Effects of antiepileptic drugs on glutamate release from rat and human neocortical synaptosomes.

Aim of this study was to learn whether the antiepileptic drugs (AEDs) carbamazepine, lamotrigine, phenytoin, gabapentin, pregabalin, levetiracetam, and valproate (all at 100 µM) presynaptically influence the neurotransmission of the excitatory transmitter glutamate. The effects of these frequently used AEDs were examined on ³H-glutamate release from superfused synaptosomes of both rat and human neocortex. Release was evoked by elevation of buffer [K(+)] from 3 to 15 mM or by the Na(+) channel activator veratridine (1, 3.2, and 10 µM). Buffer [K(+)] elevation induced ³H-glutamate exocytosis, which was Ca(²+)-, but not Na(+)-, dependent and which was accompanied only in human tissue by release through transporter reversal. In rat tissue, release was diminished by the Na(+) channel inhibitors carbamazepine, lamotrigine, and phenytoin, which therefore may also affect presynaptic Ca(²+) channels. Interestingly, levetiracetam increased ³H-glutamate release. In contrast, the tested AEDs did not affect K(+)-evoked ³H-glutamate release in human tissue, neither when the transporters were operative nor when exocytosis was isolated by transporter blockade. Veratridine-evoked ³H-glutamate release was a Na(+)-dependent transmitter efflux through reversed transporters in both species which in human synaptosomes was accompanied by exocytosis. The latter depended on external Ca(²+). Carbamazepine, lamotrigine, and phenytoin reduced this release from both rat and human tissue. There is an obvious species difference in the effects of carbamazepine, lamotrigine, and phenytoin on K(+)-evoked ³H-glutamate release while their inhibitory effects on veratridine-evoked release were similar. Thus, the depression of ³H-glutamate release by carbamazepine, lamotrigine, and phenytoin may be due to inhibited synaptosomal Na(+) or Ca(²+) influx.

Effects of ethanol and ecstasy on conditioned place preference in the rat.

The club drug ecstasy (3,4-methylenedioxymethylamphetamine or MDMA) is often taken recreationally with ethanol (EtOH). We have shown previously that EtOH potentiates the psychomotor effects of MDMA in rats. More recently, we demonstrated in striatal slices that MDMA produced preferential release of serotonin, but when combined with EtOH, the preferential release shifted to dopamine, raising the possibility that administration of EtOH may increase the reward effect of MDMA. To address this possibility, adult male Long-Evans rats were tested for conditioned place preference following treatment with saline, EtOH (0.75 g/kg), MDMA (6.6 mg/kg) or the combination. The only condition that produced a preference for the compartment associated with the drug was that of the drug combination. The current data are in line with anecdotal reports and one study in humans, indicating that EtOH alters the pharmacological effects of MDMA including self reports of enhanced or prolonged euphoria. Thus, administration of EtOH might increase the risk for compulsive use of MDMA, an issue that warrants further study.

Differential inhibitory effects of drugs acting at the noradrenaline and 5-hydroxytryptamine transporters in rat and human neocortical synaptosomes.

BACKGROUND AND PURPOSE: Although the amino acid sequences of rat and human 5-hydroxytryptamine (5-HT) and noradrenaline (NA) transporters (i.e. SERT and NET) are highly homologous, species differences exist in the inhibitory effects of drugs acting at these transporters. Therefore, comparison of the potencies of drugs acting at SERT and NET in native human and rat neocortex may serve to more accurately predict their clinical profile. EXPERIMENTAL APPROACH: Synaptosomes prepared from fresh human and rat neocortical tissues were used for [(3)H]-5-HT and [(3)H]-NA saturation and competition uptake experiments. The drugs tested included NA reuptake inhibitors (desipramine, atomoxetine and (S,S)-reboxetine), 5-HT reuptake blockers (citalopram, fluoxetine and fluvoxamine) and dual 5-HT/NA reuptake inhibitors (duloxetine and milnacipran). KEY RESULTS: In saturation experiments on synaptosomal [(3)H]-5-HT and [(3)H]-NA uptake, the dissociation constants did not indicate species differences although a smaller density of both SERT and NET was observed in human tissues. In competition experiments with the various drugs, marked species differences in their potencies were observed, especially at SERT. The rank order of selectivity ratios (SERT/NET) in human neocortex was as follows: citalopram >or= duloxetine = fluvoxamine >or= fluoxetine > milnacipran > desipramine = atomoxetine > (S,S)-reboxetine. Significant species differences in these ratios were observed for duloxetine, atomoxetine and desipramine. CONCLUSIONS AND IMPLICATIONS: This study provides the first compilation of drug potency at native human neocortical SERT and NET. The significant species differences (viz., human vs. rat) in drug potency suggest that the general use of rodent data should be limited to predict clinical efficacy or profile.

