Browsing by Author "BUSTOS, G"
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- ItemACTIVATION OF TYROSINE-HYDROXYLASE IN RAT STRIATAL SLICES BY K+-DEPOLARIZATION - EFFECT OF ETHANOL(1976) BUSTOS, G; ROTH, RH; MORGENROTH, VHSlices from rat corpus striatum were incubated for 10 min at 37.degree. C in freshly oxygenated Krebs-Ringer phosphate (KRP) media or KRP-high K+ (55 mM) media both in the presence and absence of ethanol (0.2-0.8%, wt/vol). Thereafter, the slices were homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 105,000 g supernatant fraction. The presence of K+ (55 mM) in the incubation media increased about 3-fold the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, when compared to that found in striatal slices incubated in normal KRP media. Incubation of striatal slices in a KRP-high K+ media also produced changes in the kinetic properties of tyrosine hydroxylase. The Km of the enzyme for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine HCl (DMPH4) was decreased from 0.82 to 0.09 mM and the Ki of the enzyme for dopamine (DA) was increased from 0.13 to 3.52 mM. Ethanol (0.2-0.8%, wt/vol) added directly to the KRP-high K+ media markedly blocked the K+-induced activation of tyrosine hydroxylase and the kinetic alterations in the enzyme observed after K+-depolarization of the striatal slices. In contrast, the presence of ethanol did not modify the activity and kinetic characteristics of tyrosine hydroxylase isolated from slices incubated in normal KRP media. The increase in DA synthesis observed in striatal slices after K+-depolarization might be mediated in part via an allosteric activation of tyrosine hydroxylase. This activation appeared to be mediated by an increase in the affinity of the enzyme for the pterin cofactor and a decreased affinity for the end-product inhibitor DA. The blocking effect of ethanol upon the kinetic activation of tyrosine hydroxylase after K+-depolarization seemed to offer a likely explanation for the inhibitory effect of ethanol on K+-induced increase in DA synthesis reported by Gysling et al.
- ItemCHANGES IN EXTRACELLULAR LEVELS OF GLUTAMATE AND ASPARTATE IN RAT SUBSTANTIA-NIGRA INDUCED BY DOPAMINE-RECEPTOR LIGANDS - IN-VIVO MICRODIALYSIS STUDIES(1995) ABARCA, J; GYSLING, K; ROTH, RH; BUSTOS, GThe microdialysis technique was utilized to study the local effects of D-1 and D-2 family type dopamine (DA) receptor (R) ligands on the in vivo release of endogenous glutamate (GLU) and aspartate (ASP) from rat substantia nigra (SN). Addition to the dialysis perfusion solution of either D-1-R and D-2-R agonists, such as SKF-38393 (50 and 100 mu M) and Quinpirole (5 and 10 mu M), resulted in dose-dependent increases in extracellular concentrations of GLU and ASP, respectively. The SKF-38393 and Quinpirole-induced effects were reduced by SCH-23390 (0.5 mu M), a D-1-R antagonist, and by Spiperone (1.0 mu M), a D-2-R antagonist, respectively. However, SCH-23390 and Spiperone did increase GLU and ASP extracellular concentrations. Local infusion with Tetrodotoxin (TTX) (1.0 mu M), a blocker of voltage-dependent Na+ channels, increased basal extracellular levels of GLU. In addition, co-infusion of TTX and SKF-38393 evoked increases in extracellular GLU levels higher than those observed after SKF-38393 alone. Finally, chemical lesions of nigral DA cells with 6-OH-DA. increased the basal extracellular levels of GLU. It is proposed that the release of GLU and ASP from SN may be regulated by D-1- and D-2- receptors present in this basal ganglia structure. In addition, part of the D-1 receptors present in SN might be located presynaptically on GLU-containing nerve endings.
