Cholecystokinin facilitates motor skill learning by modulating neuroplasticity in the motor cortex

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1. 

   Version published to 10.7554/elife.83897 on eLife

   May 3, 2024
2. 

   eLife

   Dec 17, 2023

   Author Response

   Reviewer #1 (Public Review):

   This paper combines an array of techniques to study the role of cholecystokinin (CCK) in motor learning. Motor learning in a pellet reaching task is shown to depend on CCK, as both global and locally targeted CCK manipulations eliminate learning. This learning deficit is linked to reduced plasticity in the motor cortex, evidenced by both slice recordings and two-photon calcium imaging. Furthermore, CCK receptor agonists are shown to rescue motor cortex plasticity and learning in knockout mice. While the behavioral results are clear, the specific effects on learning are not directly tested, nor is the specificity pathway between rhinal CCK neurons and the motor cortex. In general, the results present interesting clues about the role of CCK in motor learning, though the specificity of the …

   Author Response

   Reviewer #1 (Public Review):

   This paper combines an array of techniques to study the role of cholecystokinin (CCK) in motor learning. Motor learning in a pellet reaching task is shown to depend on CCK, as both global and locally targeted CCK manipulations eliminate learning. This learning deficit is linked to reduced plasticity in the motor cortex, evidenced by both slice recordings and two-photon calcium imaging. Furthermore, CCK receptor agonists are shown to rescue motor cortex plasticity and learning in knockout mice. While the behavioral results are clear, the specific effects on learning are not directly tested, nor is the specificity pathway between rhinal CCK neurons and the motor cortex. In general, the results present interesting clues about the role of CCK in motor learning, though the specificity of the claims is not fully supported.

   Since all CCK manipulations were performed throughout learning, rather than after learning, it is not clear whether it is learning that is affected or if there is a more general motor deficit. Related to this point, Figure 1D appears to show a general reduction in reach distance in CCK-/- mice. A general motor deficit may be expected to produce decreased success on training day 1, which does not appear to be the case in Figure 1C and Figure 2B, but may be present to some degree in Figure 5B. Or, since the task is so difficult on day 1, a general motor deficit may not be observable. It is therefore inconclusive whether the behavioral effect is learning-specific.

   Thanks for your comments and suggestions.

   We have tested the basic movement ability of CCK-/- and WT mice and we found that there were no significant difference between CCK-/- and WT in terms of stride length, stride time, step cycle ratio and grasp force (Figure S1C, S1D, S1E, S1F). Besides, we also have tested the performance of mice injected with CCKBR antagonist or injected with hM4Di together with clozapine after learned the task (Figure S2D, S8D). The performance of mice before and after antagonist injection or chemogenetic manipulation were comparable. These results suggested that all the CCK manipulations did not cause general defects to the movement ability of mice.

   The paper implicates motor cortex-projecting CCK neurons in the rhinal cortex as being a key component in motor learning. However, the relative importance of this pathway in motor learning is not pinned down. The necessity of CCK in the motor cortex is tested by injecting CCK receptor antagonists into the contralateral motor cortex (Figure 2), though a control brain region is not tested (e.g. the ipsilateral motor cortex), so the specificity of the motor cortex is not demonstrated.

   Thanks for your comments and suggestions.

   In this study, we focus on the role played by CCK from the rhinal cortex to the motor cortex, and how CCK affects motor learning. The single pellet reaching task was selected to study the role of CCK from the rhinal cortex to the motor cortex in motor skill learning and the motor cortex is considered as the main area generates motor memory when training in this task (Komiyama et al., 2010; Peters et al., 2014; Richard et al., 2019). We emphasized that the importance of the motor cortex in motor learning, not meant that other brain areas where also receive CCK-positive neural projections from the rhinal cortex, for example hippocampus (spatial memory), are not important for the performance of this task. In fact, specifically inhibiting the projection from the rhinal cortex to the contrallateral motor cortex is not enough to suppress the motor learning ability of, but inhibiting projecting in both sides (contro- and ipsi-lateral) could suppress the learning ability of mice, suggesting that the whole motor cortex is critical for motor skill learning (Figure 6, S8). In this paper, we studied the relationship between the rhinal cortex and the motor cortex and the role played by CCK in this circuit. The specificity of the motor cortex is task-dependent, not the main purpose in this study.

   The learning-related source of CCK in the motor cortex is also unclear, since even though it is demonstrated that CCK neurons in the rhinal cortex project to the motor cortex in Figure 4D, Figure 4C shows that there is also a high concentration of CCK neurons locally within the motor cortex. Likewise, the importance of the projection from the rhinal cortex to the motor cortex is not specifically tested, as rhinal CCK neurons targeted for inactivation in Figure 5 include all CCK cells rather than motor cortex-projecting cells specifically.

   Thanks for your comments and suggestions.

   The specificity of the CCK-projection from the rhinal cortex to the motor cortex for motor skill learning was studies using chemogenetic methods in the revised version of the manuscript. We first determined that over 98% of neurons in the rhinal cortex that projected to the motor cortex are CCK positive (Figure 6A, S6A, S6B). Next, we injected the retro-Cre virus in the motor cortex and the Cre-dependent hM4Di in the rhinal cortex in C57BL/6 mice to specifically inhibit the CCK neurons from the rhinal cortex to the motor cortex. Compared to two control groups, the learning ability of the experimental group was significant suppressed, suggesting that CCK projections from the rhinal cortex to the motor cortex are critical for motor skill learning (Figure 6). Detailed description was added in the part of "Result" in the manuscript.

