Monoamine Uptake Blockers Research Articles

Harmane: An atypical neurotransmitter?

Harmane is an active component of clonidine displacing substance and a candidate endogenous ligand for imidazoline binding sites. The neurochemistry of tritiated harmane was investigated in the present study examining its uptake and release properties in the rat brain central nervous system (CNS) in vitro. At physiological temperature, [3H]harmane was shown to be taken up in rat brain cortex. Further investigations demonstrated that treatment with monoamine uptake blockers (citalopram, nomifensine and nisoxetine) did not alter [3H]harmane uptake implicating that the route of [3H]harmane transport was distinct from the monoamine uptake systems. Furthermore, imidazoline ligands (rilmenidine, efaroxan, 2-BFI and idazoxan) showed no prominent effect on [3H]harmane uptake suggesting the lack of involvement of imidazoline binding sites. Subsequent analyses showed that disruption of the Na+ gradient using ouabain or choline chloride did not block [3H]harmane uptake suggesting a Na+-independent transport mechanism. Moreover, higher temperatures (50°C) failed to impede [3H]harmane uptake implying a non-physiological transporter. The failure of potassium to evoke the release of preloaded [3H]harmane from rat brain cortex indicates that the properties of this putative endogenous ligand for imidazoline binding sites do not resemble that of a conventional neurotransmitter.

Simultaneous monitoring of dopamine concentration at spatially different brain locations in vivo

When coupled with a microelectrode, background-subtracted fast scan cyclic voltammetry (FSCV) allows fast, sensitive and selective determination of analytes within a small spatial location. For the past 30 years experiments using this technique have been largely confined to recordings at a single microelectrode. Arrays with closely separated microelectrodes would allow researchers to gain more informative data as well as probe regions in close spatial proximity. This work presents one of the first FSCV microelectrode arrays (MEA) implemented in vivo with the ability to sample from different regions in close spatial proximity (equidistant within 1 mm). The array is manufactured from fused silica capillaries and a microfabricated electrode spacer. The functionality of the array is assessed by simultaneously monitoring electrically stimulated dopamine (DA) release in the striatum of anesthetized rat. As expected, heterogeneous dopamine release was simultaneously observed. Additionally, the pharmacological effect of raclopride (D 2 receptor antagonist) and cocaine (monoamine uptake blocker) on the heterogeneity of DA release, in spatially different brain regions was shown to alter neurotransmitter release at all four electrode sites.

Inhibitory effect of antidepressants on the NMDA-evoked [ 3H]noradrenaline release from rat hippocampal slices

We have previously shown that monoamine uptake blocker-type antidepressants with different chemical structure and selectivity are able to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) in concentrations observed during antidepressant treatment. The mechanism of action of these drugs is similar to that of mecamylamine, a channel blocker-type antagonist of nAChRs. Since mecamylamine has been shown to block also NMDA receptors, our aim was to investigate whether the monoamine uptake blockers may affect the function of these ionotropic glutamate receptors. We studied, therefore the effect of the two most potent nicotinic antagonist antidepressants, the tricyclic desipramine and the selective serotonin reuptake inhibitor fluoxetine on the NMDA-induced [ 3H]noradrenaline ([ 3H]NA) release from rat hippocampal slices. The NMDA-induced hippocampal [ 3H]NA release was effectively blocked by the selective, non-competitive NMDA antagonist MK-801 (IC 50 = 0.54 μM), indicating that the [ 3H]NA release was mediated through NMDA receptors. This response was also dose-dependently inhibited by desipramine (IC 50 = 14.57 μM) and fluoxetine (IC 50 = 41.06 μM). The Na +-channel blocker TTX equally inhibited both the electrical stimulation- and the NMDA-evoked [ 3H]NA release (the IC 50 was 55 nM and 66 nM, respectively), whereas the antidepressants inhibited only the NMDA-evoked response. These data suggest that the inhibitory effect of fluoxetine and desipramine on the NMDA-evoked [ 3H]NA release is exerted directly on NMDA receptors rather than indirectly on Na +-channels. Due to accumulation processes the concentration of desipramine and fluoxetine in the brain might be in the same range as the observed IC 50 values, thus our data indicate that monoamine uptake blocker-type antidepressants are able to influence the function of NMDA receptors during antidepressant treatment, and the inhibitory effect on NMDA receptors might contribute to the therapeutic effects of these drugs.