- ItemCORTICAL ABLATION REDUCES VERATRIDINE EVOKED RELEASE OF ENDOGENOUS GLUTAMATE FROM SUPERFUSED SUBSTANTIA-NIGRA SLICES(1991) FIEDLER, J; BUSTOS, GA brain slice superfusion system and HPLC coupled to fluorometric detection were used to study depolarization-evoked release of endogenous glutamate (Glu) and aspartate (Asp) from substantia nigra (SN) slices. Veratridine (15-25 mu-M) and K+ (50 mM) evoked both a Ca2+-dependent and Ca2+-independent release of Glu and Asp. However, the pattern of Glu release was different from that of Asp. In addition, cortical ablation significantly reduced the evoke release of Glu but not that of Asp. There results indicate the existence of SN of functional Glu- and Asp-releasing nerve terminals. Some of the Glu-enriched nerve terminals might belong to a cortico-nigral neuronal pathway.
- ItemL-GLUTAMIC ACID, A NEUROMODULATOR OF DOPAMINERGIC TRANSMISSION IN THE RAT CORPUS STRIATUM(1983) RUDOLPH, MI; ARQUEROS, L; BUSTOS, GA superfusion system was used to study the effects of neuroexcitatory amino acids upon spontaneous and depolarization-evoked release of exogenously taken up and newly synthesized [3H]dopamine by rat striatal slices. Neither L-glutamate nor other aminoacids such as L-aspartate and D-glutamate (5 .times. 10-5 M) modified the spontaneous release of exogenous [3H]dopamine from rat striatal slices. These neuroexcitatory aminoacids did potentiate spontaneous release of striatal [3H]dopamine newly synthesized from [3H]tyrosine. A different pattern of effects emerged when depolarization-evoked release of dopamine was studied. Only L-glutamate (5 .times. 10-6-1 .times. 10-4 M) potentiated dopamine release under these experimental conditions in a rather specific and stereoselective manner. Similar results were obtained regardless of whether depolarization-induced release of exogenous or newly synthesized [3H]dopamine was studied. The effect of L-glutamate on depolarization-induced release depended both upon the degree of neuronal depolarization and upon the presence of external Ca2+ in the superfusion medium and it was blocked by L-glutamate diethylester. This effect of L-glutamate seemed quite specific with regard to regional localization within the brain as it was only demonstrated in slices from striatum and not in slices from olfactory tubercle or hippocampus. It is suggested that during depolarization a Ca2+-dependent event occurs at the striatal membrane level which changes the sensitivity of the dopamine release process to neuroexcitatory aminoacids in such a way as to render it relatively more specific and steroselective towards L-glutamate stimulation. Evidently L-glutamic acid could play a role as a neuromodulator of dopaminergic transmission in the rat corpus striatum.
- ItemON THE MECHANISM OF PRE-SYNAPTIC AUTORECEPTOR-MEDIATED INHIBITION OF TRANSMITTER SYNTHESIS IN DOPAMINERGIC NERVE-TERMINALS(1982) BITRAN, M; BUSTOS, GThe effect of apomorphine (APO) upon dopamine (DA) synthesis and release from rat striatal slices was studied. The synthesis of DA was measured by incubating the slices in Krebs-Ringer phosphate (KRP) medium of variable ionic compositon containing L-tyrosine[14C-U] as DA precursor. A superfusion system was used to study both spontaneous and K+-induced release of labeled DA from striatal slices. The addition of APO directly to the normal KRP medium markedly blocked the formation of [14C]DA from [14C]Tyr with an IC50 [median inhibitory concentration] of 1.8 .times. 10-7 M. Haloperidol (4 .times. 10-7 M), a known DA antagonist, produced a shift to the right of the concentration-response curve for APO inhibition on DA synthesis, whereas the DA antagonist (+)butaclamol (4 .times. 10-7 M) completely reversed the inhibition caused by APO (2 .times. 10-7 M). DA uptake blockers, such as benztropine (2 .times. 10-6 M) or cocaine (1 .times. 10-5 M), did not affect the ability of APO to inhibit DA synthesis. The .alpha.2-adrenergic agonist clonidine produced only a mild inhibition and the .beta.-adrenergic agonist isoproterenol produced no inhibition of [14C]DA formation. APO was able to inhibit DA formation in the absence of Ca in the incubation medium or in the presence of high external Ca concentrations (4, 8 and 24 mM) which depress the rate of DA synthesis. Incubation conditions that cause an increase of free intraneuronal Ca concentrations, such as Na+-free medium, the presence of ouabain (1 .times. 