猪食管神经支配的神经化学研究——一氧化氮类及肽类成分在平滑肌中的分布(英文)

采用免疫荧光组织化学技术及迷走神经切断术,探讨猪食管一氧化氮类及肽类神经支配的神经化学特性。在光学显微镜下可观察到肌间神经丛及粘膜下神经丛中有部分神经元呈nNOS、VIP、GAL、NPY、PACAP、L-ENK、SP、5-HT及CB免疫阳性,但未见CGRP及SOM阳性神经元。nNOS及CB免疫阳性产物主要分布于不同的神经元胞体内。将PGP9.5作为神经元胞体的标记物,并采用免疫荧光免疫组织化学双重染色方法,分别观察了PGP9.5与nNOS、VIP、SP的双标情况。结果如下:(1)nNOS免疫阳性神经元约占PGP9.5标记神经元总数的63%,而VIP免疫阳性神经元约占36%,SP免疫阳性神经元约占28%;(2)神经节内神经元的平均数量呈现吻尾方向的递增趋势,且食管腹段神经丛内神经节数量明显高于食管其他部位;(3)食管肌层内VIP/GAL/NPY免疫阳性纤维分布最广,其中部分阳性纤维同时呈nNOS或PACAP免疫阳性;SP和/或L-ENK免疫阳性纤维在粘膜肌层的分布明显多于平滑肌层。CGRP阳性纤维非常少见,这一点不同于对其他动物的观察结果;(4)经一侧迷走神经切断后,肌间神经丛内PACAP及5-HT免疫阳性纤维明显减少,提示这些纤维可能来源于迷走神经;而平滑肌中VIP/GAL/NPY和/或nNOS免疫阳性纤维数量未发现明显变化,可能为内源性来源。     

苦参碱注射液对AD模型大鼠脑组织白介素1β、NO含量及nNOS免疫阳性神经元的影响

为探讨苦参碱注射液对老年性痴呆(AD)模型大鼠脑组织白介素1β(IL-1β)、NO含量及nNOS免疫阳性神经元的影响以及药物的作用效果及机制,本研究用SD大鼠将鹅膏蕈氨酸(IBO)双侧海马立体定位注射造模,实验分为对照组、高剂量苦参碱治疗组、低剂量苦参碱治疗组、模型组和哈伯因组。检测大鼠大脑皮质和海马内IL-1β、NO的含量变化以及nNOS免疫阳性神经元的形态改变。结果显示:模型组大鼠大脑皮质和海马内IL-1β及NO的含量均显著高于对照组(P<0.01);高剂量治疗组大脑皮质和海马内IL-1β及NO的含量均低于模型组(P<0.05);模型组nNOS免疫阳性神经元有明显变性、数目减少;高剂量治疗组大鼠nNOS免疫阳性神经元的数目与模型组比较明显增多(P<0.05),变性有所减轻;但低剂量组的nNOS免疫阳性神经元数与模型组没有显著差异(P>0.05)。本文结果提示,模型组大鼠大脑皮质和海马内IL-1β及NO的含量均显著增高,可能为导致AD神经元损伤的原因之一,苦参碱注射液对AD神经元损伤的保护作用可能是通过下调IL-1β和NO而实现的。     

大鼠桡神经钳夹伤后背根节和脊髓nNOS免疫阳性神经元的变化

目的研究大鼠桡神经钳夹伤后背根神经节(DRG)和脊髓nNOS免疫阳性神经元的变化,探讨NO是否参与大鼠桡神经钳夹伤后的DRG和脊髓水平的痛觉调制。方法用辣根过氧化物酶追踪大鼠桡神经的由来;大鼠桡神经钳夹伤结合免疫组化法,研究桡神经钳夹伤后DRG鄄和脊髓的nNOS免疫阳性结构变化。结果(1)大鼠桡神经的组成范围在C5～T1;(2)大鼠桡神经钳夹伤后,DRG内nNOS免疫阳性神经元的变化难以分析:脊髓后角nNOS免疫阳性结构数量减少、免疫强度下降。结论大鼠桡神经钳夹伤后,脊髓nNOS免疫阳性结构发生可塑性变化,NO在桡神经钳夹伤后的痛觉调制中有一定的作用。     

