The projection and synaptic organisation of NTS afferent connections with presympathetic neurons, GABA and nNOS neurons in the paraventricular nucleus of the hypothalamus

Abstract

Elevated sympathetic nerve activity, strongly associated with cardiovascular disease, is partly generated from the presympathetic neurons of the paraventricular nucleus of the hypothalamus (PVN). The PVN-presympathetic neurons regulating cardiac and vasomotor sympathetic activity receive information about cardiovascular status from receptors in the heart and circulation. These receptors signal changes via afferent neurons terminating in the nucleus tractus solitarius (NTS), some of which may result in excitation or inhibition of PVN-presympathetic neurons. Understanding the anatomy and neurochemistry of NTS afferent connections within the PVN could provide important clues to the impairment in homeostasis cardiovascular control associated with disease. Transynaptic labelling has shown the presence of neuronal nitric oxide synthase (nNOS)-containing neurons and GABA interneurons that terminate on presympathetic PVN neurons any of which may be the target for NTS afferents. So far NTS connections to these diverse neuronal pools have not been demonstrated and were investigated in this study. Anterograde (biotin dextran amine - BDA) labelling of the ascending projection from the NTS and retrograde (fluorogold - FG or cholera toxin B subunit - CTB) labelling of PVN presympathetic neurons combined with immunohistochemistry for GABA and nNOS was used to identify the terminal neuronal targets of the ascending projection from the NTS. It was shown that NTS afferent terminals are apposed to either PVN-GABA interneurons or to nitric oxide producing neurons or even directly to presympathetic neurons. Furthermore, there was evidence that some NTS axons were positive for vesicular glutamate transporter 2 (vGLUT2). The data provide an anatomical basis for the different functions of cardiovascular receptors that mediate their actions via the NTS-PVN pathways.

Fig. 1

Spinal cord and NTS injection sites and PVN labelling. (A) Micrograph of a longitudinal section of spinal cord, which shows site of pressure injection of FG (arrowhead) into the IML of the T2 segment that resulted in labelling of neurons in the PVN. (B) Coronal section of the PVN (Bregma −1.6 mm) showing neurons labelled with FG (blue, arrowhead) from the spinal cord and BDA fibres (green, arrows) from the NTS. (C) Micrograph of coronal section of NTS (Bregma −13.8 mm) showing an injection site (arrowhead) of BDA into the interstitial region of the NTS. (D) Coronal map of injection sites into NTS that resulted in axonal labelling in the PVN. Each filled circle represents a deposit of BDA into the NTS that resulted in axonal projections in and around the PVN. Abbreviations used in the figures: 3V, 3rd ventricle; 4V, 4th ventricle; Amb, ambiguus nucleus; AP, area postrema; CAA, central autonomic area; CC, central canal; chp, choroid plexus; CVRG, caudo-ventrolateral respiratory group; dp, dorsal parvocellular; DVN, dorsal vagal nucleus; GLU, glutamate; ICN, intercalated nucleus; IML, intermediolateral cell column; LF, lateral funiculus; M, magnocellular; mp, medial parvocellular; NTS, nucleus tractus solitarius; pp, periventricular parvocellular; Py, pyramidal tracts; RVRG, rostral ventral respiratory group; Sol, nucleus of solitary tract; SolC, nucleus of solitary tract, commissural; SolDM, nucleus of solitary tract, dorsomedial; SolI, nucleus of solitary tract interstitial; SolIM, nucleus of solitary tract, intermediate; SolM, nucleus of solitary tract, medial; SolVL, nucleus of solitary tract, ventrolateral; vp, ventral parvocellular. Scale bars A, C = 200 μm; B = 75 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Spinally projecting neurons as targets for axons projecting from the NTS. Views of the dorsal cap region of PVN showing NTS axons (black) with close appositions to the cell bodies (A, A′) and dendrites (B, B′) of the cholera toxin-labelled spinally projecting presympathetic neurons (brown). (A′) Multiple axons (arrowhead) wound around cells where synaptic boutons (arrow) apposed the cell soma. Similarly, axons (B′) can be observed to traverse cell bodies in their passage through the PVN. These axons had numerous boutons (arrow) interspersed by non-bouton regions (arrowhead). (C) A deconvolved image showing the course of an axon (green) as it crossed the cell soma of a FG-labelled presympathetic neuron (blue) with multi bouton appositions over the cell soma (arrow). (C′) An isosurface 3-D rendered view of (C) revealing the close association between the green NTS boutons (arrows) with the preautonomic neurons (blue). Scale bars A, B = 60 μm; A′ = 6 μm; B′ = 30 μm; C = 15 μm; C′ = 8 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

NTS originating axons course through the GABA neuronal pools adjacent to the PVN. (A) Coronal section at Bregma −1.88 mm showing the PVN-containing spinally projecting neurons (green) and GABA interneurons (box, arrowheads). (B, C) Examples of orange NTS axons coursing through the GABA neuronal pools (green, arrowhead) that had synaptic boutons (arrow) closely apposed to the cell bodies of the GABA neurons (boxed insert right hand corner B, C). (C′) deconvolved image of boxed area in (C). The course of the orange axon (asterisk) can be traced crossing the green GABA-positive cells (1–4). The arrows indicate the sites of en passent boutons across the cells. (C″) Isosurface 3-D rendered image of (C′), which provides evidence in support that axons (asterisk) with en passent boutons (arrows) could be in synaptic contact with the GABA-positive neurons 1–4. Scale bars A = 120 μm; B = 13 μm and 6 μm; C = 14 μm and 8 μm; C = 15 μm and 8 μm; C′, C″ = 5 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

