Electrophysiological Characterization of AMPA and NMDA Receptors in Rat Dorsal Striatum

The striatum receives glutamatergic afferents from the cortex and thalamus, and these synaptic transmissions are mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to characterize glutamate receptors by analyzing NMDA/AMPA ratio and rectification of AMPA and NMDA excitatory postsynaptic currents (EPSCs) using a whole-cell voltage-clamp method in the dorsal striatum. Receptor antagonists were used to isolate receptor or subunit specific EPSC, such as (DL)-2-amino-5-phosphonovaleric acid (APV), an NMDA receptor antagonist, ifenprodil, an NR2B antagonist, CNQX, an AMPA receptor antagonist and IEM-1460, a GluR2-lacking AMPA receptor blocker. AMPA and NMDA EPSCs were recorded at -70 and +40 mV, respectively. Rectification index was calculated by current ratio of EPSCs between +50 and -50 mV. NMDA/AMPA ratio was 0.20±0.05, AMPA receptor ratio of GluR2-lacking/GluR2-containing subunit was 0.26±0.05 and NMDA receptor ratio of NR2B/NR2A subunit was 0.32±0.03. The rectification index (control 2.39±0.27) was decreased in the presence of both APV and combination of APV and IEM-1460 (1.02±0.11 and 0.93±0.09, respectively). These results suggest that the major components of the striatal glutamate receptors are GluR2-containing AMPA receptors and NR2A-containing NMDA receptors. Our results may provide useful information for corticostriatal synaptic transmission and plasticity studies.     

GTPγS Incorporation in the Rat Brain: A Study on μ-Opioid Receptors and CXCR4

Chemokine and opioid receptors are G-protein-coupled receptors that play important roles in both the central nervous system and the immune system. The long-term goal of our research is to establish whether opioids regulate the activity of the chemokine receptor CXCR4 (one of the major HIV co-receptors) in the brain. In this research, we studied the anatomical distribution of functional receptors in young and adult animals by using the [³⁵S]GTPγS “binding” assay as an indication of G-protein activation by CXCL12 (the natural CXCR4 ligand) or by μ-opioid agonists. Brain slices or homogenates from Holtzmann rats of different ages (from 2 to 21 days old and adult animals) were treated with CXCL12 (0.001–100 nM), d-ala2,MePhe4,gly-ol5]enkephalin (DAMGO; 0.0003–10 μM) or morphine (0.0003–10 μM) and then processed for the assay. Our results show stimulation of both μ-OR and CXCR4 in several brain areas, including cortex and hippocampus (p < 0.001); this effect is dose and age dependent, and the magnitude of response varies among different brain regions. Furthermore, AMD3100 (100 ng/ml), a specific CXCR4 antagonist, abolished CXCL12 stimulation in all the brain regions analyzed (p < 0.001). Our findings suggest a similar pattern of expression for μ-OR and CXCR4 in the brain, supporting the possibility of an interaction between the two G-protein-coupled receptors in vivo. This might be relevant to the role of opiates in HIV neuropathogenesis. This paper was presented at the 13th SNIP Meeting, Salt Lake City (Utah), April 11–14, 2007.     

NMDA Receptors,mGluR5, and Endocannabinoids are Involved in a Cascade Leading to Hippocampal Long-Term Depression

