Appetite controlled by a cholecystokinin nucleus of the solitary tract to hypothalamus neurocircuit

Abstract

The nucleus of the solitary tract (NTS) is a key gateway for meal-related signals entering the brain from the periphery. However, the chemical mediators crucial to this process have not been fully elucidated. We reveal that a subset of NTS neurons containing cholecystokinin (CCK(NTS)) is responsive to nutritional state and that their activation reduces appetite and body weight in mice. Cell-specific anterograde tracing revealed that CCK(NTS) neurons provide a distinctive innervation of the paraventricular nucleus of the hypothalamus (PVH), with fibers and varicosities in close apposition to a subset of melanocortin-4 receptor (MC4R(PVH)) cells, which are also responsive to CCK. Optogenetic activation of CCK(NTS) axon terminals within the PVH reveal the satiating function of CCK(NTS) neurons to be mediated by a CCK(NTS)→PVH pathway that also encodes positive valence. These data identify the functional significance of CCK(NTS) neurons and reveal a sufficient and discrete NTS to hypothalamus circuit controlling appetite.

Keywords: feeding behavior; mouse; neuronal circuits; neuropeptides; neuroscience.

Figure 1.. CCK^NTSneurons are activated by feeding.

(A) Schematic of mouse crossing to generate a CCK-iCre::R26-loxSTOPlox-eYFP mouse line (Cck^YFP). (B) Representative expression of Cck^YPF in a NTS coronal section. (C) Quantification of Cck^YPF-expressing cells across the rostral-to-caudal extent of the NTS. (D) Representative c-Fos-IR in Cck^YPF NTS cells in ad libitum fed, fasted or fasted then re-fed mice (white arrows denote colocalized neurons) and (E) quantification of c-FOS-positive Cck^YPF NTS cells (n = 3–4; one-way ANOVA F_(2,7) = 39.82, p = 0.0001; Sidak’s post hoc comparison). (F) Quantification of c-FOS-IR across the rostral-to-caudal extent of the NTS in Cck^YPF cells by bregma level following intragastric delivery of water, amino acids (aa) or sucrose (n = 3–5 per group; one-way ANOVA, F_(2,21) = 7.280, p = 0.0040; Tukey’s post hoc comparison). *p<0.05, **p< 0.01, ***p< 0.001. Scale bar in B and D represents 200 μm. AP, area postrema; DMX, dorsal motor nucleus; NTSco, nucleus of the solitary tract, commissural part; NTSm,nucleus of the solitary tract, medial part; NTSl, nucleus of the solitary tract, lateral part. DOI: http://dx.doi.org/10.7554/eLife.12225.003

Figure 2.. Activation of CCK^NTS neurons reduces feeding and body weight.

(A–D) Bilateral stereotaxic injection of Cre-dependent excitatory hM3D_q-mCherry virus into the NTS of male Cck-iCre mice facilitated activation of CCK^NTS neurons. (A) Schematic and Cre-mediated recombination of hM3D_q-mCherry allele. (B) Representative image of Cre-dependent expression of hM3D_q-mCherry within the caudal aspect of the NTS of a Cck-iCre mouse (coronal sections; numbers indicate bregma levels, scale bar represents 200 μm). (C) c-Fos-IR in the NTS and co-expression (green) in hM3D_q-mCherry-transduced CCK^NTS neurons (red) (scale bar represents 200 μm). (D) Membrane potential and firing rate of Cck-iCre-hM3D_q-mCherry^NTS neurons increased upon 5 μM CNO application. (E) Cck-iCre-hM3D_q-mCherry^NTS mice exhibited a significant reduction of spontaneous feeding following CNO, compared to saline, administration (n = 6; RM ANOVA, main effect of treatment [F_(1,5) = 22.41, p = 0.0052], main effect of time [F_(47,235) = 101.6, p<0.0001], and interaction [F_(47,235) = 1.807, p = 0.0023]); tick marks on x-axis represent 3 hr, measurements collected with 30-min intervals) and (F) a reduction of post-fast re-feeding following CNO compared to saline administration (n = 6; paired t test, t₍₅₎ = 4.769, p = 0.005). (G) CNO-induced reduction in spontaneous feeding was attenuated by pre-treatment with the CCK-A-receptor antagonist, devazepide (DEV; 1 mg/kg), (n = 6; ANOVA, F_(3,20) = 16.81, p<0.0001; Sidak’s post hoc comparison ***p<0.001, *p< 0.05). (H) CNO did not change fasting glucose level or (I) glucose disposal rate following a systemic glucose load (1 g/kg, IP). (J) Repeated CNO administrations over 4 days reduced body weight (n = 6; RM ANOVA, main effect of treatment [F_(1,100) = 60.78, p<0.0001], main effect of time [F_(9,100) = 8.877, p<0.0001], and interaction [F_(9,100) = 7.483, p<0.0001]) and decreased food intake (main effect of treatment [F_(1,100) = 16.13, p = 0.0001], main effect of time [F_(9,100) = 4.106, p = 0.0002], and interaction [F_(9,100) = 3.307, p = 0.0014]; Sidak’s post hoc comparisons, *p<0.05, **p<0.01, ***p<0.001) in Cck-iCre-hM3D_q-mCherry^NTS as compared to Cck-iCre-mCherry^NTS mice. DOI: http://dx.doi.org/10.7554/eLife.12225.004