Presynaptic modulation of 5-HT release in the rat septal region.

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.

Does the release of acetylcholine in septal slices originate from intrinsic cholinergic neurons bearing p75(NTR) receptors? A study using 192 IgG-saporin lesions in rats.

In previous studies electrically-evoked release of acetylcholine in septal slices was demonstrated. The present experiment aimed at verifying if this release involved intrinsic neurons bearing p75(NTR) receptors. Long-Evans rats sustained injections of 192 IgG-saporin into the medial septum/diagonal band of Broca (0.8 microg). Sham-operated rats served as controls. Two to 3.5 weeks later, the electrically-evoked release of acetylcholine ([(3)H]ACh) was measured in slices from the lateral septum (LS), medial septum (MS) and diagonal band of Broca (DBB). Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and monoamine concentrations were measured in the septum, cortex and hippocampus. The lesion extent was also assessed by ChAT immunostaining in a separate series of rats. In the septum, the number of ChAT-positive neurons was depleted dramatically (>90% at the level of the injection site). In the hippocampus, the lesions reduced ChAT and AChE activity by 91% and 84%, respectively. In the cortex, this reduction was weaker (-55% and -47%). In the septal region, the reduction was either weak or not significant. The evoked release of acetylcholine in septal slices was not reduced, except in the slices from the LS (-64%). The effects of physostigmine and atropine confirmed the presence of autoreceptors. Our data exclude that a major part of the acetylcholine released by MS and DBB slices derived from intrinsic neurons bearing p75(NTR) receptors. In the LS, part of the released acetylcholine might be from projections of such neurons located in the LS, MS and/or DBB. These data also suggest that the MS and the DBB may be the target of extrinsic cholinergic innervation that does not bear p75(NTR) receptors.

High potassium-induced activation of choline-acetyltransferase in human neocortex: implications and species differences.

The role of electrical and potassium (K(+))-induced depolarisation on choline-acetyltransferase (ChAT) activity in human and mouse neocortical slices was studied. When [3H]-ACh release was evoked by two K(+) stimulations in human neocortex, the mean S(2)/S(1) ratio was significantly below unity. ChAT inhibitors, like bromo-acetylcholine and ocadaic acid, raised this ratio by 79 and 63%, respectively, suggesting that the diminished S(2)/S(1) value in the absence of ChAT inhibitors reflected an increased ChAT activity at S(2) following K(+) depolarisation at S(1). When stimulated electrically, however, the S(2)/S(1) ratio in human neocortex was near unity and ocadaic acid remained without effect. In parallel experiments on mouse neocortical slices, the S(2)/S(1) ratio was near unity in both electrically or K(+)-evoked [3H]-ACh release and was not altered by ChAT inhibition. ChAT activity following K(+) depolarisation was also determined directly. ChAT activation in human neocortical slices was highest at 10 and 20mM K(+). ChAT activity in mouse neocortical tissue was not altered by K(+) depolarisation. These results suggest that in human, but not in mouse, neocortex ChAT activity may be increased due to ongoing K(+) depolarisation. This increase of ChAT activity supports a cholinergic degeneration hypothesis which has been entitled "autocannibalism" by Wurtman [TINS 15 (1992) 177].

