Histochemistry of nitric oxide synthase in the nervous system

Summary Nitric oxide synthase, which generates the physiological messenger molecule nitric oxide, and its associated NADPH diaphorase (NADPHd) activity are distributed throughout selective neuronal populations of the central and peripheral nervous system. Considerable evidence has been accumulated to indicate that NADPHd activity labels cells lacking neuronal nitric oxide synthase, i.e., the specificity of the reaction has to be considered for the reliable detection of the enzyme in neuronal but also non-neuronal tissue. In the present review, critical aspects of nitric oxide synthase visualization in neurones, using its NADPHd activity, are discussed. Furthermore, the organization of the central and peripheral nitric oxide synthase-containing neuronal systems is described. Nitric oxide synthase is present in local cortical and striatal neurones, hypothalamic magnocellular neurones, mesopontine cholinergic neurones, cerebellar interneurones, preganglionic sympathetic and parasympathetic neurones, neurones in parasympathetic autonomic and enteric ganglia and primary viscero-afferent neurones. Finally, injury-related alterations in nitric oxide synthase activity are briefly outlined. In this respect, the histochemistry of nitric oxide synthase may represent a valuable marker for neurochemical, if not structural, alterations observed in neural diseases, regeneration and transplantation.     

Distribution of SchistoFLRFamide-like immunoreactivity in the adult ventral nervous system of the locust,Schistocerca gregaria

SchistoFLRFamide (PDVDHVFLRF-NH₂) is one of the major endogenous neuropeptides of the FMRF-amide family found in the nervous system of the locust,Schistocerca gregaria. To gain insights into the potential physiological roles of this neuropeptide we have examined the distribution of SchistoFLRFamide-like immunoreactivity in the ventral nervous system of adult locusts by use of a newly developed N-terminally specific antibody. SchistoFLRFamide-like immunoreactivity in the ventral nerve cord is found in a subgroup of the neurones that are immunoreactive to an antiserum raised against bovine pancreatic polypeptide (BPP). In the suboesophageal ganglion three groups of cells stain, including one pair of large posterior ventral cells. These cells are the same size, in the same location in the ganglion and have the same branching pattern as a pair of BPP immunoreactive cells known to innervate the heart and retrocerebral glandular complex of the locust. In the thoracic and abdominal ganglia two and three sets of cells, respectively, stain with both the SchistoFLRFamide and BPP antisera. In the abdominal ganglia the immunoreactive cells project via the median nerves to the intensely immunoreactive neurohaemal organs.     

Nitric oxide: a co-modulator of efferent peptidergic neurosecretory cells including a unique octopaminergic neurone innervating locust heart

Our findings suggest that nitric oxide (NO) acts as peripheral neuromodulator in locusts, in which it is commonly co-localized with RF-like peptide in neurosecretory cells. We also present the first evidence for NO as a cardio-regulator in insects. Putative NO-producing neurones were detected in locust pre-genital free abdominal ganglia by NADPH-diaphorase histochemistry and with an antibody against NO synthase (NOS). With both methods, we identified the same 14 somata in each examined ganglion: two dorsal posterior midline somata; six ventral posterior midline somata; and three pairs of lateral somata. A combination of NOS-detection methods with nerve tracing and transmitter immunocytochemistry revealed that at least 12 of these cells were efferent, of which four were identified as peptidergic neurosecretory cells with an antiserum detecting RFamide-like peptides. One of the latter was unequivocally identified as an octopaminergic dorsal unpaired median (DUM) neurone, which specifically projected to the heart (“DUM-heart”). Its peripheral projections revealed by axon tracing appeared as a meshwork of varicose endings encapsulating the heart. NOS-like immunoreactive profiles were found in the heart nerve. NO donors caused a dose-dependent increase in heart rate. This cardio-excitatory effect was negatively correlated to resting heart rate and seemed to be dependent on the physiological state of the animal. Hence, NO released from neurones such as the rhythmically active DUM-heart might exert continuous control over the heart. Possible mechanisms for the actions of NO on the heart and interactions with other neuromodulators co-localized in the DUM-heart neurone (octopamine, taurine, RF-amide-like peptide) are discussed.P.A.S. is the leading author, following the tragic death of Alexander Bullerjahn on 20.12.03.This work was supported by the Deutsche Forschungsgemeinschaft (grant number SFB 515; project of H.-J.P.).     

