NADPH-diaphorase Reactivity Research Articles

Changes of c-Fos expression and NADPH-d activity in claustrum induced by chronic muscle inflammation in cat (a preliminary study)

An investigation of the central mechanisms underlying muscle inflammation and musculoskeletal pain is an important step to find means for the prevention or treatment of muscle inflammation. One of the insufficiently studied brain structures involved in the transmission of nociceptive information is the claustrum (CL). Therefore, the aim of the study was to reveal changes in the patterns of Fos-immunoreactivity and NADPH-diaphoreactivity in the nucleus claustrum (CL) and additionally in the ventral putamen (Pu) during chronic inflammation of m. gastrocnemius-soleus in cat, induced by intramuscular injection of complete Freund’s adjuvant (CFA). Immunohistochemical and histochemical techniques were used to detect Fos-immunoreactive (Fos-ir) and NADPH-diaphorase reactive (NADPH-dr) neurons within studied structures. It was revealed that nine days after CFA-induced muscle inflammation the level of Fos-immunoreactivity and NADPH-d reactivity within the CL and in the ventral part of Pu increased two-fold in comparison with the control. Because the CL is reciprocally connected with many structures of the brain cortex and subcortical structures, all these structures can be pathways of transmission of nociceptive information, nevertheless, it can be assume that the central amygdala nucleus may make the main nociceptive contribution to the activation of neurons within the CL. It is known that CL is mutually related to Pu, but it was not possible to assess their mutual influence in this study. The results of the study of the Fos-ir neurons distribution in CL and Pu under conditions of long-term muscles inflammation indicate the active involvement of the mentioned structures in the formation of adaptive reactions. The increase in the number of neurons with NADPH-d reactivity in CL and Pu indicates that NO-signals play a significant role in the formation and amplification of the response to painful impulses from inflamed muscles. In addition, further research is needed to accurately identify all possible nociceptive inputs to the CL and to separate the emotional, motor, auditory, and visual components that may accompany nociceptive processes.

Cafeteria diet-induced obesity reduces leptin-stimulated NADPH-diaphorase reactivity in the hypothalamic arcuate nucleus of rats

Leptin is an adipokine that plays an important role in the regulation of energy homeostasis. The failure of endogenous and exogenous leptin to mediate its effects (for example, at suppressing appetite and decreasing body weight) has been termed leptin resistance. Hyperleptinemia and leptin resistance can be well demonstrated in animals in which obesity is induced by consumption of a palatable, high-calorie diet (e.g., cafeteria diet-induced obesity). Since leptin receptor signaling is known to be impaired in the hypothalamic arcuate nucleus (ARC) of obese rodents, we investigated the effect of leptin on nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in the ARC of male Wistar rats with cafeteria diet-induced obesity. Our results have shown that after intraperitoneal administration of leptin, the number of NADPH-d positive neurons in the ARC was significantly lower in obese rats compared with that observed in normal weight rats. Additionally, we have found that leptin-induced NADPH-d staining in ARC neurons and the adjacent ependyma was decreased in obese rats. The results presented here suggest that the ability of leptin to activate nitric oxide synthase in neurons within the ARC as well as tanycytes and ependymal cells of the third ventricle is reduced in rats made obese by a cafeteria diet. We speculate that impairment in leptin-induced NO production presents a potential mechanism, involved in the pathogenesis of obesity and obesity-related disease states.

Effects of methylmercury on the pattern of NADPH diaphorase expression and astrocytic activation in the rat.

