Dopamine and macromolecule synthesis in rat hippocampus

In hippocampus slices both dopamine and apomorphine lead to an increased incorporation of (³H)-fucose into total proteins, whereas the incorporation of (¹⁴C)-leucine was unchanged or decreased, respectively. Noradrenaline did not alter the incorporation of both precursors, whereas haloperidol partially reduced the dopamine induced increased in incorporation of fucose. Thus, an induction process of the observed macromolecular changes involving dopaminoceptive structures of hippocampus can be assumed.     

Hippocampal activation and incorporation of macromolecule precursors

The effect of rhythmic slow wave activity (theta rhythm) on the incorporation of ³H-leucine and ³H-fucose into the total proteins of different hippocampus areas was studied. The theta rhythm was elicited by electrical stimulation of medial septum nuclei. An increase in ³H-leucine incorporation into the total proteins of CA 3 and CA 1 sectors of the hippocampus was observed, whereas the stimulation had no influence on precursor incorporation into the complex CA 4/area dentata. In contrast to these findings ³H-fucose incorporation into hippocampal proteins was not influenced by electrical stimulation of the medial septum. These findings are discussed in comparison to the results obtained in a learning experiment, which revealed an increased incorporation of both leucine and focuse into hippocampal proteins.     

Dopamine induced changes in L-fucose incorporation into proteins of rat hippocampus and corpus striatum during postnatal development

In 60 day old (adult) male Wistar rats dopamine caused a dose-dependent increase of L-fucose incorporation into total proteins of both hippocampus and corpus striatum slices up to +47.8 +/- 6.0% (n=6) and +53.2 +/- 8.5% (n=20), respectively, when compared to corresponding controls. Under these conditions the dopamine concentration leading to a maximum stimulation of fucose incorporation was 5 X 10(-4) M in hippocampus and 1 X 10(-3) M in corpus striatum. In the latter tissue the range of dopamine concentrations causing a significant elevation in incorporation rates was larger than in hippocampal tissue. In the corpus striatum of 9 day old rats dopamine was ineffective, but by 30 days the transmitter stimulated fucose incorporation rate reached the maximum observed for any age studied. This developmental pattern seems to be related to the ontogenesis of dopamine receptor sites or dopamine sensitive adenylate cyclase formation in this brain structure. In the hippocampus the postnatal development of dopamine induced augmentation of glycoprotein synthesis showed a longer latency, but the maximum effect was also seen in 30 day old animals. These results support our assumption that at the end of the postnatal differentiation period the glycoprotein synthesis in brain tissue may be controlled (at least to some extent) by the state of dopaminergic receptors and/or of dopamine sensitive adenylate cyclase.     

Effects of ethanol on glycoprotein synthesis and secretion during inflammation-induced stimulation of hepatic glycoprotein secretion

The effects of ethanol on glycoprotein metabolism in liver slices obtained from rats whose synthesis and secretion of glycoproteins had been stimulated by turpentine-induced inflammation were investigated and compared to colchicine, a potent inhibitor of secretion. Inflammation resulted in a large stimulation in liver slices of [¹⁴C]glucosamine and [¹⁴C]leucine incorporation into medium proteins and a lesser stimulation into hepatocellular proteins. Both ethanol (10 mm) and colchicine (50 μm), when present in the incubation medium, markedly decreased the appearance of glucosamine- and leucinelabeled proteins in the medium of both turpentine-stimulated and control livers. However, when microsomes were isolated and fractionated in a fraction rich in secretory contents and another rich in membrane components, the effects of these agents on the labeling of glycoproteins in these microsomal fractions differed. Colchicine did not affect the incorporation of either glucosamine or leucine into the membrane fraction but increased the labeling of the secretory fraction, whereas, ethanol decreased the labeling of both fractions. These effects were observed for both the stimulated and control slices. When protein synthesis was inhibited by cycloheximide in both types of slices, ethanol and colchicine decreased the secretion of glucosamine-labeled protein into the medium while increasing the specific activity of the glycoproteins of the secretory fraction without altering the labeling of the membrane fraction. When fucose, a terminal sugar, was used as a label, both agents decreased the secretion of fucose-labeled proteins into the medium while also causing a retention of labeled glycoproteins in the microsomal secretory fraction. These results indicate that the ethanol-induced inhibition of glycoprotein synthesis and secretion in the normal liver persists in the liver where synthesis and secretion have been greatly stimulated by inflammation.     

