大鼠翼腭神经节、耳神经节神经元一氧化氮合酶与神经肽Y的共存

目的：为进一步阐明一氧化氮(NO)与神经肽Y(NPY)在翼腭神经节、耳神经节支配机制中的作用提供形态学资料。方法：用尼克酰胺腺嘌呤二核苷酸-黄递酶(NADPH-d)组织化学方法与免疫组织化学方法结合技术观察翼腭神经节、耳神经节中一氧化氮合酶(NOS)与NPY阳性神经元分布及共存关系。结果：翼腭神经节、耳神经节中有NOS、NPY单标神经元和NOS-NPY双标神经元，以双标神经元最多，NPY单标神经元最少。3种神经元在神经节中无区域特异性的散在分布。两神经节中，各种神经元分布特点相似。结论：NO可能对翼腭神经节、耳神经节节后神经纤维末梢NPY的释放起重要作用。     

Role of membrane depolarization in transsynaptic induction of tyrosine hydroxylase in organ cultures of sympathetic ganglia

In contrast to nicotinic cholinomimetics which generate action potentials in postganglionic adrenergic neurons, general non-specific depolarizing agents such as veratridine and batrachotoxin (which also generate action potentials) do not initiate tyrosine hydroxylase (TH) induction.Moreover, tetrodotoxin, a selective blocker of fast sodium channels, which prevents the formation of action potentials, does not interfere with TH induction mediated by carbamylcholine or nicotine.It is concluded that specific TH induction is mediated via nicotinic receptors by a mechanism which is not dependent on the activation of the fast sodium channels.     

The neuronal nitric oxide synthase (nNOS) gene contributes to the regulation of tyrosine hydroxylase (TH) by cocaine

Recently, we demonstrated that intact nitric oxide (NO) signaling is essential for the development of cocaine behavioral sensitization in adulthood [M.A. Balda, K.L. Anderson, Y. Itzhak, Differential role of the nNOS gene in the development of behavioral sensitization to cocaine in adolescent and adult B6;129S mice, Psychopharmacology (Berl) 200 (2008) 509–519]. Given the requirement of dopamine (DA) transmission in cocaine-induced behavioral sensitization and the interactions between NO and DA systems, the present study investigated the role of the neuronal nitric oxide synthase (nNOS) gene and the effect of cocaine on the expression of tyrosine hydroxylase (TH)-immunoreactive (-ir) neurons. Adult (postnatal day 80) wild type (WT) and nNOS knockout (KO) mice received saline or a sensitizing regimen of cocaine (20 mg/kg) for 5 days. After 24 h, TH immunoreactivity was assessed in the ventral tegmental area (VTA) and the dorsal striatum (dST) using stereology and Western blotting, respectively. We report that (a) nNOS KO mice express lower levels of TH-ir neurons in the VTA compared to WT counterparts, (b) cocaine administration to WT mice significantly increased striatal TH expression, and (c) the same cocaine administration to nNOS KO mice significantly decreased striatal TH expression. Thus, the nitrergic system may contribute to cocaine-induced behavioral sensitization by regulating dopaminergic neurotransmission.     

内皮型、诱导型一氧化氮合酶在乳腺癌中的表达

目的 :研究内皮型一氧化氮合酶 (eNOS)、诱导型一氧化氮合酶 (iNOS)在乳癌中表达及与淋巴结转移的关系。方法 :采用免疫组化S P法检测 60例乳癌中eNOS和iNOS的表达。结果 :eNOS和iNOS阳性在乳癌中表达率分别为 75 0 %和71 7%。在淋巴结转移组和无淋巴结转移组中eNOS阳性表达率分别为 66 7%和 83 3 % ,两组间差异无统计学意义 (χ2 =2 2 2 ,P >0 0 5) ,而iNOS在淋巴结转移和无转移组中阳性表达率分别为 53 3 %和 90 0 % ,两组间差异有统计学意义 (χ2 =9 93 ,P <0 0 1 )。结论 :内皮型、诱导型一氧化氮合酶在乳腺癌中高表达 ;iNOS的表达与乳腺癌的淋巴转移相关     

Pathological consequences of inducible nitric oxide synthase expression in hippocampal slice cultures

