Heterosynaptic plasticity of the visuo-auditory projection requires cholecystokinin released from entorhinal cortex afferents

  1. Wenjian Sun
  2. Haohao Wu
  3. Yujie Peng
  4. Xuejiao Zheng
  5. Jing Li
  6. Dingxuan Zeng
  7. Peng Tang
  8. Ming Zhao
  9. Hemin Feng
  10. Hao Li
  11. Ye Liang
  12. Junfeng Su
  13. Xi Chen
  14. Tomas Hökfelt
  15. Jufang He

  • Curated by eLife

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    eLife assessment

    This important work provides knowledge regarding how neuropeptides, which are highly expressed in the brain, can influence cortical plasticity. The conclusions are supported by compelling evidence from both in vitro and in vivo assays, although some control experiments are needed to further strengthen the conclusions. This paper will be of interest to neuroscientists studying cortical processing and neural plasticity, as well as cell biologists and biochemists interested in peptide function in general.

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Abstract

The entorhinal cortex is involved in establishing enduring visuo-auditory associative memory in the neocortex. Here we explored the mechanisms underlying this synaptic plasticity related to projections from the visual and entorhinal cortices to the auditory cortex in mice using optogenetics of dual pathways. High-frequency laser stimulation (HFS laser) of the visuo-auditory projection did not induce long-term potentiation. However, after pairing with sound stimulus, the visuo-auditory inputs were potentiated following either infusion of cholecystokinin (CCK) or HFS laser of the entorhino-auditory CCK-expressing projection. Combining retrograde tracing and RNAscope in situ hybridization, we show that Cck expression is higher in entorhinal cortex neurons projecting to the auditory cortex than in those originating from the visual cortex. In the presence of CCK, potentiation in the neocortex occurred when the presynaptic input arrived 200 ms before postsynaptic firing, even after just five trials of pairing. Behaviorally, inactivation of the CCK + projection from the entorhinal cortex to the auditory cortex blocked the formation of visuo-auditory associative memory. Our results indicate that neocortical visuo-auditory association is formed through heterosynaptic plasticity, which depends on release of CCK in the neocortex mostly from entorhinal afferents.

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  1. Version published to 10.7554/elife.83356 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Sun et al. investigated the circuit mechanism of a novel type of synaptic plasticity in the projection from the visual cortex to the auditory cortex (VC-AC), which is thought to play an important role in visuo-auditory associative learning. The key question behind this paper is what is the role of CCK positive projection from the entorhinal cortex in the plasticity of VC-AC projections? They discover that the strength of VC-AC projections does not change when pairing the stimulation of this pathway with the acoustic stimulation of the auditory cortex (AC) unless CCK is applied to the AC or CCK positive projection from the entorhinal cortex to auditory cortex (EC-AC) is optogenetically stimulated. In contrast, optogenetically stimulating VC-AC projections, which express a lower level of CCK than the EC-AC projection, do not induce such synaptic plasticity. Interestingly, the data also indicates that even if the EC-AC pathway is stimulated 500ms ahead of the pairing of stimulating VC-AC pathway and the AC, the VC-AC synaptic strength can still be potentiated, consistent with the long-lasting nature of CCK as a neuropeptide. By performing a fear conditioning assay, the authors demonstrate that the CCK signaling is indeed required for the association of visual and auditory cues.

    The proposed mechanism is interesting because it not only helps explain the heterosynaptic plasticity of the visual-auditory projection but also will provide insight into how the entorhinal cortex as an association area contributes to the association of visual and auditory cues. Nevertheless, this study suffers from the lack of a few key experiments, which prevents drawing a conclusion on the contribution of CCK release from the EC-AC projection to the plasticity of the VC→AC projection.

    We are grateful for the constructive comments provided by the reviewers and appreciate the significant effort they have dedicated to reviewing our manuscript. To enhance our study and strengthen our conclusions, we have made the following revisions in response to their feedback.

    1. One main conclusion from figures 1-3 is that CCK released from the EC-AC projection is required for the plasticity of VC-AC projection in addition to pairing VALS with noise/electrical stimulation. But the data in those figures cannot exclude alternative explanations that CCK alone or the pairing CCK with either VALS or noise are sufficient to make the VC-AC synaptic connection more potent. It concerns the mechanism underlying the effect of CCK: CCK may function simply as a neuromodulator to regulate the excitatory synaptic transmission, but not to promote long term synaptic plasticity.

