Protective Effects of a synthetic glycosaminoglycan mimetic (OTR4132) in a rat immunotoxic lesion model of septohippocampal cholinergic degeneration.

Using a partial hippocampal cholinergic denervation model, we assessed the effects of the RGTA® named OTR4132, a synthetic heparan-mimetic biopolymer with neuroprotective/neurotrophic properties. Long-Evans male rats were injected with the cholinergic immunotoxin 192 IgG-saporin into the medial septum/diagonal band of Broca (0.37 µg); vehicle injections served as controls. Immediately after surgery, OTR4132 was injected into the lateral ventricles (0.25 µg/5 µl/rat) or intramuscularly (1.5 mg/kg). To determine whether OTR4132 reached the lesion site, some rats received intracerebroventricular (ICV) or intramuscular (I.M.) injections of fluorescent OTR4132. Rats were sacrificed at 4, 10, 20, or 60 days post-lesion (DPL). Fluorescein-labeled OTR4132 injected ICV or I.M. was found in the lesion from 4 to 20 DPL. Rats with partial hippocampal cholinergic denervation showed decreases in hippocampal acetylcholinesterase reaction products and in choline acetyltransferase-positive neurons in the medial septum. These lesions were the largest at 10 DPL and then remained stable until 60 DPL. Both hippocampal acetylcholinesterase reaction products and choline acetyltransferase-positive neurons in the medial septum effects were significantly attenuated in OTR4132-treated rats. These effects were not related to competition between OTR4132 and 192 IgG-saporin for the neurotrophin receptor P75 (p75NTR), as OTR4132 treatment did not alter the internalization of Cy3-labelled 192 IgG. OTR4132 was more efficient at reducing the acetylcholinesterase reaction products and choline acetyltransferase-positive neurons than a comparable heparin dose used as a comparator. Using the slice superfusion technique, we found that the lesion-induced decrease in muscarinic autoreceptor sensitivity was abolished by intramuscular OTR4132. After partial cholinergic damage, OTR4132 was able to concentrate at the brain lesion site possibly due to the disruption of the blood-brain barrier and to exert structural and functional effects that hold promises for neuroprotection/neurotrophism.

Reactive oxygen species (ROS) in the human neocortex: role of aging and cognition.

Reactive oxygen species (ROS), formed during normal aerobic metabolism, are involved in signal transduction and cognitive functions, but highly increased ROS concentrations may also have detrimental effects. The aim of the present study was to investigate whether aging and cognitive functions are associated with ROS generation in human neocortex obtained from neurosurgical patients. ROS formation in mitochondria from fresh and re-thawed neocortical specimens was measured by monitoring ROS-mediated conversion of dihydrorhodamine 123 to fluorescent rhodamine 123. The validity of this technique was characterized in rat brain mitochondria. The increase in the concentration-response curve of the complex I inhibitor rotenone on ROS generation, as measured by rhodamine 123 (Rh123) fluorescence, was much more pronounced than that of rotenone on mitochondrial [(3)H]-choline uptake [which indicates changes in the mitochondrial membrane potential (DeltaPsi(M))]. Thus, mitochondrial ROS generation can be shown by Rh123 fluorescence although this fluorescence may also reflect changes in DeltaPsi(M) to some extent. ROS formation in human brain mitochondria positively correlated with the age of patients. Moreover, an age-corrected positive correlation of ROS formation with presurgical cognitive performance was observed. Our data suggest a mild increase in ROS formation with aging possibly reflecting a physiological compensation of mitochondrial function. Furthermore, higher cognitive performances in tests of executive functions may be paralleled by slightly increased ROS levels.

Functional characterization of muscarinic autoreceptors in rat and human neocortex.

