Effects of 2-Deoxy-d-Glucose on Methamphetamine-Induced Dopamine and Serotonin Neurotoxicity

Abstract: The mechanisms underlying the neurotoxic actions of methamphetamine (METH) and related substituted amphetamines are unknown. Previous studies with 2-deoxyglucose (2-DG) have suggested that METH-induced neurotoxicity may involve exhaustion of intracellular energy stores. However, because 2-DG also produces hypothermic effects, and because METH's neurotoxic actions are highly susceptible to thermoregulatory influence, previous findings with 2-DG are difficult to interpret. The present studies were undertaken to further examine the influence of 2-DG's glucoprivic and thermic effects in the context of METH-induced dopamine (DA) and serotonin (5-HT) neurotoxicity. 2-DG protected against METH-induced DA neurotoxicity in both rats and mice. In both species, 2-DG, alone or in combination with METH, produced hypothermic effects. METH's toxic effects on brain 5-HT neurons were either unaffected or exacerbated by 2-DG, depending on species, brain region, and dose of METH tested. These results indicate that different mechanisms may underlie METH-induced DA and 5-HT neurotoxicity, and suggest that, as compared with 5-HT neurons, DA neurons are more susceptible to temperature influence, whereas 5-HT neurons are more vulnerable than DA neurons to metabolic compromise. Additional studies are needed to further assess the role of energy stores in the neurotoxic effects of METH and related drugs.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.     

Effect of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) on monoamine neurotransmitters in mouse brain & heart

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied on dopamine (DA), norepinephrine (NE), serotonin (5HT) and γ-aminobutyric acid (GABA) neurons in mouse brain and on NE neurons of mouse heart. MPTP (45 mg/kg) was administered s.c. to mice twice daily for 2 consecutive days. This dosage regimen produced a decrease in the forebrain concentrations of DA and NE at 7 and 20 days after injection. In contrast, the forebrain concentrations of 5HT and GABA were not significantly decreased at either time. MPTP administration also produced a marked decrease in the uptake of ³H-DA into striatal slices and ³H-NE into cerebral cortical slices. In contrast, the uptake of ³H-NE into hypothalamic slices and the uptake of ³H-5HT into slices from several brain regions were not altered. MPTP initially reduced the concentration of NE in the heart, but unlike the persistent decreases in the forebrain concentrations of NE and DA, the NE concentration in the heart returned to control levels at approximately 20 days after MPTP administration. These results, showing that MPTP can produce a long lasting and selective decrease in the forebrain concentrations of NE and DA and in the uptake of radioactive DA and NE into brain slices, suggest that MPTP can cause the destruction of catecholamine neurons in mouse brain. In contrast, MPTP administration does not appear to produce long term changes in either 5HT or GABA neurons.     

Assessment of blood platelets as a model for CNS response: comparative effects of caffeine on 5-HT uptake and release mechanisms in rat platelets and rat brain serotonin neurons

We have previously reported that caffeine significantly enhanced 5-HT uptake and reduced 5-HT release from crude synaptosomal fractions obtained from rat cerebral cortex and from midbrain raphe region. Blood platelets, as reported by many laboratories and also demonstrated in our own labs, have a very active mechanism for 5-HT uptake and storage. In this regard platelets bear a high degree of similarity to brain serotonin neurons. The present experiments were, therefore, carried out to investigate the effects of caffeine on 5-HT uptake and release from rat platelets in an attempt to assess the possibility of using platelets as a model for studying the CNS effects of caffeine. Platelet rich plasma was prepared from the trunk blood of decapitated rats. Effects of caffeine were investigated at 10(-7), 10(-6), 10(-5) and 10(-4)M, on both the high affinity 3H-5-HT uptake and the spontaneous 5-HT release from 3H-5-HT preloaded platelets. The results show that caffeine did not change 5-HT uptake into platelets. In brain synaptosomes the same concentration of caffeine, however, increased 5-HT uptake dose-dependently. The results also revealed that caffeine increased 5-HT release from rat platelets in a concentration-dependent manner. The concentrations 10(-6), 10(-5), and 10(-4)M increased release significantly compared to control. This finding is also in contrast to that observed in synaptosomes of brain serotonin neurons where caffeine decreased 5-HT release. It is concluded, therefore, that the rat blood platelet is not a suitable model for studying these CNS actions of caffeine. Furthermore, our observations imply that rat platelet serotonin uptake and release mechanisms are not identical to those mechanisms in brain serotonin neurons.     

Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

DETECTION OF A SOLUBLE SEROTONIN-BINDING PROTEIN IN THE MAMMALIAN MYENTERIC PLEXUS AND OTHER PERIPHERAL SITES OF SEROTONIN STORAGE

Groups of neurons intrinsic to the mammalian myenteric plexus have been shown to have both tryptophan hydroxylase and a specific uptake mechanism for serotonin. They are probably serotonergic. A soluble protein with a high binding affinity for serotonin, similar to a protein previously found in rat brain by TAMIR & HUANG (1974), has now been found in the myenteric plexus of both rabbit and guinea pig. Partial purification of the protein from the rabbit's myenteric plexus by ammonium sulfate fractionation increased the ratio of specific to nonspecific serotonin binding almost 3-fold. Two dissociation constants for serotonin binding were obtained by equilibrium dialysis: 6.7 × 10^?10 M and 4.8 × 10^?7 M. The protein was similar to the soluble serotonin-binding protein of CNS: the indole derivatives 5, 6- and 5, 7-dihydroxytryptamine, and 6-hydroxytryptamine inhibited serotonin binding by 50% at 10^?7 M; norepinephrine was a poor inhibitor of serotonin binding; most of the serotonin-protein complex had a very high molecular weight and did not penetrate a 6.5% acrylamide gel. The appearance of the serotonin binding protein during development of the intestine in fetal rabbit correlates closely with the development of a serotonin uptake mechanism by nerves of this tissue and precedes the ingrowth of the adrenergic innervation. In-vitro administration of 6-hydroxydopamine to adult animals has no effect on the binding capacity for serotonin. Binding activity in denervated preparations is only 1/5 that of innervated tissue. It is concluded that the serotonin-binding protein, which has been found associated with serotonergic pathways in the CNS, is found associated with serotonergic neurons in the periphery as well. Since a similar serotonin-binding protein is also found in sheep thyroid, which stores but does not take up serotonin, the protein may be a component of the serotonin storage mechanism.     

Regional Differences in 5-Hydroxytryptamine and Catecholamine Uptake in Primary Astrocyte Cultures

The uptake of 3H-labelled 5-hydroxytryptamine (5-HT, serotonin) norepinephrine ([3H]NE), and 3,4-dihydroxyphenylethylamine ([ 3H]dopamine, [3H]DA) was studied in primary astrocyte cultures prepared from the cerebral cortex, corpus striatum, and hippocampal regions of neonatal rat brain. Na+-dependent uptake showed marked regional differences. For [3H]5-HT the magnitude of uptake was corpus striatum greater than or equal to cerebral cortex greater than hippocampus, whereas for [3H]NE the order was hippocampus greater than corpus striatum greater than cerebral cortex. For [3H]DA, only the hippocampal cultures showed significant Na+-dependent uptake. [3H]5-HT uptake was specifically inhibited by 10(-7) M fluoxetine whereas [3H]NE uptake was preferentially inhibited by 10(-7) M desipramine. These results may reflect regional brain specialization and/or different developmental patterns of high affinity uptake of serotonin and catecholamines by astrocytes in situ.     

5-Hydroxytryptophyl peptides: potent inhibitors of a storage compartment of serotonin.

Several analogues of 5-hydroxytryptophan were tested for their ability to inhibit the binding of serotonin to serotonin-binding protein (SBP), a protein found within serotonergic neurons which has a high affinity for serotonin. An N-substituted dipeptide, N-acetyl-5-hydroxytryptophan-5-hydroxytryptophan amide, was found to be an inhibitor of this binding. The inhibition (50% at 1.0 μM) was specific, since it did not affect other known sites of serotonin binding. The binding of serotonin to its membrane receptor was not affected by the dipeptide (up to 10 μM). Uptake of serotonin by synaptosomes was only slightly affected (9% at 10 μM), and aromatic-L-amino-acid carboxy-lyase(EC 4.1.1.28) and amine: oxygen oxidoreductase (deaminating) (flavin-containing) (EC 1.4.3.4) were not inhibited (10 μM and 5 mM respectively), The peptide was not hydrolyzed by honiogenates of brain or myenteric plexus. The ¹⁴C-labelled dipeptide was shown to be taken up by synaptosomes. However, the uptake of the peptide was not affected either by drugs that inhibit serotonin uptake or by serotonin itself although the uptake was abolished by excess 5-hydroxytryptophan. Intraventricular injection of N-acetyl dipeptide caused a biphasic effect depending on dose. Lower doses (10nmol) induced a decrease in serotonin brain levels (40%). Higher doses (300 nmol) caused a 95% increase in serotonin levels. It is suggested that 5-hydroxytryptophyl peptides may be used as potent specific inhibitors of SBP, a storage compartment of serotonin.     

