NADPH-diaphorase activity and Fos expression in brain nuclei following nitroglycerin administration

Organic nitrates are considered nitric oxide donors in that they have been shown to form nitric oxide in vitro and in vivo. Nitroglycerin is an organic nitrate which possesses peculiar activities mediated, to some extent, by the central nervous system via the noradrenergic system. Previous reports have shown that systematic nitroglycerin is able to induce Fos expression in brain nuclei which are known to contain also the nitric oxide synthesizing enzyme. Neuronal NADPH-diaphorase has been shown to be a nitric oxide synthase. Thus, in this study we used NADPH-diaphorase histochemistry to evaluate the distribution of Fos-immunoreactive cells within neurons which contain nitric oxide synthase. The data obtained showed co-localization of Fos with NADPH-diaphorase activity in numerous neurons of the paraventricular and supraoptic nuclei of the hypothalamus. In the brainstem, a few neurons were doubly labeled for Fos and NADPH-diaphorase activity, but NADPH-diaphorase positive fibers and Fos-immunoreactive neurons were consistently co-distributed in the locus coeruleus, parabrachial nucleus, nucleus tractus solitarius and spinal trigeminal nucleus caudalis. These findings demonstrate that nitroglycerin administration activates a selective group of neurons which are a source of nitric oxide or which are in close proximity with neuronal processes containing nitric oxide synthase, and suggest that the nitric oxide synthesizing pathway may be involved at various levels in the central effect of nitroglycerin.     

Nitric oxide synthase and glutamate receptor immunoreactivity in the rat spinal trigeminal neurons expressing Fos protein after formalin injection

Although recent studies implicated glutamate receptors and nitric oxide in nociception, much still needs to be known about their localisation in neurons involved in nociceptive transmission from the orofacial region. In this study, c-fos expression indicated by Fos immunohistochemistry in the caudal spinal trigeminal nucleus induced by subcutaneous injection of formalin into the lateral face of the rat was used as a marker for nociceptive neurons. The study sought to determine whether Fos-positive neurons express nitric oxide synthase, glutamate N-methyl-D-aspartate type receptor subunit 1, and glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type receptor subunit 2/3; and whether they project to the thalamus. After formalin injection, many Fos-positive nuclei appeared in the superficial laminae of the ipsilateral trigeminal nucleus. Confocal laser scanning microscope revealed that almost all neurons with Fos immunofluorescent nuclei were colocalised with N-methyl-D-aspartate receptor 1, 94% with glutamate receptor 2/3 and 14% with nitric oxide synthase. Some of them were closely related to neurons labelled by nitric oxide synthase. Lastly, some of the Fos-positive neurons were labelled by tetramethylrhodamine-dextran injected into the trigeminothalamic tract or the thalamic region. The results suggested that activation of N-methyl-D-aspartate receptor 1 and glutamate receptor 2/3 upon glutamate release in response to noxious stimulation to the orofacial region might mediate c-fos expression in neurons involved in nociception. The expression of Fos in the neurons could also be mediated by nitric oxide produced from the same, as well as neighbouring neurons, when nociceptive stimulation persisted. Fos-positive neurons in the spinal trigeminal nucleus may project to the thalamus, relaying orofacial nociception to the higher sensory centre.     

Calbindin D28k-containing neurons in the paratrigeminal nucleus receive convergent nociceptive information and project to nucleus of the solitary tract in rat

The paratrigeminal nucleus (PTN) receives orofacial somatic and visceral afferent fibers and contains many calbindin-D28k neurons (CB-containing neurons) that project to nucleus of the solitary tract (NTS). In the present study, retrograde and transganglionic tracing methods combined with immunofluorescence histochemistry and confocal laser scanning microscopy were used. After Fluoro-gold (FG) injection into the unilateral NTS, 74.4% FG-labeled neurons of ipsilateral PTN were double-labeled with CB. Furthermore, 41.0% and 32.5% FG/CB double-labeled neurons co-existed with Fos induced by nociceptive stimulation of the lips and the upper alimentary tract, respectively. In the PTN unilateral to FG injection site, 26.6% CB-LI neurons were double-labeled with PAG, 61.5% and 79.0% CB/PAG double-labeled neurons were triple-labeled with FG and Fos, and 22.9% FG/CB double-labeled neurons were triple-labeled with PAG, 84.3% FG/PAG double-labeled neurons expressed Fos induced by the upper alimentary tract stimulation. In the intact animals, 62.8% CB-LI neurons and 88.3% PAG-LI neurons co-existed with GABA(B)R, respectively. In addition, some terminals from the inferior alveolar nerve (IAN) were closely apposed to CB/Fos double-labeled or CB single-labeled neurons. These results suggested that CB-containing neurons in the PTN receive the nociceptive information converge from the orofacial area and visceral organs, and comprising the glutamatergic excitatory transmission pathway from the PTN to the NTS. This pathway might be modulated by GABA via the GABA(B) receptor.     