The generation of toxic concentrations of nitric oxide by the inducible nitric oxide synthase expressed in microglia and other brain cell types is frequently invoked as a causative factor in neurodegeneration. Experiments were carried out on slice cultures of rat hippocampus to test this hypothesis. Exposure of the slices to bacterial lipopolysaccharide plus interferon-gamma led to a time-dependent expression of functional inducible nitric oxide synthase that was found only in microglia. Microglial activation by other means, such as physical damage, was not associated with inducible nitric oxide synthase expression. Damage and cell death in slices expressing inducible nitric oxide synthase was evaluated over a period of 6 days, but none was found. Consistent with this result, cGMP measurements indicated that the average local nitric oxide concentration remained in the low nanomolar range. When the microglial population was expanded to a density three-fold above normal by applying granulocyte-macrophage colony stimulating factor, however, lipopolysaccharide plus interferon-gamma provoked neurodegeneration that could be blocked by an inducible nitric oxide synthase inhibitor. The associated nitric oxide concentration in the slices was saturating for guanylyl cyclase-coupled nitric oxide receptors, signifying at least 10 nM. It is concluded that inducible nitric oxide synthase is expressed in microglia only in response to specific stimuli involving the innate immune system, and that the resulting level of nitric oxide in intact brain tissue is normally too low to inflict damage directly. Quantities of nitric oxide sufficient to contribute directly or indirectly to pathology could be produced should the density of microglia become high enough, although caution must be exercised in extrapolating this finding to the human brain in vivo.     

Neuronal nitric oxide synthase is the dominant nitric oxide supplier for the survival of dorsal root ganglia after peripheral nerve axotomy

This study was designed to determine whether nitric oxide supply may be a major factor in the survival of dorsal root ganglia in a sciatic nerve injury model. Wild-type (WT) mice were compared with knockout (KO) mice lacking neuronal nitric oxide synthase (nNOS) or endothelial (eNOS). The NO-generating capacities were analysed by NOS immunohistochemistry and NADPH-diaphorase staining 1, 2, 6, and 12 weeks after nerve transection. The occurrence and morphological type of neuronal death were determined by TUNEL reaction and ultrastructural examination. Cell loss following nerve section, whist dependent on the availability of NO, as shown by its marked elevation in nNOS KO mice, did not correlate well with nNOS expression in WT animals. Whereas a lack of eNOS was tolerated, deficiency of nNOS led to an enhanced cell loss. The results suggest a crucial role of NO supply after transection of peripheral nerves with a particular significance of the nNOS isoform.     

The immunohistochemical localization of neuronal nitric oxide synthase in the basal forebrain of the dog

The present work describes for the first time the anatomical distribution of neuronal nitric oxide synthase (nNOS) immunoreactivity and NADPH-d activity in the basal forebrain of the dog. As in other species, small, intensely nNOS-immunoreactive cells were seen within the olfactory tubercle, caudate nucleus, putamen, nucleus accumbens and amygdala. In addition, a population of mixed large and small nNOS positive cells was found in the medial septum, diagonal band and nucleus basalis overlapping the distribution of the magnocellular cholinergic system of the basal forebrain. Our results show that the distribution of NOS containing neurons in these nuclei in the dog is more extensive and uniform than that reported in rodents and primates. When double labeling of nNOS and NADPH-d was performed in the same tissue section most neurons were double labeled. However, a considerable number of large perikarya in the diagonal band and nucleus basalis appeared to be single labeled for nNOS. Thought a certain degree of interference between the two procedures could not be completely excluded, these findings suggest that NADPH-d histochemistry, which is frequently used to show the presence of NOS, underestimates the potential of basal forebrains neurons to produce nitric oxide. In addition, a few neurons mainly localized among the fibers of the internal capsule, appeared to be labeled only for NADPH-d. These neurons could be expressing a different isoform of NOS, not recognized by our anti-nNOS antibody, as has been reported in healthy humans and AD patients.     

Expression of peptides, nitric oxide synthase and NPY receptor in trigeminal and nodose ganglia after nerve lesions

