Paolo Paudice

Autoregulation of acetylcholine release in isolated hippocampal nerve endings

The existence of presynaptic autoreceptors modulating acetylcholine release from central cholinergic nerve endings was investigated by using rat hippocampal synaptosomes in a superfusion system. The presence of exogenous acetylcholine, carbachol or oxotremorine in the superfusion fluid produced a dose-dependent inhibition of the release of [3H]acetylcholine elicited by 15 mM KCl in synaptosomes prelabeled with tritiated choline. The inhibition was counteracted by atropine. Another well known muscarinic agonist, bethanechol, had no effect on [3H]acetylcholine release. Our results indicate that central cholinergic nerve terminals possess autoreceptors of the muscarinic type for the control of acetylcholine release. Moreover, differences seem to exist between pre-and postsynaptic muscarinic receptors in the central nervous system.

Cholecystokinin release mediated by 5-HT3 receptors in rat cerebral cortex and nucleus accumbens.

1 The effects of 5‐hydroxytryptamine (5‐HT) on the release of cholecystokinin‐like immunoreactivity (CCK‐LI) were examined in synaptosomes prepared from rat cerebral cortex and nucleus accumbens and depolarized by superfusion with 15 mm KCl. 2 In both areas 5‐HT, tested between 0.1 and 100 nm, increased the calcium‐dependent, depolarization‐evoked CCK‐LI release in a concentration‐related manner. The concentration‐response curves did not differ significantly between the two brain areas (EC50: 0.4 + 0.045 nm and 0.48 ± 0.053 nm, respectively, in cortical and n. accumbens synaptosomes; maximal effect: about 60% at 10 nm 5‐HT). 3 The 5‐HT1/5‐WT2 receptor antagonist methiothepin (300 nm) did not affect the CCK‐LI release elicited by 10 nm 5‐HT. However, the effects of 10 nm 5‐HT were antagonized in a concentration‐dependent manner by the 5‐HT3 receptor antagonists (3α‐tropanyl)‐1H‐indole‐3‐carboxylic acid ester (ICS 205–930; 0.1–100 nm; IC50: 3.56 ± 0.42 nm in the cortex and 3.90 ± 0.50 nm in the n. accumbens) and ondasetron (IC50: 8.15 + 0.73 nm in the cerebral cortex). 5‐HT (10 nm) was also strongly antagonized by 100 nm 1αH, 3α5αH‐tropan‐3‐yl‐3,5‐dichlorobenzoate (MDL 72222) another blocker of the 5‐HT3 receptor. Moreover, the 5‐HT3 receptor agonist 1‐phenylbiguanide (tested in the cerebral cortex between 0.1 and 100 nm) enhanced CCK‐LI release in a manner almost identical to that of 5‐HT (EC50 = 0.64 + 0.071 nm). 4 It is concluded that 5‐HT can act as a potent releaser of CCK‐LI in rat cerebrocortex and nucleus accumbens through the activation of receptors of the 5‐HT3 type situated on the CCK‐releasing terminals. This interaction may provide a rationale for the clinical development of both 5‐HT3 and CCK receptor antagonists as novel anxiolytic drugs.

