The caudal pedunculopontine tegmental nucleus may be involved in the regulation of skeletal muscle activity by melanocortin-sympathetic pathway: a virally mediated trans-synaptic tracing study in spinally transected transgenic mice

Abstract

Understanding neuroanatomical sympathetic circuitry and neuronal connections from the caudal pedunculopontine tegmental nucleus to skeletal muscle is important to the study of possible mechanisms of pedunculopontine tegmental nucleus (PPTg) and cuneiform nucleus (CnF) that are involved in the regulation of skeletal muscle activity of the sympathetic pathway. The aim of this study was to use virus PRV-614 to trace the melanocortin-sympathetic neural pathways from PPTg and CnF to a hindlimb muscle (gastrocnemius) in spinally transected MC4R-GFP transgenic mice. PRV-614 was injected into the gastrocnemius muscle after receiving a complete spinal cord transection below the L2 level. PRV-614/MC4R-GFP and PRV-614/TPH dual-labeled neurons were found in the dissipated parts of PPTg (dpPPTg), but not between the compact parts of PPTg (cpPPTg) and CnF. It is proposed that a hierarchical pathway of neurons within the caudal pedunculopontine tegmental nucleus sends projections to the RVLM, which in turn projects onto the IML sympathetic preganglionic neurons that regulate muscle blood flow through melanocortin-sympathetic signals. Our results collectively indicate that MC4Rs expressed in caudal pedunculopontine tegmental nucleus may be involved in skeletal muscle activity of melanocortin-sympathetic pathways.

Keywords: melanocortin-4 receptor; mouse; pedunculopontine tegmental nucleus; pseudorabies virus; skeletal muscle.

Figure 1. Micrographs illustrating sections through the CnF and caudal levels of the edunculopontine tegmental nucleus (PPTg)

(A) Sections immunoreacted for PRV-614 (red). (B) Sections immunoreacted for MC4R-GFP (green). (C) Sections immunoreacted for TPH (blue). (D) Sections immunoreacted for PRV-614/MC4R-GFP dual labeled neurons (yellow, some are indicated by white arrows). (E) Sections immunoreacted for PRV-614/TPH dual labeled neurons (pink, some are indicated by white arrows). After a complete spinal cord transection, injections of PRV-614 into the gastrocnemius muscle resulted in retrograde infection of neurons in the CnF, cpPPTg and dpPPTg, and PRV-614/MC4R-GFP and PRV-614/TPH dual labeled neurons were detected in the dpPPTg. In contrast to the dpPPTg, we didn't detect dual labeled neurons in the CnF and cpPPTg. PRV-614, pseudorabies virus-614; CnF, cuneiform nucleus; Aq, aqueduct; DR, dorsal raphe; dpPPTg, the dissipated parts of PPTg; cpPPTg, the compact parts of PPTg; TPH, tryptophan hydroxylase. Scale bar: 50 μm.

Figure 2

Gastrocnemius muscle cell groups target PRV-614 positive neurons (red) of the rostral ventral medulla (A) and spinal cord (B) levels. (A1 and B1) Sections immunoreacted for MC4R-GFP (green). (A2 and B2) Sections immunoreacted for PRV-614 (red). (A3 and B3) Sections immunoreacted for PRV-614/MC4R-GFP dual labeled neurons (yellow, some are indicated by white arrows). After a complete spinal cord transection, injections of PRV-614 into the gastrocnemius muscle resulted in retrograde infection of neurons in the RVLM, RVMM, and IML. In contrast to the IML, we didn't detect labeled neurons in the VH in spinally transected transgenic mice. PRV-614, pseudorabies virus-614; IML, intermediolateral column; Py, pyramidal tract; RVLM, rostral ventrolateral medulla; RVMM, rostral ventromedial medulla; VH, ventral horn. Scale bar: 50 μm.

Figure 3. Summary diagram showed that the melanocortin-sympathetic pathway between the dpPPTg and skeletal muscle

Neurons within the dpPPTg send projections to the rostral ventral medulla (e.g., RVLM premotor neurons), which in turn projects to the sympathetic preganglionic neurons within the IML, which control skeletal muscle activity regulated by melanocortin- sympathetic signals. PRV-614, pseudorabies virus-614; dpPPTg, the dissipated parts of PPTg; IML, intermediolateral column; RVLM, rostral ventrolateral medulla; RVMM, rostral ventromedial medulla. Some drawings were taken from HB Xiang (Brain 2013).