    Thanks for the valuable comment and pointing out the weakness. In response to the comment, we have conducted additional control experiments to reinforce our conclusions. These include: For Figure 1G, we introduced three control groups: CCK alone (Figure1-figure supplement 1F-G), CCK + presynaptic activation of VC-to-AC inputs (Figure 1-figure supplement 1H-I), and CCK + postsynaptic firing induced by noise (Figure 1-figure supplement 1J-K). Our findings from these control experiments indicate that in all three scenarios, there was no potentiation of the VC-to-AC inputs. Further details can be found in Figure 1-figure supplement 1F-K.

    For Figure 2E, we introduced three control groups: HFS laser EC-to-AC alone (Figure 2-figure supplement 1H-I), HFS laser EC-to-AC + presynaptic activation of VC-to-AC inputs (Figure 2-figure supplement 1L-M), and HFS laser + postsynaptic firing induced by noise (Figure 2-figure supplement 1P-Q). And we found that in all three scenarios, the VC-to-AC inputs were not significantly potentiated. Please see details in Figure 2-figure supplement 1.

    Given that our in vivo results already demonstrated that neither HFS laser EC-to-AC alone, nor its combination with presynaptic or postsynaptic activation, potentiated the VC-to-AC inputs, we did not replicate these control groups in our ex vivo setup. These additional experiments enhance the robustness of our findings and address the initial concerns raised.

    1. Similar issue exists in Fig. 2H and 3J. Without proper controls, it is impossible to tell whether all three conditions (HFLSEA, VALA, noise/electrical stimulation) are necessary for potentiated AC responses to acoustic/electrical stimulation.

    Same as above, we have conducted additional control experiments to reinforce our conclusions. These include:

    For Figure 2H, we also tested the noise response in the above three control groups: HFS laser EC to AC alone (Figure 2-figure supplement 1J-K), HFS laser EC-to-AC + presynaptic activation of VC-to-AC inputs (Figure 2-figure supplement 1N-O), and HFS laser + postsynaptic firing induced by noise (Figure 2-figure supplement 1R-S). And we found that fEPSPs evoked by noise stimuli were significantly potentiated after HFS laser EC-to-AC + Post (Figure 2-figure supplement 1R-S). However, there was no potentiation observed following HFS laser EC-to-AC alone (Figure 2-figure supplement 1J-K) and HFS laser EC-to-AC + Pre (Figure 2-figure supplement 1N-O).

    These results suggest that both HFS laser targeting the EC-to-AC projection and noise-induced AC firing are required to potentiate the AC's response to acoustic stimuli. In contrast, activation of the VC-to-AC projection is not necessary. This finding aligns with our previous research (Li et al., 2014).

    Given the similarity in experimental design, we opted not to replicate these specific control groups in our ex vivo setup.

    These additional control experiments have been crucial in reinforcing the conclusions of our study.

    1. Fig. 2E and 3G show that the stimulation of CCK-positive EC-AC projection is required for the plasticity of VC-AC projection. Considering most EC-AC projection neurons co-release glutamate and CCK, however, we cannot tell if CCK or glutamate or both matter to this type of plasticity. Even though the long delay in Fig 5B is consistent with the neuropeptide nature of CCK, direct experimental evidence is needed, since it is where the novelty of the paper is.

    Thank you for your constructive feedback. In response to the suggestions, for Figure 2E, we have incorporated two additional experiments: one with a CCKB receptor (CCKBR) antagonist and another with ACSF infused into the AC prior to HFS laser EC-to-AC + Pre/Post Pairing (Figures 2N-P). Our findings demonstrate that the CCKBR antagonist effectively inhibited the potentiation of the VC-to-AC inputs following the HFS laser EC-to-AC + Pre/Post Pairing. Conversely, ACSF did not exhibit this inhibitory effect. For further information, please refer to Figures 2N-P. Given the similarity in experimental design, we opted not to replicate these groups in our ex vivo setup.

    1. In Fig. 6, the authors examined the necessity of CCK for the generation of the visuo-auditory association. The experimental approach of injection CCK receptor blocker or CCK-4 is not specific to the EC-AC pathway. There is neither a link between VC-AC plasticity nor this behavioral result. Thus, the explanatory power of this experiment is limited in the context set up by the first 5 figures.