Mercury (Hg) is an environmental contaminant that poses great risk to human health. However, it is still widely used in artisanal gold-mining enterprises around the world, especially in developing countries. Methylmercury (MeHg) is produced environmentally by biomethylation of inorganic Hg present in water sediments, leading to its subsequent accumulation in the aquatic food chain. Due to its high metabolic rate, the Central Nervous System (CNS) is one of the main targets of MeHg. In the present study, we investigate the impact of chronic MeHg intoxication on NADPH diaphorase (NADPH-d) activity and astrocyte mobilization in the visual cortex of the rat. After 60 days of MeHg administration by oral gavage (0.04mg/kg/day), tissue samples containing the visual cortex were submitted to measurements of Hg levels, NADPH-d activity, and GFAP immunohistochemistry for identification of astrocytes. MeHg intoxication was associated with increased Hg deposits and with reduced NADPH-d neuropil reactivity in the visual cortex. A morphometric analysis suggested that NADPH-d-positive neurons were mostly spared from MeHg harmful action and intoxicated animals had astrocytic activation similar to the control group. The decrease in NADPH-d neuropil reactivity may be due to the negative effect of chronic MeHg poisoning on both the synthesis and transport of this enzyme in afferent pathways to the visual cortex. The relative resistance of NADPH-d-reactive neurons to chronic MeHg intoxication may be associated with peculiarities in cell metabolism or to a protective role of nitric oxide, safeguarding those neurons from Hg deleterious effects.

Increased NADPH-diaphorase reactivity in the hypothalamic paraventricular nucleus and tanycytes following systemic administration of leptin in rats

Leptin, a hormone mainly produced by adipocytes in proportion to fat mass, is a key component in the regulation of energy homeostasis and reproductive, neuroendocrine, immune, and metabolic functions. Leptin binds to the leptin receptor, which is expressed throughout the central nervous system but particularly in neurons of several nuclei of the hypothalamus, such as the arcuate nucleus (ARC) and paraventricular nucleus (PVN). It has been found that nitric oxide (NO) plays an important role in mediating effects of leptin. Since PVN and ARC neurons are known to express leptin receptors, we investigated the effects of leptin on nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in the PVN and ARC of male Wistar rats. Our results have shown that systemic administration of leptin resulted in increased NADPH-d positive cell number in the PVN and ARC, suggesting that both the PVN and ARC may be important centers in the hypothalamus for the leptin action, mediated by increased NO production. In addition, we have also observed that hypothalamic tanycytes in the ventral portion of the third ventricle were NADPH-d positive. We speculate that leptin may affect the release of neurohormones and hypothalamic neurogenesis by activating nitric oxide synthase in hypothalamic tanycytes.

Does hypothyroidism augment sun-induced skin damage?

ABSTRACTObjectives: We investigated the mutual effects of overt hypothyroidism and prolonged sunlight exposure on free radical accumulation and oxidative skin damage.Methods: Free radical accumulation was evaluated by monitoring the transformation of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) into MTT-formazan. The pro-oxidant enzymes xanthine oxidase (XO) and NADPH-diaphorase were measured in the skin. XO activity was estimated based on the yield of uric acid, while NADPH-diaphorase reactivity was monitored histochemically as an indirect marker of nitric oxide synthase and nitric oxide activity. Cellular damage was determined by malondialdehyde formation, a marker for lipid peroxidation.Results: In the skin of both euthyroid and hypothyroid animals, solar simulated ultraviolet irradiance increased the activity of XO and the NADPHdiaphorase reactivity as a protective response to formation of free radicals, such as reactive oxygen or nitrogen species. These pro-oxidant enzymes diminished in hypothyroid rats. Accumulation of the same amount of free radicals led to similar peroxidation in both hypothyroid and irradiated euthyroid rats. Hypothyroid skin after UV-exposure showed even greater lipid peroxidation.Discussion: The hypothyroid state could be a risk factor for enhanced oxidative skin damage in chronic photo-exposed skin due to oxidative stress. The lipid peroxidation is one of the major pathways by which photo-oxidative stress promotes photocarcinogenesis and photo-aging.

