GLP-1 neurons form a local synaptic circuit within the rodent nucleus of the solitary tract

Abstract

Glutamatergic neurons that express pre-proglucagon (PPG) and are immunopositive (+) for glucagon-like peptide-1 (i.e., GLP-1+ neurons) are located within the caudal nucleus of the solitary tract (cNTS) and medullary reticular formation in rats and mice. GLP-1 neurons give rise to an extensive central network in which GLP-1 receptor (GLP-1R) signaling suppresses food intake, attenuates rewarding, increases avoidance, and stimulates stress responses, partly via GLP-1R signaling within the cNTS. In mice, noradrenergic (A2) cNTS neurons express GLP-1R, whereas PPG neurons do not. In this study, confocal microscopy in rats confirmed that prolactin-releasing peptide (PrRP)+ A2 neurons are closely apposed by GLP-1+ axonal varicosities. Surprisingly, GLP-1+ appositions were also observed on dendrites of PPG/GLP-1+ neurons in both species, and electron microscopy in rats revealed that GLP-1+ boutons form asymmetric synaptic contacts with GLP-1+ dendrites. However, RNAscope confirmed that rat GLP-1 neurons do not express GLP-1R mRNA. Similarly, Ca²⁺ imaging of somatic and dendritic responses in mouse ex vivo slices confirmed that PPG neurons do not respond directly to GLP-1, and a mouse crossbreeding strategy revealed that <1% of PPG neurons co-express GLP-1R. Collectively, these data suggest that GLP-1R signaling pathways modulate the activity of PrRP+ A2 neurons, and also reveal a local "feed-forward" synaptic network among GLP-1 neurons that apparently does not use GLP-1R signaling. This local GLP-1 network may instead use glutamatergic signaling to facilitate dynamic and potentially selective recruitment of GLP-1 neural populations that shape behavioral and physiological responses to internal and external challenges.

Keywords: GABA; NTS; RRID: AB_2314562; RRID: AB_300798; RRID: AB_518978; glutamate; local circuit network; noradrenergic; preproglucagon; prolactin-releasing peptide; synapse.

Figure 1

The portion of the caudal nucleus of the solitary tract (cNTS) within which GLP‐1 neurons are sequestered is illustrated for the rat brain. (a) The location of the cNTS within the hindbrain dorsal motor vagal complex is marked in red. (b–d) The cytoarchitecture of this cNTS region is depicted in three coronal sections stained with the Kluver–Barrera method. (e–g) Immunoperoxidase labeling reveals GLP‐1+ profiles in coronal sections through rostrocaudal levels comparable to those shown in panels (b–d). Arrows indicate specific regions shown at higher magnification in the panel insets. Scale bars: 50 μm in (e–g), and 20 μm for the insets within those panels. AP = area postrema; DMV = dorsal motor nucleus of the vagus; NTS = nucleus of the solitary tract; ts = solitary tract [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Immunoperoxidase localization of yellow fluorescent protein (YFP) immunoperoxidase labeling within the caudal medulla of pre‐proglucagon (PPG)‐YFP mice, in which YFP+ axons frequently form close appositions on YFP+ dendrites. In each panel, arrowheads indicate the YFP+ axonal source of YFP+ terminals that form close appositions. (a, b, b′) In the caudal nucleus of the solitary tract (a), a YFP+ terminal forms a close apposition (arrow in b, b′) on the proximal dendrite of a PPG‐YFP neuron. (c, c′) A YFP+ terminal closely apposes (right arrow) a rostral PPG‐YFP neuron that has spines (double arrowheads) on its soma and a proximal dendrite. Another YFP+ terminal forms a close apposition (left arrow) on a nearby YFP+ dendrite. (d, d′) Near the cc, a YFP+ dendrite receives a close apposition (arrow) from a YFP+ terminal. (e, f) Other close appositions (arrows) from YFP+ terminals onto YFP+ dendrites. Scale bars: 250 μm in (a); 20 μm in (b–d); 10 μm in (b′, c′, d′, e, f). cc = central canal [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