   CCK is suggested to play a role in producing reliable activity in the motor cortex through learning through two-photon imaging experiments. This is useful in demonstrating what looks like normal motor cortex activity in the presence of CCK receptor antagonist, indicating that the manipulations in Figure 2 are not merely shutting off the motor cortex. It is also notable that, as the paper points out, the activity appears less variable in the CCK manipulations (Figure 3G). However, this could be due to CCK manipulation mice having less-variable movements throughout training. The Hausdorff distance is used for quantification against this point in Figure 1E, though the use of the single largest distance between trajectories seems unlikely to give a robust measure of trajectory similarity, which is reinforced by the CCK-/- traces looking much less variable than WT traces in Figure 1D. The activity effects may therefore be expected from a general motor deficit if that deficit prevented the mice from normal exploratory movements and restricted the movement (and activity) to a consistently unsuccessful pattern.

   Thanks for your comments and suggestions.

   To totally suppress CCK receptors in the motor cortex, the antagonist is unavoidable to diffuse to the adjacent brain areas as the motor cortex is not regularly circular. But the area inhibited most should be the motor cortex. We applied the chemogenetics method to further determine the specificity of the motor cortex in the motor skill learning. Specific projection from the RC to the MC was inhibited bilaterally, which suppressed the motor learning ability.

   For a wild-type mouse, neurons were activated when it try to get the food pellet. Neuronal pattern corresponding to each trial will be remembered, and the patterns corresponding to successful movements will tend to be repeated. Manipulations of CCK prevented neurons from remembering the pattern they tried and repeated the pattern they tried before no matter it is successful or not. This is corresponding to the neuron-activation pattern showed in figure 3D, 3E and 3G, the population activities (neuronal activities) are comparable, while the trial-to-trial population correlation is a little bit higher for the CCK-manipulation groups on Day 1. In terms of the behavior, manipulations of CCK decreased the possibility to explore the best path to get food pellets and just repeating a reach for the food pellet like it was the first time. Besides, many tests including the movement ability of CCK-/-, performance of antagonist injection group and chemogenetics manipulation group after learning indicated that CCK-manipulation did not affect the basic movement ability.

   Hausdorff distance is the greatest of all the distances from a point in one set to the closest point in the other set. It is not just the largest distance between two trajectories, but comprehensively takes all points in each trajectory into consideration. Hausdorff distance is widely used to assess the variation of two trajectories. The similarity of the shapes of trajectories is not applied for analysis because it is not very effective to assess the performance of a mouse. The fixed location of the initial site and food site makes all trajectories are single lines in the same direction, thus, the shapes of the trajectories are very similar among different trials. Two trajectories with similar shape but far from each other (big Hausdorff distance) should be treated as big variation because, in terms of the final results, they are quite different (success vs. miss). Therefore, Hausdorff distance is more reliable to be applied for assessment of the performance of mice.

   Finally, slice experiments are used to demonstrate the lack of LTP in the motor cortex following CCK knockout, which is rescued by CCK receptor agonists. This is a nice experiment with a clear result, though it is unclear why there are such striking short-term depression effects from high-frequency stimulation observed in Figure 6A that are not observed in Figure 1H. Also, relating to the specificity of the proposed rhinal-motor pathway, these experiments do not demonstrate the source of CCK in the motor cortex, which may for example originate locally.

   Thanks for your comments.

   1. Because CCK4 is a small molecule, which degrades very fast with half-time less than 1 min in the rat serum and 13 min in the human serum, we injected the drug into the electrode recording dishes, while the ACSF was stopped flowing, leading to a relatively low oxygen condition. As it showed in Figure 6A, it cost about 15 min for the brain slices to recover. Compared with CCK4 manipulation, the depression of vehicle group is stronger, which could be due to the effects of CCK4 induced LTP after HFS compensated the depression.

   2. In the motor cortex, many CCK-positive neurons are γ-aminobutyric acid-ergic (GABAergic) neurons, in which the role played by CCK is not very clear (Whissell et al., 2015). However, evidence showed that GABA may inhibit the release of CCK in the neocortex (Yaksh et al., 1987). Many glutamatergic neurons in the neocortex also express CCK (Watakabe et al., 2012). In this study, the stimulation electrode was placed on the layer 1, where receives most CCK projections from the rhinal cortex, to release CCK from the rhinal cortex, but can not rule out the possibility that some CCK may release from the local CCK neurons (Figure 4B). We focused on the importance of CCK for neural plasticity in the motor cortex, but did not aim to figure out the role played by the cortical CCK-positive neurons, including inhibitory and excitatory neurons, in neuronal plasticity and motor skill learning by this experiment.

   Therefore, the specificity of the projections from the rhinal cortex to the motor cortex was further studied by chemogenetic manipulation. Inhibiting the activity of the projections suppressed the learning ability compared with two types of control manipulations, indicating the CCK projections from RC to the MC is critical for motor skill learning.

