Emergence of Functional Heart-Brain Circuits in a Vertebrate

Reviewer #1 (Public review):

Summary:

The manuscript by Hernandez-Nunez et al. provides a comprehensive characterization of how heart-brain circuits develop in a vertebrate brain, namely the zebrafish. The characterization is performed using a combination of modern and sophisticated imaging and neural manipulation techniques and achieves unprecedented clarity and detail in how the heart-brain communication develops early in life. The paper describes a three-stage program, where first an efferent-circuit from the motor vagus to the heart develops, followed by sympathetic innervation, and lastly sensory neurons innervate the heart.

Strengths:

The paper is very clearly and nicely written. The findings are novel and of high quality and relevance. The presentations are very clear and nicely interpreted. The analyses are well presented and applied.

Weaknesses:

From the heart rate traces, heart rate variability seems to be prominent and changes across days post-fertilization (dpf). That would be a useful dependent variable, considering that the variation captured by the models does not fully explain heart rate, both for sympathetic and parasympathetic efferents. Given the strong autorhythmicity of nodal tissue in neurogenic hearts, modulatory inputs could potentially predict heart rate variability with higher precision.

Reviewer #2 (Public review):

Hernandez-Nunez et al. investigate the development and function of neural circuits involved in the regulation of heart rate in larval zebrafish. Using conserved genetic markers, they identify neural pathways involved in the bidirectional control of heart rate and in providing sensory feedback, potentially enabling more precise tuning. The main observation is that the different elements of this circuit are laid down in a developmentally staggered manner.

At 4 days old, the heart rate is invariant to a range of sensory stimuli, and the vagal motor or sympathetic pathways could not be seen to innervate the heart. Progressively through development, the heart is first innervated by the vagal motor pathway, whose axons are cholinergic, before the formation of phox2bb+ intracardiac neurons (ICNs). At this stage, before the first ICNs are observed, activation of the vagal motor pathway by optogenetic activation of a localized population of cholinergic hindbrain neurons leads to bradycardia. After the vagal motor innervation begins, the sympathetic pathway innervates the heart, which could be visualized in the form of TH+ fibers from the anterior paravertebral ganglia (APG). The activity of the TH+ APG neurons was diverse and showed proportional, integral, and derivative-like relationships to the heart rate, suggesting a role in more precise tuning of the rate than what could be achieved through the vagal pathway alone. The sensory vagus innervation of the heart was identified to be the last stage to develop; however, neurons in the nodose ganglion exhibited diverse responses tuned to the heart rate well before the innervation reached the heart. The authors attribute this to the fact that other indirect sensory cues from the gills or vasculature could be used to sense heart rate prior to innervation.

This study identifies key components of the control loop required for the regulation of heart rate in zebrafish. The control mechanism appears to be independent of the cues that trigger heart rate changes, indicating that the circuit is indeed part of an interoceptive pathway for heart rate control. Evidence for the staggered development of the vagal-motor, sympathetic, and sensory pathways is conclusive, and as the authors discuss, this phenomenon progressively allows for finer-grained control of the heart rate. This could be achieved through proportional-integral-derivative-like control properties emerging in a diverse set of neurons in the APG and sensory feedback of the state of the heart. In line with these findings, the baseline variability of heart rate prior to innervation at 4 days old appears to be comparatively lower than the later stages (Figure 1C, D, Supplementary Figure 1C-F) and increases over development.

Based on this observation and the time courses of the kernels identified by the GLMs, I would expect heart rate fluctuations of a finer time scale, ultimately limited by the time course of GCaMP6s, to be captured by the models in Figures 3, 5, and 7, in addition to the stimulus-locked changes that are highlighted. While the models yield valuable insight in the form of the activation kernels and their potential roles, in one instance, this captures the potential contribution of either the motor vagus or the APG to the change in heart rate. This makes it challenging to identify where it falls short and the potential functions of pathways that are yet to be discovered.

Lastly, the proposed anatomical connectivity of the heart-brain circuit is based on tracts observed in this study as well as those inferred from function and from previous studies.

(1) It is not clear from the images presented here whether the VSNs send feedback projections to the brainstem VPN.

(2) Do the brainstem neurons identified by their functional roles send efferent projections via the motor vagus nerve? This is unclear from the results presented and needs to be clarified in the text.

(3) Add appropriate clarifying annotations to Figure 9 and a section of text discussing the potential unknowns in the proposed circuit diagram.

Author response:

We thank the reviewers for their thoughtful, constructive, and generous evaluations of our manuscript. We are encouraged by their overall assessment of the clarity, novelty, and significance of the work, and we appreciate the opportunity to further strengthen the manuscript.

Both reviewers highlight the central contribution of this study: a developmental, circuitlevel dissection of how heart–brain signaling emerges in a vertebrate. We are pleased that the evidence supporting the staggered assembly of vagal motor, sympathetic, and sensory pathways was found to be compelling, and that the computational and experimental framework was viewed as appropriate and informative.

Below, we briefly outline how we plan to address the main points raised in the reviews.

Heart rate variability and temporal structure

Both reviewers note that heart rate variability (HRV) changes across development and suggest that HRV may provide additional insight into the function of autonomic circuits. We agree that HRV is an important physiological readout and that its developmental changes are consistent with the progressive emergence of autonomic control.

In the revised manuscript, we plan to (i) discuss heart rate variability more explicitly in the context of circuit maturation and (ii) clarify the temporal scales captured by our experiments and modeling framework. In particular, we will emphasize that our analyses focus on relationships between neural activity and heart-rate trajectories at timescales accessible given imaging rate and indicator kinetics, rather than beat-to-beat variability. We will also consider adding a supplementary analysis of the variability that can be reliably measured within these constraints, and, where appropriate, how neural activity predicts that measurable variation.

Scope and interpretation of the computational models

Reviewer #2 raises thoughtful points regarding what the generalized linear models can and cannot disambiguate, particularly when multiple efferent pathways may contribute to heart-rate dynamics. We will revise the text to more clearly distinguish between functional encoding relationships inferred from the models and anatomical connectivity that is directly demonstrated.

Our intent is to frame the kernels identified in the motor and sympathetic pathways as computational motifs that capture distinct dynamical contributions, rather than as exclusive or complete explanations of heart-rate control. We will clarify these limitations explicitly in the Results and Discussion.

Circuit diagram and anatomical interpretation

We appreciate the reviewer’s careful reading of the proposed circuit schematic. In the revised manuscript, we will revise the figure and accompanying text to clearly annotate which connections are directly observed, which are functionally inferred, and which remain hypothetical. We will also expand the Discussion to explicitly address open questions, including unresolved feedback pathways and the potential for additional nodes in the circuit.

We believe these revisions will improve clarity without altering the core conclusions of the study. We thank the reviewers again for their insightful feedback and look forward to submitting a revised version of the manuscript that addresses these points in detail.