NADPH-diaphorase Positive Neurons Research Articles

The Glycolysis Inhibitor 2-Deoxy-D-Glucose Exerts Different Neuronal Effects at Circuit and Cellular Levels, Partially Reverses Behavioral Alterations and does not Prevent NADPH Diaphorase Activity Reduction in the Intrahippocampal Kainic Acid Model of Temporal Lobe Epilepsy.

Temporal lobe epilepsy is the most drug-resistant type with the highest incidence among the other focal epilepsies. Metabolic manipulations are of great interest among others, glycolysis inhibitors like 2-deoxy d-glucose (2-DG) being the most promising intervention. Here, we sought to investigate the effects of 2-DG treatment on cellular and circuit level electrophysiological properties using patch-clamp and local field potentials recordings and behavioral alterations such as depression and anxiety behaviors, and changes in nitric oxide signaling in the intrahippocampal kainic acid model. We found that epileptic animals were less anxious, more depressed, with more locomotion activity. Interestingly, by masking the effect of increased locomotor activity on the parameters of the zero-maze test, no altered anxiety behavior was noted in epileptic animals. However, 2-DG could partially reverse the behavioral changes induced by kainic acid. The findings also showed that 2-DG treatment partially suppresses cellular level alterations while failing to reverse circuit-level changes resulting from kainic acid injection. Analysis of NADPH-diaphorase positive neurons in the CA1 area of the hippocampus revealed that the number of positive neurons was significantly reduced in dorsal CA1 of the epileptic animals and 2-DG treatment did not affect the diminishing effect of kainic acid on NADPH-d+ neurons in the CA1 area. In the control group receiving 2-DG, however, an augmented NADPH-d+ cell number was noted. These data suggest that 2-DG cannot suppress epileptiform activity at the circuit-level in this model of epilepsy and therefore, may fail to control the seizures in temporal lobe epilepsy cases.

A possible novel mechanism for cognitive resistance to neurodegeneration: modulation of interneuron activity in the hippocampus after exposure to enriched environments

AbstractBackgroundExposure to an enriched environment has been shown in many studies to be beneficial to brain structure and cognition by preserving neuronal integrity and strengthening the functioning and plasticity of neural circuits. These benefits derive from the added spatial, social, and sensory complexities in an enriched environment. It has been well known for a few decades that an enriched environment stimulates neurogenesis and cell proliferation in the dentate gyrus of the hippocampus, leading to an increased number of dentate granule cells (DGCs). However, the changes to other populations of cell types, particularly interneurons, has remained elusive. Interneurons are integral regulators of neurotransmission in the hippocampus. Damage to hippocampal interneurons have serious implications and lead to a decline in cognitive health. Our study sought to elucidate possible changes in interneurons affected by exposure to an enriched environment (EE) and enriched changing (EC) environment.MethodThe topographic distribution of nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPHd) positive neurons was studied in the dentate gyrus of the rat dorsal hippocampus.ResultEnvironmental enrichment increased the number of NADPHd positive neurons found in both enrichment groups. The elevated level of neurons was represented uniformly across almost all six layers of the dentate gyrus and was most significant in the expected granular cell layer (GRCL) and subgranular zone (SGZ).ConclusionThe overall preliminary results suggest that the brain has the ability to adapt to increased amounts of sensory stimulation. These changes highlight a possible new mechanism of physiological homeostasis and physical evidence of a cognitive reserve; with an increased demand for energy from new‐born DGCs and increased sensory input, the brain will regulate its energy expenditure via an increase in either the activity of existing interneurons, or the actual number of interneurons. Further research will involve running a restudy and observing the differences to the ventral hippocampus, CA1, and CA3 regions, and the effects on specific subpopulations of interneurons.

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.

Appearance and differentiation of NADPH-d-positive neurons in rat prefrontal cortex following exposure to retinoic acid

ABSTRACT Retinoic acid (RA) is a biologically active form of vitamin A. Teratogenicity has been observed in pregnant mammals exposed to high doses of vitamin A. We investigated the distribution of nitrergic neurons in rat prefrontal cortex (PFC) at developmental stages 7 days to young adulthood under physiological conditions and after prenatal application of all trans-RA. The neurons were studied histochemically using NADPH-diaphorase, which stains neurons dark blue. We found that nitrergic neurons differentiate rapidly and reach structural maturity by the end of the second week of postnatal development. We found that the processes of the neurons of nitrergic neurons of 14-day-old rats in the RA group were shorter than those of the control group. Our findings suggest that excess RA during the prenatal period may influence the development and morphology of NADPH-diaphorase positive neurons, probably by RA-specific receptors in the PFC of 14-day-old rats. RA receptors may be the main effector molecules responsible for the changes of dendrite length induced by all-trans RA. During later development, changes are not observed, probably due to maturation of the nervous system.

