Frequency-Dependent Release of Acetylcholine and Dopamine From Rabbit Striatum: Its Modulation by Dopaminergic Receptors

Abstract: The release of [³H]dopamine (DA) and [¹⁴C]acetylcholine (ACh) was monitored from single slices of the rabbit striatum. In all cases, the evoked overflow of ACh showed a higher peak and was of shorter duration than that of ³H products. For ACh, the release per pulse showed a marked decline with increasing frequency of stimulation, whereas flat frequency-release curves were obtained for DA. At 0.1 and 1 Hz the evoked overflows of ACh were 15 and 7 times greater, respectively, than those of DA. Haloperidol (0.03 μM) and sulpiride (1 μM) produced large increases in the evoked overflow of DA and ACh at 3 and 10 Hz; little effect was observed at lower frequencies. These results indicate that the frequency-release curves for DA and ACh are different and that at high frequencies the slope of the curves is modified by activation of pre- and postsynaptic DA receptors. Apomorphine inhibited in a concentration-dependent fashion the evoked overflow of DA and ACh; greater inhibition was obtained at lower frequencies of stimulation. At 0.3 Hz the- DA agonist was two times more potent in inhibiting DA than ACh overflow (IC₅₀: 12.0 ± 2.2 versus 22.0 ± 2.8 nM; p < 0.01). The greater sensitivity of pre-than postsynaptic sites to apomorphine was also seen at higher frequencies (3 Hz). Benztropine (1/μ) reduced the evoked overflow of ACh at 10 Hz, and enhanced that of ³H products at all rates of stimulation (0.3–10 Hz). These results suggest that the release of DA and ACh is regulated by dopaminergic receptors. They also indicate that the effects of DA agonists and antagonists and of uptake inhibitors on DA and ACh release are highly dependent on the frequency of stimulation used.     

Noradrenaline Release from Cultured Mouse Postganglionic Sympathetic Neurons

The possible existence of alpha2-autoreceptors, P2-autoreceptors, and adenosine A1- or A2A-receptors was studied in cultured thoracolumbar postganglionic sympathetic neurons from mice. The cells were preincubated with [3H]noradrenaline and then superfused. The selective alpha2-adrenoceptor agonist UK 14,304 reduced the electrically evoked overflow of tritium. When the cultures were stimulated by trains of increasing pulse number, ranging from a single pulse to 72 pulses at 3 Hz, the concentration-inhibition curve of UK 14,304 was shifted progressively to the right and the maximal inhibition obtainable became progressively smaller. Six alpha-adrenoceptor antagonists shifted the concentration-inhibition curve of UK 14,304 in a parallel manner to the right. Neither ATP (3-300 microM), adenosine (0.01-100 microM), the selective A1-receptor agonist cyclopentyladenosine (1-1,000 nM), nor the selective A2A-receptor agonist CGS-21680 (1-10,000 nM) changed the basal or the electrically evoked overflow of tritium. It is concluded that the cultured neurons possess presynaptic, release-inhibiting alpha2-autoreceptors. As in intact tissues, the effectiveness of presynaptic alpha2-adrenergic inhibition depends on the "strength" of the releasing stimulus. The pK(D) values of the six antagonists against UK 14,304 indicate that the autoreceptors belong to the pharmacological alpha2D and hence the genetic alpha(2A/D) subtype of alpha2-adrenoceptor. Neither P2-autoreceptors nor receptors for adenosine, the degradation product of ATP, were detected.     