   Reviewer #2 (Public Review):

   This study aims to test whether and if so, how cholecystokinin (CCK) from the mice rhinal cortex influences neural activity in the motor cortex and motor learning behavior. While CCK has been previously shown to be involved in neural plasticity in other brain regions/behavioral contexts, this work is the first to demonstrate its relationship with motor cortical plasticity in the context of motor learning. The anatomical projection from the rhinal cortex to the motor cortex is also a novel and important finding and opens up new opportunities for studying the interactions between the limbic and motor systems. I think the results are convincing to support the claim that CCK and in particular CCK-expressing neurons in the rhinal cortex are critical for learning certain dexterous movements such as single pellet reaching. However, more work needs to be done, or at least the following concerns should be addressed, to support the hypothesis that it is specifically the projection from the rhinal cortex to the motor cortex that controls motor learning ability in mice.

   1)Because CCK is expressed in multiple brain regions, as the authors recognized, results from the CCK knock-out mice could be due to a global loss of neural plasticity. In comparison, the antagonist experiment is in my opinion the most convincing result to support the specific effect of CCK in the motor cortex. However, it is unclear to me whether the CCK knock-out mice exhibited an impaired ability to learn in general, i.e., not confined to motor skills. For instance, it would be very valuable to show whether these mice also had severe memory deficits; this would help the field to understand different or similar behavioral effects of CCK in the case of global vs. local loss of function. If the CCK knock-out mice only exhibited motor learning deficits, that would be surprising but also very interesting given previous studies on its effect in other brain areas.

   Thanks for your comments. According to the studies in our lab, we found that CCK is critical for the neural plasticity in the auditory cortex, hippocampus and the amygdala and CCK-/- mice performed much worse than wildtype mice in associative, spatial and fear memory (Li et al.,2014; Chen et al., 2019; Su et al. 2019; Feng et al. 2021).

   1. Related to my last point, I believe that normal neural plasticity should be essential to motor skill learning throughout development not just during the current task. Thus, it would be important to show whether these CCK knock-out mice present any motor deficits that could have resulted from a lack of CCK-mediated neural plasticity during development. If not, the authors should explain how this normal motor learning during development is consistent with their major hypothesis in this study (e.g., is CCK not critical for motor learning during early development).

   Thanks for your comments and suggestions.

   Development is mainly gene-guided which prepares the physical structure for learning, while learning is dependent on the neural plasticity and a period of experience (such as motor training in this research). Besides, development is deemed as "experience-expectant", using common environmental information, while learning is "experience-dependent", sensitive to the specific individual experiences (Greenough et al., 1987; Galván, 2010). Moreover, development costs longer time to form a specific ability of a species in general. The role of CCK plays in the development is not clear. Duchemin et al. (1987) studied the CCK gene expression level in the brain of rats pre- and postnatally. They found that the CCK mRNA was detectable on embryonic day 14 (E14) and gradually increased to the maximum level on postnatal day 14 (P14), indicating that CCK might participate in the development of rats. Paolo et al. (2007) mapped the expression of CCK in the mouse brain. Plentiful CCK expression was observed at E12.5 in the thalamus and spinal cord and by E17.5 CCK expression extended to the cortex, hippocampus and hypothalamus, suggesting that CCK might also regulate the development of mice. Paolo et al. (2004) found that CCK suppressed the migration of GnRH-1 through CCK-A receptor in the brain. Besides, postnatal early learning may participate in development. CCK-B receptor antagonist administration (postnatal 6 hours) suppressed the infant sheep get motor preference, indicating that CCK might be important for the development of mother preference of sheep. However, what the role CCK played in the development of motor system is not known.

   In this study, the performance of both CCK-/- and WT mice is at the same level without significant difference on Day one, in terms of the percentage of "miss", "no-grasp", "drop" and "success". Besides, the movement abilities, including stride length, stride time, step cycle ratio and grasp force, were comparable for both CCK-/- and WT mice (Figure S1C, S1D, S1E, S1F), suggesting that knockout of cck gene did not affect the basic movement ability. This could be because the development of basic movement ability is not learning-guided, but is physical structure-determined. However, all these tests were on physical level, but how CCK affected the motor system on the molecular and cellular level is not known. Therefore, we further applied CCK-BR antagonist and chemogenetic method to study the role of CCK in the motor learning.

   3)Lines 198-200 and Fig. 2C: The authors found that the vehicle group showed significantly increased "no grasp" behavior, and reasoned that the implantation of a cannula may have caused injuries to the motor cortex. In order to support their reasoning and make the control results more convincing, I think it would be helpful to show histology from both the antagonist and control groups and demonstrate motor cortical injury in some mice of the vehicle group but not the antagonist group. Otherwise, I'm a bit concerned that the methods used here could be a significant confounding factor contributing to motor deficits.

   Thanks for your comments and suggestions.

   The injury of the motor cortex can not be avoided, because the cannula was inserted below the surface of the cortex (Figure S2C). The significantly increased "no-grasp" rate is because the improvement of miss rate of the Vehicle group, which turned to "no-grasp" but failed to further improve to drop or success, while for the Antagonist group, there is no significant improving from "miss" to "no-grasp", leaving no change in the "no grasp".

   1. The authors showed that chemogenetic inhibition of CCK neurons in the rhinal cortex impaired motor skill learning in the pellet-reaching task. However, we know that the rhinal cortex projects to multiple brain regions besides the motor cortex (e.g., other cortical areas and the hippocampus). Thus, the conclusion/claim that the observed behavioral deficits resulted from inhibited rhinal-motor cortical projections is not strongly supported without more targeted loss-of-function or rescue experiments.

