Superfused Rat Striatal Slices Research Articles

Novel multifunctional pharmacology of lobinaline, the major alkaloid from Lobelia cardinalis

In screening a library of plant extracts from ~1000 species native to the Southeastern United States, Lobelia cardinalis was identified as containing nicotinic acetylcholine receptor (nicAchR) binding activity which was relatively non-selective for the α4β2- and α7-nicAchR subtypes. This nicAchR binding profile is atypical for plant-derived nicAchR ligands, the majority of which are highly selective for α4β2-nicAchRs. Its potential therapeutic relevance is noteworthy since agonism of α4β2- and α7-nicAchRs is associated with anti-inflammatory and neuroprotective properties. Bioassay-guided fractionation of L. cardinalis extracts led to the identification of lobinaline, a complex binitrogenous alkaloid, as the main source of the unique nicAchR binding profile. Purified lobinaline was a potent free radical scavenger, displayed similar binding affinity at α4β2- and α7-nicAchRs, exhibited agonist activity at nicAchRs in SH-SY5Y cells, and inhibited [3H]-dopamine (DA) uptake in rat striatal synaptosomes. Lobinaline significantly increased fractional [3H] release from superfused rat striatal slices preloaded with [3H]-DA, an effect that was inhibited by the non-selective nicAchR antagonist mecamylamine. In vivo electrochemical studies in urethane-anesthetized rats demonstrated that lobinaline locally applied in the striatum significantly prolonged clearance of exogenous DA by the dopamine transporter (DAT). In contrast, lobeline, the most thoroughly investigated Lobelia alkaloid, is an α4β2-nicAchR antagonist, a poor free radical scavenger, and is a less potent DAT inhibitor. These previously unreported multifunctional effects of lobinaline make it of interest as a lead to develop therapeutics for neuropathological disorders that involve free radical generation, cholinergic, and dopaminergic neurotransmission. These include neurodegenerative conditions, such as Parkinson's disease, and drug abuse.

Frederick Morgan

Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters (DAT and NET, respectively). Recently, bupropion has been reported to noncompetitively inhibit α3β2, α3β4, and α4β2 nicotinic...

Tris-azaaromatic quaternary ammonium salts: Novel templates as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release.

A series of tris-azaaromatic quaternary ammonium salts has been synthesized and evaluated for their ability to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) mediating nicotine-evoked [(3)H]dopamine release from superfused rat striatal slices and for inhibition of [(3)H]nicotine and [(3)H]methyllycaconitine binding to whole rat brain membranes. The 3-picolinium compound 1,3,5-tri-{5-[1-(3-picolinium)]-pent-1-ynyl}benzene tribromide (tPy3PiB), 3b, exhibited high potency and selectivity for nAChR subtypes mediating nicotine-evoked [(3)H]dopamine release with an IC(50) of 0.2 nM and I(max) of 67%.

Discovery of a novel nicotinic receptor antagonist for the treatment of nicotine addiction: 1-(3-Picolinium)-12-triethylammonium-dodecane dibromide (TMPD)

Limitations in efficacy and high relapse rates of currently available smoking cessation agents reveal the need for more efficacious pharmacotherapies. One strategy is to develop subtype-selective nicotinic receptor (nAChR) antagonists that inhibit nicotine-evoked dopamine (DA) release, the primary neurotransmitter involved in nicotine reward. Simple alkylation of the pyridino N-atom converts nicotine from a potent agonist into a potent antagonist. The classical antagonists, hexamethonium and decamethonium, differentiate between peripheral nAChR subtypes. Using a similar approach, we interconnected varying quaternary ammonium moieties with a lipophilic linker to provide N, N′-bis-nicotinium analogs, affording a lead compound, N, N′-dodecyl-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), which inhibited nicotine-evoked DA release and decreased nicotine self-administration. The current work describes a novel compound, 1-(3-picolinium)-12-triethylammonium-dodecane dibromide (TMPD), a hybrid of bPiDDB and decamethonium. TMPD completely inhibited (IC 50 = 500 nM) nicotine-evoked DA release from superfused rat striatal slices, suggesting that TMPD acts as a nAChR antagonist at more than one subtype. TMPD (1 μM) inhibited the response to acetylcholine at α3β4, α4β4, α4β2, and α1β1ɛδ receptors expressed in Xenopus oocytes. TMPD had a 2-fold higher affinity than choline for the blood–brain barrier choline transporter, suggesting brain bioavailability. TMPD did not inhibit hyperactivity in nicotine sensitized rats, but significantly and specifically decreased nicotine self-administration. Together, the results suggest that TMPD may have the ability to reduce the rewarding effect of nicotine with minimal side effects, a pharmacological profile indicative of potential clinical utility for the treatment of tobacco dependence.