Electrically evoked release of [(3)H]noradrenaline from mouse cultured sympathetic neurons: release-modulating heteroreceptors

Cultured neurons from the thoracolumbar sympathetic chain of newborn mice are known to possess release-inhibiting alpha(2)-autoreceptors. The present study was carried out in a search for release-modulating heteroreceptors on these neurons. Primary cultures were preincubated with [(3)H]noradrenaline and then superfused and stimulated by single pulses, trains of 8 pulses at 100 Hz, or trains of 36 pulses at 3 Hz. The cholinergic agonist carbachol reduced the evoked overflow of tritium. Experiments with antagonists indicated that the inhibition was mediated by M(2) muscarinic receptors. The cannabinoid agonist WIN 55,212-2 reduced the evoked overflow of tritium through CB(1) receptors. Prostaglandin E(2), sulprostone, and somatostatin also caused presynaptic inhibition. The inhibitory effects of carbachol, WIN 55,212-2, prostaglandin E(2), and somatostatin were abolished (at the highest concentration of WIN 55, 212-2 almost abolished) by pretreatment of the cultures with pertussis toxin (250 ng/ml). Several drugs, including the beta(2)-adrenoceptor agonist salbutamol, opioid receptor agonists, neuropeptide Y, angiotensin II, and bradykinin, failed to change the evoked overflow of tritium. These results demonstrate a distinct pattern of presynaptic inhibitory heteroreceptors, all coupled to pertussis toxin-sensitive G proteins. The lack of operation of several presynaptic receptors known to exist in adult mice in situ may be due to the age of the (newborn) donor animals or to the culture conditions.     

Dopamine Autoreceptors Modulate Dopamine Release from the Prefrontal Cortex

Electrical stimulation (at 0.3, 1, or 10 Hz, 120 pulses each) produced a calcium-dependent overflow of radioactivity from slices of the rabbit prefrontal cortex preloaded with [3H]3,4-dihydroxyphenylethylamine ([3H]DA, [3H]dopamine) in the presence of desipramine. Flat frequency-release curves were observed. Apomorphine and LY-171555 inhibited in a concentration-dependent fashion the evoked overflow of DA, an effect antagonized by haloperidol. Stimulation frequencies comparable to normal firing rates of mesocortical neurons (10 Hz) drastically reduced apomorphine-induced inhibition of DA overflow. Haloperidol produced greater facilitation of DA overflow at 10 than at 1 Hz. Nomifensine, a neuronal uptake inhibitor, enhanced DA overflow. These results indicate that mesocortical DA neurons projecting to the prefrontal cortex have release modulatory autoreceptors of the D2 subtype.     

Long-Term Survival of Intrastriatal Dopaminergic Grafts: Modulation of Acetylcholine Release by Graft-Derived Dopamine

The nigrostriatal dopaminergic system of rats was unilaterally lesioned with 6-hydroxydopamine. Part of the animals was grafted 2 weeks later with fetal dopaminergic cells on the lesioned side; untreated rats of the same strain served as controls. Both 3 and 12-14 months after surgery the striatal dopamine (DA) content and the in vivo rotational response following injection of D-amphetamine showed significant changes in grafted as compared to lesioned animals. At 12-14 months after transplantation, the electrically evoked release of tritiated DA and acetylcholine (ACh) in slices (preincubated with [3H]DA or [3H]choline, respectively) of striata of intact, lesioned, or grafted animals was also investigated. Electrical field stimulation of striatal slices of the lesioned side did not evoke any significant [3H]DA overflow, whereas a marked [3H]DA release was observed in slices of grafted and control striata. Moreover, both DL-amphetamine (3 microM) and nomifensine (10 microM) strongly enhanced basal 3H outflow in these slices. Electrically evoked [3H]ACh release was significantly reduced in slices from all striatal tissues by 0.01 microM apomorphine. In slices from denervated striata a clearcut hypersensitivity for this action of apomorphine was present, indicating supersensitivity of DA receptors on cholinergic terminals; this hypersensitivity was significantly reduced in graft-bearing striata. Furthermore, because this hypersensitivity was unchanged in slices of lesioned striata under stimulation conditions (four pulses/100 Hz) avoiding inhibition by endogenously released DA, it is concluded that lesion-induced DA receptor supersensitivity is caused by an increase in receptor density or efficacy rather than by a decreased competition between endogenous and exogenous agonists. Both reuptake blockade of DA with nomifensine (10 microM) and release of endogenous DA by DL-amphetamine (3 microM) potently reduced [3H]ACh release only in control and grafted but not in lesioned tissue. In experiments using potassium-evoked [3H]ACh release, tetrodotoxin had no effect on the inhibitory activity of amphetamine and nomifensine, indicating that the DA receptors involved in their indirect inhibitory action are located directly on the cholinergic terminals.     

