Push-pull Superfusion Technique Research Articles

Brain mapping: topography of neurons and their transmitters involved in various brain functions.

Use of the demanding techniques microdialysis or push-pull superfusion makes it possible to identify neurons in distinct brain areas involved in central control of peripheral functions, thus enabling brain mapping. Investigations with the push-pull superfusion technique have shown that mainly catecholaminergic neurons of the posterior and anterior hypothalamus, the locus coeruleus, and the nucleus of the solitary tract are of crucial importance for blood pressure regulation. Experimentally induced blood pressure changes also modify the release of histamine, glutamate, and taurine in the posterior hypothalamus and of serotonin in the locus coeruleus. Furthermore, histaminergic neurons of the nucleus accumbens are involved in memory, serotonergic neurons of the locus coeruleus in response to noxious stimuli, while nitric oxide of striatum has been implicated in neurotoxicity elicited by amphetamines. The involvement of several neurons in one brain function is discussed.

Neurotransmitters and nitric oxide are released in various brain areas according to ultradian and infradian rhythms

The push-pull superfusion technique was used to investigate release of endogenous neurotransmitters and nitric oxide in various brain regions. The transmitters catecholamines, histamine, GABA, glutamate, as well and nitric oxide are released according to ultradian rhythms which generate fluctuations of vegetative functions such as blood pressure and arousal. More frequent infradian oscillations of neurotransmitter and nitric oxide release fates are implicated in mutual interneural control.

Neurotransmitters and nitric oxide are released in various brain areas according to ultradian and infradian rhythms

The push-pull superfusion technique was used to investigate release of endogenous neurotransmitters and nitric oxide in various brain regions. The transmitters catecholamines, histamine, GABA, glutamate, as well and nitric oxide are released according to ultradian rhythms which generate fluctuations of vegetative functions such as blood pressure and arousal. More frequent infradian oscillations of neurotransmitter and nitric oxide release fates are implicated in mutual interneural control.

Fentanyl and naloxone effects on glutamate and GABA release rates from anterior hypothalamus in freely moving rats

Fentanyl, a μ-opioid receptor agonist, has been studied for its neuro/psycho-pharmacological effects since its first clinical use; however, its effect on the release rate of the Central Nervous System (CNS) neurotransmitters has not been yet elucidated. In the present study the influence of fentanyl on the release rates of glutamate and GABA is investigated. Specifically, we examined the effects of intravenous (10 μg/kg) as well as intrahypothalamic (0.1nmol/min) fentanyl administration on the release rates of GABA and glutamate in the superfusate of anterior hypothalamus, under tail pinch manipulation. The release rate of the neurotransmitters was monitored by the push–pull superfusion technique. To investigate the role of fentanyl the opioid antagonist, naloxone 0.1 mg/kg was administered intravenously, or 50nmol/min intrahypothalamicaly. The amino acids were determined by High Performance Liquid Chromatography (HPLC) and fluorimetric detection after NBD-Cl derivatisation. After intravenous fentanyl administration a significant decrease of glutamate and increase of GABA release rates were observed. However during the pain manipulations, the release rate of glutamate was increased. Intravenous naloxone did not affect significantly the release rates of both amino acids, while intrahypothalamic antagonist administration reversed the alterations in both neurotransmitters release rates. Our results demonstrate that there is an opioid-glutamatergic transmission pathway, located in hypothalamus and that opioids can activate NMDA receptors, thus reducing the nociceptive threshold and the opioid analgesic effect.

Nitric Oxide: A Universal Modulator of Brain Function.

