Cestode larvae excite host neuronal circuits via glutamatergic signaling

Reviewer #1 (Public Review):

In the manuscript, the authors explore the mechanism by which Taenia solium larvae may contribute to human epilepsy. This is extremely important question to address because T. solium is a significant cause of epilepsy and is extremely understudied. Advances in determining how T. solium may contribute to epilepsy could have significant impact on this form of epilepsy. Excitingly, the authors convincingly show that Taenia larvae contain and release glutamate sufficient to depolarize neurons and induce recurrent excitation reminiscent of seizures. They use a combination of cutting-edge tools including electrophysiology, calcium and glutamate imaging, and biochemical approaches to demonstrate this important advance. They also show that this occurs in neurons from both mice and humans. This is relevant for pathophysiology of chronic epilepsy development. This study does not rule out other aspects of T. solium that may also contribute to epilepsy, including immunological aspects, but demonstrates a clear potential role for glutamate.

Strengths:

- The authors examine not only T. solium homogenate, but also excretory/secretory products which suggests glutamate may play a role in multiple aspects of disease progression.
- The authors confirm that the human relevant pathogen also causes neuronal depolarization in human brain tissue
- There is very high clinical relevance. Preventing epileptogenesis/seizures possibly with Glu-R antagonists or by more actively removing glutamate as a second possible treatment approach in addition to/replacing post-infection immune response.
- Effects are consistent across multiple species (rat, mouse, human) and methodological assays (GluSnFR AND current clamp recordings AND Ca imaging)
- High K content (comparable levels to high-K seizure models) of larvae could have also caused depolarization. Adequate experiments to exclude K and other suspected larvae contents (i.e. Substance P).

Weaknesses:

- Acute study is limited to studying depolarization in slices and it is unclear what is necessary/sufficient for in vivo seizure generation or epileptogenesis for chronic epilepsy.
- There is likely a significant role of the immune system that is not explored here. This issue is adequately addressed in the discussion, however, and the glutamate data is considered in this context.

Discuss impact:

- Interfering with peri-larval glutamate signaling may hold promise to prevent ictogenesis and chronic epileptogenesis as this is a very understudied cause of epilepsy with unknown mechanistic etiology.
Additional context for interpreting significance:
- High medical need as most common adult onset epilepsy in many parts of the world.

Reviewer #2 (Public Review):

Since neurocysticercosis is associated with epilepsy, the authors wish to establish how cestode larvae affect neurons. The underlying hypothesis is that the larvae may directly excite neurons and thus favor seizure genesis.

To test this hypothesis, the authors collected biological materials from larvae (from either homogenates or excretory/secretory products), and applied them to hippocampal neurons (rats and mice) and human cortical neurons.

This constitutes a major strength of the paper, providing a direct reading of larvae's biological effects. Another strength is the combination of methods, including patch clamp, Ca, and glutamate imaging.

There are some weaknesses:

1. The main one relates to the statement: "Together, these results indicate that T. crassiceps larvae homogenate results not just in a transient depolarization of cells in the immediate vicinity of application, but can also trigger a wave of excitation that propagates through the brain slice in both space and time. This demonstrates that T. crassiceps homogenate can initiate seizure-like activity under suitable conditions."
   The only "evidence" of propagation is an image at two time points. It is one experiment, and there is no quantification. Either increase n's and perform a quantification, or remove such a statement.
   Likewise, there is no evidence of seizure genesis. A single cell recording is shown. The presence of a seizure-like event should be evaluated with field recordings.

2. Control puff experiments are lacking for Fig 1. Would puffing ACSF also produce a depolarization, and even firing, as suggested in Fig. 2D? This is needed for at least one species.

3. What is the rationale to use a Cs-based solution? Even in the presence of TTX and with blocking K channels, the depolarization may be sufficient to activate Ca channels (LVGs), which would further contribute to the depolarization. Why not perform voltage clamp recordings to directly the current?

4. Why did you use organotypic slices? Since you wish to model adult epilepsy, it would have been more relevant to use fresh slices from adult rats/mice. At least, discuss the caveat of using a network still in development in vitro.

