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.     

Neuronal nitric oxide synthase in the rabbit spinal cord visualised by histochemical NADPH-diaphorase and immunohistochemical NOS methods

The NADPH-diaphorase (NADPH-d) staining method is widely used in the investigation of both the central and peripheral nervous systems. Neuronal nitric oxide synthase (nNOS) has previously been shown to be responsible for the NADPH-d activity in neurons. However, NADPH-d activity does not always fully represent the enzyme nNOS. We investigated the distribution of NADPH-d activity and nNOS protein in the rabbit spinal cord for all groups of neurons and Rexed's laminae. In most laminae the distribution of NADPH-d activity was identical to nNOS immunoreactivity. Both were present in the dorsal horn and in pericentral areas of the spinal cord, but some differences existed. The superficial part of the dorsal horn (laminae I-III) stained more intensely for NADPH-d than for nNOS. However, the most prominent difference was seen in the lateral part of the dorsal horn--the lateral collateral pathway (LCP). The LCP stained strongly for NADPH-d activity, while nNOS staining was absent. Although there is an excellent correlation between NADPH-d staining and nNOS immunohistochemical staining in the spinal cord in general, the presence of staining differences necessitates the use of immunohistochemistry for some specialized applications.     

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.     

Partial colocalization of NADPH-diaphorase and acetylcholinesterase positivity in spinal cord neurons

The freely diffusible radical, nitric oxide (NO), has been assumed to act as a retrograde signaling molecule that modulates transmitter release. Acetylcholine (ACh) is known to function as a typical neurotransmitter. In the present work we have examined the presence of both transmitters (NO and ACh) and their possible relations in the rabbit spinal cord. In our experiments we have used histochemical methods for the visualization of acetylcholinesterase (AChE) and NADPH diaphorase (NADPH-d) which label neurons that express nitric oxide synthase (NOS). Both histochemical methods were performed separately or together on the same sections of the thoracic spinal cord. NADPH-d positive dark blue stained neurons were seen mostly in superficial and deep layers of the dorsal horn, preganglionic autonomic neurons and pericentral area. The presence of AChE positive amber yellow neurons was confirmed mostly in motoneurons located in the ventral horns and in neurons of the pericentral and intermediate zone. Besides the above mentioned neurons, also double-labeled neurons were found which contained both the yellow and dark blue histochemical product. Their presence was confirmed in the intermediate zone and in the pericentral area. Thus, the co-existence of NADPH-d and AChE occurred in the location of interneurons. Our observations suggest that NO may play a role in the control of cholinergic neuronal activity and that NO can be involved in the modulation of synaptic transmission.     

Age-Related Changes of NADPH-Diaphorase Positivity in the Rat Rostral Migratory Stream

Summary Accumulating evidence confirms that nitric oxide (NO), a versatile diffusible signaling molecule, contributes to controling of adult neurogenesis. We have previously shown the timing of NADPH-diaphorase (NADPH-d) positivity within the rat rostral migratory stream (RMS) during the first postnatal month. The present study was designed to describe further age-related changes of NO presence in this neurogenic region. The presence of NO synthesizing cells in the RMS was shown by NADPH-d histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. The phenotypic identity of nitrergic cells was examined by double labeling with GFAP and NeuN. Systematic qualitative and quantitative analysis of NADPH-d-positive cells was performed in the neonatal (P14), adult(5 months) and aging (20 months) rat RMS. 1. Nitrergic cells with different distribution pattern and morphological characteristics were present in the RMS at all ages examined. In neonatal animals, small, moderately stained NADPH-d-positive cells were identified in the RMS vertical arm and in the RMS elbow. In adult and aging rats a few labeled cells could be also detected in the RMS horizontal arm. NADPH-d-positive cells in adult and aging rats were characterized by long varicose processes and displayed dark labeling in comparison to the neonatal group. 2. Double immunolabeling has revealed that nNOS-immunoreactivity co-localized with that of NeuN. This indicates that nitrergic cells within the RMS are neurons. 3. Quantitative analysis showed that the number of NADPH-d-positive cells increases with advancing age. The presence of NO producing cells in the RMS of neonatal adult and aging rats indicates, that this proliferating and migratory area is under the influence of NO throughout the entire life of the animals.     