Presynaptic modulation of acetylcholine, noradrenaline, and serotonin release in the hippocampus of aged rats with various levels of memory impairments.

Aged (25-27 months) Long-Evans female rats were distinguished according to whether they showed no significant impairment (AU), moderate impairment (AMI), or severe impairment (ASI) in a spatial reference-memory task. Young (3-5 months) rats served as controls. Electrically evoked overflow of tritium was assessed in hippocampal slices preloaded with [3H]choline or [3H]serotonin (5-HT). Nicotine-evoked overflow of tritium was measured after preloading with [3H]noradrenaline (NA). Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and concentration of monoamines were assessed in homogenates. Aged rats exhibited reduced accumulation of [3H]choline and [3H]5-HT, increased accumulation of [3H]NA, and weaker electrically evoked overflow of [3H]acetylcholine ([3H]ACh) and [3H]5-HT. The overflow of [3H]NA was not altered consistently by aging. Roughly, drugs acting presynaptically had comparable effects in aged rats: oxotremorine and CP 93,129 inhibited the overflow of [3H]ACh, CP 93,129 and UK 14,304 reduced that of [3H]5-HT. ChAT or AChE activity, and 5-HT concentration were not changed by age; NA concentration was reduced. When significant, changes were comparable in AU, AMI, and ASI rats. Data show that aging alters cholinergic and serotonergic hippocampal innervations, release of ACh and 5-HT, but not presynaptic release-modulating mechanisms. These alterations do not account for variability in water-maze performance of aged rats.

Presynaptic regulation of neurotransmitter release in the cortex of aged rats with differential memory impairments.

Cluster analysis of water-maze reference-memory performances of 25-27-month-old (compared to 3-5-month-old) rats distinguished subpopulations of young adult rats (YOUNG), aged rats with no significant impairment (AU), aged rats with moderate impairment (AMI), and aged rats with severe impairment (ASI). In the frontoparietal cortex, we subsequently assessed the electrically evoked release of tritium in slices preloaded with [3H]choline, [3H]noradrenaline (NA), or [3H]serotonin (5-HT) and the effects of an agonist (oxotremorine, UK 14,304, and CP 93,129) of the respective autoreceptors. Cholinergic and monoaminergic markers were measured in homogenates. Overall, aged rats exhibited reduced accumulation of [3H]choline (-25%) and weaker evoked transmitter release (in % of accumulated tritium: -44%, -20%, and -34%, for [3H]acetylcholine, [3H]NA, and [3H]5-HT, respectively). In all rats, the inhibitory effects of the autoreceptor agonists on the evoked release of [3H] were comparable. Acetylcholinesterase (AChE), not choline acetyltransferase (ChAT), activity was reduced. The results suggest age-related modifications in the cholinergic, noradrenergic, and serotonergic innervation of the frontoparietal cortex, alterations of evoked transmitter release, but no interference with presynaptic autoinhibition of the release. Neither of these alterations seemed to account for the cognitive impairment assessed.

Characterization of muscarinic autoreceptors in the rabbit hippocampus and caudate nucleus.

Oxotremorine-induced inhibition of electrically evoked release of 3H-acetylcholine from brain slices preincubated with 3H-choline was used to characterize muscarinic autoreceptors in rabbit hippocampus and caudate nucleus. From the shifts to the right of the concentration-response curves of oxotremorine in the presence of muscarinic receptor antagonists, the following pKB values [95% C.I.] were determined in the hippocampus: tripinamide: 8.7 [8.5, 8.8]; himbacine: 8.4 [8.3, 8.5]; AQ-RA 741: 8.3 [8.2, 8.5]; 4-DAMP: 8.2 [8.0, 8.3]; hexahydrosiladifenidol: 7.4 [7.2, 7.5]; AF-DX 116: 7.3 [7.1, 7.4]; pirenzepine: 6.8 [6.6, 7.0]; and PD102807: 6.3 [6.0, 6.5]. In the caudate nucleus: tripinamide: 9.1 [8.9, 9.2]; 4-DAMP: 8.3 [8.2, 8.5]; himbacine: 8.1 [8.0, 8.2]; AQ-RA 741: 8.1 [8.0, 8.3]; hexahydrosiladifenidol: 7.3 [7.2, 7.4]; AF-DX 116: 7.1 [7.0, 7.2]; pirenzepine: 6.7 [6.6, 6.8]; and PD102807: 6.5 [6.2, 6.8]. These pKB values fit best to literature values for M2 receptors, suggesting that the muscarinic autoreceptor of the rabbit hippocampus and caudate nucleus is the m2 gene product.

