Properties of 3,4-diaminopyridine-evoked dopamine and acetylcholine release in rabbit caudate nucleus slices: involvement of facilitatory adenosine A2 receptors or nitric oxide?

The 3H-overflow from slices of the rabbit caudate nucleus preincubated with tritiated dopamine (DA), or choline, and then superfused and stimulated twice with 3,4-diaminopyridine (3,4-DAP; 25 microM, 1 min), was explored as an in vitro model for evoked release of DA, or acetylcholine (ACh), respectively. In both cases the 3,4-DAP-evoked 3H-overflow was tetrodotoxin-sensitive and Ca(2+)-dependent and hence most probably represents action potential-induced exocytotic release of DA or ACh, respectively. Using pairs of preferential agonists/antagonists it was shown, that evoked DA release was inhibited via presynaptic D2 autoreceptors (quinpirole/domperidone) and kappa-opioid receptors (U-50488H/norbinaltorphimine). No evidence was found for the presence of presynaptic adenosine A1 or A2 receptors on dopaminergic terminals. Moreover, 3,4-DAP-evoked DA release was unaffected by increased intracellular cyclic AMP levels or by drugs affecting the NO/guanylate cyclase pathway. In a similar manner it was shown that 3,4-DAP-evoked ACh release was inhibited via presynaptic muscarine autoreceptors (oxotremorine/atropine) and dopamine D2 heteroreceptors (quinpirole/domperidone). Again, no evidence for the involvement of the NO/guanylate cyclase system in the modulation of ACh release was found, whereas the presence of inhibitory adenosine A1 receptors, but not of facilitatory A2 receptors, could be clearly established. It is concluded, that 3,4-DAP-evoked 3H-overflow from rabbit caudate nucleus slices preincubated with [3H]DA or [3H]choline, represents a simple and useful in vitro model for action potential-induced DA or ACh release, respectively. Moreover, at least in this model or rabbit brain region, facilitatory adenosine A2 receptors and the NO/guanylate cyclase system seem not to be involved in the release of these transmitters.     

No evidence for presynaptic opioid receptors on cholinergic, but presence of kappa-receptors on dopaminergic neurons in the rabbit caudate nucleus: involvement of endogenous opioids

The effects of various opioid receptor agonists and antagonists were studied in rabbit caudate nucleus slices preincubated with either [3H]dopamine or [3H]choline, superfused with medium (containing in most experiments the D2 receptor antagonist domperidone) and subjected to electrical field stimulation. The stimulation-evoked [3H]overflow from slices prelabeled with [3H]dopamine (evoked [3H]dopamine release) was significantly reduced by preferential kappa-opioid receptor agonists, like U-50,488 H, but not by mu- or delta-opioid receptor selective drugs. Opioid receptor antagonists shifted the concentration/response curve of U-50,488 H to the right (apparent pA2-value of the kappa-selective antagonist nor-binaltorphimine: 10.1) and enhanced the evoked dopamine release in the presence of a mixture of peptidase inhibitors. On the other hand, the [3H]overflow from rabbit caudate nucleus slices prelabeled with [3H]choline (evoked acetylcholine release) remained almost unaffected by any opioid receptor agonist, as long as the presynaptic D2 heteroreceptor was blocked with domperidone: in the absence of domperidone, U-50,488 H exhibited facilitatory effects. For comparison, the effects of the preferential delta-opioid receptor agonist DPDPE was also studied in slices of the rat striatum, where it clearly inhibited the evoked acetylcholine release. From our data we conclude that in the rabbit caudate nucleus the evoked dopamine release is inhibited by both exogenous and endogenous opioids via presynaptic kappa-opioid receptors, whereas the evoked release of acetylcholine is not, or only indirectly (via released dopamine) affected by opioids.     

