Peptide Modulator of TRPV1 Channel Increases Long-Term Potentiation in the Hippocampus and Reduces Anxiety and Fear in Mice Under Acute Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The present study investigates the effects of the peptide APHC3, an antagonist of the transient receptor potential vanilloid receptor 1 (TRPV1), on pre- and post-synaptic potentiation in ex vivo mouse hippocampus, as well as on mouse behaviour under stress conditions (elevated plus maze and light/dark chamber tests). APHC3 displays a non-linear dose-response in both the in vitro and in vivo studies, and at certain concentrations has comparable efficacy to a known anxiolytic drug in the behavioural studies.

Comments:

It is stated (p. 8), ““Bell-shaped” or “inverse U” dose response is a well-known phenomenon when a higher dose of ligand can change receptor representativity on membranes or other targets could be affected.” The intranasal and intramuscular dose responses suggest opposite trends (i.e. an intermediate concentration was more effective with intranasal administration, while high and low intramuscular doses were more effective). It would be good to include more in-depth discussion about whether similar effects have been observed with AMG517 or other TRPV1 antagonists.

The results in the paired pulse experiments (Figure 1D/2D) are only statistically significant at certain timepoints, suggesting that the overall effect is weak – the discussion of this could be expanded.

From comparison of intranasal vs intramuscular dosing of APHC3, the authors suggest that TRPV1 modulation exerts anxiolytic effects via the peripheral nervous system. Given the expected low CNS exposure of intranasally-administered large peptides such as APHC3 (especially when administered at low doses e.g. 0.01 mg/kg), it is not surprising that in vivo effects are weaker with intranasal dosing.

Author Response

We are grateful for the reviewer's comment, which has helped us strengthen the rigor and clarity of manuscript. A point-by-point response to the  comments is below.

• It is stated (p. 8), ““Bell-shaped” or “inverse U” dose response is a well-known phenomenon when a higher dose of ligand can change receptor representativity on membranes or other targets could be affected.” The intranasal and intramuscular dose responses suggest opposite trends (i.e. an intermediate concentration was more effective with intranasal administration, while high and low intramuscular doses were more effective). It would be good to include more in-depth discussion about whether similar effects have been observed with AMG517 or other TRPV1 antagonists.

We thank the reviewer for this insightful comment and for pointing out the nuanced interpretation of the divergent dose-response trends. We agree that this observation merits a more detailed discussion, and we have expanded our manuscript accordingly.

The intranasal dose-response profile in our study appears as an "inverse U" (with an intermediate dose being most effective), while the intramuscular profile suggests efficacy at the highest and lowest doses tested. While a direct parallel to these specific systemic (intramuscular) trends for AMG517 has not, to our knowledge, been reported, the "inverse U" or bell-shaped dose-response curve is indeed a documented phenomenon for central administration of other TRPV1 antagonists.

For instance, capsazepine injected into the ventromedial prefrontal cortex (vmPFC) of rats exhibited a clear "inverse U" anxiolytic effect in the elevated plus maze and Vogel conflict tests, where the highest dose (60 nmol/0.2 μL) was ineffective or less effective than a middle dose [Aguiar et al., 2009]. This mirrors our intranasal finding and underscores the complexity of TRPV1-mediated signaling in limbic circuits, where excessive antagonism may paradoxically attenuate the desired behavioral output, possibly due to compensatory mechanisms, receptor internalization, or engagement of off-target effects at high concentrations.

Regarding the systemic (intramuscular) response pattern, it is more complex. While systemic capsazepine has shown linear anxiolytic dose-responses in some studies (e.g., Kasckow et al., 2004), the U-shaped pattern we observed for APHC3 may reflect the complexity of  peptide action on peripheral TRPV1 channel. This pattern differs from the more localized, direct brain-targeted profile seen with intranasal administration.

To address the reviewer's suggestion directly, we have incorporated this comparative discussion into the revised manuscript.

The anxiolytic pharmacology of TRPV1 antagonists is well-established, with efficacy shown following systemic (e.g., capsazepine; Kasckow et al., 2004) and targeted intracerebral administration to limbic structures including the hippocampus, periaqueductal gray, and prefrontal cortex (Rubino et al., 2008; Santos et al., 2008; Terzian et al., 2009; Hakimizadeh et al., 2012). Importantly, this literature provides precedent for the non-linear, "inverse U-shaped" dose-response curve observed in our intranasal data. A study by Aguiar et al. (2009) directly demonstrated this phenomenon, where microinjection of capsazepine into the rat prefrontal cortex produced maximal anxiolysis at an intermediate dose (10 nmol/0.2 μL), with significantly reduced efficacy at a higher dose (60 nmol/0.2 μL). This pattern suggests that optimal TRPV1 antagonism for anxiolytic effect may occur within a specific therapeutic window, beyond which compensatory mechanisms or off-target effects may attenuate the behavioral response.

