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Fresh specimens of human and rat neocortex were used to determine direct and indirect inhibitory potencies of choline esterase inhibitors (ChEIs) on ChE and the release of acetylcholine (ACh), respectively. Km values of ChE in homogenates of rat and human neocortex did not differ significantly, whereas the specific activity of ChE was > times higher in the rat. Butyryl ChE exhibited a higher Km and a lower specific activity than ACh esterase in human neocortex. Inhibition of ChE in rat and human tissue was similar [IC50 (nM; human): donepezil: 14, physostigmine: 22, tacrine: 95, galanthamine: 575, rivastigmine: 9120]. In neocortex slices preincubated with [3H]choline, the electrically evoked release of [3H]ACh was inhibited up to 60% by ChEIs (IC50 (nM, rat): donepezil: 30, physostigmine: 39, tacrine: 302, galanthamine: 646, rivastigmine: >10000). Similar IC50-values were also estimated for ACh release in human neocortex, although the maximal inhibitory effects were much smaller ( approximately 20%). We conclude that in comparison to rats: 1) neocortical ChE concentrations are lower and 2) that ChEIs have weaker indirect (muscarine receptor-mediated) presynaptic inhibitory effects in the human brain. We further suggest that a combination of ChEIs with brain-selective muscarine autoreceptor antagonists might help to improve their clinical efficacy.

1 Electrically evoked release of [3H]-noradrenaline ([3H]-NA) or [3H]-5-hydroxytryptamine ([3H]-5-HT) in slices of human and the rat neocortex was used to characterize presynaptic opioid receptors. 2 Release of [3H]-NA in rat neocortical slices was reduced only by the μ-receptor agonist DAMGO (pIC50: 7.27, CI95: [7.22, 7.32]; Imax: 77.6±1.6%; antagonized by naloxone: pA2: 8.88, CI95: [8.78, 8.98]). 3 Release of [3H]-NA in human neocortical slices was unaffected by DAMGO, but inhibited by the δ-receptor agonist DPDPE (Imax: 25.7±2.2%) and the κ-receptor agonist U-50,488H (19.7±2.7% inhibition at 1 μM). Both effects were antagonized by naltrindole (1 μM). 4 Release of [3H]-5-HT in rat neocortical slices, was inhibited by DAMGO (10 μM) and U-50,488H (1 and 10 μM) only in the presence of the 5-HT receptor antagonist methiotepin (1 μM). 5 Release of [3H]-5-HT in human neocortical slices was unaffected by DPDPE, but U-50,488H (Imax: 40.8±8.3%; antagonized by 0.1 μM norbinaltorphimine) and DAMGO (16.4±3.9% inhibition at 1 μM; antagonized by 0.1 μM naloxone) acted inhibitory. 6 Release of [3H]-5-HT in human neocortical slices was reduced by nociceptin/orphanin (0.1 and 1 μM). These effects were antagonized by the ORL1 antagonist J-113397 (1-[(3R,4R)-1-cyclo-octylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one; 0.1 μM). 7 This study provides evidence for significant species differences in opioid receptor-mediated modulation of NA and 5-HT-release in human vs rat neocortex. In rats, μ-opioid receptors modulate NA release, but 5-HT release is only weakly affected by μ- and κ-opioids. In contrast, NA release in human neocortex is modulated via δ-opioid receptors, but 5-HT release mainly via κ-opioid receptors. In addition also the ORL1 receptor seems to be involved in 5-HT release modulation. British Journal of Pharmacology (2006) 148, 795–806. doi:10.1038/sj.bjp.0706782

Serotonergic modulation of acetylcholine (ACh) release after neuron-specific increase of the expression of 5-HT 1B receptors by gene transfer was studied in vitro and in vivo . The increased expression of the 5-HT 1B receptor in vitro was induced by treating rat primary fetal septal cell cultures for 3 days with a viral vector inducing the expression of green fluorescent protein (GFP) vector alone, or, in addition, of 5-HT 1B receptors (HA1B/GFP vector). The transfection resulted in a high number of GFP-positive cells, part of which being immunopositive for choline acetyltransferase. In HA1B/GFP-cultures (vs. GFP-cultures), electrically evoked ACh release was significantly more sensitive to the inhibitory action of the 5-HT 1B agonist CP-93,129. Increased expression of the 5-HT 1B receptor in vivo was induced by stereotaxic injections of the vectors into the rat septal region. Three days later, electrically evoked release of ACh in hippocampal slices of HA1B/GFP-treated rats was lower than in their GFP-treated counterparts, showing a higher inhibitory efficacy of endogenous 5-HT on cholinergic terminals after transfection. Moreover, CP-93,129 had a higher inhibitory potency. In conclusion, the HA1B/GFP vector reveals a useful tool to induce a targeted increase of 5-HT 1B heteroreceptors on cholinergic neurons in selected CNS regions, which provides interesting perspectives for functional approaches at more integrated levels.