Figure 3.. Activation of CCK^NTS neurons efferent to the PVH suppresses appetite.

(A) Schematic of CCK^NTS→ PVH targeting strategy using bilateral NTS delivery of Cre-dependent ChR2-eYFP expressing vector in Cck-iCre mice. (B) Selective eYFP expression following Cre-mediated recombination in the caudal aspect of the NTS (scale bar represents 200 μm). (C) CCK^NTS efferents (green) innervate the PVH (scale bar represents 400 μm). (D) CCK^NTS axon targeting for photostimulation, positioning of the optic fiber and photostimulation parameters (scale bar represents 400 μm). (E) Current clamp recording of a CCK^NTS neuron expressing ChR2 (scale bar 20 mV/100 ms). (F) Bilaterally transduced CCK^NTS axons in the PVH and c-Fos-IR following PVH photostimulation (scale bar represents 200 μm). (G) In vivo optogenetic photostimulation of NTS^CCK→PVH terminals in Cck-iCre-ChR2-eYFP^NTS significantly reduced nocturnal feeding (n = 6, RM ANOVA: main effect of treatment (F_(1,10) = 8.663, p = 0.0147), main effect of time (F_(18,180) = 97.25, p<0.0001) and interaction (F_(18,180) = 2.788, p = 0.0003) Sidak’s post hoc comparisons, *at least p<0.05) (H) without altering locomotor activity (RM ANOVA: main effect of treatment (F_{(1, 10)} = 1.510, p = 0.0.2472), main effect of time (F_(18,180) = 1.797, p = 0.0285) and interaction (F_(18,180) = 1.198, p = 0.2671) or (I) water consumption (RM ANOVA: main effect of treatment (F_(1,10) = 0.0924, p = 0.7673), main effect of time (F_(18,180) = 50.68, p<0.0001) and interaction (F_(18,180) = 0.2666, p = 0.9989) as compared to control Cck-iCre-eYFP^NTS. Tick marks on x-axis represent 10-min intervals. (J) Real time food intake reduction following optogenetic activation of NTS^CCK→PVH terminals in fasted Cck-iCre-ChR2-eYFP^NTS and reversion following injection of CCK-A receptor antagonist (devazipide; DEV) (n = 8; RM one-way ANOVA, treatment F_{(1.809,12.66)} = 16.15, p = 0.0004; individual F_(7,14) = 0.4241, p = 0.8714, Sidak’s post hoc comparison **p<0.005, *p<0.05). (K) Neither photostimulation nor DEV treatment alter food intake in fasted Cck-iCre-eYFP^NTS control mice (n = 6; RM one-way ANOVA, treatment F_{(1.294,6.469)} = 1.486, p = 0. 2780; individual F_(5,10) = 5.089, p = 0.014). (L) Photostimulation of NTS^CCK→PVH terminals reduces total intake of chocolate pellets over 30 min following 18–20 hr of food deprivation (n = 5, paired two-tailed t-test, t₍₄₎ = 6.949, p = 0.0023), as compared to no photostimulation. (M) Representative confocal image of NTS^CCK→PVH mCherry fibers and varicosities in close apposition to putative PVH MC4R-GFP neurons (scale bar represents 20 μm; 10 μm stack, maximum intensity projection). (N) Top, IV relationship of a PVH MC4R-GFP neuron produced by the superimposition of membrane potential deflection in response to a series of current injections of constant increment (scale bar 20 mV/200 ms); bottom, current clamp recording of the above neuron following bath application of CCK-8 (500 nM; scale bar 20 mV/30 s). (O) Percentage of PVH MC4R-neurons expressing CCK-A receptor mRNA as assessed by single-cell qPCR. NTS, nucleus of the solitary tract; PVH, paraventricular nucleus of the hypothalamus; AP, area postrema; cc, central canal; 3v, third ventricle. DOI: http://dx.doi.org/10.7554/eLife.12225.005

Figure 3—figure supplement 1.. Chemogenetic activation of CCK^NTS neurons elicits c-Fos in the paraventricular nucleus of the hypothalamus (PVH).