Using immunohistochemistry and in situ hybridization, the expression of galanin (GAL)/galanin message associated peptide (GMAP)-, neuropeptide Y (NPY)-, vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucine (PHI)- and nitric oxide synthase (NOS)-like immunoreactivities and mRNAs, and NPY receptor mRNA was studied in normal trigeminal and nodose ganglia and 14 and 42 days after peripheral axotomy. In normal trigeminal ganglia about 11% of the counted neuron profiles contained GAL mRNA, 4% NOS mRNA, 5% NPY mRNA, 7% VIP mRNA, and 19% NPY receptor mRNA. Peptide mRNA- and NPY receptor mRNA-positive neuron profiles were small in size. Fourteen days after axotomy a marked increase in the number of GAL mRNA- (34% of counted neuron profiles), NPY mRNA- (54%) and VIP mRNA- (31%) positive neuron profiles, and a moderate increase in the number of NOS mRNA- (22%) positive neuron profiles were observed in the ipsilateral trigeminal ganglia. The GAL/GMAP, VIP- and NOS-positive profiles were mainly small, the NPY-positive ones mostly large. NPY receptor mRNA was expressed in some large neurons. In normal nodose ganglia, about 3% of the counted neuron profiles contained GAL mRNA, 3% NPY mRNA, 17% NOS mRNA and less than 1% VIP mRNA. Fourteen days after peripheral axotomy, a marked increase in the number of GAL mRNA- (78% of counted neuron profiles), NOS mRNA- (37%) and VIP- (46%) mRNA-positive neuron profiles was seen in the ipsilateral nodose ganglia. The number of NPY-positive (23%) neurons was moderately increased, mainly in small neuron profiles. There were no NPY receptor mRNA-positive neurons, either in normal nodose ganglia or in nodose ganglia ipsilateral to the axotomy. In contralateral nodose ganglia the number of GAL- and NPY-positive neuron profiles was slightly increased, and VIP cells showed a moderate increase. Immunohistochemical analysis revealed parallel changes in expression of peptides and NOS in both trigeminal and nodose ganglia, demonstrating that the changes in mRNA levels are translated into protein. Finally, although not quantified, similar upregulations of peptide and NOS mRNA levels were observed in both ganglia 42 days after nerve injury provided that regeneration was not allowed, suggesting that the changes are long lasting. The present results show that the effect of axotomy on peptide and NOS expression in the trigeminal and nodose ganglia is similar to that previously shown for lumbar dorsal root ganglia. However, no mRNA for the NPY Y1 receptor could be detected in the vagal system. In general the mechanism(s) for and the purpose(s) of the messenger regulation in response to axotomy may be similar in these different sensory systems (dorsal root, trigeminal and nodose ganglia).During the final part of this study Dr. Jan Arvidsson tragically died from a cerebral insult.     

Tyrosine hydroxylase immunoreactive neurons in organotypic slice cultures of the rat striatum and neocortex

Summary Organotypic slice cultures of striatum and neocortex were prepared from newborn to seven day old rats and cultured for three to 60 days. When processed for tyrosine hydroxylase (TH) immunocytochemistry medium-sized, aspiny TH immunoreactive (TH-i) neurons with a similar morphology were revealed in the striatum and the neocortex. The neurons had a very similar morphology in both tissues and were present both when the two tissues were grown separately as single cultures and when grown together either en bloc as part of the same tissue slices or as co-cultures. In order to examine whether innervation by dopaminergic fibers would affect the expression of TH-i neurons in the striatal slice cultures, co-cultures of ventral mesencephalon (VM) and striatum were prepared, but the ingrowth of TH-i fibers from the VM did not alter the expression of TH immunoreactivity by a subpopulation of striatal neurons.     

Sperm nitric oxide and motility: the effects of nitric oxide synthase stimulation and inhibition

Nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as the nitric oxide synthases (NOS). We have recently shown a NOS similar to constitutive brain NOS (bNOS) and endothelial NOS (ecNOS) to be present in spermatozoa. The aim of this study is to investigate NO production by human spermatozoa and the effects of stimulation and inhibition of NOS. This was carried out using the Iso-NO, an isolated NO meter and sensor, which provides rapid, accurate and direct measurements of NO. Semen samples with normozoospermic and asthenozoospermic profiles were prepared using a direct swim-up technique. Basal concentrations of NO and stimulated NO production were measured after exposure to the calcium ionophore (A23187; 0.01-10 microM) a potent activator of constitutive NOS. NO production in human spermatozoa was significantly increased by the addition of A23187 30 seconds after stimulation. Furthermore, this response was greatly diminished by pre-incubating the samples with competitive inhibitors of L-arginine, the substrate for NOS, before treatment with calcium ionophore. In the presence of N(G)-nitro-L-arginine methyl ester (L- NAME), N(G)-nitro-L-arginine (L-NA) or N(G)-methyl-L-arginine (L-NMMA; all at 10 microM), NO production was inhibited with a rank order of potency L-NAME > L-NMMA > L-NA which is in accordance with the inhibition of an endothelial type of constitutive NOS.      