Distribution of NADPH-diaphorase reactivity in the central nervous system of the common toad (Bufo bufo)

We examined the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-reactive elements in the central nervous system (CNS) of the common toad, Bufo bufo. The investigation involved adult male and female toads collected during the breeding season. Labeled neurons of different morphological appearances (weakly or darkly stained, unipolar, bipolar, and multipolar) and fibers were observed across all subdivisions of the amphibian brain. Overall, a similar distribution of NADPH-d-labeled neurons was observed in the brain of male and female toads. In the secondary prosencephalon NADPH-d-labeled neurons were observed in the olfactory bulbs, pallial regions, nucleus accumbens, diagonal band of Broca, septum, striatum, amygdala, suprachiasmatic and magnocellular preoptic nuclei, dorsal and ventral hypothalamus. In the diencephalon, NADPH-d-positive neurons were seen in the anterior thalamic nuclei, ventromedial and ventrolateral nuclei, central and lateral thalamic nuclei, posterior tubercle, posterodorsal division of the lateral thalamic nucleus, and in the pretectal and pretoral gray. In the mesencephalon, heavily stained neurons were present in the anterodorsal and anteroventral tegmental nuclei, magnocellular, principal and laminar nuclei of the torus semicircularis, and nucleus profundus mesencephali. In the isthmus, stained cells were observed medially and ventrally in the posterodorsal and posteroventral tegmental nuclei. In the rhombencephalon, numerous NADPH-d-stained neurons were distributed in the cerebellar nucleus, sensory and descending trigeminal nuclei, motor nuclei of the glossopharyngeal and vagus nerves, the nucleus of the solitary tract, nuclei of the hypoglossal and octaval nerves, dorsal column nucleus, central gray region, and in reticular formation. However, the complete absence of NADPH-d-stained neurons in the cerebellar cortex was an unusual feature observed in this study. The widespread distribution of NADPH-d staining in diverse cell types, belonging to a variety of neuronal systems suggests a widespread role for NADPH-d in modulating diverse functions, including sensory coding in the amphibian nervous system.

Cerebral Structures Responsible for the Formation of Autonomic Reflexes Related to Realization of Motivated Operant Movements by Rats

We studied changes in the levels of Fos immunoreactivity and NADPH diaphorase reactivity in the autonomic nuclei and catecholaminergic (CA) groups of brainstem cells in rats that realized food-procuring movements by the forelimb. Fos-immunoreactivity in the medullary nuclei of the autonomic nervous system, ANS (Sol, IRt, CVL/RVL), of these animals was appreciably higher than that in control rats. Under such conditions, a considerable part of motoneurons in the brainstem autonomic motor nuclei (10, Amb, and RAmb) were activated. In the motor nuclei of the vagus nerve, large Fos-immunoreactive (Fos-ir) neurons were observed. In rats, therefore, intense autonomic reactions closely related to realization of a motor program develop during sessions of operant food-procuring movements. The expression of protein c-Fos in brainstem CA neurons during the performance of operant movements was intensified, mostly on the contralateral side. In the brainstem CA groups А5 and А6 (LC/SC), we observed a maximum number of Fos-ir neurons, as compared with the control values. These cell groups are formed mostly of noradrenergic neurons that are the main sources of descending inhibitory inputs to the spinal thorasic and lumbar intermediolateral sympathetic nuclei. Therefore, the ANS can directly influence the functioning of muscle spindles and proprioceptors and can, as a result, influence the realization of motor program.

NADPH-diaphorase reactivity and Fos-immunoreactivity within the ventral horn of the lumbar spinal cord of cats submitted to acute muscle inflammation induced by injection of carrageenan

