Age-related decrease in apomorphine modulation of acetylcholine release from rat striatal slices

Release of [3H]acetylcholine ( [3H]ACh) was assessed in striatal slices from mature, middle-aged and senescent Wistar rats 8, 12 and 24 months of age, respectively. There was an age-related decline in basal release of [3H]ACh as a function of age which was correlated with a decline in accumulation of [3H]ACh. However, the most striking finding was the failure of apomorphine to inhibit KCl-induced [3H]ACh release in the senescent (24 months) animals. Striatal dopaminergic receptor losses in senescence apparently produce several subsequent changes in striatal function which ultimately result in the decline of motor-behavioral function.     

Modulation of the endogenous acetylcholine release from rat striatal slices

The dopaminergic modulation of the acetylcholine release from rat striatal slices has been investigated using a chemiluminescent method. Dopamine, more efficiently than apomorphine, decreased the potassium-evoked release of acetylcholine. The effect of dopamine antagonists, haloperidol and sulpiride, has been studied, and haloperidol was a better antagonist than sulpiride to the dopamine effect. Haloperidol elicited an acetylcholine release from striatal slices at 0.1 nM, probably by removing endogenous dopamine from dopaminergic receptors.     

MDMA ('ecstasy') enhances basal acetylcholine release in brain slices of the rat striatum

The pharmacological basis of acute (+/-)-MDMA (3, 4-methylenedioxymethamphetamine) intoxication still awaits full characterization. According to present knowledge, MDMA enhances the release of serotonin and dopamine in striatal slices and interacts with different types of receptors such as 5-HT2 (5-hydroxytryptamine or serotonin), M1 and M2 muscarinic acetylcholine (ACh), and histamine H1 receptors. Currently, no information is available about the influence of (+/-)-MDMA on striatal cholinergic neurotransmission. In the present study, we used the in vitro perfusion technique to investigate the effect of (+/-)-MDMA on ACh release in rat striatal slices. Perfusions with (+/-)-MDMA (10-300 microM) resulted in a dose-dependent increase of spontaneous ACh release (EC50 approximately 30 microM). The effect was reversible and Ca++- and tetrodotoxin-sensitive. To determine the neurochemical pathways underlying this response, we perfused with (+/-)-MDMA in the presence of various inhibitors of neurotransmitter receptors. Blockade of glutamate or muscarinic ACh receptors as well as 5-HT1, 5-HT2, 5-HT3C or dopamine D2 receptors did not modulate (+/-)-MDMA-induced ACh release. However, the presence of histamine H1 receptor antagonists in the perfusion medium abolished (+/-)-MDMA-induced ACh release. The present data clearly demonstrate that (+/-)-MDMA enhances the activity of striatal cholinergic neurons and suggest an involvement of histamine H1 receptors. The effect is not mediated by glutamate and does not involve the activation of receptors of dopamine D2, 5-HT1, 5-HT2, 5-HT3C or muscarinic ACh. Considering the relatively high affinity of (+/-)-MDMA for the H1 histamine receptor (Ki 6 microM), a direct activation of this type of receptor might represent a plausible mechanism for (+/-)-MDMA-induced ACh release.     

Evidence of the modulatory role of serotonin in acetylcholine release from striatal interneurons

The release of acetylcholine was studied in isolated striatal slices of the rat. The spontaneous and ouabain-stimulated release of acetylcholine was higher in those slices where serotonergic input was somehow impaired: raphe nuclei lesion orp-chlorophenylalanine pretreatment or 5,7-dihydroxytryptamine pretreatment resulted in a higher release.l-(m-chlorophenyl)-piperazine, a pure serotonin receptor stimulant andd-fenfluramine, a serotonin releaser significantly reduced the release of acetylcholine evoked by ouabain. Serotonin antagonists (cyproheptadine, mianserine and methysergide) prevented the effect of serotonin agonists. When the serotonergic neurons were destroyed either byp-chlorophenylalanine or by 5,7-dihydroxytryptamine pretreatmentd-fenfluramine had no inhibitory action; however, the effect ofl(m-chlorophenyl)-piperazine was not affected.It is suggested that there is a link between serotonergic and cholinergic neurons in the striatum: serotonin released from raphe-striatal neurons is able to inhibit the release of acetylcholine from striatal interneurons.     