Melatonin reduces induction of Bax, caspase and cell death in methamphetamine‐treated human neuroblastoma SH‐SY5Y cultured cells

Several studies demonstrated that methamphetamine (MA)-treated human neuroblastoma cells exhibit increased oxidative stress, which regulates intracellular signaling cascades leading to cell death. Melatonin has a potential as a direct free radical scavenger and protects against cell death caused by MA. The objective of this study was to investigate the neuroprotective properties of melatonin on MA-induced induction of death signaling cascade and neuronal cell degeneration in human neuroblastoma SH-SY5Y cultured cells. The results of the present study demonstrate that MA significantly reduced cell viability in SH-SY5Y cultured cells. Desipramine, a monoamine uptake blocker, and melatonin reversed the toxic effect of MA in reducing cell viability. Induction of Bax, Bcl-2 and cleaved caspase-3 protein levels were observed in SH-SY5Y cultured cells treated with MA, whereas the induction of Bax and cleaved caspase-3 was diminished by melatonin. Visualization of the induction of Bax using immunofluorescence but a reduction in mitochondrial sites using red-fluorescent mitochondria-staining dye was more obviously apparent in MA-treated cells than in untreated control cells and, again, this effect was abolished by melatonin. These findings demonstrate important roles of Bax and caspase in death signaling cascade, and the protective effects of melatonin in MA-treated SH-SY5Y cells.

Melatonin reduces induction of Bax, caspase and cell death in methamphetamine-treated human neuroblastoma SH-SY5Y cultured cells

Several studies demonstrated that methamphetamine (MA)-treated human neuroblastoma cells exhibit increased oxidative stress, which regulates intracellular signaling cascades leading to cell death. Melatonin has a potential as a direct free radical scavenger and protects against cell death caused by MA. The objective of this study was to investigate the neuroprotective properties of melatonin on MA-induced induction of death signaling cascade and neuronal cell degeneration in human neuroblastoma SH-SY5Y cultured cells. The results of the present study demonstrate that MA significantly reduced cell viability in SH-SY5Y cultured cells. Desipramine, a monoamine uptake blocker, and melatonin reversed the toxic effect of MA in reducing cell viability. Induction of Bax, Bcl-2 and cleaved caspase-3 protein levels were observed in SH-SY5Y cultured cells treated with MA, whereas the induction of Bax and cleaved caspase-3 was diminished by melatonin. Visualization of the induction of Bax using immunofluorescence but a reduction in mitochondrial sites using red-fluorescent mitochondria-staining dye was more obviously apparent in MA-treated cells than in untreated control cells and, again, this effect was abolished by melatonin. These findings demonstrate important roles of Bax and caspase in death signaling cascade, and the protective effects of melatonin in MA-treated SH-SY5Y cells.

Protection against cell death and sustained tyrosine hydroxylase phosphorylation in hydrogen peroxide‐ and MPP+‐treated human neuroblastoma cells with melatonin

Neuroprotective effects of melatonin against oxidative stress-induced neuronal cell degeneration in human SH-SY5Y neuroblastoma cells were investigated in this report. The results demonstrate that exogenous administration of H(2)O(2) and 1-methyl, 4-phenyl, pyridinium ion (MPP(+)) significantly decreased cell viability in SH-SY5Y cultured cells. Desipramine, a monoamine uptake blocker was able to abolish the toxic effects of MPP(+) but not H(2)O(2) in reduction of cell viability. Conversely, melatonin reversed the toxic effects of H(2)O(2) and MPP(+) on cell viability. In addition, the reduction of phosphorylation of tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis, and phosphorylation of cyclic AMP responsive element-binding protein by H(2)O(2) and MPP(+) was also diminished by melatonin. These results demonstrate some effective roles of melatonin on neuroprotection and its action on the modulation of tyrosine hydroxylase phosphorylation.