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.     

Immunohistochemical characterisation of dorsal root ganglia neurons supplying the porcine mammary gland

The present study investigated the chemical coding of mammary gland-projecting dorsal root ganglia (DRG) neurons using double-labelling immunohistochemistry. Earlier investigations revealed the presence of Fast blue - positive (FB+) neurons in Th9-Th12 DRG after injection of the tracer into the second, right thoracic mamma. Neurons projecting to the last right abdominal mamma were found in L1-L3 DRG. In the present study, the cryostat sections from these ganglia were stained for calcitonin gene-related peptide (CGRP), substance P (SP), nitric oxide synthase (NOS), galanin (GAL) and pituitary adenylate cyclase activating polypeptide (PACAP). Immunohistochemistry revealed that the vast majority of FB+ mammary gland-projecting neurons contained immunoreactivity to CGRP (68.87±0.7%), SP (63.4±0.9%), NOS (32.47±0.9%), GAL (16.28±0.8%) and less numerous nerve cells stained for PACAP (5.87±0.5%). The present results largely correspond with findings dealing with immunohistochemical characterization of nerve fibres supplying porcine mammary gland structures described earlier.     

Distribution of NADPH-diaphorase and nitric oxide synthase in the trigeminal ganglion and mesencephalic trigeminal nucleus of the cat. A histochemical and immunohistochemical study

The trigeminal ganglion (TrG) and mesencephalic trigeminal nucleus (MTN) neurons are involved in the transmission of orofacial sensory information. The presence of nitric oxide (NO), a putative neurotransmitter substance in the nervous system, was examined in the cat TrG and MTN using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry. In the TrG, where the majority of the trigeminal primary afferent perikarya are located, most of the intensely NADPH-d/ NOS-stained cells were small in size and distributed randomly throughout the ganglion. The medium-sized neurons were moderately stained. A plexus of pericellular varicose arborizations around large unstained ganglion cells and densely stained fibers in-between could also be observed. In the caudal part of the MTN, both NADPH-d activity and NOS immunoreactivity was present in MTN neurons. In addition, a few scattered NADPH-d/NOS-containing neurons were found in the mesencephalic-pontine junction part of the nucleus. In contrast, only nerve fibers and their terminals were present at a more rostral level in the mid- and rostral MTN. MTN neuronal perikarya were enveloped in fine basket-like NADPH-d/ NOS-positive networks. Differential expression patterns of NOS and its marker NADPH-d suggest that trigeminal sensory information processing in the cat MTN is controlled by nitrergic input through different mechanisms. We introduce the concept that NO can act as a neurotransmitter in mediating nociceptive and proprioceptive information from periodontal mechanoreceptors but may also participate in modulating the activity of jaw-closing muscle afferent MTN neurons.     

磁场对丘脑下部一氧化氮含量的影响及与丘脑下部神经内分泌核 …

By employing nitric acid reductase-spectrophotometry and NADPH-diaphorase/AVP cytochemistry technique, the effects of magnetic field on NO in hypothalamus and relations to Paraventricular Nucleus (PVN), Periventricular Nucleus (PEN), Supraoptic Nucleus (SON) and Suprachiasmatic Nucleus (SCN) were investigated. It was found that the NADPH-d positive neurons and some NADPH-d/AVP dually stained neurons existed in PVN, PEN, SON, but not in SCN, and the magnetic field induced NO (OD) increase there and the high NO (OD) level lasted for 3 hours. The results suggested that NO (OD) increase after the treatment of magnetic field in hypothalamus may result from strong expression of NOergic neurons in the PVN, PEN and SON. The coexistance of NO and AVP may play important role in the regulation of endocrine and neuroendocrine by the magnetic field.     

