Agonist-mediated regulation of presynaptic receptor function during development of rat septal neurons in culture.

Presynaptic receptors modulating the release of acetylcholine (ACh) were studied in fetal septal neurons cultured in a growth medium to which various drugs were added from day 3 in vitro (DIV 3) to DIV 14. The influence of these drugs on the function of the presynaptic muscarinic (M-) autoreceptor was determined at DIV 14 by measuring the inhibitory effect of the M-agonist oxotremorine on the electrically-evoked release of [(3)H]ACh from cultures pre-incubated with [(3)H]choline. The presence of the M-agonists oxotremorine (100 micromol/L) or carbachol (100 micromol/L) from DIV 3 to DIV 14, or from DIV 13 to DIV 14, abolished M-autoreceptor function at DIV 14, whereas the presence of the M-antagonist atropine (10 micromol/L from DIV 3 to DIV 14) during growth left M-autoreceptor function unaltered. Inhibition of ACh esterase by donepezil (1 micromol/L from DIV 3 to DIV 14) weakly decreased M-autoreceptor function at DIV 14; inhibition of neuronal firing by 0.1 tetrodotoxin (0.1 micromol/L from DIV 3 to DIV 14) did not tend to affect M-autoreceptor function at DIV 14. Co-cultivation of fetal septal and raphe neurons for 2 weeks yielded cell cultures containing both vesicular ACh transporter- and tryptophan hydroxylase-immunopositive cells. From these cultures, the release of both [(3)H]ACh and [(3)H]5-HT could be induced by electrical field stimulation. In co-cultured neurons versus septal-only ones the inhibitory effect of oxotremorine on the evoked release of [(3)H]ACh appeared almost normal, whereas that of the selective 5-HT(1B) agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrollo[3,2-b]pyrid-5-one (CP-93,129) was completely abolished. The effects of CP-93,129 were also absent on DIV 14 in septal mono-cultures grown in the presence of CP-93,129 (10 micromol/L) from DIV 3 to DIV 14. It is therefore concluded that the regulation of presynaptic receptor function strongly depends on the concentrations of endogenous transmitters in the neuronal environment.

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.

Spatial reference- (not working- or procedural-) memory performance of aged rats in the water maze predicts the magnitude of sulpiride-induced facilitation of acetylcholine release by striatal slices.

Cluster analysis of water-maze reference-memory performance distinguished subpopulations of young adult (3-5 months), aged (25-27 months) unimpaired (AU) and aged impaired (AI) rats. Working-memory performances of AU and AI rats were close to normal (though young and aged rats differed in exploration strategies). All aged rats showed impaired procedural-memory. Electrically evoked release of tritium was assessed in striatal slices (preloaded with [(3)H]choline) in the presence of oxotremorine, physostigmine, atropine+physostigmine, quinpirole, nomifensine or sulpiride. Aged rats exhibited reduced accumulation of [(3)H]choline (-30%) and weaker transmitter release. Drug effects (highest concentration) were reductions of release by 44% (oxotremorine), 72% (physostigmine), 84% (quinpirole) and 65% (nomifensine) regardless of age. Sulpiride and atropine+physostigmine facilitated the release more efficiently in young rats versus aged rats. The sulpiride-induced facilitation was weaker in AI rats versus AU rats; it significantly correlated with reference-memory performance. The results confirm age-related alterations of cholinergic and dopaminergic striatal functions, and point to the possibility that alterations in the D(2)-mediated dopaminergic regulation of these functions contribute to age-related reference-memory deficits.

Ethanol, 3,4-methylenedioxymethamphetamine (ecstasy) and their combination: long-term behavioral, neurochemical and neuropharmacological effects in the rat.