In the CA1 region of the rat hippocampus, metabotropic glutamate receptor-5 (mGluR5) and cannabinoid-1 receptors (CB1Rs) are believed to participate in long-term synaptic depression (LTD). How mGluRs and CB1Rs interact to promote LTD remains uncertain. In this study, we examined LTD induced by CB1R agonists, mGluR5 agonists, and low-frequency electrical stimulation (LFS) of the Schaffer collateral pathway. Synthetic CB1R agonists induced robust LTD that was mimicked by the endocannabinoid (EC), noladin ether (NLDE), but not by anandamide. 2-Arachidonylglycerol (2AG) produced only a small degree of LTD. The selective mGluR5 agonist, namely (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), also induced robust LTD, and CHPG and NLDE occluded each other''s effects. Similarly, CHPG and NLDE occluded LFS-induced LTD, and LTD resulting from all three treatments was blocked by a CB1R antagonist. CHPG-LTD and NLDE-LTD were insensitive to N-methyl--aspartate receptor (NMDAR) block, even though LFS-LTD requires NMDARs. LTD induced by LFS or CHPG, but not NLDE-LTD, was blocked by a selective mGluR5 antagonist. (RS)-3,5-dihydroxyphenylglycine (DHPG), a less selective group I mGluR agonist, also induced LTD, but its effects were not blocked by mGluR5 or CB1R antagonists. Furthermore, DHPG-LTD was additive with LFS-LTD and CHGP-LTD. These results suggest that NMDARs, mGluR5, and CB1Rs participate in a cascade that underlies LFS-LTD and that release of an EC and CB1R activation occur downstream of NMDARs and mGluR5. Furthermore, DHPG induces a form of LTD that differs mechanistically from LFS-induced depression.     

Forskolin Enhances Synaptic Transmission in Rat Dorsal Striatum through NMDA Receptors and PKA in Different Phases

The effect of forskolin on corticostriatal synaptic transmission was examined by recording excitatory postsynaptic currents (EPSCs) in rat brain slices using the whole-cell voltage-clamp technique. Forskolin produced a dose-dependent increase of corticostriatal EPSCs (1, 3, 10, and 30 µM) immediately after its treatment, and the increase at 10 and 30 µM was maintained even after its washout. When the brain slices were pre-treated with (DL)-2-amino-5-phosphonovaleric acid (AP-V, 100 µM), an NMDA receptor antagonist, the acute effect of forskolin (10 µM) was blocked. However, after washout of forskolin, an increase of corticostriatal EPSCs was still observed even in the presence of AP-V. When KT 5720 (5 µM), a protein kinase A (PKA) inhibitor, was applied through the patch pipette, forskolin (10 µM) increased corticostriatal EPSCs, but this increase was not maintained. When forskolin was applied together with AP-V and KT 5720, both the increase and maintenance of the corticostriatal EPSCs were blocked. These results suggest that forskolin activates both NMDA receptors and PKA, however, in a different manner.     

The Downregulation of Somatic A-Type K+ Channels Requires the Activation of Synaptic NMDA Receptors in Young Hippocampal Neurons of Rats

The downregulation of A-type K⁺ channels (I_A channels) accompanying enhanced somatic excitability can mediate epileptogenic conditions in mammalian central nervous system. As I_A channels are dominantly targeted by dendritic and postsynaptic processings during synaptic plasticity, it is presumable that they may act as cellular linkers between synaptic responses and somatic processings under various excitable conditions. In the present study, we electrophysiologically tested if the downregulation of somatic I_A channels was sensitive to synaptic activities in young hippocampal neurons. In primarily cultured hippocampal neurons (DIV 6~9), the peak of I_A recorded by a whole-cell patch was significantly reduced by high KCl or exogenous glutamate treatment to enhance synaptic activities. However, the pretreatment of MK801 to block synaptic NMDA receptors abolished the glutamate-induced reduction of the I_A peak, indicating the necessity of synaptic activation for the reduction of somatic I_A. This was again confirmed by glycine treatment, showing a significant reduction of the somatic I_A peak. Additionally, the gating property of I_A channels was also sensitive to the activation of synaptic NMDA receptors, showing the hyperpolarizing shift in inactivation kinetics. These results suggest that synaptic LTP possibly potentiates somatic excitability via downregulating I_A channels in expression and gating kinetics. The consequential changes of somatic excitability following the activity-dependent modulation of synaptic responses may be a series of processings for neuronal functions to determine outputs in memory mechanisms or pathogenic conditions.     

