in the brain, under normal conditions, blood vessels as well as different neuronal ... Pulse Voltammetry were recorded every 3 minutes using the following ...
Monitoring Molecules in Neuroscience
Anomalous correlation between regional distribution of nitric oxide synthase activity and extracellular concentration of nitric oxide in brain tissues 'Gonzalez-Mora, J.L., 'Guadalupe, T., 3Perez de la Cruz, M.A. and 2Gonzalez Hernandez, T. 1Dept, o f Physiology a n d 2Dept, o f Anatomy, University o f La Laguna, Medical School, Tenerife a n d 3Dept, o f Histology, University o f Salamanca. Spain Introduction The discovery that nitric oxide (NO), a simple gas with free radical chemical properties functioning as an intercellular signalling element in the peripheral and central nervous system has dramatically extended the conceptual framework of neurotransmission. NO is synthesized from L-arginine by activation of the enzyme nitric oxide synthase (NOS). Different isoforms of NOS have been characterized and purified from brain (nNOS or NOS-I), endothelial cells (eNOS or NOS-III) and activated macrophages (macNOS or NOS-II). They all show NADPH-diaphorase (NADPH-d) activity. Morphological studies carried out during the last decade indicate that in the brain, under normal conditions, blood vessels as well as different neuronal populations display NOS activity. Recent advances on electrochemical methods, with the development of new microsensors constitute an interesting tool to detect directly the NO levels in different tissues1. Our purpose has been to investigate if a correlation exists between the intensity of NOS activity and the extracellular concentration of NO in the brain, by comparing the histochemical (NADPH-d) and immunohistochemical (nNOS and eNOS) labelling pattern within the NO levels, using a porphyrinic-based working electrode, in two different nervous centres with a well-known NOS activity pattern: visual cortex and superior colliculus. Material and methods Voltammetry. Previously treated carbon fiber microelectrodes of 12 jam diameter and 500 pm length2 were used to electrodeposit a polymeric film of tetrakis (3-metoxy-4-hidroxyphenyl porphyrin), with nickel as the central metal, (TMHPP-Ni). The electrodeposition was performed by 10 continous scans of differential pulse voltammetry (DPV) with a potential range from 0 to IV. The porphyrinic sensors were then washed and dried and coated with a cation exchange material to reject anions by dipping the sensor in Nafion solution and then applying a constant potential of 2.8V for 3s each minute. A microprocessor controlled apparatus (Bioelectrochemical analyser, BECA, Spain) was used to monitor voltammetric signals3. Differential Normal Pulse Voltammetry were recorded every 3 minutes using the following parameters: potential range from -100 to 1000 mV; scan rate 20 mV/s; pulse amplitude 40 mV; pulse duration 40 ms; prepulse duration from 50 to 120 ms. The reference (Ag/AgCl) and auxiliary (platinum wire) electrodes were placed on the bone brain surface and kept wet with saline-soaked pads. Each sensor’s performance, like sensitivity and integrity of the Nafion film coverage was characterised before and after use. Animals. Adult male Sprague-Dawley rats weighing between 250-290g were anaesthetised with choral hydrate and kept on a sterotaxic frame and the body temperature was controlled by an homeothermic blanket throughout the experiment. The porphyrinic-based working electrode was placed over the visual cortex according to the following coordinates 6.72 mm posterior to bregma, 2 mm lateral to the midline and the electrode was located during the experiment from cortex surface to 5 mm below the brain surface. Morphological study. After the electrochemical study, animals were perfused via an intraaortic cannula with 150 ml 0.9% saline and 400 ml of ice-cold 4% paraformaldehyde in 0.1M PBS, pH 7,4. Brains were removed, stored in the same fixative at 4SC during 6 hours and coronally cut with a vibratome at 50(om. For NADPH-d histochemistry, sections were incubated for 30-60 min. at 379C in 0.1M PBS containing 2.5 mM NADPH, 0.4 mM nitro blue tetrazolium and 0.3% Triton X-100. After mounting, they were quickly dehydrated and coverslipped with Entellan. Immunohistochemistry for nNOS was performed by using a polyclonal rabbit antibody raised against the C-terminal fragment of NOS from rat cerebellum (Euro-diagnostica). For eNOS we used a monoclonal mouse antibody raised against amino-acids 1030-1209 of human eNOS (Trasduction Lab.). Immunoreactions were visualized by incubation for 1 hr. at RT in avidin-biotin-peroxidase complex (1:100, BC kit, Vector) in PBS and for 5-10 min in 0.05% 3',3'diaminobenzidine and 0.01% H20 2 in 0.05M Tris-HCl, pH 7.6. Results and discussion As shown in Figure IB and C, four different brain regions were electrochemically tested: superficial layers of the visual cortex, deep layers of the visual cortex, superficial layers of the superior colliculus and deep layers of the superior colliculus. Brains were processed for NADPH-d histochemistry and nNOS and eNOQ immunohistochemistries. In this respect, our results confirm previous studies4’5, according to which, undei optimal fixative conditions, NADPH-d histochemistry is an excellent NOS marker, exhibiting more
