Preproglucagon neurons project widely to autonomic control areas in the mouse brain

Abstract

Glucagon-like peptide 1 (GLP-1) and its analogue exendin-4 inhibit food intake, reduce blood glucose levels and increase blood pressure and heart rate by acting on GLP-1 receptors in many brain regions. Within the CNS, GLP-1 is produced only by preproglucagon (PPG) neurons. We suggest that PPG neurons mediate the central effects of GLP-1 by modulating sympathetic and vagal outflow. We therefore analysed the projections of PPG neurons to brain sites involved in autonomic control. In transgenic mice expressing yellow fluorescent protein (YFP) under the control of the PPG promoter, we assessed YFP-immunoreactive innervation using an anti-GFP antiserum and avidin-biotin-peroxidase. PPG neurons were intensely YFP-immunoreactive and axons could be easily discriminated from dendrites. YFP-immunoreactive cell bodies occurred primarily within the caudal nucleus tractus solitarius (NTS) with additional somata ventral to the hypoglossal nucleus, in raphé obscurus and in the intermediate reticular nucleus. The caudal NTS contained a dense network of dendrites, some of which extended into the area postrema. Immunoreactive axons were widespread throughout NTS, dorsal vagal nucleus and reticular nucleus with few in the hypoglossal nucleus and pyramids. The dorsomedial and paraventricular hypothalamic nuclei, ventrolateral periaqueductal grey and thalamic paraventricular nucleus exhibited heavy innervation. The area postrema, rostral ventrolateral medulla, pontine central grey, locus coeruleus/Barrington's nucleus, arcuate nucleus and the vascular organ of the lamina terminalis were moderately innervated. Only a few axons occurred in the amygdala and subfornical organ. Our results demonstrate that PPG neurons innervate primarily brain regions involved in autonomic control. Thus, central PPG neurons are ideally situated to modulate sympathetic and parasympathetic outflow through input at a variety of central sites. Our data also highlight that immunohistochemistry improves detection of neurons expressing YFP. Hence, animals in which specific populations of neurons have been genetically-modified to express fluorescent proteins are likely to prove ideal for anatomical studies.

FIGURE 1. Detection of YFP-PPG neurons in the medulla using intrinsic YFP fluorescence versus immunohistochemistry for YFP

YFP-PPG neurons in transverse sections through the medulla that were processed to reveal YFP-immunoreactivity (IR) with an anti-GFP antiserum, biotinylated secondary antibodies and Cy3-streptavidin. A, Fourteen YFP-expressing somata in the intermediate reticular nucleus at the level of the rostral area postrema (AP) that were viewed with the filter combination for the intrinsic fluorescence of YFP. Arrows indicate four cell bodies with fluorescent signals that vary from faint to intense. A’, The same 14 YFP-containing somata viewed with the filter combination for immunofluorescent detection of YFP with Cy3-streptavidin. A comparison of A and A’ shows that all of the YFP-containing cell bodies appear in both micrographs. However, many more YFP-containing axons and dendrites are detected by their YFP-immunoreactivity (YFP-IR) than by the fluorescence due to their YFP content. Bars, 50 μm. B, YFP-expressing cell bodies in the NTS and YFP-expressing axons and dendrites in the caudal AP that were visualized using the filter combination for the intrinsic fluorescence of YFP. B’, The same field of view viewed using the filter combination for immunofluorescent detection of YFP with Cy3-streptavidin. The backgrounds of the two micrographs were adjusted to the same grayscale values using Adobe PhotoShop. A comparison of B with B’ shows that many more YFP-containing axons with clearly delineated varicosities (arrowheads in B’) are visualized with immunohistochemistry than with native YFP fluorescence. More dendrites (arrows in B’) are also apparent after immunohistochemical detection of YFP and their decreasing diameters and tapering ends are much better revealed. Bars, 100 μm.