Involvement of Glutamate NMDA Receptors in the Acute,Long-Term,and Conditioned Effects of Amphetamine on Rat 50kHz Ultrasonic Vocalizations

Background:

Rats emit 50kHz ultrasonic vocalizations (USVs) in response to either natural or pharmacological pleasurable stimuli, and these USVs have emerged as a new behavioral measure for investigating the motivational properties of drugs. Earlier studies have indicated that activation of the dopaminergic system is critically involved in 50kHz USV emissions. However, evidence also exists that non-dopaminergic neurotransmitters participate in this behavioral response.

Methods:

To ascertain whether glutamate transmission plays a role in 50kHz USV emissions stimulated by amphetamine, rats received five amphetamine (1–2mg/kg, i.p.) administrations on alternate days in a test cage, either alone or combined with the glutamate N-methyl-D-aspartate receptor antagonist MK-801 (0.1–0.5mg/kg, i.p.). Seven days after treatment discontinuation, rats were re-exposed to the test cage to assess drug conditioning, and afterwards received a drug challenge. USVs and locomotor activity were evaluated, along with immunofluorescence for Zif-268 in various brain regions and spontaneous alternation in a Y maze.

Results:

Amphetamine-treated rats displayed higher 50kHz USV emissions and locomotor activity than vehicle-treated rats, and emitted conditioned vocalizations on test cage re-exposure. Rats co-administered amphetamine and MK-801 displayed lower and dose-dependent 50kHz USV emissions, but not lower locomotor activity, during repeated treatment and challenge, and scarce conditioned vocalization compared with amphetamine-treated rats. These effects were associated with lower levels of Zif-268 after amphetamine challenge and spontaneous alternation deficits.

Conclusions:

These results indicate that glutamate transmission participates in the acute, long-term, and conditioned effects of amphetamine on 50kHz USVs, possibly by influencing amphetamine-induced long-term neuronal changes and/or amphetamine-associated memories.     

Evidence for Functional δ-Opiate Receptors in the Rat Intestine

Abstract— The selective δ-opiate agonists d -Ser², Leu⁵, Thr⁶-enkephalin (DSLET), d -Ala², d -Leu⁵-enkephalin and d -Pen², d -Pen⁵-enkephalin caused inhibition of the cholinergic contraction produced by transmural stimulation of the rat isolated jejunum. Dynorphin A, which is an agonist at both κ- and δ-opioid receptors also inhibited the cholinergic contraction, as did leu- and met-enkephalin. The selective μ-receptor agonist d -Ala²-NMe-Phe⁴, Gly-ol⁵-enkephalin was the least potent of all peptides tested. In general, the order of potency of the peptides was similar to that reported for the δ-receptor-rich mouse vas deferens with potency values similar to those recorded previously for the hamster vas deferens. The selective δ-opioid antagonist naltrindole caused parallel shifts to the concentration-effect curve to DSLET giving a pA₂ value of 10·15. The results indicate that the previously identified δ-binding sites in the rat jejunum may correspond to functional δ-opiate receptors involved in attenuating acetylcholine release.     

Impairment of the Ca2+‐Permeable AMPA/Kainate Receptors by Lead Exposure in Organotypic Rat Hippocampal Slice Cultures

Previous studies have demonstrated that chronic lead exposure may impair neuronal process underlying synaptic plasticity via a direct interaction with N‐methyl‐D‐aspartate (NMDA) receptors. The present study was carried out to investigate the effects of lead exposure on non‐NMDA (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid, AMPA/kainate) receptors of rat hippocampus. Ca²⁺‐permeable AMPA/kainate receptors in organotypic slice cultures were evaluated by using cobalt uptake, a histochemical method that identifies cells expressing Ca²⁺‐permeable non‐NMDA receptors. Ten mM L‐glutamate‐induced cobalt accumulation was enriched in area CA1, area CA3 and in dentate gyrus, which was totally blocked by 100 μM DL‐2‐amino‐5‐phosphonovaleric acid (AP5) and 100 μM 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (CNQX). Three hundred μM NMDA‐induced cobalt accumulation was in area CA1, area dentate gyrus and was blocked by AP5 or CNQX. One hundred μM AMPA had effects in area CA1, area CA3 and in dentate gyrus, which were blocked by CNQX, not by AP5. Furthermore, cobalt accumulations induced by NMDA and AMPA in the lead‐exposed rats decreased significantly than those in the controls. The results indicate that AMPA receptors enriched in area CA1, area CA3, area dentate gyrus and kainate receptors enriched in area CA1, area dentate gyrus are impaired by lead exposure.     

