Barbara A. Bennett

Self-administration of cocaine analogs by rats

Abstract  Rationale: A novel scheme for the synthesis of cocaine analogs from vinylcarbenoid precursors has made available compounds that have a diverse range of affinities for the DA and 5-HT transporters. These compounds were used to explore the relationship between their biochemical properties and their reinforcing effects. Objectives: The objective was to assess the reinforcing efficacy of selected cocaine analogs and compare the results with their selectivity in binding to DA and 5-HT transporters. Methods: Rats were prepared with chronically indwelling intravenous cannulae and trained to self-administer cocaine on a progressive ratio (PR) schedule. A range of doses of seven cocaine analogs were substituted for cocaine in separate groups of animals. Results: The results demonstrate a wide range of reinforcing efficacies and potencies among the seven selected drugs. Four tropane analogs (WF-11, WF-23, WF-24, WF-55) were found to support self-administration behavior on a PR schedule while three did not (WF-31, WF-54 and WF-60). The DA/5-HT selectivity ratio was found to be a better predictor of self-administration behavior than affinity at the DA transporter alone. Conclusion: These data suggest that drugs with a higher affinity for the DA versus the 5-HT transporter are more likely to be self-administered.

HIV-1 gp120-induced neurotoxicity to midbrain dopamine cultures

HIV-1-associated cognitive/motor dysfunction is a frequent neurological complication of acquired immunodeficiency syndrome (AIDS) and has been termed AIDS dementia complex (ADC). The HIV-1 envelope glycoprotein gp120 has been implicated in producing brain injury associated with ADC. The purpose of the present study was to determine if gp120-induced neurotoxicity is associated with damage to dopaminergic systems. Exposure of rat midbrain dopamine cultures to gp120 for 3 days reduced the ability of dopaminergic cells to transport this amine and also resulted in a reduction in dopamine neuron process length while it did not alter either dopamine cell number or the total number of neuronal cells. These detrimental effects of gp120 were prevented by an NMDA receptor antagonist (MK-801) or by preincubation with anti-gp120 antibody. These results suggest that dopaminergic neuronal damage may contribute to the manifestations of AIDS dementia complex.

Release of serotonin induced by 3,4-methylenedioxymethamphetamine (MDMA) and other substituted amphetamines in cultured fetal raphe neurons: further evidence for calcium-independent mechanisms of release

The substituted amphetamines 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), p-chloro-amphetamine (PCA) and fenfluramine (FEN) all exert their effects by releasing serotonin (5-HT) from presynaptic nerve terminals. In the current study, we examined the ability of these agents to induce the release of 5-HT in culture fetal raphe neurons. The data indicate that the rank order of release potencies for these agents was (+/-)PCA>(+)MDMA=(+)MDA=(+/-)FEN. Studies examining the role fo calcium in 5-HT release demonstrate that preventing calcium influx with L- and N-type calcium channel blockers inhibits potassium-stimulated release of -3H-5-HT but has no effect on release induced by the substituted amphetamines. Furthermore, omitting calcium from the extracellular media or depleting the vesicular pool of neurotransmitter with continual potassium stimulation did not affect the release of -3H-5-HT induced by these compounds. Administration of fluoxetine prior to the substituted amphetamines significantly attenuated the releasing effects of these agents, while producing no effect on potassium-stimulated release. These results are consistent with the notion that the amphetamines induce release of cytoplasmic 5-HT via the plasma membrane transporter.

Differential regulation of calcitonin gene-related peptide and substance P in cultured neonatal rat vagal sensory neurons ☆

Nodose (inferior vagal sensory) ganglia were removed from neonatal rats, enzymatically dispersed using neutral protease, and maintained on previously dispersed rat atriacytes. After 7-10 days in culture, calcitonin gene-related peptide (CGRP) was present in 1-3 times the molar amount of substance P (SP). The content of SP was doubled by the addition of nerve growth factor (NGF) whereas CGRP was significantly less increased by 50% or less. The addition of forskolin increased SP and CGRP levels in cultures with or without NGF by 60-80 percent. Phorbol ester (PMA) did not alter SP content but significantly raised CGRP content by 40% in NGF supplemented cultures (P less than 0.001). Corticosterone, 0.01-0.1 microM, reduced SP content by 30% independently of NGF but had no effect on CGRP. These studies demonstrate that SP in vagal sensory neurons is more sensitive than CGRP to the effects of NGF or corticosterone. Both peptides are up-regulated by presumed increases in intracellular cyclic AMP, while CGRP (or CGRP neurons) may be independently regulated by protein kinase C.