Release of cholecystokinin in the central nervous system

The octapeptide cholecystokinin (CCK) is one of the most abundant neuropeptides of the central nervous system. A number of features (for instance heterogeneity of the regional distribution, subcellular localization at the nerve terminal level, calcium-dependent release upon nervous tissue depolarization) support the candidacy of CCK as a neurotransmitter. The reported co-existence of CCK and dopamine in some meso-limbic neurons has led to speculation that the neuropeptide may interact with the catecholamine in neuropsychopathologies linked to dopamine dysfunctions, like schizophrenia. Data from the experimental animals have so far generated conflicting results. It should be noted that the interactions between CCK and dopamine, and, in particular, the effects of CCK and dopamine on each other release, both in vitro and in vivo, have been poorly investigated and would require special attention. Evidence is accumulating that CCK may participate in the expression of anxiety. Indeed antagonists at the central CCK receptors exhibit anxiolytic activity in the laboratory animal. An interesting linkage appears to exist in the brain between 5-hydroxytryptamine (5-HT) and CCK. Activation of 5-HT3 receptors was found to increase CCK release from rat cortical or nucleus accumbens synaptosomes. Interestingly, antagonists at 5-HT3 receptors appear to possess anxiolytic activity. Recent studies carried out in conscious unrestrained rats show that the calcium-dependent, tetrodotoxin-sensitive release of CCK-like immunoreactivity evoked in the rat frontal cortex by veratrine infusion can be inhibited by submicromolar concentrations of 5-HT3 receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for functional native NMDA receptors activated by glycine or d‐serine alone in the absence of glutamatergic coagonist

In this study we have examined the effects of N‐methyl‐d‐aspartate (NMDA) receptor activation on the release of cholecystokinin and somatostatin from rat neocortical nerve endings. The release of cholecystokinin‐like immunoreactivity (CCK‐LI) and of somatostatin‐like immunoreactivity (SRIF‐LI) elicited by 12 mm K+ from superfused synaptosomes, but not the spontaneous release, was increased by NMDA in a concentration‐dependent manner. The effects of NMDA could be prevented by antagonists selective for the glutamate recognition site, the receptor channel and the glycine site of the NMDA receptor. In the absence of NMDA, glycine increased on its own and in a concentration‐dependent manner the depolarization‐evoked release of both CCK‐LI and SRIF‐LI. This effect of glycine was strychnine‐insensitive and could be mimicked by d‐serine, a stereoselective agonist at the NMDA receptor glycine site. Antagonists selective for the glycine site or for the NMDA receptor channel prevented the effects of glycine/d‐serine; these effects were, however, insensitive to blockade of the glutamate recognition site of the NMDA receptor, suggesting that glutamate released from synaptosomes or present as contaminant was not involved. The neuropeptide release elicited by d‐serine was strongly inhibited by ifenprodil (0.3 μm) and by Zn2+ ions (50 nm), selective ligands at the NR2B and NR2A subunits of NMDA receptors, respectively. It is concluded that nerve terminals of CCK‐ and SRIF‐releasing neurons possess non‐conventional NMDA receptors whose channels can be operated by glycine or d‐serine without apparent activation of the glutamatergic coagonist site. These receptors may display the triple subunit combination NR1/NR2A/NR2B.

Is acetylcholine release from striatal nerve endings regulated by muscarinic autoreceptors

The presence in cholinergic nerve endings of muscarinic autoreceptors regulating the release of acetylcholine elicited by depolarizing stimuli was investigated in different areas of the rat brain. Synaptosomes prepared from cerebral cortex, hippocampus or corpus striatum were prelabeled with [3H]choline and the inhibitory effect of exogenous acetylcholine on the Ca2+-dependent release of [3H]acetylcholine evoked by 15 mM KCl was analyzed by superfusion. While acetylcholine was equally active in reducing its own release in hippocampus and cortex, it was much less effective in striatal synaptosomes. In contrast the values of several presynaptic cholinergic parameters ([3H]choline uptake, [3H]acetylcholine synthesis and release) were the highest in the striatum. Since experiments with slices showed that autoregulation of acetylcholine release through muscarinic receptors appeared to occur as efficiently in the striatum as in the two other areas, the present results suggest that in the striatum the autoregulation of acetylcholine release may not necessarily require the activation of autoreceptors located on cholinergic nerve terminals.

Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

A number of presynaptic cholinergic parameters (high affinity [3H]choline uptake, [3H]acetylcholine synthesis, [3H]acetylcholine release, and autoinhibition of [3H]acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of [3H]acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca2+-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca2+-dependent release.