Effects of Ethanol and Acetate on Adenosine Production in Rat Hippocampal Slices

Abstract: Since adenosine has been shown to mediate some actions of ethanol we have examined the effect of ethanol (20 and 80 mM) or its metabolite acetate (5 and 20 mM) on the formation and release of adenosine by rat hippocampal slices. The ATP pool of the slices was radioactively labelled by preincubation with [³H]-adenine. The efflux of radioactivity under basal conditions and following ATP breakdown induced by combined hypoxia/hypoglycaemia was examined. Ethanol or acetate did not increase the total efflux of [³H]-purines, but changed the composition to a larger proportion of [³H]-adenosine. The release of endogenous adenosine was also increased. This type of effect exactly mirrors that previously reported for purine nucleoside transport inhibitors. The present results thus show that ethanol (20 mM) can increase adenosine release from a brain slice by a mechanism that probably involves transport inhibition.     

Do 5-HT4 Receptors Mediate the Intestinal Secretory Response to 5-HT in Rat In-vivo?

The involvement of the recently characterized 5-HT₄ receptor in the actions of 5-hydroxytryptamine (5-HT) on jejunal, ileal and colonic electrogenic ion secretion was investigated in the rat in-vivo. 5-HT and the 5-HT₁-, 5-HT₂- and 5-HT₄-receptor agonist 5-methoxytryptamine (5-MeOT), induced a rise in transintestinal PD in all regions of the gut. However, the 5-HT₄-receptor agonists renzapride and cisapride had no effect. Furthermore, the 5-HT₄-receptor antagonists SDZ 205–557 (2-diethylaminoethyl-[2-methoxy-4-amino-5-chloro] benzoate), tropisetron and SB 204070 ([1-butyl-4-piperidinylmethyl]-8-amino-7-chloro-1,4-benzodioxan-5-carboxylate hydrochloride) did not affect the secretory response to either 5-HT or 5-MeOT in the jejunum, but did cause a small inhibition in the ileum and colon. It is concluded that 5-HT₄ receptors do not make a contribution to the electrically monitored 5-HT intestinal secretory response in the rat jejunum in-vivo, but may play a small role in the ileum and colon.     

Receptor Binding Domains of TcdB from Clostridioides difficile for Chondroitin Sulfate Proteoglycan-4 and Frizzled Proteins Are Functionally Independent and Additive

Toxin B (TcdB) produced by Clostridioides difficile is a main pathogenicity factor that affects a variety of different cell types within the colonic mucosa. TcdB is known to utilize frizzled-1,2,7 and chondroitin sulfate proteoglycan-4 (CSPG4) as protein receptors. By using human cervical cancer cell line HeLa CSPG4 knockout (CSPG4^−/−) cells as well as TcdB mutants which do not bind to either CSPG4 or frizzled-1,2,7, or both, we evaluated the impact of the individual receptors for cytopathic and cytotoxic effects of TcdB. We compared TcdB from the reference strain {"type":"entrez-protein","attrs":{"text":"VPI10463","term_id":"1642177071","term_text":"VPI10463"}}VPI10463 (TcdB_VPI) and the endemic strain {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 (TcdB_R20) which does not interact with frizzled-1,2,7. TcdB_VPI devoid of CSPG4 binding (TcdB_VPI ΔCROP) shows identical cytopathic potency as full-length TcdB in HeLa CSPG4^−/− cells, indicating that interaction with frizzled proteins is not affected in the presence of the C-terminal CROP domain. We validated CSPG4 as cellular receptor for both TcdB toxinotypes in HeLa and HEp-2 cells. By exchange of a single phenylalanine residue, 1597 with serine, we generated a mutated TcdB_VPI variant (TcdB_VPI F1597S) that in accordance with TcdB_R20 lacks binding to frizzled-1,2,7 and showed identical potency as TcdB_R20 on HeLa cells. This enabled us to estimate the respective share of CSPG4 and frizzled-1,2,7 in the cytotoxic and cytopathic effect induced by TcdB. Our data reveal that binding to frizzled-1,2,7 and to CSPG4 occurs independently and in an additive manner.     