Mice transgenic for exon 1 of Huntington's disease: properties of cholinergic and dopaminergic pre-synaptic function in the striatum.

In Huntington's disease (HD), neuronal loss is most prominent in the striatum leading to emotional, cognitive and progressive motor dysfunction. The R6/2 mice, transgenic for exon 1 of the HD gene, develop a neurological phenotype with similarities to these features of HD. In striatal tissue, electrically evoked release of tritiated acetylcholine (ACh) and dopamine (DA) were compared in wild-type (WT) and R6/2 mice. In R6/2 mice, the evoked release of ACh, its M2 autoreceptor-mediated maximum inhibition and its dopamine D2 heteroreceptor-mediated maximum inhibition was diminished to 51%, 74% and 87% of controls, respectively. Also, the activities of choline acetyltransferase and of synaptosomal high-affinity choline uptake decreased progressively with age in these mice. In the DA release model, however, electrical stimulation elicited equal amounts of [3H]-DA both in WT and R6/2 mice. Moreover, high-affinity DA uptake into striatal slices was similar in WT and R6/2 mice. In order to confirm these findings in vivo, intrastriatal levels of extracellular DA were measured by intracerebral microdialysis in freely moving mice: striatal DA levels were found to be equal in WT and R6/2 mice. In conclusion, in the transgenic R6/2 mice changes occur mainly in striatal cholinergic neurones and their pre-synaptic modulation, but not in the dopaminergic afferent terminals. Whether similar events also contribute to the pathogenesis of HD in humans has to be established.

Neurotransmitter release and its presynaptic modulation in the rat hippocampus after selective damage to cholinergic or/and serotonergic afferents.

UNLABELLED: Male Long-Evans rats sustained injections of 5,7-dihydroxytryptamine (5,7-DHT) into the fimbria-fornix and the cingular bundle or/and intraseptal injections of 192 IgG-saporin to induce serotonergic or/and cholinergic hippocampal denervations; Sham-operated rats served as controls. Four to ten weeks after lesioning, we measured (i). the electrically evoked release of acetylcholine ([3H]ACh), noradrenaline ([3H]NA) and serotonin ([3H]5-HT) in hippocampal slices in the presence of drugs acting on auto- or heteroreceptors, (ii). the nicotine-evoked release of NA and (iii). the choline acetyltransferase (ChAT) activity and the concentration of monoamines in homogenates. Saporin lesions reduced the accumulation of [3H]choline, the release of [3H]ACh and the ChAT activity, but increased the concentration of NA and facilitated the release of [3H]NA evoked by nicotine. 5,7-DHT lesions reduced the accumulation and the release of [3H]5-HT, the concentration of 5-HT, and also facilitated the release of [3H]NA evoked by nicotine. Accumulation and electrically evoked release of [3H]NA were not altered by either lesion. The combination of both toxins resulted in an addition of their particular effects. The 5-HT(1B) receptor agonist, CP 93129, and the muscarinic agonist, oxotremorine, reduced the release of [3H]ACh in control and 5,7-DHT-lesioned rats; in rats injected with saporin, their effects could not be measured reliably. CP 93129 and the alpha(2)-adrenoceptor agonist, UK 14304, reduced the release of [3H]5-HT in all groups by about 65%. IN CONCLUSION: (i). selective neurotoxins can be combined to enable controlled and selective damage of hippocampal transmitter systems; (ii). 5-HT exerts an inhibitory influence on the nicotine-evoked release of NA, but partial serotonergic lesions do not influence the release of ACh at a presynaptic level and (iii). presynaptic modulatory mechanisms involving auto- and heteroreceptors may be conserved on fibres spared by the lesions.

