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.     

Modulation of electrically evoked acetylcholine release in cultured rat septal neurones

The electrically evoked release of acetylcholine and its modulation via auto- and heteroreceptors were studied in primary cell cultures prepared from embryonic rat septum (ED 17). Cultures were grown for 1, 2 or 3 weeks on circular, poly D-lysine-coated glass coverslips. They developed a dense network of non-neuronal and neuronal cells, only some of which were immunopositive for choline acetyltransferase. To measure acetylcholine release, the cells on the coverslips were pre-incubated with [3H]choline (0.1 micromol/L), superfused with modified Krebs-Henseleit buffer at 25 degrees C and electrically stimulated twice for 2 min (S1, S2; 3 Hz, 0.5 ms, 90-100 mA). The electrically evoked overflow of [3H] from the cells consisted of approximately 80% of authentic [3H]Ach, was largely Ca2+-dependent and tetrodotoxin sensitive, and hence represents an action potential-evoked, exocytotic release of acetylcholine. Using pairs of selective agonists and antagonist added before S2, muscarinic autoreceptors, as well as inhibitory adenosine A1- and opioid mu-receptors, could be detected, whereas delta-opioid receptors were not found. Evoked [3H] overflow from cultures grown for 1 week, although Ca2+ dependent and tetrodotoxin sensitive, was insensitive to the muscarinic agonist oxotremorine, whereas the effect of oxotremorine on cells grown for 3 weeks was even more pronounced than that in 2-week-old cultures. In conclusion, similar to observations on rat septal tissue in vivo, acetylcholine release from septal cholinergic neurones grown in vitro is inhibited via muscarinic, adenosine A1 and mu-opioid receptors. This in vitro model may prove useful in the exploration of regulatory mechanisms underlying the expression of release modulating receptors on septal cholinergic neurones.     

Effects of Acetylethylcholine Mustard on [3H]Quinuclidinyl Benzilate Binding and Acetylcholine Release in Rat Brain Synaptosomes

The effects of acetylethylcholine mustard and its aziridinium derivative (AMMA) on acetylcholine (ACh) release and [3H]quinuclidinyl benzilate (QNB) binding were studied in rat cortical synaptosomes. After incubation for 5 min at 37 degrees C, AMMA reduced [3H]QNB binding with an IC50 of 9 microM. Following incubation for 5 min with 50 microM AMMA and washing, there was a 62% reduction in the [3H]QNB binding capacity with no change in the KD value for the remaining receptors, a result indicating the irreversibility of the AMMA binding. AMMA and oxotremorine both reduced the basal and 30 mM K+-induced release of newly synthesized [3H]ACh in dose-dependent manners over a 2.5-min period. At identical 50 microM concentrations, AMMA produced a much longer inhibition of basal [3H]ACh release than oxotremorine did. The inhibition of basal and 30 mM K+-induced [3H]ACh release by AMMA (10-250 microM) was blocked by 2 microM atropine during a 2.5-min release incubation, but not during a 30-min release incubation. After synaptosomes were treated with 50 microM AMMA for 5 min and the unbound drug was washed out from the tissue, [3H]ACh release (basal and K+-induced) was reduced. AMMA (50 microM) reduced high-affinity choline uptake and ACh synthesis by greater than 90% in this tissue, but these effects did not account for the [3H]ACh release inhibition, because they were not atropine sensitive and hemicholinium-3 had no effect on [3H]ACh release under the conditions used in these studies, i.e., after extracellular [3H]choline was washed out. Taken together, these results suggest that AMMA may be an irreversible agonist at presynaptic muscarinic autoreceptors.     

Selective Expression of Factors Preventing Cholinergic Dedifferentiation

Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of LF-14, THA and physostigmine in rat hippocampus and cerebral cortex

The effects of physostigmine, tetrahydroaminoacridine (THA) and LF-14 [3,3-dimethyl-1(4- amino-3-pyridyl)urea], a 3,4-diaminopyridine derivative, were compared on inhibition of acetyl- cholinesterase (AChE) activity, and release of [³H]acetylcholine (ACh) from rat brain cortical and hippocampal slices. All three compounds caused a concentration dependent inhibition of AChE, with an order of potency physostigmine > THA > LF-14. The electrically stimulated release of ACh from hippocampal and cortical slices was decreased by 10⁻⁵M physostigmine, although the effect was significant only in cortex. THA (5 × 10⁵M) caused a slight, but not significant, decrease in ACh release from both tissues. In contrast, LF-14 (5 × 10⁻⁵ M) caused an approx. 3-fold enhancement of stimulated release. When AChE was inhibited by prior addition of physostigmine, THA caused only a slight enhancement of ACh release, whereas LF-14 greatly increased release. ACh release was also reduced by stimulation of presynaptic muscarinic receptors with oxotremorine. In this case, THA had no effect on ACh release, while LF-14 was able to reverse the inhibition. This study suggests that LF-14 acts to promote ACh release through blocking K⁺ channels, and has a less potent AChE inhibitory effect. It is possible that a compound like LF-14 could be useful in treating diseases of cholinergic dysfunction such as Alzheimer's disease, by both promoting the release of ACh and inhibiting its breakdown.     

