Cholecystokinin modulates age-dependent Thalamocortical Neuroplasticity

Reviewer #1 (Public review):

This report addresses a compelling topic. However, I have significant concerns, which necessitate a reassessment of the report's overall value.

Anatomical Specificity and Stimulation Site:
While the authors clarify that the ventral MGB (MGv) was the intended stimulation target, the electrode track (Fig. 1A) and viral spread (Fig. 2E) suggest possible involvement of the dorsal MGB (MGd) and broader area. Given that MGv-AI and MGd-AC pathways have distinct-and sometimes opposing-effects on plasticity, the reported LTP values (with unusually small standard deviations) raise concerns about the specificity of the findings. Additional anatomical verification would help resolve this issue.

Statistical Rigor and Data Variability:
The remarkably low standard deviations in LTP measurements are unexpected based on established variability in thalamocortical plasticity. The authors' response confirms these values are accurate, but further justification, such as methodological controls or replication-would bolster confidence in these results. Additionally, the comparison of in vivo vs. in vitro LTP variability requires more substantive support.

Viral Targeting and Specificity:
The manuscript does not clearly address whether cortical neurons were inadvertently infected by AAV9. Given the potential for off-target effects, explicit confirmation (e.g., microphotograph of stimulation site) would strengthen the study's conclusions.

Integration of Prior Literature:
The discussion of existing work is adequate but could be more comprehensive. A deeper engagement with contrasting findings would provide better context for the study's contributions.

Therapeutic Implications:
The authors' discussion of therapeutic potential is now appropriately cautious and well-reasoned.

Conclusion:
While the study presents intriguing findings, the concerns outlined above must be addressed to fully establish the validity and impact of the results. I appreciate the authors' efforts thus far and hope they can provide additional data or clarification to resolve these issues. With these revisions, the manuscript could make a valuable contribution to the field.

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 is 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:

There are some details that should be addressed, primarily regarding potential baseline differences in comparison groups. 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 is 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 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.

Comments on revisions:

The manuscript is much improved and many of the issues or questions have been addressed. Ideally, evidence for the degree of transsynaptic spread for AAV9-Syn-ChrimsonR-tdTomato would also be provided in some form since in the authors' response in sounds like some was observed, as expected.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

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.

Thank you for your question regarding the role of CCK and NMDA receptors (NMDARs) in thalamocortical LTP. We propose that CCK receptor (CCKR) activation enhances intracellular calcium levels, which are crucial for thalamocortical LTP induction. Calcium influx through NMDARs is also essential to reach the threshold required for activating downstream signaling pathways that promote LTP (Heynen and Bear, 2001). Thus, CCKRs and NMDARs may function in a complementary manner to facilitate LTP, with both contributing to the elevation of intracellular calcium.

However, it is important to note that the postsynaptic mechanisms of thalamocortical LTP in the auditory cortex (ACx) differ from those in other sensory cortices. Studies have shown that thalamocortical LTP in the ACx appears to be less dependent on NMDARs (Chun et al., 2013), which is distinct from somatosensory or visual cortices. Our previous studies also found that while NMDAR antagonists can block HFS-induced LTP in the inner ACx, LTP can still be induced in the presence of CCK even after the NMDARs blockade (Chen et al. 2019). These findings suggest that CCK may act through an alternative mechanism involving CCKR-mediated calcium signaling and AMPAR modulation, which partially compensates for the loss of NMDAR signaling. This distinction may reflect functional differences between the ACx and other sensory cortices, as highlighted in previous studies (King and Nelken, 2009).

While our current study focuses on the role of CCKR-mediated plasticity in the auditory system, further investigations are needed to elucidate how CCKRs and NMDARs interact within the broader framework of thalamocortical neuroplasticity across different cortical regions. Understanding whether similar mechanisms operate in other sensory systems, such as the visual cortex, will be an important direction for future research.

Heynen, A.J., and Bear, M.F. (2001). Long-term potentiation of thalamocortical transmission in the adult visual cortex in vivo. J Neurosci 21, 9801-9813. 10.1523/jneurosci.21-24-09801.2001.

Chun, S., Bayazitov, I.T., Blundon, J.A., and Zakharenko, S.S. (2013). Thalamocortical Long-Term Potentiation Becomes Gated after the Early Critical Period in the Auditory Cortex. The Journal of Neuroscience 33, 7345-7357. 10.1523/jneurosci.4500-12.2013.

Chen, X., Li, X., Wong, Y.T., Zheng, X., Wang, H., Peng, Y., Feng, H., Feng, J., Baibado, J.T., Jesky, R., et al. (2019). Cholecystokinin release triggered by NMDA receptors produces LTP and sound-sound associative memory. Proc Natl Acad Sci U S A 116, 6397-6406. 10.1073/pnas.1816833116.

King, A. J., & Nelken, I. (2009). Unraveling the principles of auditory cortical processing: can we learn from the visual system? Nature neuroscience, 12(6), 698-701.

(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.