Quantitation of age-related mitochondrial DNA deletions in rat tissues shows that their pattern of accumulation differs from that of humans

We studied the postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors in the rat hippocampus. To this end, hippocampal slices (350 microns thick) from rats of various postnatal ages (postnatal day 3 [P3] to P16) were preincubated with [3H]choline and stimulated twice (S1, S2: 360 pulses, 2 ms, 3 Hz, 60 mA) during superfusion with physiological buffer containing hemicholinium-3 (10 microM). In parallel, the activities of hemicholinium-sensitive high-affinity choline uptake (HACU, in synaptosomes) and of choline acetyltransferase (ChAT, in crude homogenates) were determined as markers for the cholinergic ingrowth. In hippocampal slices preincubated with [3H]choline, the electrically evoked overflow of 3H at S1 increased from 0.11 (P3) to 0.81% of tissue 3H (P16), the latter value being still much lower than that of hippocampal slices from adult rats (2.89% of tissue 3H). Already at P3 the evoked overflow of 3H was Ca(2+)-dependent and sensitive to tetrodotoxin, indicating an action potential-evoked exocytotic mechanism of ACh release. The muscarinic agonist oxotremorine (1 microM) significantly inhibited the evoked ACh release in hippocampal slices with increasing effectivity from P4 to P16; no significant effect was detectable at P3. The ACh esterase inhibitor physostigmine and the muscarinic antagonist atropine (1 microM, each) exhibited significant inhibitory and facilitatory effects, respectively, only at P15-16. The specific activities of both hippocampal HACU (pmoles/mg protein/min) and ChAT (nmoles/mg protein/min) continuously increased from P3 to P16. It is concluded (1) that cholinergic nerve terminals arriving at the hippocampal formation during postnatal ingrowth are already endowed with the apparatus for action potential-induced, Ca(2+)-sensitive (exocytotic) ACh release; (2) that, in contrast, the expression of presynaptic muscarinic autoreceptors on these cholinergic axon terminals is delayed; and (3) that autoinhibition due to endogenous ACh develops even later, probably when the density of presynaptic terminals in the hippocampus and hence, the concentration of released ACh has reached a suprathreshold value.     

Inhibition of human poorly differentiated nasopharyngeal carcinoma cell line CNE-3 growth by antisense EB virus LMP-1 gene]

Slices of hippocampus of the rat, preincubated with [3H]noradrenaline ([3H]NA), were used to investigate the effects of toosendanin on the release of [3H]NA. Toosendanin potently enhanced spontaneous 3H outflow. Seventy-four percent of the enhancement was inhibited by reserpine pretreatment. The toosendanin-induced 3H overflow was in a concentration-dependent manner (5-60 microM) both in the presence and absence of extracellular calcium. Under Ca(2+)-free conditions, the effect of toosendanin on 3H outflow was unchanged by TTX, but inhibited by Ca(2+)-chelator BAPTA-AM; dantrolene sodium failed to affect the toosendanin-induced 3H outflow, while 3,4-diaminopyridine showed an additive effect on the outflow with this substance. The findings suggest that in the absence of extracellular Ca2+, toosendanin enhances [3H]NA release through the liberation of intracellular Ca2+ stores.     

Adenosine A1 receptors mediate hypoxia-induced inhibition of electrically evoked transmitter release from rat striatal slices

We have examined the role of adenosine in mediating effects of mild hypoxia on electrically evoked transmitter release. Rat striatal slices, preincubated with [3H]dopamine and [14C]choline, were superfused continuously and stimulated electrically. Before and during the second stimulation, some slices were superfused with Krebs' solution with lowered oxygen. This mild hypoxia caused a significant increase of the electrically evoked outflow of endogenous adenosine, hypoxanthine and inosine into the superfusion buffer, whereas electrically evoked release of [3H]dopamine and [14C]acetylcholine was significantly decreased. The addition of 8-cyclopentyl-1,3-dipropylxanthine, a selective adenosine A1 receptor antagonist, blocked the hypoxia-induced inhibitory effect on the evoked release of these two transmitters in a concentration-dependent manner. In summary, the results show that reduction of the oxygen supply to striatal slices results in an increased release of endogenous adenosine, which, by acting on adenosine A1 receptors, decreases the electrically evoked release of dopamine and acetylcholine.     

