Kynurenic acid is a nutritional cue that enables behavioral plasticity - PubMed (original) (raw)

Kynurenic acid is a nutritional cue that enables behavioral plasticity

George A Lemieux et al. Cell. 2015.

Abstract

The kynurenine pathway of tryptophan metabolism is involved in the pathogenesis of several brain diseases, but its physiological functions remain unclear. We report that kynurenic acid, a metabolite in this pathway, functions as a regulator of food-dependent behavioral plasticity in C. elegans. The experience of fasting in C. elegans alters a variety of behaviors, including feeding rate, when food is encountered post-fast. Levels of neurally produced kynurenic acid are depleted by fasting, leading to activation of NMDA-receptor-expressing interneurons and initiation of a neuropeptide-y-like signaling axis that promotes elevated feeding through enhanced serotonin release when animals re-encounter food. Upon refeeding, kynurenic acid levels are eventually replenished, ending the elevated feeding period. Because tryptophan is an essential amino acid, these findings suggest that a physiological role of kynurenic acid is in directly linking metabolism to activity of NMDA and serotonergic circuits, which regulate a broad range of behaviors and physiologies.

Copyright © 2015 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Post-fast Hyperactive Feeding Requires Serotonin Signaling

(A) Animals were fasted for 1, 2, or 4 hours and the pharyngeal pumping rate was measured before exposure to food, and 5, 30, 60 and 120 min post-fast. To facilitate visualization of data pertaining to changes in pumping rate, all feeding data is presented with the X-axis defining the ad libitum fed wildtype rate. n = 10–14 animals per condition, ns: p > 0.05, ** p < 0.01 compared to ad libitum fed ANOVA (Dunnett) (B) Pharyngeal pumping rates of wildtype, mutant or transgenic animals at the indicated fasting and refeeding periods. n = 10 animals per condition. * p < 0.05, *** p < 0.001, **** p < 0.0001 ANOVA (Sidak). In (A) and (B) error bars = 95% c.i. See also, Figure S1 and Table S1.

Figure 2

Figure 2. Mutants With Impaired KynA Production Exhibit Constitutively Hyperactive Pharyngeal Pumping That Requires Serotonin Signaling

(A) Pharyngeal pumping rates. n = 9–13 animals per condition ** p < 0.01, **** p < 0.0001 ANOVA (Tukey). (B) Pharyngeal pumping rates of serotonin pathway mutants cultured on either vector control RNAi or _nkat- 1(RNAi)_ expressing _E. coli_.n = 10 animals per condition ns: p > 0.5 * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to wildtype animals cultured of RNAi control vector ANOVA (Dunnett). (C) Schematic of the kynurenine pathway. tdo-2: tryptophan 2,3 dioxygenase, kmo-1: kynurenine mono-oxygenase, flu-2: required for kynureninase expression (D) Comparisons of kynurenine pathway metabolite levels in wildtype versus nkat-1 mutants. Error bars = s.d. of determinations from 5 cultures per genotype. * p < 10−5 (2-tailed, t- test: wildtype vs nkat-1) (E) Effects of KynA supplementation on pharyngeal pumping. n = 10 animals per condition. **** p < 0.0001 ANOVA (Tukey). In (A), (B), and (E) error bars = 95% c.i.

Figure 3

Figure 3. KynA Depletion During Fasting Is Required For Post-fast Hyperactive Feeding

(A) Determination of tryptophan (Trp), kynurenine (Kyn), kynurenic acid (KynA) and anthranilic acid (Ant) from extracts of wildtype C. elegans fed ad libitum, after 2 hr of fasting, after 20 min or 2 hr of post-fast feeding. For each metabolite, data are normalized to the ad libitum fed condition. Error bars = s.d. of 3–4 independent cultures ns: p >0.05, * p < 0.05, ** p < 0.0001 ANOVA (Tukey) relative to _ad libitum_ fed for each metabolite. (B) Observed relationships between KynA levels, pharyngeal pumping rate, and food availability with time indicating three distinct behavioral states (1–3). (C) Effects of metabolite supplementations on pharyngeal pumping rates of wildtype _C. elegans_. During the fasting period animals were treated with either vehicle, or 100 µM of each of Kyn, KynA, 3-hydroxykynurenine (3-HKyn) and serotonin (5-HT). n=10–12 animals per condition. ns p > 0.5, * p < 0.01, ** p < 0.0001 ANOVA (Holm-Sidak) compared to _ad libitum_ fed control. (D) Pumping rates from wildtype and mutants at the indicated fasting and refeeding periods. n = 12–14 animals per condition. ns: p > 0.05, ** p < 0.0001 ANOVA (Holm-Sidak) compared to ad libitum fed for each mutant and wildtype control. In (C) and (D) error bars = 95% c.i. See also, Figure S2.