An FMRFamide antiserum differentiates between populations of antigens in the ventral nervous system of the locust,Schistocerca gregaria

Summary The distribution of FMRFamide immunoreactive neurones in the ventral nerve cord of the locust, Schistocerca gregaria, is described. These neurones are found only in the suboesophagael and thoracic ganglia, although immunoreactive processes are found in the neuropils of the abdominal ganglia. Many of these neurones also react with an antiserum raised against bovine pancreatic polypeptide (BPP), but this antiserum also reveals another population of cells in the abdominal ganglia. The staining obtained with the BPP antiserum is blocked by preabsorption of the antiserum with FMRFamide; the converse is not true: FMRFamide-like immunoreactivity is not suppressed by preincubation with BPP. These results suggest that there are at least two endogenous peptide antigens in the locust nerve cord: one is found in cells of the suboesophageal and thoracic ganglia, and the other is found in cells of the abdominal ganglia.     

NADPH-diaphorase histochemistry in the terminal abdominal ganglion of the crayfish

Nitric oxide (NO) has an important modulatory role on the processing of sensory signals in vertebrates and invertebrates. In this investigation we studied the potential sources of NO in the terminal abdominal ganglion of the crayfish, Pacifastacus leniusculus, using NADPH-diaphorase (NADPHd) histochemistry, with NADPHd acting as a marker for NO synthase (NOS). In the terminal ganglion a mean of 27 strongly labelled NADPHd-positive cell bodies were found, and of these 80% [of stained cell bodies] [corrected] occurred in three regions located in antero-lateral, central and posterior parts of the ganglion. Ventral and antero-ventral commissures as well as specific dorsal and ventral areas of the dendritic neuropil showed positive staining. Intense labelling was seen in the ventro-medial tract, and in the connective between the terminal ganglion and the 5th abdominal ganglion. In addition, some motor neurones and neurones with branches in the sensory commissures were NADPHd positive. Our finding that NADPHd-positive cells occur in consistent patterns in the terminal abdominal ganglion implies that NO may have a role in mechanosensory processing in the crayfish.     

Octopamine-like immunoreactive neurones in locust genital abdominal ganglia

Using a well characterized anti-serum, the distribution of octopamine-like immunoreactive neurones is described in the locust seventh abdominal (A7) and terminal ganglia (TG), which are associated with genital organs. Apart from 4 paired ventral somata occasionally observed in the TG, all labelled cells could be identified as efferent dorsal- and ventral unpaired median (DUM/VUM) neurones by virtue of the characteristic large size and position of their somata, projections of their primary neurites in DUM-cell tracts, and bifurcating axons which arise from dorsal T-junctions and enter peripheral nerves. For the examined ganglia our data indicate that the whole population of efferent DUM and VUM-cells, defined here as progeny of the segment specific unpaired median neuroblast with peripheral axons, are octopaminergic, and that equal numbers of these cells occur in both sexes: 8 in A7 and 11 in TG. Sex-specific differences are probably restricted to the axonal projections of 5 octopamine-like immunoreactive DUM-somata in A7, and 5 in TG, which in females project into their segment specific sternal nerves, but in males into the genital nerve of the TG. Numerous intersegmentally projecting octopamine-like immunoreactive fibres traverse both ganglia. The majority probably stem from previously described octopamine-like immunoreactive neurones in the thoracic and suboesophageal ganglia.     

Ultrastructural investigation of nitric oxide synthase-immunoreactive nerves associated with coronary blood vessels of rat and guinea-pig

Ultrastructural investigation of nitrix oxide synthase-immunoreactive nerves closely associated with blood vessels in rat and guinea-pig hearts revealed many labelled nerve fibres in the walls of the main branches of the coronary arteries, and in arterioles, capillaries and post-capillary venules. The number of nitric oxide synthase-containing nerve fibres associated with different vessels, even those of the same calibre, varied. Terminal regions of nitric oxide synthase-immunoreactive fibres were observed in the endocardium and myocardium. Nitric oxide synthase-labelled fibres displayed electrondense immunoproduct in both varicose and intervaricose regions. Immunoreactive axonal varicosities contained both small and large synaptic vesicles. The characteristics of the nitric oxide synthase-immunoreactive nerve fibres observed in the heart and the possibility that these fibres represent the processes of intracardiac neurones and/or sensory neurones of extrinsic origin are discussed.     