• The results in the paired pulse experiments (Figure 1D/2D) are only statistically significant at certain timepoints, suggesting that the overall effect is weak – the discussion of this could be expanded.

AMG517 partially facilitated PPR in the CA3-CA1 hippocampal region. Changes in the paired-pulse ratio most often indicate that synaptic plasticity results from a change in neurotransmitter release. Apparently, TRPV1 blocking leads to incomplete removal of Ca2+ ions from the synapse in the time between the first and second stimuli, and as a consequence, greater messenger concentration during the second pulse. However, the paired-pulse facilitation via TRPV1 blockade occurred at certain timepoints, which indicates the pathway complexity. The functional TRPV1 receptors are present on excitatory CA3 and CA1 pyramidal cell bodies as well as on some inhibitory interneurons (IN) of CA1 region. Thereby measured field postsynaptic potentials had a multisynaptic nature – excitation from CA3 pyramidal cells spread directly as well as via interneurons (disynaptic mode) to CA1 cells. Moreover, disynaptic mode is a complex process involving excitatory and inhibitory components, the ratio of which changes over time. Therefore, TRPV1 inhibition in hippocampal cells leads to synaptic plasticity through fluctuations in calcium concentration and following neurotransmitter release of multisynaptic pathway CA1←CA3→IN→CA1. Further experiments will help to clarify these molecular mechanisms.

Interestingly, TRPV1 activation enhances vesicle recycling and neurotransmitter release, followed by postsynaptic action potential firing. TRPV1 agonists can decrease excitatory transmission from CA3 onto IN, which innervate and inhibit CA1 pyramidal cells. Moreover, TRPV1 activation enhances LTP through the GABAergic interneuron inputs, showing that TRPV1 agonist-mediated LTP enhancement appears most likely because of pyramidal cell disinhibition via feedforward interneurons. In other words, LTP induction by TRPV1-agonists goes through the CA3→IN→CA1 pathway.

Peptide APHC3 significantly enhanced LTP at concentrations of 30 and 300 nM but showed no effect at 900 nM. Facilitation of LTP could result from the ability of APHC3 to potentiate weak stimulation of TRPV1, thus mimicking agonist action, while robust channel activation would be blocked. Our data demonstrated that at low concentrations APHC3 increased TRPV1 activation, inducing LTP. However, the rise of peptides’ concentration potentially enhanced neurons depolarization and TRPV1 activation to a critical value at which the peptide mode of action changed to inhibition and no LTP was observed. The PPR after APHC3 application changed only for the lowest used concentration that highlights the similar to AMG517 mechanism of short-term plasticity modulation. Interestingly, previous studies of LTP and PPF relationships showed that LTP can be associated with PPF decrease, or the paired-pulse ratio did not change significantly. Therefore, molecular mechanisms underlying the influence of APHC3 on LTP and PPF represent contradictory modes of action, which could be explained by diverse effects of peptide on presynaptic and postsynaptic terminals, namely blocking or potentiating, respectively, depending on the degree of channel activation. Moreover, in the PPF increase after application of 30 nM peptide could be involved in the direct CA3→CA1 pathway, which requires further studies.

• From comparison of intranasal vs intramuscular dosing of APHC3, the authors suggest that TRPV1 modulation exerts anxiolytic effects via the peripheral nervous system. Given the expected low CNS exposure of intranasally-administered large peptides such as APHC3 (especially when administered at low doses e.g. 0.01 mg/kg), it is not surprising that in vivo effects are weaker with intranasal dosing.

We thank the reviewer for raising this important point regarding the expected CNS bioavailability of a large peptide like APHC3. Indeed, we also considered the possibility that the weaker anxiolytic effect observed at the lower intranasal dose (0.01 mg/kg) might simply reflect insufficient CNS exposure.

We tested the hypothesis directly and performed an experiment with a 10- and 100-fold higher intranasal dose (0.1 and 1 mg/kg), anticipating that this would significantly increase brain concentration and, consequently, the behavioral response. Contrary to this expectation, the anxiolytic effect disappeared entirely at this higher dose, as shown in Figure 3. This result demonstrates a clear non-monotonic ("inverse U-shaped") dose-response relationship, which is not consistent with a simple model of limited CNS penetration causing the weaker effect. Therefore, we conclude that the reduced efficacy of the lower intranasal dose is not an artifact of poor CNS bioavailability but rather a distinct pharmacodynamic profile. The biphasic response suggests that optimal anxiolysis via the intranasal route occurs within a narrow concentration window. 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Pavlov et al. investigates the role of TRPV1 modulation in hippocampal synaptic plasticity and anxiety-like behaviour using a sea anemone peptide APHC3 and the small-molecule antagonist AMG517. While the study addresses an interesting question regarding peripheral versus central contributions to anxiolytic effects of TRPV1 modulators, several critical methodological and interpretational issues must be addressed before the manuscript can be considered for publication.