Figure 4

Figure 4. nkat-1 Expression Is Limited To A Few Neurons

(A) Merged DIC and green epifluorescence image of an animal containing an nkat-1p::gfp transcriptional fusion. Arrow indicates a green head neuron. Scale bar = 20 µm. (B) Maximum intensity projection of the right lateral side of the head of a young adult animal co-expressing nkat-1p::gfp and nmr-1p::mCherry transcriptional fusions. Individual neurons are identified. Scale bar = 10 µm. (C) Pharyngeal pumping rates of the indicated strains. Error bars = 95% c.i. n = 10–12 animals per condition. **** p < 0.0001 ANOVA (Dunnett) in comparison with wildtype. See also, Figure S3 and Table S1.

Figure 5

Figure 5. Hyperactive Feeding Requires An NMDA-receptor To Peptidergic Signaling Axis That Converges On A Serotonergic Sensory Neuron

(A) Pharyngeal pumping rates of wildtype, nmr-1, and nmr-2 mutants cultured on either RNAi vector control or nkat-1(RNAi).* p < 0.0001 ANOVA (Tukey). (B) Pharyngeal pumping rates at the indicated fasting and refeeding periods. ns p > 0.1, * p < 0.0001 ANOVA (Tukey) comparing fasted, post-fast animals to ad libitum fed controls. (C) Pharyngeal pumping rates of the indicated strains. Animals were cultured on bacteria expressing nkat-1(RNAi) or RNAi vector control. * p < 0.0001 ANOVA (Sidak) comparing vector control to nkat-1(RNAi) for each genotype. (D) Pharyngeal pumping rates of wildtype, flp-18 mutants and flp-18 transgenic lines used in (C) fed ad libitum, after 2 hr of fasting, and after 5 min of post-fast refeeding. * p < 0.05, ** p < 0.0001 ANOVA (Tukey) comparing fasting and post-fast measurements to that of ad libitum fed for each measurement. (E) Pharyngeal pumping rates of wildtype, npr-5 mutants and npr-5 mutants expressing transgenes directing expression of npr-5a and -5b to ADF neurons. Animals were cultured on bacteria expressing either RNAi vector control or nkat-1(RNAi). * p < 0.0001 ANOVA (Sidak) comparing vector control treated to nkat-1(RNAi) treated for each genotype. (F) Pharyngeal pumping rates of wildtype, npr-5 mutants and the npr-5 transgenic line used in (E) fed ad libitum, after 2 hr of fasting, and after 5 min of refeeding post-fast. * p < 0.05, p < 0.0001 ANOVA (Tukey) comparing fasting and post-fast measurements to that of ad libitum fed for each measurement. In (A–F), error bars = 95% c.i. n = 10–16 animals per condition. See also, Figure S4 and Table S1.

Figure 6

Figure 6. KynA Represses AVA Interneuron Activity and Secretion From ADF Through Antagonism Of The NMDA-r/FLP-18/NPR-5 Signaling Axis

(A) Sample ratiometric ΔF/F plot showing spontaneous changes over the 250 s imaging window. The area under the signal in the green shaded plot is the basis of the data in (B). The inset plot shows the portion of a spontaneous transient extracted from the 250 s imaging window aligned to a -5 to 60 s timescale axis which is the basis of the data shown in (C). See also, Figure S5. (B) The total integrated change in fluorescence intensity (ΔF/F) over the 250 s recordings. Wildtype animals were fed ad libitum, fasted 2 hr or fasted 2 hr in media supplemented with either 100 µM Kyn or 100 µM KynA. Mutants in nkat-1, nmr-1 and flp-18 were fed ad libitum or fasted 2 hr. ns: p > 0.8, * p < 0.02, ** p < 0.01 ANOVA (Sidak). (C) Averaged spontaneous Ca2+ transients from each of the conditions recorded in (B). (B, C) error bars = s.e.m., n = 10–12 animals per condition. (D–E) Measurement of DAF-28::mCHERRY fluorescence in coelomocytes from transgenic animals expressing daf-28::mCherry in ADF neurons. Error bars = s.e.m., n = 15–38 per condition. (D) Wildtype, flp-18, and npr-5 mutants expressing the transgene were cultured on bacteria expressing either nkat-1(RNAi) or an RNAi vector control. * p < 0.0001 ANOVA (Tukey) compared to RNAi vector control treated animals. (E) Lines used in (D) measured during ad libitum feeding, after a 2 hr fast and after 20 minutes of post-fast re-feeding. * p < 0.05, **** p < 0.0001 ANOVA (Dunnett) comparing all measurements to wildtype, ad libitum fed animals.

Figure 7

Figure 7. Model Of A Neural Circuit That Integrates Food Sensory Cues With Nutritional Status To Promote Experience-Dependent Plasticity of Feeding

State 1: the activity of AVA (hexagon) is attenuated by KynA synthesized from kynurenine by NKAT-1 in RIM, RMDV and/or RID (circle). Kynurenine is ultimately derived from ingested food. ADF (triangle) senses food cues leading to 5-HT secretion to stimulate pumping. State 2: fasting represses KynA production causing activation of AVA neurons that promotes FLP-18/NPR-5 signaling to serotonergic ADF. In the absence of food cues, serotonin signaling from ADF is muted. State 3: when animals re-encounter food immediately post-fast, the activated NPR-5 signaling state leads to enhanced secretion from ADF when food derived sensory cues are detected. Continued feeding leads to an accumulation of KynA returning the animals to State 1.

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