Dopaminergic modulation of footshock induced aggression in paired rats.

Footshock induced aggression (FIA) was induced in weight matched paired rats and three paradigms of aggressive behaviour was recorded, namely, the latency to fight (LF), total period of physical contact (TPP) and cumulative aggression scores (CAS). Dopamine (DA), administered centrally, and peripherally administered L-dopa (with benserazide, a peripheral decarboxylase inhibitor), a DA precursor, and the postsynaptic D2 receptor agonists, apomorphine, N-n-propyl-norapomorphine (PNA), bromocriptine, lisuride and pergolide, induced a dose-related facilitation of FIA characterized by decrease in LF and increase in TPP and CAS. However, the DA presynaptic receptor agonist, BHT-920, induced a biphasic effect with inhibition of FIA being induced by a lower dose and facilitation of the aggressive behaviour produced by a higher dose. The postsynaptic D2 receptor antagonists, haloperidol, spiperone and pimozide, induced a dose-related attenuation of FIA, an effect not seen with domperidone, a peripheral DA receptor antagonist. The results indicate that central dopaminergic postsynaptic D2 receptors have a modulatory facilitative effect on FIA, while the presynaptic DA autoreceptors mitigate aggressive behaviour. However, the presynaptic DA receptor agonist, BHT-920, appears to lose its receptor specificity on dose increment. Long term administration of haloperidol, followed by withdrawal, or desipramine, induced per se augmentation of FIA and potentiated the aggression-facilitative effects of L-dopa, apomorphine and PNA. Since both these treatments are known to induce supersensitivity of central postsynaptic dopamine D2 receptors, the effects are likely to be related to augmented function of dopamine neurones. The findings, in conjunction with a recent report from this laboratory indicating an increase in rat brain DA levels in FIA, support the contention that the central DA system has a facilitative effect on FIA.     

Modulation of electrically evoked serotonin release in cultured rat raphe neurons

Electrically evoked release of serotonin (5-HT) and its modulation via 5-HT autoreceptors and alpha(2)-heteroreceptors was studied in primary cell cultures prepared from the embryonic (ED 15) rat mesencephalic brain region comprising the raphe nuclei. Cultures were grown for up to 3 weeks on circular glass coverslips. They developed a dense network of non-neuronal and neuronal cells, some of which were positive for tryptophan hydroxylase. To measure 5-HT release, the cultures were pre-incubated with [(3)H]5-HT (in the presence of the selective noradrenaline reuptake inhibitor oxaprotiline [1 micromol/L]), superfused with modified Krebs-Henseleit medium containing 6-nitroqipazine [1 micromol/L] and electrically stimulated using two conditions. Condition A: 360 pulses, 3 Hz, 0.5 ms, 90 mA, or condition B: 4 pulses 100 Hz, 0.5 ms, 90 mA (a condition which diminishes interactions with endogenously released transmitters during ongoing stimulation). After only 1 week in culture, the electrically evoked overflow of [(3)H] was Ca(2+) dependent and tetrodotoxin sensitive, suggesting an action-potential-induced exocytotic release of 5-HT. Using stimulation condition A in cultures grown for 2 weeks, both basal and evoked 5-HT release were strongly enhanced by methiotepine (1 micromol/L) but unaffected by the 5-HT(1B) autoreceptor agonist CP-93, 129 (1 micromol/L) and the alpha(2)-adrenoceptor agonist UK-14, 304 (1 micromol/L). Conversely, using stimulation condition B, not only CP-93, 129 (IC(50) 8.1 +/- 1.4 nmol/L) and UK-14, 304 (IC(50) 14.9 +/- 1.6 nmol/L) had inhibitory effects on cells grown for 2 weeks, but also the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin. In conclusion, we describe for the first time electrically evoked release of 5-HT from primary cultures of fetal raphe cells and its modulation via 5-HT(1B) and 5-HT(1A) auto- and alpha(2)-heteroreceptors. Such cultured raphe cells may represent a suitable model to study expression and development of presynaptic receptors on serotonergic neurons in-vitro.     