Methamphetamine-induced alterations in dopamine transporter function.

Repeated methamphetamine (METH) administration has been shown to produce differing neurochemical as well as behavioral effects in rats. This study was designed to examine the effects of acute and chronic METH exposure on uptake and release of [3H]dopamine (DA) in cultured midbrain dopamine neurons to determine if persistent neuronal adaptations ensue. In addition, we have assessed DA D2 receptor function to determine if chronic METH alters this receptor. Fetal midbrain cultures were exposed to METH (1, 10 microM) for 5 days and dopaminergic function examined 1 or 7 days after drug removal. The ability of METH to release [3H]DA was compared to other releasing agents as well as several potent uptake inhibitors. Chronic exposure to a release-promoting concentration of METH resulted in either no change or a reduction in [3H]DA release upon subsequent METH challenge. Pretreatment with METH was also found to cause a decrease in the Bmax for [3H]raclopride binding, suggesting that persistently elevated DA levels cause a downregulation of DA D2 receptors. Examination of transporter kinetics utilizing initial velocity of uptake revealed that METH treatment caused a significant decrease in affinity (K(m)) for the substrate (DA), while not altering the maximal velocity of uptake (Vmax). Binding studies with [125I]RTI-55 revealed that there was no alteration in either the Bmax or Kd for this ligand, suggesting that the changes induced by METH treatment are due to alterations in K(m) and not in the number of DA transport sites. The results from these studies indicate that METH treatment produces a modification in transporter function which may be associated with both the altered uptake and release of [3H]DA. These changes have broad implications for the regulation of transporter activity not only because of the relevance to pre-synaptic mechanisms controlling neurotransmission, but also to the importance of the neuronal adaptation that occurs in response to chronic METH exposure.

Similar properties of fetal and adult amine transporters in the rat brain.

A variety of drug classes, including psychomotor stimulants and antidepressants, interact with monoamine transporters in order to exert their effects. Although these transporters have been extensively characterized in the adult brain, little is known about uptake mechanisms in the fetal system. High affinity dopamine (DA) and serotonin (5-HT) uptake in the striatum and frontal cortex, respectively, were examined in rat fetuses (embryonic day 20; E-20). These results were then compared to uptake in adult rat synaptosomal preparations of the same regions. The data indicate that the fetal (E-20) uptake mechanism is sodium-dependent. Furthermore, the potency of various agents to inhibit transporter function was assessed. These drugs produced a concentration-dependent inhibition of uptake, and the resulting IC50 values were not significantly different from those obtained in the adult preparations. Our results provide evidence that the affinity of monoamine uptake inhibitors for fetal (E-20) DA and 5-HT transporters is similar to that observed with adult transporters. This observation has broad implications when considering neuronal development and in utero exposure to drugs that exert their effects through these transporters.

Long-term cocaine administration is not neurotoxic to cultured fetal mesencephalic dopamine neurons

The psychostimulants cocaine and methamphetamine produce their euphoric effects through an interaction with the mesolimbic dopamine system. Methamphetamine, unlike cocaine, has been shown to be neurotoxic to both dopaminergic and serotonergic systems. We have previously determined that a 6 day exposure to methamphetamine causes neuronal damage to tyrosine hydroxylase-immunopositive cells in our tissue culture model of the mesencephalon. Over the same exposure period, cocaine neither impaired neuronal function nor altered dopamine cell survival. To test whether a longer exposure period to cocaine would alter dopamine function, we added cocaine (100 microM) to the cultures once daily for either 8 or 11 days and examined changes in dopamine uptake, cell survival and morphology 24 h after the last administration. Cocaine did not produce any signs of neurotoxicity in the mesencephalic cultures.

