Khem Jhamandas

Antinociceptive effects of intrathecally administered F8Famide and FMRFamide in the rat

The effects of intrathecal injections of F8Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2, 0.05-17.5 nmol) and FMRF-amide (Phe-Met-Arg-Phe-NH2, 0.002-25 nmol), known as anti-opioid agents, were investigated by using noxious thermal (tail flick) and mechanical (paw pressure) tests in the rat. Both peptides produced significant long-lasting (24-48 h) analgesia in both tests without causing detectable motor dysfunction. Pretreatment with systemic naloxone (5.5 mumol/kg i.p.) attenuated the initial antinociceptive effects (first hour) induced by both peptides (8.8 nmol) in the tail flick test and only by FMRFamide in the paw pressure test. A subeffective dose of F8Famide (0.05 nmol) enhanced both the intensity and the duration of spinal morphine (6.6 nmol) analgesia in both tests. In contrast, a subanalgesic dose of FMRFamide (0.002 nmol) decreased the intensity and enhanced the duration of the effect of morphine. These results show that, besides acting as antinociceptive agents in the spinal cord, F8Famide and FMRFamide could differentially modulate spinal opioid functions.

Differential Effects of Scopolamine on Working and Reference Memory of Rats in the Radial Maze

Anticholinergics have often been found to impair choice accuracy in the radial maze. Some researchers have suggested that this indicates involvement of cholinergically innervated structures in cognitive mapping while others argue that these structures mediate working memory. However, most results are open to either interpretation since the baiting method did not allow a distinction between reference and working memory errors. To further test these hypotheses this study examined the effects of systemic scopolamine on radial maze performance, using a 4-out-of-8 baiting procedure. Food-deprived Wistar rats were pretrained until working memory choice accuracy stabilized to a criterion of 87% or better. Scopolamine (0.1, 0.4 and 0.8 mg/kg, IP, 30 min before a session) significantly increased the number of working memory errors (re-entries into baited arms) whereas reference memory errors (entries into never baited arms) did not change significantly. Observed deficits appeared not to be attributable to a drug-induced disruption of motivational systems. Results confirm the behavioural similarities between the memorial effects of hippocampectomy and anticholinergics, and implicate cholinergically innervated structures in working memory.

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 µm) 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.

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 CGRP8‐37 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 μg), once daily, produced a progressive decline of antinociceptive effect and an increase in the ED50 value in the tailflick and paw pressure tests. Co‐administration of CGRP8‐37 (4 μg) or BIBN4096BS (0.05, 0.1 μg) with morphine (15 μg) prevented the decline of antinociceptive effect and increase in ED50 value in the tailflick test. Intrathecal administration of the CGRP receptor antagonists did not alter the baseline responses in either tests. Acute CGRP8‐37 also did not potentiate the acute actions of spinal morphine. In animals rendered tolerant to intrathecal morphine, subsequent administration of CGRP8‐37 (4 μg) with morphine (15 μg) partially restored the antinociceptive effect and ED50 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 CGRP8‐37 (4 μg). In animals already tolerant to morphine, the increase in CGRP but not substance P‐like immunostaining was partially reversed by CGRP8‐37 (4 μg). 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.

Glutamate‐evoked release of endogenous brain dopamine: inhibition by an excitatory amino acid antagonist and an enkephalin analogue