Nornicotine inhibition of dopamine transporter function in striatum via nicotinic receptor activation

Nornicotine, a tobacco alkaloid and N-demethylated nicotine metabolite, releases DA from superfused rat striatal slices in a mecamylamine-sensitive manner, indicating nicotinic receptor (nAChR) modulation of this response. The current study determined the effect of nornicotine on rat striatal dopamine transporter (DAT) function using in vivo voltammetry. In a dose-related and mecamylamine-sensitive manner, nornicotine (0.35-12.0 mg/kg, s.c.) decreased DA clearance, suggesting that nornicotine inhibits striatal DAT function via a nAChR-mediated mechanism. Furthermore, the nAChRs mediating the nornicotine-induced inhibition of DAT function appear to be different from those activated by nicotine which increases DA clearance. Understanding the actions of nornicotine in brain may have significance for emerging therapeutics and for the management of nicotine dependence.

Regulation of Amphetamine-stimulated Dopamine Efflux by Protein Kinase C β

Evidence suggests that protein kinase C (PKC) and intracellular calcium are important for amphetamine-stimulated outward transport of dopamine in rat striatum. In this study, we examined the effect of select PKC isoforms on amphetamine-stimulated dopamine efflux, focusing on Ca(2+)-dependent forms of PKC. Efflux of endogenous dopamine was measured in superfused rat striatal slices; dopamine was measured by high performance liquid chromatography. The non-selective classical PKC inhibitor Gö6976 inhibited amphetamine-stimulated dopamine efflux, whereas rottlerin, a specific inhibitor of PKC delta, had no effect. A highly specific PKC beta inhibitor, LY379196, blocked dopamine efflux that was stimulated by either amphetamine or the PKC activator, 12-O-tetradecanoylphorbol-13-acetate. None of the PKC inhibitors significantly altered [3H]dopamine uptake. PKC beta(I) and PKC beta(II), but not PKC alpha or PKC gamma, were co-immunoprecipitated from rat striatal membranes with the dopamine transporter (DAT). Conversely, antisera to PKC beta(I) and PKC beta(II) but not PKC alpha or PKCg amma were able to co-immunoprecipitate DAT. Amphetamine-stimulated dopamine efflux was significantly enhanced in hDAT-HEK 293 cells transfected with PKC beta(II) as compared with hDAT-HEK 293 cells alone, or hDAT-HEK 293 cells transfected with PKCa lpha or PKC beta(I). These results suggest that classical PKC beta(II) is physically associated with DAT and is important in maintaining the amphetamine-stimulated outward transport of dopamine in rat striatum.

N‐n‐alkylnicotinium and N‐n‐alkylpyridinium analogs inhibit the dopamine transporter: Selectivity as nicotinic receptor antagonists

AbstractN‐n‐Alkylnicotinium and N‐n‐alkylpyridinium analogs act as antagonists at nicotinic acetylcholine receptors (nAChRs) mediating nicotine‐evoked [3H]dopamine (DA) overflow from superfused rat striatal slices in the presence of a DA transporter (DAT) inhibitor. However, the potential interaction of these nAChR antagonists with DAT has not been evaluated. In the present study, analog inhibition of [3H]DA uptake into striatal synaptosomes and inhibition of [3H]GBR 12935 binding to striatal membranes was determined. N‐n‐Alkylnicotinium analogs with n‐alkyl chains of C6–12 and N‐n‐alkylpyridinium analogs with n‐alkyl chains of C7–20 inhibited [3H]DA uptake with a wide affinity range. With the exception of the C20 N‐n‐alkylpyridinium, a linear relationship between chain length and inhibition of [3H]DA uptake was found in both analog series. Similarly, these analogs inhibited [3H]GBR 12935 binding (Ki=5.7–250 μM), and a linear relationship with chain length was observed, with the exception of the C8 N‐n‐alkylnicotinium analog. Kinetic analyses of inhibition of [3H]DA uptake and [3H]GBR 12935 binding using representative C12 analogs from each series revealed decreases in maximal [3H]DA transport velocity and maximal [3H]GBR 12935 binding without alterations in affinity, indicating noncompetitive interactions with DAT. In comparison, classical nAChR antagonists (mecamylamine, dihydro‐β‐erythroidine and methyllycaconitine) did not inhibit [3H]DA uptake or [3H]GBR 12935 binding. Moreover, inhibition of DAT function occurred at analog concentrations 10–120‐fold higher than those inhibiting nAChR function. Taken together with the inability of these analogs to inhibit field‐stimulation‐evoked [3H]DA overflow, the results indicate that these analogs act selectively as antagonists at nAChRs mediating nicotine‐evoked [3H]DA overflow. Drug Dev. Res. 60:270–284, 2003. © 2003 Wiley‐Liss, Inc.