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Monitoring Molecules in Neuroscience
morphological details than immunohistochemistry and in addition, allowing to identify simultaneously both endothelial (vascular profiles) and neuronal (somata, dendrites and axons)activities. The electrochemical study showed the highest concentration of extracellular NO in the superficial cortical layers, and the lowest in the deep collicular ones (Fig. 1C and D). It is interesting to note the high levels of NO in the cerebral cortex, in comparison with the paucity of NOS positive neurons (Fig. 1A, top), while in the superficial layers of the superior colliculus, the NO levels were very low in comparison with their high density of NOS positive cells (Fig. 1A, bottom). This indicates that a heavy NOS activity does not necessarily imply a high concentration of extracellular NO. The discrepancy suggests that in the cerebral cortex, most of NO arises from non-neuronal elements. Taking into account that NO is a powerful cerebrovasodilator that participates in the maintenance of resting cerebral blood flow and the cerebrovasodilatation elicited by increased neural activity6 and that cortical vessels display an intense NADPH-d reaction, the high concentration of NO perhaps originates from the vascular endothelium. However, we must say that cortical NOS positive neurones, although sparse, display large dendritic branches and dense axonal network, wich could maintain innervated ample cortical areas. In contrast, in the superficial layers of the superior colliculus, in spite of the intense neuronal NOS activity, the NO levels are low, indicating that, in resting conditions, most of this enzymatic activity remains silent. In conclusion, our findings suggest that both sources and functions of NO in the cerebral cortex are qualitatively different from those in the superior colliculus. In the cerebral cortex, it could have a rather metabolic role, related to the control of blood flow, while in the superior colliculus, it could be produced in response to specific stimuli, just like other neurotransmitters respond.
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Figure 1. In vivo voltammetric detection o f NO by a porphyrinic-based microsensor and micrographs o f NADPH-diaphorase-stained. B Sterotaxic diagram o f visual cortex and superior colliculus. The black bars represents the relative position o f porphyrinc-based microelectrodes. C Brighfield photomontage o f NADPHdiaphorase o f the same region (part A o f the figure shows a detailed zoom o f selected areas). D Electrochemical recordings obtained with the porphyrinic microsensor in the selected areas, the shaded region shows the NO peak (~ 650 mV). Acknowledgements: This work was supported by Grant PINT 95/049 from CEE and GAC (C.de E. y H.). References 1. 2. 3. 4. 5.
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Malinski T., and Taha, Z. (1992) Nitric oxide release from a single cell measured in situ by a porphirinic-based microsensor, Nature 358 , 676-678, Gonzalez-Mora, J.L., Guadalupe T., Fumero B. and Mas M. (1991) Mathematical resolution o f mixed in vivo voltammetry signals. Models, equipment, assessment by simultaneous microdialysis sampling. J. Neurosci. Methods., 39 , 231-244. J.-L. Gonzalez-Mora and R.F. Vera. (1994), Simultaneous real-time measurements o f dopamine and serotonin levels: New methodological developments, “Monitoring Molecules in Neuroscience” A. Louilot, T. Durkin, U. Spanpinato, M. Cador, Ed. 1-3. Weinberg, R.J., Valtschanoff, J. and Schmidt, H.H.H.W. (1996) The NADPH diaphorase histochemical stain. In: Feelisch ,H., Stamler, S.S., (eds), Methods in nitric oxide research, New York, Wiley, pp 236-248. Gonzalez-Hemandez, T., Perez de la Cruz, M.A. and Mantolan-Sarmiento, B. (1996) Histochemical and immunohistochemical detection o f neurons that produce nitric oxide: effect o f different fixative parametersand immunoreactivity against non-neuronal NOS antisera. J. Histochem. Cytochem. (in press). ladecola, C. (1993) Regulation o f the cerebral microcirculation during neural activity: is nitric oxide the missing link?. Trends Neurosci., 16 , 206-214.
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