Measurement possibilities using an electronic portal imaging device

The effects of hexachlorocyclohexane (HCH) isomers and some GABAergic compounds on [3H]noradrenaline (NA) release from rat hippocampal slices prelabelled with 80 nM [3H]NA were determined. The convulsant gamma-HCH isomer facilitated (EC50 = 21 microM) and the depressant delta-HCH isomer reduced (EC50 = 48 microM) the Ca(2+)-dependent K(+)-evoked release of [3H]NA, whereas alpha- and beta-HCH isomers did not show any effect. Moreover, alpha- and delta-HCH isomers antagonized the facilitation of evoked [3H]NA release induced by the gamma-HCH isomer. The GABAergic convulsant drugs, bicuculline, picrotoxin and pentylenetetrazol, did not cause any modification of the evoked [3H]NA release even at high concentrations. Neither bicuculline nor picrotoxin blocked the effects of HCH isomers on K(+)-evoked release of [3H]NA. Exposure of slices to diazepam reduced the K(+)-evoked release of [3H]NA (EC50 = 33 microM) in a manner similar to that of the delta-HCH isomer. In addition, diazepam (50 microM) blocked the gamma-HCH effect and caused an additive inhibitory response with the delta-HCH isomer. On the other hand, diazepam and delta-HCH induced a time-dependent Ca(2+)-independent enhancement of basal [3H]NA release. The results suggest that modulation of [3H]NA release in the hippocampus by HCH isomers may be involved in the central actions of these compounds, and that sites other than the classic GABAA receptor may underlie their presynaptic mechanisms of action.     

Experience with non-immunologic hydrops fetalis]

We studied the postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors in the cell body region of the septohippocampal cholinergic pathway. To this end, septal slices (350 microns thick) from rats of various postnatal ages (postnatal day 3 [P3] to P16) were preincubated with [3H]choline and stimulated twice (S1, S2: 360 pulses, 2 ms, 3 Hz, 60 mA) during superfusion with physiological buffer containing hemicholinium-3 (10 microM). In parallel, the activities of hemicholinium-sensitive high-affinity choline uptake (HACU, in synaptosomes) and of choline acetyltransferase (ChAT, in crude homogenates) were determined as markers for the development of cholinergic functions. In septal slices preincubated with [3H]choline, the electrically evoked overflow of 3H at S1 increased from 0.31% (P3) to 2.10% of tissue 3H (P16), the latter value being still lower than that of septal slices from adult rats (3.46% of tissue 3H). Already at P3, the evoked overflow of 3H was Ca(2+)-dependent and sensitive to tetrodotoxin, indicating an action potential-evoked exocytotic mechanism of ACh release early after birth. Presence of the muscarinic agonist oxotremorine (1 microM) significantly inhibited the evoked ACh release in septal slices beginning from P5: no significant effect was detectable at P3. The ACh esterase inhibitor physostigmine (1 microM) exhibited significant inhibitory effects from P13 onwards. The muscarinic antagonist atropine (1 microM) enhanced the evoked ACh release only in septal tissue from adult rats. The specific activities of HACU, or ChAT showed a 2- or 8-fold increase, respectively, from P3 to P16. In conclusion, presynaptic cholinergic functions seem to develop almost in parallel both in the cell body and the target area of the septohippocampal projection: also in the septal region nerve terminals on axon collaterals are endowed very early (at least at P3) with the apparatus for action potential-induced, exocytotic release of ACh. In contrast, the appearance of feedback inhibition via presynaptic muscarinic autoreceptors is delayed. Autoinhibition due to endogenously released ACh can be detected only later, most probably when endogenous ACh concentrations in the septal nuclei have reached a threshold value.     

3,4-Diaminopyridine-evoked noradrenaline release in rat hippocampal slices: facilitation by endogenous or exogenous nitric oxide