Expression and colocalization of NADPH-diaphorase and Fos in the subnuclei of the parabrachial nucleus in rats following visceral noxious stimulation

To investigate whether neural nitric oxide synthase (nNOS) in the parabrachial nucleus (PB) is involved in processing visceral noxious stimulation, we mapped the distribution of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a marker for nNOS, and immunohistochemical staining for Fos, a neuronal activity marker, in the subnuclei of the PB following 2% formalin injection into the stomach of rats. NADPH-d and noxious-stimuli induced Fos staining were also examined in tissue containing PB cells labeled by the retrograde transport of fluogold (FG) injected into the central nucleus of the amygdala (CeA). We found that the number of Fos immunoreactive (Fos-IR) neurons was significantly increased in the dorsal lateral (dl), external lateral (el) and K?lliker-Fuse (KF) subnuclei of the PB. We observed that intensely labeled (type 1) NADPH-d positive neurons were mainly located in the rostral part of the PB; they extended long processes adjacent Fos-IR neurons, but no Fos/type 1 NADPH-d double-labeled neurons were seen. In contrast, lightly labeled (type 2) NADPH-d positive neurons were principally localized in the dl of the PB, in which a few Fos/type 2 NADPH-d double-labeled neurons were detected. Additionally, a large number of FG/Fos double-labeled neurons were observed to be surrounded closely by the intensive NADPH-d staining in the el of the PB. These results suggest that neurons in the el of the PB that project to the CeA are activated by visceral noxious stimulation and could be indirectly influenced by nitric oxide in the PB.     

Anatomical study of the final common pathway for vocalization in the cat

Vocalization, the nonverbal production of sound, can be elicited in many vertebrates by stimulation in several regions of the limbic system but most easily in the caudal periaqueductal gray (PAG). This study shows that a specific cell group in the lateral part of the caudal PAG and in the tegmentum just lateral to it projects bilaterally to the nucleus retroambiguus (NRA) in the caudal medulla oblongata. Similar but much weaker projections are derived from the dorsal PAG. Neurons in the NRA in turn project via a contralateral pathway through the ventral funiculus of the spinal cord to motoneuronal cell groups, innervating intercostal and abdominal muscles. These projections are stronger on the contralateral side, although at lower thoracic and upper lumbar levels, many fibers recross to terminate in the ipsilateral motoneuronal cell groups. In the brainstem NRA neurons project to the motoneuronal cell groups innervating mouth-opening and perioral muscles as well as to motoneurons innervating the pharynx, soft palate, and tongue, and probably to the larynx. All these muscles are active in vocalization. The present anatomical results, combined with the physiological results of others, indicate that the projections from PAG via NRA to vocalization motoneurons form the final common pathway in vocalization. The role of this pathway in the total framework of emotional behavior is discussed.     

The medial preoptic nucleus of the hypothalamus modulates activity of nitric oxide sensitive neurons in the midbrain periaqueductal gray