The NADPH-diaphorase activity and Fos-immunoreactivity within the ventral horn of the lumbar spinal cord were studied in cats with acute unilateral myositis following injection of carrageenan into the m.m. gastrocnemius-soleus. In carrageenan-injected cats maximum in the mean number of intensely stained NADPH-diaphorase reactive (NADPH-dr) neurons was found in lamina VII (+100%) and VIII (+33%) of the contralateral ventral horn of the L6/L7 segments as compared with control animals. The maximumal level of Fos-immunoreactivity was registered in the same laminae with ipsilateral predominance (39.3±4.6 and 7.6±0.9 cells), in comparison with the contralateral side (13.6±0.8 and 5.5±0.6 cells, respectively; P<0.05). We also visualized low-intensely stained and double labelled (Fos immunoreactive+low-intensely stained NADPH-dr) multipolar and fusiform Renshaw-like cells (RLCs) within the ventral horn on both sides of the L6/L7 segments in carrageenan-injected cats. We visualized the double labelled (Fos-ir+NADPH-dr) multipolar and fusiform Renshaw-like cells (RLCs) within the ventral horn on both sides of the L6/L7 segments in carrageenan-injected cats. A significant difference in the mean number of RLCs was recorded between the ipsi- and contralateral sides in the lamina VII (13.6±2.5 vs. 4.9±0.7 cells, respectively). We suppose that activation of inhibitory RLCs in ipsilateral lamina VII could be directed on attenuation of activation of motoneurons during muscle pain development. Our study showed that a significant contralateral increase in the number of NADPH-dr cells is accompanied by an ipsilateral increase in c-Fos expression in lamina VII. These data may suggest that NADPH-dr neurons of the contralateral ventral horn through commissural connections also involved in the maintenance of the neuronal activity associated with acute muscle inflammation. It is also hypothesized, that during acute myositis, plastic changes in the ventral horn activate the processes of disinhibition due to an increase in the number of NADPH-d-reactive neurons in the spinal gray matter.

Blockade of 5-HT2A receptors at the site of inflammation inhibits activation of spinal dorsal horn neurons in rats.

Repeated inflammation in the periphery is a major cause of chronic inflammatory pain. We have showed that blockade of 5-HT2A receptors in the periphery inhibits repeated inflammation-induced pain hypersensitivity. The present study investigated whether inhibition of 5-HT2A receptor signaling at the site of inflammation could inhibit repeated inflammation-induced neurochemical changes in spinal dorsal horn neurons. Treatment with the 5-HT2A receptor antagonist ketanserin (20μg) in the hindpaw following intraplantar injection of carrageenan inhibited the increase in nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity, a marker of nitric oxide synthase (NOS), in the spinal dorsal horn. Administration of formalin (1%) in the hindpaw following the carrageenan injection evoked a great increase in NADPH-d reactivity in spinal dorsal horn neurons on the ipsilateral and contralateral sides. These changes were abolished by the pretreatment of ketanserin (20μg). The ketanserin treatment also reduced the repeated inflammation-induced expression of protein kinase gamma (PKCγ) in the membrane of spinal dorsal horn neurons and increased PKCγ protein level in the cytosol. Following the treatment with the opioid receptor antagonist naloxone methiodide (5mg/kg, s.c.), the administration of ketanserin failed to inhibit the repeated inflammation-induced increase in NADPH-d reactivity and c-Fos expression in the spinal dorsal horn. These results suggest that 5-HT2A receptor bioactivity in the inflammatory site plays an important role in repeated inflammation-induced central sensitization. The present study supports the notion that targeting 5-HT2A receptors in the periphery can be a promising therapy for relieving chronic inflammatory pain.

Impaired expression of neuronal nitric oxide synthase in the gracile nucleus is involved in neuropathic changes in Zucker Diabetic Fatty rats with and without 2,5-hexanedione intoxication

