Delineation of the central melanocortin circuitry controlling the kidneys by a virally mediated transsynaptic tracing study in transgenic mouse model

To examine if brain neurons involved in the efferent control of the kidneys possess melanocortin-4 receptor (MC4-R) and/or tryptophan hydroxylase (TPH). Retrograde tracing pseudorabies virus (PRV)-614 was injected into the kidneys in adult male MC4R-green fluorescent protein (GFP) transgenic mice. After a survival time of 3-7 days, spinal cord and brain were removed and sectioned, and processed for PRV-614 visualization. The neurochemical phenotype of PRV-614-positive neurons was identified using double or triple immunocytochemical labeling against PRV-614, MC4R, or TPH. Double and triple labeling was quantified using microscopy. The majority of PRV-614 immunopositive neurons which also expressed immunoreactivity for MC4R were located in the ipsilateral intermediolateral cell column (IML) of the thoracic spinal cord, the paraventricular nucleus (PVN) of the hypothalamus, and raphe pallidus (RPa), nucleus raphe magnus (NRM) and ventromedial medulla (VMM) of the brainstem. Triple-labeled MC4R/PRV-614/TPH neurons were concentrated in the PVN, RPa, NRM and VMM. These data strongly suggest that central MC4R and TPH are involved in the efferent neuronal control of the kidneys.


INTRODUCTION
As an important determinant of balancing the volume and composition of body fluids, the kidneys play a critical role in the homeostatic control of water and sodium [1][2][3], and understanding the mechanisms of their central control could lead to pharmacological and behavioral therapeutic tools of renal-related disease, e.g. renal hypertension and renal diabetes. It is clear that kidneys are received from the body in the form of circulating hormones (arginine vasopressin (AVP), angiotensin AT1, etc.) [4][5][6][7], fuels (glucose, etc.) [8,9], and other factors from the internal or external stimuli (chronic cold exposure, plasma volume expansion, etc.) [10,11]. Activation of renal sympathetic nerves produces marked changes in renal hemodynamics, tubular ion, water transport and renin secretion [12,13]. In addition, many other sensory, metabolic, and emotional factors also influence renal sympathetic activity or induce the change of renal function, suggesting that between brain and kidneys comprise a complex neural circuit that integrates multiple signals relevant for this change.
A large number of significant scientific reports demonstrated that the melanocortinergic signaling pathway constitutes a major signaling system in the control of energy homeostasis [14][15][16][17][18][19][20][21][22][23][24][25][26][27]. It has been evidenced that the central melanocortin system plays an important role in the regulation of basal plasma insulin levels and glucose tolerance [28][29][30][31][32]. A growing body of literature supports that sympathetic activity are tightly interconnected via hypothalamic melanocortinergic pathways involving the melanocortin-4 receptor (MC4R), a G protein-coupled, seven-transmembrane receptor expressed in the brain [15,[33][34][35]. MC4R-knockout mice, and loss-of-function mutations in MC4R in humans, are associated with severe obesity and hyperinsulinemia, supporting an essential role for the melanocortin system Research Paper: Pathology in the regulation of glucose metabolism [36,37]. It was shown that the renal sympathoexcitatory responses to leptin and insulin are dependent on the MC4R, suggesting an important role for the MC4R in the regulation of renal sympathetic nerve outflow [38]. In addition, MC4R is found in numerous brain nuclei [33,37,[39][40][41][42], including the sites known to be in the autonomic circuitry of the kidney, such as the hypothalamic paraventricular nucleus (PVH), the dorsomedial hypothalamus (DMH), the raphe pallidus (RPa) of the brainstem, etc [43][44][45]. However, the exact neurosubstrate underlying the regulation of renal function by the central melanocortin system has not been well defined. The main objective of this study is to provide direct neuroanatomical evidence for the central melanocortin circuits connecting to renal tissues via autonomic nervous system.