10-4 M), or K+ depolarization, dramatically abolished or impaired the ability of APO to inhibit DA synthesis in striatal slices. It was not possible to demonstrate any change in spontaneous and K+ (27 mM)-induced release of DA in the presence of APO concentrations that produced a marked inhibition of DA synthesis. Tissue slices can evidently be used as a valuable experimental tool to study the inhibitory effect of APO on DA synthesis; this effect occurs through an interaction of APO with presynaptic DA autoreceptors located in striatal dopaminergic nerve terminals. The results do not correlate with the autoreceptor-mediated inhibition of DA synthesis occurring through regulation of Ca influx into the DA nerve terminals. A sensitivity to high intraneuronal Ca concentration may exist during the events that mediate APO-DA autoreceptor interaction and DA synthesis inhibition. DA-synthesis-modulating autoreceptors apparently do not participate in the modulation of DA release.
- ItemPUTATIVE GLUTAMATE RECEPTORS IN MEMBRANES OBTAINED FROM HEADS OF DROSOPHILA-MELANOGASTER(1986) FIEDLER, JL; INESTROSA, NC; BUSTOS, GThe specific binding of L-[3H]-glutamic acid (GLU) was examined in membrane preparations derived from heads of Drosophila melanogaster. L-[3H]-GLU bound to the membrane preparation with biphasic kinetics in a reversible and saturable way. The specific binding was inhibited by Cl- and Ca2+ ions. Saturation analysis of the data fit a model of two independent binding sites with dissociation constants (KD) of 29 nM and 249 nM and corresponding binding site densities (Bmax) of 5.7 and 24.6 pmol/mg protein. A series of excitatory amino acid receptor agonists and antagonists were tested as inhibitors of L-[3H]-GLU specific binding. Some compounds almost totally abolished the specific binding, whereas others reduced the binding to 45% over the concentration range used. Among the first type of inhibitors, L-GLU was the most potent, D-aspartate (D-ASP) and D-GLU were 10 times less effective than L-GLU, whereas quisqualic acid had the lowest potency. Among the second type of inhibitors, glutamate diethyl ester, D-.alpha.-amino adipate, and N-methyl-D-aspartate showed the highest potency, and DL-2-amino-4-phosphonobutyrate was the less effective. Kainic acid and .gamma.-amino butyric acid were not able to modify at any concentration used the specific binding of L-[3H]-GLU. These data suggest the presence of putative GLU receptors in the brain of Drosophila.
- ItemREGULATION OF [H-3] NOREPINEPHRINE RELEASE BY N-METHYL-D-ASPARTATE RECEPTORS IN MINISLICES FROM THE DENTATE GYRUS AND THE CA(1)-CA(3) AREA OF THE RAT HIPPOCAMPUS(1993) ANDRES, ME; BUSTOS, G; GYSLING, KIt has been reported previously that N-methyl-D-aspartic acid induces a significant release of [H-3] norepinephrine preaccumulated in slices from the hippocampus. In the present study, we investigated whether there are regional differences in the hippocampus regarding this N-methyl-D-aspartate effect. In the absence of Mg2+, N-methyl-D-aspartate (10-200 mu M) induced the release of [H-3]norepinephrine from superfused minislices containing the dentate gyrus area or the CA(1)-CA(3) region of the hippocampus. Such N-methyl-D-aspartate effects on [H-3]norepinephrine release were significantly higher in the dentate gyrus than in the CA(1)-CA(3) area. The N-methyl-D-aspartate effects in both hippocampal areas were also reduced significantly by D-2-amino-5-phosphonovaleric acid (50 mu M), an antagonist of the N-methyl-D-aspartate receptor, and by tetrodotoxin, a blocker of the voltage-dependent Na+ channels. The extent of this reduction was the same in the dentate gyrus and the CA(1)-CA(3) area. Further experiments, conducted in the presence of Mg2+, demonstrated that N-methyl-D-aspartic acid increased K+-induced release of [H-3]norepinephrine from dentate gyrus minislices but not from the CA(1)-CA(3) area. The results are consistent with the existence of a higher density and/or different subtypes of N-methyl-D-aspartate receptors modulating [H-3]norepinephrine release in the dentate gyrus as compared with the CA(1)-CA(3) hippocampal area.