1 The present study examined the effect of a selective δ‐opioid receptor agonist [d‐Ala2‐d‐Leu5] enkephalin (DADL) on the spontaneous and the l‐glutamic acid (l‐Glu)‐evoked release of endogenous dopamine from superfused slices of rat caudate‐putamen. The amount of dopamine in slice superfusates was measured by a sensitive method employing high‐performance liquid chromatography with electrochemical detection (h.p.l.c.‐e.d.) after a two‐step separation procedure. 2 The spontaneous release of endogenous dopamine was (a) partially dependent on Ca2+, (b) enhanced in Mg2+‐free superfusion medium, (c) partially reduced by tetrodotoxin (TTX, 0.3 μm), (d) partially reduced by the putative excitatory amino acid receptor antagonist dl‐2‐amino‐7‐phosphonoheptanoic acid (dl‐APH, 1 mm), and (f) increased 10 fold by the dopamine uptake blocker, nomifensine (10 μm). DADL (5 and 50 nm) did not significantly affect spontaneous dopamine release. 3 l‐Glu (0.1–10 mm) produced a concentration‐dependent release of endogenous dopamine from slices of caudate‐putamen. This effect was (a) Ca2+‐dependent, (b) strongly inhibited by 1.2 mm Mg2+, (c) attenuated by dl‐APH (1 mm), (d) attenuated by TTX (0.3 μm), and (c) enhanced by nomifensine (10 μm). In the presence of nomifensine DADL (50 nm) reduced significantly the l‐Glu‐evoked release of endogenous dopamine by 20%. The inhibitory effect of DADL was blocked by 10 μm naloxone. 4 These results indicate tht l‐Glu stimulates the Ca2+‐dependent release of endogenous dopamine in the caudate‐putamen by activation of N‐methy‐d‐aspartate‐type of excitatory amino acid receptors. This release can be selectively modified by the δ‐opioid agonist DADL in a naloxone‐sensitive manner.

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.

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

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. Repeated administration of intrathecal morphine (15 μg), 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 μg) or S(+) ibuprofen (10 μg) with morphine (15 μg) prevented the decline of antinociceptive effect and increase in ED50 value. Similar treatment with L‐NAME (100 μg) 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). 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. In animals already tolerant to intrathecal morphine, subsequent administration of ketorolac (30 μg) with morphine (15 μg) partially restored the antinociceptive effect and ED50 value of acute morphine, reflecting the reversal of tolerance. Intrathecal L‐NAME (100 μg) exerted a weaker effect. 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.

Protection against quinolinic acid-mediated excitotoxicity in nigrostriatal dopaminergic neurons by endogenous kynurenic acid

Endogenous excitotoxins have been implicated in the degeneration of dopaminergic neurons in the substantia nigra compacta of patients with Parkinsons disease. One such agent quinolinic acid is an endogenous excitatory amino acid receptor agonist. This study examined whether an increased level of endogenous kynurenic acid, an excitatory amino acid receptor antagonist, can protect nigrostriatal dopamine neurons against quinolinic acid-induced excitotoxic damage. Nigral infusion of quinolinic acid (60 nmoles) or N-methyl-D- aspartate (15 nmoles) produced a significant depletion in striatal tyrosine hydroxylase activity, a biochemical marker for dopaminergic neurons. Three hours following the intraventricular infusion of nicotinylalanine (5.6 nmoles), an agent that inhibits kynureninase and kynurenine hydroxylase activity, when combined with kynurenine (450 mg/kg i.p.), the precursor of kynurenic acid, and probenecid (200 mg/kg i.p.), an inhibitor of organic acid transport, the kynurenic acid in the whole brain and substantia nigra was increased 3.3-fold and 1.5-fold respectively when compared to rats that received saline, probenecid and kynurenine. This elevation in endogenous kynurenic acid prevented the quinolinic acid-induced reduction in striatal tyrosine hydroxylase. However, 9 h following the administration of nicotinylalanine with kynurenine and probenecid, a time when whole brain kynurenic acid levels had decreased 12-fold, quinolinic acid injections produced a significant depletion in striatal tyrosine hydroxylase. Intranigral infusion of quinolinic acid in rats that received saline with kynurenine and probenecid resulted in a significant depletion of ipsilateral striatal tyrosine hydroxylase. Administration of nicotinylalanine in combination with kynurenine and probenecid also blocked N-methyl-D-aspartate-induced depletion of tyrosine hydroxylase. Tyrosine hydroxylase immunohistochemical assessment of the substantia nigra confirmed quinolinic acid-induced neuronal cell loss and the ability of nicotinylalanine in combination with kynurenine and probenecid to protect neurons from quinolinic acid-induced toxicity. The present study demonstrates that increases in endogenous kynurenic acid can prevent the loss of nigrostriatal dopaminergic neurons resulting from a focal infusion of quinolinic acid or N-methyl-D-aspartate. The strategy of neuronal protection by increasing the brain kynurenic acid may be useful in retarding cell loss in Parkinsons disease and other neurodegenerative diseases where excitotoxic mechanisms have been implicated.