Electrically evoked overflow of [(3)H]acetylcholine in slices of rat neocortex and of human neocortex (freshly obtained during neurosurgical treatment of epilepsy or deep-seated tumors) was used to functionally characterize the muscarinic receptor subtype, which mediates autoinhibition of acetylcholine release in these tissues. In the rat neocortex, the following pK(B) values [CI(95)] were calculated from the shifts to the right of the concentration-response curves of the full agonist oxotremorine in presence of subtype preferring muscarinic receptor antagonists: tripitramine: 9.1 [8.8, 9.4], tripinamide: 8.6 [8.5, 8.7], AQ-RA 741 (11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido(2,3-b)[1,4]benzodiazepine-6-one): 8.2 [8.0, 8.4], himbacine: 8.0 [7.9, 8.1], 4-diphenylacetoxy-N-methylpiperidine methobromide: 8.0 [7.8, 8.1], methoctramine: 7.5 [7.4, 7.6], AF-DX 116 (11[[2-[(diethyl-amino)methyl]-1-piperidinyl] acetyl] 5,11-dihydro-6H-pyrido(2,3-b)[1,4]benzodiazepine-6-one): 7.1 [7.0, 7.3], hexahydro-sila-difenidol: 6.8 [6.7, 6.9], pirenzepine: 6.6 [6.4, 6.7], and 3,6a,11,14-tetrahydro-9-methoxy-2-methyl-12H-isoquino[1,2-b]pyrrolo[3,2-f] [1,3]benzoxazine-1-carboxylic acid ethyl ester (PD 102807): 6.0 [5.8, 6.2]. In the human neocortex the following values were found: tripitramine: 9.4 [9.3, 9.6], tripinamide: 9.0 [8.9, 9.2], AF-DX 116: 6.7 [6.4, 6.9], hexahydro-sila-difenidol: 6.6 [6.2, 6.9], and PD 102807: 6.5 [6.3, 6.6]. In correlation plots, these pK(B) values correspond best to published binding data on native or recombinant M(2) receptors but not to those on M(1), M(3), M(4), and M(5) receptors, suggesting that muscarinic autoreceptors of both the rat and human neocortex belong to the M(2) subtype. This observation lends further support to the development of M(2) receptor selective brain penetrating antagonists for application in Alzheimer's disease.

Inhibitory potency of choline esterase inhibitors on acetylcholine release and choline esterase activity in fresh specimens of human and rat neocortex.

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.

The modulation of striatal dopamine release correlates with water-maze performance in aged rats.

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.

Noradrenergic denervation facilitates the release of acetylcholine and serotonin in the hippocampus: towards a mechanism underlying upregulations described in MCI patients?

Patients with mild cognitive impairment (MCI), who are at risk for Alzheimer's disease (AD), or those with early AD, exhibit noradrenergic degeneration in the locus coeruleus. In MCI patients, upregulations of cholinergic and serotonergic functions were described in the hippocampus. To investigate the effects of selective noradrenergic denervation on hippocampal neurotransmitter functions, rats were treated with 50 mg/kg (i.p.) of N-2-chlorethyl-N-ethyl-2-bromobenzylamin (DSP-4). DSP-4 treatment reduced hippocampal noradrenaline (NA) by more than 90% (vs. controls), whereas dopamine and 5-HT levels were unaffected. The accumulation and electrically-evoked release (in nCi) of [(3)H]-NA in hippocampal slices were strongly reduced. Accumulation of [(3)H]-5-HT was reduced in DSP-4 rats, whereas spontaneous and electrically-evoked release of [(3)H]-5-HT was significantly enhanced, probably due to a weaker effect of endogenous NA via alpha(2)-adrenoceptors on serotonergic terminals. Accordingly, the alpha(2)-agonist UK-14,304 [5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline] more potently inhibited the evoked 5-HT release in DSP-4 rats, whereas the alpha(2)-antagonist idazoxan failed to exert facilitatory effects. Most surprisingly, the accumulation of [(3)H]-choline, and both the basal and electrically-evoked overflow of [(3)H] from hippocampal slices preincubated with [(3)H]-choline, were also significantly increased in DSP-4 rats. These observations suggest that noradrenergic damage in the locus coeruleus may facilitate cholinergic and serotonergic functions in the hippocampus. Although the current lesion model does not mimic the protracted evolution of neurodegenerative processes in MCI and AD, our data could point to an explanation for the upregulations of cholinergic and serotonergic functions described in the hippocampus of MCI patients.

Increased expression of 5-HT(1B) receptors by Herpes simplex virus gene transfer in septal neurons: New in vitro and in vivo models to study 5-HT(1B) receptor function.

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.

Effects of ethanol and 3,4-methylenedioxymethamphetamine (MDMA) alone or in combination on spontaneous and evoked overflow of dopamine, serotonin and acetylcholine in striatal slices of the rat brain.

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.

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.

Presynaptic opioid receptors on noradrenergic and serotonergic neurons in the human as compared to the rat neocortex.

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.

Presynaptic serotonergic modulation of 5-HT and acetylcholine release in the hippocampus and the cortex of 5-HT1B-receptor knockout mice.

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.

Maturation and maintenance of cholinergic medial septum neurons require glucocorticoid receptor signaling.

