Sleep deprivation inhibits expression of NADPH-d and NOS while activating microglia and astroglia in the rat hippocampus

This study investigated the expression of nitric oxide (NO)-synthesizing enzymes and the glial reaction in the rat hippocampal formation following sleep deprivation for 5 days. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity was markedly reduced in the hippocampal CA1, CA2 and CA3 sectors as well as in the dentate gyrus, suggesting a suppression of NO production in these areas. Microglial cells were hypertrophic and showed an up-regulation of complement type 3 receptors as determined by antibody OX-42. However, expression of major histocompatibility complex class I and II antigens, and antigen of monocyte/macrophage lineage marked by OX-18, OX-6 and ED1, respectively, was undetected. Astrocytes also displayed hypertrophied processes with enhanced glial fibrillary acidic protein (GFAP) immunoreactivity. Western blots of hippocampal tissues corroborated the above-mentioned morphological findings in that expression of NO-synthase (NOS) was decreased while that of OX-42 and GFAP was increased in the sleep-deprived rats. Since NO is thought to be involved in memory consolidation processes in the hippocampus during sleep, the inhibition of NADPH-d and NOS reactivities may account for the memory decline after long-term sleep deprivation. The concomitant reactions in microglia and astrocytes suggest the involvement of these cells in the deleterious effect of prolonged sleep deprivation.     

Expression of NADPH-diaphorase and nitric oxide synthase in lumbosacral motoneurons after knee joint immobilisation in the guinea pig

The expression of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and nitric oxide synthase (NOS) in spinal ventral horn neurons was studied in the guinea pig after right knee joint immobilisation (RKJI). At 1 wk after RKJI, neurons in the ipsilateral ventral horn from L4 to S1 segments showed a moderate reactivity for NADPH-d staining. At 2 wk, NADPH-d labelled neurons were also observed in the contralateral ventral horn. Ipsilateral NOS immunoreactive cells were not detectable until wk 2. The intensity of NADPH-d and NOS labelled neurons in the bilateral ventral horns was sustained, peaking at the 4th wk after RKJI. In guinea pigs subjected to 4 wk of RKJI and subsequently released from the immobilisation for 2 and 4 wk, NADPH-d and NOS reactivity in ventral horn neurons diminished. The expression of NADPH-d positive neurons differed from that of NOS labelled neurons in terms of time interval, cell number and staining intensity, the latter being later, fewer and weaker. It is suggested that the induction and upregulation of NADPH-d and NOS are attributable to reduced activity of muscles acting on the knee joint after RKJI; the changes are reversible. It is speculated that increased levels of NO production are involved in protective mechanisms against possible neuronal degeneration as a consequence of target dysfunction.     

Exposure to aluminium changes the NADPH-diaphorase/NPY pattern in the rat cerebral cortex

Aluminium (Al) impairs the glutamate-nitric oxide-cGMP pathway and reduces the number of nitroxidergic neurons in the rat somatosensory cortex. To understand better the effect of the time of exposure, we monitored the effect of aluminium administration on nitroxidergic neurons, identified by NADPH-diaphorase (NADPH-d) or by nitric oxide synthase (NOS) staining, after 0.5, 1, 2, 3, 6 and 12 months of aluminium administration. Since neuropeptide Y (NPY) is known to be colocalised with nitric oxide synthase in cortical neurons, the aim of this work was to study the effects of Al administration on the cortical expression of NADPH-d, nNOS, and NPY. NADPH-d or NOS positive neurons were found scattered in the cortex where they constituted about 1% of all neurons. Double staining using NADPH-d and NPY showed that almost all nitroxidergic neurons were co-localised with NPY neurons (NADPH-d/NPY double stained neurons) whereas some neurons were stained only with NPY (NPY single stained neurons) ; these were more numerous than NADPH-d/NPY double stained neurons. Al significantly reduced NADPH-d and nNOS positive neurons in the cerebral cortex time dependently, with the greatest effect appearing after 3 months. Also measured was the integrated optical density (IOD) of nNOS positive neurons showing a significant decrease of NOS immunostaining even in the remaining NOS positive neurons. The double staining experiment exhibited a decrease in NADPH-d/NPY double stained neurons with an apparent increase in NPY single stained neurons; these then decreased after 6-12 months. On the whole, the results confirm that Al impairs nitroxidergic pathways time dependently; moreover, the transient increase in NPY single stained neurons from 1 to 3 months suggests that there is an intraneuronal down-regulation of NOS, without affecting neuronal viability. In addition, the decrease in the NPY system found at 6 and 12 months may indicate that Al affected nitroxidergic and NPY systems at different times.     