   It would also be very informative to the field to compare the specific behavioral deficits, if any, of inhibiting specific downstream targets of the rhinal CCK neurons. As a concrete example, the hippocampus may be involved in learning more sophisticated motor skills (as the authors pointed out in the Discussion) besides the motor cortex. It would be a critical result if the authors could either show or exclude the possibility that the motor learning deficits observed in CCK-/- mice were at least partially due to the inhibition of hippocampal plasticity. This echoes my earlier point (point 1) that it is unclear whether the effect of lacking CCK in knock-out mice is specific in the motor cortex or engages multiple brain regions.

   Lastly, because Fig. 4 only showed histology in the rhinal and motor cortices, I am not sure whether the motor cortex solely receives CCK input from the rhinal cortex. A more comprehensive viral tracing result could be important to both supporting the circuit-specificity of the observed behavior in this study and providing a clearer picture of where the motor cortex receives CCK inputs.

   Thanks for your comments.

   The specificity of the CCK-projection from the rhinal cortex to the motor cortex for motor skill learning was studies using chemogenetic methods in the revised version of the paper. We first determined that over 98% of neurons in the rhinal cortex that projected to the motor cortex are CCK positive (Figure 6A, S6A, S6B). Next, we injected the retro-Cre virus in the motor cortex and the Cre-dependent hM4Di in the rhinal cortex in C57BL/6 mice to specifically inhibit the CCK neurons from the rhinal cortex to the motor cortex. Compared to two control groups, the learning ability of the experimental group was significantly suppressed, suggesting that CCK projections from the rhinal cortex to the motor cortex are critical for motor skill learning (Figure 6). Detailed description was added in the part of "Result" in the manuscript.

   In this study, we focus on the role played by CCK from the rhinal cortex, and how CCK affects motor learning. The single pellet reaching task was selected to study the role of CCK from the rhinal cortex in motor skill learning and the motor cortex is considered as the main area generates motor memory when training in this task (Komiyama et al., 2010; Peters et al., 2014; Richard et al., 2019). We emphasized that the importance of the contrallateral motor cortex in motor learning, not meant that other brain areas where also receive CCK-positive neural projections from the rhina cortex, for example hippocampus (spatial memory), are not important for the performance of this task. In fact, specifically inhibiting the projection from the rhinal cortex to the contrallateral motor cortex is not enough to suppress the motor learning ability, but inhibiting projecting in both sides (contro- and ipsi-lateral) could suppress the learning ability of mice, suggesting that the whole motor cortex is critical for motor skill learning (Figure 6, S8). In our lab, we found that CCK projection from the entorhinal cortex to the hippocampus is critical for spatial memory formation (Su et al., 2019). Impaired hippocampus, to some extent, affected the performance in single pellet reaching task (Shwuhuey et al., 2007). Therefore, manipulation of CCK projections from the rhinal cortex to the hippocampus may also affect the performance in the single pellet reaching task. In this paper, we aim to study the relationship between the rhinal cortex and the motor cortex and the role played by CCK in this circuit. Other brain areas involved in the single pellet reaching task are not the core concern in this study.

   The motor cortex also receive CCK projections from other cortices, such as the contrallateral motor cortex, the deep layer of visual cortex and auditory cortex, and thalamus (Figure S4).

   1. I am glad to see the CCK4 rescue experiment to demonstrate the sufficiency of CCK in promoting motor learning. However, the rescue experiment lacked specificity: IP injection did not allow specific "gain of function" in the motor cortex but instead, the improved learning ability in CCK knock-out mice could be a result of a global effect of CCK4 across multiple brain regions. CCK4 injection specifically targeted at the motor cortex would be necessary to support the sufficiency of CCK-regulated neuroplasticity in the motor cortex to promote motor learning.

   Thanks for your comments.

   First, the specificity of the circuit were studied by injecting a Cre virus in the MC and a Cre-dependent hM4Di virus in the RC. After injection with clozapine, the motor learning ability were significantly suppressed compared with the saline control and the control virus combined with clozapine.

   Besides, we emphasized that the importance of the motor cortex in motor learning, not meant that other brain areas where also receive CCK-positive neuronal projections from the rhinal cortex, for example hippocampus (spatial memory), are not important for the performance of this task. Specific infusion the drug into the motor cortex is hard to rescue the motor learning ability of CCK-/- mice because the motor cortex is very large, varying from AP: -1.3 to 2.46 mm and ML: ±0.5 to ±2.75 mm and other areas receiving CCK projections from the rhinal cortex also could be important for motor learning. Actually, we tried to inject CCK into the motor cortex through a drug cannula, but the result showed that it is hard to compensate the knock out of cck gene in the whole brain, and rescue the motor learning ability (Figure S11D, S11E). Moreover, cannula implantation causes inescapable injury to the motor cortex, because the cannula must be inserted into the brain, so that the drug could be infused into the brain. This injury may affect the performance in the task, as the motor cortex is very critical for motor learning. Therefore, it is not the best method to be applied for motor skill rescuing.