A large proportion of pelvic neurons innervating the corpora cavernosa of the rat penis exhibit NADPH-diaphorase activity

Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, which indicates the presence of neural nitric oxide synthase, the enzyme responsible for the generation of nitric oxide, was used in combination with retrograde labelling methods to determine, in whole-mounts and sections of rat major pelvic ganglia, whether neurons destined for the penile corpora cavernosa were able to produce nitric oxide. In whole-mount preparations of pelvic ganglia, among the 607±106 retrogradely labelled neurons innervating the penile corpora cavernosa, 84±7% were NADPH-diaphorase-positive, 30±7% of which were intensely histochemically stained. In serial sections of pelvic ganglia, out of a mean count of 451 retrogradely labelled neurons, 65% stained positively for NADPH-diaphorase. An average of 1879±363 NADPH-diaphorase positive cell bodies was counted in the pelvic ganglion. In the major pelvic ganglion, neurons both fluorescent for Fluorogold or Fast Blue and intensely stained for NADPH-diaphorase were consistently observed in the dorso-caudal part of the ganglia in the area close to the exit of the cavernous nerve and within this nerve. This co-existence was much less constant in other parts of the ganglion. In the rat penis, many NADPH-diaphorase-positive fibres and varicose terminals were observed surrounding the penile arteries and running within the wall of the cavernous spaces. This distribution of NADPH-diaphorase-positive nerve cells and terminals is consistent with the idea that the relaxation of the smooth muscles of the corpora cavernosa and the dilation of the penile arterial bed mediated by postganglionic parasympathetic neurons is attributable to the release of nitric oxide and that nitric oxide plays a crucial role in penile erection. Moreover, the existence in the pelvic ganglion of a large number of NADPH-diaphorase-positive neurons that are not destined for the corpora cavernosa suggests that nitric oxide is probably also involved in the function of other pelvic tissues.     

Histochemical methods for detecting nitric oxide synthase

Summary The three isoforms of nitric oxide synthase (NOS), neuronal (nNOS), endothelial (eNOS), and inducible (iNOS), can be visualized in cells and tissues by NADPH-diaphorase (NADPH-d) histochemistry, immunocytochemistry and in situ hybridization. Histochemical demonstration of NADPH-d shows the formazan final reaction product as a solid blue deposit. The ultrastructural localization of NADPH-d in the rat hippocampus showed an electron-dense deposit on membranes predominantly of the endoplasmic reticulum. The immunohistochemical demonstration of nNOS, using the nickel enhancement technique, shows positive reaction product over the dendrites and the soma of the nerve cell in the rat brain. Ultrastructural localization of nNOS in whole mount preparations of myenteric plexus and circular smooth muscle from guinea-pig ileum shows that NOS immunoreactivity was patchily distributed in myenteric neurones and was not specifically associated with any intracellular organelles or with plasma membranes. In situ hybridization, using radio-labelled probes, was used to study nNOS mRNA in lumbar dorsal root ganglia after peripheral transection of the sciatic nerve in rats. Labelling of the NOS mRNA-positive neurones is observed as a series of dense granules over the entire cell. NADPH-d histochemistry, immunocytochemistry and in situ hybridization each have a significant role to play in the localization of NOS. NADPH-d detects an enzyme associated with the NOS molecule, immunocytochemistry detects the NOS molecule, and in situ hybridization detects mRNA for NOS. Therefore, if each of these techniques is applied in carefully controlled experiments, consideration of the accumulated data should be valuable in revealing insights into the biology of NOS.     

Complementary distribution of NADPH-diaphorase and l-arginine in the snail nervous system

Since the interneuronal messenger nitric oxide (NO) can not be stored in neurones, the regulation of the NO-producing enzyme nitric oxide synthase (NOS) is crucial. Neuronal NOS metabolises L-arginine to nitric oxide (NO) and L-citrulline in a Ca(2+)-dependent manner. Thus, availability of L-arginine to NOS may modulate NO production. In this study, we examined the cellular distribution of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, L-arginine and L-citrulline. Using NADPH-diaphorase histochemistry to visualise putative NO-producing cells and immunocytochemistry to localise L-arginine, we showed that the distribution of L-arginine-immunoreactive neurones correlates well with those of NADPH-diaphorase-positive neurones in cerebral ganglia of the pulmonate Helix pomatia. However, substrate and enzyme were visualised in separate but adjacent neurones. We further examined whether NADPH-diaphorase-labelled cells contain the L-citrulline. Following elevation of intracellular Ca(2+) by the Ca(2+) ionophore, ionomycin, or by a high-K(+) solution, the number of L-citrulline-immunoreactive neurones in mesocerebrum and pedal lobe increased up to tenfold. Preincubation of ganglia with the NOS inhibitor N(G)-nitro-L-arginine prevented ionomycin or high-K(+) solution-induced L-citrulline synthesis. Most L-citrulline-immunoreactive neurones contain NADPH-diaphorase activity. In conclusion, these experiments indicate a complementary distribution of NOS and L-arginine and suggest an unknown signalling pathway between neurones to maintain L-arginine and NO homeostasis.     

