layers have distinct architectures.
We appreciate the Reviewer’s pointing out that neuron density may play a role in where we find TINs. For example, the …
Author Response:
Reviewer #1 (Public Review):
The primary strength of this paper is the attempt to characterize the neurons injected by Toxoplasma and the electrophysiological changes that ensue. Three major problems are however noted.
- Figure 1 attempts to identify regions of the brain more profoundly impacted by Toxoplasma and does so by normalizing the numbers of injected neurons to the size of the region. But since the reporter system used requires the parasite injected protein to interact with a neuron's nucleus, The authors claims can only be valid after normalizing not to size but to density of nuclei in a region. This is especially important in the cortex where different layers have distinct architectures.
We appreciate the Reviewer’s pointing out that neuron density may play a role in where we find TINs. For example, the limited number of neuron nuclei in/near white matter tracts is almost certainly what accounts for the “under enrichment” of TINs in white matter tracts (in a sense it is a nice built-in control that our system is working). We have noted this explanation in the results.
We also agree that neuron density could explain why TINs somas are enriched in deeper cortical layers and not in more superficial cortical layers. We will incorporate such considerations into future studies/analyses but feel that such re-analysis is beyond the scope of this paper for several reasons. First, prior T. gondii studies that have quantified cyst locations have used region size for normalization (Berenreiterova et al 2011; Boillat et al 2020). Thus, by using size ourselves, we could be consistent with these papers and draw connections to them. Second, for most of the major brain regions, relative size correlates with the number of TINs found in those regions, suggesting that size may adequately explain why a certain percentage of TINs is found in those regions (even without accounting for nuclei density). From a statistical standpoint, for both type II and type III infection, size does not correlate with the number of TINs for only two regions beyond the white matter tracts: the cortex and cerebellum. Given that the cerebellum is a relatively large brain region and has a high density of neurons in the granular layer— the lack of TINs here suggests that some unknown factor (such as difference in vascular permeability (Daniels et al 2017)) rather than area size or neuron density accounts for the cerebellum’s relative resistance to infection. Third, unlike brain region size where the Allen Institute Mouse Brain Atlas is accepted as a standard, there is no accepted standard for neuron density, in part because counts vary widely (reviewed in Keller et al 2018, Tables 1-4). Given the lack of standard counts, the most appropriate way to normalize would be to do our own counts of host nuclei in each region and ideally in both uninfected and infected mice. Counting host nuclei in infected tissue becomes complicated because of the infiltrating immune cells and dividing glia, an issue that cannot be solved by counting only neurons because of a loss of antigenicity for multiple neuron markers in inflamed brain tissue (Cekanaviciute et al 2014; David et al 2016). Pursuing such counts in uninfected tissue alone leads to a technical issue for us: when trying to count cells with high numbers using our program (Mendez, Potter et al 2018), our processing system cannot not handle the workload (i.e., the program crashes). Thus, at this time, we feel that normalizing by size is an appropriate first step and will use the recommended normalization in subsequent work which will pursue defining the mechanisms that lead to enrichment of TINs in the cortex and a lack of TINs in the cerebellum.
- The authors claim that inhibitory neurons are significantly less injected than excitatory ones. But how do they know that the inhibitory ones just don't die more quickly.
We have added this possibility to our discussion.
- All of the electrophysiological changes that are reported to happen in the injected neurons can be most easily explained by the fact that they are unhealthy due to the injection. This does not mean that the data are insignificant since increased neuronal damage/death in injected neurons is a critical finding.
We agree with that the electrophysiology findings of TINs may be due to neuronal injury but, at this time, we cannot prove this assumption. We have amended our discussion to more clearly state that we do not know if neuron healthy (or disease) is driving the TINs physiology or if the TINs physiology ultimately results in neuron death.
Reviewer #2 (Public Review):
The location and longevity of Toxoplasma infection in neurons accompanied by continuous immune infiltration to the brain provides many specific questions about the long term implications of this common infection as well as a broadly applicable model for neurotropic infections. Here Mendez and colleagues continue the use of a reporter system to reveal neurons that have been injected with parasite proteins (TINs) to determine the anatomical and cellular localization of parasite-neuron interactions and the electrophysiological properties of these neurons. This is a technically impressive piece of work first using the Allen Brain Atlas to map the location of TINs that may be a new gold standard for this type of work. Secondly, for the first time there is a record of functional data from parasite manipulated neurons suggesting that these cells ultimately die due to infection. The full consequences of this data do not seem to be fully addressed and certain limitations of the data are worthy of discussion.
- Although acknowledged on the first page of the introduction, there is a distinction between TINs and infected neurons. This important distinction is not continued later in the paper. Indeed, one conclusion is a change in neurotropic dogma regarding the long-lived nature of neurons being an attractive location for infections. The combination of methods used in this study allows major conclusions to be made regarding Toxoplasma injected neurons and as a result is an exciting body of work however it cannot distinguish what is happening in infected/cyst containing neurons.