Stimulant-induced alterations in dopaminergic and serotonergic function in fetal raphe neurons

Methamphetamine and its structural analogues have been demonstrated to be neurotoxic to CNS dopamine (DA) and serotonin (5-HT) neurons both in vivo and in vitro. Our laboratory has been actively characterizing mesencephalic cultures and the effects of methamphetamine exposure on neurochemical and immunochemical indices. The purpose of the present studies was to extend our findings with mesencephalic cultures and compare them with methamphetamine-induced alterations in fetal raphe cultures that contain both DA and 5-HT cells. Methamphetamine (10 and 100 microM) was added to the cultures 24 h after plating and fresh daily thereafter. The effects of chronic methamphetamine exposure on [3H]-DA and [3H]-5-HT uptake were determined after 5 days of drug treatment. Additional cultures were immunochemically analyzed for the presence of DA- and 5-HT-containing cells and total neuronal density. Results indicate that repeated methamphetamine exposure decreased DA and 5-HT uptake. Furthermore, repeated exposure to the higher concentration of methamphetamine (100 microM) caused a significant reduction in the number of DA and 5-HT cells as well as reducing the total neuronal density. This would suggest that this higher concentration of methamphetamine results in generalized neurotoxicity. Exposure to 10 microM methamphetamine resulted in more specific effects on dopaminergic function. These findings indicate that repeated methamphetamine administration can induce similar alterations in both dopaminergic and serotonergic neurons in raphe cultures.

Prolonged dopamine and serotonin transporter inhibition after exposure to tropanes

Cocaine and tropane analogs are known to interact with biogenic monoamine transporters by inhibiting amine uptake. Previous in vivo studies have demonstrated that some of these tropanes produce a longer lasting behavioral effect compared with cocaine. We have previously examined several tropane analogs and found a difference in their relative affinities for dopamine (DA) and serotonin (5-HT) transporters. The purpose of this study was to determine the recovery time of transporter function in vitro and in vivo comparing cocaine with the tropane analogs WF-11 (PTT, selective for DA transporters), WF-31 (selective for 5-HT transporters) and WF-23 (highly potent at both DA and 5-HT transporters). In vitro, using primary rat brain cultures of either midbrain or raphe regions, the recovery of the ability to transport either [3H]dopamine or [3H]serotonin, respectively was evaluated at 0, 3, 24, 48, 120 and 240 h after a 1 h exposure to cocaine and tropane analogs. The tropanes exhibited clearance half-lives ranging from 12 to 69 h, while cocaine, on the other hand, exhibited a clearance half-life of approximately 6 h. In studies utilizing [125I]RTI-55 binding, intraperitoneal injections of cocaine and WF-23 into the rat resulted in striatal clearance half-lives ex vivo that were almost identical to those obtained in vitro. These data suggest that the tropanes bind to and reduce transporter function for prolonged periods of time (up to 10-fold longer than cocaine) and those compounds with the highest affinity may produce a pseudo-irreversible inhibition of transporter function.

21 – Evaluation of Catecholaminergic Activity in Hypothalamic Cultures

Publisher Summary This chapter discusses the evaluation of catecholaminergic activity in hypothalamic cultures. There are two basic types of cell culture: (1) continuous cell lines in which proliferation can be maintained indefinitely and (2) primary culture, where tissue is removed directly from an animal and grown for a limited amount of time. The chapter reviews the methodology for the preparation of dissociated cultures of the medial basal hypothalamus (MBH) and the assessment of catecholaminergic activity by neurochemical and immunochemical means. The MBH consists primarily of the arcuate and periventricular nuclei (dopamine areas A12 and A14), and the median eminence. This area has been chosen because of its high concentration of dopamine cell bodies and nerve terminals. A technique was characterized for the study of catecholamine biosynthesis in vitro which involved incubation with a labeled precursor followed by separation with high-performance liquid chromatography (HPLC) and quantitation with electrochemical detection. This technique is now used for the examination of catecholamine biosynthesis in primary cell cultures.