Preproglucagon (PPG) neurons innervate neurochemically identified autonomic neurons in the mouse brainstem

Abstract

Preproglucagon (PPG) neurons produce glucagon-like peptide-1 (GLP-1) and occur primarily in the nucleus tractus solitarius (NTS). GLP-1 affects a variety of central autonomic circuits, including those controlling the cardiovascular system, thermogenesis, and most notably energy balance. Our immunohistochemical studies in transgenic mice expressing YFP under the control of the PPG promoter showed that PPG neurons project widely to central autonomic regions, including brainstem nuclei. Functional studies have highlighted the importance of hindbrain receptors for the anorexic effects of GLP-1. In this study, we assessed YFP innervation of neurochemically identified brainstem neurons in transgenic YFP-PPG mice. Immunoreactivity for YFP plus choline acetyltransferase (ChAT), tyrosine hydroxylase (TH) and/or serotonin (5-HT) was visualised with two- or three-colour immunoperoxidase labelling using black (YFP), brown and blue-grey reaction products. In the dorsal motor nucleus of the vagus (DMV), terminals from fine YFP-immunoreactive axons closely apposed a small proportion of ChAT-positive and rare TH-positive/ChAT-positive motor neurons, mostly ventral to AP. YFP-immunoreactive innervation was virtually absent from the compact and loose formations of the nucleus ambiguus. In the NTS, some TH-immunoreactive neurons were closely apposed by YFP-containing axons. In the A1/C1 column in the ventrolateral medulla, close appositions on TH-positive neurons were more common, particularly in the caudal portion of the column. A single YFP-immunoreactive axon usually provided 1-3 close appositions on individual ChAT- or TH-positive neurons. Serotonin-immunoreactive neurons were most heavily innervated, with the majority of raphé pallidus, raphé obscurus and parapyramidal neurons receiving several close appositions from large varicosities of YFP-immunoreactive axons. These results indicate that GLP-1 neurons innervate various populations of brainstem autonomic neurons. These include vagal efferent neurons and catecholamine neurons in areas linked with cardiovascular control. Our data also indicate a synaptic connection between GLP-1 neurons and 5-HT neurons, some of which might contribute to the regulation of appetite.

FIGURE 1

Three-colour immunoperoxidase labelling for YFP in YFP-PPG neurons (black), tyrosine hydroxylase (TH) in catecholamine neurons (brown) and choline acetyltransferase (ChAT) in cholinergic neurons (blue-grey) in transverse sections through the dorsal medulla of YFP-PPG mice. A, Low magnification micrograph showing the cell bodies of black, YFP-immunoreactive PPG neurons in the nucleus of the solitary tract (NTS). These neurons lie lateral to the dorsal motor nucleus of the vagus (DMV) and the majority occur at the level of the area postrema (AP). The dendrites of the YFP-PPG neurons in NTS extend dorsomedially and also run lateral to the hypoglossal nucleus (HGN) and along the border between the DMV and the HGN. cc, central canal. Bar, 100 μm. B, In the intermediate reticular nucleus (IRT), the black, YFP-immunoreactive cell bodies of PPG neurons are located dorsomedial to both the brown, TH-immunoreactive neurons of the A1 cell group and the blue-grey, ChAT-immunoreactive neurons of the nucleus ambiguus (NA). Bar, 100 μm. C, Low magnification micrograph showing the dorsal vagal complex at the level of the most rostral YFP-PPG neurons. A collection of YFP-immunoreactive dendrites travels between the DMV and HGN. Arrows, brown, TH-immunoreactive cell bodies that are shown at higher magnification in D and E. Bar, 250 μm. D and E, Single varicosities (arrows) of black, YFP-immunoreactive axons closely appose brown, TH-immunoreactive cell bodies in the NTS. Bars, 20 μm. F, Low magnification micrograph showing the AP near the level of the most rostral YFP-PPG neurons. Arrow, a brown, TH-immunoreactive cell body near the surface of AP that is shown at higher magnification in G. Bar, 100 μm. G, A single varicosity (arrow) of a black, YFP-immunoreactive axon closely apposes the brown, TH-immunoreactive cell body in AP. Bar, 10 μm. H, At the surface of rostral AP, black, varicose, YFP-immunoreactive axons and black, varicose, YFP-immunoreactive dendrites are intermixed with brown, TH-immunoreactive dendrites arising from AP neurons. Bar, 20 μm. I, Low magnification micrograph showing the DMV near the level of the most rostral YFPPPG neurons. Arrows J1 and J2, two blue-grey, ChAT-immunoreactive vagal motor neurons that are shown at higher magnification in J. Bar, 100 μm. J, Fine varicosities (arrows) of black, YFP-immunoreactive axons closely appose the blue-grey, ChAT-immunoreactive cell bodies of DMV neurons J1 and J2. Bar, 20 μm.

FIGURE 2

Maps of sections through the dorsal vagal complex of a YFP-PPG mouse showing the distribution of ChAT-immunoreactive and TH-immunoreactive neurons with and without close appositions from YFP-immunoreactive varicosities. Sections were triple-stained to reveal YFP, TH and ChAT. Every third section was mapped; maps are therefore separated by 60μm. The most caudal section is at the top left of the figure and the most rostral section is at the bottom right. TH-immunoreactive neurons in the nucleus tractus solitarius (NTS) and area postrema (AP) are represented by circles; and ChAT-immunoreactive neurons, predominantly in the dorsal motor nucleus of the vagus (DMV), by diamonds. TH-immunoreactive neurons that receive close appositions from YFP-immunoreactive varicosities are shown in red; those that do not receive appositions are shown in blue. ChAT-immunoreactive neurons that receive close appositions from YFP-immunoreactive varicosities are shown in gold; those that do not receive appositions are shown in green. The cell bodies of YFP-PPG neurons are represented by white stars. Numerical values indicate the proportion of neurons in each region that received close appositions from YFP-immunoreactive varicosities. The percentages of neurons that received YFP- immunoreactive appositions in each region are given in parentheses. The asterisk indicates an area of the DMV in which appositions could not be assessed because the dendrites of YFP-PPG neurons were too dense. cc, central canal; HGN, hypoglossal nucleus.