Biphasic Effects of Cannabinoids in Anxiety Responses: CB1 and GABAB Receptors in the Balance of GABAergic and Glutamatergic Neurotransmission

Biphasic effects of cannabinoids have been shown in processes such as feeding behavior, motor activity, motivational processes and anxiety responses. Using two different tests for the characterization of anxiety-related behavior (elevated plus-maze and holeboard), we first identified in wild-type C57BL/6N mice, two doses of the synthetic CB1 cannabinoid receptor agonist CP-55,940 with anxiolytic (1 μg/kg) and anxiogenic properties (50 μg/kg), respectively. To clarify the role of CB1 receptors in this biphasic effect, both doses were applied to two different conditional CB1 receptor knockout (KO) mouse lines, GABA-CB1-KO (CB1 receptor inactivation in forebrain GABAergic neurons) and Glu-CB1-KO (CB1 receptor inactivation in cortical glutamatergic neurons). We found that the anxiolytic-like effects of the low dose of cannabinoids are mediated via the CB1 receptor on cortical glutamatergic terminals, because this anxiolytic-like response was abrogated only in Glu-CB1-KO mice. On the contrary, the CB1 receptor on the GABAergic terminals is required to induce an anxiogenic-like effect under a high-dose treatment because of the fact that this effect was abolished specifically in GABA-CB1-KO mice. These experiments were carried out in both sexes, and no differences occurred with the doses tested in the mutant mice. Interestingly, the positive allosteric modulation of GABA_B receptor with GS-39783 was found to largely abrogate the anxiogenic-like effect of the high dose of CP-55,940. Our results shed new light in further understanding the biphasic effects of cannabinoids at the molecular level and, importantly, pave the way for the development of novel anxiolytic cannabinoid drugs, which may have favorable effect profiles targeting the CB1 receptor on glutamatergic terminals.     

抑郁症模型大鼠血清雌激素水平与海马5-HT含量之间的关系

目的:观察抑郁症模型大鼠血清雌二醇(E2)水平、海马5-羟色胺(5-HT)含量及其中缝核雌激素受体β(ERβ)和色氨酸羟化酶2(TPH2)表达的变化,探讨抑郁症发生的可能机制.方法:雌性SD大鼠20只,随机平均分为正常组和模型组,模型组给予孤养和21 d慢性不可预见性的温和应激(CUMS)制备抑郁症大鼠模型.采用放射免疫分析法(RIA)测定血清雌二醇(E2)水平、高效液相色谱-电化学检测法(HPLC-ECD)测定海马5-羟色胺(5-HT)含量、RT-PCR技术检测中缝核ERβ mRNA和TPH2 mRNA表达以及荧光免疫组化双重染色法检测中缝核ERβ蛋白和TPH2蛋白表达.结果:21 d应激后,模型组大鼠血清E2水平下降、海马5-HT含量减少、中缝核ERβ mRNA和TPH2 mRNA相对表达量下降及ERβ阳性细胞数和TPH2阳性细胞数明显减少.结论:E2与海马5-HT之间可能存在E2→中缝核5-HT能神经元ERβ→TPH2→海马5-HT的功能作用链.     

