Low doses of alpha 2-adrenoceptor antagonists augment spinal morphine analgesia and inhibit development of acute and chronic tolerance.

BACKGROUND AND PURPOSE: Ultra-low doses of opioid receptor antagonists augment spinal morphine antinociception and block the induction of tolerance. Considering the evidence demonstrating functional and physical interactions between the opioid and alpha(2)-adrenoceptors, this study investigated whether ultra-low doses of alpha(2)-adrenoceptor antagonists also influence spinal morphine analgesia and tolerance. EXPERIMENTAL APPROACH: Effects of low doses of the competitive alpha(2)-adrenoceptor antagonists-atipamezole (0.08, 0.8 ng), yohimbine (0.02, 2 ng), mirtazapine (0.02 ng) and idazoxan (0.08 ng) were investigated on intrathecal morphine analgesia, as well as acute and chronic morphine antinociceptive tolerance using the rat tail flick and paw pressure tests. KEY RESULTS: At doses markedly lower than those producing alpha(2)-adrenoceptor blockade, atipamezole, yohimbine, mirtazapine and idazoxan, prolonged the antinociceptive effects of morphine. When co-administered with repeated acute spinal injections of morphine, all four agents blocked the induction of acute tolerance. Co-injection of atipamezole with morphine for 5 days inhibited the development of tolerance in a chronic treatment paradigm. Spinal administration of atipamezole also reversed established antinociceptive tolerance to morphine as indicated by the restoration of morphine antinociceptive potency. The effects of atipamezole on spinal morphine tolerance were not influenced by treatment with 6-hydroxydopamine. CONCLUSIONS AND IMPLICATIONS: Low doses of competitive alpha(2)-adrenoceptor antagonists can augment acute morphine analgesia and block or reverse tolerance to spinal administration of morphine. These actions are interpreted in terms of their interaction with an opioid-alpha(2)-adrenoceptor complex, whose activity may have a function in the genesis of analgesic tolerance.

Augmentation of spinal morphine analgesia and inhibition of tolerance by low doses of mu- and delta-opioid receptor antagonists.

BACKGROUND AND PURPOSE: Ultralow doses of naltrexone, a non-selective opioid antagonist, have previously been found to augment acute morphine analgesia and block the development of tolerance to this effect. Since morphine tolerance is dependent on the activity of micro and delta receptors, the present study investigated the effects of ultralow doses of antagonists selective for these receptor types on morphine analgesia and tolerance in tests of thermal and mechanical nociception. EXPERIMENTAL APPROACH: Effects of intrathecal administration of mu-receptor antagonists, CTOP (0.01 ng) or CTAP (0.001 ng), or a delta-receptor antagonist, naltrindole (0.01 ng), on spinal morphine analgesia and tolerance were evaluated using the tail-flick and paw-pressure tests in rats. KEY RESULTS: Both micro and delta antagonists augmented analgesia produced by a sub-maximal (5 microg) or maximal (15 microg) dose of morphine. Administration of the antagonists with morphine (15 microg) for 5 days inhibited the progressive decline of analgesia and prevented the loss of morphine potency. In animals exhibiting tolerance to morphine, administration of the antagonists with morphine produced a recovery of the analgesic response and restored morphine potency. CONCLUSIONS AND IMPLICATIONS: Combining ultralow doses of micro- or delta-receptor antagonists with spinal morphine augmented the acute analgesic effects, inhibited the induction of chronic tolerance and reversed established tolerance. The remarkably similar effects of micro- and delta-opioid receptor antagonists on morphine analgesia and tolerance are interpreted in terms of blockade of the latent excitatory effects of the agonist that limit expression of its full activity.

Involvement of cannabinoid (CB1)-receptors in the development and maintenance of opioid tolerance.

