Cytokine-specific Neurograms in the Sensory Vagus Nerve

Abstract

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-1β-specific neurograms are eliminated in knockout mice lacking IL-1β receptor (IL-1RKO). Conversely, TNF neurograms are preserved in IL-1RKO mice, and IL-1β 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.

Figure 1

Electrophysiological recording system for the cervical vagus nerve. (A) (Top left) Surgical procedure used to place the cuff electrodes in the cervical vagus nerve of an anesthetized BALB/c mouse. (Top right) Enlargement indicating the cuff electrodes, the vagus nerve and neighboring structures. (Bottom left) Lab-made hook electrodes used to record compound action potentials (CAPs) from the vagus nerve. (Bottom right) Representative recording of the neural signals by three adjacent hook electrodes. (B) (Left) By using standard template matching and spike-sorting algorithms relying on principal component analysis, unit spikes are discriminated in the vagus nerve recordings. Most large-amplitude waveforms correspond to CAPs, but spikes in the 5–10 μV amplitude range are likely carried by single fibers. (Right) Putative single-fiber units identified by spike sorting. (C) (Left) Diagram of the axon-attached recordings triggered by a brief stimulating pulse (S1). (Right) Examples of superimposed single-fiber action potentials (shown as negative waves). Same scale as in B.

Figure 2

Recording compound action potentials in the vagus nerve. (A) Simultaneous recordings (two of three electrodes are shown) of spontaneous activity in the vagus nerve. (B) (Left) Vagus nerve excitation by KCl (4 mM, gray box) applied to the surgical field. (Top right) Period of quiescent activity before KCl (marked by i). (Middle) Period of intense spiking during KCl (marked by ii). Blue line indicates the adaptive threshold used to detect compound action potentials (CAPs). (Bottom) Isolated CAPs are aligned with the trace above. (C) (Inset) Brief electrical pulses stimulate the vagus nerve and produce evoked CAPs. The graph shows the CAP area (mean ± SD), which becomes larger with increasing stimulation intensity (pre circles). Lidocaine (2%) largely blocks the evoked CAPs (post circles). (D) (Inset) Evoked CAPs previous to treatment with tetrodotoxin (100 μM). The graph shows the CAP area (mean ± SD), which is completely blocked by the drug. (E) Representative firing of single unit fibers obtained with hook and cuff electrodes. Scale bars (x, y), A, 100 ms, 100 μV; B left, 1 sec, 20 μV; B right, 10 ms, 20 μV; C, D, 2 ms, 40 μV; E, 2 ms. 10 μV.

Figure 3

Criteria for selecting vagus nerve recordings for analysis. (A) The diagram shows the inclusion/exclusion criteria used to select experiments for further analysis. From a total of 119 cervical vagus recordings performed in anesthetized mice, 83 recordings lasting > 10 min were selected. (B) The box plot shows the spontaneous activity of the vagus nerve during baseline recording for all the included experiments. Mean CAP frequency of 5.61 ± 0.74 Hz (mean ± SEM, N = 83). Each dot represents a separate experiment.

Figure 4

Afferent fibers of the vagus nerve carry TNF-induced neurograms. (A) (Top) Trace showing the spontaneous activity of the cervical vagus nerve. (Bottom) Trace showing the injection of TNF (dose of 50 μg, marked by line). (B) Graph depicting the frequency of CAP firing for the control and TNF-induced neurograms (shown in A). The 10-min period immediately after TNF injection is used to calculate CAP firing, as well as the equivalent period from the baseline. (C) Diagrams of the surgical vagotomies employed to test the direction of flow of the TNF-induced neurogram. A proximal (Prox) cut between the electrodes and the brain isolates the sensory component, while a distal cut isolates the efferent arm. (D) Graph showing the frequency of CAP firing in 60-s bins (mean ± SEM, line ± shaded area) starting 10 min prior to TNF injection (dose of 50 μg) at time zero. Data represent N = 3 for each of proximal (filled circles) and distal (open circles) vagotomies. (E) Plot showing mean CAP frequencies for the 10-min periods before and right after TNF in individual mice. The distal cut completely abolishes the TNF effect, which is not affected by the proximal cut (P = .03 for post values, t test), indicating that afferent fibers are required.

Figure 5

Time course for TNF- and IL-1β–mediated neurograms. (A) (Top) Trace showing vagus nerve activity under vehicle (200 μL sterile saline, marked by line), which is used as control. (Bottom) Graph describing the frequency of CAP firing (mean ± SEM, gray area) for N = 6 mice starting 10 min prior to saline injection. (B) (Top) Representative neurogram for TNF (dose of 50 μg, marked by line). (Bottom) Graph showing the mean frequency ± SEM (blue area) for N = 21 mice. (C) (Top) Representative neurogram for IL-1β (dose of 350 ng, marked by line). (Bottom) Graph showing mean frequency ± SEM (green area) for N = 10 mice. (D) Plot depicting the total CAP counts (mean ± SEM) in the 10 min immediately after TNF administration at the indicated doses (N = 5–7 mice per dose). (E) Plot showing total CAP counts in the 5 min following IL-1β treatment at the indicated doses (N = 5–9 mice per dose).

Figure 6

Time domain and spectral analysis of TNF- and IL-1β–mediated neurograms. (A) Plot showing the total CAP counts in the 10-min period immediately after cytokine administration (saline, 200 μL per mouse, N = 6; TNF, 50 μg per mouse, N = 21; IL-1β, 350 ng/kg, N = 10). Values between the saline control and each cytokine are statistically significant (*, P < .05, MW test), but the cytokines are not significantly different from each other (ns, P = .07, t test). (B) Graph depicting the latency (mean ± SEM) for the TNF and IL-1β responses, with no significant difference between values (P = .16, t test). (C) Power spectral densities (PSDs) for the TNF (blue) and IL-1β (green) responses in the 0–30 Hz range for the unfiltered neurogram recordings. (Inset) PSD for the 0–200 Hz range, with the gray rectangle indicating the expanded plot. (D) The areas under the PSDs (0–400 Hz range) were calculated for TNF and IL-1β. The calculated area for each response is shown for TNF (blue) and IL-1β (green). Responses are statistically different (*, P < .05, MW test, N = 7 for each group), suggesting a potential biological substrate for cytokine discrimination within the CNS.

Figure 7

Genetic ablation of cytokine-mediated neurograms. (A, B) Plots showing the total CAP count in the 5-min period immediately after IL-1β or TNF administration in KO mice. (A) In TNFR1/2KO mice, responses for IL-1β (N = 8) and TNF (N = 5) are significantly different (*, P < .05, MW test). (B) In IL1RKO mice, CAPs for IL-1β (N = 8) and TNF (N = 8) are also statistically significant (*, P < .05, MW test). (C) Cultured nodose ganglia are stained for NeuN (left panel) to identify all cultured neurons, antibody for TNFR1 (green panel) and antibody for IL1R (red panel); merged signals are shown in the right panel. Scale bar, 50 μm. (D) Responsive neurons display a ≥ two-fold increase in Fluo-4 fluorescence in response to 100 ng/mL cytokine. (Top) Plot of neurons that respond to TNF in WT (N = 7) and TNFR1/2KO (N = 7) mice. (Bottom) Neurons that respond to IL-1β in WT (N = 5) and IL1RKO (N = 4) groups; *, P < .05, MW test.