Life Sci. 2015 Aug 1;134:79-84.

Origin of endogenous nitric oxide released in the nucleus accumbens under real-time in vivo conditions.

Prast H1, Hornick A1, Kraus MM2, Philippu A3.
  • 1Department of Pharmacology and Toxicology, University of Innsbruck, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria.
  • 22nd Laboratory of Pharmacology, School of Medicine, Aristotle University of Thessaloniki, GR-54006 Thessaloniki, Greece.
  • 3Department of Pharmacology and Toxicology, University of Innsbruck, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria. Electronic address:




Nitric oxide (NO), is a simple but multifarious molecule. It is implicated in physiological and pathological processes within the striatum, mainly in the nucleus accumbens (NAc). The aim of the present study was to determine the origin of NO in the NAc of anaesthetized rats by applying various compounds known to modulate the release of NO when applied either systemically or locally.


Real-time monitoring of NO was carried out by introducing an amperometric NO sensor into the outer tubing of a push-pull cannula. For local application of substances, the push-pull superfusion technique was used.


An overdose of urethane (i.p.) or superfusion of the NAc with tetrodotoxin (TTX) led to a fall of NO release in the NAc. The NO synthase (NOS) inhibitors 7-nitroindazolmonosodiumsalt (7-NINA, neuronal NOS selective) and N-nitro-L-arginine (L-NNA, NOS selective) decreased release of NO when applied i.p. or locally. Superfusion of the NAc with N-methyl-D-aspartate (NMDA) elicited a dose dependent increase of NO release.


Combination of an amperometric NO sensor for real-time monitoring of NO release with the push-pull superfusion technique showed that NO released in the NAc is, at least to a great extent, of neuronal origin. The enhanced release of NO elicited by locally applied NMDA demonstrates that activation of NMDA receptors facilitates NO synthesis, thus underlining the functionality of NO targets within the NAc.


Amperometric nitric oxide sensor; Neuronal nitric oxide synthase (nNOS); Nucleus accumbens (NAc); Oscillations; Push–pull superfusion technique; Real-time nitric oxide (NO) assessment

PMID: 26006039



The push-pull superfusion technique possesses the following characteristics:

1)  the continuous, qualitative and quantitative determination of neurotransmitters,

2) the good time resolution that is necessary when release of transmitters is correlated to brain functions,

3) no gliosis is developed around the superfused area when the guide cannula is implanted for chronic experiments in conscious animals. Hence, the ratio  transmitter/metabolites is not changed. Finally,

4) the unique characteristic of the push-pull superfusion technique is that microelectrodes and biosensors may be inserted into the push-pull cannula for recordings of extracellular potentials or EEG and on-line determination of nitric oxide (NO) respectively. Thus, signals are recorded in the microscopically exactly same area in which the transmitter release is determined.

Preliminary experiments have shown that combination of the push-pull cannula with a sensor for the determination of NO renders possible the simultaneous quantitative determination of acetylcholine (and consequently all neurotransmitters) and the  determination of the NO signal  under real-time in vivo conditions in the microscopically very same distinct brain area of anaesthetized and conscious, freely moving animals.



ap fig1Fig. 1 Push-pull superfusion technique. Left panel: combination of the push-pull cannula with a microelectrode for simulataneous quentitative determination of neurotransmitters (catecholamines, serotonin and its metabolite 5-HIAA, histamine, acetylcholine, adenosine, excitatory and inhibitory amino acids) and electrical stimulation of the superfused area or for recordings of evoked potentials in other distinct brain areas. Right panel: Combination of a modified push-pull cannula with a biosensor for the simultaneous quantitative determination of neurotransmitters and NO released in a distinct brain area (1)


To investigate whether a certain neurotransmitter is involved in a particular brain function, experimentally evoked changes of the brain function are carried out and the transmitter release is determined that reflects neuronal activity. For example, an experimentally induced fall of the arterial blood pressure evoked by intravenous injection of sodium nitroprusside leads to an increased release of all three catecholamines in the posterior hypothalamus while an experimentally induced rise in blood pressure exerts the opposite effect, thus pointing to the counteracting action of the catecholaminergic neurons within the hypothalamus. Besides catecholamines, GABA, serotonin, histamine, NO, are involved in central brain blood pressure control.

The above mentioned strategy has been used in mnemonic possesses, aversive stimuli and inescapable fear and has led to a bulk of knowledge concerning the involvement of neurotransmitters in brain function. For details see (1).



  1. M. M. Kraus and A. Philippu: Use of Push-Pull Superfusion Technique for Identifying Neurotransmitters Involved in  Brain Functions: Achievements and Perspectives. Current Neuropharmacology, 2015, 13, 819-829)





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