- ItemREGULATION OF TRANSMITTER SYNTHESIS AND RELEASE IN MESOLIMBIC DOPAMINERGIC NERVE-TERMINALS - EFFECT OF ETHANOL(1981) BUSTOS, G; LIBERONA, JL; GYSLING, KSlices from rat olfactory tubercle were incubated in freshly oxygenated Krebs-Ringer phosphate (KRP) and in the presence of L-tyrosine[14C-U [uniformly 14C-labeled]] as dopamine (DA) precursor. The newly synthesized [14C]DA and the [14C]DA released into the incubation media were isolated by Alumina column and ion-exchange chromatography. The presence of K+ depolarizing concentrations (25-70 mM) in the incubation media markedly increased the formation of [14C]DA from [14C]tyrosine, following a rather complex and biphasic pattern. Dibutyryl cAMP (dB-cAMP) and theophylline increased the formation of newly synthesized [14C]DA. Ethanol (0.2 to 0.4%, wt/vol) significantly blocked the stimulation of [14C]DA biosynthesis that was induced by low K+ depolarizing concentrations (25 mM) and by dB-cAMP (5 .times. 10-4 M) or theophylline (1 .times. 10-3 M. Only higher ethanol concentrations (0.8 to 1.1%, wt/vol) blocked the increase in DA formation induced by high K+ depolarizing concentrations (40 and 55 mM). K depolarization (40 mM) markedly evoked the release of newly synthesized [3H]DA or [3H]DA previously taken up by the slices. The release was dependent upon the presence of Ca2+ and inhibited by an excess of Mg2+ (12 mM). Ethanol (0.8-1.1%, wt/vol) produced no effect on K+-induced release of [3H]DA. The model described can be used as a simple experimental tool to study neurotransmitter synthesis and release from nerve terminals belonging to the mesolimbic dopaminergic system. At least 2 mechanisms exist by which neuronal depolarization increases transmitter formation in mesolimbic dopaminergic terminals. Ethanol, at relatively low concentrations, seems to produce a specific inhibitory effect upon the mechanism that predominates under low depolarizing conditions. The possibility is raised that the effects described for ethanol may play a role in the neuropharmacological responses induced by this agent in vivo.