Response of distant regions affected by diaschisis commissuralis in one of the most common models of transient focal ischemia in rats

Stroke induces widespread changes in the brain. In this paper, we monitored some markers of early (2 h) and delayed events (1, 3 and 7 days of reperfusion) initiated by middle cerebral artery occlusion in core/penumbra counterparts of the non-ischemic hemisphere (i.e. contra-core and contra-penumbra).Our results showed that a profound transient drop (2 h and 3 days) of protein synthesis was measured in the contra-core, while the contra-penumbra exhibited translation over-activity at the same time. Glutamate release was detected only in the contra-core, with a peak on the first day. Degenerating neurons became visible in the striatum (day 1), followed by cortex (day 3), earlier in contra-penumbra and later in contra-core. Moreover, the loss of NADPH diaphorase-positive neurons in the non-ischemic hemisphere was detected, with the greatest drop at the first day. Total microglia also started to fall, the earliest in the contra-penumbra region of the striatum (day 1), followed by the contra-core of the striatum and both cortex regions at the seventh day.In conclusion, transient focal ischemia affects remote regions of the brain and initiates processes involved in neuronal degeneration in an order which corresponds to the tissue sensitivity to ischemia, namely earlier in the contra-penumbra, and afterwards in the contra-core. The mechanism of secondary damage would influence the progressive neuronal loss of more distant brain regions.

Schizandrin ameliorates ovariectomy-induced memory impairment, potentiates neurotransmission and exhibits antioxidant properties.

Schizandrin (SCH) has been reported to prevent or reduce learning and memory defects. However, it is not known whether SCH ameliorates cognitive impairments induced by oestrogen deficiency. In the present study, we investigated the effect of SCH on memory in ovariectomized (OVX) and non-OVX rats. A passive avoidance test was used to evaluate the effect of SCH on memory. Field EPSPs were recorded in hippocampal slices using an electrophysiological method. In OVX rats, biochemical parameters in the bilateral hippocampus were measured; these included superoxide dismutase (SOD), malondialdehyde (MDA) and AChE. Also, the number of NADPH-diaphorase (NADPH-d) positive neurons was counted by NADPH-d histochemistry staining technique. Oral SCH improved the memory and facilitated the induction of long-term potentiation in non-OVX and OVX rats; this effect was more obvious in OVX rats. Similarly, SCH perfusion enhanced synaptic transmission in hippocampal slices from both non-OVX and OVX rats. However, SCH perfusion reduced the ratio of paired-pulse facilitation only in OVX but not in non-OVX rats. In addition, SCH decreased AChE activity and MDA level and increased SOD activity and the number of NADPH-d-positive neurons in OVX rats. SCH improves memory in OVX rats and its potential mechanisms may include a reduction in the loss of hippocampal NADPH-d positive neurons, an increase of antioxidant properties and a potentiation of synaptic transmission that possibly involves to enhance cholinergic function. Overall, our findings indicate that SCH has potential as a therapeutic strategy for the cognitive dysfunctions associated with the menopause.

Changes in the Density of Nitrergic Neurons in the Hippocampus of Rats Following Kainic Acid and Melatonin Administration

Nitric oxide (NO) may play a role in the pathophysiology of excitotoxicity. It is also possible that increase in Ca²⁺ overload and NO-mediated events are involved in neuronal loss during excitotoxicity. Using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, we have investigated the effects of melatonin on NADPH-d positive hippocampal neurons after kainic acid (KA) induced excitotoxicity in female rats of Wistar strain. Cytosolic Ca²⁺ (free calcium) in all the respective experimental groups was also studied. Kainic acid was administered, with a single dose of 10 mg/kg/bw (body weight) to the animals. KA treated rats were given melatonin at a dose of 20 mg/kg/bw (for 14 day). On the last day of treatment, animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anaesthesia. Cryostat sections (20 microm) were cut and stained for NADPH-d positive neurons. KA exposed animals showed a significantly increased number of NADPH-d positive neurons in the dorsal and ventral blade of the dentate gyrus (DG), hilus, CA1 and CA3 area of hippocampus, with a parallel increase in intracellular free Ca²⁺ ion concentration, as compared to the control group. KA + melatonin-treated animal groups showed reduced number of NADPH-d positive neurons in DG, hilus, CA1 and CA3 areas and a decline in cytosolic Ca²⁺ concentration, as compared to KA treated group. Our study suggests that the enhanced levels of cytosolic Ca²⁺ and nitric oxide (NO) play an important role in kainate induced excitotoxicity. Inhibition of NO production may be another means whereby melatonin can reduce oxidative damage and seems to play important role in neuroprotection.