Patterns of endogenous amino acid release from slices of rat and guinea-pig olfactory cortex

A study has been made of the effects of depolarizing stimuli on the release of endogenous amino acid neurotransmitter candidates (aspartate, glutamate, GABA and taurine) from in vitro preparations of rat and guinea pig olfactory cortex. Exposure of small cubes of olfactory cortex tissue from either species to potassium chloride (50 mM) was accompanied by a calcium-dependent release of aspartate, glutamate and GABA. A similar release pattern was evoked by protoveratrine A (100 μM) although the release was largely calcium-independent. Neither agent led to increased release of taurine. Electrical stimulation of the excitatory input (lateral olfactory tract) of freshly prepared, synaptically intact olfactory cortex slices of both species induced significant release of aspartate and GABA from the uncut pial surface and of aspartate, GABA and glutamate from the cut surface. Evoked taurine release occurred from both surfaces of rat olfactory cortex slices but no release was detected from guinea pig olfactory cortex slices. These patterns of release were unaffected by changes in stimulus frequency and were mimicked by protoveratrine A (100 μM) applied to one or other surface. Preincubation of slices from rats for 2 h led to loss of tissue amino acids and to changes in their release patterns; the presence of glutamine (5 mM) during preincubation prevented the loss of amino acids but did not alter their pattern of release. Because of the close similarities between both the electrophysiological properties and the patterns of amino acid release it is concluded that there is probably an identity of amino acid neurotransmitters (aspartate, glutamate and GABA) in rat and guinea pig olfactory cortex. The role of taurine in the rat olfactory cortex is unknown but would seem unlikely to be that of a neurotransmitter. The results are discussed: (i) in terms of the cellular origins of the released amino acids; and (ii) with respect to apparent experimental discrepancies which have appeared in the literature^3,10.     

The effects of ionotropic agonists of excitatory amino acids on the release of arginine vasopressin in rat hypothalamic slices

The effects of ionotropic excitatory amino acids agonists on the release of vasopressin from rat hypothalamic slices were studied. Incubation with increasing doses of NMDA, kainate or AMPA decreased the release of vasopressin in a dose-dependent manner. The values of the IC50 were 1.0, 9.6, or 3.7 x 10-8 M, respectively. The inhibitory effect of the various excitatory amino acids tested was blocked by coincubation with their respective antagonists. Vasopressin secretion was stimulated to 140.3 +/- 7.6% of controls when the slices were obtained from chronically (7 days) salt-loaded rats. Addition of 1 x 10-7 M NMDA or 1 x 10-6 M kainate to the incubation medium antagonized the salt loading-induced increase in vasopressin release. Incubation with 1 x 10-4 M tetrodotoxin did not change basal vasopressin release, but it blocked the decrease in vasopressin secretion induced by 1 x 10-7 M NMDA or 1 x 10-6 M kainate or 1 x 10-6 M AMPA. Incubation with 1 x 10-5 M phaclophen (a GABAB antagonist) and 1 x 10-5 M bicuculline (a GABAA antagonist) was without effect on basal vasopressin secretion while it reversed the inhibition of vasopressin release induced by 1 x 10-7 M NMDA. Incubation with 1 x 10-6 M GABA alone decreased vasopressin secretion to 64.6 +/- 6.9% of control values. The inhibitory effect of GABA did not change when 1 x 10-7 M NMDA was added to the incubation medium. These findings demonstrate that ionotropic excitatory amino acids agonists inhibit vasopressin secretion from hypothalamic slices. They strongly suggest that this inhibitory effect is mediated through local GABAergic interneurones.     

[H]Dopamine accumulation and release from striatal slices in young, mature and senescent rats

Examinations of [³H]dopamine ([³H]DA) release following KCl or amphetamine administration in striatal slices from young (7 month), mature (12 month) and senescent (24 month) Wistar rats showed no age-related changes. Further, the amount of [³H]DA accumulated in the striatal slices showed no changes with age. Thus, previously reported age-related deficits in motor behavior (i.e. rotational) are not produced by changes in striatal DA accumulation or release.     

Neurotensin facilitates dopamine release in vitro from rat striatal slices

Neurotensin, (0.1-10 microM) stimulated the release of [3H]dopamine from rat striatal slices in a calcium-dependent manner, and potentiated the K+-evoked release of [3H]dopamine and endogenous dopamine. This effect was dose-dependent, (1 nM-1 microM) with an EC50 of approximately 10 nM, and was mediated by means of a receptor of similar structure-activity profile to those described in other tissues.     

