Nitrergic Proprioceptive Afferents Originating from Quadriceps Femoris Muscle are Related to Monosynaptic Ia-Motoneuron Stretch Reflex Circuit in the Dog

1. The aim of the present study was to examine the occurrence of the neuronal nitric oxide synthase immunoreactivity in the stretch reflex circuit pertaining to the quadriceps femoris muscle in the dog.2. Immunohistochemical processing for neuronal nitric oxide synthase and histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase were used to demonstrate the presence of neuronal nitric oxide synthase in the proprioceptive afferents issuing in the quadriceps femoris muscle. The retrograde tracer Fluorogold injected into the quadriceps femoris muscle was used to detect the proprioceptive afferents and their entry into the L5 and L6 dorsal root ganglia.3. A noticeable number of medium-sized intensely nitric oxide synthase immunolabelled somata (1000–2000 μm² square area) was found in control animals in the dorsolateral part of L5 and L6 dorsal root ganglia along with large-caliber intraganglionic nitric oxide synthase immunolabelled fibers, presumed to be Ia axons. Before entering the dorsal funiculus the large-caliber nitric oxide synthase immunolabelled fibers of the L5 and L6 dorsal roots formed a massive medial bundle, which upon entering the dorsal root entry zone reached the dorsolateral part of the dorsal funiculus and were distributed here in a funnel-shaped fashion. The largest nitric oxide synthase immunolabelled fibers, 8.0–9.2 μm in diameter, remained close to the dorsal horn, while medium-sized fibers were seen dispersed across the medial portion of the dorsal funiculus. Single, considerably tapered nitric oxide synthase immunolabelled fibers, 2.2–4.6 μm in diameter, were seen to proceed in ventrolateral direction until they reached the mediobasal portion of the dorsal horn and the medial part of lamina VII. In lamina IX, only short fragments of nitric oxide synthase immunoreactive fibers and their terminal ramifications could be seen. Nitric oxide synthase immunolabelled terminals varying greatly in size were identified in control material at the base of the dorsal horn, in the vicinity of motoneurons ventrally and ventrolaterally in L5 and L6 segments and in Clarke’s column of L3 and L4 segments. Injections of the retrograde tracer Fluorogold into the quadriceps femoris muscle and cut femoral nerve, combined with nitric oxide synthase immunohistochemistry of the L5 and L6 dorsal root ganglia, confirmed the existence of a number of medium-sized nitric oxide synthase immunoreactive and Fluorogold-fluorescent somata presumed to be proprioceptive Ia neurons (1000–2000 μm² square area) in the dorsolateral part of both dorsal root ganglia. L5 and L6 dorsal rhizotomy caused a marked depletion of nitric oxide synthase immunoreactivity in the medial bundle of the L5 and L6 dorsal roots and in the dorsal funiculus of L5 and L6 segments.4. The analysis of control material and the degeneration of the large- and medium-caliber nitric oxide synthase immunoreactive Ia fibers in the dorsal funiculus of L5 and L6 segments confirmed the presence of nitric oxide synthase in the afferent limb of the monosynaptic Ia-motoneuron stretch reflex circuit related to the quadriceps femoris muscle. Abbreviations ABC, avidin–biotin complex; bNOS, neuronal nitric oxide synthase; bNOS-IR, neuronal nitric oxide synthase immunoreactive; bNOS-IRB_s, neuronal nitric oxide synthase immunoreactive boutons; cNOS, catalytic nitric oxide synthase; DAB, diaminobenzidine; DF, dorsal funiculus; DH, dorsal horn; DREZ-one, dorsal root entry zone; DRG_s, dorsal root ganglia; eNOS, endothelial nitric oxide synthase; FG, Fluorogold; FN, femoral nerve; mNOS, macrophage nitric oxide synthase; NADPHd, nicotinamide adenine dinucleotide phosphate diaphorase; NBT, nitroblue tetrazolium; NO, nitric oxide; NOS, nitric oxide synthase; NOS-IR, nitric oxide synthase immunoreactive; PBS, phosphate-buffered saline; VGLUT1 and VGLUT2, vesicular glutamate transporters