The vulnerability of nitrergic neurons to transient spinal cord ischemia: a quantitative immunohistochemical and histochemical study

Spinal cord ischemia belongs to serious and relatively frequent diseases of CNS. The aim of the present study was to find out the vulnerability of nitrergic neurons to 15 min transient spinal cord ischemia followed by 1 and 2 weeks of reperfusion. We studied neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in structural elements of lumbosacral spinal cord along its rostrocaudal axis. In addition, a neurological deficit of experimental animals was evaluated. Spinal cord ischemia, performed on the rabbit, was induced by abdominal aorta occlusion using Fogarty catheter introduced into the right femoral artery for a period of 15 min. After surgical intervention the animals survived for 7 and 14 days. nNOS-immunoreactivity (nNOS-IR) was measured by immunohistochemical and NADPHd-positivity by histochemical method, and both immunohistochemical and histochemical stainings were quantified by densitometric analyses. Neurological deficit was evaluated according Zivin's criteria. The number of nNOS-IR and/or NADPH-d positive neurons and the density of neuropil were markedly increased in superficial dorsal horn (laminae I-III) after 15 min ischemia and 7 days of reperfusion. However, ischemia followed by longer time of survival (14 days) returned the number of nNOS-IR and NADPH-d positive neurons to control. In the pericentral region (lamina X) containing interneurons and crossing fibers of spinal tracts, than in lamina VII and in dorsomedial part of the ventral horn (lamina VIII) we recorded a decreased number of nNOS-IR and NADPH-d positive neurons after both ischemia/reperfusion periods. In the medial portion of lamina VII and dorsomedial part of the ventral horn (lamina VIII) we observed many necrotic loci. This area was the most sensitive to ischemia/reperfusion injury. Fifteen minute ischemia caused a marked deterioration of neurological function of hind limbs, often developing into paraplegia. A quantitative immunohistochemical and histochemical study have shown a strong vulnerability of nitrergic neurons in intermediate zone to transient spinal cord ischemia.

Postnatal development of NADPH-diaphorase expression in the visual cortex of the golden hamster**

Nitric oxide is an important neuromodulator in the brain and is involved in the development of visual system. But it is not clear how nitric oxide and nitric oxide synthase (NOS) are involved in the developing visual cortex of rodents. Thus we examined the expression of NOS activity in the postnatal developing visual cortex of the golden hamster by using histochemical technique for NADPH-diaphorase (NADPH-d). A heavily stained NADPH-d band was observed in the neuropil of the visual cortex. This NADPH-d band initially appeared in the cortical plate from the day of birth (P0) to postnatal day 4 (P4). From P7 to P21, this band was confined to area 17 and migrated to the deeper layers III-IV and V-VI before it eventually disappeared at P28. Such developmental trends of the band correlated well with the process of formation and establishment of the geniculo-cortical projection patterns. Thus, the areal specific development of the band suggests that NOS is closely related to the cortical differentiation and synaptic formation of the primary visual cortex. On the other hand, monocular eye enucleation on P1 could not alter the appearance of this NADPH-d positive band, indicating a non-activity dependant role of NOS. In addition, differences in the laminar distributions and developmental sequence between the heavily and lightly stained NADPH-d positive neurons during development suggest that they play different roles in the development.

Postnatal development of nitrergic and cholinergic structures in rat spinal cord.

