Freire et al. Nitric oxide Small hydrophobic molecule with a short half-life and freely diffusible across cell membranes, with several functions in the normal and diseased central nervous system. It is synthesized by a group of enzymes called nitric oxide synthases (NOS). Pain An unpleasant sensation that can range from mild to severe, caused by injury or illness, but sometimes with no apparent etiology, generating anxiety and discomfort. are related to the excessive activation of NMDA receptors, including stroke and amyotrophic lateral sclerosis (Lipton and Rosenberg, 1994). Since NMDA receptor activation is the primary stimulus for NO synthesis, it has been suggested that it might have a role in NMDA-mediated neu- rodegeneration. This has been conﬁrmed in stud- ies of nNOS knockout mice, which are resistant to excitotoxicity following acute ischemic injury (Huang et al., 1994). All three NOS types are dependent on NADPH as a co-factor for their activity (Marletta, 1994). Since the early 1960’s, it has been known that histochemistry to reveal the activity of the oxi- doreductase enzyme nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry is associated with a sub-population of inhibitory neurons in the mammalian CNS (Thomas and Pearse, 1964). Any enzyme of the NADPH-d family catalyzes the transfer of elec- trons from NADPH to the colorless salt nitrob- lue tetrazolium (NBT), producing a colored, water-insoluble formazan dye which is visible through light microscopy (see Scherer-Singler et al., 1983 for details). Though in unﬁxed tis- sue many enzymes show a NADPH-d activity, it was demonstrated that in ﬁxed tissue most of these enzymes are inactivated and only those of the NOS family retain their NADPH-d activity (Dawson et al., 1991; Hope et al., 1991). Accordingly, NADPH-d has been widely used to detect NOS activity in aldehyde-ﬁxed tissue in both the normal (Freire et al., 2005; Sandell, 1986; Wiencken and Casagrande, 2000) and altered CNS (Freire et al., 2007; Stojkovic et al., 1998; Wallace et al., 1996), revealing two distinct types of non- pyramidal cells: a more reactive group, with a Golgi-like appearance (type I cells) and a small and more weakly reactive population (type II cells) (Luth et al., 1994). Both are co-localized with GABA in cortical neurons (Yan et al., 1996). Type I neurons have large, darkly stained cell bod- ies and dendritic trees (Luth et al., 1994) and are present in the brain of all mammals examined, from monotremes to primates (Hassiotis et al., 2005; Yan and Garey, 1997). Type II neurons, con- versely, are weakly stained, with small cell bodies and few or no labeled processes (Luth et al., 1994), and are reported to be especially numerous in the primate brain (Sandell, 1986; Yan and Garey, 1997). Both cell groups constitute an important source of NO in the CNS. The distribution of NADPH-d neurons has already been described in the CNS of several mammalian orders (Barone and Kennedy, 2000; Luth et al., 1994; Mizukawa et al., 1989; Pereira et al., 2000; Wiencken and Casagrande, 2000; Nitric oxide and pain Xiao et al., 1996). In the rat brain, for instance, it was shown that NADPH-d neurons amount to approximately 2% of the whole cortical cell population (Valtschanoff et al., 1993; Vincent and Hope, 1992). NADPH-d/NOS neurons were also detected in the spinal cord (Anderson, 1992; Dun et al., 1993; Marsala et al., 1997; Valtschanoff et al., 1992b), mainly in superﬁcial laminae (Valtschanoff et al., 1992a), with a distribution pattern very similar to PKG (Tao et al., 2000). The development of hyperalgesia in the rat’s hindpaw is followed by nNOS up-regulation in the spinal cord (Lam et al., 1996; Yonehara et al., 1997). In our research paper (Freire et al., 2008), we char- acterized the distribution and morphometric aspects of NADPH-d/NO neurons in the spinal cord of the agouti, a medium-sized terrestrial and burrowing rodent with a widespread distribution over Central and South American Neotropical forests (Silvius and Fragoso, 2003) that rely mostly on a frugivorous diet, usually manipulating seeds with high dexterity (Henry, 1999). We showed that in this species, similar to the rat (Valtschanoff et al., 1992a), NADPH-d/NOS cells are detected mainly in laminae I–IV and X along the entire length of the spinal cord and in the intermediolateral column (IML) of both the thoracic and lumbar segments. In addition, we showed that NOS/NADPH-d neuropil is usually more reactive in the dorsal horn of both the cervi- cal and lumbar enlargements than in the thoracic segments of the agouti. NOS/NADPH-d neu- rons also concentrate around the central canal (lamina X) (Freire et al., 2008), which has been shown to receive input from visceral and cutane- ous noxious inputs (Wall et al., 2002; Willis et al., 1999). Pain information from lamina X ascends to the brain through the dorsal column, blurring the lines separating the “touch” and “pain” pathways (Willis et al., 1999). HYPERALGESIA, PAIN AND NITRIC OXIDE Pain, as a sensation, is notoriously difﬁcult to deﬁne in scientiﬁc terms, mostly due to its affective-emotional component.The International Association for the Study of Pain (IASP) offers the following deﬁnition of pain: “An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage”. Damage is signaled to the brain by specialized high-threshold receptors spread all over the body, both externally and inter- nally and associated with small, slow-conducting unmyelinated ﬁbers. Pain signals enter the brain through neurons located in ganglia in the spi- nal cord and the brainstem. In the spinal cord, nociceptors make synapse with projection neu- Frontiers in Neuroscience www.frontiersin.org September 2009 | Volume 3 | Issue 2 | 177