These studies examined the influence of 2,5-hexanedione (2,5-HD) intoxication on expression of neuronal nitric oxide synthase (nNOS) in the brainstem nuclei in Zucker Diabetic Fatty (ZDF) vs. lean control (LC) rats. Functional neuropathic changes were also investigated following axonal damage and impaired axonal transport induced by the treatment. Animals were intoxicated by i.p. injection of 2,5-HD plus unilateral administration of 2,5-HD over the sciatic nerve. The mechanical thresholds and withdrawal latencies to heat and cold stimuli on the foot were measured at baseline and after intoxication. The medulla sections were examined by nNOS immunohistochemistry and NADPH-diaphorase histochemistry at the end of the treatments. The mechanical thresholds and withdrawal latencies were significantly decreased while nNOS immunostained neurons and NADPH-diaphorase positive cells were selectively reduced in the gracile nucleus at baseline in ZDF vs. LC rats. NADPH-diaphorase reactivity and nNOS positive neurons were increased in the ipsilateral gracile nucleus in LC rats following 2,5-HD intoxication, but its up-regulation was attenuated in ZDF rats. These results suggest that diabetic and chemical intoxication-induced nNOS expression is selectively reduced in the gracile nucleus in ZDF rats. Impaired axonal damage-induced nNOS expression in the gracile nucleus is involved in neuropathic pathophysiology in type II diabetic rats.

Ontogenesis of NADPH-diaphorase positive neurons in guinea pig neocortex.

In mammalian cerebrum there exist two distinct types of interneurons expressing nitric oxide synthase (NOS). Type I neurons are large in size and exhibit heavy nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemical reaction, while type II cells are small with light NADPH-d reactivity. The time of origin of these cortical neurons relative to corticogenesis remains largely unclear among mammals. Here we explored this issue in guinea pigs using cell birth-dating and double-labeling methods. Bromodeoxyuridine (BrdU) pulse-chasing (2 doses at 50 mg/kg, 12 h apart) was given to time-pregnant mothers, followed by quantification of NADPH-d/BrdU colocalization in the parietal and temporal neocortex in offspring at postnatal day 0 (P0), P30 and P60. Type I neurons were partially colabeled with BrdU at P0, P30 and P60 following pulse-chasing at embryonic day 21 (E21), E28 and E35, varied from 2–11.3% of total population of these neurons for the three time groups. Type II neurons were partially colabeled for BrdU following pulse-chasing at E21, E28, E35 and E42 at P0 (8.6%–16.5% of total population for individual time groups). At P60, type II neurons were found to co-express BrdU (4.8–11.3% of total population for individual time groups) following pulse-chasing at E21, E28, E35, E42, E49, E56 and E60/61. These results indicate that in guinea pigs type I neurons are generated during early corticogenesis, whereas type II cells are produced over a wide prenatal time window persisting until birth. The data also suggest that type II nitrinergic neurons may undergo a period of development/differentiation, for over 1 month, before being NADPH-d reactive.

7-Nitroindazole enhances c-Fos expression in spinal neurons in rats realizing operant movements

The expression of c-Fos and NADPH-diaphorase reactivity (NADPH-dr) in the cervical spinal cord was studied in adult male Wistar rats that realized operant reflexes after inhibition of neuronal nitric oxide synthase. Fos-immunoreactive neurons were visualized immunohistochemically in the C6/C7 spinal segments in the control, realized operant movements animals, and/or 7-nitroindazole (7-NI) injected rats. The mean numbers of immunoreactive interneurons and motoneurons (per section) were significantly greater in the Nucleus proprius (+240%) and motor nuclei (+600%) in rats of the 7-NI-pretreated and operant reflex realized group than in the isolated operant reflex realized group. Our study showed intensive staining of NADPH-dr axon terminals on the somata and initial parts of dendrites of motoneurons in experimental rats when the disodium salt of malic acid was added to the staining solution. Suppression of NO release is associated with potentiation of neuronal activation induced by descending supraspinal and proprioceptive signaling within the spinal cord.

Age-related neurochemical changes in the rhesus macaque superior olivary complex.