糖尿病大鼠脊髓nNOS免疫阳性神经元的变化

目的：观察糖尿病大鼠脊髓神经元型一氧化氮合酶(nNOS)免疫阳性神经元数量的变化，探讨NO在糖尿病发生和发展中的作用机制。方法：用链脲佐菌素诱导建立糖尿病大鼠模型，ABC免疫细胞化学法显示nNOS免疫阳性神经元。结果：大鼠脊髓内nNOS阳性神经元主要分布于中央管周围灰质和中间带等区域。中间带外侧核可见nNOS免疫阳性神经元较集中，细胞突起呈束状伸向中央管周围灰质方向；定量分析显示，糖尿病大鼠脊髓中央管周围灰质和中间外侧核在7w、12w时nNOS免疫阳性神经元数量明显增多。结论：糖尿病时伤害性刺激的传人增多，增多的nNC)S免疫阳性神经元可能与痛觉过敏等糖尿病周围神经病变有关。     

原发性高血压进程中下丘脑内神经元型一氧化氮合酶和血管紧张素Ⅱ1型受体的共表达

目的:观察Wistar-Kyoto(WKY)大鼠和自发性高血压大鼠(SHR)下丘脑室旁核(PVN)和视上核(SON)内神经元型一氧化氮合酶(nNOS)和血管紧张素Ⅱ1型受体(AT-1R)的共表达,以及加压素(AVP)和nNOS基因水平的表达差异.方法:应用免疫荧光双标染色结合RT-PCR技术.结果:SHR和WKY大鼠PVN和SON内都有大量的nNOS和AT-1R免疫阳性神经元分布并部分共存,但SHR组nNOS和AT-1R共存率明显高于对照组.与之一致,SHR组PVN和SON内nNOS和AVP mRNA含量都高于对照组.结论:在高血压发病进程中,NO发生代偿性增加,可部分负反馈抑制中枢肾素-血管紧张素系统,但这种抑制不能完全逆转过度激活的血管紧张素Ⅱ和AVP.     

ENK与CaBPs在PPE-GFP转基因小鼠视网膜细胞中的共存

目的:以PPE-GFP转基因小鼠为研究工具,观察绿色荧光蛋白(GFP)阳性的脑啡肽(ENK)能神经元与钙结合蛋白D28K(CB)、钙视网膜蛋白(CR)和小白蛋白(PV)等钙结合蛋白(CaBPs)成员在视网膜的分布及共存情况。方法:利用免疫组织化学和免疫荧光双标染色的方法。结果:GFP阳性的ENK能细胞主要分布在视网膜内核层内缘,少量分布在节细胞层。所有的GFP阳性细胞均与神经元标志物NSE共存,但不与星形胶质细胞标志物GFAP共存。GFP与CB、CR和PV均有部分共存,其中GFP/CB共存神经元占GFP阳性细胞的8.65%,占CB阳性细胞的5.84%;GFP/CR共存神经元占GFP阳性细胞的18.18%,占CR阳性细胞的14.28%,且共存细胞仅见于内核层;GFP/PV共存细胞占GFP阳性细胞的68.75%,占PV阳性细胞的91.67%,共存细胞主要位于内核层,少量见于节细胞层。结论:ENK能神经元在视网膜内具有板层特异性的分布特点和与钙结合蛋白成员有不同的共存模式,上述结果为深入研究小鼠视网膜ENK能神经元的功能意义提供了形态学依据。     

神经元型一氧化氮合酶在快速衰老小鼠额叶皮质中的增龄性改变

为了观察快速衰老小鼠(senescence accelerated mouse,SAM)衰老过程中大脑额叶皮质中nNOS的分布和表达变化,探讨NO/nNOS在中枢神经系统衰老中的作用。采用雄性快速衰老亚系8小鼠(senescence accelerated mouse/prone8,SAMP8)及抗快速衰老亚系1小鼠(senescence accelerated mouse/resistance1,SAMR1)为研究对象,其中SAMP8为实验组,SAMR1为对照组,每组动物再分为青年组(2月龄)和老年组(10月龄)两组。用免疫组织化学方法观察SAM额叶皮质中的nNOS神经元的形态和分布,并计数nNOS阳性神经元在额叶皮质中的数量;用RT-PCR法检测额叶皮质中nNOS mRNA表达水平。结果显示:SAMP8老年组与青年组相比,额叶皮质中nNOS阳性神经元的数量显著增加(15.8±6.3vs8.0±4.9,P<0.05);SAMP8与SAMR1比较,青年组额叶皮质nNOS阳性神经元的数量差异无统计学意义,老年组额叶皮质nNOS阳性神经元的数量显著增加(15.8±6.3vs7.5±5.3,P<0.05)。SAMP8老年组额叶皮质nNOS mRNA水平明显高于SAMP8青年组(1.00±0.17vs0.67±0.13,P<0.01)和老年组SAMR1(1.00±0.17vs0.67±0.11,P<0.01)。以上结果提示:额叶皮质中nNOS神经元的数量增加可能产生过量NO,NO可能参与了SAMP8快速衰老的过程。本研究的结果为通过调节额叶皮质NO产量来延缓衰老及衰老相关功能障碍提供了依据。     