   Furthermore, CCK4 molecules can be transported to the whole brain by i.p. injection, as CCK4 is capable to pass through brain blood barrier, which compensates the knockout of cck gene in the whole brain, leading to the rescuing of motor learning ability. Furthermore, i.p. injection is widely accepted for drug discovery because it is very convenient, simply manipulated and does not causes any direct injury on the brain. Thus, we applied i.p. injection not only for whole brain CCK compensation, but also for the further study of the application in drug discovery.

   Reviewer #3 (Public Review):

   The authors elucidated the roles of cholecystokinin (CCK)-expressing excitatory neurons, which project from the rhinal cortex to the motor cortex, in motor skill learning. The authors found CCK knock-out mice exhibited learning defects in the pellet reaching task while the baseline success rate of the knock-out mice was similar to that of the wild-type mice. Application of a CCK B receptor (CCKBR) antagonist into the motor cortex lowered the success rate in the motor task. The authors found the population activity which was observed in the in vivo calcium imaging during motor learning was elevated after motor learning, but this increase disappeared in CCK knock-out mice and animals with CCKBR antagonist administration. Anterograde and retrograde viral tracing revealed that CCK-expressing excitatory neurons in the rhinal cortex projected to the motor cortex. Chemogenetic inhibition of the CCK-expressing neurons in the rhinal cortex lowered the ability for motor learning. The application of a CCKBR agonist increased the motor learning ability of CCK knock-out animals as well as long-term potentiation (LTP) observed in the slice of the motor cortex.

   However, the manuscript contains several shortcomings:

   First, the "Discussion" has several statements that are only supported weakly by the results, for example, ll. 429-431, ll. 432-433, and ll. 447-448. In addition, most of the sentences in this section are not divided into subsections. The paragraphs should be composed in multiple subsections with appropriate subheadings, even though the initial section summarizing the results can lack a subheading.

   Thanks for your suggestions. The statements were revised and the discussion was divided into subsections.

   Second, it would be important that the authors showed which area(s) of the brain is affected by the CCKBR antagonist in the experiments described in ll. 166-206 and Fig. 2. The authors injected the drug into the motor cortex, but the chemical can spread to neighboring cortical areas (e.g. somatosensory cortex) or wider brain regions. If so, the blockade of the CCKBR in the brain areas other than the motor cortex could cause the defects of the motor task learning observed in these experiments. I think it is desirable that such a possibility should be excluded. Conversely, it is possible that the antagonist had an effect on a limited subarea of the motor cortex (e.g. only the primary motor cortex (M1)). In this case, the information about the field altered by the CCKBR blocker would be useful to interpret the results of the learning defects.

   Thanks for your comments and suggestions.

   The drug cannula was implanted in the motor cortex (coordinates: AP, 1.4 mm, ML, -/+1.6 mm, DV, 0.25 - 0.3 mm) contralateral to the dominant hand of the mice (Figure S2C). To totally inhibit CCKBR in the motor cortex, we injected over-dosage of antagonist into the motor cortex. Thus, we cannot totally exclude the possibility that some antagonist spread to the neighboring cortices. However, the fact is that the motor cortex is very large, varying from AP: -1.3 to 2.46 mm and ML: ±0.5 to ±2.75 mm. It is not easily to spread out of the motor cortex with high concentration.

   Third, the authors need to show bilateral data about their anterograde and retrograde tracking of CCK-expressing neurons in the rhinal cortex. In ll. 290-292, they described as follows: "Both anterograde and retrograde tracking results indicated that CCK-expressing neurons in the rhinal cortex projecting to the motor cortex were asymmetric, showing a preference for the ipsilateral hemisphere." However, they provided only unilateral data for the anterograde (Fig. 4B) and the retrograde (Fig. 4D) experiments.

   Thanks for your comments. Both anterograde and retrograde tracking data from bilateral hemisphere were added to the supplementary file (Figure S4).

   Fourth, unilateral (contralateral to the dominant forelimb) experiments are needed in the chemogenetic inhibition of the CCK neurons. In ll. 301-338 and Fig. 5, the authors inhibited the CCK -expressing neurons in both hemispheres by injecting the virus into both sides. However, the CCKBR antagonist injection into the motor cortex contralateral to the dominant forelimb caused defects in motor learning ability, as described in ll. 166-206. The authors also observed that the population neuronal activity in the motor cortex contralateral to the dominant forelimb changed in accordance with the improvement of the motor skill in ll. 208-269. Therefore, it may be the case that inhibition of CCK neurons only in the side contralateral to the dominant forelimb - not bilaterally, as the authors did - could cause the lowered ability of motor learning. Such unilateral inhibition can be carried out by unilateral injection of the virus. In relation to the point above, in the chemogenetic inhibition experiments, it would be important to show which neurons in which cortical area is inhibited. This could be done by examining the distributions of the mCherry-labeled somata in the rhinal cortex using histochemistry.

   Thanks for your comments and suggestions.

