Cytokine-specific Neurograms in the Sensory Vagus Nerve
The axons of the sensory, or afferent, vagus nerve transmit action potentials to the central nervous system in response to changes in the body’s metabolic and physiological status. Recent advances in identifying neural circuits that regulate immune responses to infection, inflammation and injury have revealed that vagus nerve signals regulate the release of cytokines and other factors produced by macrophages. Here we record compound action potentials in the cervical vagus nerve of adult mice and reveal the specific activity that occurs following administration of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin 1β (IL-1β). Importantly, the afferent vagus neurograms generated by TNF exposure are abolished in double knockout mice lacking TNF receptors 1 and 2 (TNF-R1/2KO), whereas IL-1 β-specific neurograms are eliminated in knockout mice lacking IL-1 β receptor (IL-1RKO). Conversely, TNF neurograms are preserved in IL-1RKO mice, and IL-1 β neurograms are unchanged in TNF-R1/2KO mice. Analysis of the temporal dynamics and power spectral characteristics of afferent vagus neurograms for TNF and IL-1β reveals cytokine-selective signals. The nodose ganglion contains the cell bodies of the sensory neurons whose axons run through the vagus nerve. The nodose neurons express receptors for TNF and IL-1β, and we show that exposing them to TNF and IL-1β significantly stimulates their calcium uptake. Together these results indicate that afferent vagus signals in response to cytokines provide a basic model of nervous system sensing of immune responses.
Sensory neurons propagate action potentials to the central nervous system (CNS) in response to changes in the chemical, mechanical and electromagnetic environment. This information provides the afferent arc of reflex circuits that maintain homeostasis in the body’s cellular metabolism and organ physiology. An emerging field of knowledge has delineated mechanisms for reflex neural circuits that modulate immune responses. A prototypical neural pathway is the inflammatory reflex (1), defined by the electrical signals transmitted in the vagus nerve to the splenic nerve, culminating in a specialized subset of T lymphocytes that release acetylcholine (2,3). This T cell-derived neurotransmitter signals via a mechanism that depends on α-7 nicotinic acetylcholine receptors (α-7nAChR) to inhibit cytokine production by splenic macrophages (2).
The vagus nerve is primarily sensory, and it is the principal conduit that relays afferent signals from the visceral organs to the brainstem. Seminal studies by Linda Watkins revealed that an intact vagus nerve is required for the development of pyrexia in response to intra-abdominal IL-1β administration (4,5). Moreover, early work by Niijima (6, 7, 8) suggested that IL-1β might activate signaling in peripheral vagus afferents. Sensory neurons express cytokine receptors, including TNF and IL-1β receptors, and change their activation thresholds when exposed to the corresponding cytokines (9, 10, 11). This work, together with other studies, suggests that the vagus nerve may be an important component of a peripheral neural network capable of reporting changes in peripheral inflammation and immunity.
Here we reasoned that the vagus nerve transmits specific neural signatures in response to specific cytokines. By analyzing compound action potentials recorded in the cervical vagus nerve that were elicited by exposure of mice to TNF and IL-1β, we lay the groundwork for deciphering cytokine-specific vagus nerve signatures.
Materials and Methods
All experimental protocols were approved by the Institutional Animal Care and Use Committee at the Feinstein Institute for Medical Research, Northwell Health, which follows National Institutes of Health guidelines for the ethical treatment of animals. We used male BALB/c and C57Bl/6 (8–12 wks, weight 20–30 g), TNFR1/2 double KO (strain B6.129S2-TnfrsflatmlImx Tnfrsf1btm1Imx/J, p55- and p75-deficient) and IL-1R KO mice (strain B6.129S7-Il1r1tm1Imx/J), which were purchased from Jackson Laboratory. Animals were housed at 25° C, with ad libitum water and chow, and acclimated to a 12 h light and dark cycle for >3 d prior to conducting experiments.
Lidocaine, tetrodotoxin (TTX) (Sigma-Aldrich) and human IL-1β (eBiosciences) were purchased. Recombinant and tagfree human TNF was produced in-house. Following expression in Escherichia coli, TNF was purified using a cation exchange column and endotoxins removed by phase separation with Triton X-114.
In Vivo Vagus Nerve Recordings
A surgical cut of the vagus nerve was completed either proximal or distal to the recording electrodes, using the brain as the point of reference. Following placement of the vagus nerve on the hook electrodes, a silk suture was passed under and secured to the vagus nerve with a single knot. Ethyl cyanoacrylate was used to adhere the vagus nerve to the silk suture and the surgical cut was completed.