大鼠脑干神经元型一氧化氮合酶免疫阳性神经元的分布

目的　观察大鼠脑干神经元型一氧化氮合酶 (nNOS)免疫阳性神经元的分布 ,为探讨nNOS的作用提供形态学资料。方法　用ABC免疫细胞化学方法显示脑干nNOS免疫阳性神经元。结果　大鼠脑干nNOS免疫阳性神经元以中脑和脑桥分布丰富 ,延髓较稀少 ;在中脑 ,nNOS免疫阳性神经元主要分布于中脑水管周围灰质的背侧部、被盖背外侧核、中缝背核、上下丘灰质等部位 ;在脑桥 ,主要分布于被盖背外侧核、脑桥中缝核、被盖脚桥核、蓝斑、臂旁核、斜方体核 ,以及脑桥网状结构 ;与中脑和脑桥相比 ,延髓nNOS免疫阳性神经元较少 ,主要分布于延髓网状结构、三叉神经脊束核和孤束核等核团。结论　分布于脑干内丰富的nNOS免疫阳性神经元可能通过其生成的NO调节其他神经递质的分泌 ,共同参与内脏活动、感觉和运动的传导 ,以及睡眠和觉醒等脑的高级整合功能的调节。     

糖尿病大鼠下丘脑视上核nNOS免疫阳性神经元的变化

目的：观察糖尿病大鼠下丘脑视上核神经元型一氧化氮合酶(nNOS)免疫阳性神经元数量的变化。方法：用链脲佐菌素诱导建立糖尿病大鼠模型；免疫细胞化学染色显示nNOS免疫阳性神经元，并进行定量分析。结果：糖尿病视上核nNOS免疫阳性神经元着色深浅不一，着色较深的阳性神经元散在分布，神经元的形态多样，突起较少。对照组大鼠视上核nNOS免疫阳性神经元较稀疏，各时期无明显改变。糖尿病2w，nNOS免疫阳性神经元数量与对照组无显著差异；7w，nNOS阳性神经元较密集，明显多于对照组；12w，nNOS免疫阳性神经元数量略低于7w，但仍多于对照组。结论：糖尿病大鼠下丘脑视上核nNOS免疫阳性神经元数量明显增多。     

猕猴胸腰段脊神经节nNOS免疫阳性神经元的分布

目的　观察正常猕猴胸腰段脊神经节神经元型一氧化氮合酶 (nNOS)免疫阳性神经元的分布。方法　用ABC免疫细胞化学方法显示nNOS免疫阳性神经元 ,并用体视学方法进行定量分析。结果　胸段和腰段脊神经节nNOS免疫阳性神经元的分布相似 ,均可见较丰富的nNOS免疫阳性神经元分布 ,神经元的大小不等 ,多呈圆形或椭圆形。胞浆着色较深 ,胞核位于细胞中央 ,不着色 ,细胞被神经纤维束分隔成群。nNOS免疫阳性神经元以中型神经元为主 ,其次为小型神经元 ,其胞浆呈强阳性染色 ,细胞直径 <50 μm ,大型神经元较少。胸、腰段脊神经节nNOS免疫阳性神经元的密度无明显差异 (P >0 0 5)。结论　在灵长类动物中 ,NO可能在感觉的传导和调节中发挥重要作用 ,但由于nNOS主要在中、小型神经元中表达 ,提示NO可能主要参与痛觉的调制     

Nocturnal motor coordination deficits in neuronal nitric oxide synthase knock-out mice