Thank you for your valuable feedback. We would like to clarify that stimulation was conducted in the medial geniculate nucleus ventral (MGv), and recording was performed in layer IV of the ACx. Targeting the MGv allows us to investigate the influence of thalamic inputs on auditory cortical responses. Layer IV of the ACx is known to receive direct thalamic projections, making it an ideal site for assessing how thalamic activity influences cortical processing. We will incorporate this clarification into the revised manuscript to enhance the robustness of our study.

Results section:

“Stimulation electrodes were placed in the MGB (specifically in the medial geniculate nucleus ventral subdivision, MGv), and recording electrodes were inserted into layer IV of ACx”

“The recording electrodes were lowered into layer IV of ACx, while the stimulation electrodes were lowered into MGB (MGv subdivision). The final stimulating and recording positions were determined by maximizing the cortical fEPSP amplitude triggered by the ES in the MGB. The accuracy of electrode placement was verified through post-hoc histological examination and electrophysiological responses.”

(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.

Thank you for your question. In our experiments, the sample size N represents the number of animals used, while n refers to the number of recordings, with each recording corresponding to a distinct stimulation and recording sites. To adhere to ethical guidelines and minimize animal usage, we often perform multiple recordings within a single animal, such as from different hemispheres of the brain. Although N may appear small, our statistical analyses are based on n, ensuring sufficient data points for reliable conclusions.

Furthermore, as our experiments are conducted in vivo, we observe lower variability in the increase of fEPSP slopes following LTP induction compared to brain slice preparations, where standard deviations exceeding 50% of the mean are common. This reduced variability likely reflects the robustness of the physiologically intact conditions in the in vivo setup.

(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.

Thank you for your question. In Figure 2A, EYFP expression indicates thalamocortical projections, while the co-expression of EYFP with PSD95 confirms the identity of thalamocortical terminals. The CCK-B receptors (CCKBR) are located on postsynaptic cortical neurons. The observed co-labeling of thalamocortical terminals and postsynaptic CCKBR suggests that CCK-expressing neurons in the medial geniculate body (MGB) can release CCK, which subsequently acts on the postsynaptic CCKBR. This evidence supports our interpretation of the functional role of CCK modulating neural plasticity between thalamocortical inputs and cortical neurons. As shown in Figure 2A, we aim to demonstrate that the co-labeling of thalamocortical terminals with CCK receptors accounts for a substantial proportion of the thalamocortical terminals. We will ensure that this clarification is emphasized in the revised manuscript to address your concerns.

Results section:

“Cre-dependent AAV9-EFIa-DIO-ChETA-EYFP was injected into the MGB of CCK-Cre mice. EYFP labeling marked CCK-positive neurons in the MGB. The co-expression of EYFP thalamocortical projections with PSD95 confirms the identity of thalamocortical terminals (yellow), which primarily targeted layer IV of the ACx (Figure 2A, upper panel). Immunohistochemistry revealed that a substantial proportion (15 out of 19, Figure 2A lower right panel) of thalamocortical terminals (arrows) colocalize with CCK receptors (CCKBR) on postsynaptic cortical neurons in the ACx (Figure 2A lower panel), supporting the functional role of CCK in modulating thalamocortical plasticity.”

(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.

Thank you for your valuable feedback. We will enhance our discussion on auditory thalamocortical LTP during early development and adulthood to provide a more comprehensive context for our study.

(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.

Thank you for your thoughtful feedback. We agree that the therapeutic applications mentioned in our study are speculative at this stage and should be regarded as a forward-looking perspective rather than definitive conclusions. Our intention was to highlight the broader potential of our findings to inspire further research, rather than to propose immediate clinical applications.

In light of your feedback, we have adjusted the language in the manuscript to reflect a more cautious interpretation. Speculative discussions are now explicitly framed as hypotheses or possibilities for future exploration. We emphasize that our findings provide a foundation for further investigations into CCK-based plasticity and its implications.

We believe that appropriately framed forward-thinking discussions are valuable in guiding the direction of future research. We sincerely hope that our current and future work will contribute to a deeper understanding of thalamocortical plasticity and, over time, potentially lead to advancements in human health.

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.

Thank you for this insightful comment. We agree that the differential roles of PV-interneurons and pyramidal neurons in CCK-dependent thalamocortical plasticity remain unclear and acknowledge this as an important limitation of our study. Our primary focus was on pyramidal neurons, as our in vivo electrophysiological recordings measured the fEPSP slope in layer IV of the auditory cortex, which primarily reflects excitatory synaptic activity. However, we recognize the critical role of the excitatory-inhibitory balance in cortical function and the potential contribution of PV-interneurons to this process. In future studies, we plan to utilize techniques such as optogenetics, two-photon calcium imaging and cell-type-specific recordings to investigate the distinct contributions of PV-interneurons and pyramidal neurons to CCK-dependent thalamocortical plasticity, thereby providing a more comprehensive understanding of how CCK modulates thalamocortical circuits.

(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.