nNOS-positive neurons within and adjacent to the PVN associated with NTS axons. (A) Coronal section (Bregma −2.12 mm) showing PVN with presympathetic neurons (blue, arrowhead) and intermingled nNOS-positive neurons (green, arrows) that lie within the PVN. The small arrows indicate presympathetic neurons that are also positive for nNOS. (B, C) NTS axons (green, arrows) intermingled with nNOS-positive neurons (orange, arrowheads) in the more ventrolateral magnocellular region, as well as in the parvocellular region of PVN and adjacent to the PVN. (b) Deconvolved images of boxed area in (B). An NTS axon (arrows) crossed the nNOS-positive neuron outside the nominal boundary of the PVN where two boutons (arrowhead) were closely apposed to the cell body. (b′) isosurface 3-D rendered image of (b) highlighting the close bouton association (arrowhead) with the nNOS cell body. The arrows indicate axons passing close by as determined from image (b). (c, c′, d, d′) Deconvolved and isosurface 3-D rendered images from (C) of NTS axonal (arrows) relationship with nNOS neurons. Numerous boutons (arrowhead) were adjacent to the cell bodies. Apparent hole in (c′) is the position of the cell nucleus that does not stain for nNOS so appears empty when viewed as an isosurface structure. Scale bars A = 60 μm; B, C = 120 μm; b = 7 μm; b′ = 9 μm; c, c′ = 8 μm; d = 7 μm, d′ = 9 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Relationship between spinally projecting, GABA neurons and vGLUT2 transporter. (A) Micrograph at Bregma −1.80 mm showing PVN with CTB-labelled presympathetic neurons (green) surrounded by varicosities stained positive for the vGLUT2 transporter (red). (a) Deconvolved image of boxed area in (A). The three presympathetic neurons (1–3) were surrounded by vGLUT2-positive varicosities (arrows). (a′, a″) Isosurface 3-D rendered views suggest some varicosities may be closely associated with the neuronal cell surface but the majority display no relationship with the presympathetic neurons. (B) Coronal section with the PVN defined by orange boutons labelled for vGLUT2 transporter (arrow) passing through GABA interneurons (boxed area, arrowhead). (C) High power of boxed area in (B) illustrating the relationship between two GABA interneurons (arrowhead) and vGLUT2 varicosities (arrows). (c, c′) Deconvolved and isosurface 3-D rendered images to show that the majority of vGLUT2-positive boutons (arrow) surround the GABA interneurons while a small number are closely apposed (arrowhead). Scale bars A, B = 60 μm; a, a″ = 11 μm; C = 11 μm; c, c′ = 5 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Relationship between nNOS neurons, NTS axons and vGLUT2 transporter. (A) Low power micrograph showing vGLUT2 transporter-IR-labelled varicosities (green, arrowhead) surrounding nNOS-IR-positive neurons (orange, arrows). (a) Boxed area (a′) from (A) showing the widespread dense distribution of vGLUT2-positive varicosities relative to an nNOS neuron. The arrows indicate varicosities not associated with the neuron (majority) and those closely apposed (arrowhead). (a′, a″) Deconvolved and isosurface images of nNOS neuron (boxed area a) surrounded by vGLUT2. The arrowheads highlight appositions and arrows indicate non-apposed boutons. (B, C) Relationship between NTS axons (arrows) and vGLUT2 transporter varicosities (arrowhead). (c′, c″) Deconvolved and isosurface 3D-rendered image from box in (C). This shows vGLUT2 transporter co-localised with an NTS bouton (yellow, big arrowhead). A number of vGLUT2 boutons are in close relationship with the NTS axon (small arrowhead). The arrows indicate boutons some distance from this particular axon. Scale bars A–C = 60 μm; a = 20 μm; a′ = 15 μm; a″ = 5 μm; c′ = 6 μm; c″ = 5 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

NTS axonal boutons and expression of the vGLUT2 transporter. (A) NTS axonal boutons (red) within the PVN that express vGLUT2 (blue). The boxed area is expanded in (B). The large arrowhead (A, B, a, b, b′) indicates a possible co-localised axonal bouton while the small arrowheads show NTS (red) only and arrows vGLUT2 (blue) boutons only. (a) Combines a deconvolved and isosurface 3-D rendered view to reveal the relationship between NTS and vGLUT2 expression. Numerous NTS and vGLUT2 boutons are apposed (small arrowheads). A proportion of NTS (asterisk) and vGLUT2 (arrows) boutons show no relationship. The large arrowhead indicates a possible co-localisation of vGLUT2 and NTS axon. (b′, b″) Isosurface 3-D rendered images presented in different planes indicating an NTS bouton that could be expressing vGLUT2 (large arrowhead). The small arrowheads indicate sites where NTS boutons and vGLUT2 transporter are closely associated. However, the majority of vGLUT2 (arrows) and NTS axons (asterisk) are not associated. Scale bars A = 15 μm; B = 10 μm; a = 14 μm; b′ = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Drawing summarising the four main axonal pathways projecting from the NTS to the PVN and surrounding region. Ascending NTS axons project to at least four neuronal targets in and around the PVN that are associated with cardiovascular control: (1) Projections to the spinally projecting neurons (green) within the parvocellular regions of the PVN. (2) Projections to the nNOS-containing magnocellular neurons (blue) of the PVN. (3) Projections to the GABA interneurons (red) surrounding the PVN, which are in synaptic contact (Watkins et al., 2009) with spinally projecting neurons. (4) Projections to the nNOS-containing interneurons (pink) that border the PVN (Watkins et al., 2009). The release of NO from these neurons could affect GABA interneurons and/or spinally projecting neurons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)