Orphanin FQ/nociceptin attenuates motor stimulation and changes in nucleus accumbens extracellular dopamine induced by cocaine in rats

RATIONALE: Orphanin FQ (OFQ; also known as nociceptin), the endogenous ligand of the opioid receptor-like receptor, injected intracerebroventricularly (i.c.v.) decreases basal motor activity and basal extracellular levels of dopamine (DA) in the nucleus accumbens (Nuc Acc) in rats. OBJECTIVE: The present study was designed to determine if OFQ similarly attenuates cocaine-induced motor stimulation and to determine if this effect is dependent on attenuation of the increase in extracellular DA. METHODS: After a 1-h adaptation period, rats were injected with either artificial cerebrospinal fluid or OFQ (3-30 nmol, i.c.v.) 5 min prior to cocaine (10 mg/kg, i.p.) or apomorphine (3 mg/kg, i.p.) administration and the total distance traveled was measured for a further 1 h. In a separate experiment, changes in extracellular DA were monitored by microdialysis following cocaine and OFQ treatment in anesthetized rats. RESULTS: OFQ dose-dependently attenuated both basal and cocaine-induced motor stimulation. OFQ (30 nmol, i.c.v.) also attenuated both the basal and the cocaine-induced increase in extracellular DA in the Nuc Acc. OFQ, at the highest dose, also decreased the motor stimulation induced by apomorphine. CONCLUSIONS: Our results suggest that the modulatory effect of OFQ on locomotor activity is not solely due to its inhibitory action on extracellular DA in the Nuc Acc.     

The endogenous OFQ/N/ORL-1 receptor system regulates the rewarding effects of acute cocaine

Previous studies have shown that orphanin FQ/nociceptin (OFQ/N), the endogenous ligand of the opioid receptor-like (ORL-1) receptor, reduces the rewarding and addictive properties of cocaine and other drugs of abuse. In the present study, using the conditioned place preference (CPP) paradigm, as an animal model of drug reward, we assessed whether the rewarding action of acute cocaine would be altered in mice lacking the ORL-1 receptor or in wild type mice treated with J-113397, an ORL-1 receptor antagonist, relative to their saline-treated controls. On day 1, mice were tested for their baseline place preferences, in which each mouse was placed in the neutral chamber of a three-chambered CPP apparatus, allowed to freely explore all the chambers and the amount of time that a mouse spent in each conditioning chamber was recorded for 15 min. On days 2-3, mice received once daily alternate-day saline/cocaine (15 or 30 mg/kg) conditioning for 30 min. On day 4, mice were tested for their postconditioning preferences, as described for day 1. In a subsequent study, the effect of J-113397 (3 mg/kg) on the rewarding action of acute cocaine (15 mg/kg) was also examined in wild type mice. Our results showed that mice lacking the ORL-1 receptor expressed greater CPP than their wild type littermates. Furthermore, the rewarding action of cocaine was enhanced in the presence of J-113397 in wild type mice. Together, the present results suggest that the endogenous OFQ/N/ORL-1 receptor system is involved in the rewarding action of acute cocaine.     

孤啡肽对大鼠顶叶皮层神经元延迟整流钾电流的抑制作用

目的研究孤啡肽(orphanin FQ/nociceptin,OFQ/N)对大鼠顶叶皮层神经元延迟整流钾电流(IK)的影响,初步探讨其干扰学习记忆过程的离子机制。方法采用全细胞膜片钳技术,观察OFQ/N对急性分离的大鼠顶叶皮层神经元IK的作用。结果①OFQ/N明显抑制IK,并呈剂量依赖性(P<0.05)。②0.1μmol.L-1OFQ/N使IK的电流-电压(I-V)曲线降低(n=8,P<0.01)。③0.1μmol.L-1OFQ/N使IK激活曲线的半数激活电压(V1/2)和斜率因子(k)分别由给药前的(-43.4±6.1)mV和(11.5±1.1)mV变为给药后的(-19.1±4.6)mV和(17.3±3.2)mV(n=8,P<0.01)。④0.1μmol.L-1OFQ/N使IK失活曲线的半数失活电压(V1/2)和斜率因子(k)分别由给药前的(-68.8±2.6)mV和(16.5±1.7)mV变为给药后的(-76.8±2.8)mV(n=5,P<0.01)和(15.7±3.1)mV(n=5,P>0.05)。结论OFQ/N可抑制大鼠顶叶皮层神经元IK,使IK激活曲线右移,失活曲线左移。     