Vagus Nerve Evoked Responses
The vagus nerve was placed on wet tissue paper laid over a custom acrylic platform and kept moist using artificial cerebral spinal fluid (126 mM NaCl, 26 mM NaHCO3, 10 mM glucose, 2.5 mM KCl, 2.4 mM CaCl2, 1.3 mM MgCl2 and 1.2 mM NaH2PO4). A recording electrode (glass pipette filled with 2M NaCl) and a stimulating electrode (FHC) were placed onto the vagus nerve, with the latter 4–5 mm away from the former (and farther from the head). Signals were amplified (×1000, model 1800, AM Systems) and digitized (30 kHz) through an acquisition system (Micro1400 unit and Spike2 version 7 software, CED). Stimuli (20 µs long) of increasing intensity (1–50 V) were delivered with a stimulator (SD9, Grass) to generate input/output curves (1 V increments in the 1–10 range, 5 V increments in the 10–50 range), which were generated first in a saline solution and then in the setting of either lidocaine (2%) or TTX (100 µM), a dose inhibitory against voltage-gated sodium channels such as Nav1.8 (14, 15, 16, 17, 18). The responses were assessed by integrating the area between the response curve and the baseline value determined 1–10 ms prior to stimulation.
Vagus Nerve Recording Data Analysis
Spike2 software (version 7, CED) was used for analysis of raw recordings, which were filtered (using high-pass filter with an edge of 160 Hz) and smoothed. Neural signals were identified by user-specified adaptive threshold methodology. Identified CAPs were reviewed and signals that might have been erroneously captured by the adaptive threshold were manually removed. We ignored all areas of signal saturation, as well as signals corresponding to cardiac and respiratory components. Following this signal processing, information regarding rate and temporal coding patterns were extracted and further analyzed using OriginPro software (version 8, OriginLab). Neurograms were defined as the first interval of time in which the CAP frequency was >3× baseline level. TNF and IL-1β neurograms were extracted and subjected to fast Fourier transform for power spectral density (PSD) analysis (frequency resolution of 0.98 Hz, using a Hanning window). Within the frequency domain, notch filters were applied (60 ± 10 and 120 ± 10 Hz) to minimize the contribution of electrical noise along with its dominant harmonic. Data were linearly interpolated between the notch-filtered intervals. The areas under the PSDs (0–400 Hz range) were calculated for each cytokine response.
Nodose Ganglion Cultures
The nodose ganglia harvest protocol was adapted from previously published reports (27). Briefly, nodose ganglia were excised from euthanized mice under optical magnification. Connective tissue was digested using collagenase and dispase (100 mg/mL) for 1 h at 37° C. Cells were washed with Neurobasal medium, then triturated prior to plating in poly-D-lysine and laminin-coated tissue culture wells. Cultures were maintained in Neurobasal medium supplemented with B27, GlutaMAX, NGF (25 ng/mL), and antibiotic for 4 d prior to use for immunocytochemistry or intracellular calcium measurements.
Intracellular Calcium Measurements
Nodose ganglia were washed with Hank’s buffered salt solution and loaded with the calcium-sensitive Fluo-4 NW with pluronic acid F-127 (Molecular Probes) for 60 min, then mounted on the stage of an Axiovert 200M inverted fluorescence microscope (Carl Zeiss Microscopy). The sample was illuminated every 10 s with light from a mercury lamp passing through an excitation filter (470 nm ± 40) before being directed at the cells via a 495 nm dichroic mirror. A cooled CCD camera (AxioCam monochromatic, Carl Zeiss Microscopy) captured the emitted light after it was passed through a 525 nm ± 50 emission filter. The acquisition hardware was controlled by Axiovision 4.8 software (Carl Zeiss Microscopy). After being placed on the microscope stage, cells were washed once and baseline fluorescence recorded for 3 min, at which time the neurons were treated with either TNF (100 ng/mL) or IL-1β (100 ng/mL). Intracellular fluorescence was monitored for an additional 10 min before concluding the experiment by treating with 55 mM KCl to depolarize the neurons. The acquired images were exported to ImageJ software for analysis. The mean fluorescence intensity values of the visualized cells were extracted for each time point, background subtracted, and normalized to their average baseline intensity. Individual neurons were identified by morphology and considered viable and appropriate for final statistical analysis if they exhibited an abrupt increase in intracellular calcium with KCl depolarization. A cell was defined as responsive to the cytokine if its fluorescence increased twofold during the first 5 min of treatment with cytokine. Each well was considered an individual experiment.
