Gut vagal afferents differentially modulate innate anxiety and learned fear

Abstract

Vagal afferents are an important neuronal component of the gut-brain axis allowing bottom-up information flow from the viscera to the CNS. In addition to its role in ingestive behavior, vagal afferent signaling has been implicated modulating mood and affect, including distinct forms of anxiety and fear. Here, we used a rat model of subdiaphragmatic vagal deafferentation (SDA), the most complete and selective vagal deafferentation method existing to date, to study the consequences of complete disconnection of abdominal vagal afferents on innate anxiety, conditioned fear, and neurochemical parameters in the limbic system. We found that compared with Sham controls, SDA rats consistently displayed reduced innate anxiety-like behavior in three procedures commonly used in preclinical rodent models of anxiety, namely the elevated plus maze test, open field test, and food neophobia test. On the other hand, SDA rats exhibited increased expression of auditory-cued fear conditioning, which specifically emerged as attenuated extinction of conditioned fear during the tone re-exposure test. The behavioral manifestations in SDA rats were associated with region-dependent changes in noradrenaline and GABA levels in key areas of the limbic system, but not with functional alterations in the hypothalamus-pituitary-adrenal grand stress. Our study demonstrates that innate anxiety and learned fear are both subjected to visceral modulation through abdominal vagal afferents, possibly via changing limbic neurotransmitter systems. These data add further weight to theories emphasizing an important role of afferent visceral signals in the regulation of emotional behavior.

Keywords: GABA; anxiety; fear; gut feelings; vagus nerve.

Figure 1.

Schematic illustration of afferent and efferent vagal fibers targeted by the SDA procedure. Afferent and efferent fibers are represented in red and blue colors, respectively. In the SDA procedure, the left (dorsal) subdiaphragmatic trunk of the vagus nerve is fully transected (indicated by the first scissor symbol), leading to a disconnection of both afferent and efferent fibers in the left (dorsal) trunk of the vagus nerve. The right (ventral) subdiaphragmatic trunk of the vagus nerve is left intact. In addition, a left-sided intracranial vagal rhizotomy is performed (indicated by the second scissor symbol) to selectively disconnect the remaining vagal afferents. This SDA procedure induces a complete (100%) disconnection of vagal afferents while leaving 50% of the vagal efferents functionally intact.

Figure 2.

Body weights and functional verification of the SDA procedure. A, The line plot shows body weights before surgery (day 0; indicated by the dashed lines) and during the postsurgery recovery period (days 1–14). Note that both Sham and SDA rats similarly regained their initial presurgery body weight within 4 d after surgery. No significant group differences were detected between SDA and Sham rats. N(Sham) = 10, N(SDA) = 12; all values are means ± SEM. B, Verification of the SDA completeness in a CCK satiation test. The bar plot shows the percentage (%) reduction in CCK-induced food intake (% baseline food intake) during a 30 min test session. Note that SDA rats displayed no reduction in food intake in response to CCK administration relative to their baseline food intake, confirming the completeness of SDA. In contrast, Sham control rats displayed a 25–40% reduction in food intake in response to CCK relative to their baseline food intake. Against these backgrounds, 1 SDA animal (with 23% reduction in food intake in response to CCK) of 12 was excluded from the final analysis and presentation of the data. ***p < 0.001; N(Sham) = 10, N(SDA) = 11. All values are means ± SEM.

Figure 3.

SDA reduces innate anxiety-like behavior in the elevated plus maze test. A, The bars show the percentage open arm time, percentage open arm distance, and total distance moved (cm) in SDA and Sham control rats. B, Computer-generated exploration paths of representative Sham and SDA rats in the elevated plus maze test. CA, enclosed (protected) arms; OA, open (unprotected) arms. N(Sham) = 10, N(SDA) = 11; *p < 0.05. All values are means ± SEM.

Figure 4.

SDA reduces innate anxiety-like behavior in the open field test. A, Distance moved (cm) in the center zone as a function of 5 min bins in SDA and Sham control rats. B, Number of center zone entries as a function of 5 min bins in SDA and Sham control rats. C, Total distance moved (cm) in the entire open field arena as a function of 5 min bins in SDA and Sham control rats. D, Computer-generated exploration paths of representative Sham and SDA rats in the open field test. CZ, center zone. N(Sham) = 10, N(SDA) = 11; *p < 0.05. All values are means ± SEM.

Figure 5.

SDA reduces innate anxiety-like behavior in the food neophobia test. The graph shows latency (s, sqrt-transformed) to eat a novel food in trial (T) 1 and 2 of the food neophobia test for both SDA and Sham control rats. N(Sham) = 10, N(SDA) = 11; *p < 0.05 and ***p < 0.001. All values are means ± SEM.

Figure 6.

SDA attenuates extinction of auditory-cued conditioned fear. A, The line plot shows percentage time freezing during presentation of five successive CS–US trials during the conditioning phase of pavlovian fear conditioning in SDA and Sham control rats. B, Expression of conditioned fear toward the context as measured by percentage time freezing during the context test. The line plot depicts percentage time freezing as a function of 30 s bins in SDA and Sham control rats. C, Expression of auditory-cued conditioned fear during the tone-CS test. The line plot shows percentage time freezing as a function of 30 s bins in SDA and Sham control rats. The bar plot depicts the mean percentage time freezing across all bins. N(Sham) = 10, N(SDA) = 11; *p < 0.05. All values are means ± SEM.

Figure 7.

SDA does not affect basal and stress-induced CORT secretion. The graph shows plasma CORT levels at basal conditions (sampling time 0) as well as 20, 40, 80, and 120 min after onset of stress exposure. Stress was applied in the form of restraint stress for 20 min (indicated by the black bar). N(Sham) = 10, N(SDA) = 11. All values are means ± SEM.

Figure 8.

SDA modulates limbic neurotransmitter levels. Basal levels of NA, 5-HT, and GABA are depicted as nm/mg protein levels for the (A) vPFC, (B) NAc, (C) AMY, and (D) HPC of SDA and Sham control rats. N(Sham) = 10, N(SDA) = 11; *p < 0.05. All values are means ± SEM.