Methylated analogues of methyl (R)-4-(3,4-dichlorophenylacetyl)- 3-(pyrrolidin-1-ylmethyl)piperazine-1-carboxylate (GR-89,696) as highly potent kappa-receptor agonists: stereoselective synthesis, opioid-receptor affinity, receptor selectivity, and functional studies.

Analogues of the kappa-receptor agonist methyl (R)-4-(3,4-dichlorophenylacetyl)-3-(pyrrolidin-1-ylmethyl)piperazine-1-carboxylate (GR-89,696, 6) bearing an additional methyl substituent in the side chain are synthesized and evaluated for their kappa-receptor affinity and selectivity. A key step in the synthesis is the stereoselective reductive amination of the ketones 9, 18, and 19 with pyrrolidine and NaBH(3)CN, which succeeds only in the presence of the Lewis acid Ti(OiPr)(4). Whereas the BOC-substituted ketone 9 affords the unlike and like diastereomers of 10 in a ratio of 70:30, the diastereoselectivity during the reductive amination of the butyl and phenyl substituted ketones 18 and 19 is enhanced to 85:15 (butyl derivative) and >95:<5 (phenyl derivative) in favor of the unlike diastereomers. In receptor binding studies using the radioligand [(3)H]U-69,593 the (S,S)-configured methyl carbamate (S,S)-14 reveals the highest kappa-receptor affinity (K(i) = 0.31 nM) within this series, even exceeding the lead kappa-agonist 6 (GR-89,696). A slightly reduced kappa-receptor affinity is observed with the propionamide (S,S)-13 (K(i) = 0.67 nM). The kappa-receptor affinity of piperazines with acyl or alkoxycarbonyl residues at both nitrogen atoms (11, 13, 14) decreases in the order (S,S) > (R,R) > (S,R) > (R,S). The methyl carbamate (S,S)-14 discloses a unique activity profile also binding at mu-receptors in the subnanomolar range (K(i) = 0.36 nM). In a functional assay, i.e., by measuring acetylcholine release in rabbit hippocampus slices, the agonistic effects of the methyl carbamate (S,S)-14 and the propionamide (S,S)-13 are demonstrated. Only weak kappa- and mu-receptor affinities are found with the butyl- and phenyl-substituted piperazines 22 and 23. However, considerable sigma(1)-receptor affinity is determined for the enantiomeric, unlike-configured butyl derivatives (R,S)-22 and (S,R)-22 with K(i)-values of 40.2 nM and 81.0 nM, respectively.

Electrically evoked release of glutamate in rat hippocampal slices: effects of various drugs and fimbria-fornix lesions.

A model of electrically evoked release of glutamate from rat hippocampus was developed and used to detect possible changes induced by lesions of hippocampal afferences. Neuronal glutamate in hippocampal slices was labelled by preincubation with [3H]glutamine. The slices were then superfused with physiological medium in the presence of the glutamate uptake inhibitor L-transpyrrolidine-2,4-dicarboxylic acid (100 microM or 3 microM) and stimulated twice electrically (S1, S2: 240 pulses, 3 Hz, 2 ms, 26-30 mA); various drugs were added before S2. In order to determine the basal and evoked outflow of [3H]glutamate only, the mixture of 3H-labelled compounds (glutamine, glutamate and GABA) was separated by ion exchange chromatography in superfusate fractions and slices. The electrically evoked overflow of [3H]glutamate was largely Ca2+-dependent and tetrodotoxin-sensitive and hence represented action potential-induced exocytotic release of [3H]glutamate. Evoked [3H]glutamate release was significantly increased by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM), suggesting the presence of endogenous inhibitory adenosine, and reduced by the A1 receptor agonist N6-cyclopentyladenosine (1 microM, antagonized by DPCPX, 0.1 microM). There was no evidence for a cholinergic, serotonergic, or adrenergic modulation of the evoked release of [3H]glutamate: the corresponding selective agonists (or antagonists) were ineffective. After aspirative lesions of the septohippocampal pathways the hippocampal noradrenaline content was markedly increased, whereas cholinergic and serotonergic markers were reduced. The evoked release of [3H]glutamate in hippocampal slices of lesioned rats was significantly increased by a mechanism which still has to be determined, but which is not related to alterations in A1 receptor function. It is concluded that the present model was able to detect lesion-induced differences in electrically evoked release of [3H]glutamate, but the relationship of these differences to changes of noradrenergic, cholinergic or serotonergic hippocampal innervations remains to be established.