The new neurokinin 1-sensitive receptor mediates the facilitation by endogenous tachykinins of the NMDA-evoked release of acetylcholine after suppression of dopaminergic transmission in the matrix of the rat striatum

Using an in vitro microsuperfusion procedure, the NMDA-evoked release of [3H]ACh was studied after suppression of dopamine (DA) transmission (alpha-methyl-p-tyrosine) in striatal compartments of the rat. The effects of tachykinin neurokinin 1 (NK1) receptor antagonists and the ability of appropriate agonists to counteract the antagonist responses were investigated to determine whether tachykinin NK1 classic, septide-sensitive and/or new NK1-sensitive receptors mediate these regulations. The NK1 antagonists, SR140333, SSR240600, GR205171 but not GR82334 and RP67580 (0.1 and 1 microM) markedly reduced the NMDA (1 mm + D-serine 10 microM)-evoked release of [3H]ACh only in the matrix. These responses unchanged by coapplication with NMDA of NK2 or NK3 agonists, [Lys5,MeLeu9,Nle10]NKA(4-10) or senktide, respectively, were completely counteracted by the selective NK1 agonist, [Pro9]substance P but also by neurokinin A and neuropeptide K (1 nM each). According to the rank order of potency of agonists for counteracting the antagonist responses ([Pro9]substance P, 0.013 nM > neurokinin A, 0.15 nM > substance P(6-11) 7.7 nM = septide 8.7 nM), the new NK1-sensitive receptors mediate the facilitation by endogenous tachykinins of the NMDA-evoked release of ACh in the matrix, after suppression of DA transmission. Solely the NK1 antagonists having a high affinity for these receptors could be used as indirect anti-cholinergic agents.     

Halothane enhances acetylcholine release by decreasing dopaminergic activity in rat striatal slices

The present study investigated the effect of halothane on acetylcholine (ACh) and dopamine (DA) release from the rat striatum. Halothane decreased DA release in a concentration-dependent manner, while increased ACh release. In our previous investigation, a volatile anesthetic, halothane, inhibited DA release from the rat striatal slices in a concentration-dependent manner. Although the release of ACh from cholinergic interneurons is tonically modulated by DA in the striatum, the effect of halothane on the relationship between the release of ACh and DA has not been discussed. Using double-labeled techniques, we investigated the effect of halothane on ACh and DA release simultaneously. The slices were incubated with [14C]-choline and [3H]-DA and superfused with modified Krebs solution containing 1 microM of hemicholinium-3. We applied electrical field stimulation (2 Hz, 240 shocks), and the amount of the release of radioactivity evoked by stimulation was calculated by subtraction of the basal radioactive outflow from the total outflow at the beginning of the respective stimulation periods. The effects of drugs on the release were expressed as the ratio of stimulation-evoked fractional releases (FR), measured in the presence and absence (FRS2/FRS1) of the drug. Halothane decreased DA release in a concentration-dependent manner (FRS2/FRS1=0.767+/-0.021, 0.715+/-0.026, 0.671+/-0.014 and 0.639+/-0.033 at the concentration of 0, 0.5, 2 and 4%, respectively), while ACh release showed a biphasic change in the presence of different concentrations of halothane. The release of ACh was significantly increased at the concentration of 2%, but not at 0.5 or 4%. Halothane failed to increase the release of ACh in striatal slices after lesion by 6-OH-dopamine. The application of amphetamine reduced the release of ACh and abolished the effect of halothane. These results indicate that the effect of halothane on ACh release is indirect: it increases the release by attenuating the inhibitory effect of DA released from the nigro-striatal pathway. The nonsynaptic interaction between DA and ACh release is involved in the effect of halothane on ACh release.     