Nitric oxide is formed in the brain primarily by neurons containing neuronal nitric oxide synthase (nNOS), though some neurons may express endothelial NOS (eNOS), and inducible NOS (iNOS) only occurs in neurons following toxic stimuli. Mice with targeted disruption of nNOS (nNOS-) display distended stomachs with hypertrophied pyloric sphincters reflecting loss of nNOS in myenteric plexus neurons. nNOS- animals resist brain damage following middle cerebral artery occlusions consistent with evidence that excess release of nitric oxide mediates neurotoxicity in ischemic stroke. Neuronal NOS- mice have no grossly evident defects in locomotor activity, breeding long-term depression in the cerebellum, long-term potentiation in the hippocampus, and overall sensorimotor function. However, nNOS- animals display excessive, inappropriate sexual behavior and dramatic increases in aggression. Because the cerebellum possesses the greatest levels of nNOS neurons in the brain, it was surprising that presumed cerebellar functions such as balance and coordination were grossly normal in nNOS- mice. These previous studies were all conducted during the day (between 1400 and 1600, lights on at 0700). We now report striking, discrete abnormalities in balance and motor coordination in nNOS-mice reflected selectively at night.     

Chronic Alcohol Consumption Affects Gastrointestinal Motility and Reduces the Proportion of Neuronal NOS‐Immunoreactive Myenteric Neurons in the Murine Jejunum

Alcohol consumption interferes with gastrointestinal transit causing symptoms in alcoholic patients. Nitric oxide (NO), synthesized by neuronal nitric oxide synthase (nNOS) plays an important role in the control of gastrointestinal motility. Our aim was to investigate whether chronic alcohol intake in a murine model induces gastrointestinal motility disturbances and affects the nitrergic myenteric neurons in the stomach and jejunum. Gastric emptying, small intestinal transit and geometric centre were measured in vivo after intragastric gavage of Evans blue. Nitrergic relaxations to electrical field stimulation (EFS) and exogenous NO were recorded in jejunal muscle strips in vitro. The proportion of nNOS‐immunopositive myenteric neurons was assessed using PGP9.5 and nNOS immunostaining. After chronic alcohol consumption, gastric emptying and small intestinal transit were delayed compared with control mice, and the nitrergic nerve‐mediated relaxations to EFS in the jejunum were decreased, whereas relaxations to exogenous NO did not differ. The proportion of nNOS‐immunoreactive neurons did not change in the stomach, whereas in the jejunum the percentage decreased from 33% to 27% (P < 0.001) after chronic alcohol intake. The total number of myenteric neurons remained unchanged. These results suggest that chronic alcohol consumption disturbs gastric and small intestinal motility in vivo and in vitro and is associated with a decrease in the proportion of nNOS‐immunoreactive myenteric neurons in the murine jejunum. Anat Rec 293:1536–1542, 2010. © 2010 Wiley‐Liss, Inc.     

Reduced number of intrinsic pulmonary nitrergic neurons in Fawn-Hooded rats as compared to control rat strains

The Fawn-Hooded rat (FHR) strain reveals a congenital predisposition to primary (idiopathic) pulmonary hypertension (PPH), and can therefore be regarded as an animal model in which to study possible mechanisms underlying an inherited susceptibility to pulmonary hypertension. Pulmonary hypertension can be induced in FHRs after a short exposure to mild hypoxia, presumably because of an altered peripheral oxygen sensitivity. Given the presence of pulmonary nitrergic neurons in rat lungs, the observed link between airway hypoxia and the expression of pulmonary neuronal nitric oxide synthase (nNOS), and the fact that nNOS appears to be involved in peripheral chemoreceptor sensitivity, we examined the intrinsic pulmonary nitrergic innervation in the FHR. In the present study the number of intrapulmonary nitrergic nerve cell bodies, detected by NADPH diaphorase (NADPHd) histochemistry, was quantified in the FHR and three control rat strains. Compared to the control rat strains, the FHR lungs revealed a highly significantly lower number of intrinsic nitrergic neurons, while no apparent differences were found in the number of enteric nitrergic neurons in the esophagus. In conclusion, the possible links between neuronal NO, hypersensitivity to airway hypoxia, and the development of PPH clearly deserve further investigation.     