磁场对丘脑下部一氧化氮含量的影响及与丘脑下部神经内分泌核团的关系

应用硝酸还原酶反应—分光光度法测定和NADPH-d组织化学技术,对磁场处理后丘脑下部一氧化氮量的变化及其可能的原因进行了研究,发现磁场可促使丘脑下部一氧化氮量(OD值)显著升高,并具有显著滞后效应。NADPH-d阳性神经细胞及NADPH-d和血管加压素(AVP)双染阳性神经细胞集中分布在丘脑下部室旁核、室周核和视上核,但不存在 于视交叉上核,提示室旁核、室周核和视上核一氧化氮能神经细胞是丘脑下部的一氧化氮的主要来源。磁场处理后大鼠丘脑下部一氧化氮含量(OD值)较正常对照组显著升高应归因于这些神经细胞受磁场作用表达增强。一氧化氮和血管加压素的共存可能对磁场调节内分泌具有一定意义。     

鞘内注射神经激肽-1受体激动剂Sar-SP增强大鼠脊髓一氧化氮合酶表达和一氧化氮生成

探讨P物质(substance P,SP)对脊髓一氧化氮合酶(nitric oxide synthase,NOS)表达和一氧化氮(nitric oxide,NO)生成的影响。实验用热甩尾法测定大鼠痛阈的变化,分别应用NADPH-d组织化学法和硝酸还原法测定大鼠脊髓内NOS表达和NO生成的变化。结果显示,鞘内注射神经激肽-1受体(neurokinin-1 receptor,NK-1)激动剂[Sar^9,Met(O2)^11]-substance P(Sar-SP)可使大鼠痛阈降低,脊髓后角浅层和中央管周围灰质内NOS表达增强,脊髓腰膨大部位NO生成增多;预先鞘内注射非选择性NK-1受体拮抗剂[D—Arg^1,D-Trp^7,9,Leu^11]-substance P(spantide)可抑制上述变化。结果表明,SP可促进脊髓内NOS表达和NO生成。     

Localization and characterization of nitric oxide synthase at the frog neuromuscular junction

Summary. The frog neuromuscular junction is sensitive to nitric oxide (NO), since exogenously applied NO reduces the release of transmitter by presynaptic terminals and the size of ATP-induced Ca²⁺ responses in perisynaptic Schwann cells. This study aimed at determining whether an NO synthase (NOS) is present at the neuromuscular junction, notably in perisynaptic Schwann cells, the glial cells at this synapse. The NADPH-diaphorase (NADPH-d) histochemical technique revealed the presence of NOS in cell bodies and presumed processes of perisynaptic Schwann cells. Incubation with NOS inhibitors, N^G-nitro-L-arginine methyl ester or N^G-monomethyl-L-arginine-acetate, abolished the NADPH-d staining. Moreover, L-arginine, the precursor of NO, impeded the blockade by NOS inhibitors, establishing the NOS specificity of NADPH-d staining in frog tissue. The pattern of labelling with a polyclonal antibody against the neuronal form of NOS was similar to the NADPH-d staining, also suggesting the presence of a neuronal NOS in perisynaptic Schwann cells. Using electron microscopy, the NOS immunostaining was found at the membrane and occasionally in the cytoplasm of perisynaptic Schwann cells and was not detected in the nerve terminal or muscle. There was no enzymatic or immunocytochemical labelling of NOS 6 days after denervation. It is concluded that NOS is present in frog perisynaptic Schwann cells. The presence of this endogenous NOS suggests that NO may act as a diffusible glial messenger to modulate synaptic activity and synapse formation at the neuromuscular junction.     

Localization of nitric oxide synthase in rat trigeminal primary afferent neurons using NADPH-diaphorase histochemistry