Purinergic Modulation of Hippocampal Acetylcholine Release Involves α-Dendrotoxin-Sensitive Potassium Channels

Modulation of acetylcholine (ACh) release from superfused hippocampal slices was examined when the release of ACh was stimulated by exposure of slices to elevated K+ concentration. Evoked release was not sensitive to inhibition by 0.1 microM tetrodotoxin, but it could be inhibited in a dose-dependent manner by a muscarinic agonist (10-100 nM oxotremorine) and a purinergic agonist (10-100 nM 2-chloroadenosine). The alpha-dendrotoxin (100 nM), which selectively blocks voltage-gated inactivating K+ channels in nerve endings, did not affect the release of ACh under resting or depolarized conditions. However, alpha-dendrotoxin reduced the 2-chloroadenosine-induced inhibition of release, but did not alter the oxotremorine-induced inhibition. These results suggest that an alpha-dendrotoxin-sensitive K+ channel may be activated as an obligatory step in the modulation of ACh release by presynaptic purinergic receptor activation, but not in the modulation by presynaptic muscarinic receptors.     

Dopamine Efflux from Striatum After Chronic Nicotine: Evidence for Autoreceptor Desensitization

We examined the effect of chronic nicotine treatment on dopaminergic activity by measuring the effects of D1 and D2 dopamine (DA) receptor agonists and antagonists on tritium release from mouse striatum preloaded with [3H]DA. The radioactivity released during superfusion was separated on alumina columns and the distribution and efflux of [3H]DA and its main 3H-labeled metabolites were quantified. After preloading by incubation with [3H]DA, the electrical stimulation-evoked tritium overflow was higher in striatum prepared from nicotine-treated mice, whereas in vitro addition of nicotine caused a similar increase in tritium release from striatum of untreated and chronic nicotine-treated mice. The overflow of [3H]DA and its 3H-metabolites exhibited similar distribution patterns in [3H]DA-preloaded striatum dissected from untreated and chronic nicotine-pretreated mice, indicating that repeated injections with nicotine did not alter the metabolism of [3H]DA taken up by the tissue. (-)-Quinpirole, a selective agonist for D2 DA receptors, and apomorphine, a nonselective D1/D2 agonist, inhibited the electrical stimulation-induced tritium efflux from striatum of untreated mice, whereas (+/-)-sulpiride, a D2 DA receptor antagonist, enhanced the evoked release of tritium. These changes in tritium efflux effected by (-)-quinpirole and (+/-)-sulpiride reflected changes in [3H]DA release and not in DA metabolism, as shown by separation of the released radioactivity on alumina columns. The D1 receptor agonist (+/-)-SKF-38393 did not affect the tritium overflow, whereas the D1 receptor antagonist (+)-SCH-23390 exerted a stimulatory action but only at a high concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopaminergic Inhibition of Prolactin Release and Calcium Influx Induced by Neurotensin in Anterior Pituitary Is Independent of Cyclic AMP System

The present study demonstrates that 3,4-dihydroxyphenylethylamine (DA, dopamine) prevents neurotensin (NT) stimulation of both prolactin (PRL) release and calcium influx by interacting with specific receptors that are functionally linked to calcium channels. As shown by the studies with dispersed cells from rat anterior pituitary, the pharmacology of the control of PRL release and calcium influx, both induced by NT, was found to be typical of a DAergic process. This was demonstrated by the order of potency of agonists in inhibiting PRL release and calcium influx (DA greater than epinephrine greater than norepinephrine much greater than isoproterenol); by the high affinity of antagonists such as haloperidol and fluphenazine for this process; and by the high degree of stereoselectivity of sulpiride. Specific D2 receptor agonists, such as bromocriptine and lisuride, and the specific D2 receptor antagonist (-)-sulpiride were found to be highly potent on the DA receptors negatively coupled with calcium channels and PRL release. DA was found to lack the capacity to change the influx of calcium induced by either the sodium channel activator veratridine or high extracellular potassium levels, thus indicating a specific action of this amine on calcium channels sensitive to NT. In a range of concentrations that are effective in inhibiting either the calcium influx or the PRL release, both induced by NT, DA did not alter the cyclic AMP generating system. DA (from 1.0 nM to 50 nM) did not affect adenylate cyclase activity in rat pituitary gland homogenates and did not modify intracellular cyclic AMP levels in pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concomitant D1 dopamine receptor activation (SKF 38393) unmasks D2 dopaminergic actions of B-HT 920 in mice.