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.

Postnatal development of opioid receptors modulating acetylcholine release in hippocampus and septum of the rat.

The postnatal development of presynaptic opioid receptors inhibiting the release of acetylcholine (ACh) was studied in rat brain hippocampus, medial septum (MS) and diagonal band of Broca (DB). To this end, the corresponding brain slices (350 microm thick) of rats of various postnatal ages (postnatal day 4 [P4] to P16, and adult) were preincubated with [(3)H]choline and stimulated twice for 2 min (S(1), S(2): at 3 Hz, 2 ms, 60 mA) during superfusion with physiological buffer containing hemicholinium-3. In parallel, the activity of choline acetyltransferase (ChAT) was determined in crude homogenates of the tissues as a marker for the development of cholinergic neurons. At any postnatal age, the electrically evoked overflow of tritium from slices preincubated with [(3)H]choline was highest in the DB, followed by the MS and the hippocampus. The evoked [(3)H]overflow increased with postnatal age, reached about 50% (MS, DB) or 30% (hippocampus) of the corresponding adult levels at P16 and correlated significantly with the corresponding ChAT activities. Presence of the preferential mu-opioid receptor agonist DAMGO during S(2) significantly inhibited the evoked overflow of tritium already at P4 in DB and MS, whereas in the hippocampus significant inhibitory effects were first observed at P8 only. Moreover, adult levels of inhibition due to DAMGO were reached at P16 in the DB and MS but not in the hippocampus. In septal areas, also the effect of the preferential delta-opioid receptor agonist DPDPE on the evoked [(3)H]overflow was studied: in contrast to DAMGO, however, significant inhibitory effects of DPDPE were first observed at P12 only. In conclusion, the postnatal development of presynaptic mu-opioid receptors on cholinergic neurons in the DB and MS starts earlier than in the hippocampus and precedes that of presynaptic delta-opioid receptors.

Characterization of opioid receptor types modulating acetylcholine release in septal regions of the rat brain.

Presynaptic opioid receptors of the delta- and mu-types have been shown to inhibit the release of acetylcholine (ACh) in the rat striatum and hippocampus, respectively, but it is unknown whether opioid receptors modulate the release of ACh also in the region of origin of the hippocampal cholinergic innervation, the septum. To answer this question, slices (350 microm) of the medial septal area and of the diagonal band of Broca, as well as (for comparison) of the hippocampus, were prepared from adult male Wistar rats. The slices were incubated with [3H]choline, superfused in the presence of hemicholinium-3 (10 microM) and stimulated twice (S1, S2) by electrical fields (360 pulses, 3 Hz, 2 ms, 60 mA); opioid receptor agonists were present during S2. The preferential mu-agonist [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) inhibited the evoked ACh release by maximally about 40% in hippocampal slices and acted even more strongly in the medial septal area, or the diagonal band of Broca (about 60% or 75% maximal inhibition, respectively). These effects were reduced or abolished by the preferential mu-antagonist naloxone, which showed no effects when given alone. Using naloxone in the presence of a cocktail of peptidase inhibitors, no evidence for an endogenous tone of opioid peptides was found in the medial septal area, diagonal band of Broca or the hippocampus. Using the preferential delta-agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) and the delta-antagonist naltrindole, a delta-opioid receptor inhibiting evoked ACh release was clearly detectable both in the medial septal area and the diagonal band of Broca, but not in the hippocampus, whereas the preferential kappa-agonist trans-3,4-dichloro-N-methyl-N-[2(1-pyrrolidinyl)cyclo-hexyl] benzeneacetamide (U50,488H) had only weak or no effects. In addition to the functional experiments, double in-situ hybridization studies were performed, in which cells containing mRNA for choline acetyltransferase (ChAT) were labeled by an antibody-linked enzymatic staining procedure, whereas mRNAs for mu- or delta-opioid receptors were detected with radioactive probes. These experiments revealed that in the septal region mainly mu-opioid receptors were expressed by neurons positive for ChAT mRNA, whereas in the rat striatum the expression of delta-opioid receptors prevailed in those neurons. We conclude that in the septal area of the rat brain, in contrast to the rat striatum and hippocampus, both presynaptic mu- and delta-opioid receptors modulate the evoked release of ACh. Whether presynaptic mu- and delta-opioid receptors occur on the same or on different septal cells or axon terminals remains to be clarified.