Extracellular calcium alters frequency modulation of [H]acetylcholine release from rat hippocampal slices

The concentration of extracellular Ca²⁺ has been shown to enhance or attenuate [³H]acetylcholine (ACh) release subsequent to a conditioning stimulus in rat brain hippocampal slices. Slices were incubated in vitro in [³H]choline solution. Subsequently the slices were subjected to two consecutive electrical stimulations separated by 15 or 30 min at 0.25, 1, 4 and 16 Hz and [³H]ACh release was assessed. It was found that a conditioning stimulus may reduce [³H]ACh release during a second stimulation. This phenomenon is frequency related and disappears when the two stimulations are 30 min apart. High extracellular Ca²⁺ (4.0 mM) further attenuated [³H]ACh release during the second stimulation whereas low Ca²⁺ (0.32 mM) abolished the decrease in [³H]ACh release following the second stimulation in all frequencies tested.     

Effects of d-aspartate on excitatory amino acid-induced release of [H]GABA from goldfish retina

The rate of release of [³H]GABA from isolated intact goldfish retinas was studied. Release of [³H]GABA is markedly stimulated by the inclusion in the incubation medium of the photoreceptor neurotransmitter candidates l-glutamate (l-Glu) and l-aspartate (l-Asp), and the glutamate analogs, kainate and quisqualate. At micromolar concentrations, kainate and quisqualate are effective releasers of [³H]GABA, whereas millimolar concentrations of l-Glu and l-Asp are required to release comparable amounts of [³H]GABA. The d-isomers of aspartate (d-Asp) and glutamate (d-Glu) are able to release [³H]GABA, but only when applied at high concentrations (3–30 mM). In the presence of 5 mM d-Asp, the effect of l-Glu in releasing [³H]GABA was markedly potentiated. This dose-response curve of l-Glu was shifted to the left in the presence of d-Asp, although the maximal amount of release was unchanged. d-Asp at 5 mM only slightly increased the GABA release induced by quisqualate, and it did not increase the GABA release induced by kainate. Finally, low concentrations of l-Asp were potentiated by d-Asp, but higher concentrations of l-Asp (3–10 mM) were clearly inhibited by this agent. This biphasic effect of d-Asp on l-Asp-induced release of [³H]GABA is a possible explanation for previously conflicting reports of d-Asp's effect on l-Asp action^2,8,29. Our data suggest that d-Asp has both pre- and postsynaptic sites of action.     

Voltage clamp analysis of the effect of excitatory amino acids and derivatives on Purkinje cell dendrites in rat cerebellar slices maintained in vitro

A voltage clamp analysis of the effects ofl-aspartate,l-glutamate and related derivatives on Purkinje cell dendrites was performed in rat cerebellar slices maintained in vitro. Short iontophoretic pulse applications ofl-aspartate andl-glutamate in the dendritic field of Purkinje cells induced dose-dependent inward currents with fast onset and recovery. Quisqualate application also gave rise to well developed inward currents with fast onset and slow recovery, whereas N-methyl-d,l-aspartate had no or little effect on Purkinje cell membranes unless prolonged (several seconds) applications were used. Steady applications of low doses of N-methyl-d,l-aspartate much more severely depressedl-aspartate thanl-glutamate mediated responses, whereas inward currents due to quisqualate were unaffected. Inward currents due to quisqualate were often more reduced than those due tol-aspartate by steady applications of 2-amino-5-phosphonovalerate, and the antagonistic action of this drug on responses due tol-glutamate was very weak. These results suggest that receptors of Purkinje cells for glutamate and aspartate are different, and are also different from N-methyl-d-aspartate and quisqualate receptors.     

Presynaptic kainate and N-methyl-d-aspartate receptors regulate excitatory amino acid release in the olfactory cortex

The potassium-evoked release of endogenous aspartate, glutamate and GABA from olfactory cortex slices has been monitored. Release of aspartate alone is significantly reduced by N-methyl-d-aspartate (NMDA) whilst kainate significantly increases release of both aspartate and glutamate. These effects, which are blocked by appropriate receptor antagonists, suggest that presynaptic NMDA and kainate receptors regulate excitatory amino acid release in the olfactory cortex.     