 

Major Concerns

1. Insufficient evidence for the proposed peripheral mechanism of anxiolytic action

The authors conclude that "the efficacy of TRPV1 modulators as anxiolytic substances most probably was the result of a modification in primary afferent activity" (lines 321-323), based primarily on the observation that intramuscular administration of APHC3 produced more robust effects than intranasal administration. However, this interpretation suffers from a fundamental confound: differential delivery efficiency versus site of action.

 

- The authors must provide direct evidence that intranasal administration achieves meaningful CNS penetration of APHC3. This should include pharmacokinetic analysis demonstrating peptide concentrations in brain tissue (hippocampus and cortex at minimum) following i.n. administration at the tested doses.

- A positive control is essential - the authors should demonstrate successful i.n. delivery using either a fluorescently-labelled version of APHC3 or a reference peptide known to achieve CNS penetration via this route.

- To definitively test the peripheral versus central hypothesis, direct CNS administration (intracerebroventricular or intrahippocampal injection) should be compared with i.m. administration at equimolar doses.

- Without these data, the conclusion that peripheral mechanisms predominate is not supported and should be substantially revised or removed.

 

2. Non-monotonic dose-response relationships require mechanistic investigation

 

The consistent loss of efficacy at 0.1 mg/kg APHC3 across multiple behavioural endpoints (Figures 4 and 6) represents a critical finding that is dismissed as a "well-known phenomenon" (lines 281) without adequate investigation.

 

- Pharmacokinetic analysis at 0.01, 0.1, and 1 mg/kg doses to determine whether the non-monotonic response reflects absorption, distribution, metabolism issues, or true pharmacodynamic properties.

- Plasma and tissue (brain, muscle) concentrations of APHC3 at these doses would clarify whether the 0.1 mg/kg dose represents a metabolic saturation point or involves distinct receptor populations.

- The authors invoke APHC3's "dual action" (“The unstable effect of APHC3 in LTP experiments and after intranasal administration could be the result of the involvement of TRPV1 in both LTD and LTP”; lines 283-285) as an explanation, but do not test this hypothesis. Dose-response curves in the slice preparation across a wider concentration range (1-1000 nM) would help determine whether similar non-monotonic effects occur in the isolated CNS preparation.

- The correlation between the 0.1 mg/kg behavioural null effect and the hypothermic effect at this dose (line 276) requires explicit investigation - temperature measurements during behavioural testing are necessary to rule out thermoregulatory confounds.

 

3. Concerns regarding LTP experimental design and interpretation

The concentration-dependent effects of APHC3 on LTP (30 and 300 nM effective, 900 nM not effective) raise questions about receptor desensitization and the temporal dynamics of peptide action that are not addressed.

 

- Demonstration that 15-minute preincubation with peptides does not alter baseline synaptic transmission across all concentrations tested. The authors should present baseline fEPSP amplitude and slope measurements for each experimental group.

- Given TRPV1's well-characterized desensitization kinetics and APHC3's complex modulatory profile, the authors should test acute peptide application (added immediately before or during HFS) versus the chronic preincubation protocol used. This would distinguish effects requiring sustained receptor occupancy from those potentially compromised by desensitization.

- Time-course experiments showing when LTP enhancement emerges during the preincubation period would strengthen mechanistic understanding.

- The authors should discuss potential off-target effects at 900 nM APHC3. Have the authors tested this concentration against other TRP channels or voltage-gated calcium channels that could influence LTP?

 

Minor Concerns

 

4. Experimental design issues

- Line 380-382: The authors state "slices obtained from one animal were used in different sets of experiments" to reduce animal numbers. Please clarify: were slices from the same animal used in both APHC3 and AMG517 experiments, or only across different concentrations of the same compound? If the former, this represents a pseudoreplication issue that violates independence assumptions.

- The PPR analysis (Figures 1D and 2D) shows effects at specific interpulse intervals. What is the biological rationale for testing these particular intervals (50, 100, 200, 250, 650, 850 ms)? A more systematic sweep of intervals would be more informative.