Neurons were fixed for 10 min in 4% paraformaldehyde and 30% sucrose solution (50:50) warmed to 37° C, then washed with PBS containing 0.1% Triton-X for 10 min, followed by three rinses in PBS only. Neurons were blocked with 10% bovine serum albumin in PBS for 1 h at room temperature. After blocking, the cultures were incubated in primary antibodies for TNFR1 (clone HM104; Thermo Scientific) or IL1R (clone H-8; Santa Cruz Biotechnology) overnight at 4° C. The cells were rinsed 3× in PBS and incubated in secondary antibodies overnight at 4° C prior to washing and mounting on slides. In control coverslips treated in the same manner except without primary antibodies, there was no immunofluorescence.
Data are presented as individual samples, mean ± standard deviation (SD), and mean ± standard error of the mean (SEM). The Shapiro-Wilk test was used to test for normality. Analysis of variance, Student t test, Mann-Whitney test and Kolmogorov-Smirnov test were used to examine for statistical significance. P values < .05 were considered significant.
All supplementary materials are available online at https://doi.org/www.bioelecmed.org.
Vagus Nerve Recordings Capture Neural Compound Action Potentials
Afferent Nature of the Vagus Neurograms
The enhanced neurogram following TNF administration might reflect sensory signals, motor signals or both traveling through the vagus nerve. To directly test the signal directionality, we carried out proximal and distal vagotomies (relative to the brain) prior to administering TNF (Figure 4C). With a proximal cut, the afferent fibers tracking toward the recording electrodes from the visceral organs remained intact while efferent fibers no longer interfaced with the electrodes. The opposite was true with the distal vagotomy, in which the recording electrodes no longer accessed afferent fibers but the efferent fibers remained intact. For each experiment, CAPs were identified and the mean CAP frequencies were plotted across recordings (Figure 4D). To evaluate the responses, 10 min intervals immediately before and after TNF injection were considered. With proximal vagotomy, the CAP frequency showed a significant increase from baseline (pre, 5.8 ± 2.8; post, 39.5 ± 9.1 Hz; mean ± SEM). In stark contrast, distal vagotomy resulted in a null enhancement (pre, 5.7 ± 1.3; post, 2.9 ± 1.5 Hz; mean ± SEM). Comparing the individual post-injection CAP frequencies (Figure 4E) further demonstrated the significant difference between proximal and distal cuts (P = .03, T = 3.28, t test). These data strongly suggest that the first response recorded in the cervical vagus neurograms following cytokine administration includes and requires the sensory function of the vagus nerve.
Vagus Neurograms Induced by Proinflammatory Cytokines
Next, the vagus neurograms were evaluated in the context of IL-1β injection (350 ng per kg in 200 µL saline i.p.). Interestingly, the peak response to IL-1β occurred earlier than that of TNF (Figure 5C). For this reason, we decided to use a 5-min interval to evaluate the responses immediately before and after IL-1β. The mean CAP frequency was significantly higher following the cytokine (pre, 6.2 ± 2.5; post, 26.1 ± 8.3 Hz, N = 10, P = .01, Z = 2.5, Mann-Whitney [MW] test), as was the peak CAP frequency (pre, 21.2 ± 9.3; post, 80.9 ± 14.7 Hz; N = 10, P = .003, Z = 3.0, MW test). Following the enhanced IL-1β neurogram, the mean CAP frequency returned to baseline level (4.4 ± 1.9 Hz). Importantly, the increased vagus activity that was triggered by cytokines did not reflect physical trauma or disruption to the peritoneal cavity and its viscera, because injection of vehicle alone (200 µL sterile saline i.p.) did not elicit a change in vagus activity (Figure 5A), as evidenced by the mean CAP frequency (pre, 5.5 ± 0.92; post, 5.3 ± 1.1 Hz; N = 6, P = .9, Z = 0.08, MW test).
To investigate the dose dependency of the cytokine-induced neurograms, we applied increasing amounts of proinflammatory cytokines. We found that TNF (0.05–50 µg) produced a dose-dependent increase in CAP activity during the 10-min interval immediately following TNF injection, with the exception of the 5-µg dose (Figure 5D). Similarly, the number of CAPs dose-dependently increased in the 5 min following IL-1β administration when the IL-1β was titrated from 3.5 to 350 ng/kg (Figure 5E). The shorter time intervals used for IL-1β compared with TNF reflected the different temporal profiles of neurogram activation to IL-1β compared with TNF. In particular, the IL-1β response peaked earlier (150 sec post-injection) as compared with TNF (300 sec post-injection).