An identified dorsal unpaired median neurone and bilaterally projecting neurones exhibiting bovine pancreactic polypeptide-like/FMRFamide-like immunoreactivity in abdominal ganglia of the migratory locust

Summary Three antisera were used to study the distribution and anatomy of bovine pancreatic polypeptide (BPP)-like/FMRFamide-like immunoreactive neurones within the unfused abdominal ganglia of the migratory locust, Locusta migratoria. All the antisera used stained two or more clusters of perikarya, localized anteriorly and posteriorly near the midline within each unfused abdominal ganglion. Double labelling experiments with intracellular dye injection, or differential backfilling, combined with subsequent immunostaining were carried out to identify these neurones. Two of the antisera (antisera 1 and 2, both raised against FMRFamide) stained three groups of midline neurones, located anterior dorsal, anterior ventral and posterior dorsal within the ganglion. Neurones of the former of these two clusters projected via the anterior median nerve to a neurohaemal organ. The posterior cluster of midline cells comprised immunopositive perikarya all but one of which also projected via the anterior median nerve to innervate the neurohaemal organ. Double labelling with Lucifer yellow and antisera 1 and 2 showed that the remaining neurone was the previously identified doral unpaired median (DUM)heart1 neurone. The third antiserum (AK141), also raised against FMRFamide, stained neurones within an anterior dorsal cluster, and in a posterior cluster. Double labelling with differential Co²⁺/Ni²⁺-backfilling and the antiserum 3 (AK141) demonstrated that the large neurones of both clusters belonged to the population of bilaterally projecting neurones (BPNs), including the DUMheart1 neurone. Since the antisera cross-react with BPP and fail to label neurones when preadsorped with BPP or FMRFamide, we conclude that the labelled neurones contain polypeptides of the FMRFamide/BPP-family.     

河北环毛蚓神经系统 一氧化氮合酶的组织化学定位

用依赖还原型辅酶Ⅱ的黄酶组织化学方法,研究了环节动物门寡毛纲种类河北环毛蚓（Pheretima tschiliensis)神经系统k 一氧化氮合酶(NOS)阳性细胞及阳性纤维的分布,结果表明,河北环毛蚓神经系统中脑神经节背侧有大量细胞呈现NO强阳性反应,胞体和突起染色明显。咽下神经中偶尔能见少数染色较浅的神经元。在脑神经节腹内侧、围咽神经、 咽下神经节外侧部及腹神经链中都有一氧化氮合酶阳性纤维存在脸染色很深,实验结果表明,在环节动物中作为信息分子的一氧化氮已广泛存在于神经系统中。     

Nitric oxide synthase-containing nerves and ganglia in the dog prostate: A comparison with other transmitters

Summary The distribution of nitric oxide synthase immunoreactive nerves in the dog prostate was compared to the total innervation (as estimated by protein gene product 9.5 immunoreactivity), and to that of adrenergic (tyrosine hydroxylase-immunoreactive), cholinergic (acetylcholinesterase-positive), and some peptidergic nerves immunoreactive towards vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and helospectin. Clusters of ganglia with cell bodies containing acetylcholinesterase, or one of these six immunoreactive components, were found in the dorsal capsule. Coarse nerve trunks expressing these immunoreactive components extended from the ganglia, and divided into varicose terminals in the capsule and intraglandular smooth muscle strands, and gave off further branches, which surrounded acini and accompanied ducts. The labelling for nitric oxide synthase generally coincided with that for vasoactive intestinal peptide within cell bodies and nerves of various types. Cell bodies, nerve trunks and varicose terminals showing labelling for pituitary adenylate cyclase-activating peptide and helospectin were generally also labelled for vasoactive intestinal peptide. The innervation pattern suggests that nitric oxide may act in concert with vasoactive intestinal peptide and related peptides in the control of prostatic smooth muscle activity and secretion.     

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.     