    Thank you for highlighting this area for improvement. To enhance the explanatory power of our behavioral experiments, we conducted the following additional studies:

    1. Assessing the Necessity of CCK+ EC-to-AC Projection in Establishing Visuo-Auditory Association:

    We bilaterally injected AAV9-syn-DIO-hM4Di-eYFP or AAV9-syn-DIO-eYFP into the EC and implanted cannulae in the AC of Cck Ires-Cre mice. During the encoding phase, we inactivated the CCK+ EC-to-AC pathway via CNO infusion into the AC. Our results show that this inactivation prevents the behavioral establishment of an association between the visual stimulus (VS) and auditory stimulus (AS), without affecting the fear conditioning memory to the AS (Figure 6B, beige).

    1. Determining the Role of VC-to-AC Projection in Establishing Visuo-Auditory Association: We bilaterally injected AAV9-syn-hM4Di-eYFP or AAV9-syn-eYFP into the visual cortex (VC) and also implanted cannulae in the AC of Cck Ires-Cre mice. Inactivating the VC-to-AC pathway during the encoding phase with CNO infusion in the AC, we observed that this inactivation hinders the establishment of a behavioral association between VS and AS, but does not interfere with the fear conditioning memory to the AS (Figure 6B, red).

    2. Investigating the Importance of CCK+ EC-to-AC Projection in Recalling Recent Visuo-Auditory Association:

    Again, AAV9-syn-DIO-hM4Di-eYFP or AAV9-syn-DIO-eYFP was injected bilaterally into the EC, and cannulae were implanted in the AC of Cck Ires-Cre mice. By inactivating the CCK+ EC-AC pathway during the retrieval phase with CNO infusion into the AC, we found that such inactivation disrupted the recall of the recent association between VS and AS behaviorally, yet did not affect the fear conditioning memory to the AS (Figure 6D, beige).

    1. Assessing the Necessity of VC-to-AC Projection in Recalling Recent Association Memory: For this experiment, AAV9-syn-hM4Di-eYFP or AAV9-syn-DIO-eYFP was injected bilaterally into the VC, and cannulae were placed in the AC of Cck Ires-Cre mice. Inactivating the VC-AC pathway during the retrieval phase with CNO infusion in the AC led to the discovery that this inactivation disrupted the behavioral recall of the recent association between VS and AS but did not disrupt the fear conditioning memory to the AS (Figure 6D, red).

    These additional experiments significantly contribute to our understanding of the roles played by the CCK+ EC-AC and VC-AC projections in both the establishment and recall of visuo-auditory associative memories.

    1. In page 16, line 322-326, the authors concluded that to induce the plasticity of VC→AC projection, Delay 1 should be longer than 10 ms and Delay 2 should be longer than 0 ms. This conclusion was not fully supported by the data from Figure 5B-D, because there is no data point between -65 ms and 10 ms for Delay 1 (for example 0 ms), and no negative values for Delay 2.

    We rewrote this paragraph and hope it is more accurate now.

    “Taken together, our study indicates that significant potentiation of the VC-to-AC inputs can be observed (Figure 5D, black cube) across five pairing trials with a 10-second inter-trial interval, under certain tested conditions: (i) the frequency of repetitive laser stimulation of the CCK+ entorhinal cortex (EC) to AC projection was maintained at 10 Hz or higher (as we did not test frequencies between 1 to 10 Hz), (ii) Delay 1 was set within the tested range of 10 to 535 ms (noting the absence of data between -65 to 10 ms), and (iii) Delay 2 was within the range of 0 to 200 ms (acknowledging that negative values for Delay 2 were not explored).”

    Reviewer #2 (Public Review):

    The manuscript by Sun et al., investigates the synaptic plasticity underlying visuo-auditory association. Through a series of in vivo and ex vivo electrophysiology recordings, the authors show that high-frequency stimulation (HFLS) of the cholecystokinin (CCK) positive neurons in the entorhino-auditory projection paired with an auditory stimulus can evoke long-term potentiation (LTP) of the visuo-auditory projection. However, LTP of the visuo-auditory projection could not be elicited by HFLS of the visuo-auditory projection itself or by an unpaired stimulus. They further demonstrate that auditory stimulus pairing with CCK is required to elicit LTP of the visuo-auditory projection as well as visuo-auditory association in a fear conditioning behavioral experiment. As they found elevated expression of CCK in entorhinal neurons which project to the auditory cortex, they conclude that HFLS of the entorhino-auditory projection causes CCK release.

    Strengths:

    The authors use an elegant approach with Chrimson and Chronos to stimulate different auditory inputs in the same mouse in vivo and also in slice and demonstrate that potentiation of the visuo-auditory projection is dependent on HFLS of the entorhino-auditory projection paired with auditory stimulus. Furthermore, they test several parameters in a systematic fashion, generating a comprehensive analysis of the plasticity changes that regulate visuo-auditory association.