Modulation of acetylcholine and 5-hydroxytryptamine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts containing cholinergic and/or serotonergic neurons.

Three-month-old Long-Evans female rats sustained aspirative lesions of the dorsal septohippocampal pathways and, 2 weeks later, received intrahippocampal suspension grafts containing fetal cells from the mesencephalic raphe (rich in serotonergic neurons; RAPHE), the medial septum and the diagonal band of Broca (rich in cholinergic neurons; SEPT), or a mixture of both (COTR). Lesion-only (LES) and sham-operated rats (SHAM) were used as controls. Hippocampal slices of these rats (5-9 month after surgery) were preincubated with [3H]choline or [3H]5-HT, superfused continuously (in the presence of hemicholinium-3 or fluvoxamine) and stimulated electrically (360 pulses, 2 ms, 3 Hz, 26-28 mA) in order to study the presynaptic modulation of acetylcholine (ACh) and serotonin (5-HT) release. The accumulation of [3H]choline and the evoked overflow of [3H]ACh were significantly reduced in slices from LES and RAPHE rats, but reached a close-to-normal level in SEPT and COTR rats. As to accumulation and overflow of [3H]5-HT, the lesion-induced reduction was compensated for only in RAPHE and COTR rats. The relative amount of evoked [3H]5-HT release (in % of tissue-3H) was significantly increased in LES and SEPT rats. Only slight differences (group LES) were found in the sensitivity of muscarinic and serotonergic autoreceptors towards oxotremorine and CP 93,129, respectively. Moreover, CP 93,129 induced a significantly weaker inhibition of ACh release in slices of COTR rats than in all other groups. Using the 5-HT1A receptor agonist 8-OH-DPAT and antagonist Way 100,635, no evidence for a modulatory influence of 5-HT1A receptors was found in RAPHE and COTR rats. It is concluded that despite substantial lesion- and graft-induced changes in the amount of ACh and 5-HT released by hippocampal slices of lesion-only or grafted rats, the presynaptic modulation of these transmitters is only slightly affected by changes in the neuronal environment.

Evaluation of autoreceptor-mediated control of [(3)H]acetylcholine release in rat and human neocortex.

In order to assess the autoinhibitory control of endogenous acetylcholine (ACh) in rat and human neocortex, slices of these tissues were prelabelled with [(3)H]choline, superfused continuously and stimulated electrically using various frequencies in the presence or absence of drugs. The autoinhibitory feedback control of [(3)H]ACh release was operative - despite the absence of blockers of ACh esterase - at stimulation frequencies >/= 3 Hz in rat and >/= 6 Hz in human neocortex tissue. At these frequencies the muscarinic antagonist atropine (0.1 microM) disinhibited the release of [(3)H]ACh in both species. Estimation of the biophase concentration of ACh near the autoreceptor in the rat neocortex from concentration-response curves of the muscarinic agonist oxotremorine revealed that at 3 Hz about 25% of the autoreceptors were activated by endogenously released ACh. This estimation is consistent with an increase in [(3)H]ACh release to about 120% of control values by complete blockade of autoreceptors with atropine. The observation that in human neocortical tissue presynaptic autoinhibition of [(3)H]ACh release is operative at stimulation frequencies >/= 6 Hz suggests that selective blockade of autoinhibition may also increase ACh release in the cortex of Alzheimer's disease patients, without additional blockade of the enzyme acetylcholinesterase.