The involvement of nitric oxide (NO) in the evoked release of noradrenaline (NA) was studied in rat hippocampal slices preincubated with [3H]NA and stimulated with 3,4-diaminopyridine (3,4-DAP; 200 microM) for 2 min. The 3,4-DAP-evoked [3H]overflow was enhanced by the NO synthase substrate L-arginine, but not by D-arginine; it was reduced by the NO synthase inhibitor NG-nitro-L-arginine, which also antagonized the effects of L-arginine. The corresponding nitro derivative of D-arginine was inactive and unable to block the effects of L-arginine. Also drugs known to produce NO in-vitro, like sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) enhanced the 3,4-DAP-evoked NA release. The NO scavenger hemoglobin showed no significant effects when given alone, but reduced or abolished, respectively, the facilitatory effects of SNP, or SNAP and L-arginine. The cyclic GMP derivatives 8-Br-cGMP and Sp-8-p-chlorophenylthioguanosine-3',5'-cyclic monophosphorothioate (Sp-8-pCPT-cGMPS) also acted facilitatory, whereas the corresponding Rp-enantiomer of the latter compound was inactive, but antagonized the effect of Sp-8-pCPT-cGMPS. NA release evoked by 3,4-DAP (10 microM) from rat hippocampus synaptosomes was not affected by L-arginine or NG-nitro-L-arginine but slightly increased by SNAP and Sp-8-pCPT-cGMPS. Antagonists at NMDA, non-NMDA and metabotropic glutamate receptors neither affected the 3,4-DAP-evoked NA release nor the facilitatory effect of L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrically evoked acetylcholine release from hippocampal slices is inhibited by the cannabinoid receptor agonist, WIN 55212-2, and is potentiated by the cannabinoid antagonist, SR 141716A

This study examined the effect of the cannabinoid receptor agonist, WIN 55212-2, on the electrically evoked release of [14C]acetylcholine (ACh) from superfused brain slices from the hippocampus, a region with a high density of cannabinoid receptors. A comparison was also made with [14C]ACh release from the nucleus accumbens, which has relatively fewer cannabinoid receptors. In the hippocampal slices, WIN 55212-2 produced a dose-dependent inhibition of [14C]ACh release, with an EC50 of 0.03 microM and a maximal inhibition of 81% at 1 microM. In the nucleus accumbens slices, WIN 55212-2 produced a weak inhibition of [14C]ACh release, which did not quite reach statistical significance. The inhibition of electrically evoked hippocampal [14C]ACh release by WIN 55212-2 could be prevented by the cannabinoid receptor antagonist, SR 141716A (EC50, 0.3-1.0 microM). In addition to antagonizing the effects of WIN 55212-2, SR 141716A alone produced a 2-fold potentiation of the electrically stimulated [14C]ACh release in this region (EC50, 0.1-0.3 microM). By contrast, in nucleus accumbens slices, no potentiation of the stimulated release of [14C]ACh release by SR 141716A was observed. Basal [14C]ACh release was unaffected by WIN 55212-2 or SR 141716A in either area. These results suggest that cannabinoid receptor activation can produce a strong inhibition of ACh release in the hippocampus. Furthermore, the potentiation of ACh release in the hippocampus by SR 141716A alone suggests either that this compound is an inverse agonist at cannabinoid receptors or it is antagonizing the actions of an endogenous ligand acting on these receptors.     

Assessment of the role of TRH in the release of [3H]-dopamine from rat nucleus accumbens-lateral septum slices

We have studied [3H]-dopamine ([3H]-DA) release from rat nucleus accumbens lateral septum slices in response to various paradigms aimed at increasing endogenous or exogenous thyrotropin releasing hormone (TRH) concentrations in the extracellular space. High KCl concentrations significantly enhanced [3H]-DA release by fourfold. TRH (10(-4) or 5 x 10(-4) M) did not affect [3H]-DA release. The release of [3H]-DA was not stimulated by TRH either in the presence of N-1-carboxy-2-phenylethyl (N(im)benzyl)-histidyl-beta naphthylamide, a specific pyroglutamyl peptidase II inhibitor, or that of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptidase I. None of the peptidase inhibitors modified the [3H]-DA release by themselves. These results suggest that the TRH stimulation of [3H]-DA release in vitro observed in previous studies is not due to peptide inactivation but may be due to a nonspecific effect. TRH enhancement of DA release in nucleus accumbens in vivo may not be the result of a direct effect of TRH on DA terminals.     

Muscarinic M1 receptor mediated inhibition of GABA release from rat cerebral cortex

Presynaptic modulation of [3H]GABA release was examined using rat cerebral cortical slices. In vitro addition of carbachol, a muscarinic receptor agonist, resulted in a significant suppression of the release of [3H]GABA evoked by high potassium (50 mM) stimulation in a dose dependent manner, while noradrenaline, isoproterenol, dopamine, 5-hydroxytryptamine, histamine and glutamic acid had no significant effect on the evoked release of [3H]GABA. This suppressive effect of carbachol was antagonized invariably by atropine. Furthermore, it was found that the suppressive action of carbachol could be antagonized by pirenzepine, a selective M1 muscarinic receptor antagonist, but not by AF-DX 116 and 4-DAMP, M2 and M3 receptor antagonists, respectively. These results suggest that the release of GABA from cerebral cortical GABA neurons may be modulated by presynaptic M1 muscarinic receptor.     

