Anna Siniscalchi

5-HT1A agonists increase and 5-HT3 agonists decrease acetylcholine efflux from the cerebral cortex of freely-moving guinea-pigs

1 The influence of 5‐hydroxytryptamine1A (5‐HT1A), 5‐HT2 and 5‐HT3 agonists and antagonists on acetylcholine (ACh) release from the cerebral cortex was studied in freely moving guinea‐pigs. 2 8‐Hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT, 0.01–1 mg kg−1, s.c.) caused the 5‐HT syndrome and dose‐dependently increased ACh release. Ru 24969 (1–10 mg kg−1, s.c.) shared the same effects, but it was less potent. (−)‐Propranolol (5 mg kg−1) and metitepine (2 mg kg−1) prevented these behavioural and neurochemical responses. 3 (±)‐1(4‐Iodo‐2,5‐dimethoxyphenyl)2‐aminopropane (DOI) up to 2 mg kg−1 did not modify ACh release and ketanserin (0.5 mg kg−1) was ineffective on 5‐HT‐induced changes of ACh outflow. 4 2‐Methyl‐5‐HT (500 μg, i.c.v.) and 5‐HT (500 μg, i.c.v.) plus metitepine (2 mg kg−1, s.c.) inhibited the gross behaviour and ACh release. ICS 205–930 (0.5 mg kg−1) prevented these responses. 5 2‐Methyl‐5‐HT, up to 10 μmoll−1, and 8‐OH‐DPAT, up to 0.1 μmoll−1, (like 5‐HT) did not change [3H]‐choline efflux from cerebral cortex slices. 6 These results suggest that exogenous 5‐HT and related selective agonists modulate guinea‐pig cortical cholinergic structures through 5‐HT1A and 5‐HT3 receptors. The stimulation of 5‐HT1A autoreceptors may lead to disinhibition of the cholinergic cells, tonically inhibited by the tryptaminergic control. Conversely, the stimulation of 5‐HT3 receptors inhibits ACh release, possibly through an interneurone. No direct 5‐HT modulation of the cholinergic nerve endings was found.

Different approaches to study acetylcholine release: endogenous ACh versus tritium efflux.

SummarySuperfused slices of guinea-pig cerebral cortex (CC), caudate nucleus (CN) and thalamus (Th) were used to compare i) the resting and electrically-evoked release of endogenous acetylcholine (ACh) in the presence of physostigmine (Phys) and ii) the resting and electrically-evoked tritium efflux (after preloading with 3H choline) in the absence or in the presence of Phys and hemicholinium (HC-3).In addition, the effect of GABA, morphine and their antagonists on both effluxes was investigated.1)By matching the ACh and tritium outflow on a molar basis, an average ratio of 100: 2–4 was found. When expressed as a percentage of tissue content, the ACh release at 2 Hz (2 min) was 4.1 in CN, 0.92 in CC and 0.44 in Th. Lower percent values in the same rank order, were found for tritium outflow with Phys. Thus, CN has the highest secretory activity.2)Tritium evoked outflow in the presence of Phys was nearly halved in comparison with the normal values (without Phys). Therefore, the autoreceptor-mediated negative feed-back seems to be similar in the three areas.3)Tritium evoked outflow in the presence of HC-3 was more than doubled in Th (less so in CC and CN) in comparison with the normal values. A second stimulation at 2 Hz (2 min) gave rise to the same outflow in Th while an evident fall in radiolabel efflux was found in CN. Therefore the blockade of high affinity choline uptake plays a different role in Th and CN.4)The ratio between two subsequent periods of stimulation at 2 Hz for 2 min (at the 45th and 75th min, St2/St1) ranged as a rule between 0.75 and 1 for ACh and tritium. However, when the evoked outflow of ACh and tritium was tested during St2 at different rates (1–2–5–10 Hz), a three-times greater increase of ACh with respect tritium was found, as a function of the stimulation frequency. This suggests an inverse relationship between specific radioactivity of released transmitter and second stimulus intensity, above all when Phys and HC-3 are not present.5)GABA 0.3–0.6 mM, added before St2, inhibited the evoked ACh and tritium efflux in CC and CN and increased the resting release in a dose-dependent manner. Conversely, picrotoxin, which prevented GABA effect, increased the evoked ACh release but not tritium efflux. Morphine 30 μM also inhibited the evoked ACh and tritium efflux from Th slices. However, the drug in the presence of naloxone enhanced ACh release but not tritium outflow.6)In conclusion, the two methods give results only in part superimposable. Tritium method allows drug-induced inhibition to be seen more readly than facilitation. This fact may depend on the reduction of ACh specific radioactivity in St2, whenever drug treatment enhances the release process, thus involving less labelled (or more diluted) neurotransmitter stores.

