Cholecystokinin modulates age-dependent Thalamocortical Neuroplasticity

Reviewer #1 (Public review):

This study offers a valuable investigation into the role of cholecystokinin (CCK) in thalamocortical plasticity during early development and adulthood, employing a range of experimental techniques. The authors demonstrate that tetanic stimulation of the auditory thalamus induces cortical long-term potentiation (LTP), which can be evoked through either electrical or optical stimulation of the thalamus or by noise bursts. They further show that thalamocortical LTP is abolished when thalamic CCK is knocked down or when cortical CCK receptors are blocked. Interestingly, in 18-month-old mice, thalamocortical LTP was largely absent but could be restored through the cortical application of CCK. The authors conclude that CCK contributes to thalamocortical plasticity and may enhance thalamocortical plasticity in aged subjects.

While the study presents compelling evidence, I would like to offer several suggestions for the authors' consideration:

(1) Thalamocortical LTP and NMDA-Dependence:
It is well established that thalamocortical LTP is NMDA receptor-dependent, and blocking cortical NMDA receptors can abolish LTP. This raises the question of why thalamocortical LTP is eliminated when thalamic CCK is knocked down or when cortical CCK receptors are blocked. If I correctly understand the authors' hypothesis - that CCK promotes LTP through CCKR-intracellular Ca2+-AMPAR. This pathway should not directly interfere with the NMDA-dependent mechanism. A clearer explanation of this interaction would be beneficial.

(2) Complexity of the Thalamocortical System:
The thalamocortical system is intricate, with different cortical and thalamic subdivisions serving distinct functions. In this study, it is not fully clear which subdivisions were targeted for stimulation and recording, which could significantly influence the interpretation of the findings. Clarifying this aspect would enhance the study's robustness.

(3) Statistical Variability:
Biological data, including field excitatory postsynaptic potentials (fEPSPs) and LTP, often exhibit significant variability between samples, sometimes resulting in a standard deviation that exceeds 50% of the mean value. The reported standard deviation of LTP in this study, however, appears unusually small, particularly given the relatively limited sample size. Further discussion of this observation might be warranted.

(4) EYFP Expression and Virus Targeting:
The authors indicate that AAV9-EFIa-ChETA-EYFP was injected into the medial geniculate body (MGB) and subsequently expressed in both the MGB and cortex. If I understand correctly, the authors assume that cortical expression represents thalamocortical terminals rather than cortical neurons. However, co-expression of CCK receptors does not necessarily imply that the virus selectively infected thalamocortical terminals. The physiological data regarding cortical activation of thalamocortical terminals could be questioned if the cortical expression represents cortical neurons or both cortical neurons and thalamocortical terminals.

(5) Consideration of Previous Literature:
A number of studies have thoroughly characterized auditory thalamocortical LTP during early development and adulthood. It may be beneficial for the authors to integrate insights from this body of work, as reliance on data from the somatosensory thalamocortical system might not fully capture the nuances of the auditory pathway. A more comprehensive discussion of the relevant literature could enhance the study's context and impact.

(6) Therapeutic Implications:
While the authors suggest potential therapeutic applications of their findings, it may be somewhat premature to draw such conclusions based on the current evidence. Although speculative discussion is not harmful, it may not significantly add to the study's conclusions at this stage.

Reviewer #2 (Public review):

Summary:

This work used multiple approaches to show that CCK is critical for long-term potentiation (LTP) in the auditory thalamocortical pathway. They also showed that the CCK mediation of LTP is age-dependent and supports frequency discrimination. This work is important because it opens up a new avenue of investigation of the roles of neuropeptides in sensory plasticity.

Strengths:

The main strength is the multiple approaches used to comprehensively examine the role of CCK in auditory thalamocortical LTP. Thus, the authors do provide a compelling set of data that CCK mediates thalamocortical LTP in an age-dependent manner.

Weaknesses:

The behavioral assessment is relatively limited but may be fleshed out in future work.