Identification and role of serotonin 5-HT1A and 5-HT1B receptors in primary cultures of rat embryonic rostral raphe nucleus neurons

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10(-8) M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), and the 5-HT(1D/1B) agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionam ide dihydrochloride quaternary-hydrate (WAY 100135) (10(7) M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-p iperazinyl]-1Hindole-2-carboxylate (SDZ 216-525) (10(-7)M), and that of CP-93,129 was blocked by methiothepin (10(-7) M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10(-6) M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.     

The activation of phosphatidylinositol turnover is not directly involved in the modulation of neurotransmitter release mediated by presynaptic muscarinic receptors

In the rat cerebral cortex, the comparative effects of various muscarinic agonists on the release of [³H]dopamine ([³H]DA), [³H]acetylcholine ([³H]ACh), and [³H]5-hydroxytryptamine ([³H]5-HT) from superfused nerve endings and on phosphatidylinositol (PI) turnover were studied. Acetylcholine (ACh) was found to be the most potent among the agonists tested on all three release systems examined, and also on the activation of PI turnover. Oxotremorine and bethanechol were very weak agonists when tested as stimulators of PI turnover. However, oxotremorine was very effective as a release modulator, while bethanechol was completely ineffective. Our data suggest that the activation of PI turnover is not directly involved in the modulation of neurotransmitter release mediated by presynaptic muscarinic receptors.     

Mechanisms of [(3)H]glycine release from mouse spinal cord synaptosomes selectively labeled through GLYT2 transporters

Glycine release has been rarely studied. The aim of this work was to characterize the release of the amino acid from spinal cord glycinergic nerve endings selectively pre-labeled through glycine transporters of the GLYT2 type. Purified mouse spinal cord synaptosomes were incubated with [(3)H]glycine in the presence of the GLYT1 blocker N-[(3R)-3-([1,1'-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine hydrochloride and exposed in superfusion to varying concentrations of KCl, 4-aminopyridine (4-AP), or veratridine. KCl (< or = 15 micromol/L), 4-AP (up to 1 mmol/L), and veratridine (< or = 0.3 micromol/L)-provoked [(3)H]glycine release by external Ca2+-dependent, botulinum toxin C(1)-sensitive, exocytosis. The overflows evoked by higher concentrations of K+ or veratridine involved external Ca2+-independent mechanisms of different nature. Only the overflow evoked by 3 or 10 micromol/L veratridine occurred totally (3 micromol/L) or in part (10 micromol/L) by transporter reversal, being sensitive to the GLYT2 blockers 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminociclopentyl)-methyl] benzamide or O-[(2-benzyloxyphenyl-3-flurophenyl)methyl]-l-serine; in contrast, the external Ca2+-independent [(3)H]glycine overflow provoked by 50 mmol/L K+ was transporter-independent. This component of K+-evoked overflow and the GLYT2-independent portion of the 10 micromol/L veratridine-evoked overflow, were largely sensitive to the vesicle depletor bafilomycin or BAPTA-AM and were prevented by blocking the mitochondrial Na+/Ca2+ exchanger with 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one, indicating the involvement of exocytosis triggered by intraterminal mitochondrial Ca2+ ions.     

Selective 5-HT1B receptor agonist reduces serotonin synthesis following acute, and not chronic, drug administration: results of an autoradiographic study

The effects of acute and chronic administration of the serotonin (5-HT)1B agonist CP-93,129, on 5-HT synthesis rates were evaluated using the alpha-[14C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method. In the acute treatment study, CP-93,129 (7 mg/kg) was injected intraperitoneally 30 min before the alpha-MTrp injection (30 microCi over 2 min). A single dose of CP-93,129 caused a significant increase in the synthesis in the median raphe nucleus (MR) without a significant influence on the dorsal raphe nucleus (DR). There was a reduction in 5-HT synthesis in almost all of the projection areas. In the chronic treatment study, CP-93,129 was administered continuously (7 mg/kg/day) for 14 days using an osmotic minipump implanted subcutaneously. The chronic treatment with CP-93,129 did not produce a significant change in 5-HT synthesis in the raphe nuclei nor in the nerve terminal structures, except for the medial frontal bundle and the visual and sensory-motor cortices. The unaltered 5-HT synthesis rates in the chronic treatment study probably reflect a normalization of the synthesis as a result of the desensitization of 5-HT1B autoreceptors and/or heteroreceptors.     