The Reviewer is absolutely correct. With the current Cre system, in vivo, we cannot distinguish between aborted invasion and invasion followed by intracellular killing of the parasite. In addition, as many cysts are in distal neuronal processes (Cabral, Tuladhar et al 2016), we often cannot determine if a TIN is infected unless we do a full neuron reconstruction, which requires thick sections rather than the 40 micron sections we used for the immunohistochemical studies. For this reason, we do not make distinctions about infection status or how an uninfected but injected TIN arose. We clarified this issue in the text (i.e. TINs refers to both infected and uninfected, injected neurons) as well as included a new figure that more clearly explains this concept. In addition, we have noted that our prior work suggests that >90% of these TINs are not infected, which is consistent with our findings in thick sections where we have done whole neuron reconstructions (unpublished data.)
- The electrophysiology study is well controlled by data from bystander neurons in the same tissue. These bystander neurons show a significant (p<0.001) increase in resting membrane potential. This striking significance seems underplayed for the rest of the study somewhat overshadowed by the extreme read outs on TINs. It would be interesting to hear what this mild depolarization functionally means for these bystander neurons. The data may suggest there is greater variation of membrane potential between neurons from uninfected mice and bystander neurons. The significance is lost later in the paper and the conclusions are summarized with bystander neurons being 'akin' to neurons from uninfected mice which seems not accurate.
As noted by the Reviewer, we have focused on the TINs as opposed to the bystander neurons. In part we have primarily focused on the TINs because our lab is interested in how neuron- Toxoplasma interactions govern parasites persistence. We also did not focus on the bystanders because, compared to MSNs in uninfected mice, the difference in resting membrane potential was the only statistically significant difference we found be in the bystander MSNs (which is also why we refer to them as akin to MSNs in uninfected mice; we have changed this word to “similar”). As you correctly point out, it is an interesting difference (and, as far as we know, the first time such studies have been done). We have amended our discussion to highlight that while the bystander physiology is relatively normal compared to TINs physiology, it is still abnormal. In addition, we have also included a new figure (Supp Fig 6) which shows an expected consequence of the depolarized nature of bystanders; they require fewer steps of input current to trigger the first action potential. In the discussion, we have noted that such changes in other neurons (e.g., cortical neurons) might lead to an increase in seizures— which is seen in patients with symptomatic toxoplasmosis (ie. congenital and recrudescent infections)— or even the various behavioral changes observed in rodents. We have expanded our discussion to include such possibilities but have stressed that these findings are speculations that require further studies to confirm or negate.
- Two pieces of data support the concept that neurons that have been infected with parasite proteins die - firstly that readings from TINs are highly depolarized and secondly there is a loss of neurons by 8 weeks post infection. This is a significant piece of new information that is not stated in the abstract. Some hesitation in making this conclusion may be from the difficulty in obtaining electrophysiology data from these cells. Another way to support this conclusion would be helpful for this shift in our thinking of the effects of Toxoplasma infection on the brain.
We have updated the abstract to reflect that TINs die. We are actively working on other ways to support this conclusion but believe these data (determining how TINs die- so we can block TINs death, etc) are beyond the scope of this paper.
- The impressive data investigating the anatomical location and the cellular specificity of TINs is further strengthened by the use of two types of parasites a Type II and Type III. The properties of these different 'strains' that may lead to alterations in neurons is not fully explored and conclusions about similarities or differences are unmade.
We agree with the Reviewer that one of the strengths of this paper is using genetically distinct strains types as it allows us to determine what findings are likely universal vs. strain-specific. We have not focused on the differences or similarities because we do not have any data to support what factors might influence these differences and similarities. As such, we prefer to save such commentary for another paper in which we can more clearly identify these factors (or even use multiple strains from several haplotypes so that we can more definitively state whether these findings are strain type-specific or simply different between these two strains).
-
Reviewer #2 (Public Review):
The location and longevity of Toxoplasma infection in neurons accompanied by continuous immune infiltration to the brain provides many specific questions about the long term implications of this common infection as well as a broadly applicable model for neurotropic infections. Here Mendez and colleagues continue the use of a reporter system to reveal neurons that have been injected with parasite proteins (TINs) to determine the anatomical and cellular localization of parasite-neuron interactions and the electrophysiological properties of these neurons. This is a technically impressive piece of work first using the Allen Brain Atlas to map the location of TINs that may be a new gold standard for this type of work. Secondly, for the first time there is a record of functional data from parasite manipulated …
Reviewer #2 (Public Review):
The location and longevity of Toxoplasma infection in neurons accompanied by continuous immune infiltration to the brain provides many specific questions about the long term implications of this common infection as well as a broadly applicable model for neurotropic infections. Here Mendez and colleagues continue the use of a reporter system to reveal neurons that have been injected with parasite proteins (TINs) to determine the anatomical and cellular localization of parasite-neuron interactions and the electrophysiological properties of these neurons. This is a technically impressive piece of work first using the Allen Brain Atlas to map the location of TINs that may be a new gold standard for this type of work. Secondly, for the first time there is a record of functional data from parasite manipulated neurons suggesting that these cells ultimately die due to infection. The full consequences of this data do not seem to be fully addressed and certain limitations of the data are worthy of discussion.