N-n-alkylpyridinium analogs, a novel class of nicotinic receptor antagonists: selective inhibition of nicotine-evoked [3H] dopamine overflow from superfused rat striatal slices.

Structural simplification of N-n-alkylnicotinium analogs, antagonists at neuronal nicotinic acetylcholine receptors (nAChRs), was achieved by removal of the N-methylpyrrolidino moiety affording N-n-alkylpyridinium analogs with carbon chain lengths of C1 to C20. N-n-Alkylpyridinium analog inhibition of [3H]nicotine and [3H]methyllycaconitine binding to rat brain membranes assessed interaction with alpha4beta2* and alpha7* nAChRs, respectively, whereas inhibition of nicotine-evoked 3H overflow from [3H]dopamine ([3H]DA)-preloaded rat striatal slices assessed antagonist action at nAChR subtypes mediating nicotine-evoked DA release. No inhibition of [3H]methyllycaconitine binding was observed, although N-n-alkylpyridinium analogs had low affinity for [3H]nicotine binding sites, i.e., 1 to 3 orders of magnitude lower than that of the respective N-n-alkylnicotinium analogs. These results indicate that the N-methylpyrrolidino moiety in the N-n-alkylnicotinium analogs is a structural requirement for potent inhibition of alpha4beta2* nAChRs. Importantly, N-n-alkylpyridinium analogs with n-alkyl chains < C10 did not inhibit nicotine-evoked [3H]DA overflow, whereas analogs with n-alkyl chains ranging from C10 to C20 potently and completely inhibited nicotine-evoked [3H]DA overflow (IC50 = 0.12-0.49 microM), with the exceptions of N-n-pentadecylpyridinium bromide (C15) and N-n-eicosylpyridinium bromide (C20), which exhibited maximal inhibition of approximately 50%. The mechanism of inhibition of a representative analog of this structural series, N-n-dodecylpyridinium iodide, was determined by Schild analysis. Linear Schild regression with slope not different from unity indicated competitive antagonism at nAChRs mediating nicotine-evoked [3H]DA overflow and a KB value of 0.17 microM. Thus, the simplified N-n-alkylpyridinium analogs are potent, selective, and competitive antagonists of nAChRs mediating nicotine-evoked [3H]DA overflow, indicating that the N-methylpyrrolidino moiety is not a structural requirement for interaction with nAChR subtypes mediating nicotine-evoked DA release.

DOPA cyclohexyl ester potently inhibits aglycemia-induced release of glutamate in rat striatal slices

Brain ischemic insult causes glutamate release and resultant neuronal cell death. We here show that l-3,4-dihydroxyphenylalanine (DOPA) is a positive regulatory factor for glutamate release elicited by a mild brain insult using in vitro superfused rat striatal slices as a model system. Glucose deprivation for 18 min elicited release of glutamate, DOPA and dopamine (DA). Either tetrodotoxin (TTX) (1 μM) or α-methyl- p-tyrosine (α-MPT) (1 mM), a tyrosine hydroxylase inhibitor reduced markedly each of these releases. NSD-1015 (20 μM), an aromatic l-amino acid decarboxylase inhibitor restored the inhibition by α-MPT of glutamate and DOPA but not DA release. DOPA cyclohexyl ester (DOPA CHE) (0.3–1 μM), a competitive DOPA antagonist, concentration-dependently suppressed aglycemia-induced glutamate release, the effect which was mimicked neither by S-sulpiride nor SCH23390, a DA D 1 or D 2 receptor antagonist, respectively. Zonisamide (1–1000 μM), an anticonvulsant or YM872 (1 μM), an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) a receptor antagonist produced no effect on aglycemia-induced glutamate release. DOPA CHE thus showed a relatively potent inhibitiory action on aglycemia-induced glutamate release among several neuroprotective agents tested.