Ethanol (EtOH) potentiates the locomotor effects of 3,4-methylenedioxymetamphetamine (MDMA) in rats. This potentiation might involve pharmacokinetic and/or pharmacodynamic mechanisms. We explored whether the latter could be local. Using a slice superfusion approach, we assessed the effects of MDMA (0.3, 3microm) and/or EtOH (2mm) on the spontaneous outflow and electrically evoked release of serotonin (5-HT), dopamine (DA) and acetylcholine (ACh) in the striatum, and for comparison, on 5-HT release in hippocampal and neocortical tissue. MDMA and less effectively EtOH, augmented the outflow of 5-HT in all regions. The electrically evoked 5-HT release was increased by MDMA at 3microm in striatal slices only. With nomifensine throughout, EtOH significantly potentiated the 0.3microm MDMA-induced outflow of 5-HT, but only in striatal slices. EtOH or MDMA also enhanced the spontaneous outflow of DA, but MDMA reduced the electrically evoked DA release. With fluvoxamine throughout superfusion, EtOH potentiated the effect of MDMA on the spontaneous outflow of DA. Finally, 3microm MDMA diminished the electrically evoked release of ACh, an effect involving several receptors (D2, 5-HT2, NMDA, nicotinic, NK1), with some interactions with EtOH. Among other results, we show for the first time a local synergistic interaction of EtOH and MDMA on the spontaneous outflow of striatal DA and 5-HT, which could be relevant to the EtOH-induced potentiation of hyperlocomotion in MDMA-treated rats. These data do not preclude the contribution of other pharmacodynamic and/or pharmacokinetic mechanisms in vivo but support the hypothesis that EtOH may affect the abuse liability of MDMA.

Cluster analysis of performance during acquisition of a place-learning task in the water maze distinguished between subpopulations of aged rats (25-27 months) classified as moderately (AMI) or severely impaired (ASI) in comparison with young adults (3-5 months). Using a slice-superfusion device, electrically or nicotine-evoked release of dopamine from striatum was assessed in the presence of GR-55,562 (5-HT(1B) receptor antagonist), methiotepin (mixed 5-HT(1/2) receptor antagonist) and/or sulpiride (D(2)/D(3) receptor antagonist). The main neuropharmacological results demonstrated age-related alterations in the 5-HT(1B)- and D(2)/D(3)-mediated modulation of electrically evoked striatal dopamine release. Regression analyses indicated a possible contribution of such alterations to the age-related behavioural deficits: the larger the deficit, the weaker the electrically evoked release under 5-HT(1B) and D(2)/D(3) receptor blockade. Extending our recent report on the modulation of striatal acetylcholine release in aged rats [Cassel et al., 2007. Neurobiol. Aging 28, 1270-1285], these new findings make dopaminergic and serotonergic functional alterations potential candidates to participate in age-related deficits in the water maze, most probably in interaction with formerly described cholinergic dysfunctions.

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.

5-HT released from serotonergic axon terminals in the septal nuclei modulates the activity of septal output neurons (e.g. septohippocampal cholinergic neurons) bearing somatodendritic 5-HT receptors. Therefore, we studied the mechanisms involved in the presynaptic modulation of 5-HT release in the lateral (LS) and medial septum (MS), and the diagonal band of Broca (DB). HPLC analysis showed that tissue concentrations of noradrenaline, dopamine and 5-HT were highest in DB (DB>MS>LS). Slices prepared from LS, MS and DB regions were preincubated with [(3)H]5-HT, superfused in the presence of 6-nitro-2-(1-piperazinyl)-quinoline (6-nitroquipazine) and electrically stimulated up to three times (first electrical stimulation period (S(1)), S(2), S(3); 360 pulses, 3 Hz, 2 ms, 26-28 mA). In all septal regions the Ca(2+)-dependent and tetrodotoxin-sensitive electrically-evoked overflow of [(3)H] was inhibited by the 5-HT(1B) agonist CP-93,129 and the alpha(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline tartrate (UK-14,304). Also the mu- and kappa-opioid receptor agonists (d-Ala(2), N-Me-Phe(4), glycinol(5))-enkephalin (DAMGO) and [trans-(1S,2S(-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzenacetamide hydro-chloride] (U-50,488H), respectively, acted inhibitory (although less potently), whereas the delta-opioid receptor agonist (d-Pen(2), d-Pen(5))-enkephalin (DPDPE), the dopamine D(2) receptor agonist quinpirole and the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine were all ineffective; the GABA(B) receptor agonist baclofen had weak effects. All inhibitory effects of the agonists were antagonized by the corresponding antagonists (3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR-55,562), idazoxan, naloxone, nor-binaltorphimine), which also significantly enhanced the evoked release of 5-HT at S(1). It is concluded that 5-HT release in septal nuclei of the rat is modulated by presynaptic 5-HT(1B) autoreceptors, as well as by alpha(2)-, mu- and kappa-opioid heteroreceptors. All of these receptors seem to be under a tonic inhibitory influence of the corresponding endogenous agonists and show qualitatively comparable modulatory properties along the dorso-ventral distribution of the 5-HT terminals.