Although acknowledged on the first page of the introduction, there is a distinction between TINs and infected neurons. This important distinction is not continued later in the paper. Indeed, one conclusion is a change in neurotropic dogma regarding the long-lived nature of neurons being an attractive location for infections. The combination of methods used in this study allows major conclusions to be made regarding Toxoplasma injected neurons and as a result is an exciting body of work however it cannot distinguish what is happening in infected/cyst containing neurons.
The electrophysiology study is well controlled by data from bystander neurons in the same tissue. These bystander neurons show a significant (p<0.001) increase in resting membrane potential. This striking significance seems underplayed for the rest of the study somewhat overshadowed by the extreme read outs on TINs. It would be interesting to hear what this mild depolarization functionally means for these bystander neurons. The data may suggest there is greater variation of membrane potential between neurons from uninfected mice and bystander neurons. The significance is lost later in the paper and the conclusions are summarized with bystander neurons being 'akin' to neurons from uninfected mice which seems not accurate.
Two pieces of data support the concept that neurons that have been infected with parasite proteins die - firstly that readings from TINs are highly depolarized and secondly there is a loss of neurons by 8 weeks post infection. This is a significant piece of new information that is not stated in the abstract. Some hesitation in making this conclusion may be from the difficulty in obtaining electrophysiology data from these cells. Another way to support this conclusion would be helpful for this shift in our thinking of the effects of Toxoplasma infection on the brain.
The impressive data investigating the anatomical location and the cellular specificity of TINs is further strengthened by the use of two types of parasites a Type II and Type III. The properties of these different 'strains' that may lead to alterations in neurons is not fully explored and conclusions about similarities or differences are unmade.
-
Reviewer #1 (Public Review):
The primary strength of this paper is the attempt to characterize the neurons injected by Toxoplasma and the electrophysiological changes that ensue. Three major problems are however noted.
Figure 1 attempts to identify regions of the brain more profoundly impacted by Toxoplasma and does so by normalizing the numbers of injected neurons to the size of the region. But since the reporter system used requires the parasite injected protein to interact with a neuron's nucleus, The authors claims can only be valid after normalizing not to size but to density of nuclei in a region. This is especially important in the cortex where different layers have distinct architectures.
The authors claim that inhibitory neurons are significantly less injected than excitatory ones. But how do they know that the inhibitory ones …
Reviewer #1 (Public Review):
The primary strength of this paper is the attempt to characterize the neurons injected by Toxoplasma and the electrophysiological changes that ensue. Three major problems are however noted.
Figure 1 attempts to identify regions of the brain more profoundly impacted by Toxoplasma and does so by normalizing the numbers of injected neurons to the size of the region. But since the reporter system used requires the parasite injected protein to interact with a neuron's nucleus, The authors claims can only be valid after normalizing not to size but to density of nuclei in a region. This is especially important in the cortex where different layers have distinct architectures.
The authors claim that inhibitory neurons are significantly less injected than excitatory ones. But how do they know that the inhibitory ones just don't die more quickly.
All of the electrophysiological changes that are reported to happen in the injected neurons can be most easily explained by the fact that they are unhealthy due to the injection. This does not mean that the data are insignificant since increased neuronal damage/death in injected neurons is a critical finding.
-
Evaluation Summary:
This study uses a new approach to map all neurons in the brain that have been infected with Toxoplasma gondii or injected with parasite proteins. The authors show that Toxoplasma injected neurons are heterogeneously distributed in murine brain tissues, that excitatory neurons are the primary targets, and that injection of parasite proteins leads to neuronal death. This work provides new insights into Toxoplasma-neuron interactions that underlie the pathology and potential changes in behaviour of infected individuals. The manuscript will be of interest to those working in neuroscience and/or parasitic infections.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained …
Evaluation Summary:
This study uses a new approach to map all neurons in the brain that have been infected with Toxoplasma gondii or injected with parasite proteins. The authors show that Toxoplasma injected neurons are heterogeneously distributed in murine brain tissues, that excitatory neurons are the primary targets, and that injection of parasite proteins leads to neuronal death. This work provides new insights into Toxoplasma-neuron interactions that underlie the pathology and potential changes in behaviour of infected individuals. The manuscript will be of interest to those working in neuroscience and/or parasitic infections.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
-