Target influences on [3H]ACh synthesis and release by ciliary ganglion neurons in vitro

The developmental influence of neuron-target interaction upon transmitter synthesis from labeled precursor and the capacity to release labeled transmitter were examined in dispersed cell cultures of embryonic ciliary ganglion neurons by comparing cultures of neurons plated alone and neurons plated upon pectoral myotubes. Of the total ACh synthesized from radiolabeled choline by neurons plated alone, more than half is via a Na+-dependent path, but a larger fraction of the synthesis is Na+ insensitive in culture than in mature neurons in vivo. In addition, at 1 week in culture the neurons lacking target failed to significantly increase ACh synthesis from the labeled choline in response to a previous high [K+]0 depolarization. Synthetic responsiveness to depolarization is a characteristic of mature nerve terminals in this preparation. One week after plating neurons onto myotube cultures, synthesis of ACh from the exogenous precursor is double that of sibling cultures lacking muscle, and prior depolarization with [K+]0 results in an increase in labeled product. Release from the labeled transmitter pool by the neurons with myotubes was also enhanced. [3H]ACh release elicited by depolarization via a Ca2+-dependent mechanism was more than fivefold higher in the cocultures. The influence of coculture with myotubes upon neuronal development is not duplicated by the neurons themselves despite formation of apparent interneuronal synapses (G. Crean, G. Pilar, J. Tuttle, and K. Vaca, 1982, J. Physiol. (London). 331, 87-104), by "fibroblasts" or medium conditioned over myotube cultures. Neurons under these conditions neither increase synthesis of [3H]ACh in response to a prior depolarization nor demonstrate enhanced basal [3H]ACh synthesis and release. Thus, coculture of embryonic ciliary ganglion neurons with a striated muscle target has a somewhat specific inductive effect, enhancing the capacity for neuronal [3H]ACh synthesis and release toward mature levels. This influence of a readily accessible target upon ciliary neuron cholinergic development in vitro may reflect a normal neuromuscular interaction occurring during embryogenesis.     

In vivo and in vitro evidence for ACh-stimulated L-arginine uptake

Whereas L-arginine is clearly recognized as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y(+) transport is established, few data exist regarding the acute regulation of this transport process. We specifically investigated the effect of ACh and isoprenaline (Iso) on L-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady-state intra-arterial infusion of [(3)H]L-arginine (100 nCi/min), the effects of ACh (9.25 and 37 microg/min), Iso (25-50 and 200 microg/min), and sodium nitroprusside (SNP) (1-2 and 8 microg/min) on forearm plasma flow (FPF), L-[(3)H]arginine uptake, and L-[(3)H]citrulline release were determined. In parallel experiments, the effects of ACh, Iso, and SNP on L-[(3)H]arginine uptake were studied in BAEC. L-Arginine uptake was inversely related to FPF (r = -0.50; P < 0.005). At a similar FPF (ACh 56.82 +/- 9.25, Iso 58.49 +/- 5.56, SNP 57.92 +/- 4.96 ml/min; P = ns), intra-arterial ACh significantly increased forearm uptake of L-[(3)H]arginine (54,655 +/- 8,018 dpm/min), compared with that observed with either Iso (40,517.23 +/- 6,841 dpm/min; P = 0.01) or SNP (36,816 +/- 4,650 dpm/min; P = 0.011). This was associated with increased ACh-induced L-[(3)H]citrulline release compared with Iso and SNP (P = 0.046). Similarly, in BAEC, ACh significantly increased L-[(3)H]arginine uptake compared with control, Iso, or SNP (ACh 12.0 x 10(7) +/- 1.83 x 10(7) vs. control 6.67 x 10(7) +/- 1.16 x 10(7) vs. Iso 7.35 x 10(7) +/- 1.63 x 10(7) vs. SNP 6.01 x 10(7) +/- 1.11 x 10(7) fmol.min(-1).mg(-1) at 300 micromol/l L-arginine; P = 0.043). Taken together, these data indicate that ACh stimulates L-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial L-arginine uptake.     