Nicotinic acetylcholine receptor antagonistic property of the selective dopamine uptake inhibitor, GBR-12909 in rat hippocampal slices

Previously we found that inhibitors of noradrenaline (NA) and/or 5-HT reuptake are able to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) in the CNS most probably by a channel blocker-type mechanism. The aim of our study was to clarify whether selective dopamine uptake inhibitors also possess this property, therefore we investigated the effect of GBR-12909 on the nicotine-evoked release of [3H]NA from rat hippocampal slices. GBR-12909, similar to selective NA and 5-HT uptake blockers, inhibited the nicotine-evoked release with an IC50 of 2.32 μM. The ability of monoamine uptake blockers to inhibit nicotine-evoked [3H]NA release (IC50) and NA reuptake (Ki) showed no correlation, indicating that the NA uptake system is not involved in the inhibition of the response to nicotine. Previously we have shown in whole cell patch clamp experiments, that GBR-12909, depending on the stimulation pattern, inhibits Na+-currents with an IC50 in the 6–35 μM concentration range [Mike A, Karoly R, Vizi ES, Kiss JP (2003) Inhibitory effect of the DA uptake blocker GBR-12909 on sodium channels of hippocampal neurons. Neuroreport 14:1945–1949]. To study whether the inhibition of Na+-channels is involved in the action of GBR-12909 on the nicotine-evoked [3H]NA release, we compared the effect of GBR-12909 and the Na+-channel blocker tetrodotoxin (TTX) on the electrical stimulation- and nicotine-evoked response. TTX prevented the release of [3H]NA induced by both types of stimulation, whereas GBR-12909 inhibited only the nicotine-induced response, indicating that under our experimental conditions the target of GBR-12909 is not the Na+-channel. These data indicate that the selective DA uptake inhibitor GBR-12909 is able to inhibit nAChRs, that is, the nAChR antagonistic property of monoamine uptake inhibitors is independent of their selectivity. The fact that monoamine uptake inhibitors with different chemical structure and selectivity are able to inhibit nAChRs may reveal some common properties of nicotinic receptors and monoamine uptake carriers.

5-HT uptake blockade prevents the increasing effect of K ATP channel blockers on electrically evoked [ 3H]-5-HT release in rat and mouse neocortical slices

To explore if prolonged – as opposed to acute – 5-HT uptake blockade can lead to changes in the function of ATP-dependent potassium (K ATP) channels, we investigated in rat and mouse neocortical slices the effects of K ATP channel blockers on electrically evoked [ 3H]-serotonin ([ 3H]-5-HT) release after short- and long-term exposure to 5-HT uptake blockers. Glibenclamide (1 μM), a K ATP channel blocker, enhanced the electrically evoked [ 3H]-5-HT release by 66 and by 77%, respectively, in rat and in mouse neocortex slices. This effect was confirmed in the rat by tolbutamide (1 μM), another K ATP channel antagonist. After short-term blockade (45 min) of 5-HT uptake, glibenclamide still increased the release of [ 3H]-5-HT in the rat. Glibenclamide, however, failed to enhance [ 3H]-5-HT release after long-term uptake blockade (210 min). In the mouse, however, both short- and long-term inhibition of 5-HT reuptake by citalopram (1 μM) prevented the facilitatory effect of glibenclamide. The Na +/K +-ATPase inhibitor ouabain (3.2 μM) abolished the glibenclamide-induced increase in [ 3H]-5-HT release in both rat and mouse, suggesting that an operative Na +/K +-ATPase is a prerequisite for activation of K ATP channels. The terminal 5-HT 1B autoreceptor-mediated feedback control was involved in the glibenclamide-induced increase in [ 3H]-5-HT release only in mouse neocortical tissue, as evident from the use of the 5-HT 1B autoreceptor ligands metitepin (1 μM) and cyanopindolol (1 μM). These results suggest that in the rat long-term uptake blockade leads to an impaired activity of the Na +/K +-ATPase, which increases intracellular ATP and consequently closes K ATP channels. In the mouse, however, short-term uptake blockade seems to already reduce the activity of the Na +/K +-ATPase and thereby the consumption of ATP. Blockade of 5-HT transporters thus may close K ATP channels through increased intracellular ATP. The following slight depolarisation seems to facilitate 5-HT release. These results may contribute to a better understanding of the mechanisms involved in the clinical time latency of antidepressant efficacy of monoamine uptake blockers.