Age-related alterations in pre-synaptic and receptor-mediated cholinergic functions in rat brain

Fractional [3H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [3H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [3H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 microM) significantly decreased stimulation-induced fractional [3H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [3H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [3H]ACh release from striatum of both age groups is modulated mainly by M3 muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-3H]inositol decreased with aging, carbachol-stimulated [3H]myo inositol-1-fosfat (IP1) accumulation was found similar in striatal, cortical and hippocampal slices.     

3H]acetylcholine release from rat amacrine-like neurons is inhibited by adenosine A1 receptor activation

We studied the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultures enriched (96.4+/-0.4%) in rat cholinergic amacrine-like neurons, as determined by labeling with an antibody against choline acetyltransferase. A small population of these cells also contained GABA. Using these cultures we observed that both [3H]ACh release, which was largely Ca2+-dependent, and 45Ca2+ influx, evoked by depolarization with 50 mM KCl, were increased when adenosine A1 receptor activation was prevented by removal of endogenous adenosine with adenosine deaminase, or by application of the A1 receptor antagonist DPCPX. Our results indicate that, in cultured rat amacrine-like neurons, the activation of A1 receptors decreases calcium influx and, thereby, inhibits [3H]ACh release.     

Effect of long-term administration of duloxetine on the function of serotonin and noradrenaline terminals in the rat brain

Duloxetine, an inhibitor of both 5-hydroxytryptamine (5-HT) and noradrenaline (NA) reuptake processes, has been developed as a potential antidepressant drug. The present study was initiated to investigate the functioning of multiple components of the 5-HT and NA systems following the long-term administration of duloxetine. In rats treated for 21 days with duloxetine (20 mg/kg/day), the recovery times of dorsal hippocampus CA3 pyramidal neurons from microiontophoretic applications of 5-HT and NA were significantly increased, indicating ongoing reuptake blockade with the minipump in place delivering the drug. The remaining experiments were performed following a 48-h washout. Electrically evoked release of [3H]5-HT from preloaded slices was enhanced in the midbrain, presumably due to a desensitization of the somatodendritic 5-HT1D and 5-HT1A autoreceptors. In addition, evoked release of [3H]5-HT was increased in the hippocampus, which could have been due to the desensitization of the alpha2-adrenergic heteroreceptors located on the 5-HT terminals. In contrast, there was no change in the evoked release of [3H]5-HT in the frontal cortex despite decreased functioning of the 5-HT transporter found in this brain region. Similar to changes in 5-HT release, electrically evoked release of [3H]NA was enhanced in the hippocampus and frontal cortex of rats treated chronically with duloxetine. These increases in [3H]NA release were most likely due to the desensitization of the alpha2-adrenergic autoreceptor in the hippocampus and to the desensitization of the NA transporter in the frontal cortex, respectively. These data suggest that long-term administration of duloxetine is able to induce changes in the 5-HT and NA systems that lead to enhanced release of both 5-HT and NA in some limbic brain areas. Duloxetine, therefore, may be a useful antidepressant compound.     

Preferential release of ATP and its extracellular catabolism as a source of adenosine upon high- but not low-frequency stimulation of rat hippocampal slices