Inhibitory effect of nociceptin on [3H]-5-HT release from rat cerebral cortex slices

The effect of nociceptin (NC) on 5‐hydroxytryptamine (5‐HT) release was studied in rat cerebral cortex slices preincubated with [3H]‐5‐HT and electrically stimulated (3 Hz, for 2 min) at the 45th (St1) and the 75th (St2) min of superfusion. NC (0.1–3 μM), present in the medium from the 70th min onward, concentration‐dependently reduced electrically evoked [3H]‐5‐HT efflux (pEC50=6.54, Emax −54%). The inhibition was not antagonized by naloxone (1 μM) ruling out the involvement of opioid receptors. Phe1ψ(CH2‐NH2)Gly2]NC(1‐13)NH2, which acts as an opioid‐like receptor (ORL1) antagonist at the peripheral level, behaved as a partial agonist in cerebral cortex slices i.e. it inhibited [3H]‐5‐HT efflux when added before St2, however, when present in the medium throughout the whole experiment, [Phe1ψ(CH2‐NH2)Gly2]NC(1‐13)NH2 prevented the action of NC added at the 70th min. The non‐selective ORL1 receptor antagonist, naloxone benzoylhydrazone (3 μM), in the presence of 10 μM naloxone, did not modify the St2/St1 ratio but completely abolished the NC effect. These findings demonstrate that NC inhibits 5‐HT release from rat cerebral cortex slices via ORL1 receptors, suggesting its involvement in central processes mediated by 5‐HT.

THE EFFECT OF NALOXONE ON OPIOID‐INDUCED INHIBITION AND FACILITATION OF ACETYLCHOLINE RELEASE IN BRAIN SLICES

1 The effect of morphine, methionine‐enkephalin (Met‐enkephalin) and d‐Ala2‐d‐Leu5‐enkephalin (DADLE) were tested on the spontaneous and electrically‐evoked release of acetylcholine (ACh) from superfused slices of guinea‐pig thalamus, caudate nucleus and cerebral cortex. 2 At no concentration did morphine, Met‐enkephalin or DADLE modify the outflow of ACh at rest but Met‐enkephalin in the presence of naloxone, reduced the resting ACh release. 3 Morphine, at a low dose (3 μm) had no effect in slices of cerebral cortex, but it enhanced the evoked release of ACh in thalamic and caudate, slices. At higher doses of morphine (10–30 μm), the ACh release evoked by electrical pulses was significantly inhibited in every area. 4 Met‐enkephalin behaved like morphine in thalamic slices, whereas DADLE, a specific < 5 agonist, produced a slight inhibition of ACh outflow only at 10 μm. 5 Naloxone antagonized the inhibitory effect of morphine in the cerebral cortex and caudate nucleus slices. Naloxone and also spiroperidol blocked the releasing effect of morphine in caudate slices. In contrast naloxone did not affect the increase of ACh release caused by morphine and Met‐enkephalin in thalamic slices. The inhibitory effect of both opioids at high doses was reversed by naloxone so that they then enhanced ACh release. 6 A two fold increase of calcium concentration in the Krebs solution prevented the inhibitory effects of morphine 10 μm. 7 It is suggested that two receptors are present in thalamic slices, one of which inhibits and the other facilitates ACh release.

Cannabinoid receptor agonist WIN 55,212-2 inhibits rat cortical dialysate gamma-aminobutyric acid levels.

The effects of the cannabinoid receptor agonist WIN 55,212‐2 (0.1–5 mg/kg i.p.) on endogenous extracellular γ‐aminobutyric acid (GABA) levels in the cerebral cortex of the awake rat was investigated by using microdialysis. WIN 55,212‐2 (1 and 5 mg/kg i.p.) was associated with a concentration‐dependent decrease in dialysate GABA levels (–16% ± 4% and –26% ± 4% of basal values, respectively). The WIN 55,212‐2 (5 mg/kg i.p.) induced‐inhibition was counteracted by a dose (0.1 mg/kg i.p.) of the CB1 receptor antagonist SR141716A, which by itself was without effect on cortical GABA levels. These findings suggest that cannabinoids decrease cortical GABA levels in vivo, an action that might underlie some of the cognitive and behavioral effects of acute exposure to marijuana. J. Neurosci. Res. 66:298–302, 2001.

Different effects of 8-OH-DPAT, a 5-HT1A receptor agonist, on cortical acetylcholine release, electrocortigram and body temperature in guinea pigs and rats

8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) dose dependently increased cortical acetylcholine (ACh) release and the body temperature of freely moving guinea pigs. Moreover, it evoked an electroencephalographic (EEG) arousal reaction in phenobarbital-pretreated animals. 8-OH-DPAT was about 10 times less effective in increasing cortical ACh release in the rat than in the guinea pig, and did not modify the phenobarbital-synchronized EEG but dose dependently decreased body temperature. The possibility that 5-HT1A receptors play different roles, depending on the animal species, is discussed.