Tyrosine hydroxylase activation and transmitter release from central noradrenergic neurons by electrical field stimulation

Summary Electrical stimulation of the noradrenergic neurons in the locus coeruleus of the rat results in a marked increase in the tyrosine hydroxylase activity of the hippocampus on the stimulated side (Roth et al., 1975). We have developed an in vitro system to further study this interesting phenomenon. Rat hippocampal slices were stimulated in an electrical field using specially built chambers which permitted a continous superfusion with Krebs Ringer Phosphate solution while stimulation was taking place. The slices were then homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 104000 g supernate fraction. Electrical field stimulation (60 V, 4 ms, 5–20 Hz, 5 min) induced a stimulus-dependent increase in tyrosine hydroxylase activity. The increase in tyrosine hydroxylase activity was directly dependent on the number of pulses of stimulation applied. Potassium depolarization of hippocampal slices also resulted in a marked increase in the activity of the enzyme. Electrical field stimulation appears to activate tyrosine hydroxylase by causing an increase in its affinity for both substrate and pteridine cofactor and by decreasing its affinity for the endproduct inhibitor, norepinephrine. No change in V_max was observed. The superfusion system was also used to study spontaneous and electrically evoked release of labelled norepinephrine from hippocampal slices. Electrical field stimulation for 1.0 min produced a marked increase in the release of label. Absence of calcium from the superfusion solution almost completely abolished the electrically evoked release of exogenously taken up ³H-norepinephrine. The release of ³H-norepinephrine was also found to be dependent on the number of pulses of stimulation used. The results reported support the hypothesis that depolarization of central noradrenergic neurons results in an increase in transmitter synthesis mediated in part via a kinetic activation of tyrosine hydroxylase. The concomitant use of superfusion and electrical field stimulation of hippocampal slices provides a simple system to study neurotransmitter synthesis and release in central noradrenergic neurons.A preliminary account of this work was reported at the eight Meeting of the Argentinian Society for Experimental Pharmacology held November 14–18, 1975, at San Luis, Argentina     

Choline uptake and permanent memory storage

The uptake of ³H-choline and its incorporation into ³H-acetylcholine was studied in vitro on hippocampus slices obtained from animals showing a good or poor long-term memory. The animals were selected on the basis of their retention performance when tested by a brightness discrimination model. The ³H-choline uptake and the incorporation of ³H-choline into ³H-acetylcholine was higher in hippocampus slices from animals showing good retention compared to those from animals with poor retention. The level of high affinity uptake of choline into hippocampus slices may serve as an indicator of the cholinergic activity in this structure under in vivo conditions. The present findings suggest that individual differences in the activity level of the hippocampal cholinergic system do exist and are capable of influencing the retention of the individual animals to a variable degree.     

Inhibition of K+-stimulated [3H]dopamine and [14C]acetylcholine release by the putative dopamine autoreceptor agonist,B-HT 920

Summary The inhibition of K¹-stimulated [³H]dopamine and [¹⁴C]acetylcholine release from preloaded rat striatal slices was used to examine the presynaptic selectivity of the putative dopamine autoreceptor agonist, B-HT 920. In the micromolar range, B-HT 920 caused a concentration-dependent inhibition of the release of both labeled neurotransmitters as evoked by 20 mM K⁺. The effect of B-HT 920 on both [³H]dopamine and [¹⁴C]acetylcholine release was completely blocked by (+) butaclamol but not by (–) butaclamol. Sulpiride, a selective D₂ antagonist, similarly blocked the inhibitory effect of B-HT 920 on the release of both labeled neurotransmitters indicating both responses were mediated by D₂ receptors. (+) Butaclamol alone elevated stimulated [³H]dopamine release suggesting a significant amount of autoreceptor occupancy by endogenously released dopamine. Experiments with tolazoline and the alpha₂ agonist, B-HT 933, did not suggest any involvement of alpha-adrenoceptor activity in the inhibitory effects of B-HT 920 on the release of either transmitter. Inhibition of release was a selective effect of B-HT 920 as the drug was without effect on the K⁺-stimulated release of [³H]serotonin. The results indicate that in vitro B-HT 920 is active of both pre-and postsynaptic dopamine receptors in contrast to the pattern of effects observed after its in vivo administration.     