The Sleep-Promoting and Hypothermic Effects of Glycine are Mediated by NMDA Receptors in the Suprachiasmatic Nucleus

The use of glycine as a therapeutic option for improving sleep quality is a novel and safe approach. However, despite clinical evidence of its efficacy, the details of its mechanism remain poorly understood. In this study, we investigated the site of action and sleep-promoting mechanisms of glycine in rats. In acute sleep disturbance, oral administration of glycine-induced non-rapid eye movement (REM) sleep and shortened NREM sleep latency with a simultaneous decrease in core temperature. Oral and intracerebroventricular injection of glycine elevated cutaneous blood flow (CBF) at the plantar surface in a dose-dependent manner, resulting in heat loss. Pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists AP5 and {"type":"entrez-protein","attrs":{"text":"CGP78608","term_id":"876863604","term_text":"CGP78608"}}CGP78608 but not the glycine receptor antagonist strychnine inhibited the CBF increase caused by glycine injection into the brain. Induction of c-Fos expression was observed in the hypothalamic nuclei, including the medial preoptic area (MPO) and the suprachiasmatic nucleus (SCN) shell after glycine administration. Bilateral microinjection of glycine into the SCN elevated CBF in a dose-dependent manner, whereas no effect was observed when glycine was injected into the MPO and dorsal subparaventricular zone. In addition, microinjection of D-serine into the SCN also increased CBF, whereas these effects were blocked in the presence of L-701324. SCN ablation completely abolished the sleep-promoting and hypothermic effects of glycine. These data suggest that exogenous glycine promotes sleep via peripheral vasodilatation through the activation of NMDA receptors in the SCN shell.     

Glutamatergic control of food intake in pigeons: effects of central injections of glutamate, NMDA, and AMPA receptor agonists and antagonists

The possible involvement of glutamatergic mechanisms in the control of food intake was studied in free-feeding and in 24-h food-deprived (FD24) pigeons for 1 h after intracerebroventricular (i.c.v.) treatment with glutamate (Glu, 0, 50, 150, 300, and 600 nmol). Glu injections dose dependently induced decreases (30-65%) in food intake (FI) and feeding duration (FD), and increases in latency to start feeding (LSF) in FD24 animals, but not in free-feeding ones. None of these treatments affected noningestive behaviors (locomotion, sleep, and preening). In FD24 pigeons, i.c.v. treatments with N-methyl-D-aspartic acid (NMDA, 0.1, 1, 4, 8, or 16 nmol) or D,L-alpha-amino-3-hydroxy-isoxazole proprionic acid (AMPA, 0.1, 1, 4, or 8 nmol) decreased FI and FD, but left LSF unchanged compared to vehicle-treated FD24 controls. Kainic acid (0.1, 0.5, and 1 nmol), or [trans-(1S,3R)-ACPD-(5NH4OH)] (ACPD, 0.1, 1, 4, 8, and 16 nmol) left unchanged the ingestive profile of FD24 pigeons. Pretreatment with the NMDA receptor antagonist MK-801 (15 nmol) and the AMPA-kainate receptor antagonist CNQX (390 nmol), 20 min before an i.c.v. injection of Glu (300 nmol) induced a partial blockade of the Glu-induced decreases in FI and FD and completely inhibited the Glu-induced increase in LSF in FD24 pigeons. I.c.v. injections of MK-801 (30 nmol) and of CNQX (780 nmol) increased FI and FD and reduced LSF in free-feeding pigeons. A lower dose of MK-801 (15 nmol) increased FI and FD, but not LSF. Conversely, a lower dose of CNQX (390 nmol) reduced LSF without changing FI or FD. These findings indicate the involvement of Glu as a chemical mediator in the regulation of food intake in the pigeon, possibly acting on multiple central mechanisms in this species through NMDA- and AMPA-sensitive Glu receptors.     