The present study attempts to demonstrate D1/D2 dopamine (DA) receptor interactions during stereotyped behaviour in mice. B-HT 920 [2-amino-6-allyl-5, 6, 7, 8-tetrahydro-4H-thiazolo-(4, 5-d)-azepine] (0.05-1.0 mg/kg), a selective D2-DA agonist, induced mild per se stereotypy consisting mainly of sniffing and rearing responses. Apomorphine, a mixed D1/D2 agonist, also produced typical stereotypic response in mice. The stereotypic response of B-HT 920 was blocked by D2-DA antagonist, sulpiride (50 mg/kg). The effect of apomorphine was not influenced by co-treatment with SKF 38393. Simultaneous administration of B-HT 920 (0.1-0.5 mg/kg) with SKF 38393 (5 mg/kg), a selective D1-DA agonist, elicited dramatic increase in stereotyped behaviours in naive as well as in 24 hr reserpinised (2 mg/kg) mice. Co-treatment of apomorphine (0.5 mg/kg) with B-HT 920 (0.1, 0.25 mg/kg) also resulted in a clearly synergistic effect on stereotyped behaviour. These potentiated responses were reduced or blocked by haloperidol, a D2-DA antagonist. The data suggest that in presence of concomitant stimulation of D1-DA receptors. B-HT 920 exhibits full expression of postsynaptic D2-DA receptor mediated behavioural effects.     

Impulse-dependent extracellular resting dopamine concentration in rat striatum in vivo

The ambient resting dopamine (DA) concentration in brain regulates cognition and motivation. Despite its importance, resting DA level in vivo remains elusive. Here, by high-frequency stimulation of the medial forebrain bundle and immediately following the stimulus-induced DA overflow, we recorded a DA “undershoot” which is a temporal reduction of DA concentration to a level below the baseline. Based on the DA undershoot, we predicted a resting DA concentration of ∼73 nM in rat striatum in vivo. Simulation studies suggested that removing basal DA by DAT during the post-stimulation inhibition of tonic DA release caused the DA undershoot, and the resting concentration of DA modulated the kinetics of the evoked DA transient. The DA undershoot was eliminated by either blocking D2 receptors with haloperidol or blocking the DA transporter (DAT) with cocaine. Therefore, the impulse-dependent resting DA concentration is in the tens of nanomolar range and is modulated by the presynaptic D2 receptors and the DAT in vivo.     

Facilitation of acetylcholine release and improvement in cognition by a selective M2 muscarinic antagonist, SCH 72788

Current treatment of Alzheimer's Disease (AD) requires acetylcholinesterase inhibition to increase acetylcholine (ACh) concentrations in the synaptic cleft. Another mechanism by which ACh levels can be increased is blockade of presynaptic M2 muscarinic autoreceptors that regulate ACh release. An antagonist designed for this purpose must be highly selective for M2 receptors to avoid blocking postsynaptic M1 receptors, which mediate the cognitive effects of ACh. Structure-activity studies of substituted methylpiperadines led to the synthesis of 4-[4-[1(S)-[4-[(1,3-benzodioxol-5-yl)sulfonyl]phenyl]ethyl]-3(R)-methyl-1-piperazinyl]-4-methyl-1-(propylsulfonyl)piperidine. This compound, SCH 72788, binds to cloned human M2 receptors expressed in CHO cells with an affinity of 0.5 nM, and its affinity at M1 receptors is 84-fold lower. SCH 72788 is a functional M2 antagonist that competitively inhibits the ability of the agonist oxotremorine-M to inhibit adenylyl cyclase activity. In an in vivo microdialysis paradigm, SCH 72788 increases ACh release from the striatum of conscious rats. The compound is also active in a rodent model of cognition, the young rat passive avoidance response paradigm. The effects of SCH 72788 suggest that M2 receptor antagonists may be useful for treating the cognitive decline observed in AD and other dementias.     

