Dorsal root ganglia (DRGs) contain the cell bodies of primary afferent neurons that convey somatic and visceral input from the body to the spinal cord. Several studies have indicated that primary afferent neurons of the DRGs can be classified in different populations on the basis of their morphological, physiological, and biochemical features (Lawson,1992; Willis and Coggeshall,2004). On the basis of cell body size, the DRG neurons of the bottlenose dolphin (Tursiops truncatus, Montagu 1821) can be categorized into three groups: small (perikaryal area < 1,000 μm2), medium (perikaryal area 1,000–2,000 μm2), and large (perikaryal area > 2,000 μm2). In the bottlenose dolphin, the majority of sensory neurons were large (49.4%), followed by medium (40.8%) and small elements (9.8%). As in other species, small- and medium-sized neurons of the DRGs of the bottlenose dolphin can express substance P (SP). However, SP-immunoreactive (IR) neurons are more represented in bottlenose dolphin DRGs (∼50% of the total primary afferent neurons) than in others mammals (Lawson,1992; Willis and Coggeshall,2004; Bombardi et al.,2010). This data suggest that in the cetaceans SP-IR DRGs neurons could be involved not only in the transmission of nociceptive information but also in the direct regulation of the blood circulation, acting on the vascular tone (Bombardi et al.,2010).
Nitric oxide (NO) is a free radical gas involved in many diverse physiological processes in the nervous system (Ahern et al.,2002; Calabrese et al.,2007). The synthesis of NO is catalyzed by the enzyme nitric oxide synthase (NOS), which converts L-arginine and nicotinamide adenine dinucleotide phosphate (NADPH) into free NO and citrulline. Three NOS isoforms have been identified: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). The nNOS (Type I) is located in a discrete population of neurons, whereas the iNOS (Type II) is expressed by astrocytes in response to inflammatory stimuli. Finally, the eNOS (Type III) is located in the endothelial cells and astrocytes. The activity of both nNOS and eNOS is Ca2+/calmodulin dependent, whereas that of iNOS is Ca2+ insensitive (Garthwaite,1991; Bredt and Snyder,1992; Dawson and Dawson,1996; Moncada et al.,1997; Bredt,1999; Maihöfner et al.,2000; Davis et al.,2001; Ahern et al.,2002). The identification of nNOS or nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) has been employed to localize nitrergic neurons in the nervous system. It has been shown that the occurrence of nNOS is almost completely homotopic with the localization of neurons stained for NADPH-d (Dawson et al.,1991; Vincent and Kimura,1992; Giraldi-Guimarães et al.,1999; Bombardi et al.,2006).
Previous studies have demonstrated NADPH-d-positive or nNOS-IR neurons in the DRGs of different mammalian species (Aimi et al.,1991; Morris et al.,1992; Zhang et al.,1993; Qian et al.,1996; Bergman et al.,1999; Thippeswamy and Morris,2002; Luo et al.,2004; Lukáčová et al.,2006; Tan et al.,2008; Russo et al.,2010). The majority of nitrergic DRG neurons are small to medium sized and also contain the nNOS in their axonal processes (Zhang et al.,1993; Qian et al.,1996; Thippeswamy and Morris,2002). Double immunofluorescence studies demonstrated that many nitrergic neurons express the neuropeptide SP (Aimi et al.,1991; Zhang et al.,1993; Tan et al.,2008; Russo et al.,2010).
NO regulates many functions in the DRGs, including transmission of visceral sensation, primary afferent neurons development and differentiation, communication between neurons and satellite glial cells, and neuroprotection (Aimi et al.,1991; Thippeswamy and Morris,2002; Tan et al.,2008; Russo et al.,2010). In addition, immunohistochemical, electrophysiological, and pharmacological studies have suggested that NO acts as a neurotransmitter or neuromodulator in the processing of nociceptive stimuli. However, the influences of NO upon nociceptive transmission at different levels of the spinal cord are opposing and complex, and the exact sites and mechanisms of these actions are controversial (Meller and Gebhart,1993; Cížkova et al.,2002; Ruscheweyh et al.,2006; Boettger et al.,2007; Ma and Eisenach,2007; Kawano et al.,2009). In fact, both hyperalgesic and analgesic effects of NO were demonstrated at different levels of the neuraxis (Hoheisel et al.,2005). Interestingly, nNOS-containing fibers that originate in the DRGs provide a dense perivascular network around the arterial system (Cao et al.,2009), and NO is believed to act as vasodilator (Holzer et al.,1995). Finally, the possibility that nNOS participates in the pathophysiology of DRGs (Lehmann et al.,2007; Ma and Eisenach,2007) and spinal cord injury (Sharma et al.,1996; Maršala et al.,2007) has been investigated.
