Identification of cytokine-specific sensory neural signals by decoding murine vagus nerve activity

Abstract

The nervous system maintains physiological homeostasis through reflex pathways that modulate organ function. This process begins when changes in the internal milieu (e.g., blood pressure, temperature, or pH) activate visceral sensory neurons that transmit action potentials along the vagus nerve to the brainstem. IL-1β and TNF, inflammatory cytokines produced by immune cells during infection and injury, and other inflammatory mediators have been implicated in activating sensory action potentials in the vagus nerve. However, it remains unclear whether neural responses encode cytokine-specific information. Here we develop methods to isolate and decode specific neural signals to discriminate between two different cytokines. Nerve impulses recorded from the vagus nerve of mice exposed to IL-1β and TNF were sorted into groups based on their shape and amplitude, and their respective firing rates were computed. This revealed sensory neural groups responding specifically to TNF and IL-1β in a dose-dependent manner. These cytokine-mediated responses were subsequently decoded using a Naive Bayes algorithm that discriminated between no exposure and exposures to IL-1β and TNF (mean successful identification rate 82.9 ± 17.8%, chance level 33%). Recordings obtained in IL-1 receptor-KO mice were devoid of IL-1β-related signals but retained their responses to TNF. Genetic ablation of TRPV1 neurons attenuated the vagus neural signals mediated by IL-1β, and distal lidocaine nerve block attenuated all vagus neural signals recorded. The results obtained in this study using the methodological framework suggest that cytokine-specific information is present in sensory neural signals within the vagus nerve.

Keywords: bioelectronic medicine; cytokines; decoding; inflammation; vagus nerve.

Fig. 1.

Nerve recording interface, experimental design, preprocessing methodological framework, and raw surface recordings before and after lidocaine administration. (A) Photograph of the bipolar cuff electrode recording activity from the surface of the cervical vagus nerve of mice. (B) Schematic diagram of the cytokine-injection experiments, with TNF injected first and IL-1β second or IL-1β injected first and TNF injected second. (C) Schematic diagram of the preprocessing data-analysis methodological framework with all the steps carried out to extract neural responses. (D) Trace of raw surface recordings during experiments where lidocaine was dropped distally on the cervical vagus nerve (the time of lidocaine administration is indicated by the blue arrow). (Upper) The complete recording. (Lower) A zoomed-in portion of the recording around the time of the lidocaine drop, with respiratory modulations colored red.

Fig. 2.

Preprocessing framework. (A) The raw recorded signal. (B) Wavelet decomposition. (C) Adaptive thresholding. (D) Dimensionality reduction through t-SNE and clustering using the DBSCAN method. (E) Resulting CAP waveforms and inter-CAP interval (ICI) histograms.

Fig. 3.

Examples of neural responses to cytokines. Each colored trace represents the response rate against time of a different CAP. Solid lines correspond to lower-firing-rate CAPs (maximum of 15 CAPs/s), and dotted lines correspond to high-firing-rate CAPs (maximum of 80 CAPs/s). Right subpanel for all panels includes a subset of detected CAP waveforms and the median of each CAP group in thicker lines. (A) A vagus nerve response curve, along with the respective decoding accuracies, in a mouse injected first with 35 ng/kg IL-1β and then with 20 µg/kg TNF. (B) A vagus nerve response curve, along with the respective decoding accuracies, in a mouse injected first with 20 µg/kg TNF and then with 35 ng/kg IL-1β. (C) An example of neural responses to the saline injections control condition, where there is no discernible response to the injections. (D) A vagus nerve response curve in a mouse vagotomized proximally to the recording electrode and injected first with 35 ng/kg IL-1β and then with 20 µg/kg TNF.

Fig. 4.

CAP waveforms often occur during respiratory modulations of vagus nerve recordings and are silenced by lidocaine. (A) Representative example of the respiratory modulation apparent in most of our vagus nerve recordings, with several CAP waveforms occurring during this modulation (two CAP waveforms are plotted at the right of the panel). (B) Moving average respiratory-modulation rate (Left) and respiratory-modulation duration (Right), calculated throughout the time course of experiments, for three different experiment groups: responders to cytokine exposure (blue traces), nonresponders (red traces), and saline injections (yellow traces). The moving average of the SD is shown in the corresponding shaded colors. (C) Normalized mean firing rate and SD (error bars) of all CAP groups (blue) and all respiratory groups (red) across the lidocaine experiments (n = 6 mice), before and after the lidocaine drop. *Two-sample t test, P < 0.001.

Fig. 5.

Decoding algorithm and illustrative example. (A) Schematic diagram of the decoder used to discriminate between no injection (baseline) and IL-1β or TNF injection. (B and C) Illustrative example of the transformation of the data from the time domain to the CAP response domain (B), where the decoder detects the two responding CAP clusters, thus grouping the response values into the three distinct classes, baseline or IL-1β or TNF injection, using threefold cross-validation (C, Left). (C, Right) The concatenated out-of-sample prediction of the algorithm from all the folds to validate our algorithm shows the result of the decoding and is indicative of its accuracy.

Fig. 6.

Examples of decoder input and output. (A and B) Indicative examples of the 20 µg/kg TNF first injection/35 ng/kg IL-1β second injection experiments (A) and 35 ng/kg IL-1β first injection/20 µg/kg TNF second injection experiments (B). It is clear that the different injections elicit different responses and thus are successfully decoded. (C) An indicative example of the saline control experiments in which the responses are overlapping, leading to evidently poor decoding performance. (D) An indicative example of the experiment in which vagotomy was performed proximal to the recording electrode. It is clear that the different injections elicit different responses and thus are successfully decoded.

Fig. 7.

Neural responses are receptor and fiber type specific. Shown are indicative examples of neural responses to different injections and control experiments. Each colored trace represents the response rate against time of a different CAP. Solid lines correspond to lower-firing-rate CAPs (maximum of 15 CAPs/s). Dotted lines correspond to high-firing-rate CAPs (maximum of 80 CAPs/s). (A) An example of neural responses from an IL-1βR–KO mouse injected first with 35 ng/kg IL-1β and second with 20 µg/kg TNF. There is no significant IL-1β response (the response does not cross the responder thresholds), and there is a clear and significant response to the subsequent TNF response. (B) An indicative example of decoder input and output in the IL-1R–KO control experiment in which the baseline and IL-1β–injection responses overlap but the TNF injection is separated and successfully decoded. (C) An example of a TRPV1-Cre/DTA mouse injected with 35 ng/kg IL-1β with no significant postinjection response. Right subpanels for A and C include a subset of detected CAP waveforms and the median of each CAP group in thicker lines.

Fig. 8.

Different doses of cytokines evoke different patterns. Indicative examples of neural responses to different doses of a cytokine. Each colored trace represents the response rate against time of a different CAP. Solid lines correspond to lower-firing-rate CAPs (maximum of 15 CAPs/s). Dotted lines correspond to high-firing-rate CAPs (maximum of 80 CAPs/s). (A, Left) An example of neural responses from double-dose IL-1β injections, where we first injected 35 ng/kg IL-1β and then injected 350 ng/kg IL-1β, showing a clear and significant response to both injections. (B) An example of neural responses from double-dose TNF injections, where we first injected 20 μg/kg TNF and then injected 200 μg/kg TNF, showing responses to both exposures. (C) Decoder output of the double-dose IL-1β injections (Upper) and TNF injections (Lower). In both cases, the two consecutive doses are successfully decoded. Right subpanels for A and B include a subset of detected CAP waveforms and the median of each CAP group in thicker lines.