Dopamine Transporter Loss in 6-OHDA Parkinson’s Model Is Unmet by Parallel Reduction in Dopamine Uptake

The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [³H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [³H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [³H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [³H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [³H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.     

Characterization of the effects of serotonin on the release of [3H]dopamine from rat nucleus accumbens and striatal slices

The effect of serotonin agonists on the depolarization (K⁺)-induced, calcium-dependent, release of [³H]dopamine (DA) from rat nucleus accumbens and striatal slices was investigated. Serotonin enhanced basal³H overflow and reduced K⁺-induced release of [³H]DA from nucleus accumbens slices. The effect of serotonin on basal³H overflow was not altered by the serotonin antagonist, methysergide, or the serotonin re-uptake blocker, chlorimipramine, but was reversed by the DA re-uptake carrier inhibitors nomifensine and benztropine. With the effect on basal overflow blocked, serotonin did not modulate K⁺-induced release of [³H]DA in the nucleus accumbens or striatum. The serotonin agonists, quipazine (in the presence of nomifensine) and 5-methoxytryptamine, did not significantly affect K⁺-induced release of [³H]DA in the nucleus accumbens. This study does not support suggestions that serotonin receptors inhibit the depolarization-induced release of dopamine in the nucleus accumbens or striatum of the rat brain. The present results do not preclude the possibility that serotonin may affect the mesolimbic reward system at a site which is post-synaptic to dopaminergic terminals in the nucleus accumbens.     

Dopamine and norepinephrine uptake and metabolism by astroglial cells in culture

Primary cultures of normal astroglia started from the cerebral hemispheres of neonatal rats took up dopamine (DA) and norepinephrine (NE) in the concentration range of 10^?7 to 10^?4M and metabolized each to their respective principal central nervous system products by the actions of both catechol-0-methyl transferase and monoamine oxidase. At 10^?7M, uptake of ³H labelled DA and NE was inhibited by omission of Na⁺, addition of ouabain or lowered temperatures. Uptake at 10^?4M was considerable but was Na⁺-independent. Only Na⁺-independent uptake was seen in primary cultures started from the meninges of neonatal rats. These data suggest that astroglial cells in the CNS have a high affinity uptake system for catecholamines, and such uptake is followed by catecholamine metabolism.     

PAF antagonists: different effects on platelets, neutrophils, guinea pig ileum and PAF-induced vasolidation in isolated rat lung

The effects of structurally different PAF receptor blockers were investigated in platelets, neutrophils, guinea pig ileum, rat isolated lung and rat isolated pulmonary artery. PAF caused serotonin release from platelets and a characteristic shape change and adhesion of neutrophils. The antagonists (CV 3988, alprazolam, 48740 RP and Merck-Sharp and Dohme L-652, 731) inhibited platelet serotonin release but not neutrophil shape change adhesion or lysosomal enzyme release. The antagonists in high concentrations (10⁻⁵ −10⁻⁴M) inhibited nonspecifically the PAF-induced (10⁻⁸M) guinea pig ileum contraction, but were ineffective at concentrations which inhibited platelet responses. In the rat lung the compounds, in high concentrations, partially inhibited the low dose PAF-induced pulmonary vasodilation and the high dose PAF induced pulmonary vasoconstriction and edema. Our data indicate that some platelet PAF antagonists may be ineffective in blocking the action of PAF on neutrophils and smooth muscle preparations and suggest either PAF-receptor independent actions of PAF or different classes of PAF receptors.     

Characteristics of synaptosomal tyrosine uptake in various brain regions: Effect of other amino acids

Tyrosine uptake by rat synaptosomes was maximal after 5–10 min of incubation and at 30°C; uptake was inhibited by dinitrophenol (10^?4 M) or ATP (10^?3 M) and increased by reducing sodium concentrations or increasing calcium or potassium. The best model for uptake is a two-carrier system, in which one carrier shows high-affinity uptake and the other may be diffusional. Both uptake mechanisms are more concentrated in catecholamine-rich brain areas, and are inhibited $\text{in vitro}$ by other large, neutral amino acids. At physiologic amino acid brain concentrations, each system probably carries about half of the tyrosine into the nerve terminal.     