肝性脑病大鼠海马CA3区神经元形态和一氧化氮合酶表达的变化

目的观察肝性脑病模型组和正常对照组大鼠脑海马CA3区神经元的变化及一氧化氮合酶(NOS)的表达;探讨海马CA3区神经元的形态学改变及一氧化氮(NO)在肝性脑病发病机制中的作用。方法雄性大鼠50只,实验开始前所有动物均进行莫里斯水迷宫测试,之后将动物分为对照组和实验组。9周后建立CCL4肝性脑病模型,分别取两组大鼠海马组织进行尼氏染色及烟酰胺腺嘌呤二核苷酸-黄递酶(NADPH-d),染色。结果尼氏染色发现,实验组大鼠海马神经元数目减少、染色较浅,胞质内尼氏体减少或消失;NADPH-d染色发现,实验组可见粗大轴突着色,树突联系广泛;对照组则少有粗大轴突着色,树突间联系不如实验组广泛。实验组NOS阳性神经元染色较对照组深,为紫蓝或深蓝色(强阳性及阳性),且阳性神经元数目较多;而对照组染色浅淡,呈浅蓝或与背景同色,为弱阳性。结论肝性脑病时海马受到损伤,NO可能介导了神经元的损伤并参与了肝硬化和肝性脑病的发病,血氨升高是肝性脑病(HE)致病因素之一。     

NADPH-diaphorase and cytosolic urea cycle enzymes in the rat accessory olfactory bulb

The nitric oxide cycle consists of nitric oxide synthase, argininosuccinate synthetase and argininosuccinate lyase to form nitric oxide. We have examined the colocalization of nitric oxide synthase and the cytosolic urea cycle enzymes (argininosuccinate synthetase, argininosuccinate lyase and arginase) in the accessory olfactory bulb of the rat by using a double labeling procedure combining reduced-nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) reaction with fluorescent immunocytochemistry. Each glomerulus showed a different NADPH-d activity, and those with the strongest NADPH-d activities were assembled in the caudomedial part of the accessory olfactory bulb. Argininosuccinate synthetase-like immunoreactive glomeruli were distributed in the caudomedial part of the accessory olfactory bulb, and most of them were also strongly NADPH-d positive. The mitral or tufted cells were argininosuccinate synthetase-, argininosuccinate lyase- and arginase-like immunoreactive, but were not NADPH-d positive. The granule cells were NADPH-d positive or argininosuccinate lyase-like immunoreactive, but were not argininosuccinate synthetase- or arginase-like immunoreactive. Some granule cells were both NADPH-d positive and argininosuccinate lyase-like immunoreactive. The results indicate the heterogeneity of glomeruli of the accessory olfactory bulb with respect to the distribution of these enzymes. The granule cells have nitric oxide synthase and argininosuccinate lyase, and thus may efficiently produce nitric oxide.     

Corticosterone and phenytoin reduce neuronal nitric oxide synthase messenger RNA expression in rat hippocampus.

The production and release of the corticosteroids, namely the glucocorticoids and the mineralocorticoids, are regulated by various stimuli, including stress. Previous studies from our laboratory have shown that chronic exposure to stress or to stress levels of glucocorticoids produces atrophy of the apical dendrites of CA3 pyramidal neurons in the hippocampus. This stress-induced dendritic remodeling is blocked by the anti-epileptic drug phenytoin, which suppresses glutamate release, and also by N-methyl-D-aspartate receptor antagonists. These results suggest an interaction between glucocorticoids and excitatory amino acids in the development of stress-induced atrophy of CA3 pyramidal neurons. Since nitric oxide is proposed to play an important role in mediating both the physiological and pathophysiological actions of excitatory amino acids, we examined the regulation of neuronal nitric oxide synthase messenger RNA expression by corticosterone and phenytoin in the rat hippocampus. The expression of neuronal nitric oxide synthase messenger RNA in hippocampal pyramidal neurons and granule neurons of the dentate gyrus was unaffected by 21-day administration of corticosterone (40 mg/kg), phenytoin (40 mg/kg) or the combination of corticosterone and phenytoin. However, in hippocampal interneurons, corticosterone/ phenytoin co-administration led to a significant reduction in neuronal nitric oxide synthase messenger RNA levels when compared with vehicle controls. These results suggest that, during exposure to stress levels of corticosterone, phenytoin inhibits glucocorticoid-induced atrophy of CA3 pyramidal neurons by reducing neuronal nitric oxide synthase expression in hippocampal interneurons. Moreover, these results may provide another example of synaptic plasticity in the hippocampus mediated by nitric oxide synthase.     