Protein kinase C activity, translocation, and selective isoform subcellular redistribution in the rat cerebral cortex after in vitro ischemia

Protein kinase C (PKC) involvement in ischemia‐induced neuronal damage has been investigated in superfused rat cerebral cortex slices submitted to 15 min of oxygen‐glucose deprivation (OGD) and in primary cultures of rat cortical neurons exposed to 100 μM glutamate (GLU) for 10 min. OGD significantly increased the total PKC activity in the slices, mostly translocated in the particulate fraction. After 1 hr of reperfusion, the total PKC activity was reduced and the translocated fraction dropped by 84% with respect to the control. Western blot analysis of OGD samples showed an increase in total β2 and ϵ PKC isoform levels. After reperfusion, the total levels of α, β1, β2 and γ isoforms were significantly reduced, whereas the ϵ isoform remained at an increased level. Endogenous GLU release from OGD slices increased to about 15 times the basal values after 15 min of oxygen‐glucose deprivation, and to 25 and 35 times the basal level in the presence of the PKC inhibitors staurosporine (0.1 μM) and bisindolylmaleimide (1 μM), respectively. Western blot analysis of GLU‐treated cortical neurons showed a significant decrease only in the total level of β2 isoforms. Cell survival was reduced to 31% in GLU‐treated neuronal cultures; PKC inhibitors were not able to modify this effect. These findings demonstrate that the cell response to OGD and GLU involves PKC in a complex way. The net role played by PKC during OGD may be to reduce GLU release and, consequently, neurotoxicity. The isoforms β2 and ϵ are affected the most and may play a significant role in the mechanisms underlying neurotoxicity/neuroprotection.

Influence of N-allyl-normetazocine on acetylcholine release from brain slices: involvement of muscarinic receptors

SummaryThe effects of (±)N-allyl-normetazocine on the release of acetylcholine from different areas of guinea-pig and rat brain were investigated. 1. The drug did not modify the electrically (2 Hz) evoked tritium efflux from guinea-pig cerebral cortex, thalamus and caudate nucleus slices, preloaded with 3H-choline 0.1 μmol/l and superfused with Krebs solution containing hemicholinium-3 10 μmol/l. 2. (±)N-allyl-normetazocine 10 μmol/l. enhanced the evoked 3H efflux from guinea-pig brain slices superfused with Krebs solution containing physostigmine 30 μmol/l or oxotremorine 0.3 -1 gmol/l; the effect was naloxone-insensitive and was abolished by atropine 0.15 μmol/l, but not by pirenzepine 1 μmol/l. 3. (±)N-allyl-normetazocine 5 μmol/l enhanced the electrically evoked release of endogenous acetylcholine as well, in a naloxone-insensitive way. 4. Both (±) and (+)N-allyl-normetazocine were without effect on 3H efflux from rat caudate nucleus slices electrically stimulated at 0.2 Hz frequency, after preloading with 3H-choline and during superfusion with hemicholinium-3. 5. The results are discussed in view of the antimuscarinic properties of the drug.

Effect of 5-hydroxytryptamine on [3H]-acetylcholine release from guinea-pig striatal slices.

1 The effect of 5‐hydroxytryptamine (5‐HT) on spontaneous and electrically‐evoked tritium efflux was studied in guinea‐pig caudate nucleus slices preloaded with [3H]‐choline. 2 5‐HT, 10–300 μmol1−1, temporarily increased the spontaneous tritium efflux (as well as the endogenous acetylcholine (ACh) release) and, after 15 min perfusion, inhibited it. The facilitatory effect of 5‐HT on spontaneous efflux was increased while the inhibitory effect did not occur in slices taken from dopamine‐depleted guinea‐pigs. 3 The increase in spontaneous tritium efflux by 5‐HT was blocked by methiothepin, methysergide (pA2 8.7) and by the selective 5‐HT2 antagonist, ritanserin (pA2 6.7). 4 The inhibition of spontaneous tritium efflux by 5‐HT was prevented by methysergide and methiothepin but not by ritanserin and (−)‐propranolol. 5 5‐HT, 100 μmol1−1, inhibited the electrically‐evoked tritium efflux and this effect was unchanged in dopamine‐depleted slices. 6 The inhibition of electrically‐evoked tritium efflux by 5‐HT was blocked by methiothepin and methysergide but not by (−)‐propranolol or ritanserin. 7 These results suggest that 5‐HT may exert a rapid and transient (excitatory) and a more prolonged (inhibitory) control over striatal cholinergic neurones.

Neurotensin increases endogenous glutamate release in rat cortical slices

In the present study, the effects of the tridecapeptide neurotensin [NT(1-13)] and its fragments, NT(1-7) and NT(8-13), on endogenous glutamate release from rat cortical slices, were evaluated. NT(1-13) (100-1000 nM) slightly increased spontaneous glutamate release, while it was ineffective at 1 and 10 nM concentrations. Neither the biologically active NT fragment NT(8-13) nor the inactive one NT(1-7) affected basal glutamate release. NT(1-13) (1-1000 nM) enhanced potassium (35 mM)-evoked glutamate release displaying a bell-shaped concentration response curve. In addition NT(8-13) (10 nM) increased K+-evoked-glutamate release similarly to the parent peptide (10 nM), while the biologically inactive fragment NT(1-7) (10-100 nM) was ineffective. The effects of NT(1-13) and NT(8-13) were fully counteracted by the selective neurotensin receptor antagonist SR48692 (100 nM). These findings suggest that NT plays a role in regulating cortical glutamate transmission.