The ORL-1 (NOP1) receptor ligand nociceptin/orphanin FQ (N/OFQ) inhibits neurogenic dural vasodilatation in the rat

The effects of the ORL-1 (NOP(1)) receptor ligand nociceptin (N/OFQ) and the nociceptin antagonists [Nphe(1)]N/OFQ-(1-13)-NH(2) (Nphe) and nocistatin (NST) on neurogenic dural vasodilatation (NDV) in the rat dura mater evoked by electrical stimulation of a closed cranial window were studied. The middle meningeal artery was visualised using intravital microscopy, and the vessel diameter analysed using a video dimension analyser. N/OFQ (1, 10, 100 nmol kg(-1); i.v., n=10) significantly and dose-dependently suppressed NDV maximally by 65% (P<0.01). Neither Nphe (100 nmol kg(-1); n=5) nor NST (100 nmol kg(-1); n=4) alone had an effect on NDV (P>0.05). Baseline vessel diameter was not significantly affected by application of N/OFQ, NST or Nphe. Application of the selective N/OFQ antagonist Nphe (10, 100 nmol kg(-1) i.v., n=8) dose-dependently and significantly (P<0.01) reversed the inhibition of NDV induced by application of N/OFQ (10 nmol kg(-1)). NST (10, 100 nmol kg(-1); n=7) failed to reverse the effects elicited by N/OFQ. Application of N/OFQ elicited a dose-dependent transient decrease in arterial blood pressure (P<0.01). Nphe dose-dependently reversed the cardiovascular effects induced by application of N/OFQ (10 nmol kg(-1)) (P<0.01),while NST did not alter the blood pressure reaction elicited by N/OFQ. The results show that N/OFQ inhibits NDV, an effect which is antagonised by Nphe, but not by NST. ORL-1 (NOP(1)) receptors located on trigeminal sensory fibres may be involved in the regulation of dural vessel diameter and hence may play a role in migraine pathophysiology.     

Functional antagonism between nociceptin/orphanin FQ (N/OFQ) and corticotropin-releasing factor (CRF) in the rat brain: evidence for involvement of the bed nucleus of the stria terminalis

Rationale Nociceptin/orphanin FQ (N/OFQ) has been proposed to be a functional antagonist of corticotropin-releasing factor (CRF) in relation to its anti-stress action and its ability to antagonize the anorectic effect of CRF in rats without exhibiting affinity for CRF receptors. The bed nucleus of the stria terminalis (BST) is highly sensitive to the inhibitory effect of N/OFQ on CRF-induced anorexia. Objective The present study was aimed at further evaluating the role of the BST in the functional antagonism between N/OFQ and CRF by examining it at molecular level and in the context of CRF-induced anxiety in the rat. Materials and methods First, in situ hybridization experiments investigated the expression of the pro-N/OFQ precursor and of NOP receptors in several brain areas 6 h after injection of CRF (0.2 and 1 μg/rat) into the lateral cerebroventricle (LV). Second, the elevated plus maze test was used to evaluate whether N/OFQ, injected into the BST (0.05 and 0.5 μg/rat) or into the LV (0.5, 1.8, and 2.4 μg/rat), inhibits the anxiogenic-like effect evoked by LV injection of CRF (1 μg/rat) in rats. Results The in situ hybridization study showed that LV injection of CRF 1 μg/rat increases NOP receptor expression in the BST, while no change of the N/OFQ precursor was observed. On the other hand, N/OFQ injection into the BST blocks the anxiogenic effect of CRF at doses lower than those required by LV injection (0.5 vs 1.8 μg/rat, respectively). Conclusion These data provide further support for the hypothesis that N/OFQ may behave as functional antagonist of CRF and suggest that this antagonism may occur within the BST.     