大鼠耳神经节和基底动脉壁神经纤维一氧化氮合酶表达的增龄变化

目的:观察大鼠耳神经节及基底动脉壁神经纤维一氧化氮合酶(NOS)神经细胞的年龄变化.方法:黄递酶组织化学法及图像分析对不同年龄组耳神经节和基底动脉壁神经纤维NOS表达进行观察.结果: NOS神经细胞弥散分布在神经节内,细胞密度(细胞数/面积)在出生后1周达到高峰,以后逐渐下降,至成年降到最低值,至老年无明显改变;在发育中,NOS神经元胞体大小逐渐增加,核质比下降,至成年时最大,至老年无明显改变.基底动脉神经纤维密度从幼年到成年逐渐增高,从成年起维持较高密度直到老年.结论:出生后耳神经节NOS神经细胞的发育变化有明显的规律性,对其发育研究有临床意义.     

叶酸对去卵巢大鼠抗氧化酶、一氧化氮合酶和一氧化氮的影响

 目的: 观察叶酸对去卵巢大鼠抗氧化酶、一氧化氮合酶和一氧化氮的影响。方法: 40只3月龄健康雌性SD大鼠,随机分成5组:假手术组、去卵巢组、二乙基己烯雌酚(0.03 mg·kg^-1·d^-1)组、低剂量(5 mg·kg^-1·d^-1)叶酸组和高剂量(20 mg·kg^-1·d^-1)叶酸组。各组大鼠于术后1周开始灌胃给药,假手术组和去卵巢组给予蒸馏水,10周后,取L₅椎体和右股骨行骨密度(BMD)检测;测定血浆和骨匀浆总抗氧化能力(TAC)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、一氧化氮合酶(NOS)和一氧化氮(NO)水平。结果: 与假手术组比较,去卵巢组大鼠L₅椎体和股骨BMD显著降低(P < 0.01),血浆GSH-Px、NO和骨匀浆TAC、GSH-Px、NOS及NO水平明显降低(P < 0.01),MDA浓度升高显著(P < 0.01);与去卵巢组大鼠比较,高剂量叶酸组大鼠L₅椎体和股骨BMD增加(P < 0.01),骨匀浆TAC、GSH-Px、NOS和NO水平升高(P < 0.01),MDA浓度降低(P < 0.01),血浆GSH-Px和NO水平升高。结论: 去卵巢大鼠体内抗氧化酶、NOS和NO水平降低,氧化应激参与了去卵巢大鼠骨质疏松的发生;高剂量叶酸能提升去卵巢大鼠腰椎和股骨BMD,提高其体内抗氧化酶、NOS和NO水平,改善氧化应激,这可能是高剂量叶酸防治去卵巢大鼠骨质疏松的机制之一。     

一氧化氮/诱导型一氧化氮合酶在动脉粥样硬化过程中的作用

 目的：探讨一氧化氮（NO）/诱导型一氧化氮合酶（iNOS）在动脉粥样硬化（atherosclerosis,AS）过程中的动态变化,分析其对动脉粥样硬化形成过程的影响。方法：将60只SD大鼠随机分成2组：对照组及AS组,每组30只。AS组采用维生素D3腹腔注射联合高脂饲料饲养的方法构建动脉粥样硬化模型。用相关生化方法检测血清各项生化指标：总胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、空腹血糖和钙离子,比色法检测血清NO浓度,并对主动脉行HE染色,免疫组化技术检测iNOS蛋白表达,将所得数据进行统计分析,用简单线性相关分析NO与钙离子及动脉粥样硬化指数的相关性。结果：90 d后成功构建了主动脉中膜钙化型动脉粥样硬化模型。血清NO浓度在动脉粥样硬化过程中逐步下降,各组间差异均有统计学意义（均P<0.05）。动脉粥样硬化过程中动脉粥样硬化指数与钙离子呈正相关,与NO呈负相关。在90 d的AS组粥样斑块区免疫组化技术检测到iNOS蛋白表达。结论:在动脉粥样硬化形成过程中,主动脉粥样斑块区iNOS蛋白高表达,但血清NO浓度逐渐降低,NO抗动脉粥样硬化作用减弱。     

Putative dopamine-containing cells in the retina of seven species demonstrated by tyrosine hydroxylase immunocytochemistry