Glucocorticoids have been shown to influence trophic processes in the nervous system. In particular, they seem to be important for the development of cholinergic neurons in various brain regions. Here, we applied a genetic approach to investigate the role of the glucocorticoid receptor (GR) on the maturation and maintenance of cholinergic medial septal neurons between P15 and one year of age by using a mouse model carrying a CNS-specific conditional inactivation of the GR gene (GRNesCre). The number of choline acetyltransferase and p75NTR immuno-positive neurons in the medial septum (MS) was analyzed by stereology in controls versus mutants. In addition, cholinergic fiber density, acetylcholine release and cholinergic key enzyme activity of these neurons were determined in the hippocampus. We found that in GRNesCre animals the number of medial septal cholinergic neurons was significantly reduced during development. In addition, cholinergic cell number further decreased with aging in these mutants. The functional GR gene is therefore required for the proper maturation and maintenance of medial septal cholinergic neurons. However, the loss of cholinergic neurons in the medial septum is not accompanied by a loss of functional cholinergic parameters of these neurons in their target region, the hippocampus. This pinpoints to plasticity of the septo-hippocampal system, that seems to compensate for the septal cell loss by sprouting of the remaining neurons.

Dopamine release in human neocortical slices: characterization of inhibitory autoreceptors and of nicotinic acetylcholine receptor-evoked release.

The autoinhibitory control of electrically evoked release of [3H]-dopamine and the properties of that induced by nicotinic receptor (nAChR) stimulation were studied in slices of the human neocortex. In both models [3H]-dopamine release was action potential-induced and exocytotic. The selective dopamine D2 receptor agonist (-)-quinpirole reduced electrically evoked release of [3H]-dopamine, yielding IC50 and I(max) values of 23 nM and 76%, respectively. Also, the effects of several other subtype-selective dopamine receptor ligands confirmed that the terminal dopamine autoreceptor belongs to the D2 subtype. The autoinhibitory feedback control was slightly operative under stimulation conditions of 90 pulses and 3 Hz, with a biophase concentration of endogenous dopamine of 3.6 nM, and was enhanced under blockade of dopamine reuptake. [3H]-dopamine release evoked in an identical manner in mouse neocortical slices was not inhibited by (-)-quinpirole, suggesting the absence of dopamine autoreceptors in this tissue and underlining an important species difference. Also, nAChR stimulation-induced release of [3H]-dopamine revealed a species difference: [3H]-dopamine release was evoked in human, but not in rat neocortical slices. The nAChRs inducing [3H]-dopamine release most probably belong to the alpha3/beta2subtype, according to the potencies and efficacies of subtype-selective nAChR ligands. Part of these receptors may be located on glutamatergic neurons.

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.

Characterization of nicotinic receptors inducing noradrenaline release and absence of nicotinic autoreceptors in human neocortex.

UNLABELLED: Presynaptic facilitatory nicotinic receptors (nAChRs) on noradrenergic axon terminals were studied in slices of human or rat neocortex and of rat hippocampus preincubated with [3H]noradrenaline ([3H]NA). During superfusion of the slices, stimulation by nicotinic agonists for 2 min only slightly increased [3H]NA outflow in the rat neocortex, but caused a tetrodotoxin-sensitive. Ca(2+)-dependent release of [3H]NA in rat hippocampus and human neocortex. In both tissues a similar rank order of potency of nicotinic agonists was found: epibatidine >> DMPP > nicotine approximately cytisine > or = acetylcholine; choline was ineffective. In human neocortex, the effects of nicotine (100 microM) were reduced by mecamylamine, methyllycaconitine, di-hydro-beta-erythroidine (10 microM, each) and the alpha3beta2/alpha6betax-selective alpha-conotoxin MII (100/200 nM). The alpha3beta4 selective alpha-conotoxin AuIB (1 microM), and the alpha7 selective alpha-conotoxin ImI (200 nM) as well as alpha-bungarotoxin (125 nM) were ineffective. Glutamate receptor antagonists (300 microM AP-5, 100 microM DNQX) acted inhibitory, suggesting the participation of nAChRs on glutamatergic neurons. On the other hand, nAChR agonists were unable to evoke exocytotic release of [3H]acetylcholine from human and rat neocortical slices preincubated with [3H]choline. IN CONCLUSION: (1) alpha3beta2 and/or alpha6 containing nAChRs are at least partially responsible for presynaptic cholinergic facilitation of noradrenergic transmission in human neocortex; (2) nicotinic autoreceptors were not detectable in rat and human neocortex.

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.