Anti-allodynic and anti-hyperalgesic effects of nociceptin receptor antagonist, JTC-801, in rats after spinal nerve injury and inflammation

The effects of nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist JTC-801 on allodynia and hyperalgesia were examined in rats in order to explore the involvement of N/OFQ system in these pathological pain states. Tactile allodynia induced by L5/L6 spinal nerve ligation was reversed by both systemic (3–30 mg/kg) and spinal (22.5 and 45 pg) JTC-801 in a dose-dependent manner. Concerning hyperalgesia induced by formalin injection into the hindpaw, JTC-801 dose-dependently suppressed the second phase, but not the first phase, of the licking behavior. Furthermore, systemic JTC-801 reduced Fos-like immunoreactivity in the dorsal horn of the spinal cord (laminae I/II). In conclusion, N/OFQ receptor antagonist JTC-801 exerted anti-allodynic and anti-hyperalgesic effects in rats, suggesting that N/OFQ system might be involved in the modulation of neuropathic pain and inflammatory hyperalgesia.     

Recent advances towards the discovery of ORL-1 receptor agonists and antagonists

Since their discovery a decade ago, remarkable progress has been made toward understanding the biological function and significance of the opioid receptor-like-1 (ORL-1) receptor and its endogenous peptide ligand, nociceptin. The human nociceptin receptor, herein referred to as ORL-1, but also known as OP4 (the fourth member of opioid peptide receptor family) or nociceptin/orphanin FQ peptide (NOP) receptor, was first identified as an orphan opioid receptor with close homology to the classical μ-, κ-, and δ-opioid receptors. ORL-1 does not bind endogenous ligands of the other opioid receptors with high affinity, but instead prefers the 17 amino acid peptide nociceptin. The obvious homologies of ORL-1 to opioid receptors, and its ligand nociceptin to opioid peptide ligands, led to a period of intense investigation that resulted in a number of significant reports describing the biology of the receptor and ligand. The emerging pharmacological evidence from these reports suggests that ORL-1 agonists may be clinically useful for treatment of stress, anxiety, substance abuse (opioid and alcohol), anorexia, cachexia, cough, asthma, and possibly neuropathic pain/allodynia. The peripheral effects of nociceptin suggest that agonists may have utility in the treatment of gastrointestinal motility disorders, water retention, and hypertension. ORL-1 antagonists may be useful in enhancing cognitive function and treating locomotor disorders such as Parkinsonism. In addition to research into the fundamental biology of ORL-1 and nociceptin, noteworthy advances have been made in the discovery of new peptide and non-peptide agonists and antagonists of the ORL-1 receptor leading to a better understanding of its involvement in a variety of biological processes. This review highlights the rationale for the development of ORL-1 ligands and recent progress made by different research groups towards the development of peptidic and non-peptidic ORL-1 agonists or antagonists over the last four years. To add perspective on the commercial potential of this research area, the development status of advanced new molecules is addressed together with any pharmacological characterisation of these entities.     

Alterations of N/OFQ and NOP receptor gene expression in the substantia nigra and caudate putamen of MPP and 6-OHDA lesioned rats

It has been suggested that the opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOPr) may contribute to Parkinson's disease. Based on this idea, the aim of our study was to investigate the involvement of the N/OFQ-NOPr system in an animal model of Parkinson's disease and to evaluate if this neuropeptidergic system is acting through mechanisms involving glutamate and/or GABA. We injected the neurotoxins MPP⁺ or 6-OHDA into the cerebral ventricles and 10 days later measured N/OFQ and NOPr gene expression in caudate putamen (CP) and substantia nigra (SN), by RT-PCR. A large reduction in N/OFQ and NOPr mRNAs was observed in the CP of rat treated with either MPP⁺ or 6-OHDA, MPP⁺ being more effective than 6-OHDA. Both the neurotoxins induced an increase in N/OFQ gene expression in the SN, but only MPP⁺ evoked a significant down-regulation of NOPr in this area, showing a slight trend of reduction in 6-OHDA treated rats. Moreover, a reduction in the levels of glutamic acid decarboxylase (GAD_65/67), an enzyme that converts the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), was also observed in the SN following 6-OHDA. These data suggest that DA modulates N/OFQ-NOPr system gene expression in SN and CP, strengthening the hypothesis that this neuropeptidergic system could be implicated in the mechanisms underlying Parkinson's disease. Our data might also suggest that the GABAergic system plays a role in the regulation of nigral function, although further studies are necessary to confirm this hypothesis. In agreement with previous studies, we also support the hypothesis of a potential value for NOP receptor antagonists to attenuate symptoms related to the degeneration of nigrostriatal dopaminergic pathway.     