Role of opioid receptors in the spinal antinociceptive effects of neuropeptide FF analogues

1 Neuropeptide FF (NPFF) has been shown to produce antinociceptive effects and enhance morphine‐induced antinociception after intrathecal (i.t.) injection. In this study, the spinal effects of two NPFF analogues, [D‐Tyr1, (NMe)Phe3]NPFF (1DMe) and [D‐Tyr1, D‐Leu2, D‐Phe3]NPFF (3D), which are resistant to degradation and exhibit a high affinity for NPFF binding sites, were examined in tests of thermal and mechanical nociception. 2 1DMe and 3D produced potent dose‐dependent spinal antinociception in the tail‐flick test. On a molar basis, 1DMe was 20 and 50 times more potent than 3D and morphine, respectively, and high doses of 1DMe and 3D produced a sustained antinociceptive effect without visible signs of motor impairment. 3 Spinal antinociceptive effects produced by 1DMe (0.86 nmol) or 3D (8.6 nmol) were significantly reduced by i.t. co‐administration of naloxone (11 nmol) or i.t. pre‐administration of D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP, 9.25 nmol) or β‐funaltrexamine (β‐FNA, 2 nmol) or naltrindole (2.2 nmol). The doses of the μ‐antagonists (CTOP and β‐FNA) or the δ‐antagonist (naltrindole) used in 1DMe and 3D experiments blocked the antinociceptive effects of μ‐or δ‐receptor‐selective agonists. 4 When administered in combination with antinociceptive doses of the μ‐receptor agonist, morphine (13.2 nmol) or the δ‐receptor agonist, [D‐Ala2]deltorphin I (20 nmol), sub‐effective dose of 1DMe or 3D (0.009 nmol) enhanced and prolonged the spinal effects of these opioid agonists. 5 The results of this study show that spinal μ‐and δ‐opioid receptors play a role in antinociception produced by NPFF analogues. These results also suggest a role for NPFF in modulation of nociceptive signals at the spinal level.

Inhibition of neurokinin-1–substance P receptor and prostanoid activity prevents and reverses the development of morphine tolerance in vivo and the morphine-induced increase in CGRP expression in cultured dorsal root ganglion neurons

Chronic treatment with opioid drugs such as morphine leads to the development of tolerance, which manifests as a loss of drug potency. The mechanisms underlying this phenomenon are poorly understood, but recent evidence suggests that increased activity of nociceptive sensory transmitters [calcitonin gene‐related peptide (CGRP) and substance P] and other signalling messengers (prostaglandins) contribute to its development. Chronic intrathecal morphine administration to rats for 7 days produced analgesic tolerance. Co‐administration of SR140333, a selective substance P receptor (neurokinin‐1) antagonist, or nimesulide, a cyclooxygenase‐2‐selective inhibitor, augmented the acute effects of morphine, prevented morphine tolerance and reversed established tolerance. In cultured adult dorsal root ganglion neurons, exposure to morphine for 5 days increased the number of neurons expressing CGRP immunoreactivity. Co‐exposure with the peptide CGRP receptor antagonist CGRP8‐37, SR140333 or nimesulide prevented the morphine‐induced increase in the expression of CGRP immunoreactivity. Additionally, BIBN4096BS, a nonpeptide CGRP receptor antagonist, stereoselectively produced similar effects. In summary, this investigation demonstrates that activity of CGRP and substance P contributes to both the induction and expression of opioid analgesic tolerance. Additionally, it highlights the involvement of prostaglandins generated by spinal cyclooxygenase‐2 activity in the genesis of opioid tolerance. The neuropeptide and prostanoid activity contributing to tolerance is expressed at the level of the primary afferents terminating in the spinal cord. The combination of opioids with agents that block this activity may represent a useful strategy for the prevention as well as the reversal of clinical opioid tolerance.