Thank you for this thoughtful comment. We acknowledge that our study did not directly address the fidelity of temporal processing, which is indeed a critical aspect of auditory function. Our behavioral experiments primarily focused on linking frequency discrimination to the role of CCK in synaptic strengthening within the auditory thalamocortical pathway. However, we agree that enhanced responsivity of the system could also impact temporal processing dynamics, such as the precise timing of auditory responses. Whether this modulation improves or reduces the fidelity of temporal processing remains an open and important question.

As you noted, understanding these dynamics will require a deeper investigation into the interactions between different cell types, particularly the balance between excitatory and inhibitory neurons. Exploring how CCK modulation affects both the circuit and cellular levels in temporal processing is an important direction for future research, which we plan to pursue. Thank you again for raising this important point.

Disscusion section:

“While we focused on homosynaptic plasticity at thalamocortical synapses by recording only fEPSPs in layer IV of ACx, it is essential to further explore heterosynaptic effects of CCK released from thalamocortical synapses on intracortical circuits, particularly its role in modulating the excitatory-inhibitory balance. PV-interneurons, as key regulators of cortical inhibition, may contribute to the temporal fidelity of sensory processing, which is critical for auditory perception (Nocon et al., 2023; Cai et al., 2018). Additionally, CCK may facilitate cross-modal plasticity by modulating heterosynaptic plasticity in interconnected cortical areas. Future studies would provide valuable insights into the broader role of CCK in shaping sensory processing and cortical network dynamics.”

Nocon, J.C., Gritton, H.J., James, N.M., Mount, R.A., Qu, Z., Han, X., and Sen, K. (2023). Parvalbumin neurons enhance temporal coding and reduce cortical noise in complex auditory scenes. Communications Biology 6, 751. 10.1038/s42003-023-05126-0.

Cai, D., Han, R., Liu, M., Xie, F., You, L., Zheng, Y., Zhao, L., Yao, J., Wang, Y., Yue, Y., et al. (2018). A Critical Role of Inhibition in Temporal Processing Maturation in the Primary Auditory Cortex. Cereb Cortex 28, 1610-1624. 10.1093/cercor/bhx057.

(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.

Thank you for your insightful comment. In our in vivo electrophysiological experiments on LTP induction, we recorded neural activity for over 1.5 hours to assess changes in neuronal responses over time, both prior to and following the induction. While single neuron firing data can provide valuable insights, such measurements are inherently more variable due to factors like cortical state fluctuations and the condition of nearby neurons, which makes them less reliable for long-term analysis. For this reason, we focused on fEPSP, as it offers a more stable and robust readout of synaptic activity over extended periods.

We appreciate your suggestion and recognize the value of single-neuron data in understanding how CCK and HFS affect temporal processing and excitability. In future studies, we will consider to incorporate single-neuron analyses to complement our synaptic-level findings and provide a more comprehensive understanding of these mechanisms.

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

Thank you for your comment. Data from the CCK-KO mice are presented in Figure 3A (far right) and in the upper panel of Figure 3B (far right). In the lower panel of Figure 3B, data from the CCK-KO group are not shown because the normalized values for this group were essentially zero, as expected due to the absence of CCK mRNA.

(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.

Thank you for raising this important point. Pre-pulse inhibition (PPI) of the acoustic startle response indeed involves multiple brain regions, with the ascending auditory pathway playing a key role (Gómez-Nieto et al., 2020). Within the auditory cortex, layer IV neurons receive tonotopically organized inputs from the medial geniculate nucleus and are critical for integrating thalamic inputs and shaping auditory processing.

In our behavioral experiments, mice were required to discriminate pre-pulses of varying frequencies against a continuous background sound. Given the role of auditory cortical neurons in integrating thalamic inputs and shaping auditory processing, it is likely that synaptic plasticity in these neurons contributes to the enhanced discrimination of pre-pulses. Supporting this idea, our previous work demonstrated that local infusion of CCK, paired with weak acoustic stimuli, significantly increased auditory responses in the auditory cortex (Li et al., 2014). In the current study, we further showed that CCK release during high-frequency stimulation of the thalamocortical pathway induced LTP in layer IV of the auditory cortex. Together, these findings suggest that CCK-dependent synaptic plasticity in layer IV may amplify the cortical representation of weak auditory inputs, thereby improving pre-pulses detection and enhancing PPI performance.

It is also worth noting that aged mice with hearing loss typically exhibit PPI deficits due to impaired auditory processing (Ouagazzal et al., 2006 and Young et al., 2010). We propose that enhanced plasticity in the thalamocortical pathway, mediated by CCK, might partially compensate for these deficits by amplifying residual auditory signals in aged mice. However, the precise mechanisms by which layer IV synaptic plasticity modulates PPI behavior remain to be fully understood. Given the complex dynamics of sensory processing, future studies could explore how layer IV neurons interact with other cortical and subcortical circuits involved in PPI, as well as the specific contributions of excitatory and inhibitory cell types. These investigations will help provide a more comprehensive understanding of the role of CCK in modulating sensory gating and auditory processing.