Studies on the metabolic pathway of the acetyl group for acetylcholine synthesis

Glucose and pyruvate are the most effective precursors of the acetyl moiety of acetylcholine in mammalian brain; the metabolic intermediates between pyruvate and acetylcholine, however, are unknown. The following data suggest that citrate is not the sole intermediate of the acetyl group for acetylcholine synthesis in rat brain slices or synaptosomes: (1) 2.5 mM (?)-hydroxycitrate decreased acetylcholine synthesis from [U-¹⁴C]glucose by only 25 per cent; (2) inhibition of citrate transport out of mitochondria by n-butylmalonate or 1,2,3-benzenetricarboxylate variably affected acetylcholine synthesis; and (3) high concentrations of nonradioactive citrate decreased the synthesis of acetylcholine but did not decrease the specific activity of the acetylcholine synthesized from [U-¹⁴C]glucose. even though the uptake of citrate into the synaptosomes under these experimental conditions was approximately five times greater than the uptake of glucose. Other possible acetyl donors altered acetylcholine synthesis. Acetylcarnitine stimulated synthesis in brain slices, and carnitine stimulated synthesis by synaptosomes.The specificactivity of the acetylcholine synthesized from [U-^14Cglucose by synaptosomes was decreased by N-acetyl-l-aspartate (10mM), acetyl CoA (1 mM), and acetyl phosphate (10mM) which is consistent with these compounds acting as direct acetyl donors. Acetate (10 mM) did not affect either the amount or specific activity of the acetylcholine synthesized. Further evidence of compartmentation of cytoplasmic acetyl CoA is presented. The cytoplasmic acetyl CoA for acetylcholine synthesis is distinguishable from the cytoplasmic acetyl CoA for lipid synthesis. (?)-Hydroxycitrate inhibited acetylcholine synthesis without inhibiting lipid synthesis from [U-¹⁴C]glucose. However, when 3-hydroxy[3-¹⁴C]butyrate was used as substrate, (?)-hydroxycitrate inhibited incorporation into lipids twice as much as incorporation into acetylcholine. [U-¹⁴C]Glucose metabolism by infant brain slices was more sensitive than adult brain slices to (?)-hydroxycitrate. However, the response to the other compounds which interfere with citrate metabolism was similar in slices from adult and infant brains.     

Effect of chronic treatment with the GABA transaminase inhibitors γ-vinyl GABA and ethanolamine O-sulphate on the in vitro GABA release from rat hippocampus

1. The effects of 2, 8 and 21 day oral treatment with the specific γ-aminobutyric acid transaminase (GABA-T) inhibitors γ-vinyl GABA (GVG) and ethanolamine O-sulphate (EOS) on brain GABA levels, GABA-T activity, and basal and stimulated GABA release from rat cross-chopped brain hippocampal slices was investigated.
2. Treatment with GABA-T inhibitors lead to a reduction in brain GABA-T activity by 65–80% compared with control values, with a concomitant increase in brain GABA content of 40–100%.
3. Basal hippocampal GABA release was increased to 250–450% of control levels following inhibition of GABA-T activity. No Ca²⁺ dependence was observed in either control or treated tissues.
4. GVG and EOS administration led to a significant elevation in the potassium stimulated release of GABA from cross-chopped hippocampal slices compared with that of controls. Although stimulated GABA release from control tissues was decreased in the presence of a low Ca²⁺ medium, GVG and EOS treatment abolished this Ca²⁺ dependency.
5. GABA compartmentalization, Na⁺ and Cl⁻ coupled GABA uptake carriers and glial release may provide explanations for the loss of the Ca²⁺ dependency of stimulated GABA release observed following GVG and EOS treatment.
6. Administration of GABA-T inhibitors led to increases in both basal and stimulated hippocampal GABA release. However, it is not clear which is the most important factor in the anticonvulsant activity of these drugs, the increased GABA content ‘leaking'' out of neurones and glia leading to widespread inhibition, or the increase in stimulated GABA release which may occur following depolarization caused by an epileptic discharge.