Positive immunoreactivity to the calcium-binding protein parvalbumin (PV) and nitric oxide synthase NADPH diaphorase (NADPHd) is well documented within neurons of the central auditory system of both rodents and primates. These proteins are thought to play roles in the regulation of auditory processing. Studies examining the age-related changes in expression of these proteins have been conducted primarily in rodents but are sparse in primate models. In the brainstem, the superior olivary complex (SOC) is crucial for the computation of sound source localization in azimuth, and one hallmark of age-related hearing deficits is a reduced ability to localize sounds. To investigate how these histochemical markers change as a function of age and hearing loss, we studied eight rhesus macaques ranging in age from 12 to 35 years. Auditory brainstem responses (ABRs) were obtained in anesthetized animals for click and tone stimuli. The brainstems of the sesame animals were then stained for PV and NADPHd reactivity. Reactive neurons in the three nuclei of the SOC were counted, and the densities of each cell type were calculated. We found that PV and NADPHd expression increased with both age and ABR thresholds in the medial superior olive but not in either the medial nucleus of the trapezoid body or the lateral superior olive. Together these results suggest that the changes in protein expression employed by the SOC may compensate for the loss of efficacy of auditory sensitivity in the aged primate.

Endothelial and inducible nitric oxide synthase (NOS) immunoreactivity and NOS-associated NADPH-diaphorase histochemistry in the domestic cat (Felis catus) testis

In this study, the cellular localization of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and the endothelial (eNOS) and inducible (iNOS) forms of nitric oxide (NO) synthase in the cat testis were studied using enzyme histochemical and immunohistochemical techniques. Stage-dependent nuclear and cytoplasmic eNOS/iNOS immunoreactivity and cytoplasmic NADPH-d reactivity were found in all germ cells, including spermatogonia, primary spermatocytes (preleptotene, zygotene, and pachytene spermatocytes), and round (Sa, Sb1) and elongating spermatids (Sb2, Sc) of the seminiferous epithelium. The pachytene spermatocytes exhibited strong positive reactions at all spermatogenic stage. Interestingly, in elongated spermatids (Sd1) at stages VI to VII, eNOS and iNOS immunostainings was observed only in the cytoplasm but not in the nuclei. eNOS and iNOS immunolabeling was observed in the acrosomal vesicle of some round spermatids (Sb1) at stages I, VII, and VIII, and in the acrosomal cap of elongating spermatids (Sb2) at stage II. Furthermore, eNOS, iNOS, and NADPH-d reactions in elongated spermatids (Sd2) just before spermiation at stage VIII were restricted only to the middle and principal pieces of the tail. Positive reactions were also observed in the Sertoli and Leydig cells as well as in other tissues including vascular endothelial and smooth muscle cells and peritubular myoid cells. These results suggest that NO may play an important role in chromatin condensation, spermatid shaping, and the final release of sperm from the spermatogenic epithelium. Furthermore, NO may also be involved in spermiogenesis, steroidogenesis, and apoptotic cell death.

Effects of sciatic nerve transection on ultrastructure, NADPH-diaphorase reaction and serotonin-, tyrosine hydroxylase-, c-Fos-, glucose transporter 1- and 3-like immunoreactivities in frog dorsal root ganglion

Frogs have been used as an alternative model to study pain mechanisms. Since we did not find any reports on the effects of sciatic nerve transection (SNT) on the ultrastructure and pattern of metabolic substances in frog dorsal root ganglion (DRG) cells, in the present study, 18 adult male frogs (Rana catesbeiana) were divided into three experimental groups: naive (frogs not subjected to surgical manipulation), sham (frogs in which all surgical procedures to expose the sciatic nerve were used except transection of the nerve), and SNT (frogs in which the sciatic nerve was exposed and transected). After 3 days, the bilateral DRG of the sciatic nerve was collected and used for transmission electron microscopy. Immunohistochemistry was used to detect reactivity for glucose transporter (Glut) types 1 and 3, tyrosine hydroxylase, serotonin and c-Fos, as well as nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase). SNT induced more mitochondria with vacuolation in neurons, satellite glial cells (SGCs) with more cytoplasmic extensions emerging from cell bodies, as well as more ribosomes, rough endoplasmic reticulum, intermediate filaments and mitochondria. c-Fos immunoreactivity was found in neuronal nuclei. More neurons and SGCs surrounded by tyrosine hydroxylase-like immunoreactivity were found. No change occurred in serotonin- and Glut1- and Glut3-like immunoreactivity. NADPH-diaphorase occurred in more neurons and SGCs. No sign of SGC proliferation was observed. Since the changes of frog DRG in response to nerve injury are similar to those of mammals, frogs should be a valid experimental model for the study of the effects of SNT, a condition that still has many unanswered questions.