   The specific of the CCK-projection from the rhinal cortex to the motor cortex for motor skill learning was studied using chemogenetic methods in the revised version of the paper. We first determined that over 98% of neurons in the rhinal cortex that projected to the motor cortex are CCK positive by retrograde virus injection and immunostaining (Figure 6A, S6A, S6B). Next, we injected the retro-Cre virus in the motor cortex and the Cre-dependent hM4Di in the rhinal cortex in C57BL/6 mice to specifically inhibit the CCK neurons from the rhinal cortex to the motor cortex. Compared to two control groups, the learning ability of the experimental group was significant suppressed, suggesting that CCK projections from the rhinal cortex to the motor cortex are critical for motor skill learning (Figure 6). Furthermore, we also injected the retro-Cre virus into the single site of the motor cortex controlateral to the dominant forelimb together with Cre-dependent hM4Di virus in the rhinal cortex. The result showed that after injection of clozapine, the motor learning ability was not significantly suppressed, suggesting that the bilateral motor cortex is important for motor skill learning. This is consistent with the previous findings that the increased GluA1 expression were observed bilaterally in the motor cortex after training in the single pellet reaching task. Detailed description was added in the part of "Result" in the manuscript.

   Fifth, it would be valuable to further examine differences in task performance across sessions and groups. The paragraph in ll. 138-153 needs a comparison of the "miss" rates of CCK-/- animals between Day 1 vs. Day 6 (related to ll. 429- 431). This paragraph also needs comparisons of the "no-grasp" and "drop" rates of CCK-/- animals between Day 1 vs. Day 6 (related to ll. 432- 433). The paragraph in ll. 175-190 needs comparisons of success rates between Day 1 and Day 5/6 within the antagonist group (related to ll. 447-448).

   Thanks for your comments. The comparisons were made in the revised manuscript.

   Read the original source
3. 

   eLife

   Dec 3, 2023

   eLife assessment

   This valuable paper examines the link between the neuropeptide cholecystokinin (CCK) and motor learning and neural plasticity in the motor cortex. While CCK was known to be involved in neural plasticity in other brain regions and behavioral contexts, this study is the first to provide evidence that CCK manipulation causes deficits in motor learning. However, the evidence for specific effects regarding behavior, activity, and pathways is currently incomplete.

   Read the original source
4. 

   eLife

   Dec 3, 2023

   Reviewer #1 (Public Review):

   This paper combines an array of techniques to study the role of cholecystokinin (CCK) in motor learning. Motor learning in a pellet reaching task is shown to depend on CCK, as both global and locally targeted CCK manipulations eliminate learning. This learning deficit is linked to reduced plasticity in the motor cortex, evidenced by both slice recordings and two-photon calcium imaging. Furthermore, CCK receptor agonists are shown to rescue motor cortex plasticity and learning in knockout mice. While the behavioral results are clear, the specific effects on learning are not directly tested, nor is the specificity pathway between rhinal CCK neurons and the motor cortex. In general, the results present interesting clues about the role of CCK in motor learning, though the specificity of the claims is not fully …

   Reviewer #1 (Public Review):

   This paper combines an array of techniques to study the role of cholecystokinin (CCK) in motor learning. Motor learning in a pellet reaching task is shown to depend on CCK, as both global and locally targeted CCK manipulations eliminate learning. This learning deficit is linked to reduced plasticity in the motor cortex, evidenced by both slice recordings and two-photon calcium imaging. Furthermore, CCK receptor agonists are shown to rescue motor cortex plasticity and learning in knockout mice. While the behavioral results are clear, the specific effects on learning are not directly tested, nor is the specificity pathway between rhinal CCK neurons and the motor cortex. In general, the results present interesting clues about the role of CCK in motor learning, though the specificity of the claims is not fully supported.

   Since all CCK manipulations were performed throughout learning, rather than after learning, it is not clear whether it is learning that is affected or if there is a more general motor deficit. Related to this point, Figure 1D appears to show a general reduction in reach distance in CCK-/- mice. A general motor deficit may be expected to produce decreased success on training day 1, which does not appear to be the case in Figure 1C and Figure 2B, but may be present to some degree in Figure 5B. Or, since the task is so difficult on day 1, a general motor deficit may not be observable. It is therefore inconclusive whether the behavioral effect is learning-specific.

   The paper implicates motor cortex-projecting CCK neurons in the rhinal cortex as being a key component in motor learning. However, the relative importance of this pathway in motor learning is not pinned down. The necessity of CCK in the motor cortex is tested by injecting CCK receptor antagonists into the contralateral motor cortex (Figure 2), though a control brain region is not tested (e.g. the ipsilateral motor cortex), so the specificity of the motor cortex is not demonstrated. The learning-related source of CCK in the motor cortex is also unclear, since even though it is demonstrated that CCK neurons in the rhinal cortex project to the motor cortex in Figure 4D, Figure 4C shows that there is also a high concentration of CCK neurons locally within the motor cortex. Likewise, the importance of the projection from the rhinal cortex to the motor cortex is not specifically tested, as rhinal CCK neurons targeted for inactivation in Figure 5 include all CCK cells rather than motor cortex-projecting cells specifically.

   CCK is suggested to play a role in producing reliable activity in the motor cortex through learning through two-photon imaging experiments. This is useful in demonstrating what looks like normal motor cortex activity in the presence of CCK receptor antagonist, indicating that the manipulations in Figure 2 are not merely shutting off the motor cortex. It is also notable that, as the paper points out, the activity appears less variable in the CCK manipulations (Figure 3G). However, this could be due to CCK manipulation mice having less-variable movements throughout training. The Hausdorff distance is used for quantification against this point in Figure 1E, though the use of the single largest distance between trajectories seems unlikely to give a robust measure of trajectory similarity, which is reinforced by the CCK-/- traces looking much less variable than WT traces in Figure 1D. The activity effects may therefore be expected from a general motor deficit if that deficit prevented the mice from normal exploratory movements and restricted the movement (and activity) to a consistently unsuccessful pattern.