5. Please include both the number of slices and number of cells recorded in each condition. This is the standard (the number of cells is not enough).

6. Please provide a table with the basic properties of cells (Rin, Rs, etc.). This is standard to assess the quality of the recordings.

7. Please provide a table on patient's profile. This is standard when using human material. Were these TLE cases (and "control" cortex) or epileptogenic cortex?

Globally, the authors achieved their aims. They show convincingly that larvae material can depolarize neurons, with glutamate (and aspartate) as the most likely candidates.
This is important not only because it provides mechanistic insight but also potential therapeutic targets. The result is impactful, as the authors use quasi-naturalistic conditions, to assess what might happen in the human brain. The experimental design is appropriate to address the question. It can be replicated by any interested person.

Reviewer #3 (Public Review):

This paper has high significance because it addresses a prevalent parasitic infection of the nervous system, Neurocysticercosis (NCC). The infection is caused by larvae of the parasitic cestode Taenia solium It is a leading cause of epilepsy in adults worldwide

To address the effects of cestode larvae, homogenates and excretory/secretory products of larvae were added to organotypic brain slice cultures of rodents or layer 2/3 of human cortical brain slices from patients with refractory epilepsy.

A self-made pressure ejection system was used to puff larvae homogenate (20 ms puff) onto the soma of patched neurons. The mechanical force could have caused depolarizaton so a vehicle control is critical. On line 150 they appear to have used saline in this regard, and clarification would be good. Were the controls here (and aCSF elsewhere) done with the low Mg2+o aCSF like the larvae homogenates?

They found that neurons depolarized after larvae homogenate exposure and the effect was mediated by glutamate but not nicotinic receptors for acetylcholine (nAChRs), acid-sensing channels or substance P. To address nAChRs, they used 10uM mecamyline, and for ASICs 2mM amiloride which seems like a high concentration. Could the concentrations be confirmed for their selectivity? Glutamate receptor antagonists, used in combination, were 10uM CNQX, 50uM DAP5, and 2mM kynurenic acid. These concentrations are twice what most use. Please discuss. Also, it would be very interesting to know if the glutamate receptor is AMPA, Kainic acid, or NMDA. Were metabotropic antagonists ever tested? That would be logical because CNQX/DAPR/Kynurenic acid did not block all of the depolarization.

They also showed the elevated K+ in the homogenate (~11 mM) could not account for the depolarization. However, the experiment with K+ was not done in a low Mg2+o buffer (Or was it -please clarify). They also confirmed that only small molecules led to the depolarization after filtering out very large molecules. That supports the conclusion that glutamate - which is quite small - could be responsible.

It is logical to test substance P because the Intro points out prior work links the larvae and seizures by inflammation and implicates substance P. However, why focus on nAChRs and ASIC?

The depolarizations caused seizure-like events in slices. The slices were exposed to a proconvulant buffer though- low Mg2+o. This buffer can cause spontaneous seizure-like events so it is important to know what the buffer did alone.

They suggest the effects could underlie seizure generation in NCC. However, there is only one event that is seizure-like in the paper and it is just an inset. Were others similar? How frequency were they? How long?

Using Glutamate-sensing fluorescent reporters they found the larvae contain glutamate and can release it, a strength of the paper.

Fig. 4. Could an inset be added to show the effects are very fast? That would support an effect of glutamate.

Why is aspartate relatively weak and glutamate relatively effective as an agonist?

Could some of the variability in Fig 4G be due to choice of different cell types? That would be consistent with Fig 5B where only a fraction of cells in the culture showed a response to the larvae nearby.

On what basis was the ROI drawn in Fig. 5B.

Also in 5B, I don't see anything in the transmitted image. What should be seen exactly?

Human brain slices were from temporal cortex of patients with refractory epilepsy. Was the temporal cortex devoid of pathology and EEG abnormalities? This area may be quite involved in the epilepsy because refractory epilepsy that goes to surgery is often temporal lobe epilepsy. Please discuss the liitations of studying the temporal cortex of humans with epilepsy since it may be more susceptible to depolarizations of many kinds, not just larvae.

Please discuss the limitations of the cultures - they are from very young animals and cultured for 6-14 days.