Nitric oxide (NO) is known to be a freely diffusible gaseous neurotransmitter that is not requiring synaptic connection to exert its effects. Nitric oxide synthase (NOS), the enzyme responsible for NO synthesis can be visualised by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Other neurotransmitter is a classical neurotransmitter acetylcholine (ACh), regulated by enzyme acetylcholinesterase (AChE) that hydrolyses the acetylcholine after its releasing. This work is presenting results of histochemical study of the NADPH-d and AChE expression (nitrergic and cholinergic neurons) in the spinal cord (SC) during various periods in its development. Specimens from Wistar rat pups in the age ranging from 1st to 21st postnatal days (P1-P21) have been compared with those of adult rats (P90). Transverse sections of the SC were evaluated by light microscope. In adults, the NADPH-d positivity was detectable in the neurons of superficial and deep layers of the dorsal horn, pericentral area and in the area of preganglionic autonomic nuclei. AChE positive structures were seen in the same locations as previous ones with the exception of two locations: in superficial layers of the dorsal horn AChE staining was absent, while in the ventral horn the groups of AChE positive motoneurons were found. At the perinatal period both NADPH-d and AChE positive neurons were stained from slight to moderate intensity only. During later developmental periods the staining gradually increased and achieved adult level of intensity on the day P21. Our results confirmed the presence of nitrergic and cholinergic neurons in investigated areas of the SC and indicated their fully functioning of NADPH-d and AChE positive structures in SC from the third postnatal week.

Comparative analysis of NADPH-diaphorase positive neurons in the rat, rabbit and pheasant thoracic spinal cord. A histochemical study

The distribution of NADPH-diaphorase (NADPH-d) activity was investigated and compared in the rat, rabbit and pheasant thoracic spinal cord. The investigation of all spinal cord regions (laminae) in three experimental species revealed marked differences in the distribution of NADPH-d activity. Cross sectional analysis of the spinal cord of the rat, rabbit and pheasant confirmed differences in the shape of the gray matter in all examined species. More detailed investigation of Rexed's laminas showed similar distribution of NADPH-d activity in the spinal cord of the rat and rabbit, which were different when compared with the spinal cord of the pheasant. Ventral horn of the rat and rabbit showed no labelling whereas in pheasant this area possessed a number of scattered, intensively stained neurons. In the location of autonomic preganglionic neurons, differences were found as well. In the rat there was seen a number of densely packed, clearly dark blue coloured neurons. Similarly, these neurons were present in the rabbit spinal cord but they were less numerous. No staining was found in this region of pheasant. Pericentral area (lamina X) and intermediate zone (laminaVII) revealed the presence of NADPH-d positive neurons in all examined species although they differed in number and shape of their bodies. The dorsal horn showed the presence of NADPH-d staining in all three animals but its distribution was different in medio-lateral direction. It can be suggested that observed differencies in the presence and distribution of NADPH-d activity across the examined species may reflect different fylogenetic development.

Sex differences in NADPH-diaphorase activity in the rat posterodorsal medial amygdala

The rat posterodorsal medial amygdala (MePD) is a sexually dimorphic area implicated in the control of reproduction. Interestingly, nitric oxide (NO) synthetizing neurons are widely distributed in brain regions involved with the modulation of sexual behavior. Here we studied the NADPH-diaphorase (NADPH-d) activity and the number of positive cells in the MePD of adult males and adult females either across the estrous cycle (diestrus, proestrus, estrus, and metaestrus) or following ovariectomy and substitutive therapy (consisting of oil, estradiol alone, the combination of estradiol and progesterone, or progesterone alone). The NADPH-d histochemical technique was followed by a semi-quantitative analysis using optical densitometry. Males showed a higher MePD regional optical density and neuronal optical density than females across the estrous cycle, with the exception of the diestrus phase ( P < 0.01). No differences were found in these parameters during the ovarian cycle ( P > 0.05). There were no statistically significant differences among males and cycling females in the number of NADPH-d positive cells ( P > 0.05). Additionally, no statistically significant difference was found in the regional optical density, in the neuronal optical density, or in the number of NADPH-d positive neurons when comparing the data from ovariectomized females that received vehicle or the three different hormonal replacement therapies ( P = 0.07, P = 0.18, and P = 0.95, respectively). Results suggest that NADPH-d activity in the rat MePD is different between sexes but in females it is not affected by changing levels of circulating gonadal hormones in physiological or supraphysiological conditions.

