Excitatory cholecystokinin neurons in CA3 area regulate the navigation learning and neuroplasticity

Reviewer #1 (Public review):

Summary:

CCK is the most abundant neuropeptide in the brain, and many studies have investigated the role of CCK and inhibitory CCK interneurons in modulating neural circuits, especially in the hippocampus. The manuscript presents interesting questions regarding the role of excitatory CCK+ neurons in the hippocampus, which has been much less studied compared to the well-known roles of inhibitory CCK neurons in regulating network function. The authors adopt several methods, including transgenic mice and viruses, optogenetics, chemogenetics, RNAi, and behavioral tasks to explore these less-studied roles of excitatory CCK neurons in CA3. They find that the excitatory CCK neurons are involved in hippocampal-dependent tasks such as spatial learning and memory formation, and that CCK-knockdown impairs these tasks.

However, these questions are very dependent on ensuring that the study is properly targeting excitatory CCK neurons (and thus their specific contributions to behavior).

There needs to be much more characterization of the CCK transgenic mice and viruses to confirm the targeting. Without this, it is unclear whether the study is looking at excitatory CCK neurons or a more general heterogeneous CCK neuron population.

Strengths:

This field has focused mainly on inhibitory CCK+ interneurons and their role in network function and activity, and thus, this manuscript raises interesting questions regarding the role of excitatory CCK+ neurons, which have been much less studied.

Weaknesses:

(1a) This manuscript is dependent on ensuring that the study is indeed investigating the role of excitatory CCK-expressing neurons themselves and their specific contribution to behavior. There needs to be much more characterization of the CCK-expressing mice (crossed with Ai14 or transduced with various viruses) to confirm the excitatory-cell targeting. Without this, it is unclear whether the study is looking at excitatory CCK neurons or a more general heterogeneous CCK neuron population.

(1b) For the experiments that use a virus with the CCK-IRES-Cre mouse, there is no information or characterization on how well the virus targets excitatory CCK-expressing neurons. (Additionally, it has been reported that with CaMKIIa-driven protein expression, using viruses, can be seen in both pyramidal and inhibitory cells.)

(2) The methods and figure legends are extremely sparse, leading to many questions regarding methodology and accuracy. More details would be useful in evaluating the tools and data. More details would be useful in evaluating the tools and data. Additionally, further quantification would be useful-e.g. in some places, only % values are noted, or only images are presented.

(3) It is unclear whether the reduced CCK expression is correlated, or directly causing the impairments in hippocampal function. Does the CCK-shRNA have any additional detrimental effects besides affecting CCK-expression (e.g., is the CCK-shRNA also affecting some other essential (but not CCK-related) aspect of the neuron itself?)? Is there any histology comparison between the shRNA and the scrambled shRNA?

Reviewer #2 (Public review):

Summary:

In this study, the authors have demonstrated, through a comprehensive approach combining electrophysiology, chemogenetics, fiber photometry, RNA interference, and multiple behavioral tasks, the necessity of projections from CCK+ CAMKIIergic neurons in the hippocampal CA3 region to the CA1 region for regulating spatial memory in mice. Specifically, authors have shown that CA3-CCK CAMKIIergic neurons are selectively activated by novel locations during a spatial memory task. Furthermore, authors have identified the CA3-CA1 pathway as crucial for this spatial working memory function, thereby suggesting a pivotal role for CA3 excitatory CCK neurons in influencing CA1 LTP. The data presented appear to be well-organized and comprehensive.

Strengths:

(1) This work combined various methods to validate the excitatory CCK neurons in the CA3 area; these data are convincing and solid.

(2) This study demonstrated that the CA3-CCK CAMKIIergic neurons are involved in the spatial memory tasks; these are interesting findings, which suggest that these neurons are important targets for manipulating the memory-related diseases.

(3) This manuscript also measured the endogenous CCK from the CA3-CCK CAMKIIergic neurons; this means that CCK can be released under certain conditions.

Weaknesses:

(1) The authors do not mention which receptors of the CCK modulate these processes.

(2) This author does not test the CCK gene knockout mice or the CCK receptor knockout mice in these neural processes.

(3) The author does not test the source of CCK release during the behavioral tasks.

Reviewer #3 (Public review):

Summary:

Fengwen Huang et al. used multiple neuroscience techniques (transgenetic mouse, immunochemistry, bulk calcium recording, neural sensor, hippocampal-dependent task, optogenetics, chemogenetics, and interfer RNA technique) to elucidate the role of the excitatory cholecystokinin-positive pyramidal neurons in the hippocampus in regulating the hippocampal functions, including navigation and neuroplasticity.

Strengths:

(1) The authors provided the distribution profiles of excitatory cholecystokinin in the dorsal hippocampus via the transgenetic mice (Ai14::CCK Cre mice), immunochemistry, and retrograde AAV.

(2) The authors used the neural sensor and light stimulation to monitor the CCK release from the CA3 area, indicating that CCK can be secreted by activation of the excitatory CCK neurons.

(3) The authors showed that the activity of the excitatory CCK neurons in CA3 is necessary for navigation learning.

(4) The authors demonstrated that inhibition of the excitatory CCK neurons and knockdown of the CCK gene expression in CA3 impaired the navigation learning and the neuroplasticity of CA3-CA1 projections.

Weaknesses:

(1) The causal relationship between navigation learning and CCK secretion?

(2) The effect of overexpression of the CCK gene on hippocampal functions?

(3) What are the functional differences between the excitatory and inhibitory CCK neurons in the hippocampus?

(4) Do CCK sources come from the local CA3 or entorhinal cortex (EC) during the high-frequency electrical stimulation?