Gómez-Nieto, R., Hormigo, S., & López, D. E. (2020). Prepulse inhibition of the auditory startle reflex assessment as a hallmark of brainstem sensorimotor gating mechanisms. Brain sciences, 10(9), 639.

Li, X., Yu, K., Zhang, Z., Sun, W., Yang, Z., Feng, J., Chen, X., Liu, C.-H., Wang, H., Guo, Y.P., and He, J. (2014). Cholecystokinin from the entorhinal cortex enables neural plasticity in the auditory cortex. Cell Research 24, 307-330. 10.1038/cr.2013.164.

Ouagazzal, A. M., Reiss, D., & Romand, R. (2006). Effects of age-related hearing loss on startle reflex and prepulse inhibition in mice on pure and mixed C57BL and 129 genetic background. Behavioural brain research, 172(2), 307-315.

Young, J. W., Wallace, C. K., Geyer, M. A., & Risbrough, V. B. (2010). Age-associated improvements in cross-modal prepulse inhibition in mice. Behavioral neuroscience, 124(1), 133.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

Major concerns:

(1) In Figure 1, the authors used different metrics for fEPSP strength. In Figure 1D, the authors used the slope, while they used the amplitude in Figure 1G. It is known that the two metrics are different from each other. While the slope is calculated from the linear regression between the voltage change per time of the rising phase of the fEPSP, the amplitude represents the voltage value of the fEPSP's peak. Please clarify here and in the method what metric you used, because the two terms are not interchangeable.

Thank you for pointing out this oversight in our manuscript. We confirm that we used the slope of the fEPSP as the metric for assessing synaptic strength throughout the study, including both Figure 1D and Figure 1G. We will make the necessary corrections to ensure clarity and consistency. Thank you for bringing this to our attention.

(2) It is not mentioned in the details of the methods about the CCK-KO mice. Please give such details. Although the authors used the CCK-KO mouse model as a control, I think that it is not a good choice to test the hypothesis mentioned in lines 165 and 166. The experiment was supposed to monitor the CCK-BR activity after HFS of the MGB and answer whether the CCK-BR will get activated by thalamic stimulation, but the CCK-KO mouse does not have CCK to be released after the optogenetic activation of the Chrimson probe. Therefore, it is expected to give nothing as if the experimenter runs an experiment without intervention. I think that the appropriate way to examine the hypothesis is to compare mice that were either injected with AAV9-Syn-FLEX-ChrimsonR-tdTomato or AAV9-Syn-FLEX-tdTomato. However, CCK-OK would be a perfect model to confirm that LTP can be only generated dependently on CCK, by simply running the HFS of the MGB that would be associated with the cortical recording of the fEPSP. This also will rule out the assumption that the authors mentioned in lines 191 and 192.

Thank you for your valuable feedback. The rationale behind our experimental design was to validate the newly developed CCK sensor and confirm its specificity. We aimed to verify CCK release post-HFS by comparing the responses of the CCK sensor in CCK-KO mice and CCK-Cre mice. This comparison allowed us to determine that the observed increase in fluorescence intensity post-HFS was specifically due to CCK release, rather than other neurotransmitters induced by HFS.

We appreciate your suggestion to compare mice injected with AAV9-Syn-FLEX-ChrimsonR-tdTomato and AAV9-Syn-FLEX-tdTomato, as it is indeed a valuable approach for directly testing the hypothesis regarding CCK-BR activation. However, we prioritized using the CCK-KO model to validate the CCK sensor's efficacy and specificity. The validation can be inferred by comparing the CCK sensor activity before and after HFS.

Regarding concerns mentioned in lines 191 and 192 about potential CCK release from other projections via indirect polysynaptic activation, CCK-KO mice were not suitable for this aspect due to their global knockout of CCK. To address this limitation, we utilized shRNA to specifically down-regulate Cck expression in MGB neurons. This approach focused on the necessity of CCK released from thalamocortical projections for the observed LTP and effectively ruled out the possibility of indirect polysynaptic activation.

We also acknowledge that the methods section lacked sufficient details about the CCK-KO mice, which may have caused confusion. In the revised methods section, we will add the following details:

(1) The genotype of the CCK-KO mice used in this study (CCK-ires-CreERT2, Jax#012710).

(2) A brief description of the CCK-KO validation, emphasizing the absence of CCK mRNA in these mice (as shown in Figure 3A and 3B).

(3) The experimental purpose of using CCK-KO mice to validate the specificity of the CCK sensor.

We believe these additions will clarify the rationale for using CCK-KO mice and their role in this study. Thank you again for highlighting these important points.

(3) Figure 3C: The authors should examine if there is a difference in the baseline of fEPSPs across different age groups as the dependence on the normalization in the analysis within each group would hide if there were any difference of the baseline slope of fEPSP between groups which could be related to any misleading difference after HFS. Also, I wonder about the absence of LTP in P20, which is a closer age to the critical period. Could the authors discuss that, please?