Dicyclomine- and pirenzepine-sensitive muscarinic receptors mediate inhibition of [3H]serotonin release in different rat brain areas

The effects of acetylcholine (ACh) on the release of [3H]5-hydroxytryptamine ([3H]5-HT) were investigated in synaptosomes prepared from rat cerebral cortex, hypothalamus and hippocampus and depolarized with 15 mM KCl under superfusion conditions. ACh inhibited the release of [3H]5-HT in all three brain areas. This effect was not modified by hexamethonium but was antagonized by atropine and by the non-classical antagonists pirenzepine and dicyclomine.

The HIV-1 coat protein gp120 and some of its fragments potently activate native cerebral NMDA receptors mediating neuropeptide release

The objective of this study was to investigate the effects of the HIV‐1 envelope protein gp120 and its peptide fragments on the function of N‐methyl‐ d‐aspartate (NMDA) receptors mediating release of cholecystokinin (CCK) and somatostatin (SRIF). These are nonconventional NMDA receptors recently found to be activated by glycine or d‐serine ‘only’. The release of cholecystokinin‐like immunoreactivity (CCK‐LI) and of somatostatin‐like immunoreactivity (SRIF‐LI) elicited by 12 mm K+ from superfused rat neocortex synaptosomes was potently increased by gp120, its cyclic V3 loop and the linear V3 sequence BRU‐C‐34‐A, but not by RP‐135 (a central portion of BRU‐C‐34‐A). The EC50 values of gp120 were 0.02 nm (CCK‐LI release) and 0.01 nm (SRIF‐LI release). The releasing effect of gp120 was prevented by blocking the glycine site or the ion channel of NMDA receptors, but not the glutamate recognition site; in addition, the gp120 effect was strongly inhibited by nanomolar concentrations of Zn2+ ions and by low micromolar concentrations of ifenprodil. It is concluded that gp120 acts as a very potent agonist at the glycine site of NMDA receptors sited on CCK‐ and SRIF‐releasing nerve endings; the protein is able to activate the receptor channel in the absence of glutamate. Gp120 activates the receptors through its V3 loop as peptide fragments related to V3 retain near‐maximal activity. The sensitivity of the gp120 effect to both Zn2+ and ifenprodil would not be incompatible with the idea that these NMDA receptors contain the triple subunit combination NR1/NR2A/NR2B.

Presynaptic muscarinic receptor activation enhances striatal dopamine release evoked by depolarization but not that induced by non-depolarizing stimuli

The release of [(3)H]dopamine stimulated by depolarization with 15 mM KCl of superfused rat striatal synaptosomes was potentiated by acetylcholine through the activation of presynaptic muscarinic receptors. In contrast, acetylcholine did not potentiate the release of [(3)H]dopamine elicited by d-amphetamine nor that caused by the calcium ionophore A23187. The dopamine carrier blocker nomifensine prevented the releasing action of amphetamine but not that of acetylcholine. The results suggest that the activation of muscarinic receptors on dopamine terminals in the rat corpus striatum selectively affects the calcium-dependent depolarization-induced release of the [(3)H]catecholamine. Moreover, the [(3)H]dopamine release caused by acetylcholine seems to occur independently of the membrane dopamine carrier.

The activation of phosphatidylinositol turnover is not directly involved in the modulation of neurotransmitter release mediated by presynaptic muscarinic receptors.

In the rat cerebral cortex, the comparative effects of various muscarinic agonists on the release of [3H]dopamine ([3H]DA), [3H]acetylcholine ([3H]ACh), and [3H]5-hydroxytryptamine ([3H]5-HT) from superfused nerve endings and on phosphatidylinositol (PI) turnover were studied. Acetylcholine (ACh) was found to be the most potent among the agonists tested on all three release systems examined, and also on the activation of PI turnover. Oxotremorine and bethanechol were very weak agonists when tested as stimulators of PI turnover. However, oxotremorine was very effective as a release modulator, while bethanechol was completely ineffective. Our data suggest that the activation of PI turnover is not directly involved in the modulation of neurotransmitter release mediated by presynaptic muscarinic receptors.