Activation of glutamate receptors promotes a calcium-dependent and transporter-mediated release of purines in cultured avian retinal cells: possible involvement of calcium/calmodulin-dependent protein kinase II

Calcium-dependent release of purines was previously demonstrated in cultures of chick retinal cells stimulated with high potassium concentrations but there is no evidence for an exocytotic mechanism of adenosine release from presynaptic terminals. Here we show that activation of NMDA or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate glutamate ionotropic receptors promotes a two- to three-fold increase in the release of purines from these cultures. Approximately 96% of intracellular radioactivity is found as nucleotides after incubation with [(3)H]adenosine, but more than 85% of glutamate-stimulated released material is found as inosine (60%), hypoxanthine (19.9%) and adenosine (7.8%). The release is prevented by removal of extracellular calcium, by the transporter blocker nitrobenzylthioinosine, or inhibitors of calcium/calmodulin-dependent protein kinase II (CAMK II). The uptake of [(3)H]adenosine, but not of [(3)H]GABA or [(3)H]choline, is also blocked by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN62), N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl)phenyl-N-[2-hydroxiethyl]-4-methoxybenzenesulfonamide (KN93) or the myristoylated autocamtide-2-related inhibitory peptide, suggesting that the enzyme modulates the nucleoside transporter. The distribution of intracellular purines was not affected by KN62. These results indicate that activation of glutamate receptors triggers the release of purines from retinal cells by a mechanism involving calcium influx, CAMK II and the nitrobenzylthioinosine-sensitive nucleoside transporter. The regulation of adenosine release by glutamate receptors and CAMK II could have important consequences in the presynaptic control of glutamate release.     

Effects of Nucleus Basalis Lesions on the Muscarinic and Nicotinic Modulation of [3H] Acetylcholine Release in the Rat Cerebral Cortex

Presynaptic muscarinic and nicotinic receptors in the cerebral cortex reportedly inhibit and increase acetylcholine (ACh) release, respectively. In this study, we investigated whether these receptors reside on cholinergic nerve terminals projecting to the cerebral cortex from the nucleus basalis magnocellularis (nbm). Adult male rats received unilateral infusions of ibotenic acid (5 micrograms/1 microliter) in the nbm. Two weeks later, cerebral cortical cholinergic markers (choline acetyltransferase activity, high-affinity choline uptake, and coupled ACh synthesis) were significantly reduced in synaptosomes prepared from the lesioned hemispheres compared to contralateral controls. The depolarization-induced release of [3H]ACh from these synaptosomes was also reduced in the lesioned hemispheres, reflecting the reduced synthesis of transmitter. However, the nbm lesions had no effect on the inhibition of release induced by 100 microM oxotremorine. Synaptosomal [3H]ACh release was not altered by nicotine or the nicotinic agonists anabaseine and 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine. Nicotine (10-100 microM) did increase [3H]ACh release in control and lesioned hemispheres in cortical minces, but to a similar extent. These results suggest that neither muscarinic nor nicotinic receptors modulating ACh release reside on nbm-cholinergic terminals.     

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.     

Phorbol 12,13-dibutyrate enhances electrically stimulated neuromessenger release from rat dorsal hippocampal slices in vitro

The protein kinase C activator 4 beta-phorbol 12,13-dibutyrate (PDB) enhanced the electrically stimulated release of radiolabelled noradrenaline (NA), acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) from dorsal hippocampal slices of the rat in vitro in a concentration-dependent manner. 4 alpha-Phorbol 12,13 didecanoate did not have an effect on the electrically stimulated release of any of the neuromessengers. Carbachol, which when present in the superfusion medium alone inhibited [14C]ACh release, significantly reduced the effect of PDB on the release of this neuromessenger. In the presence of either clonidine or [Leu5]enkephalin, which by themselves inhibited the electrically stimulated release of [3H]NA, the effect of PDB was significantly reduced. The enhancing effects of yohimbine and PDB on the electrically stimulated release of [3H]NA were additive. In all three cases, thus, the net effects of PDB were of a similar magnitude, whether the various compounds were present or not. Taken together, the present data suggest that the diacylglycerol/protein kinase C pathway is involved in the stimulus-evoked release of NA, ACh and 5-HT from dorsal hippocampal nerve terminals. Protein kinase C seems not to be involved in the modulation of the release of NA via presynaptic alpha 2-adrenoceptors and delta-opioid receptors and in that of ACh via presynaptic ACh receptors in that brain region.     