Thank you for your insightful feedback. To address your concern regarding baseline differences in fEPSP slopes across age groups, we conducted additional analysis. Baseline fEPSP across the three groups (P20, 8w, 18m), normalized to the 8w group, were 64.8± 13.1%, 100.0 ± 20.4%, and 58.8± 10.3%, respectively. While there was a trend suggesting smaller fEPSP slopes in the P20 and 18m groups compared to the young adult group, these differences were not statistically significant due to data variability (P20 vs. 8w, P = 0.319; 8w vs. 18m, P=0.147; P20 vs. 18m, P = 1.0, one-way ANOVA). These results suggest that baseline variability is unlikely to confound the observed differences in LTP after HFS. Furthermore, we ensured that normalization minimized any potential baseline effects.

Regarding the absence of LTP in P20, this likely reflects developmental regulation of CCKBR expression in the auditory cortex (ACx). The HFS-induced thalamocortical LTP observed in our study is CCK-dependent and mechanistically distinct from the NMDA-dependent thalamocortical LTP during the critical period. Specifically, correlated pre- and postsynaptic activity can induce NMDA-dependent thalamocortical LTP only during an early critical period corresponding to the first several postnatal days, after which this pairing becomes ineffective starting from the second postnatal week (Crair and Malenka, 1995; Isaac et al., 1997; Chun et al., 2013). In contrast, the CCK-dependent Thalamocortical LTP induced by HFS is robust in adult mice but appears absent in P20, likely due to the lack of postsynaptic CCKBR expression in the ACx at this developmental stage.

We will include these clarifications in the revised manuscript, particularly in the Discussion section, to provide a more comprehensive explanation of our findings. Thank you for your valuable comments and suggestions.

Crair, M.C., and Malenka, R.C. (1995). A critical period for long-term potentiation at thalamocortical synapses. Nature 375, 325-328. 10.1038/375325a0.

Isaac, J.T.R., Crair, M.C., Nicoll, R.A., and Malenka, R.C. (1997). Silent Synapses during Development of Thalamocortical Inputs. Neuron 18, 269-280. https://doi.org/10.1016/S0896-6273(00)80267-6.

Chun, S., Bayazitov, I.T., Blundon, J.A., and Zakharenko, S.S. (2013). Thalamocortical Long-Term Potentiation Becomes Gated after the Early Critical Period in the Auditory Cortex. The Journal of Neuroscience 33, 7345-7357. 10.1523/jneurosci.4500-12.2013.

(4) Figure 4F: It is noticed that the baseline fEPSP of the CCK group and ACSF groups were different, which raises a concern about the baseline differences between treatment groups.

Thank you for your valuable feedback and for pointing out this important detail. We apologize for any confusion caused by the presentation of the data. As noted in the figure legend, the scale bars for the fEPSPs were different between the left (0.1 mV) and right panels (20 µV). This difference in scale may have created the perception of baseline differences between the CCK and ACSF groups. To enhance clarity and avoid potential misunderstanding, we will unify the scale bar values in the revised figure. This adjustment will provide a clearer and more accurate comparison of fEPSPs between groups. Thank you again for bringing this issue to our attention.

(5) From Figure S2D, it seems that different animals were injected with the drug and ACSF. Therefore, how the authors validate the position of the recording electrode to the cortical area of certain CF and relative EF. Also, there is not enough information about the basis of the selection of the EF. Should it be lower than the CF with a certain value? Was the EF determined after the initial tuning curve in each case? To mitigate this difference, it would be appropriate if the authors examined the presence of a significant difference in the tuning width and CFs between animals exposed to ACSF and CCK-4. This will give some validation of a balanced experiment between ACSF and CCK-4. I wonder also why the authors used rats here not mice, as it will be easier to interpret the results came from the same species.

Thank you for your thoughtful comments. The effective frequency (EF) was determined after measuring the initial tuning curve for each case. The EF was selected to elicit a clear sound response while maintaining a sufficient distance from the characteristic frequency (CF) to allow measurable increases in response intensity. Specifically, EF was selected based on the starting point of the tuning peak, which corresponds to the onset of its fastest rising phase. From this point, EF was determined by moving 0.2 or 0.4 octaves toward the CF. While there were individual differences in EF selection among animals, the methodology for determining EF was standardized and applied consistently across both the ACSF and CCK-4 groups.

Regarding the use of rats in these experiments, these studies were conducted prior to our current work with mice. The findings in rat provide valuable insights that support our current results in mice. Since the rat data are supplementary to the primary findings, we included them as supplementary material to provide additional context and validation. Furthermore, in consideration of animal welfare, we chose not to replicate these experiments in mice, as the findings from rats were sufficient to support our conclusions.

Methods section:

“The tuning curve was determined by plotting the lowest intensity at which the neuron responded to different tones. The characteristic frequency (CF) is defined as the frequency corresponding to the lowest point on this curve. The effective frequency (EF) was determined to elicit a clear sound response while maintaining a sufficient distance from the CF to allow measurable increases in response intensity. Specifically, EF was selected based on the starting point of the tuning peak, which corresponds to the onset of its fastest rising phase. From this point, EF was determined by moving 0.2 or 0.4 octaves toward the CF.”