Nitric oxide synthase in the spinal cord of the frog, Xenopus laevis

The expression of nitric oxide synthase was investigated in the spinal cord of the South African clawed frog by NADPH diaphorase histochemistry and immunohistochemistry. The dorsal field contained many strongly positive neurons and a dense plexus of processes. Only few nitric oxide synthase-positive cells occurred in the lateral and central field. Motoneurons were negative. A dense accumulation of stained neurons was located dorsal and dorsomedial to the motoneurons. The white matter harbored many positive fibers. These were most abundant in the dorsal funiculus, and obviously consist of nonprimary projections to the brainstem. These results suggest that nitric oxide represents a widely used messenger molecule in the frog spinal cord, in particular with respect to the processing of sensory information.     

Immunohistochemical, Histochemical and Radioassay Analysis of Nitric Oxide Synthase Immunoreactivity in the Lumbar and Sacral Dorsal Root Ganglia of the Dog

Summary In this study, immunohistochemistry for neuronal nitric oxide synthase (bNOS-IR), nicotinamide adenine dinucleotide phosphate diaphorase histochemistry (NADPHd) and nitric oxide synthase radioassay were used to study the occurrence, number and distribution pattern of nitric oxide synthesizing neurons in the lumbar (L1–L7) and sacral (S1–S3) dorsal root ganglia of the dog. Nitric oxide synthase immunolabelling was present in a large number of small- (area <1000 μm²) and medium-sized (area 1000–2000 μm²) as well as in a limited number of large-sized (area >2000 μm²) neurons. Although neuronal nitric oxide synthase immunolabelling and histochemical staining provided intense staining of multiple small- and medium-sized neurons in all lumbar and sacral dorsal root ganglia, immunolabelled or histochemically stained somata exhibited little topographic distribution in individual dorsal root ganglia. Great heterogeneity was noticed in the immunolabelling of medium-sized nitric oxide synthase immunopositive neurons ranging from lightly immunolabelled somata to heavily immunoreactive ones with completely obscured nuclei. Both staining procedures proved to be highly effective in visualizing intraganglionic fibers of various diameters. In general, the largest fibers revealed at the peripheral end of lumbar and sacral dorsal root ganglia were larger, 6.49–9.35 μm in diameter, while those running centrally and proceeding into the dorsal roots were about 30% reduced, ranging between 5.32 and 8.67 μm in diameter. Peripherally, the occurrence of nitric oxide synthase detected in axonal profiles, and confirmed histochemically, in the specimens of the femoral and sciatic nerves, is the first indication of the presence of nitric oxide synthase in the peripheral processes of somata located in L4–S2 dorsal root ganglia. Large and thin central nitric oxide synthase immunoreactive processes of L1–S3 dorsal root ganglion neurons segregate shortly before entering the spinal cord, the former making a massive medial bundle in the dorsal root accompanied by a slim lateral bundle penetrating Lissauer's tract. Quantitative assessment of the distribution of bNOS-IR and/or NADPHd-stained neurons showed a peculiar pattern in relation to spinal levels. Apparent incongruity was found in the total number of NADPHd-stained versus bNOS-IR neurons, demonstrating a clear prevalence of small bNOS-IR somata in all lumbar ganglia, while medium-sized NADPHd-stained somata clearly prevailed all along the rostrocaudal axis with a peak in L5 ganglion. While the number of small bNOS-IR neurons clearly outnumbered NADPHd-stained and NADPHd-unstained somata in S1–S3 ganglia, an inverse relation appeared comparing the total number of medium-sized NADPHd-stained and NADPHd-unstained somata compared with the number of moderate and intense bNOS-IR neurons. Densitometry of bNOS-IR and NADPHd-stained neurons in lumbar and sacral ganglia revealed two distinct subsets of densitometric profiles, one relating to more often found medium-sized bNOS immunolabelled and the other, characteristic for moderately bNOS immunoreactive somata of the same cell size. Considerable differences in catalytic nitric oxide synthase activity, determined by conversion of [³H]arginine to [³H]citrulline were obtained in lumbosacral dorsal root ganglia all along the lumbosacral intumescence, the lowest (0.898± 0.2 dpm/min/μg protein) being in the L4 dorsal root ganglion and the highest (4.194± 0.2 dpm/min/μg protein) in the S2 dorsal root ganglion.     