     

Changes in activities of fucokinase and fucosyltransferase in rat hippocampus after acquisition of a brightness discrimination reaction

Activities of enzymes involved in utilization of the glycoprotein precursor L-fucose (fucokinase and fucosyltransferase) were studied in rat hippocampal tissue after acquisition of a brightness discrimination reaction. Fucokinase activity was increased immediately after training, while fucosyltransferase revealed decreased values. However, 7 hr after training fucokinase activity showed normal values, while fucosyltransferase activity rose in trained animals over active and passive controls. The results are discussed in the light of a regulatory role that fucokinase and fucosyltransferase may play in fucose utilization under altered functional conditions.     

Nicotine-induced release of catecholamines from rat hippocampus and striatum

The present report is a comparative study of [³H]catecholamine release from noradrenergic and dopaminergic neuron terminals of the central nervous system, induced by nicotine and potassium depolarization. Striatal and hippocampal slices of rat brain that had incorporated [³H]catecholamines in previous incubations with the radioamines were superfused and stimulated by nicotine and high potassium. Nicotine produced a marked [³H]catecholamine release from these two brain areas, this effect being much greater in the striatum. The time course of radioactive efflux released by nicotine was different from that induced by high potassium and similar to that evoked by tyramine. Nicotine induced released was dose dependent, inhibited by low temperature, independent of extracellular calcium and not inhibited by an excess of magnesium. Studies with newly synthesized [³H]dopamine and [¹⁴C]urea suggested that nicotine acted on the storage vesicles. [³H]catecholamine release was continuous during prolonged nicotine stimulation, in contrast to the transient efflux induced by prolonged stimulation by potassium, and additive when the slices were superfused simultaneously with nicotine plus potassium. Previous nicotine superfusion did not modify the typical potassium release, and the response to nicotine was also not altered when the slices were exposed previously to high potassium. Based on these results, the mechanism of nicotine release of catecholamines from central catecholaminergic neuron terminals is discussed.     

Influence of choline,hemicholinium-3 and naphthylvinylpyridine on uptake and acetylation of 3H-labeled choline into hippocampus slices

In slices of rat hippocampus the influence of choline (Ch), hemicholinium-3 (HC-3), and naphthylvinylpyridine (NVP) on the uptake of [³H]-Ch and its incorporation into acetylcholine (ACh) was studied. Increasing concentrations of Ch, added to the incubation medium, led to an elevation of the uptake of labeled precursor and its acetylation. The release of radioactive labeled ACh from slices of hippocampal tissue was also enhanced. A kinetic analysis of the transport of Ch revealed two distinct uptake systems for the precursor with K_m-values of 0.87 μM and 30.3 μM, respectively. The uptake of tritiated Ch into hippocampal slices could be inhibited by HC-3. NVP, an inhibitor of choline acetyltransferase, reduced the uptake of Ch to a similar degree as observed by HC-3, whereas the subsequent acetylation to ACh was not affected. Using an intraventricular application of 400 nmoles unlabeled Ch, the influence of the precursor on uptake and incorporation of [³H]-Ch was also tested in vivo. Both the uptake of Ch and its subsequent acetylation were enhanced, whereas no changes in the ACh content of the hippocampus were observed. The data suggest that the increase of the precursor supply yields an elevation of Ch transport, its acetylation and the transmitter release.     

Alterations of the dopaminergic and glutamatergic neurotransmission in adult rats with postnatal ibotenic acid hippocampal lesion

In 6-week and 8-week-old rats (pre- and postpubertally) with neonatal excitotoxic lesions of the ventral hippocampus with ibotenic acid (IBO), we have studied apomorphine-induced motor activity and glutamate and dopamine D₁ and D₂ binding sites in the hippocampus, striatum, nc. accumbens and frontal cortex as well as K⁺-stimulated (³H)-D-aspartate release from hippocampal and frontal cortical slices. Specific glutamate binding was enhanced in the frontal cortex of 8-week-old IBO-treated animals, whereas that in other brain regions remained unchanged. Both D₁ and D₂ binding sites were downregulated in the striatum without changes in other brain structures. In 6-week-old rats, neither the glutamate nor the dopamine binding sites were altered. The amino acid release from hippocampal and frontal cortical slices of adult IBO treated rats was significantly decreased in comparison to controls, whereas in 6-week-old rats, no significant alterations were detectable. The additionally monitored motor activity was enhanced only in adult IBO-lesioned rats after apomorphine pretreatment. The present data are in agreement with the hypothesis of hyperactive dopamine and hypoactive glutamate systems in schizophrenia and are discussed in the light of schizophrenia-like behavioral changes in rats after postnatal hippocampal IBO lesion. Received: 10 August 1998/Final version: 11 February 1999     