Summary Nitric oxide (NO) is a ubiquitous gaseous neurotransmitter that has been ascribed to a large number of physiological roles in sensory neurons. It is produced by the enzyme nitric oxide synthase (NOS). To identify the NOS-containing structures of rat trigeminal primary afferent neurons, located in the trigeminal ganglion (TrG) and mesencephalic trigeminal nucleus (MTN), histochemistry to its selective marker nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) was applied in this study. In the TrG approximately half of the neuronal population was NADPH-d reactive. Strongly positive were neurons mainly of small-to-medium size. Neuronal profiles of large diameter were less intensely stained. In addition, NADPH-d-positive nerve fibers were dispersed throughout the ganglion. Nitrergic neurons were located in the caudal part and mesencephalic-pontine junction of the MTN. Most of them were large-sized pseudounipolar cells. In a more rostral aspect, the reactive psedounipolar MTN profiles gradually decreased in number and intensity of staining. There, only a fine meshwork of stained thin fibers and perisomatic terminal arborizations, and also some isolated perikarya of NADPH-d stained multipolar MTN neurons, were observed. The predominant NADPH-d localization in smaller in size TrG neurons, which are considered nociceptive, suggests that NO may play a role in the pain transmission in the rat trigeminal afferent pathways. In addition, the wide distribution of NADPH-d activity in large pseudounipolar and certain multipolar MTN neurons provides substantial evidence that NO may also participate in mediating proprioceptive information from the orofacial region. The differential expression patterns of nitrergic fibers in the TrG and MTN suggest that trigeminal sensory information processing is controlled by nitrergic input through different mechanisms.     

Sustained neurochemical plasticity in central terminals of mouse DRG neurons following colitis

Sensitization of dorsal root ganglia (DRG) neurons is an important mechanism underlying the expression of chronic abdominal pain caused by intestinal inflammation. Most studies have focused on changes in the peripheral terminals of DRG neurons in the inflamed intestine but recent evidence suggests that the sprouting of central nerve terminals in the dorsal horn is also important. Therefore, we examine the time course and reversibility of changes in the distribution of immunoreactivity for substance P (SP), a marker of the central terminals of DRG neurons, in the spinal cord during and following dextran sulphate sodium (DSS)-induced colitis in mice. Acute and chronic treatment with DSS significantly increased SP immunoreactivity in thoracic and lumbosacral spinal cord segments. This increase developed over several weeks and was evident in both the superficial laminae of the dorsal horn and in lamina X. These increases persisted for 5 weeks following cessation of both the acute and chronic models. The increase in SP immunoreactivity was not observed in segments of the cervical spinal cord, which were not innervated by the axons of colonic afferent neurons. DRG neurons dissociated following acute DSS-colitis exhibited increased neurite sprouting compared with neurons dissociated from control mice. These data suggest significant colitis-induced enhancements in neuropeptide expression in DRG neuron central terminals. Such neurotransmitter plasticity persists beyond the period of active inflammation and might contribute to a sustained increase in nociceptive signaling following the resolution of inflammation.     

Moderately Different NADPH-Diaphorase Positivity in the Selected Peripheral Nerves after Ischemia/ Reperfusion Injury of the Spinal Cord in Rabbit

1. To vicariously investigate the nitric oxide synthase (NOS) production after spinal cord injury, NADPH-d histochemistry was performed on the selected peripheral nerves of adult rabbits 7 days after ischemia. The effect of transient spinal cord ischemia (15 min) on possible degenerative changes in the motor and mixed peripheral nerves of Chinchilla rabbits was evaluated.2. The NADPH-diaphorase histochemistry was used to determine NADPH-diaphorase activity after ischemia/reperfusion injury in radial nerve and mediane nerve isolated from the fore-limb and femoral nerve, saphenous nerve and sciatic nerve separated from the hind-limb of rabbits. The qualitative analysis of the optical density of NADPH-diaphorase in selected peripheral nerves demonstrated different frequency of staining intensity (attained by UTHSCSA Image Tool 2 analysis for each determined nerve).3. On the seventh postsurgery day, the ischemic spinal cord injury resulted in an extensive increase of NADPH-d positivity in isolated nerves. The transient ischemia caused neurological disorders related to the neurological injury—a partial paraplegia. The sciatic, femoral, and saphenous nerves of paraplegic animals presented the noticeable increase of NADPH-d activity. The mean of NADPH-diaphorase intensity staining per unit area ranged from 134.87 (±32.81) pixels to 141.65 (±35.06) pixels (using a 256-unit gray scale where 0 denotes black, 256 denotes white) depending on the determined nerve as the consequence of spinal cord ischemia. The obtained data were compared to the mean values of staining intensity in the same nerves in the limbs of control animals (163.69 (±25.66) pixels/unit area in the femoral nerve, 173.00 (±32.93) pixels/unit area in saphenous nerve, 186.01 (±29.65) pixels/unit area in sciatic nerve). Based on the statistical analysis of the data (two-way unpaired Mann–Whitney test), a significant increase (p≤0.05) of NADPH-d activity in femoral and saphenous nerve, and also in sciatic nerve (p≤0.001) has been found. On the other hand, there was no significant difference between the histochemically stained nerves of fore-limbs after ischemia/reperfusion injury and the same histochemically stained nerves of fore-limbs in control animals.4. The neurodegenerative changes of the hind-limbs, characterized by damage of their motor function exhibiting a partial paraplegia after 15 min spinal cord ischemia and subsequent 7 days of reperfusions resulted in the different sensitivity of peripheral nerves to transient ischemia. Finally, we suppose that activation of NOS indirectly demonstrable through the NADPH-d study may contribute to the explanation of neurodegenerative processes and the production of nitric oxide could be involved in the pathophysiology of spinal cord injury by transient ischemia.     