LPS对大鼠海马CA2～3区nNOS和FOS蛋白表达的影响

目的 探讨NO和c-fos在SD大鼠海马CA2～3区免疫调节中的作用。方法 腹腔注射LPS600μg／kg建立免疫激发模型，对照组注射等量的生理盐水，用免疫组化方法和图像分析技术，观察两组大鼠海马CA2～3区nNOS和FOS蛋白的表达，检测OD值并进行统计学分析。结果 nNOS和FOS蛋白在大鼠海马各区均有散在分布，LPS刺激组海马CA2～3区nNOS和c-fos免疫阳性产物的OD值较对照组高，差异具有统计学意义。结论海马CA2～3区可能通过NO和(或)c-fos途径参与调节LPS诱导的免疫反应过程。     

Distribution of NADPH-diaphorase and nitric oxide synthase-containing neurons in the intramural ganglia of guinea pig urinary bladder

The cell population and distribution of NADPH-diaphorase positive and NOS immunoreactive intramural ganglion cells were examined on stretched whole-mount preparations of the guinea pig urinary bladder which was divided into 3 regions: base, body and dome. The results showed that the highest frequency both of NADPH-d and NOS positive neurons was observed in the bladder base. Cell counts in the whole bladder showed that the number of NADPH-d positive neurons was much more than that of NOS immunoreactive neurons. Using neuron specific enolase (NSE) positive neurons as a reference (100%), NADPH-d positive neurons accounted for 84% while NOS immunoreactive neurons only made up 45% of the total neuronal population. These results, along with previous studies on the function of nitric oxide, suggest that nitric oxide may be involved in the relaxation activity in the bladder base during micturition. The significant difference in the number of NADPH-d positive and NOS immunoreactive neurons suggests that the localisation of one enzyme does not necessarily reflect the presence of the other.     

睫状神经营养因子对严重烫伤大鼠纹状体及海马 NOS 阳性神经元的影响

目的：观察大鼠严重烫伤后纹状体和海马ＮＯＳ阳性神经元数目和阳性反应面积的变化及睫状神经营养因子（ＣＮＴＦ）对其的影响。方法：应用ＮＡＤＰＨ－ｄ酶组织化学的方法。结果：大鼠体表烫伤后３天,纹状体ＮＯＳ阳性神经元数目明显增加,梁色呈强阳性,阳性反应面积增加。海马ＮＯＳ阳性神经元数变化不明显,仅见阳性反应面积增加,睫状神经营养因子可降低纹状体的ＮＯＳ阳性神经元数目、阳性反应面积,ＮＯＳ阳性神经元着色较淡     

局部皮质内注射海人酸对皮质和海马一氧化氮合酶阳性神经元的影响

本实验用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶组织化学方法结合ＧＦＡＰ免疫组化方法，对微量海人酸皮质内注射后的一氧化氮合酶阳性神经元和胶质细胞等的变化进行了研究．结果发现：注射区内的一氧化氮合欧阳性神经元很快消失；注射侧皮质和海马的旧阳性神经元和神经末梢溃变；至注射海人酸后８ｈ，这些变化波及到对侧皮质和海马．随着皮质内一氧化氮合酶阳性神经元的溃变，在注射侧皮质和海马内的一些神经元、星形胶质细胞出现新的一氧化氮合酶活性，这些新的酶活性可能是诱导型的一氧化氮合酶．这些结果表明：表达一氧化氮合酶的皮质和海马神经元对海人酸的毒性很敏感，神经细胞和非神经细胞中出现的诱导型的一氧化氮合酶可能是海人酸引起脑损伤时机体的适应性反应．     