Electroconvulsive shock blocks the opioid-mediated inhibition of dopamine release in rat striatal slices

Summary The fractional release technique was applied to investigate the effects of single electroshock (ECS) on the opioid-mediated inhibition of dopamine release in rat striatal slices. Animals were submitted to ECS 24h before the experiments. The results demonstrate that pre-treatment with ECS suppresses the inhibition of dopamine release mediated by opioid receptors. These data suggest that single ECS treatment modifies the sensitivity of the opioid receptors located on the presynatic dopamine terminals in the rat striatum.     

The distribution of [H]kainic acid binding sites in rat CNS as determined by autoradiography

The distribution of [³H]kainic acid (KA) binding sites in the rat CNS was determined by in vitro autoradiography. KA sites are distributed throughout the CNS gray matter in an anatomically specific pattern with telencephalic structures and the cerebellum accounting for the majority of the binding. These results, together with our previous finding that KA sites are greatly enriched at the synapse, suggest that KA binding sites are associated with select terminal fields, and hence may be involved in neurotransmission in certain CNS pathways.     

The distribution of [3H]kainic acid binding sites in rat CNS as determined by autoradiography

The distribution of [3H]kainic acid (KA) binding sites in the rat CNS was determined by in vitro autoradiography. KA sites are distributed throughout the CNS gray matter in an anatomically specific pattern with telencephalic structures and the cerebellum accounting for the majority of the binding. These results, together with our previous finding that KA sites are greatly enriched at the synapse, suggest that KA binding sites are associated with select terminal fields, and hence may be involved in neurotransmission in certain CNS pathways.     

Uptake and release of neurotransmitter candidates, [3H]serotonin, [3H]glutamate,and [3H]γ-aminobutyric acid,in taste buds of the mudpuppy,Necturus maculosus

Neurotransmitters in vertebrate taste buds have not yet been identified with confidence. Serotonin, glutamate, and γ-aminobutyric acid (GABA) have been postulated, but the evidence is incomplete. We undertook an autoradiographic study of [³H]serotonin, [³H]glutamate, and [³H]GABA uptake in lingual epithelium from the amphibian, Necturus maculosus, to determine whether taste bud cells would accumulate and release these substances. Lingual epithelium containing taste buds was incubated in low concentrations (0.4–6 μM) of these tritiated transmitter candidates and the tissue was processed for light microscopic autoradiography. Merkel-like basal taste cells accumulated [³H]serotonin. When the tissue was treated with 40 mM K⁺ after incubating the tissue in [³H]serotonin, cells released the radiolabelled transmitter. Furthermore, depolarization (KCl)-induced release of [³H]serotonin was Ca-dependent: if Ca²⁺ was reduced to 0.4 mM and 20 mM Mg²⁺ added to the high K⁺ bathing solution, Merkel-like basal cells did not release [³H]serotonin. In contrast, [³H]glutamate was taken up by several cell types, including non-sensory epithelial cells, Schwann cells, and some taste bud cells. [³H]glutamate was not released by depolarizing the tissue with 40 mM K⁺. [³H]GABA uptake was also widespread, but did not occur in taste bud cells. [³H]GABA accumulated in non-sensory epithelial cells and Schwann cells. These data support the hypothesis that serotonin is a neurotransmitter or neuromodulator released by Merkel-like basal cells in Necturus taste buds. The data do not support (nor rule out) a neurotransmitter role for glutamate or GABA in taste buds. J. Comp. Neurol. 392:199–208, 1998. © 1998 Wiley-Liss, Inc.     

β-Amyloid peptide fragment 25–35 potentiates the calcium-dependent release of excitatory amino acids from depolarized hippocampal slices