The role of 5-HT1B receptors in the regulation of serotonin cell firing and release in the rat brain

The release of 5-HT in terminal areas of the rodent brain is regulated by 5-HT1B receptors. Here we examined the role of 5-HT1B receptors in the control of 5-HT output and firing in the dorsal raphe nucleus (DR), median raphe nucleus (MnR) and forebrain of the rat in vivo. The local perfusion (30-300 microM) of the selective 5-HT1B receptor agonist CP-93,129 to freely moving rats decreased 5-HT release in the DR and more markedly in the MnR. Likewise, 300 microM CP-93,129 reduced 5-HT output in substantia nigra pars reticulata, ventral pallidum, lateral habenula and the suprachiasmatic nucleus. The effect of CP-93,129 was prevented by SB-224289, but not by WAY-100635, selective 5-HT1B and 5-HT1A receptor antagonists, respectively. SB-224289 did not alter dialysate 5-HT in any raphe nuclei. The intravenous administration of the brain-penetrant selective 5-HT1B receptor agonist CP-94,253 (0.5-2.0 mg/kg) to anesthetized rats decreased dialysate 5-HT in dorsal hippocampus and globus pallidus, increased it in MnR and left it unaltered in the DR and medial prefrontal cortex. SB-224289, at a dose known to block 5-HT1B autoreceptor-mediated effects (5 mg/kg), did not prevent the effect of CP-94,253 on MnR 5-HT. The intravenous administration of CP-94,253 (0.05-1.6 mg/kg) to anesthetized rats increased the firing rate of MnR, but not DR-5-HT neurons. The local perfusion of CP-94,253 in the MnR showed a biphasic effect, with 5-HT reductions at 0.3-3 microM and increase at 300 microM. These results suggest that 5-HT cell firing and release in midbrain raphe nuclei (particularly in the MnR) are under control of 5-HT1B receptors. The activation of 5-HT1B autoreceptors (possibly located on 5-HT nerve endings and/or varicosities within DR and MnR) reduces 5-HT release. The effects of higher concentrations of 5-HT1B receptor agonists seem more compatible with the activation of 5-HT1B heteroreceptors on inhibitory neurons.     

Nerve Growth Factor Increases the Intracellular Content of Acetylcholine in Cultured Septal Neurons from Developing Rats

The effects of nerve growth factor (NGF) on the intracellular content of acetylcholine (ACh) in cultured septal neurons from developing rats have been examined. The content of ACh could be measured by using HPLC and electrochemical detection (HPLC-ECD), coupled with an immobilized enzyme column. This method of determination is very simple and rapid, and is highly sensitive. The content of ACh and the activity of choline acetyltransferase (ChAT) in cultured postnatal day 1 (P1) septal neurons grown on an astroglial "feeder" layer was increased during the period of cultivation by the addition of NGF. The activities of ChAT and the content of ACh increased in a dose-dependent manner in direct relationship to the different amounts of NGF employed. These effects of NGF, i.e., elevating the intracellular content of ACh, accompanied by an increase in activity of ChAT, also were confirmed in the P1 septal organotypic cultures. Additionally, embryonic day 17 (E17) septal neurons in a serum-free medium displayed a similar responsiveness to NGF with respect to the elevation in the content of ACh and the increase in activity of ChAT. These results suggest that intracellular levels of ACh are likely to be regulated by NGF in a fashion similar to that of the activity levels of the biosynthetic enzyme.     

Time course of decline of radiolabeled acetylcholine formed following intracerebroventricular administration of tritiated choline: Effects of oxotremorine and scopolamine

Rats were injected intracerebroventricularly with 5 Ci of [methyl-³H]choline. The time course of decline of the rediolabeled acetylcholine (ACh) formed was estimated in the ispilateral cerebral cortex and striatum. The [³H]ACh levels declined biphasically from the cerebral tissue. The initial decline proceeded rapidly, after which labeled ACh declined more slowly. Scopolamine (1 mg/kg, i.v.) caused a significant increase in the rat of [³H]ACh disappearance, which can be interpreted as an enhancement of ACh release. By contrast, oxotremorine (0.8 mg/kg, i.v.) markedly reduced the [³H]ACh disappearance. The results show that drug-induced changes in cholinergic neuronal activities can be estimated from the disappearance of radioactive ACh after labeling the endogenous transmitter through intracerebroventricular administration of labeled choline.     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible existence of dopaminergic receptors on cholinergic nerve terminals in the guinea-pig Auerbach's plexus.

In the Auerbach's plexus of guinea-pig ileum lower concentrations of oxotremorine (Oxo-T) produced an increase, whereas higher concentrations of Oxo-T caused inhibition of evoked acetylcholine (ACh) release, measured by bioassay using dorsal muscle of leech. Dopamine inhibited the increase in evoked release of ACh induced by Oxo-T as a function of its concentration and this inhibitory effect was nullified in presence of pimozide, the dopamine receptor antagonist. The results demonstrate existence of presynaptic dopamine receptors having inhibitory influence on excitatory presynaptic muscarinic receptors on regulation of ACh release. However, no physiological role of dopamine could be observed on ACh release in this preparation.