Sustained exposure to opioid agonists such as morphine increases levels of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn, a response implicated in the development of opioid tolerance and physical dependence. Recent evidence suggests that both the opioid-induced increase in CGRP and the development of opioid physical dependence are suppressed by blockade of spinal cannabinoid (CB1)-receptors. The present study examined whether CB1-receptor activity also has a role in the development of opioid tolerance. In rats implanted with spinal catheters, repeated acute injections of morphine (15 microg) delivered over 4 h resulted in a rapid decline of thermal and mechanical antinociception and a significant loss of analgesic potency, reflecting development of acute opioid tolerance. In another set of experiments, chronic administration of spinal morphine (15 microg) once daily for 5 days produced a similar loss of analgesic effect and a marked increase in CGRP-immunoreactivity in the superficial laminae of the dorsal horn. Consistent with the in vivo findings, primary cultures of adult dorsal root ganglion (DRG) neurons exposed to morphine for 5 days showed a significant increase in the number of CGRP-immunoreactive neurons. Co-administration of acute or chronic morphine with a CB1-receptor antagonist/inverse agonist, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM-251), inhibited the development of both acute and chronic analgesic tolerance. In animals already exhibiting tolerance to morphine, intervention with AM-251 restored morphine analgesic potency. Co-administration with AM-251 attenuated the morphine-induced increase in CGRP-immunoreactivity in the spinal cord and in DRG cultured neurons. Collectively, the results of this study suggest that activity of endocannabinoids, mediated via CB1-receptors, contributes to both the development and maintenance of opioid tolerance by influencing the opioid-induced increase in spinal CGRP.

Facilitation of spinal morphine analgesia in normal and morphine tolerant animals by neuropeptide SF and related peptides.

Neuropeptide FF and related synthetic amidated peptides have been shown to elicit sustained anti-nociceptive responses and potently augment spinal anti-nociceptive actions of spinal morphine in tests of thermal and mechanical nociception. Recent studies have described the occurrence of another octapeptide, neuropeptide SF (NPSF) in the spinal cord and the cerebrospinal fluid and demonstrated its affinity for the NPFF receptors. This study examined the effects of NPSF and two putative precursor peptides, EFW-NPSF and NPAF, on the spinal actions of morphine in normal and opioid tolerant rats using the tailflick and pawpressure tests. In normal rats, NPSF demonstrated weak intrinsic activity but sub-effective doses of the peptide significantly increased the magnitude and duration of spinal morphine anti-nociception in both tests. A low-dose of NPSF also augmented the spinal actions of a delta receptor agonist, deltorphin. The morphine-potentiating effect of NPSF was shared by EFW-NPSF and the octadecapeptide NPAF. In animal rendered tolerant by continuous intrathecal infusion of morphine for 6 days, low dose NPSF itself elicited a significant anti-nociceptive response and potently increased morphine-induced response in both tests. In animals made tolerant by repeated injections of intrathecal morphine, administration of NPSF, EFW-NPSF, and NPAF with morphine reversed the loss of the anti-nociceptive effect and restored the agonist potency. The results demonstrate that in normal animals NPSF and related peptides exert strong potentiating effect on morphine anti-nociception at the spinal level and in tolerant animals these agents can reverse the loss of morphine potency.

Localization and modulation of calcitonin gene-related peptide-receptor component protein-immunoreactive cells in the rat central and peripheral nervous systems.

Calcitonin gene-related peptide (CGRP) is widely distributed in the central and peripheral nervous system. Its highly diverse biological activities are mediated via the G protein-coupled receptor that uniquely requires two accessory proteins for optimal function. CGRP receptor component protein (RCP) is a coupling protein necessary for CGRP-receptor signaling. In this study, we established the anatomical distribution of RCP in the rat central and peripheral nervous system and its relationship to CGRP immunoreactivity. RCP-immunoreactive (IR) perikarya are widely and selectively distributed in the cerebral cortex, septal nuclei, hippocampus, various hypothalamic nuclei, amygdala, nucleus colliculus, periaqueductal gray, parabrachial nuclei, locus coeruleus, cochlear nuclei, dorsal raphe nuclei, the solitary tractus nucleus and gracile nucleus, cerebellar cortex, various brainstem motor nuclei, the spinal dorsal and ventral horns. A sub-population of neurons in the dorsal root ganglia (DRG) and trigeminal ganglia were strongly RCP-IR. Overall, the localization of RCP-IR closely matched with that of CGRP-IR. We also determined whether RCP in DRG and dorsal horn neurons can be modulated by CGRP receptor blockade and pain-related pathological stimuli. The intrathecal injection of the antagonist CGRP(8-37) markedly increased RCP expression in the lumbar DRG and spinal dorsal horn. Carrageenan-induced plantar inflammation produced a dramatic bilateral increase in RCP expression in the dorsal horn while a partial sciatic nerve ligation reduced RCP expression in the ipsilateral superficial dorsal horn. Our data suggest that the distribution of RCP immunoreactivity is closely matched with CGRP immunoreactivity in most of central and peripheral nervous systems. The co-localization of RCP and CGRP in motoneurons and primary sensory neurons suggests that CGRP has an autocrine or paracrine effect on these neurons. Moreover, our data also suggest that RCP expression in DRG and spinal cord can be modulated during CGRP receptor blockade, inflammation or neuropathic pain and this CGRP receptor-associated protein is dynamically regulated.