The pioneering work of Robert F. Furchgott, Luis J. Ignaro and Ferid Murad has led us to investigate whether nitric oxide (NO) is present in the brain, its origin and whether it possesses a functional role in brain structures. This review is mainly an outline of own findings obtained by using the push-pull superfusion technique. We have used the push-pull superfusion technique that makes it possible to determine quantitatively endogenous transmitters released from their neurons in the synaptic cleft. In some experiments, a NO sensor was inserted into the pushpull cannula for online determination of NO released in the synaptic cleft together with neurotransmitters. The release rates of endogenous NO are not constant but oscillate according to an ultradian rhythm with an apparent frequency of about 24 min per cycle. Similar rhythmic changes have been found in the release of neurotransmitters in several brain regions, as well as in the EEG delta band. Endogenous NO modulates the release of acetylcholine, glutamate, aspartate, GABA, serotonin, histamine in distinct brain areas. The release of adenosine is also increased by NO suggesting the synchronous release of ATP. Endogenous NO influences various brain functions such as blood pressure regulation and responses to stress. Recordings of evoked potentials revealed that NO plays a crucial role in the integration of afferent signals. Furthermore, NO in involved in amphetamine-induced neurotoxicity. The multifarious influences of endogenous NO on central neuronal activity, brain functions and integration of afferent signals underpin its universal modulatory role in the brain.

Use of Push-Pull Superfusion Technique for Identifying Neurotransmitters Involved in Brain Functions: Achievements and Perspectives.

The push-pull superfusion technique (PPST) is a procedure for in vivo examination of transmitter release in distinct brain areas. This technique allows to investigate dynamics of transmitter release both under normal and experimentally evoked conditions. The PPST can be modified so that it is possible to determine release of endogenous transmitters simultaneously with electroencephalogram (EEG) recordings, recordings of evoked potentials or the on-line determination of endogenous nitric oxide (NO) released into the synaptic cleft. Because of the good time resolution, the method provides further the possibility to modify the collection periods of superfusates depending on the neuronal function that is analyzed. For instance, investigation of central cardiovascular control, behavioral tasks or mnemonic processes requires very short collection periods, because changes in transmitter release occur within seconds. Even more important is the time resolution when rates of transmitter release are correlated with evoked extracellular potentials or EEG recordings. This review provides an overview of the different devices which might be combined with the PPST and perspectives for future work.

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

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.

Influence of the hippocampus on amino acid utilizing and cholinergic neurons within the nucleus accumbens is promoted by histamine viaH1receptors

The influence of the neurotransmitter histamine on spontaneous and stimulation-evoked release of glutamate, aspartate, GABA and ACh in the nucleus accumbens (NAc) was investigated in vivo. Using the push-pull superfusion technique, histaminergic compounds were applied to the NAc and neurotransmitter release was assessed. In some experiments, the fornix/fimbria of the hippocampus was electrically stimulated by a microelectrode and evoked potentials were monitored in the NAc. Superfusion of the NAc with the H1 receptor antagonist triprolidine (50 μM) decreased spontaneous outflow of glutamate, aspartate and ACh, while release of GABA remained unaffected. Superfusion with histamine elevated release of ACh, without influencing that of the amino acids. Electrical stimulation of the fornix/fimbria enhanced the output of amino acids and ACh within the NAc. The evoked outflow of glutamate and ACh was diminished on superfusion with triprolidine, while release of aspartate and GABA was not affected. Superfusion of the NAc with histamine intensified the stimulation-evoked release of glutamate and Ach. Histamine also elevated the stimulation-induced release of aspartate, without influencing that of GABA. Presuperfusion with triprolidine abolished the reinforced effect of histamine on stimulation-evoked transmitter release within the NAc. Neuronal histamine activates H1 receptors and increases spontaneous release of glutamate, aspartate and ACh within the NAc. Stimulation of the hippocampal fornix/fimbria tract also enhances release of glutamate and ACh within the NAc and this effect is intensified by H1 receptor stimulation within the NAc. The latter effects, which are mediated by hippocampal afferences, might play an important role in mnemonic performance and in emotional processes such as anxiety and stress disorders.