肝性脑病大鼠海马齿状回神经元形态及一氧化氮合酶表达的变化

目的:观察肝性脑病模型组大鼠海马齿状回内神经元的变化及一氧化氮合酶(NOS)的表达,探讨海马神经元的形态学改变及一氧化氮(NO)在肝硬化和肝性脑病发病机制中的作用。方法:先对50只雄性大鼠进行Morris水迷宫测试,之后将动物分为正常对照组和实验模型组。9周后建立CCL4肝性脑病模型,分别取两组大鼠肝、海马组织进行HE染色、Nissl染色及NADPH-d染色。结果:(1)肉眼下可见模型组肝脏普遍呈坏死性肝硬化;(2)HE模型组血氨浓度明显高于正常对照组(P<0.05);(3)Nissl染色结果显示实验组大鼠海马神经元数目减少、染色较浅,胞浆内Nissl体减少或消失;(4)NADPH-d染色结果显示实验组可见粗大轴突着色,树突联系广泛;对照组则少有粗大轴突着色,树突间联系不如实验组广泛。实验组一氧化氮合酶(NOS)阳性神经元染色较对照组深,为紫蓝或深蓝色,且阳性神经元的数目较多。结论:(1)血氨增高是肝性脑病发病机制之一;(2)肝性脑病时海马受到损伤,并且一氧化氮(NO)可能介导了神经元的损伤。     

急性缺氧小鼠海马CAl区NOS活力和nNOS蛋白表达的时程变化

目的:观察急性缺氧小鼠海马CAl区一氧化氮合酶(NOS)和神经元型一氧化氮合酶(nNOS) 阳性神经元的时程变化,探讨NO在脑缺氧中的作用并为抗脑缺氧提供依据。方法:复制小鼠急性缺氧模型,采用NADPH-d组织化学和nNOS免疫组织化学方法,研究急性缺氧后不同时程点小鼠海马CAl区NADPH-d 和nNOS阳性神经元数量的变化。结果:与正常对照组相比较,急性缺氧后0.5h组小鼠海马CAl区NADPH-d 和nNOS阳性神经元的数量无明显变化,差异无显著性(P>0.05),3h、6h和12h组逐渐增多并于12h升高达到最高峰,差异有显著性(P<0.05),而于24h后开始降低,48h恢复正常。结论:急性缺氧后早期海马CAl区NOS和nNOS水平明显增多,NO在缺氧所致早期脑损伤中起重要作用。     

Neurodegeneration and neuroinflammation in cdk5/p25-inducible mice: a model for hippocampal sclerosis and neocortical degeneration

The cyclin-dependent kinase cdk5 is atypically active in postmitotic neurons and enigmatic among the kinases proposed as molecular actors in neurodegeneration. We generated transgenic mice to express p25, the N-terminally truncated p35 activator of cdk5, in forebrain under tetracycline control (TET-off). Neuronal expression of p25 (p25(ON)) caused high mortality postnatally and early in life. Mortality was completely prevented by administration of doxycycline in the drinking water of pregnant dams and litters until P42, allowing us to study the action of p25 in adult mouse forebrain. Neuronal p25 triggered neurodegeneration and also microgliosis, rapidly and intensely in hippocampus and cortex. Progressive neurodegeneration was severe with marked neuron loss, causing brain atrophy (40% loss at age 5 months) with nearly complete elimination of the hippocampus. Neurodegeneration did not involve phosphorylation of protein tau or generation of amyloid peptide. Degenerating neurons did not stain for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling or activated caspase-3 but were marked by FluoroJadeB in early stages. Diseased neurons were always closely associated with activated microglia already very early in the disease process. Primary neurons derived from p25 embryos were more prone to apoptosis than wild-type neurons, and they activated microglial cells in co-culture. The inducible p25 mice present as a model for neurodegeneration in hippocampal sclerosis and neocortical degeneration, with important contributions of activated microglia.     

尼古丁对PD大鼠黑质多巴胺能神经元变性的影响

目的 探讨尼古丁对帕金森病(PD)大鼠黑质多巴胺能神经元变性的影响及其机制. 方法 45只大鼠随机分为PBS对照组(CON)、生理盐水+ 脂多糖(NS)组、尼古丁+脂多糖(NIC)组,每组15只.黑质内立体定向注射脂多糖(LPS)或PBS后24h,免疫印迹法检测黑质诱导性一氧化氮合酶(iNOS)蛋白表达变化;黑质注射药物后14d,采用免疫组织化学法观察大鼠黑质酪氨酸羟化酶(TH)阳性神经元数量及OX-42阳性细胞形态学变化,RT-PCR及免疫印迹检测黑质TH mRNA及TH蛋白的表达水平. 结果 与CON组相比,NS组大鼠黑质iNOS表达明显增多,TH阳性神经元、TH mRNA及TH蛋白明显减少,小胶质细胞大多呈胞体大突起短粗的形态;NIC组黑质iNOS表达明显少于NS组,黑质TH阳性神经元、TH mRNA及TH蛋白表达较NS组明显增多,大部分小胶质细胞呈胞体小,突起细长的形态. 结论 尼古丁可以减轻LPS介导的多巴胺能神经元变性,对多巴胺能神经元有保护作用,其保护机制与抑制小胶质细胞激活、减少iNOS的表达有关.     