Expression of c-Fos Protein and Activity of NO Synthase in the Mesolimbic Dopaminergic Structures and Dorsolateral Striatum of Rats Realizing Operant Food-Procuring Reflexes

We studied the quantitative distributions of c-Fos-immunopositive (Fos-ip) and NADPH-diaphorasereactive (NADPH-dr) neurons in the mesolimbic and nigrostriatal dopaminergic (DAE) midbrain structures (at levels −5.3 to −6.04 mm caudally from the bregma) and in the dorsolateral striatum (−1.4 to −2.56 mm from the bregma) of the rat brain. Measurements were performed on intact animals, those after 24 h-long food deprivation, and rats after repetitive realization of operant food-procuring reflexes (n = 4 in all the three groups). In rats realizing operant reflexes for 30–40 min each day during 12 days, the mean number of Fos-ip neurons within frontal sections of the lateral part of the substantia nigra (SNL) significantly (P < 0.05) exceeded that in control and starving animals at all the levels examined. Such exceedance was also observed within borders of the ventral tegmental area (VTA). In rats realizing operant reflexes, Fos-ip neurons were grouped bilaterally in the mediodorsal part of the dorsolateral striatum (CPu), while distribution of the labeled cells in control and starving animals was relatively even across the entire area of frontal sections of this structure. The intensity of c-Fos expression in the CPu after realization of operant reflexes observed at the levels from −1.4 to −2.56 caudally from the bregma was significantly (P < 0.05) smaller than that in control animals. Significant numbers of NADPH-dr neurons were found only in the SNL and CPu; the mean numbers of such neurons in the examined structures showed no considerable differences in animals of all experimental groups. Relatively specific patterns of c-Fos expression and NADPH-diaphorase activity in the SN, TA, CP, and also in the central amygdalar nuclei found after realizations of operant reflex movements by the animals confirm the existence of clear correlations between motor and motivational/affective phenomena in the subcortical structures involved in the performance of complex emotionally charged motor acts.

Chronic inhibition of nitric oxide synthase activity by NG-nitro-l-arginine induces nitric oxide synthase expression in the developing rat cerebellum

Studies on chronic inhibition of NO synthase (NOS) in the CNS suggest a plastic change in NO synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor N G -nitro- l -arginine (L-NNA) was examined on NO production, NO-sensitive cGMP levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5–200 mg/kg) to 21-day old rats reduced NOS activity and NO concentration dose dependently by 70–90% and the tissue cGMP level by 60–80%. By contrast, chronic application of L-NNA between postnatal days 4–21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10–50%. Chronic treatment of 10 mg/kg L-NNA induced nNOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50 mg/kg) of L-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced iNOS expression and activity in glial cells. Treatments did not affect eNOS expression. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced chronic inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.

Chronic inhibition of nitric oxide synthase activity by NG-nitro-l-arginine induces nitric oxide synthase expression in the developing rat cerebellum

Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change in nitric oxide (NO) synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor NG-nitro-l-arginine (l-NNA) was examined on NO production, NO-sensitive cyclic guanosine monophosphate (cGMP) levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5–200mg/kg) to 21-day-old rats reduced NOS activity and NO concentration dose dependently by 70–90% and the tissue cGMP level by 60–80%. By contrast, chronic application of l-NNA between postnatal days 4–21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10–50%. Chronic treatment of 10mg/kg l-NNA induced neuronal (n)NOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50mg/kg) of l-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced inducible (i)NOS expression & activity in glial cells. Treatments did not affect endothelial (e)NOS expression. The administration of the biologically inactive isomer D-NNA (50mg/kg) or saline was ineffective. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced cronich inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.

