Projections from the hypothalamic paraventricular nucleus and the nucleus of the solitary tract to prechoroidal neurons in the superior salivatory nucleus: Pathways controlling rodent choroidal blood flow

Abstract

Using intrachoroidal injection of the transneuronal retrograde tracer pseudorabies virus (PRV) in rats, we previously localized preganglionic neurons in the superior salivatory nucleus (SSN) that regulate choroidal blood flow (ChBF) via projections to the pterygopalatine ganglion (PPG). In the present study, we used higher-order transneuronal retrograde labeling following intrachoroidal PRV injection to identify central neuronal cell groups involved in parasympathetic regulation of ChBF via input to the SSN. These prominently included the hypothalamic paraventricular nucleus (PVN) and the nucleus of the solitary tract (NTS), both of which are responsive to systemic BP and are involved in systemic sympathetic vasoconstriction. Conventional pathway tracing methods were then used to determine if the PVN and/or NTS project directly to the choroidal subdivision of the SSN. Following retrograde tracer injection into SSN (biotinylated dextran amine 3K or Fluorogold), labeled perikarya were found in PVN and NTS. Injection of the anterograde tracer, biotinylated dextran amine 10K (BDA10K), into PVN or NTS resulted in densely packed BDA10K+terminals in prechoroidal SSN (as defined by its enrichment in nitric oxide synthase-containing perikarya). Double-label studies showed these inputs ended directly on prechoroidal nitric oxide synthase-containing neurons of SSN. Our study thus establishes that PVN and NTS project directly to the part of SSN involved in parasympathetic vasodilatory control of the choroid via the PPG. These results suggest that control of ChBF may be linked to systemic blood pressure and central control of the systemic vasculature.

Fig. 1

Line drawing of coronal sections at the levels of PVN (Bregma −2.12 mm), SSN (Bregma −11.00 mm) and NTS (Bregma −13.24 mm and Bregma −14.08 mm), with schematics based on the rat atlas of Paxinos and Watson (Paxinos and Watson, 1994), showing the distribution of PRV+ neurons in rat R3-07, in which the PRV injection was well confined to the right choroid and yielded extensive higher-order labeling in PVN and NTS. Each filled circle represents one PRV+ neuron.

Fig. 2

Illustration of Fluorogold injection sites targeting SSN by microsyringe pressure injection for six cases (RBPH37–RBPH42). Image A shows the cores of the Fluorogold injection sites for RBPH41 and RBPH40. In RBPH41, the injection site is centered on SSN, while for RBPH40 it was more rostral and lateral to that in RBPH41. Image B shows the cores of the Fluorogold injection sites for RBPH39 and RBPH42. Image C shows the cores of the Fluorogold injection sites for RBPH38 and RBPH37. The location and spread of the injection sites in RBPH41, RBPH39, and RBPH38 are very similar. The injection sites and resulting retrograde labeling for these cases, plus five control pressure injection cases for SSN and six iontophoretic injection cases for SSN, are also described in Table 1.

Fig. 3

Images of Fluorogold - labeled neurons from a rat that had survived 6 days after pressure injection of Fluorogold into the right SSN (RBPH41), with the labeled neurons detected by DAB peroxidase-antiperoxidase immunohistochemistry. All images are of coronal sections. Image A shows the Fluorogold injection site into the right superior salivatory nucleus (SSN). Image B presents a low power view through the caudal hypothalamus on the right side of the brain, showing a scattering of FG+ neurons on that side in the paraventricular nucleus (PVN). Image C shows a high power view of the PVN presented in image B, in which the labeled neurons can be seen more clearly. Image D and E show low power views of FG+ neurons at two levels of the nucleus of the solitary tract (NTS) near the obex. FG+ neurons are scattered throughout NTS at these levels. Image F shows a high power view of the NTS presented in image E. The scale bar in B also provides the magnification for D and E. The scale bar in C also provides the magnification for F.

Fig. 4

Illustration of cases in which PVN was targeted by iontophoresis or microsyringe pressure. Image A shows the injection site of a successful case RBP39 and those of three control cases (RBP28, RBP34, and RBP35). Image B shows the injection site of another successful case RBP38 and that of one control case RBP26. Image C shows the injection sites of two control cases, RBP29 and RBP30, at a more rostral level. Image D shows the injection sites of five control cases. Of the five control cases, RBP23 was a pressure injection case, while the remaining four were iontophoretic cases. Image E shows the injection sites for two cases with pressure injection. The injection sites and resulting anterograde labeling for these cases, and additional cases with injections targeting PVN, are also described in Table 2.

Fig. 5

Images of BDA10K+ terminals from a rat that had survived 13 days after an iontophoretic delivery of BDA10K into the right PVN (RBP39), with the labeling detected by the ABC procedure with nickel intensified DAB. All images are of coronal sections. Image A shows the BDA10K injection site into the right PVN. Image B presents a camera lucida drawing of the brain stem at the level of the facial motor nucleus (7) and SSN. The dots represent NOS+ neurons. Images C and D show SSN in adjacent sections in which prechoroidal SSN neurons have been immunostained for NOS (C) and BDA10K+ terminals from PVN neurons visualized by the ABC method (D), respectively. Note that the location of NOS+ neurons within SSN overlaps with the location of BDA10K+ terminals from PVN neurons. Image E shows a high-power view of a section with two-color DAB double labeling to highlight the overlap between NOS+ neurons (visualized by a brown DAB reaction) and BDA10K+ terminals (visualized by a black nickel intensified DAB reaction). Image F is an enlargement of part of image E to show more clearly that BDA10K+ terminals are juxtaposed to NOS+ neurons. The scale bar in C provides the magnification for C and D.

Fig. 6

Illustration of central injection sites for the BDA10K injection cases targeting NTS by iontophoresis or microsyringe. Image A shows the injection site of a successful case (RBP95) and that of a control case (RBP92). Image B shows the injection site of another successful case (RBP94) and those of two control cases. Image C shows the injection sites of one partially successful case (RBP83) and a control case (RBP30). Image D shows the injection sites for three control cases at a more rostral level. Image E show the injection sites for two cases with pressure injection. The injection sites and resulting anterograde labeling for these cases are also described in Table 3.

Fig. 7

Images of BDA10K+ terminals from a rat that survived 11 days after an iontophoretic injection of BDA10K into the right NTS (RBP95). All images are of coronal sections. Image A shows the BDA10K injection site in the right NTS at the level of the obex. Image B shows an intermediate magnification CLSM image of NOS+ neurons (green) and BDA10K+ terminals (red) at the level of the facial somatomotor nucleus and SSN, showing the extensive overlap of NTS terminals with the NOS+ neurons of prechoroidal SSN. Image C shows a low-power view of NOS+ neurons and BDA10K+ fibers and boutons within SSN by using a rendered z-stack obtained by CLSM. Densely distributed BDA10K+ fibers are juxtaposed to the NOS+ neurons. Image D is high-power view centering on one of the NOS+ neurons shown in C. Note that many BDA10K+ boutons can be seen surrounding the dendrites and perikarya of this NOS+ neuron, presumably forming direct synaptic contacts.