1. Electrically evoked release of [3H]-noradrenaline ([3H]-NA) or [3H]-5-hydroxytryptamine ([3H]-5-HT) in slices of human and the rat neocortex was used to characterize presynaptic opioid receptors. 2. Release of [3H]-NA in rat neocortical slices was reduced only by the mu-receptor agonist DAMGO (pIC50: 7.27, CI95: [7.22, 7.32]; Imax: 77.6+/-1.6%; antagonized by naloxone: pA2: 8.88, CI95: [8.78, 8.98]). 3. Release of [3H]-NA in human neocortical slices was unaffected by DAMGO, but inhibited by the delta-receptor agonist DPDPE (Imax: 25.7+/-2.2%) and the kappa-receptor agonist U-50,488H (19.7+/-2.7% inhibition at 1 microM). Both effects were antagonized by naltrindole (1 microM). 4. Release of [3H]-5-HT in rat neocortical slices, was inhibited by DAMGO (10 microM) and U-50,488H (1 and 10 microM) only in the presence of the 5-HT receptor antagonist methiotepin (1 microM). 5. Release of [3H]-5-HT in human neocortical slices was unaffected by DPDPE, but U-50,488H (Imax: 40.8+/-8.3%; antagonized by 0.1 microM norbinaltorphimine) and DAMGO (16.4+/-3.9% inhibition at 1 microM; antagonized by 0.1 microM naloxone) acted inhibitory. 6. Release of [3H]-5-HT in human neocortical slices was reduced by nociceptin/orphanin (0.1 and 1 microM). These effects were antagonized by the ORL1 antagonist J-113397 (1-[(3R,4R)-1-cyclo-octylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one; 0.1 microM). 7. This study provides evidence for significant species differences in opioid receptor-mediated modulation of NA and 5-HT-release in human vs rat neocortex. In rats, mu-opioid receptors modulate NA release, but 5-HT release is only weakly affected by mu- and kappa-opioids. In contrast, NA release in human neocortex is modulated via delta-opioid receptors, but 5-HT release mainly via kappa-opioid receptors. In addition also the ORL1 receptor seems to be involved in 5-HT release modulation.

Lesioning of serotonergic afferents increases hippocampal ACh release and attenuates memory deficits produced by cholinergic lesions. Improved memory performance described in 5-HT1B-knockout (KO) mice might thus be due to a weaker 5-HT1B-mediated inhibitory influence of 5-HT on hippocampal ACh release. The selective delay-dependent impairment of working memory observed in these KO mice suggests, however, that cortical regions also participate in task performance, possibly via indirect influences of 5-HT on ACh release. To provide neuropharmacological support for these hypotheses we measured evoked ACh and 5-HT release in hippocampal and cortical slices of wild-type (WT) and 5-HT1B KO mice. Superfused slices (preincubated with [3H]choline or [3H]5-HT) were electrically stimulated in the absence or presence of 5-HT1B receptor ligands. In hippocampus and cortex, 5-HT1B agonists decreased and antagonists increased 5-HT release in WT, but not in 5-HT1B KO mice. In 5-HT1B KO mice, 5-HT release was enhanced in both structures, while ACh release (in nCi) was reduced. ACh release was inhibited by 5-HT1B agonists in hippocampal (not cortical) slices of WT but not of 5-HT1B KO mice. Our data (i) confirm the absence of autoinhibition of 5-HT release in 5-HT1B-KO mice, (ii) demonstrate a reduced release of ACh, and the absence of 5-HT1B-receptor-mediated inhibition of ACh release, in the hippocampus and cortex of 5-HT1B-KO mice, and (iii) are compatible with an indirect role of cortical ACh in the working memory impairment observed in these KO mice.