FIGURE 3

Two-colour immunoperoxidase labelling for YFP in YFP-PPG neurons (black) and tyrosine hydroxylase (TH) in catecholamine neurons (brown) in transverse sections through the rostral ventrolateral medulla (RVLM) and dorsal pons of YFP-PPG mice. A, Low magnification micrograph showing the RVLM. Arrows, brown, TH-immunoreactive cell bodies that are shown at higher magnification in D and E. py, pyramidal tract. Bar, 100 μm. B and C, Single varicosities (arrows) of black, YFP-immunoreactive axons closely appose brown, TH-immunoreactive cell bodies in the RVLM. Bar in B, 10 μm; bar in C, 20 μm. D, Low magnification micrograph showing the region of the locus coeruleus (LC) and Barrington’s nucleus (Bar). Left is medial and the ventricle appears at top left. The region in Box E is shown at higher magnification in E. Arrow F, a brown, TH-immunoreactive neuron in the subcoeruleus region (SubC) that is shown at higher magnification in F. Bar, 250 μm. E, There are many black, varicose YFP-immunoreactive axons in Bar. However, only a few, black, varicose YFP-immunoreactive axons travel amongst the brown, TH-immunoreactive neurons of LC. Of all of the TH-immunoreactive cell bodies in this region of LC, only 6 received close appositions from YFP-immunoreactive varicosities, one of which (lower right) is marked by an arrowhead. There were no close appositions on the TH-immunoreactive dendrites of LC neurons that penetrated Bar. Bar, 50 μm. F, A single varicosity (arrow) of a black, YFP-immunoreactive axon closely apposes the brown, TH-immunoreactive cell body in SubC. Bar, 20 μm.

FIGURE 4

Maps of sections through the ventral medulla of a YFP-PPG mouse showing the distribution of TH-immunoreactive neurons with and without close appositions from YFP-immunoreactive varicosities. Sections were triple-stained to reveal YFP, TH and ChAT. Every sixth section was mapped; maps are therefore separated by 150μm. The most caudal section is at the top left of the figure and the most rostral section is at the bottom right. TH-immunoreactive neurons that receive close appositions from YFP-immunoreactive varicosities are represented by red circles; TH-immunoreactive neurons that do not receive appositions are represented by blue circles. The cell bodies of YFP-PPG neurons are represented by white stars. Numerical values indicate the proportion of neurons in each section that received close appositions from YFP-immunoreactive varicosities. The percentages of neurons that received YFP- immunoreactive appositions in each section are given in parentheses. The insets show the entire section from which each map was constructed, allowing the dorsoventral tilt of the section can be assessed. FN, facial nucleus; IO, inferior olive; NAc, compact formation of the nucleus ambiguus; py, pyramidal tract; pyx, pyramidal decussation.

FIGURE 5

Two-colour immunoperoxidase labelling for YFP in YFP-PPG neurons (black) and 5-HT in serotonin neurons (brown) in transverse sections through the caudal ventral medulla of YFP-PPG mice. A, Low magnification micrograph showing the ventral medulla from the midline through the RVLM, Arrows B and C, brown, groups of 5-HT-immunoreactive cell bodies that are shown at higher magnification in B and C. py, pyramidal tract. Bar, 100 μm. B, In the parapyramidal region (PPY), a cluster of serotonin-immunoreactive cells bodies receives close appositions from many YFP-immunoreactive axons. Bar, 20 μm. C, The cell bodies of two brown, 5-HT immunoreactive neurons in raphé pallidus (RPa) receive several close apposition from varicosities (arrows) of black, YFP-immunoreactive axons. Bar, 10 μm. D, A black, YFP-immunoreactive varicosity (arrow) forms a close apposition on a brown, 5-HT immunoreactive cell body that lies near the ventral surface (VS) in PPY. Bar, 10 μm. E, Two black varicosities on the same YFP-immunoreactive axon (arrows) closely appose a brown, 5-HT-immunoreactive cell body in RPa. Bar, 10 μm.

FIGURE 6

Maps of sections through the ventral medulla of a YFP-PPG mouse showing the distribution of 5-HT-immunoreactive neurons with and without close appositions from YFP-immunoreactive varicosities. Sections were double-stained to reveal YFP and 5-HT. Every sixth section was mapped; maps are therefore separated by 150μm. The most caudal section is at the top left of the figure and the most rostral section is at the bottom right. 5-HT-immunoreactive neurons that receive close appositions from YFP-immunoreactive varicosities are represented by red circles; 5-HT-immunoreactive neurons that do not receive appositions are represented by blue circles. The cell bodies of YFP-PPG neurons are represented by white stars. Numerical values indicate the proportion of 5-HT-immunoreactive neurons in different region or the entire mapped population that received close appositions from YFP-immunoreactive varicosities. The percentages of neurons that received YFP-immunoreactive appositions are given in parentheses. The insets show the entire section from which each map was constructed, allowing the dorsoventral tilt of the section to be assessed. FN, facial nucleus; IO, inferior olive; NAc, compact formation of the nucleus ambiguus; py, pyramidal tract; RPa, raphé pallidus; VS, ventral surface..