Differences Between Neurograms Induced by TNF and IL-1β
Spectral Power of the Neurograms Induced by TNF and IL-1β
Given that TNF and IL-1β elicited similar, but not identical, enhancements in the vagus nerve neurograms and that these signals involved afferent fibers, we sought to examine whether the vagus nerve is capable of sending distinct signals about TNF and IL-1β to the CNS. To investigate this possibility, the PSDs of the TNF and IL-1β responses were analyzed (Figure 6C) using unfiltered neurogram recordings. As described above, the response was defined as the first interval of time at which the CAP frequency resided at 3× the baseline level. Next, each filtered PSD was integrated (0–400 Hz) to compare the individual areas of the TNF (N = 14) and IL-1β (N = 7) responses (Figure 6D). Statistical analysis revealed a significant difference between TNF and IL-1β groups (P = .04, Z = 2.05, MW test). Together, the differences in the response temporal characteristics and frequency domain power analyses indeed demonstrated selective cytokine neural sensory signaling through the vagus nerve.
Receptor Requirement for the Neurograms Induced by TNF and IL-1β
Sensory Vagus Neurons from Nodose Ganglion Express Cytokine Receptors That Respond to Exogenous Cytokines
We next sought mechanistic insight into how TNF and IL-1β enhance vagus nerve activity. In our in vivo preparation, proinflammatory cytokines may directly activate sensory vagus nerve fibers within the peritoneum. Alternatively, intermediate populations of receptor-expressing somatic cells may be required to sense the cytokine and, in turn, stimulate the neurons. Several studies have demonstrated the presence of functional cytokine receptors within neuronal populations such that activation of these receptors is capable of modulating neuronal excitability (9, 10, 11). A recent report has similarly shown that bacterial products can directly activate a specific population of sensory neurons (13). Accordingly, we hypothesized that the cytokines directly engage their respective receptors on vagus nerve afferents, leading to increased nerve activity. To address this possibility, we cultured nodose ganglia neurons from adult mice and analyzed cytokine receptor expression. The cultures contained only neurons and were devoid of immune or other supportive cells. By immunocytochemistry, nodose ganglia neurons from wild type (WT) animals showed surface expression of the TNF and IL-1β receptors (Figure 7C), supporting the notion that the cytokines may directly bind receptors on the neurons.
We next evaluated cytokine-dependent nodose ganglia neuronal activation by intracellular calcium measurements with fluorescence microscopy. Cultured neurons were loaded with the calcium-sensitive dye Fluo-4 and imaged before and after the addition of either TNF (100 ng/mL) or IL-1β (100 ng/mL). Both cytokines elicited increased intracellular calcium in subpopulations of neurons harvested from WT but not TNFR1/2RKO or IL1RKO animals, respectively (Figure 7D; TNF1/2RKO versus WT, P = .002, Z = 3.03; IL1RKO versus WT, P = .01, Z = 2.5, MW test). Together, these data support a direct mechanism of vagus nerve activation through cytokine-receptor engagement on the neuron membrane.
The work presented here adds to the rapidly expanding literature describing the interface between neuroscience and immunology, delineating for the first time the afferent arc of the inflammatory reflex. We established methodologies for recording cytokine-induced CAPs in the cervical vagus nerve. By electrically stimulating or suppressing the vagus with the sodium channel blockers lidocaine and TTX, we confirmed that the recorded activity is a function of neuronal response. Interestingly, the cell bodies of nodose sensory neurons contain Nav1.8, a TTX-resistant sodium channel (14, 15, 16, 17, 18), but the dose of TTX (100 µM) we used leads to complete suppression of vagus nerve activity, in accordance with previous reports (14, 15, 16, 17, 18) and implying that the axons running through the cervical vagus predominantly express TTX-sensitive sodium channels. Furthermore, using surgical vagotomies, we verified the postulate that afferent fibers of the vagus nerve can function as cytokine sensors that relay information to the CNS (6, 7, 8). Moreover, analysis of neurograms for TNF and IL-1β reveal selective, specific, afferent signaling of the vagus nerve in response to proinflammatory cytokines.