Sinoaortic denervation abolishes blood pressure-induced GABA release in the locus coeruleus of conscious rats

Male Sprague–Dawley rats underwent sinoaortic denervation (SAD) or sham operation. We examined changes in the release rates of GABA, glutamate and arginine in the locus coeruleus (LC) elicited by experimental blood pressure increases (i.v. noradrenaline infusion for 3 min, 4 μg kg −1 min −1) or decreases (i.v. sodium nitroprusside infusion for 3 min, 150 μg kg −1 min −1). The release of the neurotransmitters was monitored by the push–pull superfusion technique. Mean blood pressure did not differ between sham-operated and SAD rats but blood pressure lability was greatly enhanced in SAD rats and accompanied by increased basal release of glutamate in the LC. GABA release was not affected. A rise in blood pressure induced by noradrenaline enhanced GABA release in the LC of sham-operated rats. This effect was abolished by SAD. Glutamate release did not respond to hypertension either in SAD or in sham-operated rats. Nitroprusside led to a fall in blood pressure which was more pronounced and lasted longer in SAD than in sham-operated rats. In SAD rats, glutamate release was enhanced by nitroprusside. The depressor response had no effect on glutamate release in sham-operated rats. GABA release did not respond to this stimulus in either SAD or sham-operated rats. SAD and blood pressure changes did not influence the release rate of arginine. In conclusion, experimental hypertension increases GABAergic activity in the LC by stimulating peripheral baroreceptors. In SAD rats, augmented blood pressure lability seems to be at least partly due to elevated glutamate outflow within the LC.

Role of nitric oxide in the stress-induced release of serotonin in the locus coeruleus.

Serotonergic mechanisms within the locus coeruleus (LC) are thought to be important in various functions including the stress response. In this study we investigated a possible role of nitric oxide (NO) as an intermediary messenger in the regulation of the serotonin (5-HT) neurotransmission within the LC. Using the push-pull superfusion technique coupled with HPLC and electrochemical detection, the in vivo release of 5-HT was determined in time periods of 10 min in the LC of freely moving rats. Superfusion with three different NO donors, SIN-1 (linsidomine), S-nitroso-N-penicillamine (SNAP) or 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPANO) increased 5-HT release in the LC. Superfusion with the precursor of NO, L-arginine, for 1 h led to a sustained increase in 5-HT release. On the other hand, the NOS inhibitor N-methyl-L-arginine methyl ester (L-NAME) did not significantly change the release of 5-HT. Infusion of N-methyl-D-aspartate (NMDA) or kainic acid, as well as exposure of rats to noise stress or tail pinch increased the release of 5-HT in the LC. Superfusion with L-NAME prevented the increase in 5-HT outflow by all these procedures, while the inactive isomer D-NAME had no effect. Taken together, the results of this study suggest that the release of 5-HT in the LC is facilitated by NO. Under resting conditions inhibition of NOS does not appear to substantially influence the release of 5-HT in the LC. However, there seems to be a facilitatory nitrergic influence on serotonergic responses evoked by excitatory amino acid receptor stimulation or various stress stimuli.

Release of glutamate and GABA in the amygdala of conscious rats by acute stress and baroreceptor activation: differences between SHR and WKY rats

To reveal the functional importance of amino acid neurotransmission in the amygdala (AMY) of conscious spontaneously hypertensive rats (SHR) and Wistar–Kyoto (WKY) rats, the in vivo release of glutamate (GLU) and GABA in this brain structure was studied using the push–pull superfusion technique. Basal GLU and GABA release rates in the AMY were comparable in SHR and WKY rats, although arterial blood pressure (BP) in SHR (152±6 mmHg) was higher than in WKY rats (102±4 mmHg). Neuronal depolarization by superfusion with veratridine enhanced the release of GLU and GABA to a similar extent in both rat strains. On the other hand, exposure to noise stress (95 dB) for 3 min led to a tetrodotoxin-sensitive increase in GLU release in the AMY of SHR, but not WKY rats. The concurrent pressor response to noise was enhanced in SHR as compared to WKY rats. A rise in BP induced by intravenous infusion of phenylephrine for 9 min had no effect on amino acid release in the AMY of both strains. The data suggest an exaggerated stress response of glutamatergic neurons in the AMY of SHR as compared with WKY rats, which might be of significance for the strain differences in the cardiovascular and behavioural responses to stress. The results also show that, in both rat strains, glutamatergic and GABAergic neurons in the AMY are not modulated by baroreceptor activation. Moreover, hypertension in adult SHR does not seem to be linked to a disturbed synaptic regulation of glutamatergic or GABAergic transmission in the AMY.