Stimulation of the medial preoptic nucleus of the hypothalamus (MPO) has been shown to produce decreases in mean arterial pressure (MAP) by a pathway involving the periaqueductal gray region of the midbrain (PAG). Previous studies have shown that the injection of nitric oxide (NO) donating compounds into the dorsal PAG also decreases MAP, while the injection of nitric oxide synthase (NOS) inhibitors increases MAP. Collectively these studies suggest that the MPO elicited hypotensive response may involve NO production in PAG neurons. In this study, we investigated this hypothesis. We found that: (1) Bilateral injection of the NOS inhibitor 7-nitro indazole (7-NI) into the dorsolateral PAG cell columns produced elevations in MAP in a highly consistent and site specific fashion. (2) Microinjection of 7-NI in quantities that were too low to directly influence MAP blocked the MPO evoked hypotensive response in 9/11 cases. (3) While 41% of dorsal PAG neurons had baseline firing rates that were sensitive to 7-NI, 69% of PAG neuronal responses to MPO stimulation were blocked by 7-NI. (4) Inhibitory responses that were not blocked by 7-NI had significantly shorter latencies to onset in the presence of 7-NI. (5) PAG neurons that projected to the medulla exhibited similar electrophysiologic response patterns. Our results suggest the following: (1) The dorsolateral PAG contains a NO producing hypotensive network. (2) The MPO elicited hypotensive response may utilize this network. (3) Stimulation of the MPO elicits NO dependent responses from PAG neurons, some of which do project to medullary-cardiovascular control centers.     

Laryngeal afferent inputs during vocalization in the cat.

To reveal the kind of information about the larynx which is transmitted to the central nervous system during vocalization, we studied discharge patterns of single fibers of the laryngeal afferent nerve during electrically induced vocalization in ketamine-anesthetized cats. Recorded fibers were classified into four types based on their discharge patterns. Type A fibers responded to vocal fold vibration during vocalization. Type B fibers increased their activity during vocalization without synchronization with vocal fold vibration. Type C fibers decreased their activity during vocalization. Type D fibers discharged only at the onset of vocal fold adduction and abduction. We discuss the functional properties of these afferents and the possibility that these afferent inputs participate in the feedback control of vocalization.     

On the relation of PAG neurons to laryngeal and respiratory muscles during vocalization in the monkey

Despite evidence from previous unit recording, microstimulation, lesioning and anatomical studies, the functions of the midbrain periaqueductal gray (PAG) remain unclear. We attempted to clarify the function of the PAG by recording activity of PAG units along with laryngeal and respiratory electromyograms (EMG) during vocalization in awake monkeys. PAG units were classified with respect to vocalization on the basis of their discharge patterns as 'early burst', 'late burst', 'tonic-increase' and 'tonic-off', with the vast majority being of the early- and late-burst type. Early-burst cells were correlated most frequently with inspiratory muscles of the respiratory system and laryngeal abductor muscles. Late-burst cells were most clearly correlated with laryngeal adductor and expiratory respiratory muscles. Data from spike-triggered averaging and parametric correlations indicate that most cells are related to single muscles, but a significant number were related to functionally related groups of two or more muscles. The results suggest that the PAG determines qualitative aspects of vocalization by the multisynaptic action its cells have on laryngeal and respiratory motoneurons.     

The physiological motor patterns produced by neurons in the nucleus retroambiguus in the rat and their modulation by vagal,peripheral chemosensory,and nociceptive stimulation

The nucleus retroambiguus (NRA) is a neuronal cell group in the medullary ventrolateral tegmentum, rostrocaudally between the obex and the first cervical spinal segment. NRA neurons are premotor interneurons with direct projections to the motoneurons of soft palate, pharynx, and larynx in the nucleus ambiguus in the lateral medulla as well as to the motoneurons in the spinal cord innervating diaphragm, abdominal, and pelvic floor muscles and the lumbosacral motoneurons generating sexual posture. These NRA premotor interneurons receive very strong projections from the periaqueductal gray (PAG) in the context of basic survival mechanisms as fight, flight, freezing, sound production, and sexual behavior. In the present study in rat we investigated the physiological motor patterns generated by NRA neurons, as the result of vagal, peripheral chemosensory, and nociceptive stimulation. The results show that the NRA contains phasic respiratory modulated neurons, as well as nonphasic tonically modulated neurons. Stimulation in the various rostrocaudal levels of the NRA generates site‐specific laryngeal, respiratory, abdominal, and pelvic floor motor activities. Vagal and peripheral chemosensory stimulation induces both excitatory and inhibitory modulation of phasic NRA‐neurons, while peripheral chemosensory and nociceptive stimulation causes excitation and inhibition of nonphasic NRA‐neurons. These results are in agreement with the concept that the NRA represents a multifunctional group of neurons involved in the output of the emotional motor system, such as vomiting, vocalization, mating, and changes in respiration.     