    Weaknesses:

    In their previous publications (Chen et al., 2019; Li et al., 2014; Zhang et al., 2020), it has been established that HFLS of the entorhino-auditory projection and CKK release are important for visuo-auditory association via electrophysiology and behavioral experiments. The Chrimson and Chronos approach was applied by Zhang et al., 2020, where they already found that the visuo-auditory projection was potentiated through HFLS of entorhino-neocortical fibers. This manuscript extends those findings by testing different parameters of pairing, which may not represent a major conceptual advance. Unlike the electrophysiological recordings, drug infusion is used in behavioral manipulations to show that HFLS of the entorhino-auditory projection is important for visuo-auditory association. While the use of drugs to inhibit CKK receptors is important, it does not directly demonstrate that CCK release from the entorhino-auditory is necessary.

    We deeply appreciate the reviewer's constructive and insightful feedback. Building on our previous work (Zhang et al., 2020), which highlighted the potentiation of the VC-to-AC projection through high-frequency laser stimulation (HFS laser) of entorhino-neocortical fibers, our current study probes further into the intricacies of this process. We have thoroughly explored the specific conditions necessary for the potentiation of the VC-to-AC projection, assessing a wide range of parameters.

    A significant advancement in our current research is the elucidation of why HFS of the VC-to-AC pathway alone fails to induce potentiation, whereas HFS of the EC-to-AC pathway, coupled with Pre/Post Pairing, is effective. This critical distinction is linked to the heightened expression of CCK in EC neurons projecting to the AC, in contrast to those from the VC. In this revised version of our study, we have also demonstrated that HFS laser stimulation of the EC-to-AC CCK+ projection induces the release of endogenous CCK in the AC using a combination of a CCK sensor and fiber photometry.

    Behaviorally, our revised research emphasizes the vital role of the CCK+ EC-AC projection in both establishing and retrieving visuo-auditory memories, thereby highlighting its fundamental importance in memory processing. Moreover, our study confirms that the CCK+ EC-AC projection is not only crucial for memory formation and retrieval but also indicates that the VC-to-AC projection is the anatomical basis for establishing visuo-auditory associations and serves as the principal storage site for visuo-auditory associative memory. These findings represent significant strides in our understanding of synaptic plasticity and memory mechanisms.

    For the behavioral part, to build the link that HFS laser of the EC-to-AC CCK+ projection is important for visuo-auditory association in the behavioral context, we conducted the following additional behavioral studies (for details please see the response to comment 4 of reviewer 1):

    1. Assessing the Necessity of CCK+ EC-to-AC Projection in Establishing Visuo-Auditory Associative memories, by inactivating the pathway with inhibitory DREADD during the encoding phase.

    2. Investigating the Importance of CCK+ EC-to-AC Projection in Recalling Visuo-Auditory Association, by inactivating the pathway with inhibitory DREADD during the retrieving phase.

  3. eLife

    eLife assessment

    This important work provides knowledge regarding how neuropeptides, which are highly expressed in the brain, can influence cortical plasticity. The conclusions are supported by compelling evidence from both in vitro and in vivo assays, although some control experiments are needed to further strengthen the conclusions. This paper will be of interest to neuroscientists studying cortical processing and neural plasticity, as well as cell biologists and biochemists interested in peptide function in general.

  4. eLife

    Reviewer #1 (Public Review):

    Sun et al. investigated the circuit mechanism of a novel type of synaptic plasticity in the projection from the visual cortex to the auditory cortex (VC-AC), which is thought to play an important role in visuo-auditory associative learning. The key question behind this paper is what is the role of CCK positive projection from the entorhinal cortex in the plasticity of VC-AC projections? They discover that the strength of VC-AC projections does not change when pairing the stimulation of this pathway with the acoustic stimulation of the auditory cortex (AC) unless CCK is applied to the AC or CCK positive projection from the entorhinal cortex to auditory cortex (EC-AC) is optogenetically stimulated. In contrast, optogenetically stimulating VC-AC projections, which express a lower level of CCK than the EC-AC projection, do not induce such synaptic plasticity. Interestingly, the data also indicates that even if the EC-AC pathway is stimulated 500ms ahead of the pairing of stimulating VC-AC pathway and the AC, the VC-AC synaptic strength can still be potentiated, consistent with the long-lasting nature of CCK as a neuropeptide. By performing a fear conditioning assay, the authors demonstrate that the CCK signaling is indeed required for the association of visual and auditory cues.