- Figure 2C: The LTP magnitude at 300 nM APHC3 appears lower than at 30 nM, yet both are statistically significant versus control. Was a direct comparison between 30 nM and 300 nM performed? This could indicate the beginning of the concentration-dependent decline in efficacy.

 

5. Incomplete characterization of APHC3's CNS effects

- The authors demonstrate LTP enhancement but do not assess whether APHC3 affects basal synaptic transmission, input-output relationships, or other forms of plasticity (LTD, depotentiation). These data would provide important context for understanding the peptide's overall effects on hippocampal function.

- Given the proposed role of TRPV1 in both LTP and LTD at different synapses (lines 51-52), have the authors considered that APHC3 might differentially affect excitatory versus inhibitory synapses? This could explain some of the behavioural complexity.

 

6. Presentation and clarity

- The manuscript would benefit from a schematic diagram illustrating the proposed mechanisms by which peripheral TRPV1 modulation might influence CNS function and anxiety-like behaviour.

 

This study addresses an important question about TRPV1's role in anxiety and synaptic plasticity, and the peptide modulator APHC3 represents a potentially valuable tool. However, the central claim - that peripheral rather than central TRPV1 modulation drives anxiolytic effects - is not adequately supported by the current data. Major revisions addressing pharmacokinetics, delivery validation, and mechanistic investigation of the non-monotonic dose responses are essential. With these additions, this could become a strong contribution to understanding TRPV1's complex roles in CNS function.

 

Author Response

We are grateful for the reviewer's comment, which has helped us strengthen the rigor and clarity of the manuscript. A point-by-point response to the comments is below.

• Insufficient evidence for the proposed peripheral mechanism of anxiolytic action
• The authors conclude that "the efficacy of TRPV1 modulators as anxiolytic substances most probably was the result of a modification in primary afferent activity" (lines 321-323), based primarily on the observation that intramuscular administration of APHC3 produced more robust effects than intranasal administration. However, this interpretation suffers from a fundamental confound: differential delivery efficiency versus site of action.

Response:

We thank the reviewer for this crucial point regarding the confound between delivery efficiency and site of action. We agree that this is a fundamental consideration in interpreting our comparative dosing data.

To address this, we have expanded the Discussion section to explicitly analyze this confound. Our conclusion favoring a peripheral site of action is based on a convergence of evidence, not solely on the intramuscular vs. intranasal efficacy comparison:

1. APHC3 is a 56-residue peptide (~6.1 kDa). Peptides of this size are generally excluded from the CNS by the intact blood-brain barrier (BBB) following systemic (intramuscular) administration. Therefore, the robust anxiolytic effect observed after intramuscular injection strongly suggests a mechanism initiated outside the CNS.
2. If lower intranasal efficacy were solely due to poor CNS delivery, we would expect a linear dose-response where higher intranasal doses yield stronger effects. Our data contradict this: a higher intranasal doses (0.1 and 1 mg/kg) abolished the anxiolytic effect (Fig. 3), revealing a non-monotonic, "inverse U-shaped" curve. This complex pharmacodynamic profile is difficult to attribute purely to increasing CNS bioavailability.
3. We appreciate the reviewer's point about delivery efficiency. While intranasal delivery of large peptides to the CNS is indeed inefficient, it represents the only non-invasive, clinically feasible route for peptides to directly target the brain. Our study aimed to explore this practical route. The finding that its efficacy profile differs fundamentally from the systemic (intramuscular) route provides critical insight: it suggests that therapeutic outcomes may be route-dependent, with intramuscular administration engaging a primarily peripheral mechanism of action.

In the revised manuscript, we have reframed our conclusion to state more precisely that the weight of evidence supports a significant role for peripheral TRPV1 modulation in the observed anxiolysis after intramuscular administration of APHC3. We added the above points to the Discussion.

• The authors must provide direct evidence that intranasal administration achieves meaningful CNS penetration of APHC3. This should include pharmacokinetic analysis demonstrating peptide concentrations in brain tissue (hippocampus and cortex at minimum) following i.n. administration at the tested doses.

 Response:

Intranasal administration is a highly promising non-invasive method for delivering therapeutic peptides to the CNS, as extensively reviewed (e.g., Alabsi et al., 2022; Meredith et al., 2015). APHC3 (~6.1 kDa) is comparable in size to peptides successfully delivered via this route, such as insulin (5.8 kDa) and insulin-like growth factor-I (IGF-I; 7.65 kDa) (Nedelkovych et al., 2017; Thorne et al., 2004). This supports the biological plausibility of its CNS penetration following intranasal administration, a principle well-established by prior proof-of-concept studies with various neuropeptides (Meredith et al., 2015).