Time course of multisynaptic CNS projections to the kidney
The entire spinal cord and brain were examined for PRV-614 and MC4R-GFP immunoreactivity. Viral immunoreactive labeling was monitored at 1-day intervals, up to 6 days after renal injection. Postinjection intervals shorter than 3 days (1~2 day) did not result in viral labeling of any neurons in the CNS.
After 3 days of renal PRV-614 infection (n = 3, phase I), colocalization of PRV-614 and MC4R-GFP immunopositive neurons was seen and largely confined to At 4 days after kidney injection (n = 3, phase II), PRV-614 immunopositive neurons were detected in the brainstem (e.g., rostral ventrolateral medulla (RVLM), A5 noradrenergic cell region (A5) and nucleus of the solitary tract (NTS), Figure 2) and the paraventricular nucleus of the hypothalamus (PVN) that give rise to direct descending projections to IML of the thoracic spinal cord. The majority of MC4R-GFP/PRV-614 double-labeled neurons were found in the PVN, raphe nuclei (RPa) and NTS, and with a less extent in the RVLM and A5.
At 5 days postinjection (n = 4, phase III), the number of PRV-614 infected neurons in regions shown at 4 days was increased and the infection spread transneuronally to neurons in other regions of the medulla (e.g., nuclei raphes,   Figure  3,4, locus coeruleus (LC)), midbrain (e.g., periaquaductal gray (PAG)) and hypothalamus. Of particular note, colocalization of PRV-614 and MC4R-GFP began to become more widespread. Colocalization was still apparent in the RPa, NTS and PVN as well as in several new regions. Double-labeled neurons were concentrated in the rostral ventromedial medulla (e.g., raphe nuclei, NRM, and NGCα) , Figure 3, 4, the dorsal brainstem (e.g., NTS), the dorsolateral pons (e.g., Barrington's nucleus and SubC), midbrain (e.g., PAG), and hypothalamus (e.g., PVN, LH). In the PVN ( Figure 5), coexpression in all the animals was observed at d5, as well as a large increase in the number of PRV-614-infected neurons, primarily in the ventromedial parvocellular PVN and lateral parvicellular PVN (coexpression in 8% of all PRV-614-infected neurons and 30% of all MC4R neurons in the PVN). The number of singly labeled PRV-614 neurons and the number of colocalized neurons at d5 were increased than that at d4 in the RVMM (coexpression in 12% of all PRV-614-infected neurons and 35% of all MC4R-expressing neurons at d5), in the RPa (coexpression in 12% of all PRV-614-infected neurons and 30% of all MC4R-expressing neurons at d5) and the NTS (coexpression in 10% of all PRV-614-infected neurons and 16% of all MC4R-expressing neurons at d5). The first colocalized neurons were detected in the lateral hypothalamus at d5, and in spite of being small in number, they were observed in all animals (coexpression in 12% of all PRV-614-infected neurons and 20% of all MC4Rexpressing neurons at d5). Areas

Infection of serotonergic neurons following injections of PRV-614 into the kidney
Because a portion of neurons that participate in regulating sympathetic outflow expresses serotonin [46][47][48][49][50], we ascertained whether MC4R-GFP-positive neurons infected after the injection of PRV-614 into the left kidney were serotonergic (5-HT and TPH immunoreactivity positive neurons). Previous studies suggested that TPH-expressing neurons were observed within distinct subdivisions of the CNS, including raphe, VMM, lateral paragigantocellular cell group (LPGi), dorsal raphe (DR), and PVN [51]. In this study, the analysis was limited to the five brain areas containing an appreciable number of PRV-614-infected cells: raphe, VMM, LPGi, DR and PVN. PRV-614/TPH dual-labeled cells and MC4R/PRV-614/TPH triple-labeled cells were present in raphe, VMM, LPGi, and PVN, and the number of dual-labeled cells was much higher than that of triple-labeled cells (Figure 4 and  5). No triple-labeled cells were observed in DR.

DISCUSSION
To date, this is the first study to explore the neurochemical phenotype of the neurons belonging to the central melanocortinergic efferent pathways innervating the kidneys. PRV-614 retrogradely labeled neurons were found in numerous CNS regions [43,45,50,52], but the proportion of these neurons possessing MC4R-GFP were mainly located in IML of the thoracic spinal cord, the PVN of the hypothalamus, and RPa, NRM and VMM of the brainstem which were believed to be related to the control of energy homeostasis [53].
In this study, we focused principally on those PRV-614-infected areas (neurons) that also express MC4R. Transsynaptic tracing by injection of attenuated PRV-614 into the kidneys in MC4R-GFP transgenic mice, coupled with dual or triple-labeling immunohistochemical techniques, reveals a specific subset of dual or triplelabeled MC4R-GFP/PRV-IR neurons in multiple nuclei at various levels of the CNS. The majority of MC4R-GFP/PRV-614 double-labeled neurons were found in the RPa, NRM and VMM, and with a less extent in the RVLM, A5, and LC. Most of the double-labeled PRV-614/TPH neurons were located in the RPa, NRM and VMM. Accordingly, it seems logical that most of the triple-labeled MC4R-GFP/PRV-614/TPH neurons were located in the same brain regions, i.e., RPa, NRM and VMM. The data presented support previous tracing and neurophysiological investigations that the MC4R signaling pathway, constituting a major signaling system in the control of energy homeostasis [15], is functionally distributed both in the hypothalamus and the brainstem [53], suggesting a broad central melanocortinergic neurons involved in kidney control. The PVN is now commonly recognized as an important brain region exerting prominent influences over endocrine and autonomic regulation [52]. A large number of significant scientific reports demonstrated MC4R in the PVN is important in regulating energy balance and sympathetic activity [54]. The existence of double-and triple-labeled neurons in the PVN is consistent with the proposed role of this brain region.
A considerable amount of literature had demonstrated that serotonin and leptin shared a similar signaling mechanism regulating energy balance and glucose homeostasis via actions within the CNS [55]. In the absence of leptin receptors, the renal sympathoexcitatory effects of melanocortin system stimulation were attenuated [38]. 5-HT2CR agonists improved glucose homeostasis via increased production and release of endogenous melanocortin agonists, with subsequent activation of sympathetic preganglionic neurons in the IML via stimulation of MC4R [56] . These data suggested the major rodent brain regions that contain MC4R and TPH, involved in the descending pathways that control the kidneys.
In conclusion, the present results reveal a hierarchical organization of the descending MC4R neuronal circuits controlling the kidney, thus providing the neuroanatomical support for the role of the MC4R, and to a less extent TPH, in the control of kidneys.