Despite the previous studies, no information is known on the exact distribution of nitrergic neurons in the DRGs of the bottlenose dolphin. In this study, immunoperoxidase and double immunofluorescence techniques were used to determine the distribution and the number of nitrergic neurons in the DRGs. As nitrergic neurons of the DRGs contain tachykinins (Aimi et al.,1991; Zhang et al.,1993; Ruscheweyh et al.,2006; Tan et al.,2008; Russo et al.,2010) in others mammals, double immunofluorescence was used to determine if nNOS is colocalized with SP also in Tursiops truncatus.
MATERIALS AND METHODS
Left and right DRGs were obtained from three specimens of bottlenose dolphins obtained from the Mediterranean marine mammal tissue bank of the University of Padova (Italy). The tissue was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS), pH 7.4, for at least 24 hr. After rinsing in PBS (pH 7.4) for 12 hr, DRGs were cryoprotected in 30% sucrose solution in PBS (pH 7.4) at 4°C for at least 1 week. Thereafter, DRGs were transferred to a mixture of PBS–sucrose and OCT compound (Tissue Tek; 4583; Sakura) at a ratio 1:1 for 24 hr before being embedded in 100% OCT. To obtain cryosections, the DRGs were frozen in isopentane and liquid nitrogen and then subsequently cut longitudinally with a cryostat. We obtained 15-μm-thick sections that were mounted in gelatin-coated glass slides. We have analyzed the left and right thoracic (T1, T5, T9, and T12), lumbar (L1, L5, L9, L13, and L16), and caudal (Ca1, Ca5, Ca9, Ca13, Ca17, Ca21, and Ca25) DRGs of each subject.
After washing in PBS (three times for 10 min each), sections (10 sections for each ganglion) were incubated with 1% H2O2 in PBS for 30 min at room temperature (T) to eliminate endogenous peroxidase activity. Sections were rinsed in PBS (three times for 10 min each) and incubated in a solution containing 10% normal goat serum (Colorado Serum, Denver, CO, #CS 0922) and 0.5% Triton X-100 (Merck, Darmstadt) in PBS for 2 hr at room temperature to block nonspecific binding. Thereafter, the sections were incubated in a solution containing mouse monoclonal antibody anti-nNOS (1:500; sc-5302; Santa Cruz Biotechnology, CA) for 48 hr at 4°C. The primary antibody was diluted in a solution (1.8% NaCl in 0.01 M PBS containing 0.1% sodium azide) containing 1% normal goat serum and 0.5% Triton X-100. After washing in PBS (three times for 10 min each), the sections were incubated in goat biotinylated anti-mouse (1:200; BA-9200, Vector Laboratories, Burlingame, CA) for 2 hr at room temperature. The secondary antibody was diluted in PBS containing 1% normal goat serum and 0.5% Triton X-100. The sections were then transferred to avidin–biotin complex (ABC kit Vectastain, PK-6100, Vector Laboratories, Burlingame, CA) for 45 min, and the immunoperoxidase reaction was developed by 3,3′-diaminobenzidine (DAB kit, SK-4100, Vector Laboratories, Burlingame, CA). Slides were dried overnight, dehydrated in ethanol, cleared in xylene, and coverslipped with Entellan (Merck, Darmstaldt, Germany). All the incubations were performed in a humid chamber.