This study investigated long-term behavioral, neurochemical, and neuropharmacological effects of ethanol-(+/-)-3,4-methylenedioxymethamphetamine (MDMA, ecstasy) combinations. Over 4 consecutive days, male Long-Evans rats received 1.5 g/kg ethanol and/or 10 mg/kg MDMA, or saline. Rectal temperatures were taken in some rats. Starting 4 days after the last injection, we tested working memory, sensory-motor coordination, and anxiety. Subsequently, we measured cortical, striatal, septal, and hippocampal monoamines (last MDMA injection-euthanasia delay: 20 days), or electrically evoked release of serotonin (5-HT) in cortical and hippocampal slices, and its modulation in the presence of CP 93,129 (3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrollo[3,2-b]pyrid-5-one) or methiotepin (last MDMA injection-euthanasia delays: 3-6 weeks). Ethanol attenuated the MDMA-induced hyperthermia, but only on the first day. In the long-term, MDMA reduced 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) content in most brain regions. The behavioral and neurochemical effects of the ethanol-MDMA combination were comparable to those of MDMA alone; sensory-motor coordination was altered after ethanol and/or MDMA. In hippocampal slices from rats given ethanol and MDMA, the CP 93,129-induced inhibition and methiotepin-induced facilitation of 5-HT release were stronger and weaker, respectively, than in the other groups. This is the first study addressing long-term effects of repeated MDMA and EtOH combined treatments in experimental animals. Whereas the drug combination produced the same behavioral and neurochemical effects as MDMA alone, our neuropharmacological results suggest that MDMA-EtOH interactions may have specific long-term consequences on presynaptic modulation of hippocampal 5-HT release, but not necessarily related to MDMA-induced depletion of 5-HT. Thus, it is likely that the psycho(patho)logical problems reported by ecstasy users drinking alcohol are not solely due to the consumption of MDMA.

Cognitive deficits in aged rats correlate with levels of L-arginine, not with nNOS expression or 3,4-DAP-evoked transmitter release in the frontoparietal cortex.

Aging is associated with altered neurotransmitter function in the brain. In this study, we measured release parameters for acetylcholine (ACh), norepinephrine and serotonin in the frontoparietal cortex of young and aged rats. We also determined cortical amino acid concentrations and nitric oxide (NO) synthase function. Prior to sacrifice, the rats had been tested for Morris water-maze performance. In aged, compared with young rats, we observed a reduction in both uptake of choline and acetylcholine release. Serotonin release and L-arginine concentrations (a precursor of NO) showed an aging-related increase; however, L-citrulline/L-arginine ratios were decreased in aged rats. Moreover, while most age-related changes in transmitter release or neurochemical markers were not related to the learning performance, L-arginine concentrations were positively correlated to cognitive deficits. NO synthase concentrations were not affected by aging. It is suggested that events related to L-arginine-to-L-citrulline/NO metabolism in the frontoparietal cortex may take part in age-related cognitive deficits.

3,4-DAP-evoked transmitter release in hippocampal slices of aged rats with impaired memory.

Based on a slice superfusion technique, this study investigated the release of acetylcholine, noradrenaline and serotonin in the hippocampus of aged rats (25-27 months) showing no or severe deficits in a spatial reference-memory task (water maze). Young adults (3-5 months) were used as controls. 3,4-diaminopyridine (3,4-DAP), a potassium channel antagonist which increases neuronal excitability, was used to evoke the overflow of the three neurotransmitters. The release of [3H]noradrenaline induced by stimulation of presynaptic nicotinic receptors was also assessed. The experiment compared the accumulation and 3,4-DAP-evoked (or nicotine-evoked) overflow of [3H] in hippocampal slices preincubated with [3H]choline, [3H]noradrenaline, or [3H]serotonin. In aged rats, only the accumulation of [3H]serotonin was reduced significantly (-17%). In percent of tissue-[3H], the 3,4-DAP-evoked overflow of [3H]serotonin was increased (+28%), and that of [3H]acetylcholine was reduced (-23%) in the aged rats. The nicotine-evoked overflow of [3H]noradrenaline was not altered in aged rats. There was a significant correlation of water-maze performance (distance to platform) and evoked overflow of [3H]serotonin. It is concluded that hippocampal cholinergic functions are more altered by aging than noradrenergic or serotonergic ones. Excessive excitability of serotonergic terminals, perhaps in addition to cholinergic dysfunction, might be a crucial factor accounting for age-related cognitive deficits in the present population of rats.