Development and persistence of long-lasting behavioral sensitization to cocaine in female mice: Role of the nNOS gene

Our recent studies have shown that the neuronal nitric oxide synthase (nNOS) gene is required for the development and persistence of psychomotor sensitization to cocaine in adult but not adolescent male mice (Balda, M.A., Anderson, K.L., Itzhak, Y., 2008. Differential role of the nNOS gene in the development of behavioral sensitization to cocaine in adolescent and adult B6;129S mice. Psychopharmacology (Berlin) 200, 509-519.). The aim of the present study was to investigate the contribution of the nNOS gene to cocaine-induced behavioral sensitization in adolescent and adult female mice. Adolescent and adult wild type (WT) and nNOS knockout (KO) mice received saline or cocaine (20 mg/kg) for 5 days and then were challenged with cocaine (20 mg/kg) after a drug-free period of either 10, 30, or 90 days. Context-dependent sensitization was determined by measuring saline-induced locomotor activity in the previously cocaine-paired environment. Results show that adolescent females of both genotypes, like their adult counterparts, developed long-lasting behavioral sensitization to cocaine (a three-month period), suggesting high vulnerability of females to cocaine regardless of age. An effect of genotype was observed in the initiation of sensitization, e.g., delayed onset in the absence of the nNOS gene. The only age-dependent difference observed was that adult, but not adolescent mice developed context-dependent sensitization. The present study suggests that long-term expression of cocaine-induced behavioral sensitization in females (adolescent and adult) is nNOS-independent, unlike our previous findings in adult males.     

Pharmacological reversal of behavioral and cellular indices of cocaine sensitization in the rat

Rationale and objectives: Behavioral sensitization has been proposed as an animal model for the intensification of drug craving in cocaine addiction. Interactions between dopamine and glutamate systems are important for the induction and maintenance of sensitization. The goal of this study was to determine if established cocaine sensitization could be reversed by pharmacological manipulation of these transmitter systems. Methods: Rats received 15 mg/kg cocaine (IP) on days 1–10 and were challenged with cocaine (10 mg/kg) on day 13 to verify that sensitization had occurred. On days 14–20, separate groups of sensitized rats received daily injections of dopamine D₁- or D₂-class agonists, an NMDA receptor antagonist, or a dopamine agonist with an NMDA antagonist. Three days or 2 weeks later, all rats were again tested for their response to cocaine to determine if sensitization had been reversed. Results: Reversal of sensitization was produced by repeated administration of either a D₁-class agonist (SKF 81297) or the combination of an NMDA receptor antagonist and a D₂-class agonist. Effective combinations were cocaine+MK-801, quinpirole+MK-801, quinpirole+CGS 19755, and pergolide+memantine. The latter drugs are approved for human use. Reversal of sensitization persisted for at least 2 weeks after cessation of drug treatment. Electrophysiological studies revealed that these drug treatments also reversed dopamine D₁ receptor supersensitivity in the nucleus accumbens, a cellular correlate of sensitization. Conclusions: These results demonstrate that pharmacotherapy can reverse behavioral and cellular adaptations associated with repeated cocaine administration, and may do so without the need for continued medication. Received: 1 October 1999 / Accepted: 19 March 2000     

Lead generation and lead optimisation approaches in the discovery of selective,non-peptide ORL-1 receptor agonists and antagonists

The discovery of the opioid receptor like-1 (ORL-1) receptor and of its endogenous agonist nociceptin/orphanin FQ has attracted great interest in the scientific community giving rise, in the last five years, to a flurry of biological studies aimed at elucidating the role of this new receptor. Hence, the involvement of the ORL-1 receptor in many important processes, such as antinociception, learning and memory, feeding and anxiety, has been well documented. However, a clear understanding of the potential therapeutic value associated with the modulation of the ORL-1 receptor needs the development of selective non-peptide agonists and antagonists allowing systemic routes of administration. This review addresses the advances made by several research groups in the discovery of such compounds and discusses the medicinal chemistry strategies which, starting from the first non-selective ligands NalBzoH and lofentanil, led to the disclosure of highly potent and selective agonists and antagonists, such as Ro 64-6198, J-113397 and JTC-801. Efforts have also focussed on the pharmacological characterisation of the newly discovered non-peptide tools, which represent a significant step forward in the understanding of the involvement of the ORL-1 receptor in a number of possible pathophysiological conditions.     