The absolute CNS bioavailability for intranasally administered peptides is typically low (<1% of the dose), with a variable fraction also reaching the systemic circulation (Meredith et al., 2015). For instance, intranasal insulin achieves a brain-to-blood concentration ratio of approximately 10:1 (Nedelkovych et al., 2017). Given the difficulty in predicting exact brain concentrations , we employed a multi-dose regimen to empirically identify an effective window. Our data (Figs. 3 & 5) demonstrate a clear anxiolytic effect of APHC3 at 0.01 mg/kg (i.n.). However, a higher dose (0.1 mg/kg) abolished this effect, revealing a non-monotonic, "inverse U-shaped" dose-response relationship. This indicates that efficacy is not simply a function of increasing CNS delivery but occurs within a specific concentration range.

• - A positive control is essential - the authors should demonstrate successful i.n. delivery using either a fluorescently-labelled version of APHC3 or a reference peptide known to achieve CNS penetration via this route.

Response:

The use of a fluorescently-labeled version of APHC3 to directly quantify distribution was considered, but this approach presents significant interpretative challenges: (1) the hydrophobic fluorophore can alter the peptide's penetration and distribution properties; (2) conjugation can modify the peptide's activity and selectivity, compromising pharmacodynamic relevance; and (3) detection methods (e.g., fluorescence) cannot distinguish between the intact labeled peptide, its fluorescent metabolites, and free dye. Therefore, we relied on the established precedent of comparable peptides and the clear, dose-dependent behavioral readout to infer central activity.

• - To definitively test the peripheral versus central hypothesis, direct CNS administration (intracerebroventricular or intrahippocampal injection) should be compared with i.m. administration at equimolar doses.

Response:

Integrating Central and Peripheral Mechanisms: We acknowledge that both central and peripheral mechanisms of action for TRPV1 antagonists are plausible and may not be mutually exclusive. Each pathway, however, has the potential to exhibit a biphasic dose-response, as reported in the literature. Indeed, direct intracerebral administration of TRPV1 antagonists like capsazepine into limbic structures—including the ventral hippocampus, dorsolateral periaqueductal gray (dlPAG), and prefrontal cortex—produces well-documented anxiolytic-like effects (Rubino et al., 2008; Santos et al., 2008; Terzian et al., 2009). The "inverse U-shaped" dose-response was observed for capsazepine in the ventromedial prefrontal cortex (vmPFC), where the highest dose tested was ineffective (Aguiar et al., 2009).

 Our experimental findings with APHC3 are interpreted within this dual-mechanism framework:

1. Following intramuscular (i.m.) injection, APHC3 is unequivocally present in systemic circulation. Its robust anxiolytic effect at this site, given its size and the blood-brain barrier, strongly implies a peripheral site of action.
2. For intranasal (i.n.) administration, we utilized a range of doses to empirically identify an effective CNS concentration window. The observation of anxiolytic activity at the lower i.n. dose (0.01 mg/kg) is consistent with direct peptide delivery to the brain, especially considering the typically low systemic absorption via this route. However, a higher i.n. doses (0.1 and 1 mg/kg), intended to increase CNS exposure, abolished the anxiolytic effect—mirroring the biphasic pattern seen in central electrophysiological assays (PPR and LTP). This non-monotonic response argues against a simple pharmacokinetic failure (i.e., insufficient CNS delivery) and instead points to a specific pharmacodynamic interaction within the CNS, where efficacy is lost at higher concentrations.

Therefore, our data suggest that APHC3 can engage TRPV1-mediated pathways via both peripheral (i.m.) and central (i.n.) routes, with each route exhibiting a distinct and potentially biphasic efficacy profile. The abstract and discussion were modified.

• - Without these data, the conclusion that peripheral mechanisms predominate is not supported and should be substantially revised or removed.

Response:

Intramuscular administration provides the effect on the peripheral nervous system. To reject this hypothesis, we should provide data on the ability of the peptide to cross the blood-brain barrier. We revised the text.

• Non-monotonic dose-response relationships require mechanistic investigation
• The consistent loss of efficacy at 0.1 mg/kg APHC3 across multiple behavioural endpoints (Figures 4 and 6) represents a critical finding that is dismissed as a "well-known phenomenon" (lines 281) without adequate investigation.

Response:

Agree with you that the loss of activity at 0.1 mg/kg needs further investigation. But we have no idea what could be done.

• - Pharmacokinetic analysis at 0.01, 0.1, and 1 mg/kg doses to determine whether the non-monotonic response reflects absorption, distribution, metabolism issues, or true pharmacodynamic properties.
• - Plasma and tissue (brain, muscle) concentrations of APHC3 at these doses would clarify whether the 0.1 mg/kg dose represents a metabolic saturation point or involves distinct receptor populations.