Controlled Release of Lidocaine from Injectable Gels and Efficacy in Rat Sciatic Nerve Block

Purpose. Methods of delaying the action of local anesthetics are important, since short duration of action limits their use in the treatment of postoperative and chronic pain. The present study evaluated the use of low-viscosity gels in prolonging the release of lidocaine. Methods. Release of lidocaine from 2% lidocaine-HC1 containing methylcellulose (MC), hydroxypropylmethylcellulose (HPMC), sodiumcarboxymethyl cellulose (CMC), and poloxamer 407 (PO) gels was studied in phosphate buffer, pH 7.4, at 37°C. Commercial metylcellulose gel (MC_com) served as control. The in vivo efficacy of the respective gel formulations were evaluated in rats. The gel was injected into the vicinity of the sciatic nerve and nociception and motor function were tested. Results. The cumulative amount of lidocaine released during 8 hr was slowest from the PO gel, followed by the CMC, HPMC and MC gels. The antinociceptive effect was not prevented by the motor block and lasted longest with the PO gel. Good linear and rank order correlation was obtained between in vitro and in vivoresults. The microscopic examination of the tissue samples revealed only mild or no irritation of the skeletal muscle tissue by the PO, HPMC, and CMC gels. Conclusions. Based on these results poloxamer gel proved to be the most promising carrier for lidocaine.     

Effects of Remoxipride's Metabolites on Dopamine D2 Receptors and Receptor Functions in the Rat

Abstract: The main metabolites of remoxipride formed in rat and man were examined for their affinities for the [³H] SCH 23390-labelled DA D₁ and [³H]-raclopride-labelled D₂ receptors in rat striatal homogenates. In addition, their effectiveness in blocking postsynaptic DA receptor activity in vivo was measured by the use of several different test models in the male rat. Phenolic metabolites formed mainly in the rat retained (similar to remoxipride) their selectivity for the D₂ receptor with very low affinities for the D₁ receptor. The pyrrolidone metabolites formed mainly in man showed very low affinities for both the D₁ and D₂ receptors. The ability of the metabolites to block postsynaptic DA receptor activity in vivo correlated with their affinities for the D₂ receptor. Among the metabolites tested, the phenolic compounds FLA 797 (–) and FLA 908 (–) were much more effective than remoxipride in inducing catalepsy, which is consistent with a higher affinity for [³H] raclopride labelled striatal D₂ receptors. However, analysis of the effectiveness of the DA receptor blockade (blockade of d-amphetamine locomotion or DA agonist hypothermia) after intraperitoneal or subcutaneous administration suggested that FLA 797 (–)/FLA 908 (–) may only contribute marginally to the D₂ receptor-blocking activity of remoxipride in the rat. This conclusion was further supported by the observation that the atypical antipsychotic profile of remoxipride was not mimicked by the active metabolites. The weak DA D₂ blocking effect of the pyrrolidone metabolites indicated that remoxipride is responsible for the clinical action.     

The Peristaltic Reflex in the Rat Ileum: Evidence for Functional μ- and δ-Opiate Receptors

The potency order of opiate agonists at decreasing the rate of peristalsis in the rat isolated ileum was: difenoxin > loperamide > DADLE (d -Ala²-d -Leu⁵-enkephalin) > morphine > DSLET (d -Ser², Leu⁵-Thr⁶-enkephalin). U-50488 (trans 3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl) cyclohexyl) benzeneacet-amide methane sulphonate) was inactive at 300 nm . Naloxone (400 nm ) caused a significant 1·52-fold increase in the rate of peristaltic contractions and inhibited the effects of the active opiate agonists. The apparent pA₂ values of naloxone were similar using difenoxin, loperamide and morphine as agonists, but the value was slightly, though significantly lower when DADLE was the agonist. It is suggested that the previously identified δ-opiate receptors of the rat small intestine have a functional role in suppressing the peristaltic reflex. The same response is subserved by μ-opiate receptors and either of these opiate-receptor subtypes could be activated by endogenous enkephalins.