For double-label immunofluorescence staining, 10 sections (different from those used in immunoperoxidase experiments) for each ganglion were rehydrated in 0.01 M PBS and then processed for immunostaining. To block nonspecific binding, the sections were preincubated for 2 hr in a solution containing 10% normal goat serum (Colorado Serum, Denver, CO, #CS 0922) and 0.5% Triton X-100 in PBS. Thereafter, the sections were incubated for 48 hr at 4°C with a specific primary antibodies solution described in the following. As the neurofilament 200 kDa is virtually expressed by every DRG neuron of the bottlenose dolphin (Bombardi et al.,2010), we used this protein as pan-neuronal marker to determine the percentage of nNOS-IR neurons. In particular, we colocalized mouse monoclonal antibody anti-nNOS (1:100; sc-5302; Santa Cruz Biotechnology, CA) with rabbit polyclonal antibody anti-NF 200 kDa (1:2,000; N4142; Sigma, Saint Louis, MO). To reveal the colocalization of nNOS with SP, the sections were incubated in a cocktail of mouse monoclonal antibody anti-nNOS (1:100; sc-5302; Santa Cruz Biotechnology, CA) and rat monoclonal antibody anti-SP (1:400; 10-S15; Fitzgerald). All the primary antibodies were diluted in a solution (1.8% NaCl in 0.01 M PBS containing 0.1% sodium azide) containing 1% normal goat serum and 0.5% Triton X-100. Sections were then rinsed in three changes of PBS (10 min each) and then incubated or in a cocktail of goat anti-mouse Alexa-488 (1:100; #A11029; Molecular Probes, Leiden, the Netherlands) and goat anti-rabbit Alexa-594 (1:100; #A11037; Molecular Probes, Leiden, the Netherlands) or in a cocktail of goat anti-mouse Alexa-488 (1:100; #A11029; Molecular Probes, Leiden, the Netherlands) and donkey anti-rat Alexa-594 (1:100; #A21209; Molecular Probes, Leiden, the Netherlands). All the secondary antibodies were diluted in PBS containing 1% normal goat serum and 0.5% Triton X-100. After washing in PBS (three times for 10 min each), the sections were coverslipped with buffered glycerol (pH 8.6). All the incubations were performed in a humid chamber.
Procedures of Staining Control
In the present experiments, control sections incubated without the primary antibodies resulted in a complete disappearance of stained profiles. The omission as well as the replacement of the secondary antibodies with inappropriate secondary antibodies resulted in elimination of all immunohistochemical staining. The specificity of the rabbit polyclonal antibody anti-NF 200 kDa was determined in immunoperoxidase, immunofluorescence, and immunoblotting studies conducted by the manufacturer, which tested this antibody in a wide range of species. The SP is highly conservative in mammals, and the specificity of antigen targeting has been evaluated by Western blot at the origin. Specificity of the mouse anti-nNOS was obtained by detecting a NADPH-d histochemical reaction in sections initially stained against nNOS. In particular, five sections for each ganglion were incubated in 0.1 M Tris buffer (pH 8.0) containing 1 mM β-NADPH (Sigma N-1630), 0.1 mM nitroblue tetrazolium (Sigma N-6876), and 0.3% Triton X-100 for 1 hr at 37°C. After washing in 0.3% Triton X-100 in Tris-buffered saline, the sections were dried overnight, dehydrated in ethanol, cleared in xylene, and coverslipped with Entellan (Merck, Darmstaldt, Germany). The NADPH-d reaction and the mouse anti-nNOS marked the same somata. Incubation without the substrate NADPH or without the electron acceptor NBT reduced NADPH-d staining. This aspect demonstrated the specificity of NADPH-d histochemical staining.