Responses of neurons containing VGLUT3/nNOS-cGMP in the rVLM to cardiac stimulation

Responses of glutamatergic neurons in the rostral ventrolateral medulla to stimulation of cardiac sympathetic afferents have not been defined. Nitric oxide influences neural function of glutamate. We evaluated the relationship between vesicular glutamate transporter 3, c-Fos and neuronal nitric oxide synthase/soluble guanylyl cyclase in the rostral ventrolateral medulla following cardiac stimulation. In anesthetized cats with barodenervation and vagotomy, epicardial application of bradykinin, but not its vehicle, caused pressor responses. More vesicular glutamate transporter 3-containing neurons colocalized with c-Fos or c-Fos and neuronal nitric oxide synthase in the rostral ventrolateral medulla in bradykinin-treated cats (n = 6; P < 0.05) than in control animals (n = 4). Colocalization of neuronal nitric oxide synthase, soluble guanylyl cyclase and c-Fos or vesicular glutamate transporter 3 was noted following bradykinin stimulation. Findings indicate activation of rostral ventrolateral medulla glutamatergic neurons by cardiac stimulation, which may be influenced by nitric oxide via cGMP.     

Nitric oxide synthase immunoreactive neurons anatomically define a longitudinal dorsolateral column within the midbrain periaqueductal gray of the rat: analysis using laser confocal microscopy

Nitric oxide has recently been proposed as a neuronal messenger in both the central and peripheral nervous system. Antibodies against nitric oxide synthase (NOS), the synthesizing enzyme for nitric oxide, were used in combination with immunocytochemistry and confocal laser microscopy to analyze the distribution of this enzyme in the midbrain periaqueductal gray (PAG) of the rat. NOS immunoreactive neurons were localized predominantly in a longitudinally oriented column in the dorsolateral PAG. NOS immunoreactive fibers and processes were scattered throughout the PAG but were most prevalent in the dorsolateral column and in the juxta-aqueductal column. This study provides neurochemical support for the existence of longitudinal columns in the PAG which are postulated to underlie the functional organization of this complex brainstem region.     

Peripheral purinergic receptor modulation influences the trigeminal ganglia nitroxidergic system in an experimental murine model of inflammatory orofacial pain

ATP plays an important role as an endogenous pain mediator generating and/or modulating pain signaling from the periphery to the central nervous system. The aim of this study was to analyze the role of peripheral purinergic receptors in modulation of the nitroxidergic system at a trigeminal ganglia level by monitoring changes in nitric oxide synthase isoforms. We also evaluated Fos‐positive neurons in brainstem (spinal trigeminal nucleus) and pain‐related behavior. We found that local administration of the P2 purinergic receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) decreased face‐rubbing activity, nitric oxide synthase isoform expression in trigeminal ganglia, and Fos expression in spinal trigeminal nucleus after subcutaneous injection of formalin. These results suggest a role for peripheral P2 purinergic receptors in orofacial pain transmission through modulation of the nitroxidergic system. © 2010 Wiley‐Liss, Inc.     

大鼠导水管周围灰质注射谷氨酸可使Barrington核神经元表达Fos

形态学研究已发现大鼠内侧视前区（ＭＰＯ）和导水管周围灰质（ＰＡＧ）发出大量轴突,投射至与排尿反射密切相关的Ｂａｒｒｉｎｇｔｏｎ 核。本研究试图通过注射谷氨酸钠到ＭＰＯ或ＰＡＧ后,观察Ｂａｒｒｉｎｇｔｏｎ 核内的Ｆｏｓ表达情况,来了解以上两通路的性质。将谷氨酸钠注射到ＭＰＯ后,只有少量Ｆｏｓ阳性神经元出现在Ｂａｒｒｉｎｇ－ｔｏｎ 核。而将谷氨酸钠注射到ＰＡＧ后,Ｂａｒｒｉｎｇｔｏｎ 核内出现大量的Ｆｏｓ阳性神经元。此结果提示,ＰＡＧ可能对大鼠脑桥排尿反射活动具有兴奋性调节作用。     

Fos expression induced by changes in arterial pressure is localized in distinct,longitudinally organized columns of neurons in the rat midbrain periaqueductal gray