Characterization of the 5-Hydroxytryptamine Receptor Modulating the Release of 5-[3H]Hydroxytryptamine in Slices of the Human Neocortex

In the rat brain, the presynaptic 5-hydroxytryptamine (5-HT) autoreceptors located on 5-HT terminals correspond to the 5-HT1B subtype. The presence of a 5-HT receptor probably located on 5-HT nerve endings and modulating transmitter release in the human neocortex has been reported, but its detailed pharmacological characterization is not yet available. On the other hand, receptor binding and autoradiographic results indicate that the 5-HT1B receptor subtype is not present in the human brain. We, therefore, studied the modulation of the electrically evoked release of [3H]5-HT by various 5-HT receptor agonists and antagonists in preloaded slices of human neocortex obtained from 18 patients undergoing neurosurgery. The nonselective 5-HT1A/1B/1D receptor agonist 5-carboxamidotryptamine produced a potent inhibition (70% at 0.03 microM) of the electrically evoked release of [3H]5-HT which was blocked by 5-HT receptor antagonists with the following relative order of potency: methiothepin greater than metergoline = methysergide greater than propranolol. The selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin at 0.1 microM did not modify the electrically evoked release of [3H]5-HT. The 5-HT1A/1B receptor agonist RU 24969 was 10 times more potent at inhibiting [3H]5-HT overflow in the rat frontal cortex than in the human neocortex. The potent 5-HT1B receptor antagonist cyanopinodolol did not modify the 5-carboxamidotryptamine-induced inhibition of the electrically evoked release of [3H]5-HT in slices of the human neocortex, but produced by itself a small inhibition of [3H]5-HT overflow.(ABSTRACT TRUNCATED AT 250 WORDS)     

A(2A) adenosine receptor facilitation of neuromuscular transmission: influence of stimulus paradigm on calcium mobilization

The influence of stimulus pulse duration on calcium mobilization triggering facilitation of evoked [(3)H]acetylcholine ([(3)H]ACh) release by the A(2A) adenosine receptor agonist CGS 21680C was studied in the rat phrenic nerve-hemidiaphragm. The P-type calcium channel blocker omega-agatoxin IVA (100 nM) decreased [(3)H]ACh release evoked with pulses of 0.04-ms duration, whereas nifedipine (1 microM) inhibited transmitter release with pulses of 1-ms duration. Depletion of intracellular calcium stores by thapsigargin (2 microM) decreased [(3)H]ACh release evoked by pulses of 1 ms, an effect observed even in the absence of extracellular calcium. With short (0.04-ms) stimulation pulses, when P-type calcium influx triggered transmitter release, facilitation of [(3)H]ACh release by CGS 21680C (3 nM) was attenuated by both thapsigargin (2 microM) and nifedipine (1 microM). With longer stimuli (1 ms), a situation in which both thapsigargin-sensitive internal stores and L-type channels are involved in ACh release, pretreatment with either omega-agatoxin IVA (100 nM) or nifedipine (1 microM) reduced the facilitatory effect of CGS 21680C (3 nM). The results suggest that A(2A) receptor activation facilitates ACh release from motor nerve endings through alternatively mobilizing the available calcium pools (thapsigargin-sensitive internal stores and/or P- or L-type channels) that are not committed to the release process in each stimulation condition.     

Rate and Duration of Stimulation Determine Presynaptic Effects of Haloperidol on Dopaminergic Neurons

Abstract: Superfused rabbit neostriatal slices prelabeled with [³H]dopamine ([³H]DA) were depolarized with electrical pulses (12 V, 1 ms). Although transmitter release showed a proportional increase with a greater number of pulses (30-360 pulses), flat frequency-release curves were obtained. Haloperidol (0.03–0.3 μ m ) enhanced ³H overflow without affecting its metabolism or time course, and antagonized apomorphine-induced inhibition of transmitter release. Maximal enhancement of release by haloperidol was obtained with 30–60 pulses delivered at a rate of 3 Hz, whereas much less facilitation of release was seen at 0.3 and 1 Hz (30–90 pulses) or with 360 pulses at either of the three frequencies. Therefore, the slope of the frequency-release curve was markedly increased by haloperidol. These results indicate that activation of presynaptic DA receptors, and thus facilitation of release by haloperidol was highly dependent on the rate and duration of stimulation of striatal dopaminergic terminals. In these neurons the feedback loop seems to act physiologically to depress the slope of the frequency-release curve.     