Epifluorescent dual localization of GLP‐1+ (green) and PrRP+ (magenta) neurons and processes in two different fields (a, b) within the rat caudal nucleus of the solitary tract, demonstrating the overlapping distribution of these separate neural populations. Both neural populations give rise to thick dendritic processes and thin varicose axons (arrows) [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 4

Confocal imaging of dual PrRP (magenta) and GLP‐1 (green) immunofluorescence in the rat cNTS. (a) Maximum projection (z‐stack) 3D image, slightly rotated in (b) to reveal the same region. (c) A “side view” image stack of the same PrRP+ neuron indicated by # in panels (a and b). (d) Higher‐magnification view of the same # neuron, revealing close appositions formed by GLP‐1+ terminals (green) onto the PrRP neuron. The smaller boxed region (arrow) is depicted at even higher magnification in (e) (“top down” view) and (f) (“side view”), revealing no apparent gap between the green and magenta profiles (arrow). Scale bars: (b) 20 μm; (d) 5 μm. GLP‐1 = glucagon‐like peptide‐1; PrRP = prolactin‐releasing peptide [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 5

The strategy used for electron microscopic analysis of GLP‐1‐labeled profiles within the rat caudal nucleus of the solitary tract (cNTS) is illustrated. (a) Plastic‐embedded 100 μm‐thick vibratome section through the cNTS which was labeled for GLP‐1 immunoperoxidase before embedding. Transillumination reveals the location of GLP‐1+ neurons (white boxed area); this area was subsequently trimmed (red trapezoid in (a), same region depicted in (b)). (c) An ultramicrotome was used to generate an alternating series of two thick (0.35 μm) followed by 15 ultrathin (600 Å) sections. One thick section between each ultrathin series was left unstained (b, e) and the other thick section was stained with toluidine blue (d, f). This systematic approach optimized identification of GLP‐1+ profiles in all regions of the cNTS, permitting identification of the same profiles in thick and ultrathin sections. For example, panel (g) shows the ultrastructure of the same cell identified at the light microscopic level (red arrows in panels (e) and (f)). TEM = transmission electron microscopy [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 6

Electron micrographs illustrating the cellular localization of GLP‐1 immunoperoxidase reaction within the rat caudal nucleus of the solitary tract. (a) GLP‐1+ neurons had a long axis of 10–15 μm and a short axis of 7–10 μm with a prominent centrally placed cell nucleus. (b) Immunoperoxidase labeling within neurons was densely concentrated in Golgi complexes surrounding the cell nucleus and in large vesicles associated with the cis face of the Golgi (arrows in (b)) or distributed among cisternae of the rough endoplasmic reticulum in the cell cytoplasm (arrows in (c)). Scale bars: 2 μm in each panel

Figure 7

Electron micrographs illustrating GLP‐1+ processes in the rat caudal nucleus of the solitary tract. (a) Immunopositive vesicles derived from the Golgi complexes of the cell soma extended into the dendritic arbor to produce discrete labeling (arrows). (b, c) Immunopositive axon terminals displayed a range of labeling density that was directly correlated with the proximity of the ultrathin section to the surface of the flat embedded tissue. Profiles within the top few microns of the tissue (b) contained dense concentrations of immunoperoxidase reaction product that filled the profile, while those deeper in the tissue (c) displayed more punctate labeling. GLP‐1+ terminals were densely filled with 40 nm lucent spherical vesicles and occasional dense core vesicles. Immunopositive terminals formed appositions with both unlabeled (b–d) and labeled (e–h) dendrites. (f, g) When synapses (open arrowheads) were present within the plane of section they were asymmetric in character and, in many cases, formed multiple synapses with the same profile. Immunopositive dendrites also were recipient of synaptic input from immunonegative terminals that exhibited both asymmetric/excitatory (i) and symmetric/inhibitory characteristics. (j) GLP‐1+ axon varicosities often were closely apposed to unlabeled axon terminals (ut) filled with 40 nm lucent spherical vesicles. Scale bars: (a) 2 μm; (b) 500 nm; (c, f) 600 nm; (d) 800 nm; (g–i) 1 μm. d, dendrite; ut, unlabeled terminal