Reticular groove and reticulum are innervated by myenteric neurons with different neurochemical codes

The reticulum and the reticular groove are functional distinct compartments within the ovine forestomach. While the reticulum takes part in various motor functions, such as mixing, retaining, and rejecting the forestomach ingesta, the reticular groove serves mainly as a bypass between the esophagus and the abomasum. To accomplish these different tasks, the compartments develop specific motility patterns that are controlled by intrinsic neural circuits. In this study the intrinsic innervation by myenteric neurons was analyzed by quadruple immunohistochemistry against cholineacetyl transferase (ChAT), nitric oxide synthase (NOS), substance P (SP), and vasoactive intestinal peptide (VIP). Four neurochemically different subpopulations of myenteric neurons were found in the reticulum and the floor of the reticular groove: ChAT/-, ChAT/SP, NOS/-, and NOS/VIP. The neuronal proportions were calculated relative to all myenteric neurons. Neurons of the reticulum were mostly immunoreactive for ChAT (89% +/- 3%), whereas neurons adjacent to the reticular groove predominantly expressed a nitrergic phenotype (62% +/- 4%). ChAT-positive neurons were also immunoreactive for SP (ChAT/SP: 64% +/- 3% reticulum; 25% +/- 1% reticular groove) or were purely cholinergic (ChAT/-: 25% +/- 4% reticulum; 13% +/- 3% reticular groove). NOS-positive neurons colocalized VIP (NOS/VIP: 10% +/- 3% reticulum; 46% +/- 1% reticular groove) or none of the other neurotransmitters (NOS/-: 1% +/- 1% reticulum; 17% +/- 3% reticular groove). Analysis of the soma sizes revealed that in both compartments the nitrergic neurons were significantly larger than the cholinergic neurons. It is suggested that the specific neurochemical code in combination with a specific morphology leads to a precise regulation of the specialized tasks of the reticulum and reticular groove by subpopulations of myenteric neurons.     

神经型一氧化氮合酶与血红素氧合酶-2在应激后大鼠结肠的表达

目的探讨神经型一氧化氮合酶（nNOS）与血红素氧合酶-2（HO-2）在应激后大鼠结肠的表达.方法采用水浸-束缚应激（WRS）动物模型,用免疫组织化学SABC法检测nNOS和HO-2在大鼠结肠中的表达,并通过图像分析系统进行定量测定.结果对照组大鼠nNOS主要表达于结肠黏膜下神经丛和肌间神经丛的神经元,HO-2主要表达于结肠黏膜固有层黏膜肌、肌层环行肌及黏膜下层的血管内皮和平滑肌.应激组黏膜下神经丛和肌间神经丛的nNOS阳性神经元的平均灰度值较对照组明显增加,阳性神经元的平均数高于对照组,且在黏膜上皮细胞、固有层淋巴细胞也有nNOS表达;应激组HO-2阳性黏膜肌的平均灰度值较对照组增加,环行肌阳性单位（PU）明显高于对照组,在部分大肠腺也有HO-2表达.与应激组比较,应激＋L-NAME组的nNOS阳性神经元的平均灰度值减少,阳性神经元的平均数下降,应激＋ZnPP组HO-2阳性黏膜肌平均灰度值减少,环行肌PU下降.结论一氧化氮（NO）和一氧化碳（CO）均是结肠重要的气体信号分子和神经递质,两者在应激所致的结肠功能失调中可能具有协同作用.     

nNOS阳性神经元支配雄性大鼠球海绵体肌和坐骨海绵体肌

为了证实一氧化氮是否参与调节球海绵体肌和坐骨海绵体肌的功能 ,本研究用 CB-HRP逆行追踪结合 n NOS免疫细胞化学的技术 ,对支配雄性大鼠球海绵体肌和坐骨海绵体肌的神经元进行了研究 ,并结合 NADPH-d组化技术对雄性大鼠腰、骶髓的 NADPH-d阳性细胞和突起的分布进行了观察。结果发现 :( 1)支配球海绵体肌和坐骨海绵体肌的 Onuf核内存在着 CB-HRP与 n NOS双重反应细胞、CB-HRP阳性细胞、n NOS阳性细胞 ;n NOS阳性细胞还可见于后连合核、骶副交感核、背角等部位。( 2 ) NADPH-d阳性神经元在腰、骶髓主要位于中央管周围、后连合核、骶副交感核、背角等部位。本研究为 NO作为神经活性物质参与球海绵体肌和坐骨海绵体肌功能的调节提供了形态学证据     