Localization of nitric oxide synthase in canine ileocolonic and pyloric sphincters

The distribution of neurons containing NADPH-diaphorase (NADPH-d) activity and nitric oxide synthase-like immunoreactivity (NOS-LI) in the canine pyloric and ileocolonic sphincters was studied. Cells within the myenteric and submucosal ganglia were positive for NADPH-d. These cells generally had the morphology of Dogiel type-I enteric neurons, however, there was some diversity in the morphology of NADPH-d-positive neurons in the myenteric plexus of the pylorus. Intramuscular ganglia were observed in both sphincters, and NADPH-d was found in a sub-population of neurons within these ganglia. Dual staining with an antiserum raised against nitric oxide synthase (NOS) demonstrated that almost all cells with NOS-LI were also NADPH-d positive. Varicose fibers within ganglia and within the circular and longitudinal muscle layers also possed NOS-LI and NADPH-d activity. Dual staining with anti-VIP antibodies showed that some of the NADPH-d-positive cells in the myenteric and submucosal ganglia also contained VIP-LI, but all VIP-LI-positive cells did not express NADPH-d activity. These data are consistent with recent physiological studies suggesting that nitric oxide serves as an inhibitory neurotransmitter in the pyloric and ileocolonic sphincters. The data also suggest that VIP is expressed in a sub-population of NADPH-d-positive neurons and may therefore act as a co-transmitter in enteric inhibitory neurotransmission to these specialized muscular regions.     

Expression and colocalization of NADPH-diaphorase and heme oxygenase-2 in trigeminal ganglion and mesencephalic trigeminal nucleus of the rat

Carbon monoxide (CO) and nitric oxide (NO) are two endogenously produced gases that can function as second messenger molecules in the nervous system. The enzyme systems responsible for CO and NO biosynthesis are heme oxygenase (HO) and nitric oxide synthase (NOS), respectively. The present study was undertaken to examine the distribution of HO-2 and NOS of the trigeminal primary afferent neurons of the rat, located in the trigeminal ganglion (TG) and mesencephalic trigeminal nucleus (MTN), using histochemistry and immunohistochemistry. NADPH-d staining was found in most neurons in TG. The intensely NADPH-d-stained neurons were small- or medium-sized, while the large-sized neurons were less intensely stained. Immunocytochemistry for HO-2 revealed that almost all neurons in TG expressed HO-2, but they did not appear cell size-specific pattern. NADPH-d and HO-2 positive neurons appeared the same pattern, which was NADPH-d activity and HO-2 expression progressively declined from the caudal to rostral part of the MTN. A double staining revealed that the colocalization of NADPH-d/HO-2 neurons was 97.3% in TG and 97.6% in MTN. The remarkable parallels between NADPH-d and HO-2 suggest that NO and CO are likely neurotransmitters and mediate the orofacial nociception and sensory feedback of the masticatory reflex arc together.     