The distribution of neurons expressing Fos within the periaqueductal gray (FAG) following pharmacologically induced high or low blood pressure was examined to determine (1) if PAG neurons are responsive to changes in arterial pressure (AP) and (2) the relationship of these cells to the functionally defined hypertensive and hypotensive columns in PAG. Changes in AP differentially induced robust Fos expression in neurons confined to discrete, longitudinally organized columns within PAG. Increased AP produced extensive Fos-like immunoreactivity within the lateral PAG, beginning at the level of the oculomotor nucleus. At the level of the dorsal raphe, Fos expression induced by increased AP shifted dorsally, into the dorsolateral division of PAG; this pattern of Fos labeling was maintained throughout the caudal one-third of PAG. Double-labeling for Fos and nicotinamide adenine dinucleotide phosphate diaphorase confirmed that Fos-positive cells induced by increased AP were located in the dorsolateral division of PAG at these caudal levels. Fos positive cells were codistributed, but not colocalized, with nicotinamide adenine dinucleotide phosphate diaphorase-positive cells. Decreased AP evoked a completely different pattern of Fos expression. Fos-positive cells were predominantly located within the ventrolateral PAG region, extending from the level of the trochlear nucleus through the level of the caudal dorsal raphe. Double-labeling studies for Fos and serotonin indicated that only 1–2 double-labeled cells per section were present. Saline infusion resulted in very few Foslike immunoreactive cells, indicating that volume receptor activation does not account for Fos expression in PAG evoked by changes in AP. These results indicate that (1) substantial numbers of PAG neurons are excited by pharmacologically induced changes in AP and (2) excitatory barosensitive PAG neurons are anatomically segregated based on their responsiveness to a specific directional change in AP. © 1995 Wiley-Liss, Inc.     

Involvement of adenosine and glutamate receptors in the induction of c-fos in the striatum by haloperidol

The psychostimulant drugs amphetamine and cocaine induce the expression of immediate early genes, such as c-fos, in the striatum via D₁ dopamine receptor activation. This occurs primarily in the striato-nigral neurons. Conversely, neuroleptic drugs, such as haloperidol, which block D₂-type dopamine receptors, induce c-fos expression in striatal neurons projecting to the globus pallidus. In order to gain insight into the neurochemical substrates of neuroleptic-induced c-fos expression, we examined the effects of adenosine A₂ and N-methyl-D-aspartate (NMDA) receptor antagonists as well as inhibition of nitric oxide synthase, on haloperidol-induced Fos immunoreactivity in the striatum. While blockade of D₁ receptors had no effect on haloperidol-induced Fos expression, adenosine A₂ receptor antagonists decreased the number of neurons in the striatum expressing haloperidol-induced Fos by half. NMDA receptor antagonists also potently blocked the induction of Fos immunoreactivity by haloperidol, while inhibition of nitric oxide synthase activity had no effect. These results indicate that in the presence of a dopamine D₂ antagonist, Fos expression in striato-pallidal neurons is mediated in part through activation of A₂ receptors by adenosine, and via NMDA receptor activation by glutamate. © 1996 Wiley-Liss, Inc.     

Fos-like immunoreactivity in the periaqueductal gray of rats exposed to a natural predator

In the present study we examined, in rats exposed to a predator (cat), the distribution of neurons expressing Fos along the continuum formed by the central gray surrounding the caudal pole of the third ventricle and the periaqueductal gray (PAG). After the predatory encounter, a distinct cluster of Fos-immunoreactive cells was observed in the precommissural nucleus. In the rostral two-thirds of the PAG, Fos expression was mostly seen in the dorsomedial and dorsolateral regions. In contrast, at caudal levels of the PAG, most of the Fos-labelled neurons were distributed in the lateral and ventrolateral PAG. These results are discussed and compared with the pattern of the PAG activation after fear conditioned to a context or elicited by aversive foot shock.     

Transmission of afferent information from urinary bladder, urethra and perineum to periaqueductal gray of cat

The micturition reflex pathway is a supraspinal pathway. Anatomical tracing evidence is compatible with an involvement of the periaqueductal gray (PAG) in the ascending limb of this reflex. We tested the involvement of the PAG in receiving urinary tract- or perineum-related information and attempted to characterize this ascending path in terms of what type of information is being conveyed. Electrical stimulation of the pelvic nerves, which carry afferent information from the urinary bladder, evoked maximum field potentials in the caudal third of the PAG, primarily in the dorsal part of the lateral PAG and in the ventrolateral PAG. Since the regions activated by pelvic nerve stimulation differed from those activated by stimulation of the sensory pudendal or superficial perineal nerves, it is possible that specific pathways for different nerve inputs to the PAG exist. Sacral spinal cord neurons ascending to the PAG were identified by antidromic activation and then tested for inputs from pelvic, sensory pudendal or superficial perineal nerves. Of 18 units identified, only five received inputs from any of the peripheral nerves tested and only two projecting neurons received a pelvic nerve input. Thus the PAG may receive inputs from bladder and perineum, but the small proportion of cells with direct projections to the PAG receiving inputs from our test nerves implies that the major part of this pathway is not directly related to lower urinary tract function.     