- ItemRELEASE OF D-[H-3] ASPARTIC ACID FROM THE RAT STRIATUM - EFFECT OF VERATRIDINE-EVOKED DEPOLARIZATION, FRONTO-PARIETAL CORTEX ABLATION, AND STRIATAL LESIONS WITH KAINIC ACID(1985) ARQUEROS, L; ABARCA, J; BUSTOS, GThe spontaneous and depolarization-evoked release of radiolabeled D-aspartic acid, previously taken up by rat striatal slices, was studied by using a superfusion system. Veratridine (10-50 .mu.M), electrical field stimulation (20 Hz, 1.0 V, 60 s), and K (53 mM) markedly potentiated the release of D-[3H]aspartate from striatal slices. The release of L-[3H]glutamate was also increased by veratridine, according to a pattern and time course of release similar to that of D-[3H]aspartate. The ratio of D-[3H]aspartic acid release evoked by veratridine over-spontaneous levels of release was much higher when compared to that of radiolabeled L-glutamate. Omission of Ca from the superfusion medium almost completely suppressed D-[3H]aspartate release evoked by veratridine or by electrical stimulation whereas high K+-evoked release of the [3H]amino acid was only slightly reduced. Increasing Mg2+ concentration of 12 mM in the superfusion medium did substantially block D-[3H]aspartate release induced by K+-depolarization. Tetrodotoxin (1 .mu.M), a blocker of voltage-dependent Na+ channels, totally abolished veratridine-evoked release of D-[3H]aspartate from striatal slices. Lesion studies showed that unilateral ablation of the frontoparietal cortex was accompanied by a significant decrease in the high-affinity uptake of striatal D-[3H]aspartate and by a large and parallel loss from striatal slices in D-[3H]aspartate release evoked by either veratridine or high K+. In contrast, unilateral injection of kainic acid into the striatum did not influence depolarization-evoked release of D-[3H]asparate from striatal slices. D-[3H]aspartic acid may be taken up preferentially and then released, in a Ca2+-dependent manner, by veratridine and electrical stimulation from nerve terminals belonging to the cortico-striatal pathway. Excitatory amino acids may act as neurotransmitters at the cortico-striatal nerve fibers.
- ItemRELEASE OF D-[H-3] ASPARTIC ACID FROM THE RAT SUBSTANTIA NIGRA - EFFECT OF VERATRIDINE-EVOKED DEPOLARIZATION AND CORTICAL ABLATION(1985) ABARCA, J; BUSTOS, GThe spontaneous and veratridine-evoked release of radioactive D-aspartic acid, previously taken up by rat substantia nigra slices, was studied by using a superfusion system. Veratridine (25 .mu.M, 1 min) markedly produced a 14-fold increase in D-[3H]aspartic acid release from nigral slices. Omission of Ca2+ and increasing Mg2+ concentration to 12 mM in the superfusion medium did substantially block D-[3H]aspartate release induced by veratridine depolarization. Veratridine was able to evoke [3H]amino acid release which seemed to be, at least, 30% Ca2+-independent. Tetrodotoxin (0.01-0.1 .mu.M), a blocker of voltage-dependent Na+ channels, totally abolished veratridine-evoked release of D-[3H]aspartate from nigral slices. Lesion studies were performed to learn about the nature of the neuronal compartment in the substantia nigra upon which veratridine-depolarization acted to induce D-[3H]aspartate release. Unilateral ablation of the fronto-parietal cortex was accompanied by a significant decrease in the accumulation of nigral D-[3H]aspartate and by a large loss from ipsilateral nigral slices in D-[3H]aspartate release evoked by veratridine. Both the accumulation and veratridine-evoked release of [3H]dopamine, remained unchanged in the ipsilateral substantia nigra slices to the lesion. D-[3H]aspartic acid may be taken up and then released, in a Ca2+-dependent manner, by nerve terminals located in the substantia nigra. L-glutamate and/or L-aspartate may act as neurotransmitters at the cortico-nigral neuronal pathway.