Neurosteroids and glutamate toxicity in fibroblasts expressing human NMDA receptors.

We characterized glutamate receptor-mediated toxicity in mouse fibroblasts expressing the human NR1a/2A or NR1a/2B NMDA receptor. After induction of NMDA receptors, cells in both lines died over a 24 h time period. This toxicity was associated with a progressive increase in the glutamate content of the media. Cell death could be prevented by including either the non-competitive NMDA receptor antagonist ketamine or the competitive antagonist D,L-AP5. Cells expressing NR1a/2A receptors were maximally protected by 0.5 mM D,L -AP-5, while those expressing NR1a/2B receptors required 2 mM D,L -AP-5 for maximal protection. The neurosteroid pregnenolone sulfate, which negatively modulates NMDA receptor function, partially protected fibroblasts containing NR1a/2A or NR1a/2B NMDA receptor constructs. However, the neurosteroid pregnenolone sulfate, which has been reported to act as a positive allosteric modulator of the NMDA receptor, had no effect on the toxicity caused by endogenous glutamate. Our results on cells expressing human NMDA receptors suggest that certain neurosteroids may protect against NMDA induced toxicity while having low neurotoxic liabilities of their own.

Neonatal exposure to the glutamate receptor antagonist MK-801: effects on locomotor activity and pre-pulse inhibition before and after sexual maturity in rats.

Neonatal lesions of the ventral hippocampus in rats lead to post- but not pre-pubertal behavioral changes suggesting adolescent onset of dopaminergic hypersensitivity and providing an animal model of schizophrenia. Neonatal exposure to glutamate receptor antagonists produces accelerated apoptosis leading to neuronal loss in central nervous system structures including the hippocampus. This suggested that neonatal MK-801 might lead to behavioural changes like those reported following ventral hippocampal lesions. Thus, rats received MK-801 (0, 0.5, 1.0 mg/kg ip) on postnatal day 3 (P3) and were tested pre- (P35) and post-pubertally (P56). MK-801 produced an increase in TUNEL staining in the hippocampus and other forebrain structures, confirming the induction of apoptosis. Results showed little difference in locomotor activity between neonatal saline- and MK-801-treated groups during habituation or following saline injection but increased activity was seen in the 0.5 mg/kg MK-801 group following amphetamine (1.5 mg/kg i.p.) at P35 but not P56. In tests of pre-pulse inhibition (PPI), neonatal saline and MK-801 groups showed stable startle amplitudes, minimal responding to the pre-pulse stimuli alone, an increase in PPI with increases in pre-pulse intensity, and reduced PPI with apomorphine (0.1 mg/kg s.c.). At P56, neonatal MK-801 groups tested following vehicle showed less sensitivity to changes in pre-pulse intensity. It was concluded that neonatal MK-801 increases apoptotic cell loss in the hippocampus but does not produce behavioural effects like those seen after neonatal ventral hippocampal lesions. However, neonatal MK-801 did lead to increases in locomotor activity in juveniles but not adults and reduced sensitivity to pre-pulse intensity in PPI tests in adulthood.

Chronic morphine exposure increases the phosphorylation of MAP kinases and the transcription factor CREB in dorsal root ganglion neurons: an in vitro and in vivo study.