Nitric oxide regulation of calcitonin gene-related peptide gene expression in rat trigeminal ganglia neurons

Calcitonin gene-related peptide (CGRP) and nitric oxide are involved in the underlying pathophysiology of migraine and other diseases involving neurogenic inflammation. We have tested the hypothesis that nitric oxide might trigger signaling mechanisms within the trigeminal ganglia neurons that would coordinately stimulate CGRP synthesis and release. Treatment of primary trigeminal ganglia cultures with nitric oxide donors caused a greater than four-fold increase in CGRP release compared with unstimulated cultures. Similarly, CGRP promoter activity was also stimulated by nitric oxide donors and overexpression of inducible nitric oxide synthase (iNOS). Cotreatment with the antimigraine drug sumatriptan greatly repressed nitric oxide stimulation of CGRP promoter activity and secretion. Somewhat surprisingly, the mechanisms of nitric oxide stimulation of CGRP secretion did not require cGMP or PI3-kinase signaling pathways, but rather, nitric oxide action required extracellular calcium and likely involves T-type calcium channels. Furthermore, nitric oxide was shown to increase expression of the active forms of the mitogen-activated protein kinases Jun amino-terminal kinase and p38 but not extracellular signal-related kinase in trigeminal neurons. In summary, our results provide new insight into the cellular mechanisms by which nitric oxide induces CGRP synthesis and secretion from trigeminal neurons.     

Nitric oxide mediates Fos expression in the spinal cord induced by mechanical noxious stimulation.

Immunocytochemical localization of Fos protein was used to analyze the involvement of nitric oxide (NO) in the expression of Fos in the spinal cord, induced by mechanical noxious stimulation (NS). Mechanical NS was applied to the left hindpaw 30 minutes after intrathecal administration of the NO synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME) and the resulting Fos expression in the spinal cord dorsal horn was compared with that obtained in rats exposed only to the mechanical NS. Pretreatment with L-NAME but not its stereoisomer N omega-nitro-D-arginine methyl ester (D-NAME), produced a dose-dependent suppression of Fos expression induced by mechanical noxious stimulation. These results indicate that NO modulates the expression of Fos in the dorsal horn induced by mechanical noxious stimulation and further support the hypothesis that NO is involved in nociceptive events occurring in the spinal cord in response to a peripheral noxious stimulus.     

Nitric Oxide Signalling is Required for the Generation of Anoxia-induced Long-term Potentiation in the Hippocampus

The involvement of nitric oxide in anoxia-induced long-term potentiation (anoxic LTP) of synaptic transmission was investigated in CA1 neurons of rat hippocampal slices using intracellular recording techniques in vitro . In response to superfusion of an anoxic artificial cerebral spinal fluid saturated with 95% N₂-_-5% CO₂, the excitatory postsynaptic potential (EPSP) generated in hippocampal CA1 neurons by stimulation of the Schaffer collateral/commissural afferent pathway was completely abolished within 10 min of anoxia. On return to reoxygenated medium, the EPSP returned to the control value within 10 min and was subsequently and progressively potentiated to reach a plateau 15–20 min after return to oxygen. This anoxia-induced persistent increase in synaptic transmission lasted for more than 1 h. Application of the nitric oxide synthase inhibitors 7-nitroindazole (7-NI) or l- N ^G-nitroarginine (NOARG) produced no effects on the baseline EPSP amplitude, but effectively attenuated the anoxic LTP. The inhibitory effects of both 7-NI and NOARG on the anoxic LTP were blocked by l-arginine, a substrate for nitric oxide synthase. These results suggest that nitric oxide is required for the generation of anoxia-induced LTP of glutamatergic synaptic transmission in the CA1 region of the rat hippocampus.