The release of adenosine and ATP evoked by electrical field stimulation in rat hippocampal slices was investigated with the following two patterns of stimulation: (1) a brief, high-frequency burst stimulation (trains of stimuli at 100 Hz for 50 ms applied every 2 s for 1 min), to mimic a long-term potentiation (LTP) stimulation paradigm, and (2) a more prolonged (3 min) and low-frequency (5 Hz) train stimulation, to mimic a long-term depression (LTD) stimulation paradigm. The release of ATP was greater at a brief, high-frequency burst stimulation, whereas the release of [3H]adenosine was slightly greater at a more prolonged and low-frequency stimulation. To investigate the source of extracellular adenosine, the following two pharmacological tools were used: alpha, beta-methylene ADP (AOPCP), an inhibitor of ecto-5'-nucleotidase, to assess the contribution of the catabolism of released adenine nucleotides as a source of extracellular adenosine, and S-(4-nitrobenzyl)-6-thioinosine (NBTI), an inhibitor of adenosine transporters, to assess the contribution of the release of adenosine, as such, as a source of extracellular adenosine. At low-frequency stimulation, NBTI inhibited by nearly 50% the evoked outflow of [3H]-adenosine, whereas AOPCP inhibited [3H]adenosine outflow only marginally. In contrast, at high-frequency stimulation, AOPCP inhibited by 30% the evoked release of [3H]adenosine, whereas NBTI produced a 40% inhibition of [3H]adenosine outflow. At both frequencies, the kinetics of evoked [3H]adenosine outflow was affected in different manners by AOPCP and NBTI; NBTI mainly depressed the rate of evoked [3H]adenosine outflow, whereas AOPCP mainly inhibited the later phase of evoked [3H]adenosine accumulation. These results show that there is a simultaneous, but quantitatively different, release of ATP and adenosine from rat hippocampal slices stimulated at frequencies that can induce plasticity phenomena such as LTP (100 Hz) or LTD (5 Hz). The source of extracellular adenosine is also different according to the frequency of stimulation; i.e., at a brief, high-frequency stimulation there is a greater contribution of released adenine nucleotides for the formation of extracellular adenosine than at a low frequency with a more prolonged stimulation.     

Nitric oxide involvement in regulating the dopamine transport in the striatal region of rat brain

Spontaneous [3H]dopamine ([3H]DA) overflow was measured from striatal slices in the presence of different glutamate (Glu) receptor agonists such as N-methyl-D-aspartate (NMDA), kainate (KA) and quisqualate (QA) and their corresponding antagonists, Dizocilpine maleate (MK-801), D-gamma-glutamyl-aminomethanesulfonic acid (GAMS) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. [3H]DA uptake and release in the presence of L-Arginine (L-Arg) and NG-nitro-arginine (L-N-Arg), an inhibitor of nitric oxide (NO) synthesis were also evaluated. L-N-Arg alone or combined with L-Arg significantly reduced [3H]DA uptake at 10 and 100 microM from 33% to 44% from striatal slices. Whereas, in brain synaptosomal fractions L-Arg induced a biphasic effect on that [3H]DA uptake in a dose dependent manner, and L-N-Arg showed an absolute inhibition in 80-90% of this [3H]DA uptake at 1-500 microM. The amino acids, lysine, valine and histidine (100 microM) had a little effect inhibitory on [3H]DA uptake from synaptosomal fractions. Glu agonists, NMDA (10 microM) and KA (10 microM) importantly increased the spontaneous [3H]DA overflow, which was blocked by MK-801 (10 microM) and GAMS (10 microM), respectively. QA had no effect on [3H]DA release. L-Arg (10-200 microM) potentiated the spontaneous [3H]DA overflow in a dose dependent fashion from striatal slices, being reverted by 10 microM L-N-Arg alone or in combination with all other compounds; whereas, lysine, histidine and valine did not modify that spontaneous [3H]DA overflow. Results support the hypothesis related to the participation of NO on DA transport possibly synthesized at the dopaminergic (DAergic) terminals in the striatum; also that L-Arg concentration may determine alternative mechanisms to regulate the DAergic activity at the striatum.     

Modulation of [3H]acetylcholine release from cultured amacrine-like neurons by adenosine A1 receptors

We have investigated the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultured chick amacrine-like neurons. The release of [3H]ACh evoked by 50 mM KCl was mostly Ca2+ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist. The effect of adenosine on [3H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca2+ channels. Ligand binding studies using [3H]DPCPX confirmed the presence of adenosine A1 receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A1 receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca2+-dependent release of [3H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A1 receptors. The effect of adenosine on the [3H]ACh release may be due to a direct inhibition of N-type Ca2+ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Dopamine and serotonin release-regulating autoreceptor sensitivity in A9/A10 cell body and terminal areas after withdrawal of rats from continuous infusion of cocaine