(6) Lines 384-386: There are no figures named 5H and I.

Thank you for pointing this out. The references to Figures 5H and 5I were incorrect and should have referred to Figures 5C and 5D. We sincerely apologize for this oversight and will correct these errors in the revised manuscript to ensure clarity and accuracy. Thank you again for bringing this to our attention.

(7) The authors should mention the sex of the animals used.

Thank you for your comment and for highlighting this important detail. The sex of the animals used in this study is specified in the Animals section of the Methods: "In the present study, male mice and rats were used to investigate thalamocortical LTP." We appreciate your careful attention to this point and will ensure that this detail remains clearly stated in the manuscript.

(8) Lines 534 and 648: These coordinates are difficult to understand. Since the experiment was done on both mice and rats, we need a clear description of the coordinates in both. Also, I think that you should mention the lateral distance from the sagittal suture as the ventral coordinates should be calculated from the surface of the skull above the AC and not from the sagittal suture.

Thank you for your valuable feedback and for pointing out this important issue. We apologize for any confusion caused by our description of the coordinates. The term “ventral” was deliberately used because the auditory cortex is located on the lateral side of the skull, which may have caused some misunderstanding.

To provide a clearer and more accurate descriptions of the coordinates, we will revise the text in the manuscript as follows: “A craniotomy was performed at the temporal bone (-2 to -4 mm posterior and -1.5 to -3 mm ventral to bregma for mice; -3.0 to -5.0 mm posterior and -2.5 to -6.5 mm ventral to bregma for rats) to access the auditory cortex.'

We appreciate your attention to these details and will ensure that the revised manuscript includes this clarification to improve accuracy and eliminate potential confusion. Thank you again for bringing this to our attention.

(9) Line 536: The author should specify that these coordinates are for the experiment done on mice.

Thank you for your valuable feedback. We will revise the manuscript to explicitly specify that these coordinates refer to the experiments conducted on mice. This clarification will help improve the clarity and precision of the manuscript. We greatly appreciate your attention to this point and your effort to enhance the quality of our work.

Methods section:

“and a hole was drilled in the skull according to the coordinates of the ventral division of the MGB (MGv, AP: -3.2 mm, ML: 2.1 mm, DV: 3.0 mm) for experiments conducted on mice.”

(10) Line 590: Please add the specifications of the stimulating electrode. Is it unipolar or bipolar? What is the cat.# provided by FHC?

Thank you for your valuable feedback. The electrodes used in the experiments are unipolar. We will include the catalog number provided by FHC in the revised manuscript for clarity. The revised text will be updated as follows:

“In HFS-induced thalamocortical LTP experiments, two customized microelectrode arrays with four tungsten unipolar electrodes each, impedance: 0.5-1.0 MΩ (recording: CAT.# UEWSFGSECNND, FHC, U.S.), and 200-500 kΩ (stimulating: CAT.# UEWSDGSEBNND, FHC, U.S.), were used for the auditory cortical neuronal activity recording and MGB ES, respectively.”

We appreciate your attention to this detail, and we will ensure that the revised manuscript reflects this clarification accurately.

(11) Lines 612-614: There are no details of how the optic fiber was inserted or post-examined. If there is a word limitation, the authors may reference another study showing these procedures.

Thank you for your insightful comment and for highlighting this important aspect of the methodology. To address this, we will reference the study by Sun et al. (2024) in the revised manuscript, which provides detailed procedures for optic fiber insertion and post-examination. We believe that this reference will help enhance the clarity and completeness of the methods section.

Sun, W., Wu, H., Peng, Y., Zheng, X., Li, J., Zeng, D., Tang, P., Zhao, M., Feng, H., Li, H., et al. (2024). Heterosynaptic plasticity of the visuo-auditory projection requires cholecystokinin released from entorhinal cortex afferents. eLife 13, e83356. 10.7554/eLife.83356.

We appreciate your valuable suggestion, which will contribute to improving the quality of the manuscript.

Minor concerns:

(1) The definition of HFS was repeated many times throughout the manuscript. Please mention the defined name for the first time in the manuscript only followed by its abbreviation (HFS).

Thank you for your suggestion and for pointing out this important detail. We will revise the manuscript to ensure that all abbreviations are defined only upon their first mention in the manuscript, with subsequent mentions using the abbreviations consistently. We appreciate your careful attention to detail and your effort to help improve the manuscript.

(2) Line 173: There is a difference between here and the methods section (620 nm here and 635 nm there) please correct which wavelength the authors used.

Thank you for your careful review and for bringing this discrepancy to our attention. We have corrected the inconsistency, and the wavelength has been unified throughout the manuscript to ensure accuracy and clarity. The revised text now reads as follows:

“The fluorescent signal was monitored for 25s before and 60s after the HFLS (5~10 mW, 620 nm) or HFS application.”