Analysis of the Sections
The sections were observed with a Zeiss Axioplan microscope (Carl Zeiss, Oberkochen, Germany) equipped with the appropriate filter cubes for immunofluorescence. We used filter set 10 for Alexa 488 (450- to 490-nm excitation filter and 515- to 565-nm emission filter) and filter set 00 for Alexa 594 (530- to 585-nm excitation filter and 615-nm emission filter). Images were recorded by using a Polaroid DMC digital photocamera (Polaroid Corporation, Cambridge, MA) and DMC2 software. Contrast and brightness images were adjusted by using Adobe Photoshop CS3 Extended 10.0 software (Adobe Systems, San Jose, CA). KS 300 Zeiss software (Kontron Elektronik, Germany) was used for morphometric analysis of the nNOS-IR neurons located in the thoracic, lumbar, and caudal right and left DRGs. Only the neurons with an evident nucleus were included in the perikaryal area analysis of the immunoperoxidase preparations. The perikaryal areas of all nNOS-IR cell bodies of five nonconsecutive sections obtained from each ganglion (of each animal) were measured after manual tracing of the cell bodies outline. Data were expressed as mean ± standard deviation (SD).
The proportion of nitrergic neurons to the total DRG neurons and the colocalization of nNOS with SP were assessed using double immunofluorescence techniques. In each DRG (of each subject), the percentage of single- and double-immunolabeled cells was calculated in 10 sections counting all immunoreactive cell bodies (five nonconsecutive sections to colocalize NF 200 kDa with nNOS and five nonconsecutive sections to determine whether there was colocalization of nNOS and SP). Cell counting was carried out with a 40× magnification. Only neuronal profiles with a visible nucleus were considered in each section. For immunofluorescence analysis, the cells were first spotted by the presence of a fluorophore, which labeled one antigen, and then the filter was switched to a fluorophore specific for a different wavelength to determine whether or not the neuron was labeled also for a second antigen. In this way, the proportions of neurons labeled for pairs of antigens or for a single antigen were determined.
nNOS-IR cell bodies and nerve fibers were detected in every DRG, in which they appeared evenly distributed (Fig. 1A–C). The immunoreaction product was located at the intracellular level, but the nuclear region was unlabeled (Fig. 1D). The intensity of immunostaining was similar in thoracic, lumbar, and caudal DRGs and varied from moderate to strong. The cell bodies of the nitrergic neurons had a mean area of 1298.6 μm2 ± 296.1 (SD) (N = 924; minimum area 489.4 μm2 and maximum area 2399.2 μm2). Morphometric analysis revealed that small neurons (perikaryal area < 1,000 μm2) constituted 14.9% (N = 138), medium-sized neurons (perikaryal area 1,000–2,000 μm2) 84% (N = 776), and large neurons (perikaryal area > 2,000 μm2) 1.1% (N = 10) of the total number (N = 924) of nNOS-IR neurons considered. Interestingly, the mean size of the immunoreactive cell bodies remained constant throughout the ganglia. Double immunofluorescence analysis revealed that about 9% of the DRG neuronal population were immunoreactive for nNOS (Fig. 2; Table 1). The distribution of nNOS-IR neurons varied at different spinal levels, being the lowest in the caudal DRGs (Table 1). Double-label immunofluorescence staining showed that the great majority (81.3%; 870 double-labeled neurons/1,070 nNOS-IR neurons) of nNOS-IR neurons contained SP (Fig. 3). On the contrary, only 12.6% (870 double-labeled neurons/6,912 SP-IR neurons) of SP-IR neurons were nNOS positive (Fig. 3). The distribution of double-immunopositive cells was similar in the different DRGs. However, their number decreased at levels posterior to the 17th caudal spinal segment. Double-immunolabeled cells were sparsely scattered throughout every DRG. In each double immunofluorescence experiment, analysis revealed a similar percentage of colocalized neurons in all bottlenose dolphin used.