Exercise reduces inhibitory neuroactivity and protects myenteric neurons from age-related neurodegeneration

The practice of regular exercise is indicated to prevent some motility disturbances in the gastrointestinal tract, such as constipation, during aging. The motility alterations are intimately linked with its innervations. The goal of this study is to determine whether a program of exercise (running on the treadmill), during 6 months, has effects in the myenteric neurons (NADH- and NADPH-diaphorase stained neurons) in the colon of rats during aging. Male Wister rats 6 months (adult) and 12 months (middle-aged) old were divided into 3 different groups: AS (adult sedentary), MS (middle-aged sedentary) and MT (middle-aged submitted to physical activity). The aging did not cause a decline significant (p>0.05) of the number of NADH-diaphorase stained neurons in sedentary rats (AS vs. MS group). In contrast, a decline of 31% was observed to NADPH-diaphorase stained neurons. Thus, animals that underwent physical activity (AS vs. MT group) rescued neurons from degeneration caused by aging (total number, density and profile of neurons did not change with age – NADH-diaphorase method). On the other hand, physical activity augmented the decline of NADPH-diaphorase positive neurons (total number, density and profile of neurons decreased). Collectively, the results show that exercise inhibits age-related decline of myenteric neurons however, exercise augments the decline of neurons with inhibitory activity (nitric oxide) in the colon of the rats.

Somatostatin in Medium-Sized Aspiny Interneurons of Striatum is Responsible for Their Preservation in Quinolinic Acid and N-Methyl-d-Asparate-Induced Neurotoxicity

Somatostatin (SST) is a multifunctional peptide and involves in several neurodegenerative diseases. N-Methyl-D-asparate (NMDA) receptor agonist quinolinic acid (QUIN)-induced neurotoxicity mimics an experimental model of Huntington's disease that is characterized by the selective preservation of medium-sized aspiny interneurons and degeneration of medium-sized spiny projection neurons in striatum. In QUIN- and NMDA-induced neurotoxicity, increased expression of SST and messenger RNA levels along with SST release in culture medium is generally observed. However, the molecular mechanisms and the functional consequences of increased SST are still obscure. In the present study, the role of SST was determined using immunoneutralization and immunoblockade of SST in cultured striatal neurons upon QUIN- and NMDA-induced neurotoxicity. The immunoblockade of SST with antisense oligonucleotides and immunoabsorption of released SST with specific antibodies potentiate QUIN- and NMDA-induced neuronal cell death. NADPH-diaphorase positive neurons that are selectively spared in several processes of neurodegeneration result in severe damage upon immunoblockade or immunoabsorption of SST. In addition, exogenous SST along with QUIN and NMDA provides selective preservation of projection neurons, which are selectively susceptible in excitotoxicity. Neuroprotective effect of SST is completely blocked by pertussis toxins, suggesting the role of somatostatin receptors. Taken together, these results provide first evidence that the presence of SST is a unique feature for the selective sparing of medium sized aspiny interneurons in excitotoxicity.

653 Adenosine 2b (A2b)Receptor Knock Out Mice Have Impaired Colonic Motility

Aim : Adenosine receptors play an important role in the motor and secretory functions of enteric nervous system. Aim of the present study was to understand the role of adenosine 2b receptor in the gastrointestinal motility. Methods: Distal colon from age matched wild type and Adenosine 2b receptor knock out mice(A2bR KO) were stained for NADPH diaphorase neurons and scored per unit area. Distal colonic motility was examined by the bead expulsion test. Neuronally-mediated colonic relaxation and contraction was assessed by isometric muscle recording in isolated circular muscle strips from the distal colon. The role of A2b receptor in inhibiting the contraction was assessed by the addition of adenosine receptor agonist, adenosine-5'-N-ethyl carboxamide (NECA, 10 μM). Results : : A2bR KO mice showed significant decrease in the number of NADPH Diaphorase positive neurons per unit area (12.2 ± 0.54) compared to WT mice (19.03 ± 0.52, p<0.001, n=3). Consistent with this loss of nitrergic neurons, colonic relaxation was significantly reduced in the distal colon of A2b KO mice (16.97 ± 4.9%)compared to the WT (54.01 ± 5.2%) mice. Loss of nitrergic neurons can contribute to delayed colonic emptying. Distal colonic emptying, assessed by bead test, was significantly delayed in the A2bR KO mice (8.4 ± 1.0 min) as compared to the WT mice (4.98 ± 0.6 min, p<0.001, n=6). Addition of NECA caused significant reduction in the amplitude of baseline as well as EFS induced contractions (n = 3, p <0.05) in the WT mice. This inhibitory effect of NECA was not seen in the A2b KO mice, indicating the role of A2b receptor in mediating inhibitory neurotransmission in the distal colon. Conclusions : These data suggest that A2b receptor in the distal colon has a role in enteric inhibitory neurotransmission and can impact GI motility. Targeting the A2b receptor might be of therapeutic interest in treating motility disorders.

Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum

Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 approximately 30 nmol/L; calcium mobility assay) and to be 10,000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 approximately 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.