Differential role of the nNOS gene in the development of behavioral sensitization to cocaine in adolescent and adult B6;129S mice

Rationale  Previous studies have suggested the involvement of neuronal nitric oxide synthase (nNOS) in the development of sensitization to psychostimulants. Ontogeny-dependent differences in the response to psychostimulants have been reported. Objective  The objectives were to investigate (a) the short- and long-term consequences of adolescent and adult cocaine exposure on behavioral sensitization and (b) the role of the nNOS gene in behavioral sensitization in adolescent and adult mice. Materials and methods  Adolescent and adult wild type (WT) and nNOS knockout (KO) mice received saline or cocaine (20 mg/kg) for 5 days and then were challenged with cocaine (20 mg/kg) after a drug-free period of 10 or 30 days. Locomotor activity was recorded by infrared beam interruptions. nNOS immunoreactive (ir) neurons in the dorsal and ventral striatum were quantified 24 h after repeated administration of cocaine to adolescent and adult WT mice. Results  Repeated administration of cocaine to either WT or nNOS KO mice during adolescence resulted in locomotor sensitization, which persisted into adulthood. WT but not KO adult mice developed long-term sensitization to cocaine. Repeated cocaine administration resulted in a 96% increase in the expression of nNOS-ir neurons in the dorsal striatum of adult but not adolescent WT mice. Conclusions  The nNOS gene is essential for the induction of behavioral sensitization to cocaine in adulthood but not in adolescence. The increased expression of nNOS-ir neurons in the dorsal striatum may underlie the induction of behavioral sensitization in adulthood. Thus, the NO-signaling pathway has an ontogeny-dependent role in the neuroplasticity underlying cocaine behavioral sensitization.     

Bidirectional modulatory effect of orphanin FQ on morphine-induced analgesia: antagonism in brain and potentiation in spinal cord of the rat

1. The present study was designed to investigate further the effects of the newly discovered orphanin FQ (OFQ)–the endogenous ligand for the orphan opioid receptor (called, e.g., ORL₁ and LC132)–on pain modulation in the rat. We used the tail-flick assay as a nociceptive index.
2. When injected into a cerebral ventricle, OFQ (4 fmol–10 nmol) has no effect on basal tail-flick latency by itself at any dose, but dose-dependently antagonizes systemic morphine analgesia (400 fmol–50 nmol).
3. Injected intrathecally, OFQ (3 and 10 nmol) displayed an analgesic effect without producing motor dysfunction, and potentiated morphine analgesia (1 and 10 nmol).
4. The anti-opioid effect of OFQ in rat brain and the high level of expression of LC132/ORL₁ receptor in the locus coeruleus indicated a possible role of OFQ in the precipitation of opiate withdrawal symptoms. However, no such precipitation was observed by OFQ in morphine-dependent rats.

     

Prenatal stress affects insulin-like growth factor-1 (IGF-1) level and IGF-1 receptor phosphorylation in the brain of adult rats

It has been shown that stressful events occurring in early life have a powerful influence on the development of the central nervous system. Insulin-like growth factor-1 (IGF-1) promotes the growth, differentiation and survival of both neurons and glial cells and is thought to exert antidepressant-like activity. Thus, it is possible that disturbances in the function of the IGF-1 system may be responsible for disturbances observed over the course of depression. Prenatal stress was used as a valid model of depression. Adult male offspring of control and stressed rat dams were subjected to behavioural testing (forced swim test). The level of IGF-1 in the blood and the expression of IGF-1, IGF-1R, and IRS-1/2 in the hippocampus and frontal cortex using RT-PCR, ELISA and western blotting were measured. In addition the effect of intracerebroventricularly administered IGF-1 and/or the IGF-1R receptor antagonist JB1 in the forced swim test was studied. Prenatally stressed rats showed depressive like behaviour, including increased immobility time as well as decreased mobility and climbing. Intracerebroventricular administration of IGF-1 reversed these effects in stressed animals, whereas concomitant administration of the IGF-1R antagonist JB1 completely blocked the effects. Biochemical analysis of homogenates from the hippocampus and frontal cortex revealed decreases in IGF-1 level and IGF-1R phosphorylation along with disturbances in IRS-1 phosphorylation. These findings reveal that prenatal stress alters IGF-1 signalling, which may contribute to the behavioural changes observed in depression.     