Figure 8

Confocal imaging of dual RNAscope fluorescent in situ hybridization labeling in rat caudal nucleus of the solitary tract (cNTS). (a) Maximum projection z‐stack showing PPG mRNA (green) and GLP1R mRNA (magenta) transcripts. (b) Rotated image stack from the same area to depict views from both sides of one PPG mRNA‐expressing neuron (marked with # in panel (a)), revealing magenta GLP1R mRNA transcripts that lie adjacent to (but not within) the PPG‐expressing neuron. (c) Maximum projection z‐stack showing GAD1 mRNA (green) and GLP1R mRNA (magenta) transcripts, with the rotated 3D side view (inset) demonstrating intracellular colocalization of both transcripts. (d) Maximum projection z‐stack showing GAD1 mRNA (green) and GLP1R mRNA (magenta) transcripts in a different field within the cNTS, demonstrating lack of colocalization of the two transcripts. Scale bars: (a) 10 μm; (c, d) 5 μm. GLP‐1R = GLP‐1 receptor, PPG = pre‐proglucagon [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 9

Confocal imaging of PrRP immunolabeling (green) and GLP1R mRNA (magenta) within the rat caudal nucleus of the solitary tract. (a) “Side view” of a rotated image stack depicting a double‐labeled neuron. The same neuron is shown from a “top view” in a maximum projection z‐stack image in panel (c), and from an “end‐on” image stack view in panel (d). Within the same field of view (visible in each panel), other double‐labeled PrRP+ neurons are located nearby, although some lack intracellular colocalization of GLP1R mRNA (e.g., the neuron located on the right in panels (a) and (c)). Scale bar: (b) 20 μm. GLP‐1R = GLP‐1 receptor; PrRP = prolactin‐releasing peptide [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 10

Photomicrographs showing yellow fluorescent protein (YFP)‐positive pre‐proglucagon (PPG) neurons (green) and tdRFP‐positive GLP‐1R neurons (magenta) in coronal NTS sections from transgenic mice cross‐bred to display both reporters. (a) At Bregma −7.6 mm, most PPG neurons are located in the lateral part of the NTS. Some GLP‐1R neurons are observed in the NTS, but a higher density is seen in the AP. The lower panel shows the PPG neurons and GLP‐1R neurons separately to confirm lack of colocalization of YFP and tdRFP in the same cells. (b) At Bregma −8.0 mm, NTS PPG neurons are located more medially than at more rostral levels. This section shows a rare case of colocalization of YFP and tdRFP in the same cell (white arrowheads in the magnified view panels located below (b)), suggesting that this lone PPG neuron expresses GLP‐1R. Scale bars = 100 μm. AP = area postrema; NTS = nucleus of the solitary tract; cc = central canal [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 11

Optical recordings of the intracellular Ca²⁺ concentration in pre‐proglucagon (PPG) neurons reveal a lack of response to GLP‐1. (a–c) Pseudocolored micrographs showing Ca²⁺ levels in eight PPG neurons under different conditions: control (a), 100 nM GLP‐1 (b), 100 μM glutamate (c). (d) The gray traces show the individual somatic responses of 27 PPG neurons (from three mice) to 100 nM GLP‐1 and 0.1 mM glutamate, and the magenta trace shows the mean response from these cells. (e) Gray traces show the individual responses of 23 PPG dendrites (from three mice) to 100 nM GLP‐1 and 0.1 mM glutamate, and the magenta trace shows the mean response from these dendrites. In both (d) and (e), statistical analysis of the responses expressed as AUC is shown as a box plot with whiskers, with the median indicated and whiskers marking the 10th and 90th percentile, and outliers represented by black filled circles. Significance was determined with a Wilcoxson test. ****p < .0001. AUC = area under the curve; GLP‐1 = glucagon‐like peptide‐1 [Color figure can be viewed at http://wileyonlinelibrary.com]