Biochemical and Pharmacological Characterization of [3H]GBR 12935 Binding In Vitro to Rat Striatal Membranes: Labeling of the Dopamine Uptake Complex

Abstract: Binding of the selective dopamine (DA) uptake inhibitor [³H]GBR 12935 to rat striatal membranes was characterized biochemically and pharmacologically. [³H]-GBR 12935 binding at 0°C was reversible and saturable and Scatchard analysis indicated a single binding site with a K_D of 5.5 nM and a B_max of 760 pmol/mg tissue. [³H]GBR 12935 labeled two binding sites. One binding site was identified as the classic DA uptake site, since methylphenidate, cocaine, diclofensine, and Lu 19–005 potently inhibited [³H]GBR 12935 binding to it. Binding to the second site was inhibited by high concentrations of the above compounds. IC₅₀ values for inhibition of [³H]GBR 12935 binding to the DA uptake site were proportional to IC50 values for inhibition of DA uptake. However, substrates of DA uptake, e.g., DA and 1-methyl-4-phenylpyridine, and DA releasers, e.g., the amphetamines, inhibited [³H]GBR 12935 binding less than DA uptake. Rate experiments excluded the possibility that these “weak” inhibitors affected the binding by alloste-ric coupled binding sites. The second binding site was not a noradrenergic, serotonergic, or GABAergic uptake site. Neither was it a dopaminergic, acetylcholinergic, histaminic, serotonergic, or adrenergic receptor. However, [³H]GBR 12935 was potently displaced from it by disubstituted piper-azine derivatives, i.e., flupentixol and piflutixol. DA uptake and the DA uptake binding site of [³H]GBR 12935 were located primarily in the striatum, but the piperazine acceptor site was distributed uniformly throughout the brain. Also only the DA uptake binding site was destroyed by 6-OH-DA. Thus, [³H]GBR 12935 labels the classic DA uptake site in rat striatum and also a piperazine acceptor site. Substrates for DA uptake and releasers of DA inhibited [³H]GBR 12935 binding with low potency, but did not alter the rate constants for [³H]GBR 12935 binding. Therefore inhibitors of DA uptake label the carrier site and prevent the carrier process.     

Effect of ascorbic acid on release of acetylcholine from synaptic vesicles prepared from different species of animals and release of noradrenaline from synaptic vesicles of rat brain.

Low concentrations of L-ascorbic acid caused release of acetylcholine from isolated synaptic vesicles (rat, guinea-pig and rabbit) in the presence of 2mM ATP, 2 mM MgCl₂ and 10^?5 M CaCl. The half maximum effect was obtained with about 2 to 2.5 ωM L-ascorbic acid, and the effect was inhibited by addition of 1mM EGTA. The release of noradrenaline from rat synaptic vesicles was also enhanced by L-ascorbic acid, but the concentration for half maximal stimulation was about 20 ωM, indicating that noradrenaline release was less sensitive to L-ascorbic acid than acetylcholine release. The physiological function of L-ascorbic acid in the brain is discussed in relation to release of transmitters.     

Acute effects of heroin and morphine on newly synthesized serotonin in rat brain.

Heroin and morphine, in acute intraperitoneal doses of 2 and 10 mg/kg respectively, produced significant increments in the formation of newly formed brain serotonin from tritiated (³H)-L-tryptophan to ³H-serotonin. Opiate analgesia, Straub tail sign and catatonia, were observed during the increase in the synthesis of serotonin. The transport of radio-labelled tryptophan into the rat brain was not increased by the acute injection of the opiates, but brain levels of ³H-serotonin and of its main metabolite, 5-hydroxyindoleacetic acid, were significantly elevated. These opiates do not interfere with the accumulation of serotonin or with the transport of its metabolite in serotonergic neurons after inhibition of monoamine oxidases with Pargyline. An increase in the activity of tryptophan hydroxylases was more pronounced in the forebrain than in the brain stem. Stimulation of newly synthesized serotonin is probably mediated by an increase in tryptophan hydroxylase activity and not by an increase in the transport of tryptophan into the brain.     

Effects of cadmium and mercury on Na(+)-K+, ATPase and uptake of 3H-dopamine in rat brain synaptosomes.

Effects in vivo of cadmium (Cd), mercury (Hg) and methylmercury (CH3Hg) on Na(+)-K+ ATPase and uptake of 3H-dopamine (DA) in rat brain synaptosomes were studied. These heavy metals significantly inhibited the Na(+)-K+ ATPase activity in a dose-dependent manner. Similarly, inhibition of DA uptake by synaptosomes was also observed in rats treated with these metals. Intraperitoneal route of metal administration was found to be more effective than per os treatment. Mercuric compounds compared to Cd elicited a higher inhibition of Na(+)-K+ ATPase and DA uptake in rat brain synaptosomes.     