- ItemRELEASE OF ENDOGENOUS CATECHOLAMINES FROM THE STRIATUM AND BED NUCLEUS OF STRIA TERMINALIS EVOKED BY POTASSIUM AND N-METHYL-D-ASPARTATE - IN-VITRO MICRODIALYSIS STUDIES(1995) ALIAGA, E; BUSTOS, G; GYSLING, KInduced release of endogenous dopamine and noradrenaline from coronal slices containing the striatum and the bed nucleus of the stria terminalis, respectively, was studied by means of in vitro microdialysis, A Ca+2-dependent and reserpine-sensitive K+-induced release of catecholamines was detected in both nuclei, We confirmed that N-methyl-D-aspartate (2.5 and 5.0 mM in the dialysis perfusion solution) induces the release of dopamine from the striatum, and this effect was blocked by prior dialysis perfusion with 500 mu M MK-801, a noncompetitive N-methyl-D-aspartate receptor antagonist, Infusion of N-methyl-D-aspartate (1-10 mM) or glutamate through the dialysis probe did not produce any detectable modification in the extracellular levels of noradrenaline in the bed nucleus of the stria terminalis, In addition, perfusion with D-serine (100 mu M) alone or in the presence of desipramine (10 mu M), resulted in a slight increase in extracellular noradrenaline in the bed nucleus of the stria terminalis. However, N-methyl-D-aspartate in the presence of D-serine and desipramine produced a marked increase in extracellular noradrenaline from the bed nucleus of the stria terminalis. These results indicate that N-methyl-D-aspartate receptors might regulate the release of noradrenaline from the bed nucleus of the stria terminalis as is the case of dopamine release in the striatum, The in vitro microdialysis seems to be a suitable complement to the in vivo microdialysis for the study of catecholamine release in discrete regions of the central nervous system and its local regulation by excitatory amino acid receptors. (C) 1995 Wiley Liss, Inc.
- ItemSEARCH OF A L-GLUTAMATE RECEPTOR RELATED TO MODULATION OF NEUROTRANSMISSION IN THE RAT CORPUS STRIATUM(1986) RUDOLPH, MI; BUSTOS, GThe specific binding of L-[3H]glutamic acid to crude membrane preparations obtained from rat striatum was studied with the aid of radioligand binding techniques. Saturation isotherms and binding of 40 nM L-[3H]glutamate in the presence of increasing concentrations of unlabeled L-glutamate revealed an anomalous kinetic pattern which suggested the presence of three populations of L-[3H]glutamate affinity binding sites in fresh striatal membranes. The presence of three binding sites became more evident in frozen membranes and in membranes prepared from striatal slices previously subjected to K+-depolarization; in addition, these experimental manipulations increased the specific binding of L-[3H]glutamate. A number of glutamate analogs were tested with the aim to discriminate among the three binding sites. Of the analogs tested, D-aspartate displaced membrane-bound L-[3H]glutamate from only two of the three binding sites. This finding permitted to investigate the characteristics of the D-aspartate-insensitive affinity site, independently to the other sites, by just including 1 mM D-aspartate in the binding assays. Specific binding of L-[3H]glutamate to the D-aspartate-insensitive site occurred in a reversible manner. A saturation curve with sigmoidal characteristic was generated when fresh, frozen and depolarized membranes were incubated with increasing concentrations of L-[3H]glutamate. Hill plots revealed slopes close or higher than 2 in all these membrane preparations. KD, and Hill slopes values were found not to differ significantly among the different membrane preparations used. However, frozen membranes as well as membranes obtained from striatal slices previously depolarized by high K+ (55 mM), showed a significant increase in Bmax as compared to fresh and non-depolarized membranes, respectively. Only L-glutamate, quisqualate and L-glutamate diethylester were relatively potent in inhibiting the specific binding of L-[3H]glutamate. L-Aspartate and N-methyl-D,L-aspartate competed partially and with relatively less potency whereas D-glutamate and kainate, even when tested at 0.5 mM, produced only a slight displacement of membrane-bound radioligand. The characteristics and pharmacological specificity of this binding site correlates very well with previous observations from us (Rudolph and Bustos, 1983; Rudolph et al., 1983) when studying the effects that L-glutamate and structural analogs produce upon depolarization-evoked release of [3H]dopamine from rat striatal slices. The proposal is made that the D-aspartate-insensitive binding site, reported in this work, might be related to a quisqualate-type receptor which becomes predominantly exposed following depolarization and might exert then a modulation of neuronal transmission in the rat striatum.