Colocalization of neuropeptides and NADPH-diaphorase in the intra-adrenal neuronal cell bodies and fibres of the rat

Colocalization of vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and tyrosine hydroxylase, respectively, with NADPH-diaphorase staining in rat adrenal gland was investigated using the double labelling technique. All vasoactive intestinal peptide- and some neuropeptide Y-immunoreactive intrinsic neuronal cell bodies seen in the gland were double stained with NADPH-diaphorase. Double labelling also occurred in some nerve fibres immunoreactive to vasoactive intestinal peptide and neuropeptide Y in the medulla and cortex. No colocalization of calcitonin gene-related peptide, substance P or tyrosine hydroxylase immunoreactivity with NADPH-diaphorase staining was observed. However, nerve fibres with varicosities immunoreactive for all the neuropeptides examined were closely associated with some of the NADPH-diaphorase-stained neuronal cell bodies. Thus, in rat adrenal gland, nitric oxide is synthesized in all ganglion cells containing vasoactive intestinal peptide and in some containing neuropeptide Y, but not in those containing calcitonin gene-related peptide, substance P or tyrosine hydroxylase.     

Comparison of NADPH diaphorase activity in the brains of hamsters infected with scrapie strains 139H or 263K or with normal hamster brain homogenate

Previous studies showed that the histopathological changes found in the brains of scrapie-infected animals included amyloid plaque formation, vacuolation, gliosis and neuronal and neurite degeneration. There were differences in the histopathological findings as a function of the scrapie strain-host combination. NADPH-diaphorase (NADPH-d) has been shown to be a selective histochemical marker for neurons containing nitric oxide (NO) synthase. Neuronal cell damage caused by NOS in brain has been reported to be associated with many neurodegenerative diseases. In this study, we used NADPH-d histostaining to investigate changes in the NOS system in brains of 139H- and 263K-infected hamsters and compared the results to normal hamster brain (NHB) injected animals. We observed that some of the NADPH-d histostaining neurons in the cortex of scrapie-infected hamsters appeared to be atrophic: the neurons were smaller and had fewer neurites. The NADPH-d histostaining intensity of neurons or astrocytes in septum, thalamus, hypothalamus and amygdala of 139H- and 263K-infected hamsters was greater than in control hamsters. Astrocytes in the thalamus, hypothalamus and lower part of the cortex (layers 4 to 6) in 263K-infected hamsters were more intensely stained for NADPH-d than in either 139H-infected hamsters or controls. Our results suggest that changes in NADPH-d system might play a role in the diversity of scrapie induced neurodegenerative changes.     

Co-administration of Monosialoganglioside and Skeletal Muscle Cells on Dorsal Root Ganglion Neuronal Phenotypes In Vitro

The neuropeptide-immunoreactive (IR) and neurofilament-IR neurons are two major phenotypical classes in dorsal root ganglion (DRG). Targets of neuronal innervation play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. Monosialoganglioside (GM1) has been considered to have a neurotrophic factor-like activity. Both GM1 and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons. However, whether target SKM cells and GM1, alone or associated, generate neuropeptide or neurofilament expression remains unclear. The aim of the present study is to investigate the effects of GM1 or/and SKM on DRG neuronal phenotypes. DRG neurons containing the neuropeptide substance P (SP) and neurofilament 200 (NF-200) were quantified using immunofluorescent labeling in cultures of DRG, which was dissected out at times before (at embryonic days 12.5, E12.5) and after (at E19.5) sensory neurons contact peripheral targets in vivo. DRG neurons were cultured in absence or presence of GM1 or/and SKM cells. In this experiment, we found that: (1) GM1 promoted expression of SP and NF-200 in E12.5 DRG cultures; (2) SKM cells promoted expression of NF-200 but not SP in E12.5 DRG cultures; (3) GM1 and target SKM cells had additive effects on expression of SP and NF-200 in E12.5 DRG cultures; and (4) SKM or/and GM1 did not have effects on expression of SP and NF-200 in E19.5 DRG cultures. These results suggested that GM1 could influence DRG, two major neuronal phenotypes, before sensory neurons contact peripheral targets in vivo. Target SKM cells could only influence neurofilament-expressed neuronal phenotype before sensory neurons contact peripheral targets in vivo. GM1 and SKM cells had the additive effects on two major DRG neuronal classes, which express neuropeptide or neurofilament when DRG cells were harvested before sensory neurons contact peripheral targets in vivo. These results offered new clues for a better understanding of the association of GM1 or/and SKM with neuronal phenotypes.