Modulation of electrically evoked serotonin release in cultured rat raphe neurons

Abstract

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.

Phosphorylation of CaMKII in the rat dorsal raphe nucleus plays an important role in sleep–wake regulation

“…; Birthelmer et al . ), which can increase 5‐HT function in neuronal synapses. Based on the present results and previous studies, we hypothesize that the role of the Ca 2+ /CaMKII signaling cascade in the DRN in sleep–wake regulation might be related to its induction of hyperactive serotonergic function.…”

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

“…The tissue pieces obtained were dissociated using the same steps of trituration, trypsinization, and centrifugation as described previously [see: (Ehret et al. 2001) and (Birthelmer et al. 2007)].…”

“…Mono‐cultures of cholinergic neurons or serotonergic neurons were characterized by immunocytochemical techniques as described previously [see: (Ehret et al. 2001) and (Birthelmer et al. 2007)].…”

“…Oxaprotilin was routinely present during the pre‐incubation phase of 5‐HT release experiments to prevent false labeling of noradrenergic neurons. These might be present in the culture as a result of the anatomical proximity of the L coeruleus and the raphe region in the embryonic brain [see (Birthelmer et al. 2007) and Fig.…”

“…To further study such questions on a more cellular level, we have developed primary cell cultures from the fetal [embryonic day 17 (ED 17)] septal (Ehret et al. 2001) or the fetal (ED 15) raphe region (Birthelmer et al. 2007).…”