5,7-Dihydroxytryptamine lesions enhance and serotonergic grafts normalize the evoked overflow of acetylcholine in rat hippocampal slices.

Adult rats were subjected to intracerebroventricular injections of 5,7-dihydroxytryptamine (5,7-DHT; 150 micro g) and, 15 days later, to intrahippocampal grafts of fetal raphe cell suspensions. About 11 months later, we assessed baseline and electrically evoked release of tritium ([3H]) in hippocampal slices, preloaded with tritiated ([3H])choline or [3H]serotonin (5-HT), in the presence or absence of the 5-HT1B receptor agonist CP-93,129 and the 5-HT receptor antagonist methiothepine. HPLC determinations of monoamine concentrations were also performed. The lesions reduced the concentration of 5-HT (-90%) and the accumulation (-80%) as well as the evoked release (-90%) of [3H]5-HT. They also decreased the inhibitory effects of CP-93,129 on the evoked release of [3H]5-HT. Most interestingly, they facilitated the evoked release of [3H]acetylcholine (+20%). In slices from rats subjected to lesions and grafts, the responsiveness of the serotonergic autoreceptors (presumably located on the terminals of the grafted neurons) and the release of acetylcholine were close to normal. These results confirm that grafts rich in serotonergic neurons may partially compensate for the dramatic effects of 5,7-DHT lesions on serotonergic hippocampal functions. The lesion-induced reduction of the 5-HT1B autoreceptor-mediated inhibition of evoked 5-HT release may be an adaptation enhancing serotonergic transmission in the (few) remaining terminals. The facilitated release of acetylcholine is probably caused by a reduced serotonergic tone on the inhibitory 5-HT1B heteroreceptors of the cholinergic terminals. When related to data in the literature, this facilitation may be of particular interest in terms of transmitter-based strategies developed to tackle cognitive symptoms related to neurodegenerative diseases.

Raphé grafts and 3,4-diaminopyridine-evoked overflow of serotonin in the rat hippocampus after 5,7-dihydroxytryptamine lesions: evidence for 5-HT1A autoreceptors.

A first experiment verified that the overflow of 5-HT evoked by 75 microM 3,4-diaminopyridine in superfused hippocampal slices was calcium-dependent, tetrodotoxin-sensitive and modulable by drugs acting on 5-HT autoreceptors. Subsequently, the technique was used in rats to investigate the effects of 5,7-dihydroxytryptamine lesions and intrahippocampal serotonergic grafts. The lesions reduced the accumulation (-81%) and relative evoked overflow (-23%; absolute evoked overflow -86%) of [ H]5-HT, but increased the relative baseline overflow (+23%; absolute baseline overflow -78%). Grafts partially compensated for these effects. In slices from grafted rats, the evoked overflow was reduced by application of a 5-HT receptor agonist (8-OH-DPAT), a response not found in sham-operated and lesion-only rats. Although the graft-induced effects were less marked than in previous studies, they were beneficial and modulated by a mechanism that normally does not operate in the intact hippocampus.

Septal grafts and evoked acetylcholine release in the rat hippocampus after 192 IgG-saporin lesions.

Adult rats received intraseptal injections of 192 IgG-saporin and intrahippocampal grafts of septal cells. Between 6 and 10 months later, we assessed baseline and electrically-evoked release of tritium in hippocampal slices preloaded with [(3)H]choline, and the uptake of [(3)H]choline, [(3)H]noradrenaline and [(3)H]serotonin by hippocampal synaptosomes. The lesions reduced the accumulation of [(3)H]choline by approximately 40%, the evoked release of [(3)H]acetylcholine by approximately 90%, and the uptake of [(3)H]choline by synaptosomes by 90% in the dorsal hippocampus, but increased the relative baseline release of [(3)H]choline by +43%, and the synaptosomal uptake of [(3)H]noradrenaline (66%) and [(3)H]serotonin (58%) in the ventral hippocampus. The increased noradrenaline uptake may account for sympathetic ingrowth. Although the grafts of fetal septal neurons produced modest cholinergic effects, these effects were positive and significant.