Response:

Unfortunately, pharmacokinetic parameters of APHC3 could not be assessed for doses below 1 mg/kg (i.m.). Much higher doses are needed for evaluation of peptide concentrations in the brain after intranasal administration. Pharmacodynamic properties have already been published for analgesic and anti-inflammatory properties: 0.01 mg/kg minimal significant effect, 0.1 - 1 mg/kg plateau of maximal efficacy [Logashina 2021].  

• - The authors invoke APHC3's "dual action" (“The unstable effect of APHC3 in LTP experiments and after intranasal administration could be the result of the involvement of TRPV1 in both LTD and LTP”; lines 283-285) as an explanation, but do not test this hypothesis. Dose-response curves in the slice preparation across a wider concentration range (1-1000 nM) would help determine whether similar non-monotonic effects occur in the isolated CNS preparation.

Response:

Thank you for the comment. Our future research will definitely be connected with more detailed investigation of AHPC3 influence on LTP and anxiety behavior. According to our previous research, IC50 of APHC3 value was 18±4 nM with a plateau of effect at 100-1000 nM. A concentration of 900 nM was measured to confirm the concentration plateau. Obtained data were unexpected, so we tested the animal models to verify in vitro results.

• - The correlation between the 0.1 mg/kg behavioural null effect and the hypothermic effect at this dose (line 276) requires explicit investigation - temperature measurements during behavioural testing are necessary to rule out thermoregulatory confounds.

Response:

As described in the discussion, APHC3 (0.1 mg/kg) causes a moderate decrease in core body temperature. An increase or decrease of the dose leads to the disappearance of the effect.  A rectal measurement of temperature was used to detect changes in core body temperature. Analysis of the temperature on the skin had great variation and was not significant compared to the control group for all doses of APHC3 tested. It is impossible to combine rectal temperature measurement with behavioral tests. At least we do not have such equipment.  We rewrote the text to make it clearer.

• Concerns regarding LTP experimental design and interpretation
• The concentration-dependent effects of APHC3 on LTP (30 and 300 nM effective, 900 nM not effective) raise questions about receptor desensitization and the temporal dynamics of peptide action that are not addressed. -

Response:

 Peptide APHC3 significantly enhanced LTP at concentrations of 30 and 300 nM but showed no effect at 900 nM. Facilitation of LTP could result from the ability of APHC3 to potentiate weak stimulation of TRPV1, thus mimicking agonist action, while robust channel activation would be blocked. Our data demonstrated that at low concentrations APHC3 increased TRPV1 activation, inducing LTP. However, the rise of peptides’ concentration potentially enhanced neurons depolarization and TRPV1 activation to critical value at which the peptide mode of action changed to inhibition and no LTP was observed. The PPR after APHC3 application changed only for the lowest used concentration that highlights the similar to AMG517 mechanism of short-term plasticity modulation. Interestingly, previous studies of LTP and PPF relationships showed that LTP can be associated with PPF decrease, or the paired-pulse ratio did not change significantly. Therefore, molecular mechanisms underlying the influence of APHC3 on LTP and PPF represent contradictory modes of action, which could be explained by diverse effects of peptide on presynaptic and postsynaptic terminals namely blocking or potentiating, respectively, depending on the degree of channel activation. Moreover, the direct CA3→CA1 pathway could be involved in the PPF increase after application of 30 nM peptide, which requires further studies. The discussion was modified.

• - Demonstration that 15-minute preincubation with peptides does not alter baseline synaptic transmission across all concentrations tested. The authors should present baseline fEPSP amplitude and slope measurements for each experimental group.

Response:

According to previous studies, TRPV1 channels do not alter neurotransmission at the CA3-CA1 pyramidal cell synapse (D.Bennion et al, 2011). Measured with peptide preincubation, fEPSPs had the same tendencies as amplitude values. Data without preincubation was not obtained because peptide perfusion into slices occurs gradually and takes time (at least 15 min) due to the molecule size.

• - Given TRPV1's well-characterized desensitization kinetics and APHC3's complex modulatory profile, the authors should test acute peptide application (added immediately before or during HFS) versus the chronic preincubation protocol used. This would distinguish effects requiring sustained receptor occupancy from those potentially compromised by desensitization.

Response:

 Peptide perfusion into slices occurs gradually and takes time (at least 15 min) due to the molecule size; that’s why acute peptide application was not tested in this study.

• - Time-course experiments showing when LTP enhancement emerges during the preincubation period would strengthen mechanistic understanding.