Intrahippocampal grafts containing cholinergic and serotonergic fetal neurons ameliorate spatial reference but not working memory in rats with fimbria-fornix/cingular bundle lesions.

Three-month-old Long-Evans female rats sustained aspirative lesions of the dorsal septohippocampal pathways and, 2 weeks later, received intrahippocampal suspension grafts containing cells from the mesencephalic raphe, cells from the medial septum and the diagonal band of Broca, or a mixture of both. Lesion-only and sham-operated rats were used as controls. All rats were tested for locomotor activity 1 week, 3 and 5 months after lesion surgery, for spatial working memory in a radial maze from 5 to 9 months, and for reference and working memory in a water tank during the 9th month after lesioning. Determination of hippocampal concentration of acetylcholine, noradrenaline, and serotonin was made after completion of behavioral testing. Compared to sham-operated rats, all rats with lesions, whether grafted or not, exhibited increased levels of locomotor activity and made more errors in the radial maze. The lesioned rats were also impaired in the probe trial (30 first seconds) of the water-tank test made according to a protocol requiring intact reference memory capabilities. While rats with septal or raphe grafts were also impaired, the rats with co-grafts showed performances not significantly different from those of sham-operated rats. With a protocol requiring intact working memory capabilities, all lesioned rats, whether grafted or not, were impaired in the water-tank test. In the dorsal hippocampus of lesion-only rats, the concentration of acetylcholine and serotonin was significantly reduced. In rats with septal grafts or co-grafts, the concentration of acetylcholine was close to normal, as was that of serotonin in rats with raphe grafts or co-grafts. These results confirm previous findings showing that co-grafts enabled the neurochemical properties of single grafts to be combined. Data from the water-tank test suggest that cholinergic and serotonergic hippocampal reinnervations by fetal cell grafts may induce partial recovery of spatial reference, but not working memory capabilities in rats.

The potentiation of amphetamine-induced hyperlocomotion by fimbria-fornix lesions in rats is abolished by intrahippocampal grafts rich in serotonergic neurons.

Three-month old Long-Evans female rats were submitted to aspirative lesions of the fimbria-fornix and intrahippocampal grafts of a cell suspension prepared from a region of the fetal brain including the septum and the diagonal band of Broca (rich in cholinergic neurons) or the raphe (rich in serotonergic neurons). A group of lesioned rats was grafted with both suspensions mixed. Lesion-only and sham-operated rats served as controls. Four months after the lesions, all rats were tested daily for locomotor activity in their home cage, 1 day without being injected, 2 days with an injection of NaCl and 5 days with an injection of 1 mg/kg (i.p.) d-amphetamine. The effects of the lesions and grafts were assessed by measuring the accumulation of [3H]-choline or [3H]-5-hydroxytryptamine (5-HT) by hippocampal slices, and the electrically-evoked release of tritium. Amphetamine injections produced hyperlocomotion which was potentiated by the lesion. This lesion-induced potentiation was also found in rats with septal grafts, but not in those with raphe or co-grafts. The uptake and electrically-evoked release of [3H]-acetylcholine or [3H]-5-HT were reduced in hippocampal slices from lesion-only rats. In rats which received grafts of septal cells or co-grafts, but not in those with raphe grafts, uptake and release of [3H]-acetylcholine were close to normal. Uptake and release of [3H]-5-HT were close to normal in rats with raphe grafts or with co-grafts, but not in those with septal grafts. Altogether, these data suggest that damage to the serotonergic afferents of the hippocampus might play some role in the potentiation of amphetamine-induced hyperlocomotion associated with fimbria-fornix lesions.