The effects of dopamine (DA) and 5-hydroxytryptamine (5-HT) autoreceptor agents on electrically induced [3H]DA and [3H]5-HT release from superfused slices of striatum, nucleus accumbens and ventral mesencephalon (VM) containing A9 and A10 neurons were investigated in rats made tolerant to the stimulatory effect of cocaine on locomotor behavior by a 14-day continuous infusion of cocaine (29 mg/kg/day) by s.c. implanted osmotic minipumps followed by a 7-day drug-free period. In VM, electrically induced [3H]DA was increased, the ability of pergolide to inhibit this release was abolished, but the ability of sulpiride to facilitate the release was potentiated, implicating a higher concentration of synaptic DA modifying the responsiveness of somatodendritic D2 autoreceptors to D2 agents. Both electrically induced [3H]5-HT release from VM and the stimulatory effect of in vitro cocaine on this release were enhanced whereas the effects of both 5-methoxytryptamine and methiothepin were attenuated, indicating that subsensitivity of 5-HT autoreceptors developed in DA cell body regions. In striatum and nucleus accumbens, no significant changes were observed in [3H]DA and [3H]5-HT release, except for a modest reduction in the effects of both pergolide and sulpiride on electrically induced [3H]DA release from striatum. These results emphasize the importance of pretreatment-induced changes in DA cell body regions, rather than terminal areas, under the present conditions. The observed increase in DA autoinhibitory tone and subsensitivity of 5-HT release-regulating autoreceptors in the VM may contribute to the locomotor tolerance upon cocaine challenge after continuous cocaine.     

Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors

The effects of external pH (pHout) variations on the Na+ and on the Ca2+ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [3H]GABA loaded mouse brain synaptosomes, the external acidification (pHout 6.0) markedly decreased the Na+ dependent fraction of [3H]GABA release evoked by veratridine (10 microM) in the absence of external Ca2+, as well as the Ca2+ dependent fraction of [3H]GABA release evoked by high (20 mM) K+ in the absence of external Na+. The depolarization-induced elevation of [Na(i)] (monitored in synaptosomes loaded with the Na+ indicator dye, SBFI) and the depolarization-induced elevation of [Ca(i)] (monitored in synaptosomes loaded with the Ca2+ indicator dye fura-2) were also markedly decreased at pHout 6. On the contrary, the external alkalinization (pHout 8) facilitated all the above responses. A slight increase of the baseline release of the [3H]GABA was observed when pHout was changed from 7.4 to 8. This effect was only observed in the presence of Ca2+. pHout changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pHout variations on [3H]GABA release were independent on the presence of HCO3-. It is concluded that external H+ regulate amino acid neurotransmitter release by their actions on presynaptic Na+ channels, as well as on presynaptic Ca2+ channels.     

Voltage-activated calcium channels involved in veratridine-evoked [3H]dopamine release in rat striatal slices

The present study explored the role of different sub-types of voltage-activated Ca2+ channels (VACCs) in mediating veratridine-evoked [3H]dopamine (DA) release from rat striatal slices. The release of [3H]DA evoked by veratridine (25 microM) decreased by 50.6+/-2.9% (n=8) in the absence of calcium and was completely abolished by 1 microM tetrodotoxin. The L-type Ca2+ channel blockers nifedipine (10 microM), nitrendipine (10 microM), diltiazem (10 microM) and verapamil (10 microM) did not modulate this release. Similarly, [3H]DA release was affected neither by the N-type VACC blocker omega-conotoxin-GVIA (1 microM) nor by the selective P-type channel blockers omega-agatoxin-IVA and omega-agatoxin-TK at low nM concentrations (30 nM), indicating no involvement of N- and P-type Ca2+ channels. In contrast, higher concentrations of omega-agatoxin-IVA that would also inhibit Q-type VACCs, blocked the release of [3H]DA by 27.9+/-8.1% (n=5) and 37.5+/-13.6% (n=3) at 0.3 and 1 microM, respectively. In addition, application of the Q-type Ca2+ channel blocker omega-conotoxin-MVIIC (0.01-3 degrees M) reduced [3H]DA release in a concentration-dependent manner, with maximum inhibition of 35.3+/-4.1% at 3 microM (n=5). On the basis of these results, it is concluded that the Ca2+ channels that participate in veratridine-evoked [3H]DA release are Q-type Ca2+ channels.