The putative polyamine antagonists ifenprodil and SL 82.0715 enhance dopamine efflux from rat striatal slices independent of NMDA receptor activation

NMDA stimulated the release of endogenous or tritiated dopamine from rat striatal slices and tritiated norepinephrine from cortical and hippocampal slices. The putative polyamine antagonists ifenprodil and SL 82.0715 inhibited [³H]norepinephrine release from cortical and hippocampal slices but enhanced the basal efflux of endogenous and tritiated dopamine from striatal slices. Incubation of striatal slices in a calcium-free buffer did not ameliorate these effects suggesting that the increase in dopamine efflux was not due to a calcium-dependent release process. Superfusion of striatal slices with 10 μM of either ifenprodil, cocaine, or amphetamine resulted in a significant release of tritiated dopamine which was reversed when the slices were again superfused with non-drug-containing buffer. The release observed in the presence of 10 μM ifenprodil (7-fold increase over basal) was intermediate between that observed for cocaine (3-fold increase) and amphetamine (12-fold increase). Both ifenprodil and SL 82.0715 also blocked the uptake of [³H]dopamine into striatal synaptosomes with IC₅₀ values of approximately 1.5 μM. This was again intermediate between the values obtained for cocaine (0.43 μM) and amphetamine (4.2 μM). These results suggest that ifenprodil and its analog SL 82.0715 may act as indirect dopamine agonists by both blocking presynaptic dopamine uptake and by directly increasing the basal efflux of dopamine.     

Adenosine A2A receptor stimulation increases release of acetylcholine from rat hippocampus but not striatum,and does not affect catecholamine release

Rat striatal and hippocampal slices, preincubated with [³H] dopamine (DA) {or [³H] noradrenaline (NA)} and [¹⁴C] choline, were superfused continuously and stimulated electrically. 2-chloroadenosine (2-CADO 0.001–100 μM), a non-selective adenosine receptor agonist, produced a concentration-dependent inhibition of the electrically evoked DA and acetylcholine (ACh) release from the striatal slices and of the electrically evoked NA and ACh release from the hippocampal slices. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 3, 30 and 200 nM), a selective adenosine A₁ receptor antagonist, caused a concentration-dependent, parallel, rightward shift of the 2-CADO concentration-response curve, consistent with competitive antagonism. The pA₂ values ranged between 8.4 and 8.8. In the case of ACh release from the hippocampus, but in no other case, was there an increase in release of radioactivity at low concentrations of 2-CADO in the presence of DPCPX. The stimulation in the hippocampus could be blocked by a selective adenosine A_2A receptor antagonist KF 17837. By itself KF 17837 (0.1–100 μM) had no effect on electrically evoked NA release from hippocampal slices, but decreased electrically evoked ACh release. This inhibition was counteracted by DPCPX (1 μM). These results show that, under the conditions used, DA release in the striatum, and NA release in the hippocampus, as well as ACh release from the striatum are regulated by adenosine A₁ but not by adenosine A_2A receptors. By contrast, ACh release from the hippocampus is tonically regulated both by adenosine A₁ receptors, which inhibit release, and by adenosine A_2A receptors which stimulate release. Received: 25 April 1996 / Accepted: 30 August 1996     

The influence of dopamine on the incorporation of different sugars into total proteins of hippocampal slices

Dopamine increases the incorporation of L-fucose and of D-mannose to a similar significant degree, whereas the incorporation of D-galactose as well as of N-acetyl-D-glucosamin into the total proteins of hippocampal slices was only slightly enhanced. The incorporation of N-acetyl-neuraminic acid was not influenced by dopamine. The results suggest that the effect of dopamine on glycoprotein formation seems mainly to depend on the kind of nucleotides necessary for activation of sugars and not on the sugar's final position in the glycan chain.     

Effect of bioamines on the cellular differentiation of Hartmannella culbertsoni

Epinephrine, 5-hydroxytryptamine, dopamine and tyramine induce axenic encystation of the free living amoeba Hartmannella culbertsoni. Cycloheximide inhibits encystation of amoeba mediated by the amines. The amines uniformly stimulate 3 to 4-fold the ability of the cells to synthesize cyclic 3′,5′-adenosine monophosphate from adenosine triphosphate formed in situ. Epinephrine[¹⁴C] is bound to membranes of the amoeba (K_mof binding = 6.5 mM epinephrine). When amoebae are exposed to epinephrine, tyramine, dopamine or cAMP in a non-nutrient medium for 6 hr protein kinase activity is stimulated as evident from increased incorporation of ³²P into cellular proteins.     