Tolerance to opiates reduces their effectiveness in the treatment of severe pain. Although the mechanisms are unclear, overactivity of pro-nociceptive systems has been proposed to contribute to this phenomenon. We have reported that the development of morphine tolerance significantly increased calcitonin-gene-related-peptide-like immunoreactivity (CGRP-IR) in primary sensory afferents of the spinal dorsal horn, suggesting that changes in pain-related neuropeptides in the dorsal root ganglion (DRG) neurons may be involved (Menard et al., 1996, J. Neurosci., 16, 2342-2351). Recently, we have shown that repeated morphine treatments induced increases in CGRP- and substance P (SP)-IR in cultured DRG, mimicking the in vivo effects (Ma et al., 2000, Neuroscience, 99, 529-539). In this study, we investigated the intracellular signal transduction pathways possibly involved in morphine-induced increases in CGRP- and SP-IR in DRG neurons. Repeated morphine exposure (10-20 microm) for 6 days increased the number of neurons expressing phosphorylated (p) mitogen-activated protein (MAP) kinases, including the extracellular signal-regulated kinase (pERK), c-jun N-terminal kinase (pJNK) and P38 (pP38 MAPK). The number of neurons expressing phosphorylated cAMP responsive element binding protein (pCREB) was also markedly increased in morphine-exposed cultured DRG neurons. pERK-, pP38-, pJNK- and pCREB-IR were colocalized with CGRP-IR in cultured DRG neurons. Naloxone effectively blocked these actions of morphine, whereas a selective MEK1 inhibitor, PD98059, inhibited the morphine-induced increase in the phosphorylation of ERK and CREB, and the expression of CGRP and SP. Moreover, in morphine-tolerant rats, the number of pCREB-, CGRP- and SP-IR neurons in the lumbar DRG was also significantly increased. These in vitro and in vivo data suggest that the phosphorylation of MAP kinases and CREB plays a role in the morphine-induced increase in spinal CGRP and SP levels in primary sensory afferents, contributing to the development of tolerance to opioid-induced analgesia.

Cognitive effects of neurotoxic lesions of the nucleus basalis magnocellularis in rats: differential roles for corticopetal versus amygdalopetal projections.

The cholinergic hypothesis states that cholinergic neurons of the basal forebrain nucleus basalis magnocellularis (nbm) that project to cortical and amygdalar targets play an important role in memory. Biochemical studies have shown that these target areas are differentially sensitive to different excitotoxins (e.g., ibotenate vs. quisqualate). This observation might explain the finding from many behavioural studies of memory that different excitotoxins affect memory differentially even though they produce about the same level of depletion of cholinergic markers in the cortex and similar cortical electrophysiological effects. Thus, the magnitude of mnemonic impairment might be related to the extent of damage to cholinergic projections to the amygdala more than to the extent of damage to corticopetal cholinergic projections. This explanation might similarly apply to the observation that the immunotoxin 192 IgG-saporin produces mild effects on memory when injected into the nbm. This is because it damages cholinergic neurons projecting to the cortex but not those projecting to the amygdala. Studies comparing the effects on memory of ibotenic acid vs. quisqualic acid lesions of the nbm are reviewed as are studies of the mnemonic effects of 192 IgG-saporin. Results support the cholinergic hypothesis and suggest that amygdalopetal cholinergic neurons of the nbm play an important role in the control of memory.

Neurosteroids and reward: allopregnanolone produces a conditioned place aversion in rats.

The neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) has been reported to have rewarding properties in mice tested for place conditioning. Another study found that allopregnanolone reduced dopamine (DA) output in the nucleus accumbens (NAc) of rats. As many rewarding stimuli increase accumbens DA, these results may appear contradictory. Thus, the present study examined the rewarding properties of allopregnanolone in rats tested for place conditioning using an unbiased conditioning procedure. In control studies, a place preference was observed following conditioning with intraperitoneal (2.0 mg/kg) or intracerebroventricular (i.c.v.) (100 microg/0.5 microl) amphetamine. Conditioning with i.c.v. allopregnanolone produced a significant aversion at a dose of 5.0 microg (in 5.0 microl) and a near aversion at 25.0 microg (in 8.3 microl); doses of 0 microg (i.e., vehicle alone, in 10 microl) or 30.0 microg (in 10 microl) produced little effect on place preference. During conditioning, locomotor activity was stimulated by amphetamine using either route of administration, but allopregnanolone had no significant main effect on locomotor activity. Thus, there was a dissociation between the effects of drugs on locomotor activity vs. place conditioning. Results show that i.c.v. amphetamine produces a place preference, whereas allopregnanolone produces either no effect or an aversion, depending on the dose.

Blockade and reversal of spinal morphine tolerance by peptide and non-peptide calcitonin gene-related peptide receptor antagonists.

This study examined the effects of the peptide CGRP receptor antagonist CGRP(8-37) and the newly-developed non-peptide CGRP receptor antagonist BIBN4096BS for their potential to both inhibit the development and reverse tolerance to the antinociceptive action of morphine. Repeated administration of intrathecal morphine (15 microg), once daily, produced a progressive decline of antinociceptive effect and an increase in the ED(50) value in the tailflick and paw pressure tests. Co-administration of CGRP(8-37) (4 microg) or BIBN4096BS (0.05, 0.1 microg) with morphine (15 microg) prevented the decline of antinociceptive effect and increase in ED(50) value in the tailflick test. Intrathecal administration of the CGRP receptor antagonists did not alter the baseline responses in either tests. Acute CGRP(8-37) also did not potentiate the acute actions of spinal morphine. In animals rendered tolerant to intrathecal morphine, subsequent administration of CGRP(8-37) (4 microg) with morphine (15 microg) partially restored the antinociceptive effect and ED(50) value of acute morphine, reflecting the reversal of tolerance. Animals tolerant to intrathecal morphine expressed increased CGRP and substance P-like immunostaining in the dorsal horn of the spinal cord. The increase in CGRP, but not substance P-like immunostaining, was blocked by a co-treatment with CGRP(8-37) (4 microg). In animals already tolerant to morphine, the increase in CGRP but not substance P-like immunostaining was partially reversed by CGRP(8-37) (4 microg). These data suggest that activation of spinal CGRP receptors contributes to both the development and expression of spinal opioid tolerance. CGRP receptor antagonists may represent a useful therapeutic approach for preventing as well as reversing opioid tolerance.

Role of adenosine in the spinal antinociceptive and morphine modulatory actions of neuropeptide FF analogs.

The neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH(2)) and its synthetic analogs bind to specific receptors in the spinal cord to produce antinociceptive effects that are partially attenuated by opioid antagonists, and at sub-effective doses neuropeptide FF receptor agonists augment spinal opioid antinociception. Since adenosine plays an intermediary role in the production of spinal opioid antinociception, this study investigated whether this purine has a similar role in the expression of spinal effects produced by neuropeptide FF receptor agonists. In rats bearing indwelling spinal catheters, injection of adenosine receptor agonists, N6-cyclohexyladenosine (CHA, 1.72 nmol) and N-ethylcarboxiamidoadenosine (NECA, 1.95 nmol), as well as morphine (13.2 nmol) elicited antinociception in the tail-flick and paw-pressure tests. Pretreatment with intrathecal 8-phenyltheophylline (5.9 and 11.7 nmol), an adenosine receptor antagonist, blocked the effect of all three agents without influencing baseline responses. Administration of two synthetic neuropeptide FF (NPFF) analogs, [D-Tyr(1),(NMe)Phe(3)]NPFF (1DMe, 0. 86 nmol) and [D-Tyr(1),D-leu(2),D-Phe(3)]NPFF (3D, 8.6 nmol) produced sustained thermal and mechanical antinociception. Pretreatment with doses of intrathecal 8-phenyltheophylline (5.9, 11. 7 and 23.5 nmol), producing adenosine receptor blockade, significantly inhibited the antinociceptive effects of 1DMe or 3D. Injection of a sub-antinociceptive dose of 1DMe (0.009 nmol) significantly augmented the antinociceptive effect of intrathecal morphine (13.2 nmol) in the tail-flick and paw-pressure tests. Intrathecal 8-phenyltheophylline (11.7 nmol) reduced the effect of this combination. Administration of low dose of 1DMe (0.009 nmol) or 3D (0.009 nmol) very markedly potentiated the antinociceptive actions of the adenosine receptor agonist, N6-cyclohexyladenosine (0. 43, 0.86 and 1.72 nmol) in the tail-flick and paw-pressure tests 50 min after injection. The results suggest that the antinociceptive and morphine modulatory effects resulting from activation of spinal NPFF receptors could be due to an increase in the actions or availability of adenosine.