Morphological changes in the hippocampus following nicotine and kainic acid administration

Using histochemical analysis (NADPH-diaphorase, Fluoro-Jade B dye and bis-benzimide 33,342 Hoechst) we studied the influence of intraperitoneal administration of nicotine (NIC), kainic acid (KA) and combination of both these substances on hippocampal neurons and their changes. In experiments, 35-day-old male rats of the Wistar strain were used. Animals were pretreated with 1 mg/kg of nicotine 30 min prior to the kainic acid application (10 mg/kg). After two days, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the CA1 and CA3 areas of the hippocampus, in the dorsal and ventral blades of the dentate gyrus and in the hilus of the dentate gyrus. Fluoro-Jade B positive cells were examined in the same areas in order to elucidate a possible neurodegeneration. In animals exposed only to nicotine the number of NADPH-diaphorase positive neurons in the CA3 area of the hippocampus and in the hilus of the dentate gyrus was higher than in controls. In contrast, KA administration lowered the number of NADPH-diaphorase positive cells in all studied hippocampal areas and in both blades of the dentate gyrus. Massive cell degeneration was observed in CA1 and CA3 areas of the hippocampus and in the hilus of the dentate gyrus after kainic acid administration. Animals exposed to kainic acid and pretreated with nicotine exhibited degeneration to a lesser extent and the number of NADPH-diaphorase positive cells was higher compared to rats, which were exposed to kainic acid only.

Nitric Oxide in Neurodegeneration

Nitric oxide NO is an atypical neurotransmitter and is formed from L-arginine by the enzyme nitric oxide synthase (NOS), which is of three types, namely neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). nNOS and eNOS are constitutive, calcium-calmodulin dependent and release NO from neurons and endothelium, while iNOS is inducible, calcium independent and formed in activated immune cells. NO plays a modulatory role in brain, controlling release of neurotransmitters and is involved in synaptogenesis, synaptic plasticity, memory function and neuroendocrine secretion. Availability of animal models, use of NOS inhibitors and mutant mice lacking each NOS isoforms have provided evidence for the injurious effects of NO. NADPH-diaphorase staining is used to localise NOS containing neurons and 3-NT staining gives an idea of generation of NO. NO may also have some neuroprotective effects as NOS (NADPH-diaphorase positive) neurons are selectively resistant to NMDA mediated toxicity, ischaemic hypoxic insults and the degenerative processes of HD, AD, PD and ALS. Use of selective NOS inhibitors is beneficial in animal models, but has met with little or no success in the clinical treatment of neurodegenerative diseases perse. doi: 10.5214/ans.0972.7531.2007.140104

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 Kölliker–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.

The Effect of Long-Term Reduction of Aortic Blood Flow on Spinal Cord Gray Matter in the Rabbit. Histochemical Study of NADPH-Diaphorase

1. The aim of this work was to study the influence of reduced aortic blood flow on NADPH-diaphorase (NADPH-d) staining in the gray matter of L4-S3 spinal cord segments. 2. Surgery was performed on the abdominal aorta of the rabbit. Spinal cord ischemia was introduced by infrarenal aortic constriction to 30% from the normal blood flow. Animals were allowed to survive 1 week, 1 month and 3 months after surgery. Neurological outcome was studied in relation to the duration of aortic occlusion. The NADPH-d histochemistry was used for the visualisation of spinal cord sections. 3. The most affected area of the spinal cord was pericentral region, and slight changes were seen in the NADPH-d activities of both dorsal and ventral horns. One week after surgery, NADPH-d positive pericentral neurons were almost unchanged in their shape and intensity of staining, the only difference was seen in slightly increased staining of the background around the central canal. One month following surgery neurons exhibited shrinkage or were swollen, NADPH-d staining was less intensive in the pericentral zone and positively stained vessels were present. 4. Three months of ischemia influenced the NADPH-d activity: (a) In the pericentral region were seen intensively NADPH-d stained neurons almost normal in shape of their bodies but with shortened processes or without them; (b) NADPH-d staining of neuropil was greatly enhanced mostly around the central canal and in the dorsal commissure; (c) Numerous vessels were present in the pericentral zone and in the location of the ventral horn. 5. It can be concluded that the reduction of blood flow in the abdominal aorta makes most changes in the pericentral region of the rabbit spinal cord. Increased NADPH-d staining of neuropil and the presence of positively stained vessels reflect increased NADPH-d/NOS production in the spinal cord gray matter after long-term incomplete aortic occlusion.