Table 1. Percentage of nNOS-immunoreactive neurons in the dorsal root ganglia
This study demonstrated that a subpopulation of primary afferent neurons located in the DRGs of the bottlenose dolphin exhibit immunoreactivity for the nNOS. Previous studies have demonstrated nitrergic neurons in the DRGs of different species including the mouse (Tan et al.,2008), rat (Aimi et al.,1991; Morris et al.,1992; Zhang et al.,1993; Bergman et al.,1999; Thippeswamy and Morris,2002), sheep (Luo et al.,2004; Russo et al.,2010), dog (Lukáčová et al.,2006), and monkey (Zhang et al.,1993). Several studies performed on the rat DRGs (Aimi et al.,1991; Morris et al.,1992; Zhang et al.,1993; Bergman et al.,1999; Thippeswamy and Morris,2002) indicated that the nitrergic neurons show a peculiar distribution pattern in relation to spinal levels. In fact, these neurons decreased in number from the middle to the more caudal spinal levels. Similarly to the results obtained in the rat, the lowest percentage of nitrergic neurons was located in the caudalmost DRGs also in the bottlenose dolphin. However, the nitrergic neurons of this latter species appeared more homogenously distributed throughout the different spinal levels than in the rat. As observed in other mammals (Thippeswamy and Morris,2002; Lukáčová et al.,2006; Russo et al.,2010), nitrergic neurons of the DRGs belonged especially to small- and medium-sized cell classes also in our bottlenose dolphin series. The present double immunofluorescence data agree with what has been reported in the mouse (Tan et al.,2008) and rat (Aimi et al.,1991; Ruscheweyh et al.,2006), in which the majority of nNOS-IR DRG neurons contained the SP. Superficial laminae are a major site for primary afferent neurons containing nNOS (Meller and Gebhart,1993; Thippeswamy and Morris,2002) and SP (Barber et al.,1979; Hunt et al.,1981; Willis and Coggeshall,2004). In fact, the influence of NO on nociceptive transmission may suggest a possible role for this molecule in the modulation of the response of thoracic, lumbar, and caudal dorsal horns to SP.
The number of nitrergic neurons was slightly higher in the thoracolumbar than in the caudal levels. The presence of nNOS-IR neurons in the DRGs of the thoracolumbar levels indicates also the possibility that NO could be implicated in the transmission of visceral afferent information. Double immunofluorescence staining demonstrated that most of the primary afferent neurons in the thoracolumbar DRGs showed intense SP immunoreactivity, suggesting that the interaction between NO and SP could regulate the visceral neurotransmission. This is consistent with evidence from a recent study suggesting that the majority of visceral afferents from the mouse intestine are provided by primary afferent neurons where nNOS is extensively colocalized with SP (Tan et al.,2008). Furthermore, the presence of SP in many visceral afferent neurons is well documented in many species (Green and Dockray,1987; Wang et al.,1998; Dinh et al.,2004).
Cetaceans have anatomical and physiological features that reflect an adaptation to life in the water. In particular, the circulatory system of cetaceans is characterized by a diffuse network of anastomotic vessels establishing a perispinal retia mirabilia. These structures act as huge blood reservoirs that regulate the oxygenation of the vital organs during diving (McFarland,1979; Pfeiffer and Kinkead,1990; Melnikov,1997). The abdominal cavity of cetaceans contains also countercurrent vascular heat exchange systems associated to the reproductive systems that regulate the temperature of the blood flow to the uterus (Rommel et al.,1993) and the testes (Rommel et al.,1992). Ganglion neurons are generally considered as purely afferent neurons that convey sensory information from the periphery to the spinal cord. However, they also hold the capacity to act in an efferent manner releasing NO (Cao et al.,2007,2009) and some neuropeptides, such as SP, from their peripheral processes (Holzer and Maggi,1998). Interestingly, a dense network of nerves containing nNOS (Cao et al.,2009) and SP (Supowit et al.,2005) was located around the blood vessels. As NO (Holzer et al.,1995; Cao et al.,2009) and SP (Pernow,1983; Supowit et al.,2005) can play an important role in the regulation of vasodilation, nNOS-IR single-labeled and nNOS/SP-IR double-labeled neurons located at the thoracic and lumbar levels may be involved in the control of blood flow in the different retia mirabilia and in the countercurrent vascular heat exchange systems. In the caudal region of the spinal cord, the number of nitrergic neurons of the DRGs decreased but remained significant. As ganglion nitrergic neurons of the caudal DRGs could not provide an extensive visceral innervation, their presence at this level could be related to the regulation of the blood flow in the retia mirabilia that surround the caudalmost tract of the spinal cord.
The authors thank the Mediterranean marine mammal tissue bank of the University of Padova for providing specimens of the spinal cord of bottlenose dolphins.