We appreciate your valuable feedback, which has helped us improve the precision and consistency of the manuscript.

(3) Line 185: I think the authors should refer to Figure 2G before mentioning the statistical results.

Thank you for your careful review and for pointing out this oversight. We have now added a reference to Figure 2G at the appropriate location to ensure clarity and logical flow in the manuscript, as recommended..

(4) Line 202: I think the authors should refer to Figure 2J before mentioning the statistical results.

Thank you again for your careful review and for highlighting this point. We have revised the manuscript to include a reference to Figure 2J before mentioning the statistical results.

We appreciate your valuable feedback, which has helped us improve the accuracy and presentation of the results.

(5) Line 260: Please add appropriate references at the end of the sentence to support the argument.

Thank you for your valuable suggestion. To address this, we have add appropriate references to support the statement regarding the multiple steps involved between mRNA expression and neuropeptide release. Additionally, we have revised the statement to adopt a more cautious interpretation. The revised text is as follows:

“It is widely recognized that mRNA levels do not always directly correlate with peptide levels due to multiple steps involved in peptide synthesis and processing, including translation, post-translational modifications, packaging, transportation, and proteolytic cleavage, all of which require various enzymes and regulatory mechanisms (38-41). A disruption at any stage in this process could lead to impaired CCK release, even when Cck mRNA is present.”

We have included the following references to support this statement:

38. Mierke, C.T. (2020). Translation and Post-translational Modifications in Protein Biosynthesis. In Cellular Mechanics and Biophysics: Structure and Function of Basic Cellular Components Regulating Cell Mechanics, C.T. Mierke, ed. (Springer International Publishing), pp. 595-665. 10.1007/978-3-030-58532-7_14.

39. Gualillo, O., Lago, F., Casanueva, F.F., and Dieguez, C. (2006). One ancestor, several peptides post-translational modifications of preproghrelin generate several peptides with antithetical effects. Mol Cell Endocrinol 256, 1-8. 10.1016/j.mce.2006.05.007.

40. Sossin, W.S., Fisher, J.M., and Scheller, R.H. (1989). Cellular and molecular biology of neuropeptide processing and packaging. Neuron 2, 1407-1417. https://doi.org/10.1016/0896-6273(89)90186-4.

41. Hook, V., Funkelstein, L., Lu, D., Bark, S., Wegrzyn, J., and Hwang, S.R. (2008). Proteases for processing proneuropeptides into peptide neurotransmitters and hormones. Annu Rev Pharmacol Toxicol 48, 393-423. 10.1146/annurev.pharmtox.48.113006.094812.

We greatly appreciate your helpful feedback, which has allowed us to improve both the accuracy and the depth of discussion in the manuscript.

(6) Line 278: The authors mentioned "due to the absence of CCK in aged animals", which was not an appropriate description. It should be a reduction of CCK gene expression or a possible deficient CCK release.

Thank you for your careful review and for pointing out the inaccuracy in our description. We agree with your suggestion and have revised the statement to more appropriately reflect the findings.

“Our findings revealed that thalamocortical LTP cannot be induced in aged mice, likely due to insufficient CCK release, despite intact CCKBR expression.”

This revision ensures a more accurate and precise description of the potential mechanisms underlying the observed phenomenon. We greatly appreciate your valuable feedback, which has helped us improve the clarity and accuracy of the manuscript.

(7) Line 291: The authors mentioned that "without MGB stimulation", which is confusing. The MGB was stimulated with a single electrical pulse to evoke cortical fEPSPs. Therefore it should be "without HFS of MGB".

Thank you for pointing this out and for highlighting the potential confusion caused by our original phrasing. Upon review, we recognize that our original phrasing "without MGB stimulation" may have been unclear and could have led to misinterpretation. To clarify, our intention was to describe the period during which CCK was present without any stimulation of the MGB.

It is important to note that, in the presence of CCK, LTP can be induced even with low-frequency stimulation, including in aged mice. This observation underscores the potent effect of CCK in facilitating thalamocortical LTP, regardless of the specific stimulation protocol used.

To address this issue, we have revised the sentence for improved clarity as follows::

" To investigate whether CCK alone is sufficient to induce thalamocortical LTP without activating thalamocortical projections, we infused CCK-4 into the ACx of young adult mice immediately after baseline fEPSPs recording. Stimulation was then paused for 15 min to allow for CCK degradation, after which recording resumed."

We believe this revision resolves the misunderstanding and provides a clearer and more accurate description of the experimental context. We greatly appreciate your insightful feedback, which has helped us refine the manuscript for clarity and precision.

Reviewer #3 (Recommendations for the authors):

Minor comments:

(1) Line 99, 134, possibly other locations: "site" to "sites".

Thank you for your careful review. We appreciate your attention to detail and have made the necessary corrections in the manuscript.

(2) Throughout the manuscript there are some minor issues with language choice and subtle phrasing errors and I suggest English language editing.