Monoamine neurotoxins-induced apoptosis in lymphocytes by a common oxidative stress mechanism: involvement of hydrogen peroxide (H 2O 2), caspase-3, and nuclear factor kappa-B (NF-κB), p53, c-Jun transcription factors

The destruction of dopaminergic and serotonergic nerve cells by selective 6-hydroxydopamine (6-OHDA), 5,6-dihydroxytryptamine (5,6-DHT) and 5,7-dihydroxytryptamine (5,7-DHT), respectively, is a commonly used tool to investigate the mapping of neuronal pathways, elucidation of function and to mimic human neurodegenerative disease such as Parkinson’s and Alzheimer’s diseases. Despite intense investigations, a complete picture of the precise molecular cascade leading to cell death in a single cellular model is still lacking. In this study, we provide evidence that 6-OHDA, 5,6- and 5,7-DHT toxins-induced apoptosis in peripheral blood lymphocytes cells in a concentration-dependent fashion by a common oxidative mechanism involving: (1) the oxidation of toxins into quinones and production of the by-product hydrogen peroxide, reflected by desipramine—a monoamine uptake blocker—and antioxidants inhibition, (2) activation and/or translocation of nuclear factor-κB, p53 and c-Jun transcription factors, showed by immunocytochemical diaminobenzidine-positive stained nuclei, (3) caspase-3 activation, reflected by caspase Ac-DEVD-CHO inhibition, (4) mRNA and protein synthesis de novo according to cycloheximide and actinomycin D cell death inhibition. These results are consistent with the notion that uptake and intracellular autoxidation of those toxins precede the apoptotic process and that once H 2O 2 is generated, it is able to trigger a specific cell death signalisation. Thus, taken together these results, we present an ordered cascade of the major molecular events leading peripheral blood lymphocytes to apoptosis. These results may contribute to explain the importance of H 2O 2 as a second messenger of death signal in some degenerative diseases linked to oxidative stress stimuli.

Cocaine or Selective Block of Dopamine Transporters Influences Multisecond Oscillations in Firing Rate in the Globus Pallidus

Previous studies have shown that direct-acting dopamine agonists modulate the multisecond oscillations which are present in globus pallidus spike trains in vivo in awake rats. To investigate possible modulation by endogenous dopamine and by other monoamines, and by drugs with abuse potential, cocaine or selective monoamine uptake blockers were injected systemically during extracellular recording of single globus pallidus neurons and the results analyzed with spectral and wavelet methods. Both cocaine and the selective dopamine uptake blocker GBR-12909 significantly shortened the period of multisecond oscillations, as well as increasing overall firing rate. Cocaine effects were blocked by dopamine antagonist pretreatment, as well as by N-methyl-d-aspartate receptor antagonist (MK-801) pretreatment. Desipramine and fluoxetine (blockers of norepinephrine and serotonin uptake, respectively) had no significant effects on multisecond oscillations. The results suggest that dopamine has a primary role among monoamines in modulating multisecond oscillations in globus pallidus activity, and that tonic dopaminergic and glutamatergic transmission is necessary for normal slow oscillatory function.

Reinforcing and discriminative stimulus effects of RTI 111, a 3-phenyltropane analog, in rhesus monkeys: interaction with methamphetamine.

The neuronal actions of methamphetamine (MA) include an increase in extracellular levels of monoamines, presumably via reverse transport involving the monoamine transporters. This action is thought to play an important role in the effects of MA. Therefore, in the present experiment, it was hypothesized that a monoamine uptake blocker would block behavioral effects of MA related to its abuse. RTI 111, a newly synthesized 3-phenyltropane analog with high affinity for the dopamine, norepinephrine, and serotonin transporters, was evaluated alone and in combination with MA for its ability to block the reinforcing and discriminative stimulus effects of MA in rhesus monkeys. RTI 111 (0.0003-0.03 mg/kg, i.v.) was made available to four rhesus monkeys for self-administration under a fixed-ratio 25 (FR 25) schedule of reinforcement. RTI 111 (0.01-0.1 mg/kg, i.m.) was also administered as a pretreatment (15 min prior) to four monkeys self-administering MA (0.0-0.3 mg/kg per injection, i.v.) on a progressive-ratio schedule of reinforcement. MA (0.01-1.0 mg/kg, i.m.), RTI 111 (0.001-0.1 mg/kg, i.m.), or the combination of MA and RTI 111 were administered to four monkeys trained to discriminate (+)-amphetamine (AMPH; 1.0 or 1.7 mg/kg, intragastric) from saline. When RTI 111 was made available for self-administration under an FR 25 schedule it functioned as a positive reinforcer in all four monkeys tested. When RTI 111 was given as a pretreatment to monkeys self-administering MA under a progressive-ratio schedule, the MA dose-response function shifted to the left and down. When RTI 111 or MA were given to monkeys trained to discriminate AMPH from saline, full AMPH-like responding was observed for both drugs. Given in combination, RTI 111 shifted the MA dose-response function to the left. These data suggest that RTI 111 is behaviorally similar to traditional psychomotor stimulants that act at the DA transporter and that it increases, rather than blocks, the behavioral potency of MA.