Suppression of acute and chronic opioid withdrawal by a selective soluble guanylyl cyclase inhibitor.

Previous studies have shown that activation of N-methyl-D-aspartate (NMDA) receptors and formation of nitric oxide (NO) contributes to the hyperactivity of locus coeruleus (LC) noradrenergic neurons and behavioural symptoms seen during opioid withdrawal. However, the role of soluble guanylyl cyclase (sGC), the 'physiological' target of NO, in this phenomenon is unclear. In this study, the effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a highly selective sGC inhibitor, on the naloxone-precipitated morphine withdrawal was examined using differential normal pulse voltammetry (DNPV) to measure LC activity, in vivo microdialysis to measure glutamate/aspartate release response, and behavioural assessment to evaluate withdrawal symptoms. In halothane-anaesthetized rats, acute intracerebroventricular (i.c.v.) morphine (10 microg) reduced the catecholamine oxidation current (CA.OC) (54.5+/-4.9% of baseline). Naloxone (2 mg/kg, i.v.) reversed this action of morphine and produced a rebound increase in CA.OC (136.1+/-6.0% of baseline), representing acute morphine withdrawal. Administration of ODQ (200 nmol, i.c.v.) blocked this response without affecting acute morphine action. In animals chronically treated with morphine (15 microg/microl/h, i.c.v., 5 days), naloxone significantly increased both the CA.OC signal (270.0+/-19.6% of baseline) and the release of L-glu (193+/-30.4%) and L-asp (221.5+/-28.4%) above baseline. These responses were attenuated in animals pretreated with ODQ. In unanaesthetized chronic morphine dependent rats, ODQ treatment suppressed the signs of withdrawal precipitated by naloxone (10 mg/kg). Taken together, the results of this study suggest that sGC plays an intermediary role in the genesis of LC neuronal hyperactivity and behavioural signs of morphine withdrawal.

Excitotoxicity of quinolinic acid: modulation by endogenous antagonists.

Quinolinic acid (QUIN), a product of tryptophan metabolism by the kynurenine pathway, produces excitotoxicity by activation of NMDA receptors. Focal injections of QUIN can deplete the biochemical markers for dopaminergic, cholinergic, gabaergic, enkephalinergic and NADPH diaphorase neurons, which differ in their sensitivity to its neurotoxic action. This effect of QUIN differs from that of other NMDA receptor agonists in terms of its dependency on the afferent glutamatergic input and its sensitivity to the receptor antagonists. The enzymatic pathway yielding QUIN produces metabolites that inhibit QUIN-induced neurotoxicity. The most active of these metabolites, kynurenic acid (KYNA), blocks NMDA and non-NMDA receptor activity. Treatment with kynurenine hydroxylase and kynureinase inhibitors increases levels of endogenous KYNA in the brain and protects against QUIN-induced neurotoxicity. Other neuroprotective strategies involve reduction in QUIN synthesis from its immediate precursor, or endogenous synthesis of 7-chloro-kynurenic acid, a NMDA antagonist, from its halogenated precursor. Several other tryptophan metabolites--quinaldic acid, hydroxyquinaldic acid and picolinic acid--also inhibit excitotoxic damage but their presence in the brain is uncertain. Picolinic acid is of interest since it inhibits excitotoxic but not neuroexcitatory responses. The mechanism of its anti-excitotoxic action is unclear but might involve zinc chelation. Neurotoxic actions of QUIN are modulated by nitric oxide (NO). Treatment with inhibitors of NO synthase can augment QUIN toxicity in some models of excitotoxicity suggesting a neuroprotective potential of endogenous NO. In recent studies, certain nitroso compounds which could be NO donors, have been reported to reduce the NMDA receptor-mediated neurotoxicity. The existence of endogenous compounds which inhibit excitotoxicity provides a basis for future development of novel and effective neuroprotectants.