Immunocytochemistry with antibodies to catecholamine synthesizing enzymes has revealed cells in the retina of chick, mouse, hamster, rat, guinea-pig, piglet and marmoset which contain tyrosine hydroxylase but not dopamine beta-hydroxylase. These findings suggest that the cells in question produce dopamine but that catecholamine synthesis does not proceed further to noradrenaline. Tyrosine hydroxylase-containing amacrine cells, located in the innermost part of the inner nuclear layer, were present in all the species studied. Some species showed atypically located amacrine cells in the inner plexiform or ganglion cell layer. In the rodents, the existence of tyrosine hydroxylase-containing interplexiform cells was suggested by the presence of a few short immunoreactive ascending processes. Three different morphological types of putative dopamine-containing cells were classified according to the level of ramification.     

糖尿病新西兰兔心、肾NOS和抗氧化酶的变化

目的：探讨糖尿病新西兰兔心、肾一氧化氮合酶（NOS）活力和抗氧化酶活力变化以及它们在糖尿病发病机制中的作用。方法：采用高脂高糖饲料喂养新西兰兔，建立一种新的糖尿病动物模型。观察糖尿病新西兰兔心、肾一氧化氮水平，一氧化氮合酶（NOS）活力及抗氧化酶活力的变化。结果：糖尿病新西兰兔出现大量蛋白尿（P<0.05）；肌酐清除率(CCr)明显高于对照组（P<0.01）；心脏组织中NO₂^-水平和NOS活力明显低于对照组（P<0.05），而肾脏组织中NO₂^-水平和NOS活力明显高于对照组（P<0.01）；血糖及胰岛素明显高于对照组（P<0.01）；超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化氢酶（catalase）活性都明显低于对照组（P<0.01）。结论：糖尿病新西兰兔心、肾中的NO₂^-水平和NOS活力异常，抗氧化酶活力明显降低，糖尿病的心、肾并发症可能与它们的异常变化有关。     

小肠内NOS分布及NO作用研究进展

一氧化氮作为一种细胞间和细胞内的信息物质 ,在小肠的生理病理过程中有重要作用。本文着重介绍小肠内的一氧化氮合酶分布NO与小肠运动、消化吸收、粘膜保护的关系等方面的研究进展。     

吸烟大鼠一氧化氮合酶和一氧化氮的变化

目的:观察吸烟对大鼠肺组织iNOS、eNOSmRNA和蛋白表达以及支气管肺泡灌洗液(BALF)中NO的影响, 探讨不同类型的NOS在吸烟所致慢性气道炎症中的作用。方法:选用Wistar大鼠80只随机分为对照组, 被动吸烟组, iNOS抑制剂L-NIL干预组及NOS抑制剂L-NAME干预组。用免疫组化法检测iNOS及eNOS的蛋白表达, 用RT-PCR检测iNOS及eNOSmRNA的表达, 用Griess法测定BALF中的NO^-₂/NO^-₃含量。结果:吸烟大鼠肺组织中iNOSmRNA及其蛋白表达增加, eNOSmRNA及蛋白表达下降, BALF中细胞总数及NO^-₂/NO^-₃显著增加(P＜0.05)。在体实验发现, L-NIL使BALF中细胞总数及NO^-₂/NO^-₃下降(P＜0.05);L-NAME对BALF中细胞总数及NO^-₂/NO^-₃无显著影响(P＞0.05)。结论:吸烟大鼠肺组织iNOSmRNA和蛋白表达增加, eNOSmRNA和蛋白表达减少。活化的iNOS产生大量NO促进炎症发展。     

Tyrosine hydroxylase and neuropeptide Y are increased in ciliary ganglia of sympathectomized rats.