A comparison of the patterns of striatal Fos-like immunoreactivity induced by various dopamine agonists in rats

In contrast to the highly patchy patterns of Fos-like immunoreactivity seen in the rostral striatum after administration of a number of dopamine agonists, the monoamine uptake blocker cocaine has been reported to produce a relatively homogenous pattern of gene expression. In the current study we extended these observations by using a quantitative technique to demonstrate that while amphetamine and apomorphine produce patchy striatal Fos expression, the selective dopamine uptake inhibitors amfonelic acid, nomifensine and GBR-12909 all, like cocaine, produce near random patterns of gene expression. These findings suggest that the production of relatively ‘non-patchy’ patterns of immediate early gene expression may be a general property of dopamine transport inhibitors unrelated to any unique pharmacological properties of cocaine.

Nicotinic acetylcholine receptor antagonistic activity of monoamine uptake blockers in rat hippocampal slices.

The aim of our study was to investigate the effect of different monoamine uptake blockers on the nicotine-evoked release of [3H]noradrenaline ([3H]NA) from rat hippocampal slices. We found that desipramine (DMI), nisoxetine, cocaine, citalopram, and nomifensine inhibit the nicotine-evoked release of [3H]NA with an IC50 of 0.36, 0.59, 0.81, 0.93, and 1.84 microM, respectively. These IC50 values showed no correlation with the inhibitory effect (Ki) of monoamine uptake blockers on the neuronal NA transporter (r = 0.17, slope = 0.02), indicating that the NA uptake system is not involved in the process. In whole-cell patch clamp experiments neither drug blocked Na+ currents at 1 microM in sympathetic neurons from rat superior cervical ganglia, and only DMI produced a pronounced inhibition (52% decrease) at 10 microM. Comparison of the effect of DMI and tetrodotoxin (TTX) on the electrical stimulation- and nicotine-evoked release of [3H]NA showed that DMI, in contrast to TTX, inhibits only the nicotine-induced response, indicating that the target of DMI is not the Na+ channel. Our data suggest that monoamine uptake blockers with different chemical structure and selectivity are able to inhibit the nicotinic acetylcholine receptors in the CNS. Because these compounds are widely used in the therapy of depressed patients, our findings may have great importance in the evaluation of their clinical effects.

Endogenous catecholamine synthesis, metabolism, storage, and uptake in human peripheral blood mononuclear cells

Evidence has been obtained that peripheral blood mononuclear cells contain dopamine, norepinephrine, epinephrine, and their metabolites. Pharmacologic inhibition of tyrosine hydroxylase or monoamine oxidase profoundly affected intracellular catecholamines (CTs) and their metabolites, indicating that these cells are able to synthesize and breakdown CTs. The sensitivity of intracellular CTs to reserpine and the presence of CTs in the extracellular medium suggest that CTs are stored and released. Moreover, the increase of extracellular CTs in the presence of monoamine uptake blockers point to the presence of functional uptake mechanisms. Altogether, these results indicate the existence of a CT lifecycle in human mononuclear cells and warrant further studies to investigate the role of adrenergic autoregulatory mechanisms in modulation of the immune response and in the pathogenesis of diseases involving the immune system.

Cocaine and Selective Monoamine Uptake Blockers (Sertraline, Nisoxetine, and GBR 12935) Prevent the d-Fenfluramine-Induced Head-Twitch Response in Mice