Does high affinity [3H] imipramine binding label serotonin reuptake sites in brain and platelet?

Recent studies have demonstrated the presence of high affinity binding sites for [³H] imipramine in membrane preparations derived from rat brain, human platelet, and human brain. Although initial reports concluded that there was no relationship between these binding sites and the reuptake sites for biogenic amines, subsequent studies in our laboratory suggested a close relationship between the high affinity imipramine binding site and the serotonin uptake or transport site (cf. ref. 9). To further establish whether these binding sites are associated with either platelet or neuronal uptake of serotonin, the relative potencies of a series of tricyclic antidepressants in inhibiting [³H] imipramine binding and serotonin uptake were determined under identical assay conditions. A close correlation between inhibition of serotonin uptake and [³H] imipramine binding was observed (r = 0.99, p < 0.001). In addition, electrolytic lesions of the midbrain raphe produced a decrease in [³H] imipramine binding in hypothalamic synaptosomes that paralleled the decrease in [³H] serotonin uptake. Finally incubation of synaptosomal membranes with 2,8-dinitroimipramine, an irreversible inhibitor of [³H] imipramine binding, produced a dose-dependent decrease in serotonin uptake, without altering the uptake of nonrepinephrine or dopamine. Taken together our results strongly suggest that high affinity binding of [³]] imipramine selectively labels serotonin uptake sites in brain and platelet.     

Studies on the specificity of uptake and release of radiolabelled histamine in rat brain slices

Previously it has been shown that radiolabelled histamine is taken up by brain slices and may subsequently be released by depolarizing stimuli in a calcium-dependent manner, indicating the involvement of neurons in uptake and release of histamine.The present study demonstrates that after incubation of brain slices with low (nM) concentrations of [³H]histamine the amine may be taken up by (and released from) dopaminergic and serotonergic neurons (nerve terminals). Thus 6-hydroxydopamine- and 5,7-dihydroxytryptamine-induced lesions not only reduced the uptake of [³H]dopamine (in striatal slices) and [³H]serotonin (in hippocampal slices), but also, though to a lesser extent, that of [³H]histamine. Immunocytochemical findings revealed that the neurotoxins did not visibly affect histaminergic neurons. Lesioning of noradrenergic neurons appeared not to alter significantly the uptake of [³H]histamine. Further, various drugs acting on either catecholamine-, serotonin- or opioid-receptors and known to cause presynaptic inhibition of the release of [³H]dopamine or [³H]wrotonin from striatal or hippocampal slices also inhibited [³H]histamine release.It is concluded that incubation of brain slices with low concentrations of [³H]histamine does not result in a selective labelling of histaminergic neurons. The possibility that, unlike other monoamines, histamine is not subject to high-affinity uptake by the nerve terminals from which it was released, is discussed.     

Presynaptic malfunction: the neurotoxic effects of cadmium and lead on the proton gradient of synaptic vesicles and glutamate transport

Exposure to Cd²⁺ and Pb²⁺ has neurotoxic consequences for human health and may cause neurodegeneration. The study focused on the analysis of the presynaptic mechanisms underlying the neurotoxic effects of non-essential heavy metals Cd²⁺ and Pb²⁺. It was shown that the preincubation of rat brain nerve terminals with Cd²⁺ (200 μM) or Pb²⁺ (200 μM) resulted in the attenuation of synaptic vesicles acidification, which was assessed by the steady state level of the fluorescence of pH-sensitive dye acridine orange. A decrease in l-[¹⁴C]glutamate accumulation in digitonin-permeabilized synaptosomes after the addition of the metals, which reflected lowered l-[¹⁴C]glutamate accumulation by synaptic vesicles inside of synaptosomes, may be considered in the support of the above data. Using isolated rat brain synaptic vesicles, it was found that 50 μM Cd²⁺ or Pb²⁺ caused dissipation of their proton gradient, whereas the application of essential heavy metal Mn²⁺ did not do it within the range of the concentration of 50-500 μM. Thus, synaptic malfunction associated with the influence of Cd²⁺ and Pb²⁺ may result from partial dissipation of the synaptic vesicle proton gradient that leads to: (1) a decrease in stimulated exocytosis, which is associated not only with the blockage of voltage-gated Ca²⁺ channels, but also with incomplete filling of synaptic vesicles; (2) an attenuation of Na⁺-dependent glutamate uptake.