An atlas of brain-bone sympathetic neural circuits in mice

Abstract

There is clear evidence that the sympathetic nervous system (SNS) mediates bone metabolism. Histological studies show abundant SNS innervation of the periosteum and bone marrow-these nerves consist of noradrenergic fibers that immunostain for tyrosine hydroxylase, dopamine beta-hydroxylase, or neuropeptide Y. Nonetheless, the brain sites that send efferent SNS outflow to the bone have not yet been characterized. Using pseudorabies (PRV) viral transneuronal tracing, we report, for the first time, the identification of central SNS outflow sites that innervate bone. We find that the central SNS outflow to bone originates from 87 brain nuclei, sub-nuclei, and regions of six brain divisions, namely the midbrain and pons, hypothalamus, hindbrain medulla, forebrain, cerebral cortex, and thalamus. We also find that certain sites, such as the raphe magnus (RMg) of the medulla and periaqueductal gray (PAG) of the midbrain, display greater degrees of PRV152 infection, suggesting that there is considerable site-specific variation in the levels of central SNS outflow to the bone. This comprehensive compendium illustrating the central coding and control of SNS efferent signals to bone should allow for a greater understanding of the neural regulation of bone metabolism, and importantly and of clinical relevance, mechanisms for central bone pain.

Keywords: bone pain; brain-bone connection; central regulation of bone metabolism; medicine; mouse; viral tracing.

Figure 1.. PRV152 transneuronal viral tract tracing.

(A) As a control for viral injection, no expresses enhanced green fluorescent protein (EGFP) signal was detected in the paraventricular nucleus (PVH), known to possess main sympathetic pre-autonomic neurons, and the RPa, when PRV152 was placed on the bone surface. (B) By contrast, PRV152 injections into the periosteum or metaphyseal bone resulted in positive EGFP immunoreactivity in the PVH. In addition, we found PRV152-infected neurons in the intermediolateral cell column (IML) of the spinal cord at T13-L2 levels, suggesting specific bone-sympathetic nervous system (SNS) ganglia-IML-brain route of infection which are in concordance with our previous findings where PRV152 individually infected the classic SNS spinal cord neurons. Also shown are representative microphotographs illustrating PRV152 immunolabeling in the PAG (midbrain and pons), RPa (medulla), LH (hypothalamus), MPOM (forebrain), S1HL (cerebral cortex), and pv (thalamus). PVH, paraventricular hypothalamic nucleus; PAG, periaqueductal gray; RPa, raphe pallidus; LH, lateral hypothalamus; MPOM, medial preoptic nucleus, medial part; S1HL, primary somatosensory cortex, hindlimb region; pv, periventricular fiber system. Scale bar = 50 µm. Also shown is a representative low-magnification image at the hypothalamus neuroanatomical level (scale bar = 500 µm).

Figure 2.. PRV152 immunolabeling in brain regions, sub-regions, and nuclei.

Numbers, and heat map representation of PRV152-labeled neurons in brain regions, namely, hypothalamus, midbrain and pons, medulla, forebrain, cerebral cortex, and thalamus, as well as their sub-regions and nuclei, following viral injections into bone. n=4. Statistics: Mean ± SEM, two-tailed Student’s t-test, *p<0.05, **p<0.01, ***p<0.001, ns (no significance).

Figure 3.. Diagrammatic outline of the sympathetic nervous system (SNS) brain–bone neuroaxis relevant to pain.

The central SNS brain-bone circuit starts in the hypothalamic paraventricular nucleus (PVH) known to home SNS pre-autonomic neurons projecting to the SNS neurons of the periaqueductal gray (PAG) in the midbrain. From the PAG the SNS outflow is further relayed to the raphe pallidus-raphe magnus (RPa-RMg) neurons that are terminated in the dorsal horn of spinal gray matter, where they regulate the release of enkephalins that inhibit pain sensation by attenuating substance P (SP) release. In turn, opiates produce antinociception via the µ-opiate receptors, in part, through modulation of responses to SP. Neurons in the RMg are involved in the central modulation of noxious stimuli, therefore, the RMg-PAG could be the part of the ascending hierarchical circuit relating to the perception of bone pain.