Serotonin release subsequent to 5-HT precursor loading mainly occurs via exocytosis. Acute cocaine or sertraline administration promote the ability of 5-HT precursors (e.g. l-tryptophan) to induce the 5-HT 2A receptor-mediated head-twitch response (HTR) in rodents. The 5-HT releaser, d-fenfluramine, at behaviorally active doses, can induce the head-twitch response in rodents by releasing cytoplasmic 5-HT via the serotonin uptake carrier working in reverse. The purpose of the present study was to utilize the d-fenfluramine–induced HTR to determine the serotonergic and nonserotonergic components of cocaine’s actions on the d-fenfluramine–sensitive pool of cytoplasmic 5-HT. Because a dramatic differential potentiation in HTR frequency is obtained when cocaine is administered prior relative to after l-tryptophan injection, the effects of varying doses of cocaine and the selective serotonin (sertraline), dopamine (DA) (GBR 12935), and norepinephrine (NE) (nisoxetine) uptake blockers were investigated on the d-fenfluramine–induced behavior in two experimental protocols. Thus, each uptake inhibitor was administered either 10 min following (protocol 1) or 10 min prior to (protocol 2) d-fenfluramine injection. All the tested uptake inhibitors attenuated the d-fenfluramine–induced HTR in a dose-dependent manner in both experimental protocols. However, their order of potency in either protocol 1 (nisoxetine > GBR 12935 > cocaine > sertraline) or protocol 2 (cocaine > GBR 12935 > nisoxetine = sertraline) does not agree with in vitro affinity of these drugs for the 5-HT transporter. In addition, the potency order for cocaine and nisoxetine in protocol 1 was significantly reversed in protocol 2. The inhibitory effects of the cited drugs on the d-fenfluramine–induced HTR are discussed in terms of: 1) high doses of selective monoamine uptake blockers may not exhibit as much selectivity for their target uptake sites as indicated by in vitro tests; and 2) possible pharmacokinetic interactions between d-fenfluramine and the monoamine uptake blockers.

49] In Vivo microdialysis for measurement of extracellular monoamine levels following inhibition of monoamine transporters

Publisher Summary This chapter discusses in vivo microdialysis for measurement of extracellular monoamine levels following the inhibition of monoamine transporters. Intracerebral microdialysis is a technique to sample endogenous substances from and to introduce exogenous substances into the brain extracellular fluid by implanting a dialysis fiber in discrete brain areas. Once removed by dialysis from the brain, the endogenous substance is analyzed with various analytic techniques. Various monoamine uptake blockers, which are either selective or nonselective according to the in vitro studies, have been used as tools to explore the in vivo relationship between monoamine transporter function and monoamine neurotransmission. The chapter explains the use of microdialysis to identify the monoamine profile arising from inhibition of monoamine transporters in the ventral tegmental area (VTA)-nucleus accumbens (NAC) DA pathway.

Antidepressants and seizures: Clinical anecdotes overshadow neuroscience

Pharmacological treatment of depression in persons with epilepsy has been an area of controversy because some drugs commonly are perceived specifically to induce or exacerbate seizures in patients with seizure disorders. This prevailing misconception is unjustified by scientific studies, yet it continues to prevent afflicted persons from receiving appropriate therapy. The scientific literature shows that tricyclic antidepressant drugs cause seizures in overdose in both animals and humans. In lower doses, these drugs have anticonvulsant activity in humans and animals. Thus, the antidepressant drugs are like several antiepileptic drugs that can both prevent and cause seizures. The anticonvulsant activity of antidepressant drugs has been studied extensively in animals and almost certainly stems from their capacity to block norepinephrine and/or serotonin reuptake. The pharmacodynamic action responsible for their convulsant effects has not been well studied but may be due to their local anesthetic, antihistaminic, or antimuscarinic activity. The newer, more selective monoamine uptake blockers have very low convulsant liability, and it is suggested that their anticonvulsant activity, which is well documented in animals, be investigated further in humans. If their effects in humans are analogous to those in animals, these drugs can be used safely in epileptic patients with depression, and it is possible that their anticonvulsant activity can be exploited for use in the treatment of epilepsy.

Differential potentiation of L-tryptophan-induced head-twitch response in mice by cocaine and sertraline