Ability of dopamine antagonists to inhibit the locomotor effects of cocaine in sensitized and non-sensitized C57BL/6 mice depends on the challenge dose

Rationale Studies in rats examining the ability of selective dopamine D₂ receptor class antagonists to attenuate the effects of a cocaine challenge have suggested that these agents are less potent in attenuating sensitized as opposed to non-sensitized locomotion. A potential issue with these studies is that the same challenge dose is used in sensitized and control conditions even though that dose may occupy different positions on the respective dose-response curves.Objectives To examine whether the ability of dopamine antagonists to attenuate cocaine-induced locomotion differs between sensitized and non-sensitized animals if they are challenged with the same dose of cocaine, and with the lowest dose to maximally increase locomotion, which is lower in sensitized than in non-sensitized animals.Methods Mice were treated repeatedly with 20 mg/kg cocaine or saline (for 3 consecutive days) and then challenged (after an 11-day drug-free interval) with different challenge doses of cocaine after pretreatment with a dopamine antagonist or saline.Results Using the same challenge dose of cocaine in both repeated treatment conditions (i.e. 20 mg/kg), the D₂ class antagonists eticlopride and raclopride were less potent in attenuating the locomotor effects of cocaine in sensitized than those in non-sensitized animals. In contrast, when the lowest doses to maximally increase locomotion in each of the repeated treatment conditions were used (10 and 40 mg/kg), the D₂ class antagonists attenuated the locomotor effects of cocaine in sensitized and non-sensitized animals with similar potencies. The ability of the D₁ class antagonist SCH23390 to attenuate the effects of cocaine demonstrated a similar dependency on the challenge dose.Conclusions These results show that, under the present conditions, the ability of dopamine antagonists to attenuate cocaine-induced locomotion is similar in sensitized and non-sensitized animals when challenged with pharmacologically equivalent doses of cocaine, but not when challenged with the same dose.     

Autoradiographic study on the pharmacological characteristics of [H]3-OH-PCP binding sites in rat brain

The pharmacological characteristics and the regional distribution of [³H]3-OH-PCP (1-[1(3-hydroxyphenyl)-cyclohexyl]piperidine) binding were investigated in rat brain by quantitative autoradiography. Kinetic analysis of [³H]3-OH-PCP binding revealed fast and slow components, in the association and dissociation studies. The regional distribution of binding closely corresponded to those of binding sites labeled by [³H]N-[1-(2-thienyl)-cyclohexyl]3,4-piperidine (TCP) and [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK 801). High densities of [³H]3-OH-PCP binding sites were found in the stratum radiatum and oriens of field CA1 in the hippocampus and in the outer layers of cerebral cortices. In contrast, low levels of binding were seen in the brain stem and the granular cell layer of the cerebellum. [³H]3-OH-PCP binding was strongly inhibited by MK 801 and 3-OH-PCP, while the potency of (+)-SKF 10047 in inhibiting [³H]3-OH-PCP binding was less in the cerebral cortex and hippocampus. The antagonists for the glutamate, glycine and polyamine recognition sites at the NMDA/PCP receptor complex displaced [³H]3-OH-PCP binding sites with a potency similar to that of [³H]MK 801. These findings suggest that the [³H]3-OH-PCP binding site is similar or identical to the PCP binding site labeled by [³H]TCP and [³H]MK 801.     

The discriminative stimulus properties of cocaine: effects of microinfusion of cocaine, a 5-HT1A agonist or antagonist, into the ventral tegmental area

 Serotonin (5-HT) afferents may modulate the dopamine mesoaccumbens circuit, which has been shown to be critically involved in the locomotor stimulatory, discriminative stimulus, and rewarding properties of cocaine. In the present study, we investigated the role of 5-HT_1A receptors in the ventral tegmental area (VTA) in mediating the discriminative stimulus effects of cocaine. Male Sprague-Dawley rats were trained to discriminate cocaine (10 mg/kg) from saline in a two-lever, water-reinforced FR 20 task. After acquiring the cocaine-saline discrimination, rats were stereotaxically implanted with bilateral guide cannulae into the VTA or adjacent substantia nigra reticulata (SNR). Intraperitoneal administration of cocaine (0.625–10 mg/kg) produced a dose-related increase in drug-lever responding. Both intra-VTA and intra-SNR infusion of cocaine (12.5–50 μg/0.5 μl/side) engendered primarily saline-like responding. Microinjection of the 5-HT_1A agonist 8-hydroxy-2-(di-N-propylamino) tetralin (DPAT; 0.1–10 μg/0.5 μl/side) or the 5-HT_1A antagonist WAY 100635 (0.01–1.0 μg/0.5 μl/side) into the VTA or SNR did not substitute for the systemic cocaine cue. Further, intra-VTA or intra-SNR DPAT or WAY 100635 in combination with systemic doses of cocaine did not alter (i.e., attenuate or potentiate) the systemic cocaine cue. Overall, these data indicate that 5-HT_1A receptors in the VTA do not mediate or modulate the discriminative stimulus effects of cocaine in the rat. Received: 15 April 1997 / Final version: 21 October 1997     