The distribution of neurones immunoreactive for β-tyrosine hydroxylase,dopamine and serotonin in the ventral nerve cord of the cricket,Gryllus bimaculatus

The cellular localization of the biogenic amines dopamine and serotonin was investigated in the ventral nerve cord of the cricket, Gryllus bimaculatus, using antisera raised against dopamine, -tyrosine hydroxylase and serotonin. Dopamine-(n<-70) and serotonin-immunoreactive (n<-120) neurones showed a segmental arrangement in the ventral nerve cord. Some neuromeres, however, did not contain dopamine-immunoreactive cell bodies. The small number of stained cells allowed complete identification of brain and thoracic cells, including intersegmentally projecting axons and terminal arborizations. Dopamine-like immunostaining was found primarily in plurisegmental interneurones with axons descending to the soma-ipsilateral hemispheres of the thoracic and abdominal ganglia. In contrast, serotonin-immunostaining occurred predominantly in interneurones projecting via soma-contralaterally ascending axons to the thorax and brain. In addition, serotonin-immunoreactivity was also present in efferent cells and afferent elements. Serotonin-immunoreactive, but no dopamine-immunoreactive, varicose fibres were observed on the surface of some peripheral nerves. Varicose endings of both dopamine-and serotonin-immunoreactive neurones occurred in each neuromere and showed overlapping neuropilar projections in dorsal and medial regions of the thoracic ganglia. Ventral associative neuropiles lacked dopamine-like immunostaining but were innervated by serotonin-immunoreactive elements. A colocalization of the two amines was not observed. The topographic representation of neurone types immunoreactive for serotonin and dopamine is discussed with respect to possible modulatory functions of these biogenic amines in the central nervous system of the cricket.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

两种软体动物神经系统一氧化氮合酶的组织化学定位

运用一氧化氮合酶(NOS)组织化学方法研究了软体动物门双壳纲种类中国蛤蜊和腹足纲种类嫁Qi神经系统中NOS阳性细胞以及阳性纤维的分布。结果表明：在蛤蜊脑神经节腹内侧,每侧约有10-15个细胞呈强NOS阳性反应,其突起也呈强阳性反应,并经脑足神经节进入足神经节的中央纤维网中;足神经节内只有2个细胞呈弱阳性反应,其突起较短,进入足神经节中央纤维网中,但足神经节中,来自脑神经节阳性细胞和外周神经系统的纤维大多呈NOS阳性反应;脏神经节的前内侧部和后外侧部各有一个阳性细胞团,其突起分别进入后闭壳肌水管后外套膜神经和脑脏神经索。脏神经节背侧小细胞层以及联系两侧小细胞层的纤维也呈NOS阳性反应。嫁Qi中枢神经系统各神经节中没有发现NOS阳性胞体存在;脑神经节、足神经节、侧神经节以及脑—侧、脑—足、侧—脏连索中均有反应程度不同的NOS阳性纤维,这些纤维均源于外周神经。与已研究的软体动物比较,嫁Qi和前鳃亚纲其它种类一样,神经系统中NO作为信息分子可能主要存在于感觉神经。而中国蛤蜊的神经系统中一氧化氮作为信息分子则可能参与更广泛的神经调节过程。     

Neurotransmitter release regulated by nitric oxide in PC-12 cells and brain synaptosomes

BACKGROUND: Nitric oxide is a messenger molecule of the nervous system, which is produced by the enzyme nitric oxide synthase, which may regulate cyclic guanosine monophosphate levels and which has been implicated in the control of neurotransmitter release. PC-12 pheochromocytoma cells differentiate to form neuronal cells in culture when they are exposed to nerve growth factor. The levels of cyclic guanosine monophosphate in the cells and their ability to release acetylcholine in response to K(+)-depolarization are both maximal after eight days of treatment with nerve growth factor. We set out to assess a possible role for nitric oxide in the processes that occur in differentiating PC-12 cells. RESULTS: Nitric oxide synthase is first evident in differentiating PC-12 cells eight days after beginning treatment with nerve growth factor, coinciding with the marked increase in K(+)-depolarization-induced release of acetylcholine. The release of both acetylcholine and dopamine in response to K(+)-depolarization is blocked by inhibitors of nitric oxide synthase and by hemoglobin, which binds nitric oxide. Providing l-arginine, a precursor required for nitric oxide synthesis, reverses the effects of the inhibitors. In synaptosomal preparations from the corpus striatum, inhibitors of nitric oxide synthase prevent the release of glutamate in response to the glutamate derivative N-methyl-d-aspartate but not in response to K(+)-depolarization. CONCLUSION: Nitric oxide may mediate the release of acetylcholine and dopamine in response to K(+)-depolarization in PC-12 cells and the release of glutamate in response to N-methyl-d-aspartate in striatal synaptosomes. Nitric oxide synthase expression is induced after eight days of treating PC-12 cells with nerve growth factor, coinciding with a marked enhancement of the release of neurotransmitters in response to K(+)-depolarization.