Age-related alteration of neurturin receptor GFRa2 and nNOS in pelvic ganglia

Neurturin is a neurotrophic factor that is widely expressed in cavernous tissue and retrogradely transported to penis-projecting neurons via its receptor, glial cell line derived neurotrophic factor family receptor alpha-2 (GFRa2). To investigate the influence of aging on neural function on the penis, we examined the expression of GFRa2 mRNA in the major pelvic ganglion and its relationship to neuronal nitric oxide synthase (nNOS)- and tyrosine hydroxylase (TH)-positive neurons. GFRa2 and nNOS mRNA expression levels in RT-PCR showed age-related decreases in 1-, 3-, 6-, 12-, 18- and 24-month-old rats. In situ hybridization also revealed that the number of GFRa2-positive neurons in pelvic ganglia decreased with aging. A double-labeling study revealed the co-expression of GFRa2 and nNOS, which simultaneously decreased in old adult (24 months) and young castrated rats compared with young adult rats (3 months). These results suggest that aging and castration influence the numbers of nNOS- and GFRa2-positive neurons. Higher age might affect not only cavernous tissue but also the neural plasticity of the cavernous nerve related to erectile function.     

Anatomical evidence of non-parasympathetic cardiac nitrergic nerve fibres in rat

Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) plays a major role in the neural control of circulation and in many cardiovascular diseases. However, the exact mechanism of how NO regulates these processes is still not fully understood. This study was designed to determine the possible sources of nitrergic nerve fibres supplying the heart attempting to imply their role in the cardiac neural control. Sections of medulla oblongata, vagal nerve, its rootlets and nodose ganglia, vagal cardiac branches, Th₁-Th₅ spinal cord segments, dorsal root ganglia of C₈-Th₅ spinal nerves, and stellate ganglia from 28 Wistar rats were examined applying double immunohistochemical staining for nNOS combined with choline acetyltransferase (ChAT), peripherin, substance P, calcitonin gene-related peptide, tyrosine hydroxylase or myelin basic protein. Our findings show that the most abundant population of purely nNOS-immunoreactive (IR) neuronal somata (NS) was observed in the nodose ganglia (37.4 ± 1.3%). A high number of nitrergic NFs spread along the vagal nerve and entered its cardiac branches. All nitrergic neuronal somata (NS) in the nucleus ambiguus were simultaneously immunoreactive (IR) to ChAT and composed only a small subset of neurons (6%). In the dorsal nucleus of vagal nerve, biphenotypic nNOS-IR/ChAT-IR neurons composed 7.0 ± 1.0%, while small purely nNOS-IR neurons were scarce. Nitrergic NS were plentifully distributed within the nuclei of solitary tract. In the examined dorsal root and stellate ganglia, a few nitrergic NS were sporadically present. The majority of sympathetic NS in the intermediolateral nucleus were simultaneously immunoreactive for nNOS and ChAT. In conclusion, an abundant population of nitrergic NS in the nodose ganglion implies that neuronal NO is involved in afferent cardiac innervation. Nevertheless, nNOS-IR neurons identified within vagal nuclei may play a role in the transmission of preganglionic parasympathetic nerve impulses.     

Distribution of Nitrergic Neurons in the Dorsal Root Ganglia of the Bottlenose Dolphin (Tursiops truncatus)

Dorsal root ganglia (DRGs) contain the cell bodies of primary afferent neurons that transmit sensory information from the periphery into the spinal cord. Distinct populations of DRG neurons have been characterized by a variety of different immunohistochemical markers. A subpopulation of ganglionic neurons containing neuronal nitric oxide synthase (nNOS), an enzyme known to generate nitric oxide, has been detected in a number of mammalian species. Despite previous studies, no information is known on the presence and exact distribution of nNOS‐immunoreactive neurons in the DRGs of the bottlenose dolphin. In this investigation, immunoperoxidase for nNOS was used to determine the distribution and the perikaryal size of nitrergic neurons in the DRGs of this species. Double immunofluorescence protocol was used to determine the percentage of nNOS‐immunoreactive (IR) neurons over the total primary afferent neurons. In addition, double immunostaining was used to verify whether there was colocalization of nNOS with substance P (SP). In all DRGs, a subpopulation of small‐ and medium‐sized neurons (about 9%) exhibited nNOS immunoreactivity. Data analysis revealed that the majority of nNOS‐IR neurons (81.3%) expressed SP. The density of nNOS‐immunoreactive and nNOS/SP‐double immunopositive cells was relatively constant throughout the ganglia. However, as observed in others mammals, the number of nitrergic neurons decreased in the caudalmost DRGs. Our results, in conjunction with previous observations, suggest that nNOS‐IR neurons may be involved in the afferent transmission of visceral and nociceptive information as well as in the regulation of the vascular tone. Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.