Dual localization of neuropeptide FF receptors in the rat dorsal horn.

Although neuropeptide FF (NPFF) is generally considered an anti-opioid, its intrathecal administration produces analgesia. In the present study, the stable analog 1DMe ([D.Tyr(1), (NMe)Phe(3)]neuropeptide FF) was used in quantitative autoradiographic experiments in combination with surgical and chemical lesions to precisely localize NPFF receptors in the rat spinal cord. Ligation of lumbar dorsal spinal roots revealed the presence of NPFF receptors in dorsal root fibers and it induced a significant accumulation of [(125)I]1DMe-specific binding on the side peripheral to the ligature, demonstrating that a population of NPFF receptors is synthesized in dorsal root ganglia and migrates anterogradely towards primary afferent nerve endings. Complete mid-thoracic spinal cord transection failed to modify the [(125)I]1DMe labeling density in the dorsal horn, indicating that NPFF receptors are not located on the descending fiber terminals. In contrast, unilateral microinjections of kainic acid into the dorsal horn dramatically reduced [(125)I]1DMe-specific binding in the superficial layers, revealing localization of a population of NPFF receptors on the spinal intrinsic neurons. NPFF receptor binding was not modified during the development of spinal opioid tolerance. The pre- and postsynaptic localization of spinal NPFF receptors provide further support for heterogeneity in the pain modulation by NPFF and related agonists.

Nociceptin receptors in the rat spinal cord during morphine tolerance.

The anatomical localization of nociceptin receptors was examined by in vitro quantitative autoradiography techniques in rat spinal cord sections by using [(125)I-Tyr(14)]nociceptin. [(125)I-Tyr(14)]nociceptin appeared to interact with a single class of binding sites (K(D)=0.1 nM) present in the grey matter in all laminae of the spinal cord from cervical to sacral levels. Pre-incubation of sections in the presence of 150 mM NaCl, did not modify the radioligand affinity but significantly augmented the number of accessible binding sites and increased specific binding of [(125)I-Tyr(14)]nociceptin differentially on each laminae. In particular, the superficial layers of the dorsal horn exhibited the highest density of sites after pre-wash. Continuous intrathecal infusion of morphine produced a tolerance accompanied by a significant increase in nociceptin site density in the superficial layers. Thus, nociceptin binding sites may have different properties dependent upon the layer and may be up-regulated during the process of opioid-induced tolerance.

Comparative effects of cyclo-oxygenase and nitric oxide synthase inhibition on the development and reversal of spinal opioid tolerance.

1. This study examined the effects of the COX inhibitors, ketorolac and ibuprofen, and the NOS inhibitor L-NAME for their potential to both inhibit the development and reverse tolerance to the antinociceptive action of morphine. 2. Repeated administration of intrathecal morphine (15 micrograms), once daily, resulted in a progressive decline of antinociceptive effect and an increase in the ED50 value in the tailflick and paw pressure tests. Co-administration of ketorolac (30 and 45 micrograms) or S(+) ibuprofen (10 micrograms) with morphine (15 micrograms) prevented the decline of antinociceptive effect and increase in ED50 value. Similar treatment with L-NAME (100 micrograms) exerted weaker effects. Administration of S(+) but not R(-) ibuprofen (10 mg kg-1) had similar effects on systemic administration of morphine (15 mg kg-1). 3. Intrathecal or systemic administration of the COX or NOS inhibitors did not alter the baseline responses in either tests. Acute keterolac or S(+) ibuprofen also did not potentiate the acute actions of spinal or systemic morphine, but chronic intrathecal administration of these agents increased the potency of acute morphine. 4. In animals already tolerant to intrathecal morphine, subsequent administration of ketorolac (30 micrograms) with morphine (15 micrograms) partially restored the antinociceptive effect and ED50 value of acute morphine, reflecting the reversal of tolerance. Intrathecal L-NAME (100 micrograms) exerted a weaker effect. 5. These data suggest that spinal COX activity, and to a lesser extent NOS activity, contributes to the development and expression of opioid tolerance. Inhibition of COX may represent a useful approach for the prevention as well as reversal of opioid tolerance.