Ischemia-related changes of adrenocorticotropic hormone immunoreactivity and its protective effect in the gerbil hippocampus after transient forebrain ischemia

In the present study, the temporal and spatial alterations of adrenocorticotropic hormone (ACTH) immunoreactivity in the gerbil hippocampus after 5 min transient forebrain ischemia were investigated as followed up 7 days after ischemic insult, and the effects of ACTH after ischemic insult were also investigated 4 days after ischemic insult. The ectopic expression of ACTH (1-24 fragments) immunoreactive neurons in the cornus ammonis 1 (CA1) region of hippocampus and hilar region of the dentate gyrus 1 day after the ischemic insult was observed. Judging from the double immunofluorescence study, these neurons contain GABA. Four days after ischemic insult, the ACTH immunoreactivity was localized in CA1 pyramidal cells and glia near the stratum pyramidale, which normally do not express ACTH. In addition, in the saline-treated groups, the percentage of the detected Cresyl Violet positive neurons was 11.2% compared with the sham-operated group 4 and 7 days after ischemic insult. In these groups, the OX-42 immunoreactive microglia were detected in the strata pyramidale, oriens and radiatum. However, in the Org2766 (analog of ACTH)-treated group, 57.8% neurons compared with the sham-operated group were stained with Cresyl Violet 4 and 7 days after ischemic insult. In these groups, the OX-42 immunoreactive microglia were significantly reduced in the stratum pyramidale. These results suggest that transient forebrain ischemia may provoke selective ectopic and enhanced expression of ACTH in the hippocampus, and further suggest that ACTH plays an important role in reducing the ischemic damage.     

Acute and delayed restraint stress-induced changes in nitric oxide producing neurons in limbic regions

RATIONALE: Microinjection into the dentate gyrus of the hippocampus of N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME), a nitric oxide synthase (NOS) inhibitor, induces antinociceptive effect 5 days after a single restraint episode. The mechanisms of this stress-antinociceptive modulatory effect have not been investigated but may involve plastic changes in the hippocampal formation (HF). OBJECTIVE: The objective of the present study was to investigate possible mechanisms of the stress-modulating effect on antinociception induced by NOS inhibition in the hippocampus. We analyzed the effects of restraint stress on neuronal NOS (nNOS) expression and nicotinamide adenine dinucleotide phosphate-diaphorase histochemical activity (NADPH-d) in the HF and related brain regions. METHODS: Male Wistar rats (n=6-11/group) were submitted to a single (acute stress) or repeated (5 days) episodes of 2-h restraint. Control animals remained in their home cages being all animals daily handled during this period. In the fifth day, animals received unilateral microinjection of l-NAME (150 nmol/0.2 microl) or saline (control) into the dentate gyrus of the dorsal hippocampus (DG). Immediately before and after drug microinjection tail-flick reflex latency or hotplate licking reaction was measured. Animals were killed i. immediately; ii. 5 days after acute stress; or iii. after repeated stress. NADPH-d and nNOS expression were quantified in the HF, caudate-putamen, secondary somatosensorial, entorhinal and piriform cortices and amygdaloid complex. RESULTS: Five days after one or five restraint episodes l-NAME microinjection into the DG elicited antinociceptive effect (analysis of variance [ANOVA], P<0.05). Acute restraint stress induced a significant increase in the density of neurons expressing NADPH-d and nNOS in the amygdaloid nuclei. nNOS expression increased also in the DG and piriform cortex. Five days after a single or repeated restraint stress there was an additional increase in NADPH-d- and nNOS-positive neurons in CA1, CA3, and entorhinal cortex. No changes were seen in non-limbic regions such as the caudate-putamen and secondary somatosensorial cortex. CONCLUSION: The results confirm that the dorsal hippocampus participates in the modulation of stress consequences. They also show that a single stress episode causes acute changes in nitric oxide system in the amygdala complex and delayed modifications in the HF. The delayed (5 days) antinociceptive effect of NOS inhibition in the HF after a single restraint episode suggests that those latter modifications may have functional consequences. It remains to be tested if the acute amygdala and delayed hippocampal changes are causally related.     