Histaminergic neurons modulate acetylcholine release in the ventral striatum: role of H1 and H2 histamine receptors.

To investigate whether H1 and H2 histamine receptors are implicated in the modulation of acetylcholine release by endogenous histamine, the ventral striatum of the conscious, freely moving rat was superfused by the push-pull superfusion technique with drugs and the release of acetylcholine was determined in the superfusate. Superfusion with the H1 receptor agonist 2-thiazolylethylamine (TEA, 50 micromol/l) enhanced the release of acetylcholine, while the H1 receptor antagonist triprolidine (50 micromol/l) reduced acetylcholine outflow and abolished the TEA-evoked release of the neurotransmitter. The inhibitory effect of triprolidine was not influenced either on simultaneous superfusion with 10 micromol/l (+/-)-7-bromo-1-(fluoresceinylthioureido)phenyl-8-hydroxy-3-methyl -2,3,4,5-tetrahydro-1H-benzazepine (SKF-83566, D1 dopamine receptor antagonist) and 50 micromol/l quinpirole (D2/D3 dopamine receptor agonist) or on superfusion with the GABAA receptor antagonist bicuculline (50 micromol/l). The H2 receptor antagonists ranitidine or famotidine (50 micromol/l each) greatly enhanced acetylcholine release rate in the ventral striatum. Presuperfusion with alpha-fluoromethylhistidine (FMH, 1 mmol/l), which inhibits neuronal synthesis of histamine, abolished the famotidine-induced release of acetylcholine. The releasing effect of famotidine was also abolished on simultaneous superfusion with 10 micromol/l SKF-83566 and 50 micromol/l quinpirole. The release of acetylcholine elicited by famotidine was reversed to a decreased acetylcholine outflow when the striatum was superfused with the GABA(A) receptor antagonist bicuculline (50 micromol/l) prior to famotidine. Superfusion with the H2 receptor agonist impromidine (1 micromol/l) decreased acetylcholine outflow, while the H2 agonist dimaprit (50 micromol/l) exerted the opposite effect. The releasing effect of dimaprit was not influenced by FMH (1 mmol/l), but it was abolished in the presence of SKF-83566 (10 micromol/l) and quinpirole (50 micromol/l). In the presence of bicuculline the release of acetylcholine by dimaprit was enhanced and prolonged. It seems possible that dimaprit and impromidine stimulate different subtypes of H2 receptors. The findings suggest that the release of acetylcholine in the striatum is modulated by neighbouring histaminergic neurons in a complex way. Stimulation of H1 histamine receptors, probably located on cholinergic neurons, enhances acetylcholine release. Stimulation by histamine of H2 receptors located on cholinergic or GABAergic neurons enhances the release of acetylcholine, while stimulation of H2 receptors located on dopaminergic neurons exerts the opposite effect.

Dependence of serotonin release in the locus coeruleus on dorsal raphe neuronal activity.

The serotonergic innervation of the locus coeruleus paetly derives from the dorsal raphe nucleus (DRN). Using the push-pull superfusion technique, we investigated whether and to what extent the release of serotonin and the extracellular concentration of its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the locus coeruleus are influenced by the neuronal activity of the DRN. In anaesthetized rats, a push-pull cannula was inserted into the locus coeruleus, which was continuously superfused with artificial cerebrospinal fluid (aCSF). Serotonin and 5-HIAA levels in the superfusate were determined by HPLC combined with electrochemical detection. Electrical stimulation (5 Hz, 300 microA, 1 ms) of the DRN for 5 min, or its chemical stimulation by microinjection of glutamate (3.5 nmol, 50 nl), led to an increased release of serotonin in the locus coeruleus and to a slight (2 mmHg) decrease in blood pressure. Superfusion of the locus coeruleus with tetrodotoxin (1 microM) abolished the increase in the release rate of serotonin evoked by electrical stimulation of the DRN, while the slight fall in blood pressure was not influenced. Thermic lesion (75 degrees C, 1 min) of the DRN elicited a pronounced decline in serotonin release rate within the locus coeruleus, the maximum decrease being 52%. The decrease in the release of serotonin was associated with a long-lasting rise in blood pressure. Microinjection of the serotonin neurotoxin 5,7-dihydroxytryptamine (5 microg, 250 nl) into the DRN led to an initial increase in the serotonin release rate that coincided with a short-lasting fall in blood pressure. Subsequently, the release of serotonin was permanently reduced and was associated with hypertension. Microinjection of the 5-HT1A receptor agonist (+/-)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT; 7.5 nmol, 50 nl) into the DRN led to a long-lasting reduction of the release rate of serotonin in the locus coeruleus. Microinjection of 8-OH-DPAT into the DRN also slightly lowered blood pressure (3 mmHg). Neither stimulations nor lesion of the DRN, nor microinjection of 8-OH-DPAT into this raphe nucleus, altered the extracellular concentration of 5-HIAA. Judging from the present biochemical results it appears that the serotonergic afferents to the locus coeruleus originate to more than 50% from cell bodies located in the DRN. The neuronal serotonin release in the locus coeruleus is modulated by 5-HT1A receptors lying within the DRN. Changes in blood pressure and release of serotonin elicited by stimulating or lesioning the DRN point to the importance of serotonergic neurons extending between this raphe nucleus and the locus coeruleus in central cardiovascular control.