- ItemSPECIFIC BINDING OF L-[H-3]-GLUTAMIC ACID TO RAT SUBSTANTIA-NIGRA SYNAPTIC-MEMBRANES(1986) FIEDLER, JL; ARQUEROS, L; BUSTOS, GThe specific binding of L-[3H]-glutamic acid (GLU) was investigated in synaptic membranes from rat substantia nigra. L-[3H]-GLU binding to the membrane preparations occurred in a reversible and saturable way. The specific binding was stimulated by the presence of CaCl2 and was reduced by freezing and thawing the membranes. Scatchard analysis of the saturation isotherms yielded a non-linear plot suggesting that the binding reaction does not occur through a simple biomolecular association. Assuming non-interacting binding sites, a high (KD1, 139 nM; Bmax1, 3.5 pmoles/mg protein) and a low (KD2, 667 nM; Bmax2, 15.1 pmoles/mg protein) affinity L-[3H]-GLU binding site were obtained. The kinetics of dissociation of bound L-[3H]-GLU was biphasic; the respective dissociation rate constant (k-1) being 0.20 min-1 and 0.013 min-1. A series of amino acid receptor agonists and antagonists were tested as inhibitors of L-[3H]-GLU specific binding. Quisqualic acid, L-GLU and D-.alpha.-aminoadipate (D-.alpha.-AA) were the most potent inhibitors. DL-2-amino-4-phosphonobutyrate (APB), N-Methyl-D-aspartate (NMDA) and D-GLU were moderate inhibitors, whereas diaminopimelic acid (DAPA) and glutamate diethyl ester (GDEE) exhibited the lowest relative potency. Kainic acid (KA), .gamma.-aminobutyric acid (GABA) and bicuculline were not able to modify at any concentration used the specific binding of L-[3H]-GLU. These data demonstrate the presence of specific GLU binding sites in synaptic structures at substantia nigra level and support the idea that excitatory amino acids may play a role in synaptic transmission in this brain region.
- ItemTYROSINE-HYDROXYLASE ACTIVATION AND TRANSMITTER RELEASE FROM CENTRAL NORADRENERGIC NEURONS BY ELECTRICAL-FIELD STIMULATION(1978) BUSTOS, G; MORGENROTH, VH; HANCKE, JL; ROTH, RHElectrical stimulation of the noradrenergic neurons in the locus coeruleus of the rat results in a marked increase in the tyrosine hydroxylase activity of the hippocampus on the stimulated side (Roth et al., 1975). An in vitro system was developed to further study this interesting phenomenon. Rat hippocampal slices were stimulated in an electrical field using specially built chambers which permitted a continuous superfusion with Krebs Ringer Phosphate solution while stimulation was taking place. The slices were then homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 104000 g supernate fraction. Electrical field stimulation (60 V [voltage], 4 ms, 5-20 Hz, 5 min) induced a stimulus-dependent increase in tyrosine hydroxylase activity. The increase in tyrosine hydroxylase activity was directly dependent on the number of pulses of stimulation applied. K depolarization of hippocampal slices also resulted in a marked increase in the activity of the enzyme. Electrical field stimulation appears to activate tyrosine hydroxylase by causing an increase in its affinity for both substrate and pteridine cofactor and by decreasing its affinity for the endproduct inhibitor, norepinephrine. No change in Vmax was observed. The superfusion system was also used to study spontaneous and electrically evoked release of labeled norepinephrine from hippocampal slices. Electrical field stimulation for 1.0 min produced a marked increase in the release of label. Absence of Ca from the superfusion solution almost completely abolished the electrically evoked release of exogenously taken up 3H-norepinephrine. The release of 3H-norepinephrine was also found to be dependent on the number of pulses of stimulation used. The hypothesis that depolarization of central noradrenergic neurons results in an increase in transmitter synthesis mediated in part via a kinetic activation of tyrosine hydroxylase is supported. The concomitant use of superfusion and electrical field stimulation of hippocampal slices provides a simple system to study neurotransmitter synthesis and release in central noradrenergic neurons.