Proportional inhibition of acetylcholine synthesis accompanying impairment of 3-hydroxybutyrate oxidation in rat brain slices

The synthesis of acetylcholine from 3-hydroxy[3-¹⁴C]butyrate has been studied in brain slices from 19-day-old and adult rats. In the brains of 19-day-old rats, the synthesis of acetylcholine from 3-hydroxybutyrate was stimulated by the addition of physiological concentrations of glucose but CO₂ production was not. Conversely, physiological concentrations of unlabeled 3-hydroxybutyrate reduced the production of acetylcholine and of ¹⁴CO₂ from [U-¹⁴C]glucose. Results with adult brains were qualitatively similar, except that glucose stimulated the production of CO₂ from 3-hydroxy[3-¹⁴C]butyrate. Methylmalonic acid inhibited the oxidation of 3-hydroxybutyrate but did not affect the oxidation of either pyruvate or glucose. Addition of methylmalonic acid to slices incubated with both 3-hydroxybutyrate and glucose decreased the synthesis of acetylcholine from 3-hydroxybutyrate with a compensatory increase in synthesis from glucose. These studies demonstrate that impairing the oxidation of 3-hydroxybutyrate to CO₂ is accompanied by a proportional decrease in the incorporation of 3-hydroxybutyrate into acetylcholine, even though the flux through oxidative pathways is over 100 times that to acetylcholine.     

In vitro synthesis of noradrenaline in ganglia and salivary glands after in vivo preganglionic stimulation

The extent of synthesis of [¹⁴C]noradrenaline from [¹⁴C]tyrosine and from [¹⁴C]dopamine was assessed in slices of superior cervical ganglia, representing cell bodies, and in submaxillary salivary glands, representing terminals of noradrenergic neurons of the cat. Immediately and 6 h after preganglionic stimulation for 3 h the rate of synthesis of noradrenaline from tyrosine and dopamine was not altered in ganglia. In salivary glands, however, synthesis of noradrenaline from both tyrosine and dopamine was increased at both times. These results suggest that acute periods of increased neural activity results in the acceleration of noradrenaline synthesis in the terminals but not in the cell bodies of noradrenergic neurons.     

Blockade of accumbens 5-HT3 receptor down-regulation by ondansetron administered during continuous cocaine administration

The present experiment examined whether ondansetron, co-administered with continuous cocaine, would block the down regulation of accumbens 5-HT₃ receptors. Rats were exposed to a 14-day pretreatment regimen that involved the continuous infusion of 40 mg kg⁻¹ day⁻¹ cocaine or 0.9% saline via a subcutaneously implanted osmotic minipump. In addition to the continuous cocaine or saline administration, all subjects received daily subcutaneous (s.c.) injections of either vehicle or 0.1 mg kg⁻¹ ondansetron for the entire 14-day pretreatment regimen. The rats were then withdrawn from this pretreatment regimen for seven days, and slices from the nucleus accumbens obtained. The slices were perfused with 25 mM K⁺ in the absence and presence of 0, 12.5, 25, or 50 μM m-Chlorophenyl-biguanide HCl (mCPBG). The efflux samples were assayed for dopamine content by high pressure liquid chromatography (HPLC) with electrochemical detection. Continuous cocaine administration significantly attenuated the ability of mCPBG to facilitate K⁺-induce dopamine overflow compared to saline control rats. In addition, the rats that received ondansetron and cocaine during the 14-day pretreatment period, the ability of mCPBG to enhance K⁺ stimulated dopamine release was not significantly different from the saline control subjects. For all groups except the cocaine alone group, the effects of mCPBG on K⁺ stimulated dopamine release were Ca²⁺ dependent, suggesting that these effects are receptor mediated. These results suggest that continuous cocaine administration functionally down-regulates 5-HT₃ receptors in the nucleus accumbens, and that this down-regulation can be blocked by chronic ondansetron administration. Hence, a functional down regulation of accumbens 5-HT₃ receptors represents a significant contribution to the tolerance induced by continuous cocaine administration.