Hyperforin enhances the extracellular concentrations of catecholamines, serotonin and glutamate in the rat locus coeruleus

Hyperforin is the main antidepressant component of hypericum perforatum (St. John's Wort). Using the push-pull superfusion technique we tested whether hyperforin influences extracellular concentrations of neurotransmitters in the rat locus coeruleus. Hyperforin (10 mg/kg, i.p.) not only enhanced the extracellular levels of the monoamines dopamine, noradrenaline and serotonin, but also that of the excitatory amino acid glutamate. The levels of the main serotonin metabolite 5-hydroxyindolacetic acid, as well as those of the amino acids GABA, taurine, aspartate, serine and arginine, were not influenced. Together with in vitro studies, our findings suggest that the antidepressant property of hyperforin is due to enhanced concentrations of monoamines and glutamate in the synaptic cleft, probably as a consequence of uptake inhibition.

Glial cells participate in histamine inactivation in vivo.

The ability of glial cells to take up histamine in vitro suggests that these cells may be involved in histamine inactivation. This prompted us to study the possible interactions between neuronal and glial processes which determine the histamine concentration in the synaptic cleft. In vitro experiments showed that the glial metabolic toxin, fluoroacetate (20 and 40 mmol/l) depressed histamine uptake into cultured astroglial cells and dissociated hypothalamic cells of rats. For in vivo experiments, the push-pull superfusion technique was used. In anaesthetized rat, the anterior hypothalamic area was superfused through the push-pull cannula with artificial cerebrospinal fluid (aCSF) or with aCSF which contained fluoroacetate and the release of endogenous histamine was determined in the superfusate. Hypothalamic superfusion with fluoroacetate (20 mmol/l) led to a pronounced increase in extracellular histamine. The effect of fluoroacetate was inhibited by 5 micromol/l tetrodotoxin. Superfusion with Ca++-free, Mg++-rich (12 mmol/l) aCSF inhibited the basal release rate of histamine. Under these conditions, 20 mmol/l fluoroacetate did not modify the level of the amine in the superfusate. These data demonstrate that depression of glial function enhances the concentration of histamine in the extracellular space by slowing down the uptake of the amine into the glial cells. Thus, under in vivo conditions, glial cells are directly involved in the continuous removal of neuronal histamine from the synaptic cleft.

Nitric oxide modulates the release of acetylcholine in the ventral striatum of the freely moving rat.