Thank you for your suggestion. In response, we have thoroughly reviewed the manuscript and addressed issues related to language choice and phrasing. The text has been carefully edited to ensure clarity, precision, and consistency. We believe these revisions have significantly enhanced the overall quality of the manuscript. We greatly appreciate your feedback, which has been invaluable in improving the presentation of our work.

(3) Based on the experimental configurations, I do not think it is a problematic caveat, but authors should be aware of the high likelihood of AAV9 jumping synapses relative to other AAV serotypes.

Thank you for bringing up the potential of AAV9 crossing synapses, a recognized characteristic of this serotype. We appreciate your observation regarding its relevance to our experimental design. In our study, we carefully considered the possibility of trans-synaptic transfer during both the experimental design and data interpretation phases. To minimize the likelihood of significant trans-synaptic spread, we implemented several measures, including controlling the injection volume, using a slow injection rate, and limiting the viral expression time. Post-hoc histological analyses confirmed that the expression of AAV9 was largely confined to the intended regions, with limited evidence of synaptic jumping under our experimental conditions.

While we acknowledge the inherent potential for AAV9 to cross synapses, we believe this effect does not substantially confound the interpretation of our findings in the current study. To address this concern, we have added a brief discussion on this point in the revised manuscript to enhance clarity. We greatly appreciate your insightful comment, which has helped us further refine our work.

Discussion section：

“ One potential limitation of our study is the trans-synaptic transfer property of AAV9. To mitigate this, we carefully controlled the injection volume, rate, and viral expression time, and conducted post-hoc histological analyses to minimize off-target effects, thereby reducing the likelihood of trans-synaptic transfer confounding the interpretation of our findings.”

(4) The trace identifiers (1-4) do not seem correctly placed/colored in Figure S1D. Please check others carefully.

Thank you for your careful review and for bringing this issue to our attention. We have corrected the trace identifiers in Figure S1D. Additionally, we have carefully reviewed all other figures to ensure their accuracy and consistency. We greatly appreciate your attention to detail, which has helped improve the overall quality of the manuscript.

(5) Please provide a value of the laser power range based on calibrated values.

Thank you for your suggestion. We have included the calibrated laser power range in the revised manuscript as follows:

“The laser stimulation was produced by a laser generator (5-20 mW(30), Wavelength: 473 nm, 620 nm; CNI laser, China) controlled by an RX6 system and delivered to the brain via an optic fiber (Thorlabs, U.S.) connected to the generator.”

We appreciate your feedback, which has helped improve the clarity and precision of our methodological description.

(6) It would be useful to annotate figures in a way that identifies in which transgenic mice experiments are being performed.

Thank you for your valuable suggestion. We will add annotations to the figures to explicitly identify the type of mice used in each experiment. We believe this enhancement will improve the clarity and accessibility of our results. We greatly appreciate your input in making our manuscript more informative.

(7) Please comment on the rigor you use to address the accuracy of viral injections. How often did they spread outside of the MGB/AC?

Thank you for raising this important question regarding the accuracy of viral injections and the potential spread outside the MGB or AC. Below, we provide details for each set of experiments:

shRNA Experiments:

For the shRNA experiments targeting the MGB, our primary goal was to achieve comprehensive coverage of the entire MGB. To this end, we used larger injection volumes and multiple injection sites, which inevitably resulted in some viral spread beyond the MGB. However, this approach was necessary to ensure robust knockdown effects that were representative of the entire MGB. While strict confinement to specific subregions could not be guaranteed, this strategy allowed us to prioritize the effectiveness of the knockdown within the target region.

Fiber photometry Experiments:

For the fiber photometry experiments targeting the auditory cortex (AC), we used larger injection volumes and multiple injection sites to cover its relatively large size. Although this approach might have resulted in some CCK-sensor virus spread outside the AC, the placement of the optic fiber was guided by the location of the auditory cortex. Consequently, any minor viral expression outside the AC would not affect the experimental results, as recordings were confined to the intended area through precise fiber placement.

Optogenetic Experiments:

For the optogenetic experiments targeting the MGB, we specifically injected virus into the MGv subregion. To minimize viral spread, we employed several strategies, including the used fine injection needles, waiting for tissue stabilization (7 minutes post-needle insertion), delivering small volumes at a slow rate to prevent backflow, aspirating 5 nL of the solution post-injection, and raising the needle by 100 μm before waiting an additional 5 minutes prior to full retraction. These measures significantly reduced the risk of viral leakage to adjacent regions.

Histological Validation:

After the electrophysiological experiments, we systematically verified the accuracy of viral expression by examining histological sections to ensure that the expression was primarily localized within the intended regions.

Terminology in the Manuscript:

In the manuscript, we deliberately used the term "MGB" in the manuscript rather than specifically "MGv" to transparently acknowledge the potential for viral spread in some experiments.

We hope this explanation clarifies the strategies we employed to address the accuracy of viral injections, as well as how we managed potential viral spread. We have also added a brief information in the revised manuscript to reflect these points and acknowledge the inherent variability in viral delivery.