FIGURE 2. Distribution of YFP-immunoreactive neurons in the lower brainstem

YFP-PPG neurons in transverse sections through the medulla were visualised by immuno-peroxidase staining with nickel-intensified diaminobenzidine A, Montage of low magnification micrographs showing YFP-immunoreactive cell bodies located in the caudal nucleus tractus solitarius, in the intermediate reticular nucleus, at the ventral border of the hypoglossal nucleus (HGN, arrow) and in raphé obscurus (arrow). There are many YFP-positive dendrites running towards the central canal (cc) along the border between the dorsal vagal nucleus and the HGN. Immunoreactive axons were widespread throughout the NTS, the dorsal vagal nucleus and the reticular nucleus (except for the parvicellular section) but the HGN is virtually devoid of YFP-positive axons. Bar, 250 μm. B. The caudal NTS contained YFP-immunoreactive cell bodies and a dense network of dendrites, some of which extended into the area postrema (AP) and the dorsal vagal nucleus. Many axons extend ventrolaterally and some travel along the lateral edge of the HGN. There are also axons that extend into AP and the dorsal vagal nucleus. Bar, 100 μm. C. The intermediate reticular nucleus (IRT) contains another substantial group of YFP-immunoreactive cell bodies. Bar, 100 μm.

FIGURE 3. YFP-immunoreactive axons in the medulla and midbrain A

The ventral medulla contains both varicose and non-varicose axons. The majority of the axons in the rostral ventrolateral medulla (RVLM) and along the ventral surface of the medulla are varicose. The arrow indicates the location of the inset, which shows these axons at higher magnification. In contrast, in the ventral medullary reticular nucleus, non-varicose axons of passage (arrows) are the most prominent. YFP-positive axons in raphé obscurus are a mixture of varicose and non-varicose whereas YFP-containing axons in raphé pallidus are mainly varicose. Bar, 250 μm. B. A dense accumulation of varicose, YFP-immunoreactive axons occur near the fourth ventricle (4V) in the region of Barrington’s nucleus and the locus coeruleus (LC). Bar, 100 μm. C. At the level of the aqueduct (Aq) many YFP-immunoreactive axons are present in subregions of the periaqueductal gray (PAG). The ventrolateral PAG (VLPAG) contains a high density of varicose YFP-positive axons, whereas there is a moderate to low density of YFP-containing axons in the dorsomedial PAG (DMPAG). Bar, 250 μm.

FIGURE 4. YFP-immunoreactive axons in hypothalamic and thalamic nuclei

A, The paraventricular nucleus of the hypothalamus (PVN) is densely innervated by YFP-immunoreactive axons. Bar, 100 μm. B, More caudally in the hypothalamus, the dorsomedial nucleus of the hypothalamus (DMH) receives dense supply of YFP-immunoreactive axons and the arcuate nucleus is moderately innervated. There are very few YFP-containing axons in the ventromedial hypothalamic nucleus (VMH) and the median eminence (ME) is free of innervation. Bar, 250 μm. C, YFP-immunoreactive axon heavily innervate the paraventricular nucleus of the thalamus (PVT). Occasional axons also occur in the subfornical organ (SFO). Bar, 250 μm. D, There is a moderate innervation of the arcuate nucleus (Arc) and no axons in the median eminence (ME). Bar, 250 μm. E, The organum vasculosum of the lamina terminalis (OVLT) contains a substantial number of YFP-immunoreactive axons. Bar, 250 μm. 3V, 3^rd ventricle; 3Vd, dorsal 3^rd ventricle.