(A) Schematic and Cre-mediated recombination of hM3D_q-mCherry vector. (B) Cck-iCre-hM3D_q-mCherry^NTS mice were food deprived overnight and then treated with a single systemic injection of CNO (0.3 mg/kg; IP) or vehicle. 2 hr following CNO injection, mice were transcardially perfused with saline then fixative, brains extracted and processed for c-Fos immunohistochemistry (c-Fos-IR). Compared to saline, CNO administration increased c-Fos-IR in the (C) NTS and (D) PVH of Cck-iCre::hM3D_q-mCherry^NTS mice. AP, area postrema; NTS, nucleus of the solitary tract; cc, central canal; DMX, dorsal motor nucleus of the vagus; PVH, paraventricular nucleus of the hypothalamus. Scale bar represents 200 μm. DOI: http://dx.doi.org/10.7554/eLife.12225.006

Figure 3—figure supplement 2.. Hypothalamic projections of CCKNTS neurons.

Representative hypothalamic coronal sections from a Cck-iCre-ChR2-eYFP^NTS mouse. Scale bar represents 200 μm. 3v, third ventricle; AHA, anterior hypothalamic (hyp) area, anterior part; AHP, anterior hyp area, posterior part; Arc, arcuate hyp nucleus; DM, dorsomedial hyp nucleus; DMD, dorsomedial hyp nucleus dorsal; DMV, dorsomedial hyp nucleus ventral; f, fornix; LA, lateroanterior hyp nucleus; LH, lateral hyp area; ME, median eminence; mt, mamillothalamic tract; opt, optic tract; Pe, periventricular hyp; PVH, paraventricular nucleus of the hypothalamus; SCh, suprachiasmatic nucleus; VMH, ventromedial hyp nucleus; ZI, zona incerta. DOI: http://dx.doi.org/10.7554/eLife.12225.007

Figure 3—figure supplement 3.. Subset of PVH MC4R neurons express CCKA receptor and are responsive to CCK.

(A) Schematic of mouse crossing to generate a Mc4r-t2a-Cre::tdTomato mouse line. (B) Schematic of PVH location in a coronal hypothalamic section. (C) C_T values for individual Mc4r-t2a-Cre::tdTomato PVH neurons used for single-cell qPCR of isolated cells. Cckar mRNA was detected in 4 out of 15 analyzed cells. Cell No. 9 was considered negative. (D) Representative traces from current clamp recording of a Mc4r-t2a-Cre::tdTomato PVH neuron activated following bath application of CCK-8 (100 nM; 6/20 cells) and blockade in presence of the CCK-A receptor antagonist SR 27,897 (250 nM; 0/11 cells). Scale bar 20 mV/1 min. DOI: http://dx.doi.org/10.7554/eLife.12225.008

Figure 4.. CCK^NTS neurons signal positive valence via the PVH.

(A and D) Experimental strategies for the interrogation of the valence encoded by NTS^CCK→PVH terminals. (B) Food-deprived Cck-iCre-ChR2-eYFP^NTS mice exhibited a significant place preference for the photostimulation-paired chamber during a real-time place preference assay, as compared to control Cck-iCre-eYFP^NTS mice (n = 5–6; Two-way ANOVA – fed: no effect of photostimulation (F_(1,18) = 0.3244, p = 0.5760), no effect of ChR2 (F_(1,18) = 0.001145, p = 0.9734) or interaction (F_(1,18) = 0.5007, p = 0.4883); food deprived: main effect of photostimulation (F_(1,18) = 5.289, p = 0.0336), no effect of ChR2 (F_(1,18) = 0.08811, p = 0.7700) and interaction (F_(1,18) = 12.63, p = 0.0023); Sidak’s post hoc comparisons,*p = 0.05). (C) Representative real-time place preference location plots one representative mouse per condition. (E) Cck-iCre-ChR2-eYFP^NTS developed preference for the photostimulation-paired arm in a three-trial T-maze test (n = 5–6, main effect of treatment (F_(1,27) = 15.93, p = 0.0005) main effect of trials (F_(2,27) = 3.36, p = 0.0498) and interaction (F_(2,27) = 4.36, p = 0.0228); Sidak’s post hoc comparisons, ***p<0.001), as compared to Cck-iCre::eYFP^NTS. (F) Representative T-maze location plots from one representative mouse per condition. NTS, nucleus of the solitary tract; PVH, paraventricular nucleus of the hypothalamus. DOI: http://dx.doi.org/10.7554/eLife.12225.009