    The proposed mechanism is interesting because it not only helps explain the heterosynaptic plasticity of the visual-auditory projection but also will provide insight into how the entorhinal cortex as an association area contributes to the association of visual and auditory cues. Nevertheless, this study suffers from the lack of a few key experiments, which prevents drawing a conclusion on the contribution of CCK release from the EC-AC projection to the plasticity of the VC→AC projection.

    1. One main conclusion from figures 1-3 is that CCK released from the EC-AC projection is required for the plasticity of VC-AC projection in addition to pairing VALS with noise/electrical stimulation. But the data in those figures cannot exclude alternative explanations that CCK alone or the pairing CCK with either VALS or noise are sufficient to make the VC-AC synaptic connection more potent. It concerns the mechanism underlying the effect of CCK: CCK may function simply as a neuromodulator to regulate the excitatory synaptic transmission, but not to promote long term synaptic plasticity.

    2. Similar issue exists in Fig. 2H and 3J. Without proper controls, it is impossible to tell whether all three conditions (HFLSEA, VALA, noise/electrical stimulation) are necessary for potentiated AC responses to acoustic/electrical stimulation.

    3. Fig. 2E and 3G show that the stimulation of CCK-positive EC-AC projection is required for the plasticity of VC-AC projection. Considering most EC-AC projection neurons co-release glutamate and CCK, however, we cannot tell if CCK or glutamate or both matter to this type of plasticity. Even though the long delay in Fig 5B is consistent with the neuropeptide nature of CCK, direct experimental evidence is needed, since it is where the novelty of the paper is.

    4. In Fig. 6, the authors examined the necessity of CCK for the generation of the visuo-auditory association. The experimental approach of injection CCK receptor blocker or CCK-4 is not specific to the EC-AC pathway. There is neither a link between VC-AC plasticity nor this behavioral result. Thus, the explanatory power of this experiment is limited in the context set up by the first 5 figures.

    5. In page 16, line 322-326, the authors concluded that to induce the plasticity of VC→AC projection, Delay 1 should be longer than 10 ms and Delay 2 should be longer than 0 ms. This conclusion was not fully supported by the data from Figure 5B-D, because there is no data point between -65 ms and 10 ms for Delay 1 (for example 0 ms), and no negative values for Delay 2.

  5. eLife

    Reviewer #2 (Public Review):

    The manuscript by Sun et al., investigates the synaptic plasticity underlying visuo-auditory association. Through a series of in vivo and ex vivo electrophysiology recordings, the authors show that high-frequency stimulation (HFLS) of the cholecystokinin (CCK) positive neurons in the entorhino-auditory projection paired with an auditory stimulus can evoke long-term potentiation (LTP) of the visuo-auditory projection. However, LTP of the visuo-auditory projection could not be elicited by HFLS of the visuo-auditory projection itself or by an unpaired stimulus. They further demonstrate that auditory stimulus pairing with CCK is required to elicit LTP of the visuo-auditory projection as well as visuo-auditory association in a fear conditioning behavioral experiment. As they found elevated expression of CCK in entorhinal neurons which project to the auditory cortex, they conclude that HFLS of the entorhino-auditory projection causes CCK release.

    Strengths:

    The authors use an elegant approach with Chrimson and Chronos to stimulate different auditory inputs in the same mouse in vivo and also in slice and demonstrate that potentiation of the visuo-auditory projection is dependent on HFLS of the entorhino-auditory projection paired with auditory stimulus. Furthermore, they test several parameters in a systematic fashion, generating a comprehensive analysis of the plasticity changes that regulate visuo-auditory association.

    Weaknesses:

    In their previous publications (Chen et al., 2019; Li et al., 2014; Zhang et al., 2020), it has been established that HFLS of the entorhino-auditory projection and CKK release are important for visuo-auditory association via electrophysiology and behavioral experiments. The Chrimson and Chronos approach was applied by Zhang et al., 2020, where they already found that the visuo-auditory projection was potentiated through HFLS of entorhino-neocortical fibers. This manuscript extends those findings by testing different parameters of pairing, which may not represent a major conceptual advance. Unlike the electrophysiological recordings, drug infusion is used in behavioral manipulations to show that HFLS of the entorhino-auditory projection is important for visuo-auditory association. While the use of drugs to inhibit CKK receptors is important, it does not directly demonstrate that CCK release from the entorhino-auditory is necessary.

  6. Version published to 10.1101/2022.10.04.510820v1 on bioRxiv