We have examined the expression of tyrosine hydroxylase and neuropeptide Y in ciliary ganglia of normal adult rats and of adult rats in which the environment of these neurons was altered by sympathectomy at birth. Following neonatal 6-hydroxydopamine treatment, the proportion of tyrosine hydroxylase-immunoreactive and neuropeptide Y-immunoreactive neurons in ciliary ganglia was significantly increased. In ciliary neurons of both control and sympathectomized rats, neuropeptide Y immunoreactivity was preferentially co-localized with tyrosine hydroxylase. Immunoblot analysis confirmed the presence of tyrosine hydroxylase and its increase following sympathectomy. In situ hybridization studies revealed that many ciliary neurons contain mRNA for tyrosine hydroxylase and for neuropeptide Y. Like tyrosine hydroxylase immunoreactivity, the number of ciliary neurons containing tyrosine hydroxylase mRNA and the amount of mRNA per cell were increased in 6-hydroxydopamine-treated rats. In contrast, neuropeptide Y mRNA levels were the same in control and 6-hydroxydopamine-treated rats. Nerve growth factor is a candidate for mediating the effects of sympathectomy and most ciliary neurons in control and sympathectomized rats expressed immunoreactivity for the low-affinity nerve growth factor receptor. In addition, ciliary neurons from 6-hydroxydopamine-treated animals possessed increased nerve growth factor receptor immunoreactivity. These studies indicate that both tyrosine hydroxylase and neuropeptide Y in the ciliary ganglion are regulated by alterations in their environment. Their expression was enhanced by chemical sympathectomy which does not affect ciliary neurons directly but, rather, removes sympathetic innervation of shared targets, including the iris. In situ hybridization analysis suggests that the increased tyrosine hydroxylase and neuropeptide Y levels result from different mechanisms and provides evidence that neuropeptide levels can be regulated without changes in mRNA levels.     

Endothelial nitric oxide synthase, caveolae and the development of atherosclerosis

Early hypercholesterolaemia-induced vascular disease is characterized by an attenuated capacity for endothelial production of the antiatherogenic molecule nitric oxide (NO), which is generated by endothelial NO synthase (eNOS). In recent studies we have determined the impact of lipoproteins on eNOS subcellular localization and action, thereby providing a causal link between cholesterol status and initial abnormalities in endothelial function. We have demonstrated that eNOS is normally targeted to cholesterol-enriched caveolae where it resides in a signalling module. Oxidized low density lipoprotein (LDL; oxLDL) causes displacement of eNOS from caveolae by binding to endothelial cell CD36 receptors and by depleting caveolae cholesterol content, resulting in the disruption of eNOS activation. The adverse effects of oxLDL are fully prevented by high density lipoprotein (HDL) via binding to scavenger receptor BI (SR-BI), which is colocalized with eNOS in endothelial caveolae. This occurs through the maintenance of caveolae cholesterol content by cholesterol ester uptake from HDL. As importantly, HDL binding to SR-BI causes robust stimulation of eNOS activity in endothelial cells, and this process is further demonstrable in isolated endothelial cell caveolae. HDL also enhances endothelium- and NO-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, lipoproteins have potent effects on eNOS function in caveolae via actions on both membrane cholesterol homeostasis and the level of activation of the enzyme. These processes may be critically involved in the earliest phases of atherogenesis, which recent studies suggest may occur during fetal life.     

New insights on the neuropeptide Y system in the larval lamprey brain: neuropeptide Y immunoreactive neurons,descending spinal projections and comparison with tyrosine hydroxylase and GABA immunoreactivities

Lampreys are useful models for studying the evolution of the nervous system of vertebrates. Here we used immunofluorescence and tract-tracing methods to study new aspects of the neuropeptide Y-immunoreactive (NPY-ir) system in larval sea lampreys. NPY-ir neurons were observed in brain nuclei that contain NPY-ir cells in other lamprey species. Moreover, a group of NPY-ir cells that migrated away the periventricular layer was observed in the lateral part of the dorsal hypothalamus, which suggests a role for NPY in feeding behavior in lampreys. We also report NPY-ir cells in the dorsal column nucleus, which appears to be unique among vertebrates, and in the habenula. A combination of tract-tracing and immunohistochemical labeling demonstrated the presence of spinal projecting NPY-ir reticular cells in the anterior rhombencephalic reticular formation, and the relationships between the NPY-ir system and the reticulospinal nuclei and some afferent systems. The colocalization of catecholamines and GABA in lamprey NPY-ir neurons was investigated by double immunofluorescence methods. Colocalization of tyrosine hydroxylase (TH) and NPY immunoreactivities was not observed in any brain neuron, although reported in amphibians and mammals. The frequent presence of NPY-ir terminals on TH-ir cells suggests that NPY modulates the activity of some dopaminergic nuclei in lampreys. Colocalization of NPY and GABA immunoreactivities was frequently observed in neurons of different rhombencephalic and diencephalic NPY-ir populations. These results in lampreys suggest that the coexpression of NPY and GABA in neurons appeared early on in the brains of vertebrates.