Response:

– Thank you for the comment. We will definitely take your valuable suggestions into account in our further research.

• - The authors should discuss potential off-target effects at 900 nM APHC3. Have the authors tested this concentration against other TRP channels or voltage-gated calcium channels that could influence LTP?

Response:

– APHC3 has no effect on potassium channels ( Kv1.1 and Kv1.3), other TRP channels, ASIC channels, p2X3 receptors, or on membranes of CHO cells and X.laevis oocytes. The text was added to the introduction.

• - Line 380-382: The authors state "slices obtained from one animal were used in different sets of experiments" to reduce animal numbers. Please clarify: were slices from the same animal used in both APHC3 and AMG517 experiments, or only across different concentrations of the same compound? If the former, this represents a pseudoreplication issue that violates independence assumptions.

Response:

Slices from the same animal were used across different concentrations of the same compound and in other experiments of our research group.

• The PPR analysis (Figures 1D and 2D) shows effects at specific interpulse intervals. What is the biological rationale for testing these particular intervals (50, 100, 200, 250, 650, 850 ms)? A more systematic sweep of intervals would be more informative.

Response:

 – The use of optimal PPR intervals (up to 100 ms) reflects accumulated calcium boosting release, longer intervals (up to 400 ms) show facilitation decays, and final intervals (up to 850 ms) demonstrate synapse recovery (A.F.Bartley, 2015).

• - Figure 2C: The LTP magnitude at 300 nM APHC3 appears lower than at 30 nM, yet both are statistically significant versus control. Was a direct comparison between 30 nM and 300 nM performed? This could indicate the beginning of the concentration-dependent decline in efficacy.

Response:

Thank you for the comment. A direct comparison between 30 nM and 300 mM was not significant. Molecular mechanisms underlying the influence of APHC3 on LTP and PPF represent contradictory modes of action, which could be explained by diverse effects of the peptide on presynaptic and postsynaptic terminals, namely blocking or potentiating, respectively, depending on the degree of channel activation.

• Incomplete characterization of APHC3's CNS effects
• - The authors demonstrate LTP enhancement but do not assess whether APHC3 affects basal synaptic transmission, input-output relationships, or other forms of plasticity (LTD, depotentiation). These data would provide important context for understanding the peptide's overall effects on hippocampal function.
• - Given the proposed role of TRPV1 in both LTP and LTD at different synapses (lines 51-52), have the authors considered that APHC3 might differentially affect excitatory versus inhibitory synapses? This could explain some of the behavioural complexity.

Response:

The unstable effect of APHC3 in field postsynaptic potential experiments and after intranasal administration could be the result of the involvement of TRPV1 in multisynaptic pathways comprising of excitatory (CA3→CA1) and inhibitory (CA3→IN→CA1) components and the ability of APHC3 both to potentiate weak activation and inhibit high-intensity stimulation of TRPV1.

• Presentation and clarity
• - The manuscript would benefit from a schematic diagram illustrating the proposed mechanisms by which peripheral TRPV1 modulation might influence CNS function and anxiety-like behaviour.

Response:

Figures 7 and 8 were added to the manuscript. 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

To ensure the manuscript maintains scientific rigor, the language should shift from definitive conclusions to more cautious, hypothesis-driven interpretations. Since the authors admitted to a lack of direct pharmacokinetic (PK) data and equipment for thermoregulatory monitoring, the text should reflect these limitations rather than framing the results as a settled mechanism.

Here is the suggested wording to provide to the authors for their next revision:

 

1. Toning Down the Peripheral vs. Central Claim

The authors currently state that the anxiolytic effect "was the result of a modification in primary afferent activity" based on peptide size. Because they did not perform direct CNS administration or PK analysis to confirm this, the language needs to be more speculative.

• Current Wording: "The efficacy of TRPV1 modulators as anxiolytic substances most probably was the result of a modification in primary afferent activity." ⁴⁴⁴⁴

 

• Suggested Revision: "While the molecular size of APHC3 (~6.1 kDa) suggests restricted entry across the blood-brain barrier following systemic administration, our results are consistent with a potential role for peripheral TRPV1 modulation in driving anxiolysis⁶. However, in the absence of direct brain pharmacokinetic data or comparative intracerebroventricular administration, a central contribution following intramuscular injection cannot be entirely ruled out."

 

2. Toning Down the 0.1 mg/kg "Non-monotonic" Effect

In their response, the authors admitted they "have no idea" what caused the loss of activity at 0.1 mg/kg and acknowledged equipment limitations regarding temperature and PK monitoring. The manuscript should openly acknowledge these gaps.