β-Amyloid protein (βAP) has been frequently associated with the neuropathology of Alzheimer's disease (AD), although the mechanisms by which it can induce neurodegeneration are still unknown. Some studies in hippocampal cultured neurons suggest that βAP, particularly its fragment 25ndash;35, may induce neural growth or render neurons more vulnerable to excitotoxic insults by a mechanism involving intracellular Ca²⁺ dyshomeostasis. We have studied the effect of fragment 25–35 on the release of endogenous amino acids from hippocampal slices of young adult (3–3.5-month-old) and aged (23–25-month-old) rats, under basal, K⁺ -depolarization, and post-depolarization conditions, in the presence and absence of Cat²⁺. In both young and aged tissue, the basal release of amino acids was not affected by the peptide. By contrast, 1-hr preincubation of slices from young animals with 10 μM 25–35 fragment resulted in a 140% increase of glutamate and aspartate release stimulated by K⁺ depolarization, compared with the control-stimulated release. These effects were strictly dependent on external Ca²⁺ Neither the K⁺ -stimulated release of γ-amino butyric acid (GABA) nor the release of glycine, glutamine, taurine, or alanine, which was not stimulated by high K⁺, were affected. Substance P and a scrambled sequence of the 25–35 fragment were without any effect per se, but substance P blocked the stimulatory effect of fragment 25–35 on glutamate and aspartate release. In slices from aged rats the basal release of glutamate was significantly higher (260%) than that in young tissue, and the K⁺ -induced release of both aspartate and glutamate was also higher. Fragment 25–35 also potentiated the K⁺ -induced release of these two amino acids, although to a lesser extent than in young tissue. These results indicate that glutamate is retained less by the aged hippocampus and that fragment 25–35 is able to augment the release of glutamate and aspartate under excitatory conditions, an effect that could be involved in the mechanisms of neurotoxicity of β-amyloid peptides. © 1995 Wiley-Liss, Inc.     

Development of excitatory amino acid induced cytotoxicity in cultured neurons

The neurotoxicity of the excitatory amino acids (EAAs) L-glutamate (L-glu), L-aspartate (L-asp), N-methyl-D-aspartate (NMDA), kainate (KA), quisqualate (QA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA) was followed as a function of development in primary cultures of cerebral cortex neurons and cerebellar granule cells. These two types of neurons express, respectively, glutamate receptor subtypes with sensitivity to all of these excitatory amino acids or only to glutamate and aspartate. None of the EAAs were toxic in cerebral cortex neurons at 2 days in culture, whereas at culture day 4 the neurons became sensitive to glutamate, at day 5 to KA followed by sensitivity to QA at day 6, and finally to NMDA, L-asp and AMPA at day 7. The rank order of potency of the EAAs was in cerebral cortex neurons cultured for 12 days: L-asp (ED50 = 0.5 microM) = L-glu (ED50 = 1 microM) greater than AMPA (ED50 = 10 microM) greater than NMDA (ED50 = 65 microM) greater than QA = KA (ED50 = 100 microM). Cerebellar granule cells were insensitive to all of the EAAs at 3 and 5 days in culture but at day 8 the cells became sensitive to toxicity induced by L-glu (ED50 = 70 microM) and L-asp (ED50 = 30 microM). In order to determine ED50 values for L-asp and L-glu accurately, media in these experiments also contained 500 microM of the glutamate uptake inhibitor L-aspartate-beta-hydroxamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of [H]L-glutamate to cerebellar membranes: Evidence for recognition site heterogeneity

The kinetics of interaction of excitatory amino acid analogues and reputed antagonists with a specific binding site for [3H]L-glutamate, having a Kd of 600 nM, were examined in washed cerebellar membranes incubated at 37 degrees C. Displacement curves were analyzed by an iterative computer program for a non-cooperative two-site competitive inhibition model. L-Glutamate, D-glutamate and D-aspartate exhibited simple, mass action kinetics with Hill coefficients near unity and Kis of 1.1, 9.3 and 23.3 microM, respectively. Quisqualate, ibotenate and cyclopentylglutamate had Hill coefficients less than 0.85 and bound to an high affinity component with KHS of 0.4, 0.8 and 1.7 microM, respectively. Neither N-methyl-D-aspartate nor derivatives of kainate, with the exception of a-keto kainate, had KiS less than 0.1 mM. Linear analogues of glutamate with reputed antagonistic properties all exhibited shallow displacement curves with Hill coefficients less than 0.6 and KHS varying from 0.5 to 6.6 microM. Notably, 2-amino-6-phosphonocaproic acid had negligible affinity for the site in contrast to the valeric and pimelic phosphono analogues. The results indicate that [3H]L-glutamate labels a single class of sites that can be resolved into subpopulations by agonists and antagonists and provide additional evidence of excitatory amino acid receptor heterogeneity.