FIGURE 5. Distribution of YFP-immunoreactive cell bodies and axons in the brains of YFP-PPG mice

Diagrams of coronal sections showing the distribution of YFP-immunoreactive cell bodies and axons in the brains of YFP-PPG mice. Mapped sections were located between the spinomedullary junction and the rostral end of the third ventricle and were matched as closely as possible to the locations of the micrographs shown in Figures 1-4. Filled circles, YFP-immunoreactive somata; asterisks, varicose YFP-immunoreactive axons; diamonds, non-varicose YFP-immunoreactive axons. For all symbols, the size of the symbol indicates the relative density of the YFP-positive structure. Grey symbols indicate that the immunoreactive structure is rare in that location. Although varicose, YFP-immunoreactive axons are present in the caudal NTS, it was difficult to assess their density because of the extensive network of YFP-positive dendrites that occurred in this region. Abbreviations: 10, dorsal motor nucleus of the vagus; 12, hypoglossal nucleus; 3V, third ventricle; 4V, fourth ventricle; 7n, facial nerve; A5, A5 noradrenaline neurons; A7, A7 noradrenaline neurons; ac, anterior commissure; AHC, anterior hypothalamic area, central part; Amb, nucleus ambiguus; AP, area postrema; Aq, aqueduct; Arc, arcuate nucleus; AVPe, anteroventral periventricular nucleus; Bar, Barrington’s nucleus; BNST, bed nucleus of the stria terminalis; cal, corpus callosum; cc, central canal; CeA, central nucleus of the amygdala; CPu, caudate putamen; Cu, cuneate nucleus; D3V, dorsal third ventricle; DA, dorsal hypothalamic area; DLPAG, dorsolateral periaqueductal gray; DMH, dorsomedial hypothalamic nucleus; DMPAG, dorsomedial periaqueductal gray; DPGi, dorsal paragigantocellular nucleus; DRN, dorsal raphé nuclei; eml, external medullary lamina; fi, fimbria of the hippocampus; fx, fornix; Gi, gigantocellular reticular nucleus; GiV, gigantocellular reticular nucleus, ventral part; GP, globus pallidus; Gr, gracile nucleus; Hb, habenula; Hip, hippocampus; ic, internal capsule; IO, inferior olive; IRt, intermediate reticular nucleus; isRt, isthmic reticular formation; LC, locus coeruleus; LHA, lateral hypothalamic area; LPAG, lateral periaqueductal gray; LPGi, lateral paragigantocellular nucleus; LRt, lateral reticular nucleus; LS, lateral septum; LV, lateral ventricle; mcp, middle cerebellar peduncle; MdD, medullary reticular nucleus, dorsal part; MdV, medullary reticular nucleus, ventral part; ME, median eminence; mfb, medial forebrain bundle; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MnPO, median preoptic nucleus; MnR, median raphé nucleus; MS, medial septal nucleus; mt, mammillothalamic tract; NTS, nucleus of the solitary tract; opt, optic tract; OVLT, organum vasculosum of the lamina terminalis; Pe, periventricular hypothalamic nucleus; Pir, piriforn cortex; PnO, pontine reticular nucleus, oral part; Pr, prepositus nucleus; Pr5, principal sensory trigeminal nucleus; PRN, pontine reticular nucleus; PVN, paraventricular hypothalamic nucleus; PVT, paraventricular thalamic nucleus; py, pyramidal tract; RIP, raphé interpositus nucleus; RMg, raphé magnus nucleus; ROb, raphé obscurus nucleus; RPA, raphé pallidus nucleus; RVLM, rostral ventrolateral medulla; SC, superior colliculus; SCh, suprachiasmatic nucleus; SFO, subfornical organ; sm, stria medullaris; SO superior olive; sol, solitary tract; SON, supraoptic nucleus; sp5, spinal trigeminal tract; Sp5, spinal trigeminal nucleus; st, stria terminalis; SuG, superficial gray layer of the superior colliculus; Tg, tegmental nuclei; Tu, olfactory tubercle; VC, ventral cochlear nucleus; VLPAG, ventrolateral periaqueductal gray; VMH, ventromedial hypothalamic nucleus; VMPO, ventromedial preoptic nucleus; ZI, zona incerta. Diagrams have been reproduced from Franklin & Paxinos (2007), with kind permission.