 

• Current Wording: Frames the 0.1 mg/kg null effect as a "well-known phenomenon" or a specific "pharmacodynamic interaction".

 

• Suggested Revision: "We observed a consistent loss of efficacy at the 0.1 mg/kg dose across multiple behavioral endpoints. While this non-monotonic response might suggest complex pharmacodynamic interactions or receptor desensitization, the underlying mechanism remains unclear. Due to technical limitations in assessing peptide concentrations at low doses and the inability to monitor core temperature during behavioral testing, we cannot definitively distinguish between metabolic saturation, thermoregulatory confounds, or specific receptor kinetics. Further studies are required to resolve this dose-response complexity."

 

3. Addressing Methodological Limitations

The authors should include a brief "Limitations of the Study" paragraph in the Discussion to provide transparency about the inferential nature of their findings.

 

• Suggested Text: "It is important to note that our conclusions regarding the site of action for APHC3 are inferred from the route of administration and the known properties of the blood-brain barrier rather than direct quantification of the peptide in brain tissue. Additionally, the mechanistic basis for the observed 'inverse U-shaped' dose-response curve requires further investigation using more sensitive pharmacokinetic assays and integrated physiological monitoring."

 

Finally, I would like to emphasize that while the "Author Response" provided helpful clarifications and candid admissions regarding experimental limitations, these insights must be clearly and explicitly reflected within the manuscript itself.

Author Response

We are grateful for the reviewer's comment, which has helped us strengthen the rigor and clarity of the manuscript. We added the suggested text to the Discussion with minor corrections.  Additionally, we reorganized the Discussion section to make it more clear.

Comments and Suggestions for Authors

Comments 1. To ensure the manuscript maintains scientific rigor, the language should shift from definitive conclusions to more cautious, hypothesis-driven interpretations. Since the authors admitted to a lack of direct pharmacokinetic (PK) data and equipment for thermoregulatory monitoring, the text should reflect these limitations rather than framing the results as a settled mechanism.

 Here is the suggested wording to provide to the authors for their next revision:

Toning Down the Peripheral vs. Central Claim

The authors currently state that the anxiolytic effect "was the result of a modification in primary afferent activity" based on peptide size. Because they did not perform direct CNS administration or PK analysis to confirm this, the language needs to be more speculative.

 Current Wording: "The efficacy of TRPV1 modulators as anxiolytic substances most probably was the result of a modification in primary afferent activity." 4444

Suggested Revision: "While the molecular size of APHC3 (~6.1 kDa) suggests restricted entry across the blood-brain barrier following systemic administration, our results are consistent with a potential role for peripheral TRPV1 modulation in driving anxiolysis. However, in the absence of direct brain pharmacokinetic data or comparative intracerebroventricular administration, a central contribution following intramuscular injection cannot be entirely ruled out."

 Response 1: Added to the text.

 Comments 2.Toning Down the 0.1 mg/kg "Non-monotonic" Effect

In their response, the authors admitted they "have no idea" what caused the loss of activity at 0.1 mg/kg and acknowledged equipment limitations regarding temperature and PK monitoring. The manuscript should openly acknowledge these gaps.

 Current Wording: Frames the 0.1 mg/kg null effect as a "well-known phenomenon" or a specific "pharmacodynamic interaction".

 Suggested Revision: "We observed a consistent loss of efficacy at the 0.1 mg/kg dose across multiple behavioral endpoints. While this non-monotonic response might suggest complex pharmacodynamic interactions or receptor desensitization, the underlying mechanism remains unclear. Due to technical limitations in assessing peptide concentrations at low doses and the inability to monitor core temperature during behavioral testing, we cannot definitively distinguish between metabolic saturation, thermoregulatory confounds, or specific receptor kinetics. Further studies are required to resolve this dose-response complexity."

Response 2: Added to the text.

Comments 3. Addressing Methodological Limitations

The authors should include a brief "Limitations of the Study" paragraph in the Discussion to provide transparency about the inferential nature of their findings.

Suggested Text: "It is important to note that our conclusions regarding the site of action for APHC3 are inferred from the route of administration and the known properties of the blood-brain barrier rather than direct quantification of the peptide in brain tissue. Additionally, the mechanistic basis for the observed 'inverse U-shaped' dose-response curve requires further investigation using more sensitive pharmacokinetic assays and integrated physiological monitoring."

Response 3: Added to the text.

Comments 4. Finally, I would like to emphasize that while the "Author Response" provided helpful clarifications and can did admissions regarding experimental limitations, these insights must be clearly and explicitly reflected within the manuscript itself.

Response 4. We tried to add as much as possible about the limitations of this study. We thank the reviewer for the important corrections to the text.