A tale of two circuits: CCKNTS neuron stimulation controls appetite and induces opposing motivational states by projections to distinct brain regions

Abstract

Cholecystokinin (CCK)-expressing neurons within the nucleus of the solitary tract (CCK^NTS) of the mouse are responsive to satiety signals and their chemogenetic activation suppresses appetite. Optogenetic activation of CCK^NTS axon terminals within either the parabrachial nucleus (PBN) or the paraventricular nucleus of the hypothalamus (PVH) is sufficient to suppress feeding. An interesting dichotomy has been revealed when assessing the motivational valence of these two circuits. Activating CCK^NTS cell bodies is aversive as demonstrated by conditioned taste aversion and place-preference assays. Activation of the CCK^NTS→PBN pathway is also aversive; however, stimulating the CCK^NTS→PVH pathway is appetitive when assayed using a real-time, place-preference task. Thus, these two projections from CCK^NTS neurons reduce food intake through opposite motivational states; one pathway signals positive valence (CCK^NTS→PVH) and the other signals negative valence (CCK^NTS→PBN).

Keywords: chemogenetics; cholecystokinin; conditioned place preference; conditioned taste aversion; nucleus tractus solitarius; optogenetics.

Fig. 1

CCK^NTS neurons suppress appetite and encode negative valence. (A) Depiction of viral targeting site for the excitatory DIO-hM3Dq:mCherry or DIO-mCherry virus in a coronal section through the medial NTS within the mouse brainstem. (B) In food-restricted animals expressing DIO-hM3Dq:mCherry, activation of CCK^NTS neurons with CNO decreased food intake at 2 h compared to intake after saline injection (n = 7). (C) Food-restricted mice expressing hM3Dq in CCK^NTS neurons display decreased preference for the CNO-paired side of a box during the final test after conditioning (n = 5). (D) Representative coronal section of the NTS in a Cck^Cre/+ mouse showing viral expression of the hM3Dq receptor in white and DAPI in blue. Scale bar, 100 µm. (E) Mice expressing DIO-hM3Dq:mCherry display CTA and avoided consumption of a high-fat diet that was previously paired with CNO in a choice test two days (left panel) and 3 weeks (right panel) after high-fat diet/CNO pairings (mCherry: n = 4, hM3Dq n = 5). All data represent means ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001. AP, area postrema; cc, central canal.

Fig. 2

A CCK^NTS→PBN circuit suppresses appetite and elicits avoidance in a RTPP test. (A) Diagram of a sagittal mouse brain depicting site of viral injections and placement of fiber optic cannulae for photostimulation of axon terminals in the PBN. (B) Representative immunohistological section demonstrating expression of ChR2:mCherry within fibers and Fos within nuclei in the lateral PBN after 30-Hz photoactivation. Scale bar, 100 µm. (C) 30-Hz photoactivation of CCK^NTS axon terminals in the PBN decreased food intake in ChR2-expressing mice (ChR2 n = 5, mCherry n = 7). (D) ChR2-expressing mice display a decreased preference for the 30-Hz, photostimulation-paired side in a RTPP assay, as compared to control, mCherry-expressing mice (ChR2 n = 10, mCherry n = 6). (E) ChR2-expressing mice did not display CTA and, similar to mCherry controls, chose to consume a high-fat diet that was previously paired with 30-Hz photoactivation in a choice test. All data represent means ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001. scp, superior cerebellar peduncle.

Fig. 3

A CCK^NTS→PVH circuit suppresses appetite and signals positive valence in a repeated RTPP test. (A) Diagram of a sagittal mouse brain depicting site of viral injections and placement of fiber optic cannula for photostimulation of axon terminals in the PVH. (B) Representative immunohistological section demonstrating expression of ChR2:mCherry within fibers and Fos within nuclei in the PVH after 30-Hz photoactivation. (C) 30-Hz photoactivation of CCK^NTS axon terminals in the PVH decreased food intake in ChR2:mCherry-expressing mice when measured 2 h after lights out compared to non-stimulated (no light) intake (n = 7). (D) ChR2:mCherry-expressing mice did not display a preference for either side in a single RTPP assay when one side was paired with 30-Hz photostimulation of CCK^NTS axon terminals in the PVH (n = 7). (E) After repeated RTPP sessions, animals increased their preference for the side of the chamber paired with photostimulation in the PVH (n = 7). (F) Immunohistological section demonstrating expression of ChR2:mCherry within fibers and absence of Fos immunoreactivity within nuclei in the lateral PBN after 30-Hz photoactivation of PVH afferents. All data represent means ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001. 3v, 3^rd ventricle. Scale bars, 100 µm.