Changes in the Parameters of Quantal Acetylcholine Release after Activation of PAR1-Type Thrombin Receptors at the Mouse Neuromuscular Junctions

In mature and newly formed neuromuscular synapses of mouse skeletal muscles, miniature endplate potentials (MEPPs) and multiquantal endplate potentials (EPPs) evoked by a single stimulation of the nerve were recorded using intracellular microelectrode technique. The mechanisms underlying the changes in spontaneous and evoked acetylcholine (ACh) release caused by the activation of PAR1-type muscle receptors induced by their peptide agonist TRAP6-NH₂ were studied. It has been shown for the first time that, in either mature or newly formed motor synapses, the activation of PAR1 that lack presynaptic localization causes a sustained increase in the MEPP amplitude due to the increase in the ACh quantal size at the presynaptic level. It was found that phospholipase C (PLC) participates in the signaling mechanism triggered by the PAR1 activation. Exogenously applied brain-derived neurotrophic factor (BDNF) mimics the effect of activation of PAR1 by TRAP6-NH₂. Moreover, an increase in the MEPP amplitude caused by the peptide PAR1 agonist was fully prevented by blocking the BDNF receptors–tropomyosin receptor kinases B (TrkB). Thus, it has been shown for the first time that the increase in ACh quantal size due to the activation of PAR1 in motor synapses is mediated by a complex signaling cascade that starts at the postsynaptic level of the motor synapse and ends at the presynaptic level. It is expected that the activation of PAR1 at the muscle fiber membrane followed by the PLC upregulation results in the release of neurotrophin BDNF as a retrograde signal. Its effect on the presynaptic TrkB receptors triggers the cascade leading to an increase in the quantal size of ACh.     

Presynaptic kainate receptors modulating glutamatergic transmission in the rat hippocampus are inhibited by arachidonic acid

Kainate receptors are ionotropic glutamate receptors located postsynaptically, mediating frequency-dependent transmission, and presynaptically, modulating transmitter release. In contrast to the excitatory postsynaptic kainate receptors, presynaptic kainate receptor can also be inhibitory and their effects may involve a metabotropic action. Arachidonic acid (AA) modulates most ionotropic receptors, in particular postsynaptic kainate receptor-mediated currents. To further explore differences between pre- and postsynaptic kainate receptors, we tested if presynaptic kainate receptors are affected by AA. Kainate (0.3-3 microM) and the kainate receptor agonist, domoate (60-300 nM), inhibited by 19-54% the field excitatory postsynaptic potential (fEPSP) slope in rat CA1 hippocampus, and increased by 12-32% paired-pulse facilitation (PPF). AA (10 microM) attenuated by 37-72% and by 62-66% the domoate (60-300 nM)-induced fEPSP inhibition and paired-pulse facilitation increase, respectively. This inhibition by AA was unaffected by cyclo- and lipo-oxygenase inhibitors, indomethacin (20 microM) and nordihydroguaiaretic acid (NDGA, 50 microM) or by the free radical scavenger, N-acetyl-L-cysteine (0.5 mM). The K+ (20 mM)-evoked release of [3H]glutamate from superfused hippocampal synaptosomes was inhibited by 18-39% by domoate (1-10 microM), an effect attenuated by 35-63% by AA (10 microM). Finally, the KD (40-55 nM) of the kainate receptor agonist [3H]-(2S,4R)-4-methylglutamate ([3H]MGA) (0.3-120 nM) binding to hippocampal synaptosomal membranes was increased by 151-329% by AA (1-10 microM). These results indicate that AA directly inhibits presynaptic kainate receptor controlling glutamate release in the CA1 area of the rat hippocampus.