Nucleus basalis injections of N-methyl-D-aspartate enhance memory of rats in the double Y-maze.

N-methyl-D-aspartate (NMDA) receptors have been implicated in learning and memory. Many findings show that NMDA receptor antagonists impair memory. Few studies, however, have investigated the role of NMDA receptor agonists in mnemonic function. The present study examined the effects of nucleus basalis magnocellularis (nbm) injections of NMDA on memory. Rats were trained in a two-component double Y-maze task consisting of a spatial discrimination and a delayed alternation. Rats (n = 7) were surgically implanted with bilateral cannulae in the nbm prior to maze training. Once trained, animals received bilateral nbm injections (0.5 microl) of saline (0.9%), NMDA (50, 75, and 100 ng/side), and the benzodiazepine receptor partial inverse agonist N-methyl-beta-carboline-3-carboxamide (FG 7142; 200 ng/side), in a counterbalanced order. During testing, delays (0, 30, 60 s) were introduced. Nbm FG 7142 or NMDA (50 ng/side) produced an improvement in the delayed alternation task. Results support the hypothesis that nbm NMDA receptors are involved in cognitive processes mediating memory.

Quinolinic acid lesion of the nigrostriatal pathway: effect on turning behaviour and protection by elevation of endogenous kynurenic acid in Rattus norvegicus.

Endogenous excitotoxins have been implicated in degeneration of nigral dopaminergic neurons in Parkinson's disease. It may be possible to reduce neurodegeneration by blocking the effects of these endogenous agents. The present study shows that contralateral turning seen following quinolinic acid-induced lesions of the nigrostriatal dopaminergic pathway was reversed by a treatment that increased brain levels of kynurenic acid, an endogenous excitatory amino acid antagonist. The treatment consisted of nicotinylalanine (5.6 nmol/5 microl i.c.v.), an inhibitor of kynureninase and kynurenine hydroxylase plus the precursor kynurenine (450 mg/kg i.p.) plus probenencid (200 mg/kg i.p.), an inhibitor of organic acid transport. Thus, neuroprotection by increasing brain kynurenic acid in vivo may be useful in retarding cell loss in Parkinson's and other neurodegenerative diseases involving excitotoxicity.

Modulation of quinolinic acid-induced depletion of striatal NADPH diaphorase and enkephalinergic neurons by inhibition of nitric oxide synthase.

The present study was designed to examine the role of nitric oxide (NO) in quinolinic acid (QUIN)-induced depletion of rat striatal nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase and enkephalinergic neurons. Intrastriatal injection of QUIN produced a dose-dependent decrease in NADPH diaphorase and enkephalin positive cells, with cell loss being evident following the injection of 6 and 18 nmol QUIN, respectively. To evaluate the role of NO in QUIN-induced toxicity, animals were pretreated with the non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine (l-NAME) or the selective neuronal NOS inhibitor, 7-nitro indazole (7-NI). l-NAME (2x250 mg/kg, i.p. 8 h apart) maximally inhibited striatal NOS activity by 85%, while 7-NI (50 mg/kg, i.p.) maximally inhibited striatal NOS activity by 60%. Pretreatment with l-NAME or 7-NI potentiated the loss of NADPH diaphorase neurons resulting from intrastriatal injection of low doses of QUIN (18 nmol). Neither NOS inhibitor had any effect on the loss of striatal NADPH diaphorase neurons induced by a higher dose of QUIN (24 nmol). In contrast, 7-NI partially prevented the QUIN (18 and 24 nmol)-induced loss of enkephalinergic neurons, while l-NAME had no effect. These results indicate that NO formation may play a role in QUIN-induced loss of enkephalinergic neurons, but not in the loss of NADPH diaphorase neurons.