Using selective monoamine uptake blockers and appropriate selective monoamine receptor antagonists, we have previously shown that cocaine enhances the frequency of 5-HT 2A receptor-mediated 5-hydroxytryptophan (5-HTP)-induced head-twitch response (HTR) in mice via inhibition of serotonin uptake. Concomitantly, cocaine prevented the maximal producible HTR frequency via simultaneous indirect stimulation of the inhibitory presynaptic 5-HT 1A and postsynaptic α 2 receptors. In the present study, we have investigated the effects of cocaine and the selective 5-HT (sertraline), norepinephrine (nisoxetine) and dopamine (GBR 12935) uptake inhibitors on the L-tryptophan-induced HTR in the presence of a nonselective monoamine oxidase inhibitor, tranylcypromine. We utilized two experimental protocols where cocaine or sertraline were administered either after (protocol 1) or prior to (protocol 2) L-tryptophan injection. Cocaine potentiated the ability of L-tryptophan to induce HTR to a greater extent in protocol 1, whereas sertraline induced a greater effect in protocol 2. However, in our earlier study cocaine (and also sertraline) up to 10 mg/kg produced a similar degree of potentiation in both experimental protocols on the 5-HTPinduced HTR. Furthermore, as in the latter study on the 5-HTP-induced HTR, in the present investigation nisoxetine potently attenuated whereas GBR 12935 did not modulate the induced HTR. The results show that the respective serotonergic and noradrenergic effects of cocaine also operate on the L-tryptophan-induced HTR. The differential effects of cocaine and sertraline in experimental protocols 1 and 2 on the L-tryptophan- versus 5-HTP-induced HTRs suggest that cocaine has additional effects on the conversion of L-tryptophan to 5-HT.

Extracellular Dopamine, Norepinephrine, and Serotonin in the Nucleus Accumbens of Freely Moving Rats During Intracerebral Dialysis with Cocaine and Other Monoamine Uptake Blockers

Monoamine-uptake blockers were applied focally (0.1-1,000 microM) through a dialysis probe in the nucleus accumbens of freely moving rats, and the extracellular concentrations of dopamine, norepinephrine, and serotonin were measured. The selective dopamine-uptake blocker GBR 12935 increased dopamine preferentially with only a small effect on norepinephrine, whereas the selective serotonin-uptake blocker fluoxetine increased serotonin output preferentially. In contrast, the selective norepinephrine-uptake blockers desipramine and nisoxetine enhanced not only norepinephrine, but also serotonin and dopamine appreciably. Cocaine increased all three amines with the greatest effects on dopamine and serotonin. As in our previous study on the ventral tegmental area, there was a positive association between dopamine and norepinephrine output when all blocker data were taken together. The present results suggest a contribution of the increase in norepinephrine, but not serotonin, to the enhancement of dopamine after cocaine applied focally in the nucleus accumbens.

Biologically Based Dose‐Response Model for Neurotoxicity Risk Assessment

The regulation of neurotoxicants has usually been based upon setting reference doses by dividing a no observed adverse effect level (NOAEL) by uncertainty factors that theoretically account for interspecies and intraspecies extraploation of experimental results in animals to humans. Recently, we have proposed a four-step alternative procedure which provides quantitative estimates of risk as a function of dose. The first step is to establish a mathematical relationship between a biological effect or biomarker and the dose of chemical administered. The second step is to determine the distribution (variability) of individual measurements of biological effects or their biomarkers about the dose response curve. The third step is to define an adverse or abnormal level of a biological effect or biomarker in an untreated population. The fourth and final step is to combine the information from the first three steps to estimate the risk (proportion of individuals exceeding on adverse or abnormal level of a biological effect or biomarker) as a function of dose. The primary purpose of this report is to enhance the certainty of the first step of this procedure by improving our understanding of the relationship between a biomarker and dose of administered chemical. Several factors which need to be considered include: 1) the pharmacokinetics of the parent chemical, 2) the target tissue concentrations of the parent chemical or its bioactivated proximate toxicant, 3) the uptake kinetics of the parent chemical or metabolite into the target cell(s) and/or membrane interactions, and 4) the interaction of the chemical or metabolite with presumed receptor site(s). Because these theoretical factors each contain a saturable step due to definitive amounts of required enzyme, reuptake or receptor site(s), a nonlinear, saturable dose-response curve would be predicted. In order to exemplify this process, effects of the neurotoxicant, methlenedioxymethamphetamine (MDMA), were reviewed and analyzed. Our results and those of others indicate that: 1) peak concentrations of MDMA and metabolites are ochieved in rat brain by 30 min and are negligible by 24 hr, 2) a metabolite of MDMA is probably responsible for its neurotoxic effects, and 3) pretreatment with monoamine uptake blockers prevents MDMA neurotoxicity. When data generated from rats administerde MDMA were plotted as bilolgical effect (decreases in hippocampal serotonin concentrations) versus dose, a saturation curve best described the observed relationship. These results support the hypothesis that at least one saturable step is involved in MDMA neurotoxicity. We conclude that the mathematical relationship between biological effect and dose of MDMA, the first step of our quantitative neurotoxicity risk assessment procedure, should reflect this biological model information generated from the whole of the dose-response curve.