   Finally, slice experiments are used to demonstrate the lack of LTP in the motor cortex following CCK knockout, which is rescued by CCK receptor agonists. This is a nice experiment with a clear result, though it is unclear why there are such striking short-term depression effects from high-frequency stimulation observed in Figure 6A that are not observed in Figure 1H. Also, relating to the specificity of the proposed rhinal-motor pathway, these experiments do not demonstrate the source of CCK in the motor cortex, which may for example originate locally.

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5. 

   eLife

   Dec 3, 2023

   Reviewer #2 (Public Review):

   This study aims to test whether and if so, how cholecystokinin (CCK) from the mice rhinal cortex influences neural activity in the motor cortex and motor learning behavior. While CCK has been previously shown to be involved in neural plasticity in other brain regions/behavioral contexts, this work is the first to demonstrate its relationship with motor cortical plasticity in the context of motor learning. The anatomical projection from the rhinal cortex to the motor cortex is also a novel and important finding and opens up new opportunities for studying the interactions between the limbic and motor systems. I think the results are convincing to support the claim that CCK and in particular CCK-expressing neurons in the rhinal cortex are critical for learning certain dexterous movements such as single pellet …

   Reviewer #2 (Public Review):

   This study aims to test whether and if so, how cholecystokinin (CCK) from the mice rhinal cortex influences neural activity in the motor cortex and motor learning behavior. While CCK has been previously shown to be involved in neural plasticity in other brain regions/behavioral contexts, this work is the first to demonstrate its relationship with motor cortical plasticity in the context of motor learning. The anatomical projection from the rhinal cortex to the motor cortex is also a novel and important finding and opens up new opportunities for studying the interactions between the limbic and motor systems. I think the results are convincing to support the claim that CCK and in particular CCK-expressing neurons in the rhinal cortex are critical for learning certain dexterous movements such as single pellet reaching. However, more work needs to be done, or at least the following concerns should be addressed, to support the hypothesis that it is specifically the projection from the rhinal cortex to the motor cortex that controls motor learning ability in mice.

   1. Because CCK is expressed in multiple brain regions, as the authors recognized, results from the CCK knock-out mice could be due to a global loss of neural plasticity. In comparison, the antagonist experiment is in my opinion the most convincing result to support the specific effect of CCK in the motor cortex. However, it is unclear to me whether the CCK knock-out mice exhibited an impaired ability to learn in general, i.e., not confined to motor skills. For instance, it would be very valuable to show whether these mice also had severe memory deficits; this would help the field to understand different or similar behavioral effects of CCK in the case of global vs. local loss of function. If the CCK knock-out mice only exhibited motor learning deficits, that would be surprising but also very interesting given previous studies on its effect in other brain areas.

   2. Related to my last point, I believe that normal neural plasticity should be essential to motor skill learning throughout development not just during the current task. Thus, it would be important to show whether these CCK knock-out mice present any motor deficits that could have resulted from a lack of CCK-mediated neural plasticity during development. If not, the authors should explain how this normal motor learning during development is consistent with their major hypothesis in this study (e.g., is CCK not critical for motor learning during early development).

   3. Lines 198-200 and Fig. 2C: The authors found that the vehicle group showed significantly increased "no grasp" behavior, and reasoned that the implantation of a cannula may have caused injuries to the motor cortex. In order to support their reasoning and make the control results more convincing, I think it would be helpful to show histology from both the antagonist and control groups and demonstrate motor cortical injury in some mice of the vehicle group but not the antagonist group. Otherwise, I'm a bit concerned that the methods used here could be a significant confounding factor contributing to motor deficits.

   4. The authors showed that chemogenetic inhibition of CCK neurons in the rhinal cortex impaired motor skill learning in the pellet-reaching task. However, we know that the rhinal cortex projects to multiple brain regions besides the motor cortex (e.g., other cortical areas and the hippocampus). Thus, the conclusion/claim that the observed behavioral deficits resulted from inhibited rhinal-motor cortical projections is not strongly supported without more targeted loss-of-function or rescue experiments.

   It would also be very informative to the field to compare the specific behavioral deficits, if any, of inhibiting specific downstream targets of the rhinal CCK neurons. As a concrete example, the hippocampus may be involved in learning more sophisticated motor skills (as the authors pointed out in the Discussion) besides the motor cortex. It would be a critical result if the authors could either show or exclude the possibility that the motor learning deficits observed in CCK-/- mice were at least partially due to the inhibition of hippocampal plasticity. This echoes my earlier point (point 1) that it is unclear whether the effect of lacking CCK in knock-out mice is specific in the motor cortex or engages multiple brain regions.

   Lastly, because Fig. 4 only showed histology in the rhinal and motor cortices, I am not sure whether the motor cortex solely receives CCK input from the rhinal cortex. A more comprehensive viral tracing result could be important to both supporting the circuit-specificity of the observed behavior in this study and providing a clearer picture of where the motor cortex receives CCK inputs.