The influence of nitric oxide on acetylcholine release in the ventral striatum was investigated by the push-pull superfusion technique in the conscious, freely moving rat. Superfusion with the nitric oxide donors S-nitroso-N-acetylpenicillamine or with 3-morpholino-sydnonimine caused a pronounced increase in striatal acetylcholine release. This effect was prevented by superfusion with tetrodotoxin. Pre-superfusion with the guanylyl cyclase inhibitor methylene blue abolished the effect of 3-morpholino-sydnonimine. Superfusion of the ventral striatum with the guanylyl cyclase inhibitor LY83583 decreased acetylcholine release by 60% of basal release, whereas the less specific guanylyl cyclase inhibitor methylene blue was ineffective in this respect. Superfusion of the ventral striatum with inhibitors of nitric oxide synthase also led to different effects on basal acetylcholine release. Superfusion with L-NG-methylarginine did not influence basal acetylcholine release, whereas superfusion with L-NG-nitroarginine or with L-NG-nitroarginine methyl ester led to a substantial decrease in acetylcholine output, the latter compound being more effective. The effect of L-NG-nitroarginine was abolished by simultaneous superfusion with L-arginine. The effects of NO donors and of LY83583 suggest that NO increases acetylcholine release, probably by a cGMP-dependent mechanism. The effectiveness of nitric oxide synthase inhibitors shows that the activity of striatal neurons is under the permanent influence of nitric oxide, that leads, via a direct or indirect mechanism, to continuous enhancement of acetylcholine release. In conclusion, our findings suggest that NO synthesized in the ventral striatum acts as an intracellular messenger which modulates acetylcholine release.

Taurine release in the hypothalamus is altered by blood pressure changes and neuroactive drugs

The release of endogenous taurine was determined in the posterior hypothalamus of the conscious, freely moving rat by using the push-pull superfusion technique. At the start of superfusion, the outflow of endogenous taurine declined rapidly with a half-time of 7.9 min and reached a steady state after approximately 1 h. Thereafter, the release rate was constant and amounted to 2.6±0.3 pmol/min. During depolarization either with K + (50 or 90 mM) or veratridine (1 or 10 μM), taurine outflow was increased in a concentration-dependent way. Hypothalamic superfusion with tetrodotoxin (1 μM) elicited a sustained decrease in the taurine release to 60% of the control values. Intravenous infusion of noradrenaline led to a rise in blood pressure (45 mm Hg) and enhanced the release of taurine in the hypothalamus. A fall of blood pressure (30 mm Hg) caused by an intravenous infusion of nitroprusside diminished taurine outflow. The results suggest that a considerable amount of the taurine detected is released from hypothalamic neurons. Changes in the release rate of taurine by experimentally induced alterations of blood pressure indicate that, in the posterior hypothalamus, the amino acid might play an important role as a neurotransmitter or neuromodulator possessing a hypotensive function.

Evidence that neurokinin A (substance K) neurons project from the striatum to the substantia nigra in rats

The striatonigral neurokinin A (NKA) pathway in rats was examined with an antiserum specific to the N-terminal region of NKA. Thermal lesions of the striatum decreased both substance P-like immunoreactivity (SP-LI) and NKA-like immunoreactivity (NKA-LI) in the substantia nigra (SN). The releases of SP-LI and NKA-LI in the SN were measured by a push pull superfusion technique. Addition of 50 mM K + to the superfusion medium increased the releases of both SP-LI and NKA-LI to 1.5 2.0 times the basal values. These results suggest that NKA neurons as well as SP neurons in the striatum project to the SN in rats.

Involvement of intraterminal dopamine compartments in the amine release in the cat striatum

A push pull superfusion technique has been used in the anesthetized cat to study the simultaneous release of tritiated and total dopamine (DA) during continuous labelling with tritiated tyrosine. The concentration of tritiated and total DA (1.3 and 70 nM respectively) and dihydroxyphenylacetic acid (1 μM) have been measured in the extracellular space under our experimental conditions. The specific activity of spontaneously released DA was found to be 0.76 Ci/mmol. The release of tritiated and total amine following 3 h superfusion with [ 3H]tyrosine did not occur in parallel in response to the local application of either α-methyl- p-tyrosine (0.1 mM) or amphetamine (1 μM). Amphetamine induced an increase followed by a decrease in the specific activity of released DA which reflects an initial release of newly synthesized DA followed by the release of stored amine. The transfer between intraterminal pools of the amine is thus clearly evidenced. The results show that the simultaneous determination of tritiated and total DA release allows the relative contribution of the two intraterminal pools to the amine release to be monitored.