TNF activates astrocytes and catecholaminergic neurons in the solitary nucleus: implications for autonomic control

Abstract

Tumor necrosis factor [TNF] produces a profound anorexia associated with gastrointestinal stasis. Our work suggests that the principal site of action of TNF to cause this change in gastric function is via vagal afferents within the nucleus of the solitary tract [NST]. Excitation of these afferents presumably causes gastric stasis by activating downstream NST neurons that, in turn, suppress gastric motility via action on neurons in the dorsal motor nucleus of the vagus that project to the stomach. Results from our parallel studies on gastric vago-vagal reflexes suggest that noradrenergic neurons in the NST are particularly important to the generation of reflex gastroinhibition. Convergence of these observations led us to hypothesize that TNF action in the NST may preferentially affect putative noradrenergic neurons. The current study confirms our observations of a dose-dependent TNF activation of cells [as indicated by cFOS production] in the NST. The phenotypic identity of these TNF-activated neurons in the NST was approximately 29% tyrosine hydroxylase [TH]-positive [i.e., presumably noradrenergic neurons]. In contrast, less than 10% of the nitrergic neurons were activated after TNF exposure. Surprisingly, another 54% of the cFOS-activated cells in the NST were phenotypically identified to be astrocytes. Taken together with previous observations, the present results suggest that intense or prolonged vagal afferent activity [induced by visceral pathway activity, action of gut hormones or cytokines such as TNF] can alter local astrocyte immediate early gene expression that, in turn, can provoke long-term, perhaps permanent changes in the sensitivity of vagal-reflex circuitry.

Figure 1

cFOS activation of cells in the NST by TNF. Direct unilateral nanoinjection of TNF_α into the DVC provokes cFOS activation in a dose-dependent fashion both ipsi- and contralaterally to the injection site [ANOVA: F_7,532 = 95.98; P < 0.0001]. cFOS activation counts in the NST induced by either 70 or 700pg TNF were significantly greater than that seen with PBS [Dunnett’s post-hoc test with PBS = control, * = ipsilateral; # = contralateral comparisons; p < 0.05].

Figure 2

TNF activation of astrocytes in the NST A: Low power photomicrograph of TNF injection site in the medial NST illustrating both cFOS-activated nuclei [red-brown] and S100+ labeled astrocytes [blue-black]. B: Control PBS injection site [portion of the pipette track indicated by chevrons] in the NST. Abbreviations: AP = area postrema; gr = gracile fasciculus; sol = solitary tract. C: Field of cFOS+ nuclei and S100+ astrocytes activated in response to TNF injection. Examples of cFOS-activated astrocytes [i.e., dual labeled cFOS+/S100+] are indicated by arrows; non-activated astrocytes [S100+ only] indicated by open triangle; un-identified cFOS+ nuclei indicated by filled triangles. D, E: higher magnification views of cFOS+/S100+ double labeled astrocytes [arrows] as well as un-identified cFOS+ nuclei [filled triangle] F, G: comparison views of non-activated [i.e., not cFOS+] S100-identified astrocytes. H, I, J: confocal examples of fluorescently labeled cells in the NST following TNF injection. H, S100 signal; I, cFOS signal; J; composite image. Double-labeled cells appear yellow/orange. Scale bars: A,B = 200micron; C, H, I,J = 75 micron; D,E,F,G = 10micron

Figure 3

TNF activation of neurons [TH+ or nNOS+] in the NST A: Example of a field of double-labeled TH+ [blue-black] and cFOS [red-brown] neurons in the NST activated following TNF injection. A relatively high percentage of TH+ neurons in the NST were activated [i.e., cFOS+] by TNF; examples indicated by arrows. Non-activated TH+ neurons are indicated by open triangles. BCD: confocal examples of fluorescently labeled TH+ neurons in the NST following TNF injection. B = TH+ signal; C = cFOS signal; D = composite image. E: Example of a field of double-labeled nNOS+ [blue-black] and cFOS [red-brown] neurons in the NST activated following TNF injection. A relatively low percentage of nNOS+ neurons in the NST were activated [i.e., cFOS+] by TNF. Double-labeled neurons are indicated by arrows; non-activated nNOS+ neurons are indicated by open triangles. FGH: confocal examples of fluorescently labeled nNOS+ neurons in the NST following TNF injection. F = nNOS+ signal; G = cFOS signal; H = composite image. Double-labeled cells appear yellow/orange. Scale bars: A = 75micron; B,C,D,E,F,G,H = 150micron

Figure 4

Identified and activated phenotypes in the NST A: The average number of each of these three cellular phenotypes per histological section through each side of the NST is presented (i.e., both left and right NST areas were tabulated). There are approximately twice as many astrocytes than either TH+ or nNOS+ neurons within the NST in any given histological section. B: Activation of cells (i.e., cFOS+) in response to nanoinjection of either PBS or TNF into the DVC provokes different distribution patterns across these three phenotypes. Clearly, more cells are activated as a consequence of TNF nanoinjection [see Figure 1], but this increase is only reflected in statistically significant increases in the number of TH+ and astrocytes+ cells that are activated. Indeed, nearly all of the increase [~89%] in cFOS+ cells can be accounted for by these two phenotypes [Table 1].

Figure 5

Cartoon illustrating potential relationship between vagal afferent fibers, astrocytes, and neurons to elicit specific motor or behavioral responses such as we have observed with TNF and gastric stasis. Our previous work demonstrated that TNFR1 receptors are located predominately on vagal afferents (Hermann et al., 2004) and that glutamate antagonism blocks activation of cells [apparently both neural and glial] in NST by TNF (Emch et al., 2001). Activation of catecholaminergic [i.e., TH-ir]-NST neurons (Rinaman, 2003) would explain the gastric stasis and anorexia evoked by TNF. Perhaps this tri-partite relationship may cause long term change in synaptic sensitivity that could produce chronic reflex hypersensitivity?