   1. I am glad to see the CCK4 rescue experiment to demonstrate the sufficiency of CCK in promoting motor learning. However, the rescue experiment lacked specificity: IP injection did not allow specific "gain of function" in the motor cortex but instead, the improved learning ability in CCK knock-out mice could be a result of a global effect of CCK4 across multiple brain regions. CCK4 injection specifically targeted at the motor cortex would be necessary to support the sufficiency of CCK-regulated neuroplasticity in the motor cortex to promote motor learning.

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6. 

   eLife

   Dec 3, 2023

   Reviewer #3 (Public Review):

   The authors elucidated the roles of cholecystokinin (CCK)-expressing excitatory neurons, which project from the rhinal cortex to the motor cortex, in motor skill learning. The authors found CCK knock-out mice exhibited learning defects in the pellet reaching task while the baseline success rate of the knock-out mice was similar to that of the wild-type mice. Application of a CCK B receptor (CCKBR) antagonist into the motor cortex lowered the success rate in the motor task. The authors found the population activity which was observed in the in vivo calcium imaging during motor learning was elevated after motor learning, but this increase disappeared in CCK knock-out mice and animals with CCKBR antagonist administration. Anterograde and retrograde viral tracing revealed that CCK-expressing excitatory neurons …

   Reviewer #3 (Public Review):

   The authors elucidated the roles of cholecystokinin (CCK)-expressing excitatory neurons, which project from the rhinal cortex to the motor cortex, in motor skill learning. The authors found CCK knock-out mice exhibited learning defects in the pellet reaching task while the baseline success rate of the knock-out mice was similar to that of the wild-type mice. Application of a CCK B receptor (CCKBR) antagonist into the motor cortex lowered the success rate in the motor task. The authors found the population activity which was observed in the in vivo calcium imaging during motor learning was elevated after motor learning, but this increase disappeared in CCK knock-out mice and animals with CCKBR antagonist administration. Anterograde and retrograde viral tracing revealed that CCK-expressing excitatory neurons in the rhinal cortex projected to the motor cortex. Chemogenetic inhibition of the CCK-expressing neurons in the rhinal cortex lowered the ability for motor learning. The application of a CCKBR agonist increased the motor learning ability of CCK knock-out animals as well as long-term potentiation (LTP) observed in the slice of the motor cortex.

   However, the manuscript contains several shortcomings:

   First, the "Discussion" has several statements that are only supported weakly by the results, for example, ll. 429-431, ll. 432-433, and ll. 447-448. In addition, most of the sentences in this section are not divided into subsections. The paragraphs should be composed in multiple subsections with appropriate subheadings, even though the initial section summarizing the results can lack a subheading.

   Second, it would be important that the authors showed which area(s) of the brain is affected by the CCKBR antagonist in the experiments described in ll. 166-206 and Fig. 2. The authors injected the drug into the motor cortex, but the chemical can spread to neighboring cortical areas (e.g. somatosensory cortex) or wider brain regions. If so, the blockade of the CCKBR in the brain areas other than the motor cortex could cause the defects of the motor task learning observed in these experiments. I think it is desirable that such a possibility should be excluded. Conversely, it is possible that the antagonist had an effect on a limited subarea of the motor cortex (e.g. only the primary motor cortex (M1)). In this case, the information about the field altered by the CCKBR blocker would be useful to interpret the results of the learning defects.

   Third, the authors need to show bilateral data about their anterograde and retrograde tracking of CCK-expressing neurons in the rhinal cortex. In ll. 290-292, they described as follows: "Both anterograde and retrograde tracking results indicated that CCK-expressing neurons in the rhinal cortex projecting to the motor cortex were asymmetric, showing a preference for the ipsilateral hemisphere." However, they provided only unilateral data for the anterograde (Fig. 4B) and the retrograde (Fig. 4D) experiments.

   Fourth, unilateral (contralateral to the dominant forelimb) experiments are needed in the chemogenetic inhibition of the CCK neurons. In ll. 301-338 and Fig. 5, the authors inhibited the CCK -expressing neurons in both hemispheres by injecting the virus into both sides. However, the CCKBR antagonist injection into the motor cortex contralateral to the dominant forelimb caused defects in motor learning ability, as described in ll. 166-206. The authors also observed that the population neuronal activity in the motor cortex contralateral to the dominant forelimb changed in accordance with the improvement of the motor skill in ll. 208-269. Therefore, it may be the case that inhibition of CCK neurons only in the side contralateral to the dominant forelimb - not bilaterally, as the authors did - could cause the lowered ability of motor learning. Such unilateral inhibition can be carried out by unilateral injection of the virus.

   In relation to the point above, in the chemogenetic inhibition experiments, it would be important to show which neurons in which cortical area is inhibited. This could be done by examining the distributions of the mCherry-labeled somata in the rhinal cortex using histochemistry.

   Fifth, it would be valuable to further examine differences in task performance across sessions and groups. The paragraph in ll. 138-153 needs a comparison of the "miss" rates of CCK-/- animals between Day 1 vs. Day 6 (related to ll. 429- 431). This paragraph also needs comparisons of the "no-grasp" and "drop" rates of CCK-/- animals between Day 1 vs. Day 6 (related to ll. 432- 433). The paragraph in ll. 175-190 needs comparisons of success rates between Day 1 and Day 5/6 within the antagonist group (related to ll. 447-448).

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7. 

   Version published to 10.1101/2022.11.21.517378v1 on bioRxiv

   Nov 22, 2022