Localization of nitric oxide synthase in rat trigeminal primary afferent neurons using NADPH-diaphorase histochemistry I. I. Stoyanova* & N. E. Lazarov Department of Anatomy, Faculty of Medicine, Thracian University, 11 Armejska Street, P.O. Box 1025, 6010 Stara Zagora, Bulgaria *Author for correspondence (e-mail: stoyanovai@yahoo.co.uk) Received 13 September 2004 and in revised form 2 February 2005 Summary Nitric oxide (NO) is a ubiquitous gaseous neurotransmitter that has been ascribed to a large number of physiological roles in sensory neurons. It is produced by the enzyme nitric oxide synthase (NOS). To identify the NOS-containing structures of rat trigeminal primary afferent neurons, located in the trigeminal ganglion (TrG) and mesencephalic trigeminal nucleus (MTN), histochemistry to its selective marker nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) was applied in this study. In the TrG approximately half of the neuronal population was NADPH-d reactive. Strongly positive were neurons mainly of small-to-medium size. Neuronal profiles of large diameter were less intensely stained. In addition, NADPH-d-positive nerve fibers were dispersed throughout the ganglion. Nitrergic neurons were located in the caudal part and mesencephalic-pontine junction of the MTN. Most of them were large-sized pseudounipolar cells. In a more rostral aspect, the reactive psedounipolar MTN profiles gradually decreased in number and intensity of staining. There, only a fine meshwork of stained thin fibers and perisomatic terminal arborizations, and also some isolated perikarya of NADPH-d stained multipolar MTN neu- rons, were observed. The predominant NADPH-d localization in smaller in size TrG neurons, which are considered nociceptive, suggests that NO may play a role in the pain transmission in the rat trigeminal afferent pathways. In addition, the wide distribution of NADPH-d activity in large pseudounipolar and certain multipolar MTN neurons provides substantial evidence that NO may also participate in mediating proprioceptive information from the oro- facial region. The differential expression patterns of nitrergic fibers in the TrG and MTN suggest that trigeminal sensory information processing is controlled by nitrergic input through different mechanisms. Introduction The identification of nitric oxide (NO) as a neuronal chemical messenger raised a particular interest because of its chemical identity, wide distribution and broad spectrum of effects (Moncada et al. 1991, Batten et al. 2000). NO is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). Currently, there exist at least three main isoforms of NOS, which are encoded by different genes and have a different molecular weight. Two of them are constitutive (neuronal and endothelial NOS) and one is inducible. The formation of neuronal NOS (nNOS) is Ca2+/calmodulin-dependent and requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen as co-sub- strates (Bredt & Snyder 1992). Besides, the fixation of tissue with paraformaldehyde appears to inactivate all NADPH-dependent oxidative enzymes, except for NOS and, therefore, NADPH-diaphorase (NADPH-d) selectively staines NOS neurons (Bredt et al. 1991, Dawson et al. 1991). In most cases NADPH-d activity completely overlaps with nNOS-immunoreactivity, and thus, this enzyme may be considered as a reliable histochemical marker for the in situ identification of nNOS in neurons (Bredt et al. 1990, Dawson et al. 1991). Therefore, the NADPH-d histochemistry after aldehyde fixation may be used as an indirect indicator of NO production; hence NADPH-d-positive neurons in this study will be referred to as ‘‘nitrergic neurons’’. Orofacial sensation is principally derived from tri- geminal innervation (for a review, see Light 1992). The trigeminal sensory system is unique because it comprises two populations of primary afferent neurons, the majority of which are located in the trigeminal ganglion (TrG), and the rest are situated in the CNS, in the me- sencephalic trigeminal nucleus (MTN; Lazarov 2002). The TrG mainly innervates mechanoreceptors, thermo- receptors and nociceptors in the orofacial region (Davies 1988), while the MTN supplies proprioceptors in the masticatory (Walberg 1984) and extrinsic ocular muscles J Mol Hist (2005) 36: 187–193 Ó Springer 2005 DOI 10.1007/s10735-005-1694-3