Effects of moderate and chronic exercise on the nitrergic system and behavioral parameters in rats

Several reports suggest that nitric oxide (NO) could play a critical role on synaptic plasticity related to physical activity improving learning and memory; thus, physical exercise would have important effects on cerebral health. In order to analyze the long-term effects of chronic moderate physical training on the morphology and activity of nitrergic neurons belonging to the cerebral cortex, hippocampus and striatum, and their relationship with behavioral parameters. Wistar rats were aerobically trained (AT) up to the age of 18 months and compared to sedentary controls (SC). At the end of the training protocol behavioral parameters were analyzed in an eight-arms radial maze. Rats were sacrificed by perfusion fixation with 4% paraformaldehyde. Brains were dissected out and coronal sections containing the three mentioned areas were obtained. The neurons expressing nitric oxide synthase (NOS) were stained using the technique of NADPH-diaphorase (NADPH-d) and their morphological and densitometric parameters were quantified by image analysis. Afterwards, the isoforms of NOS were determined by immunofluorescence. Results revealed AT rats learned faster, performed less mistakes and were more successful than SC rats in the maze. The nitrergic neurons of the cerebral cortex were larger and they had an increased number of dendrites. The NADPH-d reactivity in the cortex and striatum was upregulated. Colocalization was significant for the neuronal nitric oxide synthase (nNOS), in both groups. In conclusion, moderate and chronic exercise had a positive effect on cognitive performance and anxiety related behavior. An upregulation of the nitrergic system was detected in AT rats and this fact could be involved in this beneficial action on the aged subjects.

Activation of Neurons of the Medullary Centers of the Autonomic Nervous System Related to Motivated Operant Movements Realized by Rats

Using the corresponding techniques, we visualized Fos-immunoreactive (Fos-ir) and NADPH diaphorasereactive (NADPH-dr) neurons in the medullary centers of the autonomic nervous system (ANS) of rats, which performed repetitive operant movements (catching of food globules from the manger by the left forelimb under conditions of high food motivation). Animals were trained to perform operant movements in 30 min-long everyday sessions during 12 days. The duration of a single food-procuring movement was about 600 msec. Realization of the operant reflex was accompanied by clearly expressed motivational/affective reactions. The heart rate (HR) in the course of each operant movement sharply dropped (on the 10th day of training, by 12%, on average) with subsequent recovery of this parameter within 3–4 sec. In the course of 30-min-long training sessions, the mean HR gradually decreased (in the examined group, 7%, on average) within an interval from the 5th to the 20th min with subsequent recovery until the end of the training session. The mean numbers of Fos-ir neurons in the medullary nuclei of the ANS (Sol, IRt, CVL, RVL, Amb, 10, and MdD) of rats performing food-procuring movements (n = 4) were significantly (P IRt > CVL+RVL/CVL > RVL. Large Fos-ir neurons were observed in the dorsal motor nucleus of the n. vagus (10) and in the Amb/RAmb nuclei. In a considerable proportion of neurons of the Sol and single cells of the 10 and Amb, we observed double labeling (Fos-ir + NADPH-dr). Thus, operant food-procuring movements are accompanied by episodes of bradycardia related to each separate realization; in addition, a long-lasting tonic decrease in the HR developed. These autonomic reactions are mediated by the abovementioned medullary ANS nuclei. It is supposed that the respective weakening of inhibitory sympathetic effects on spindle receptors of the muscles involved in realization of the above operant movements can provide facilitation of generation of proprioceptive impulsation, facilitation of monosynaptic spinal reflexes, and, finally, an increase in the efficacy of targeted limb movements directed toward food procurement.