Reviewer #3 (Public review):

Summary:

Cholecystokinin (CCK) is highly expressed in auditory thalamocortical (MGB) neurons and CCK has been found to shape cortical plasticity dynamics. In order to understand how CCK shapes synaptic plasticity in the auditory thalamocortical pathway, they assessed the role of CCK signaling across multiple mechanisms of LTP induction with the auditory thalamocortical (MGB - layer IV Auditory Cortex) circuit in mice. In these physiology experiments that leverage multiple mechanisms of LTP induction and a rigorous manipulation of CCK and CCK-dependent signaling, they establish an essential role of auditory thalamocortical LTP on the co-release of CCK from auditory thalamic neurons. By carefully assessing the development of this plasticity over time and CCK expression, they go on to identify a window of time that CCK is produced throughout early and middle adulthood in auditory thalamocortical neurons to establish a window for plasticity from 3 weeks to 1.5 years in mice, with limited LTP occurring outside of this window. The authors go on to show that CCK signaling and its effect on LTP in the auditory cortex is also capable of modifying frequency discrimination accuracy in an auditory PPI task. In evaluating the impact of CCK on modulating PPI task performance, it also seems that in mice <1.5 years old CCK-dependent effects on cortical plasticity are almost saturated. While exogenous CCK can modestly improve discrimination of only very similar tones, exogenous focal delivery of CCK in older mice can significantly improve learning in a PPI task to bring their discrimination ability in line with those from young adult mice.

Strengths:

(1) The clarity of the results along with the rigor multi-angled approach provide significant support for the claim that CCK is essential for auditory thalamocortical synaptic LTP. This approach uses a combination of electrical, acoustic, and optogenetic pathway stimulation alongside conditional expression approaches, germline knockout, viral RNA downregulation, and pharmacological blockade. Through the combination of these experimental configures the authors demonstrate that high-frequency stimulation-induced LTP is reliant on co-release of CCK from glutamatergic MGB terminals projecting to the auditory cortex.

(2) The careful analysis of the CCK, CCKB receptor, and LTP expression is also a strength that puts the finding into the context of mechanistic causes and potential therapies for age-dependent sensory/auditory processing changes. Similarly, not only do these data identify a fundamental biological mechanism, but they also provide support for the idea that exogenous asynchronous stimulation of the CCKBR is capable of restoring an age-dependent loss in plasticity.

(3) Although experiments to simultaneously relate LTP and behavioral change or identify a causal relationship between LTP and frequency discrimination are not made, there is still convincing evidence that CCK signaling in the auditory cortex (known to determine synaptic LTP) is important for auditory processing/frequency discrimination. These experiments are key for establishing the relevance of this mechanism.

Weaknesses:

(1) Given the magnitude of the evoked responses, one expects that pyramidal neurons in layer IV are primarily those that undergo CCK-dependent plasticity, but the degree to which PV-interneurons and pyramidal neurons participate in this process differently is unclear.

(2) While these data support an important role for CCK in synaptic LTP in the auditory thalamocortical pathway, perhaps temporal processing of acoustic stimuli is as or more important than frequency discrimination. Given the enhanced responsivity of the system, it is unclear whether this mechanism would improve or reduce the fidelity of temporal processing in this circuit. Understanding this dynamic may also require consideration of cell type as raised in weakness #1.

(3) In Figure 1, an example of increased spontaneous and evoked firing activity of single neurons after HFS is provided. Yet it is surprising that the group data are analyzed only for the fEPSP. It seems that single-neuron data would also be useful at this point to provide insight into how CCK and HFS affect temporal processing and spontaneous activity/excitability, especially given the example in 1F.

(4) The authors mention that CCK mRNA was absent in CCK-KO mice, but the data are not provided.

(5) The circuitry that determines PPI requires multiple brain areas, including the auditory cortex. Given the complicated dynamics of this process, it may be helpful to consider what, if anything, is known specifically about how layer IV synaptic plasticity in the auditory cortex may shape this behavior.