To study the receptor dependency of the sensory component of the inflammatory reflex, we used receptor KO mice. The absence of neurogram enhancement in response to TNF or IL-1β in the respective receptor KO animals implicates the cytokine receptor in cytokine-induced activation of the vagus nerve. Specifically, the cytokine-receptor interactions mediate the neurogram response. At the cellular level, these finding were corroborated in vitro by direct activation of a subset of cultured nodose ganglia neurons by exogenous cytokines. Consistent with this finding, our immunofluorescence studies demonstrate that vagus sensory neurons express TNF and IL-1β receptors. Together, our results support a mechanism of vagus nerve activation by cytokines. Whether this occurs in vivo remains speculative. Conditional receptor KO mice would be useful to address whether cytokines activate the neurons directly in vivo, or whether they mediate their effect through an intermediate player.
Remarkably, the differences between TNF and IL-1β suggest the intriguing possibility that the CNS might be able to discriminate among a diverse set of inflammatory mediators. This notion has a strong teleological basis, because the CNS receives a continuous sensory flow of information pertaining to the internal body environment, of which the signals that relay systemic inflammation must constitute a key element for an animal’s homeostasis and survival. With the rapid delivery of signals specific to peripheral inflammatory (and immune) status, an organism would be better able to initiate appropriate physiological and behavioral responses to immunological and environmental challenges. Similarly, the vagus nerve sensory fibers may serve as an early monitoring system against invading microorganisms. This notion is consistent with reports of sensory neuron activation by bacterial products (13). Future work directed at generating a compendium of neural recordings in the context of endogenous and exogenous inflammatory stimuli will define the full extent to which the vagus nerve monitors the body’s immunological status.
In addition to the observed temporal differences in neurogram enhancement between TNF and IL-1β, the spectral densities of the individual responses (Figures 6C, D) provide further evidence for the existence of discriminating features between cytokine-elicited vagus nerve activities that could be interpreted by CNS centers, such as the nucleus tractus solitarius. There is ample evidence that many cortical and subcortical structures within the CNS can distinguish the spectral characteristics of signals and use them in processes as diverse as memory encoding, decision making and switching between sleep states (19, 20, 21). We propose that either alone or in combination, the observed differences in time and frequency domain metrics may represent the biological substrate for discrimination of peripheral cytokines by the CNS. While the specific information currency by which the brain differentially receives input about proinflammatory cytokines remains elusive, ongoing work in the development of more sensitive peripheral nerve recording systems, along with data science methodology, will likely lead to more refined definitions of cytokine-specific neural signatures through which the CNS monitors peripheral inflammation states.
The interface between neuroscience, immunology and clinical medicine is increasingly moving to the fore, especially since the use of electrical devices as therapeutic agents for disease becomes a reality and shapes the emerging field of bioelectronics medicine (22, 23, 24). For instance, a recent clinical trial that used vagus nerve stimulation to treat rheumatoid arthritis proved successful (25,26). This approach represents the first step in the development of closed-loop bioelectric therapeutics. At present, electrical stimulation therapies involve preset, empirically derived stimulation paradigms. Stimulation protocols that are able to read, interpret and respond to a patient’s active state of disease would afford a greater degree of therapeutic benefit by delivering patient-specific electric doses. An understanding of the sensory code of these neural reflexes is required to develop such a closed-loop system.
The interface between neuroscience, immunology and clinical medicine is increasingly moving to the fore. In this context, the vagus (tenth cranial) nerve plays a pivotal role as it connects the visceral organs with brain centers that respond to changes in the physiological and immunological status of the organism. In this study, we show that two pro-inflammatory cytokines (TNF and IL-1β) produce significant enhancements of neural activity in the vagus nerve. These results provide mechanistic insight into the immunological sensory neural code.
The authors declare that they have no competing interests as defined by Bioelectronic Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
We thank Thomas Faust and Stephen Frattini for their help in pilot studies. We thank Yaakov Levine for suggestions about hook electrodes, and Jianhua Li for providing purified recombinant TNF. BES is supported by a Clinician Investigator Program fellowship from the Ministry of Health (Canada), the Joseph M. West Memorial Fund and the Javenthey Soobbiah Scholarship. PTH acknowledges National Institutes of Health/National Institute of Allergy and Infectious Diseases funding (grant 5P01-AI073693). This study was completed with grant support from the Defense Advanced Research Projects Agency (W911NF-09-1-0125) to KJT.
- 24.Huerta PT, Olofsson PS. (2016) Bioelectronic medicine: Harnessing the electric patterns of neurons for therapy. Technical workshop W06 at the 10th Forum of Neuroscience, Copenhagen, Denmark. FENS; c.2016 [updated 2016 Jun 30; cited 2016 Dec 8]. Available from: https://doi.org/forum2016.fens.org/forum-programme/preliminary-programme/
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