The intra-arterial injection of microglia protects hippocampal CA1 neurons against global ischemia-induced functional deficits in rats

In the present study, we have attempted to elucidate the effects of the intra-arterial injection of microglia on the global ischemia-induced functional and morphological deficits of hippocampal CA1 neurons. When PKH26-labeled immortalized microglial cells, GMIR1, were injected into the subclavian artery, these exogenous microglia were found to accumulate in the hippocampus at 24 h after ischemia. In hippocampal slices prepared from medium-injected rats subjected to ischemia 48 h earlier, synaptic dysfunctions including a significant reduction of synaptic responses and a marked reduction of long-term potentiation (LTP) of the CA3-CA1 Schaffer collateral synapses were observed. At this stage, however, neither significant neuronal degeneration nor gliosis was observed in the hippocampus. At 96 h after ischemia, there was a total loss of the synaptic activity and a marked neuronal death in the CA1 subfield. In contrast, the basal synaptic transmission and LTP of the CA3-CA1 synapses were well preserved after ischemia in the slices prepared from the microglia-injected animals. We also found the microglial-conditioned medium (MCM) to significantly increase the frequency of the spontaneous postsynaptic currents of CA1 neurons without affecting the amplitude, thus indicating that MCM increased the provability of the neurotransmitter release. The protective effect of the intra-arterial injected microglia against the ischemia-induced neuronal degeneration in the hippocampus was substantiated by immunohistochemical and immunoblot analyses. Furthermore, the arterial-injected microglia prevented the ischemia-induced decline of the brain-derived neurotrophic factor (BDNF) levels in CA1 neurons. These observations strongly suggest that the arterial-injection of microglia protected CA1 neurons against the ischemia-induced neuronal degeneration. The restoration of the ischemia-induced synaptic deficits and the resultant reduction of the BDNF levels in CA1 neurons, possibly by the release of diffusible factor(s), might thus contribute to the protective effect of the arterial-injection of microglia against ischemia-induced neuronal degeneration.     

脑缺血后大鼠海马及纹状体一氧化氮合酶阳性神经元的变化

夹闭大鼠双侧颈总动脉２ｈ后，用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶反应观察了大鼠海马及纹状体一氧化氮合酶阳性神经元的变化及神经损伤。结果显示：在缺血损伤严重的ＣＡ１区及齿状回一氧化氮合酶阳性神经元较正常动物明显增多并深染，而在同样严重受损的纹状体一氧化氮合酶阳性神经元较正常者明显减少。结合文献及本结果提示：一氧化氮在脑缺血所致的神经损伤中起着重要作用，而海马、纹状体的一氧化氮合酶阳性神经元本身可能具有不同的抗伤害机制．     

Association study of the INPP1, 5HTT, BDNF, AP-2beta and GSK-3beta GENE variants and restrospectively scored response to lithium prophylaxis in bipolar disorder

The mesencephalic dorsolateral periaqueductal gray (dlPAG) mediates different modalities of aversive behaviors including pain and nociception and is anatomically delineated from other columns of the PAG by its content of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d). In many brain regions, neuronal NADPH-d is a nitric oxide (NO) synthase (NOS) and NO production mediates many nociceptive and aversive behavioral responses. The aim of this study was to determine how the noxious stimulant capsaicin affects intracellular dynamics in the dlPAG evidenced by Fos protein immunoreactivity (index of intracellular activation) and the NADPH-d reactivity. The basic hypothesis tested was that the effect of systemic capsaicin administration involved activation of the NO-producing machinery in the dlPAG. Compared to vehicle, capsaicin (50mg/kg, subcutaneous) significantly increased NADPH-d reactivity and Fos expression along the dlPAG neuraxis. However, less than one percent of the capsaicin-induced Fos activation occurred in NADPH-d-positive cells. This suggests that different intracellular mechanisms involving NO and activation of at least one other transmitter substance underlie the effects of capsaicin in the dlPAG. Although NADPH-d is a marker for constitutive NOS, only about two-thirds of the NADPH-d-positive neurons in the dlPAG were colocalized with neuronal NOS immunoreactive cells. This observation suggests that in contrast to other brain regions, neuronal NOS is unlikely to account for all NADPH-d activity in the dlPAG. Taken together, the present results show that the effect of capsaicin requires activation of at least one other transmitter and NADPH-d-dependent NO synthesis involving, but not limited to, the neuronal NOS isoform.