Animal care
The transgenic mice carrying green fluorescent protein (GFP) under the control of the melanocortin-4 receptor promoter coupled with the cytomegalovirus (CMV) immediate early enhancer, were first obtained from Dr. Joel Elmquist (UT Southwestern Medical Center) and then bred to generate male and female mice. Genotyping was performed by PCR as described in previous studied [17,57]. All mice used were weighted between 25-30 g and maintained in a standard 12h light-dark cycle with ad libitum access to food and water in a temperaturecontrolled room. All studies involving the animals were performed following the National Guides for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology University, Wuhan, China.

Surgical procedures, PRV-614 injection and postsurgical care
To provide a description of the pattern and temporal progression of viral infection resulting from injection of PRV-614 into the kidney, virus was injected into the left kidneys of mice. PRV-614 is a specific trans-synaptic retrograde tracer from axon terminals to the cell nucleus, where replication occurs [2,27,58]. Under full anesthesia with isoflurane, the skin overlying the kidney was incised by ventral midline laparotomy to expose the upper pole of the kidney [59,60]. Sixteen mice received a series of injections with PRV-614 (2×10 8 pfu/ml in a total of 1 µl per injection at five injection sites per kidney) into the upper pole of the visualized left kidney using a 30-gauge needle connected to a Hamilton syringe (10 µl) under microscopic guidance. After the final injection, the renal surface was rinsed twice with sterile saline-soaked swabs and blotted dry. The kidney was then returned to the abdominal cavity. The abdominal muscle incision was closed with silk sutures, and the skin incision was closed with stainless steel wound clips. The time course of infection was empirically determined by observing the pattern of infection at exactly 3-(n = 3), 4-(n = 3), 5-(n = 4), 6-day (n = 3) and 7-day (n = 3) survival time. The mice were provided with analgesia with an intramuscular injection of a mixture of ketamine (10 mg/ kg) and ketoprofen (3 mg/kg) just before the surgery and every 12 hours subsequently during a post-surgical period of 72 hours. Mice infected with PRV-614 remained asymptomatic until 5-6 days after virus injection, and they were euthanized either before or when they showed apparent traits of illness or distress (i.e. 6-7 days after inoculation).

Perfusion and fluorescence immunohistochemistry
After a survival time of 3-6 days, mice were deeply anesthetized with an overdose of urethane and perfused with 0.9% saline, which was followed by 4% paraformaldehyde-borate fixative (pH 9.5) via the left ventricle. Spinal cord and brain were removed and postfixed in 4% paraformaldehyde-borate overnight at room temperature and in a 30% sucrose solution for 2 days at 4°C. Post-fixed spinal cord and brains were blocked, and sliced at 30 µm/coronal section on a freezing-stage sledge microtome, and collected into four serially ordered sets of sections through its rostrocaudal extent. Tissue sections were stored at 4°C until they were processed for immunohistochemical visualization.
Brain sections were also processed for the triple localization of PRV-614, MC4R-GFP, and TPH. The processing of this tissue was similar to that described above. For TPH IHC, first antibody used for immunohistochemical analysis was sheep anti-TPH (1:2000; Chemicon International, Temecula, CA) and second antibody included biotinylated Donkey antisheep Ig (H+L) (Lot no.68003, Jackson ImmunoResearch Lab., West Grove, PA) and streptavidin Alexa Fluor 350 conjugate (Lot no.49248A, S-11249, Invitrogen, Molecular Probes, Eugene, OR). After the completion of immunohistochemical processing, the sections were mounted on gelatin-coated slides, dehydrated, cleared, and cover slipped with the use of mounting media. In negative control incubations, the primary antiserums were omitted from the immunohistochemical reaction. This procedure completely eliminated neuronal staining.

Tissue analysis
The regions in which infected cells were located were defined with reference to the atlases of Franklin KB and Paxinos G [62] and a study by Molander and colleagues [63] that were used to guide the spinal cord tissue analysis. Immunofluorescence photomicroscope was achieved by using the Leica Microsystems (DM400B, Wetzlar, Germany) with a filter set for visualization of Alexafluor 488 (excitation range, 425-525 nm; emission range, 500-600 nm), Cy3 (excitation range, 500-560 nm; emission range, 560-650 nm) and Alexafluor 350 (excitation range, 300-400 nm; emission range, 440-490 nm). PRV-614 infected neurons are identified with red fluorescence, MC4R-expressing neurons by green fluorescence, and TPH-expressing neurons by blue fluorescence. Images were overlaid using Adobe Photoshop, and double-labeled neurons are presented as yellow (red/green) or pink (red/blue). High-magnification analysis was used to determine whether overlapping yellow or pink images were due to colocalization in the same neuron or overlap of independently labeled neurons.