The effects of chronic administration of tranylcypromine and rimonabant on behaviour and protein expression in brain regions of the rat

Recent findings indicate that CB1 receptor blockade might be relevant to the action of antidepressant drugs as inhibition of endocannabinoid function can increase synaptic availability of neurotransmitters; an effect also seen with chronic antidepressant drug treatment. Chronic treatments with established antidepressants also lead to raised brain BDNF levels. The aim of this study was to compare the effects of rimonabant (an inverse agonist/antagonist of CB1 receptors) with those of the antidepressant tranylcypromine (TCP), on behaviour and expression of BDNF/CREB signalling pathways in rat brain.Daily (i.p.) injections of vehicle or TCP (10 mg/kg) or rimonabant (2 mg/kg) were given for 14 days. Locomotor activity (LMA) and a conditional emotional response (CER) were measured in addition to levels of BDNF and CREB/phospho-CREB, using immunoblotting, in the frontal cortex, hippocampus, striatum and cerebellum. The velocity of movement was increased significantly on the 3rd, but not 9th, day of TCP treatment versus vehicle-treated rats (p < 0.05) while rimonabant had no effect. There were no significant changes in grooming or freezing behaviours after rimonabant or TCP compared to vehicle-treated rats. Rearing was significantly reduced by TCP treatment on the 3rd, but not 9th, day of treatment (p < 0.001) while rimonabant had no effect. BDNF levels were significantly increased in the frontal cortex after TCP (p < 0.05) but not by rimonabant. Neither TCP nor rimonabant significantly affected CREB or p-CREB expression.In conclusion, chronic administration of TCP to rats increased BDNF expression in the frontal cortex but no similar effect was observed with rimonabant indicating that rimonabant does not show antidepressant drug-like responses after chronic treatment.     

大鼠心肌多胺代谢限速酶ODC、SSAT活性分析

目的建立大鼠心肌多胺代谢限速酶鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰转移酶(SSAT)活性分析方法。方法以Langendorff离体灌流心肌为实验材料,制备心肌组织匀浆;分别以dl[114C]Ornithine及[114C]acetylCoenzymeA为底物,以液体闪烁计数仪记录生成的14CO2及[14C]acetylspermidine的放射活度,并以其代表ODC,SSAT的活性;计算大鼠心肌ODC、SSAT的酶促反应动力学参数,筛选出适宜的底物浓度;同时观察一氧化氮(NO)供体硝普钠(SNP)对酶活性的影响。结果①大鼠心肌ODC、SSAT基础活性分别为:(9.67±3.09)nmol·mg-1Pro·h-1;(3.59±0.91)nmol·mg-1Pro·min-1。②ODC催化LOrnithine的酶促反应动力学参数Km=(54.95±8.14)μmol·L-1;Vmax=(2.364±0.37)nmol·mg-1·h-1;SSAT催化AcetylCoenzymeA的酶促反应动力学参数Km=(12.87±1.88)μmol·L-1;Vmax=(0.50±0.07)nmol·mg-1·min-1。③大鼠心肌ODC、SSAT活性检测的底物浓度分别为:90μmol·L-1(18.5kBq)DL[114C]Ornithine及36μmol·L-1(2.96kBq)[114C]acetylCoA。④SNP呈浓度依赖性地抑制ODC的活性、诱导SSAT的活性。结论建立了大鼠心肌多胺代谢限速酶鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰转移酶(SSAT)活性的分析方法,该方法简便易行;根据Km值确定测定大鼠心肌ODC及SSAT?