Lobeline attenuates locomotor stimulation induced by repeated nicotine administration in rats

Lobeline inhibits [ 3H]nicotine binding to rat brain membranes and nicotine-induced [ 3H]dopamine release from superfused rat striatal slices, indicating that lobeline acts as a nicotinic receptor antagonist. To determine whether lobeline also inhibits the effects of nicotine in vivo, the present study assessed the effect of lobeline pretreatment on nicotine-induced hyperactivity and sensitization. For 12 consecutive days, rats were injected subcutaneously with lobeline (3 mg/kg) or saline, followed 10 min later by nicotine (0.3 mg/kg) or saline injection, and activity was monitored. To determine if lobeline inhibits induction of sensitization to nicotine, 1 or 28 days later, rats were pretreated with saline followed by nicotine or saline. Lobeline attenuated nicotine-induced hyperactivity when both drugs were administered repeatedly. Although an initial injection of lobeline produced hypoactivity, tolerance to this effect developed. Importantly, tolerance did not develop to the lobeline-induced attenuation of nicotine hyperactivity. Lobeline attenuated the induction of sensitization to nicotine 1 day, but not 28 days, after the cessation of lobeline treatment. These results demonstrate that systemic administration of lobeline attenuates the locomotor-activating effects of repeated nicotine injection and the sensitization to nicotine, consistent with lobeline inhibition of nicotinic receptors and/or neurotransmitter transporters.

Bupropion inhibits nicotine-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine and from rat hippocampal slices preloaded with [(3)H]norepinephrine.

Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters (DAT and NET, respectively). Recently, bupropion has been reported to noncompetitively inhibit alpha3beta2, alpha3beta4, and alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes or established cell lines. The present study evaluated bupropion-induced inhibition of native alpha3beta2* and alpha3beta4* nAChRs using functional neurotransmitter release assays, nicotine-evoked [(3)H]overflow from superfused rat striatal slices preloaded with [(3)H]dopamine ([(3)H]DA), and nicotine-evoked [(3)H]overflow from hippocampal slices preloaded with [(3)H]norepinephrine ([(3)H]NE). The mechanism of inhibition was evaluated using Schild analysis. To eliminate the interaction of bupropion with DAT or NET, nomifensine or desipramine, respectively, was included in the superfusion buffer. A high bupropion concentration (100 microM) elicited intrinsic activity in the [(3)H]DA release assay. However, none of the concentrations (1 nM-100 microM) examined evoked [(3)H]NE overflow and, thus, were without intrinsic activity in this assay. Moreover, bupropion inhibited both nicotine-evoked [(3)H]DA overflow (IC(50) = 1.27 microM) and nicotine-evoked [(3)H]NE overflow (IC(50) = 323 nM) at bupropion concentrations well below those eliciting intrinsic activity. Results from Schild analyses suggest that bupropion competitively inhibits nicotine-evoked [(3)H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of bupropion inhibition of nicotine-evoked [(3)H]NE overflow. Thus, bupropion acts as an antagonist at alpha3beta2* and alpha3beta4* nAChRs in rat striatum and hippocampus, respectively, across the same concentration range that inhibits DAT and NET function. The combination of nAChR and transporter inhibition produced by bupropion may contribute to its clinical efficacy as a smoking cessation agent.

Synthesis and evaluation of conformationally restricted pyridino N‐alkylated nicotine analogs as nicotinic acetylcholine receptor antagonists

AbstractPrevious work has shown that quaternization of the pyridine‐N atom of S‐(–)‐nicotine (NIC) affords compounds such as N‐n‐octylnicotinium iodide (NONI) and N‐n‐decylnicotinium iodide (NDNI) that act as competitive nicotinic acetylcholine receptor (nAChR) antagonists at α3β2* and α4β2* subtypes, respectively. To ascertain the rotameric preference about the C3‐C2′ bond of NONI and NDNI for interaction with several nAChR subtypes, two classes of bridged analogs representing extreme rotameric conformations (syn and anti) of NONI and NDNI were synthesized. NIC‐evoked [3H]dopamine ([3H]DA) release from superfused rat striatal slices was used to determine the activity of the analogs at the α3β2* nAChR. [3H]NIC and [3H]methyllycaconitine ([3H]MLA) binding to rat brain membranes were used to determine affinity for α4β2* and α7* nAChRs, respectively. With the exception of BCDD (IC50 value = 1,580 nM), all analogs potently and selectively inhibited NIC‐evoked [3H]DA release (IC50 values = 30–660 nM), indicating antagonism of α3β2* nAChRs. None of the analogs inhibited either [3H]NIC or [3H]MLA binding, indicating a lack of interaction with α4β2* and α7* nAChR subtypes. Interestingly, the C10 N‐alkyl chain analogs, ACD and BCD, had negligible affinity for the α4β2* subtype compared to the high affinity exhibited by NDNI, suggesting that the α4β2* subtype does not recognize the unique stereochemistry of these conformationally restricted analogs. Thus, conformational restriction of N‐n‐alkylnicotinium iodides eliminated inhibitory activity at α4β2* nAChRs, but more importantly afforded high affinity and selectivity for α3β2* nAChRs. Conformational restriction of N‐n‐alkyl analogs of NIC appears to be a viable approach for the development of α3β2*‐selective nAChR antagonists. Drug Dev. Res. 55:172–186, 2002. © 2002 Wiley‐Liss, Inc.

Calcium channels involved in the inhibition of acetylcholine release by presynaptic muscarinic receptors in rat striatum.

1. The mechanism of the inhibitory action of presynaptic muscarinic receptors on the release of acetylcholine from striatal cholinergic neurons is not known. We investigated how the electrically stimulated release of [3H]-acetylcholine from superfused rat striatal slices and its inhibition by carbachol are affected by specific inhibitors of voltage-operated calcium channels of the L-type (nifedipine), N-type (omega-conotoxin GVIA) and P/Q-type (omega-agatoxin IVA). 2. The evoked release of [3H]-acetylcholine was not diminished by nifedipine but was lowered by omega-conotoxin GVIA and by omega-agatoxin IVA, indicating that both the N- and the P/Q-type (but not the L-type) channels are involved in the release. The N-type channels were responsible for approximately two thirds of the release. The release was >97% blocked when both omega-toxins acted together. 3. The inhibition of [3H]-acetylcholine release by carbachol was not substantially affected by the blockade of the L- or P/Q-type channels. It was diminished but not eliminated by the blockade of the N-type channels. 4. In experiments on slices in which cholinesterases had been inhibited by paraoxon, inhibition of [3H]-acetylcholine release by endogenous acetylcholine accumulating in the tissue could be demonstrated by the enhancement of the release after the addition of atropine. The inhibition was higher in slices with functional N-type than with functional P/Q-type channels. 5. We conclude that both the N- and the P/Q-type calcium channels contribute to the stimulation-evoked release of acetylcholine in rat striatum, that the quantitative contribution of the N-type channels is higher, and that the inhibitory muscarinic receptors are more closely coupled with the N-type than with the P/Q-type calcium channels.

Excitability of neural elements within the rat corpus striatum

The excitability of cholinergic, glutamatergic and dopaminergic elements within the rat neostriatum was studied in both in vivo and in vitro preparations. In vivo, the microdialysis technique was used to measure the release of striatal acetylcholine and dopamine under basal and electrically evoked conditions. For comparison, acetylcholine, dopamine and glutamate release was assayed in media obtained from superfused rat striatal slices. Electrical stimulation was used to derive the strength-duration functions and their chronaxies of stimulated elements containing the three neurotransmitter types. The chonaxies for experiments in vitro and in vivo were similar: the chronaxy values for elements containing acetylcholine were the shortest, the values for glutamate were intermediate, and the values for those containing dopamine were the longest. Based on the chronaxy estimates, it is proposed that the elements containing acetylcholine are the large cholinergic interneurons of striatum, and the elements containing glutamate and dopamine are the terminals of corticostriatal and nigrostriatal neurons, respectively. These results indicate that electrical stimulation of neural elements surrounding a microdialysis probe can be an additional tool to examine the factors that regulate neurotransmitter release. Likewise, investigators can activate specific striatal elements by using pulse durations that coincide with their chronaxies.

Modulation of potassium-evoked [3H]dopamine release from rat striatal slices by voltage-activated calcium channel ligands: effects of omega-conotoxin-MVIIC.

We examined the involvement of voltage-activated Ca2+ channels (VACCs) on K+(50 mM)-evoked [3H]dopamine ([3H]DA) release from superfused rat striatal slices. Neither nifedipine nor nitrendipine modified K(+)-evoked [3H]DA release, indicating that L-type VACCs are not involved. K(+)-evoked [3H]DA release was partially inhibited by omega-CTx-GVIA and omega-Aga-IVA, and was abolished by 3 microM omega-CTx-MVIIC (IC50 approximately 128 nM), suggesting the involvement of N-, P-, or Q-type VACCs, respectively. Moreover, even subnanomolar concentrations of omega-CTx-MVIIC (0.1-0.5 nM) inhibited K(+)-evoked [3H]DA release by approximately 25%, suggesting the possible involvement of a still not classified (perhaps O-type?) Ca2+ channel subtype. The effects of omega-CTx-MVIIC (10-100 nM) and omega-CTx-GVIA (1 microM) were additive, suggesting that low nanomolar concentrations of omega-CTx-MVIIC does not interact with N-type VACCs. In conclusion, the K(+)-evoked [3H]DA release from rat striatal slices is mediated by entry of Ca2+ through omega-CTx-GVIA sensitive (N-type) as well as through omega-Aga-IVA (P-type) and omega-CTx-MVIIC (probably Q-type) sensitive VACCs.

Endogenously released dopamine inhibits the binding of dopaminergic PET and SPECT ligands in superfused rat striatal slices.

Pharmacologically induced changes in synaptic levels of dopamine (DA) have been found, in some studies, to affect the in vivo binding of dopaminergic radioligands. In the present study we used a superfused brain slice preparation to examine the effect of synaptically released dopamine on the binding of some commonly used PET and SPECT radioligands under more controlled conditions than those present in vivo. The release of DA was evoked by electrical stimulation of striatal slices and the sensitivity of binding of the D1 receptor ligand, [3H]SCH 23390, the D2 receptor ligands [3H]raclopride and [123I]epidepride, and the DA uptake transporter ligands, [3H]WIN 35,428 and [123I]RTI-55, to the frequency of stimulation examined. Most affected by stimulation was the specific binding of [3H]SCH 23390, which was fully inhibited at 2.5 Hz. This was followed by [3H]raclopride and [123I]epidepride, respectively, the binding of the latter showing only a 50% reduction at the highest frequency of 10 Hz. [3H]WIN 35,428 and [123I]RTI-55 binding was unaffected by stimulation. The effects of stimulation on [3H]raclopride binding were prevented by reserpine pretreatment of the rat, when combined with inclusion of the dopamine synthesis inhibitor, alpha-methyl-p-tyrosine, in the superfusate medium. We conclude that, in brain slices, the binding of D1 and D2 receptor ligands but not that of DA uptake transporter ligands is readily inhibited by DA released into the synaptic cleft. Brain slices may prove to be a useful model system for the investigation of factors affecting competition between radioligand binding and endogenous neurotransmitters.

Intermittent morphine treatment causes long-term desensitization of functional dopamine D 2 receptors in rat striatum

3 weeks following cessation of intermittent morphine administration (10 mg/kg, s.c., once daily for 14 days), [ 3H]dopamine and [ 14C]acetylcholine release induced by 10 μM N-methyl- d-aspartate (NMDA) from superfused rat striatal slices appeared to be significantly higher than the release from striatal slices from saline-treated rats. A similar adaptive increase of the NMDA-evoked release of these neurotransmitters was observed in slices of the nucleus accumbens, whereas that of [ 3H]noradrenaline from hippocampal slices remained unchanged. Blockade of dopamine D 2 receptors by 10 μM (−)-sulpiride enhanced NMDA-induced [ 3H]dopamine and [ 14C]acetylcholine release from striatal slices from saline-treated animals, but was found to be ineffective in this respect following intermittent morphine treatment. Moreover, morphine administration appeared to cause a profound decrease in the apparent affinity of the full dopamine D 2 receptor agonist LY171555 (quinpirole) for these release-inhibitory dopamine D 2 receptors, indicating the occurrence of dopamine D 2 receptor desensitization. It is suggested that such a desensitization of dopamine D 2 receptors on dopaminergic nerve terminals as well as on cholinergic interneurons may play a pivotal role in the long-lasting nature of behavioural sensitization upon cessation of treatment with morphine and possibly other drugs of abuse.

Complex involvement of nitric oxide and cGMP at N-methyl- d-aspartic acid receptors regulating γ-[ 3H]aminobutyric acid release from striatal slices

Whilst the depolarization of postsynaptic N-methyl- d-aspartic acid (NMDA) receptors leads to an influx of Ca 2+ and subsequent synthesis of nitric oxide (NO), we examined roles for NO at striatal NMDA receptors regulating transmitter release. In superfused rat striatal slices, NMDA-evoked release of γ-[ 3H]aminobutyric acid ([ 3H]GABA) was investigated in the presence of nitrergic drugs. NMDA-induced release of [ 3H]GABA was attenuated by d-2-aminophosphonopentanoate, tetrodotoxin and omission of Ca 2+. l-Arginine enhanced NMDA-evoked release of [ 3H]GABA, but exogenous NO donors were ineffective. Inhibitors of NO synthase ( N G -nitro- and N G-amino- l-arginine) and guanylate cyclase (LY83583) elevated release. Since NMDA-evoked release of [ 3H]GABA was partially tetrodotoxin-sensitive, nitrergic-linked NMDA receptors regulating the release are both pre- and extrasynaptic. Thus not only does NO arise from multiple sites, and involve NMDA receptors with their redox site insensitive to exogenous NO donors, but the NMDA receptors are under the influence of nitrergic and cGMP-linked negative feedback mechanisms.

Minor alkaloids of tobacco release [ 3H]dopamine from superfused rat striatal slices

In addition to S(−)-nicotine, several minor tobacco alkaloids ((±)-nornicotine, anabaseine, S(−)-anabasine, and S(−)- N-methylanabasine) are present in tobacco smoke. This study demonstrates that these alkaloids increase fractional 3H release in a concentration-dependent manner from rat striatal slices preloaded with [ 3H]dopamine, with desensitization of this response. The rank order of EC 50 values was S(−)-nicotine (3.0 ± 2.2 μM) > (±)-nornicotine (6.7 ± 2.1 μM) > anabaseine (15.4 ± 6.1 μM) = S(−)- N-methylanabasine (16.3 ± 4.7 μM) = S(−)-anabasine (19.3 ± 3.2 μM). The alkaloids did not modulate fractional 3H release evoked by electrical-field depolarization. Thus, minor tobacco alkaloids may contribute to the apparent neuroprotective effects of smoking in neurodegenerative diseases.

Repeated and chronic morphine administration causes differential long-lasting changes in dopaminergic neurotransmission in rat striatum without changing its δ- and κ-opioid receptor regulation

Repeated, once daily morphine treatment (14 days) as well as chronic morphine administration (6 days) caused a rebound reduction in the electrically evoked release of [ 3H]dopamine from superfused rat striatal slices 1 day after the last subcutaneous injection. Interestingly, whereas [ 3H]dopamine release remained significantly reduced for at least 3 weeks following morphine withdrawal in chronically treated (tolerant/dependent) rats, neurotransmitter release from dopaminergic nerve terminals gradually increased above control values following cessation of repeated morphine administration. Postsynaptically, dopamine D 1 receptor-stimulated adenylate cyclase appeared to be sensitized 1–3 days but was unchanged 3 weeks after chronic morphine treatment. In contrast, such an enhanced postsynaptic dopamine D 1 receptor efficacy did not occur 1–3 days following repeated morphine administration, but appeared to develop slowly resulting in a profound increase of dopamine-sensitive adenylate cyclase 3 weeks after the last injection. The inhibitory effect of dynorphin A-(1–13) on [ 3H]dopamine release, as well as that of [Met 5]enkephalin on dopamine D 1 receptor-stimulated adenylate cyclase appeared to be unchanged subsequent to repeated or chronic morphine treatment. These data indicate that, long after cessation of drug treatment, chronic morphine treatment causes a reduction whereas repeated morphine administration gradually induces an enhancement of opioid receptor-regulated dopaminergic neurotransmission due to local adaptive changes within the rat striatum. Such distinct long-lasting alterations of dopaminergic neurotransmission induced by different temporal patterns of morphine administration in projection areas of mesencephalic dopaminergic neurons may be related to the enduring effects of drug abuse such as behavioural sensitization and drug craving.