Des-tyrosine(1) dynorphin A-(2-13) improves carbon monoxide-induced impairment of learning and memory in mice

The effects of des-tyrosine(1) dynorphin A-(2-13) (dynorphin A-(2-13)) on carbon monoxide (CO)-induced impairment of learning and memory in mice were investigated using a Y-maze task and a passive avoidance test. The lower percentage alternation and shorter step-down latency of the CO-exposed group indicated that learning and/or memory impairment occurred in mice 5 and 7 days after CO exposure, respectively. Administration of dynorphin A-(2-13) (1.5 and/or 5.0 nmol/mouse, intracerebroventricularly (i.c.v.)) 30 min before behavioral tests improved the CO-induced impairment in alternation performance and the CO-induced shortened step-down latency. We previously reported that dynorphin A-(1-13) improved the impairment of learning and/or memory via kappa opioid receptor mediated mechanisms. To determine whether the effect of dynorphin A-(2-13) was also mediated via kappa opioid receptors, we attempted to block its action using a selective kappa opioid receptor antagonist, nor-binaltorphimine (nor-BNI). Nor-BNI (4.9 nmol/mouse, i.c.v.) did not block the effects of dynorphin A-(2-13) on the CO-induced impairment of learning and/or memory. These results indicate that dynorphin A-(2-13) improves impairment of learning and/or memory via a non-opioid mechanism.     

Dynorphin A-(1-13) and (2-13) improve beta-amyloid peptide-induced amnesia in mice

The anti-amnesic effects of dynorphins on beta-amyloid peptide-(25-35)-induced impairment of learning and/or memory in mice were investigated using a Y-maze task and a passive avoidance test. Administration of beta-amyloid peptide-(25-35) (betaAP(25-35); 8.2 nmol, i.c.v.) 7 and 14 days before behavioral tests induced a decrease in both alternation behavior and latency in passive avoidance tests. Dynorphin A-(1-13) and A-(2-13) (0.5 and/or 1.5 nmol, i.c.v.) 30 min before behavioral tests improved the beta-amyloid peptide-(25-35)-induced impairment of alternation performance and shortened the step-down latency. Nor-binaltorphimine (4.9 nmol, i.c.v.) partially blocked the effects of dynorphin A-(1-13), but not A-(2-13). These results indicate that dynorphin A-(1-13) and A-(2-13) improve amnesia induced by betaAP-(25-35) via not only kappa opioid receptors, but also by non-opioid mechanisms.     

Behavioral evidence for a modulating role of σ ligands in memory processes. II. Reversion of carbon monoxide-induced amnesia

This study examined the effect of low doses of σ ligands amnesia induced in mice by successive carbon monoxide (CO) exposure. Mice were exposed three consecutive times to CO (10 ml/min, 30–50 s) at 38°C. Spatial working memory impairment was investigated 5 days later by monitoring spontaneous alternation behavior in a Y-maze. Delayed amnesia was examined 7 days after CO exposure by using a step-down passive avoidance test. The preadministration of the σ ligand 1,3-di-(2-tolyl)guanidine (DTG), at doses of 1 to 1000 μg/kg, s.c., 30 min before CO exposure did not affect the resulting amnesia in either test. However, when administered 30 min before the test, i.e., 5 or 7 dats after CO exposure, this agent completely reversed the CO-induced decrease in alternation performance, at doses of 10 to 100 μg/kg. The same effect was observed with (+)-N-allylnormetazocine ((+)-SKF 10,047), at doses of 100 to 300 μg/kg, but not with (-)-SKE 10,047. DTG, at the same dose range that reversed the decrease in alternation, also totally reversed the CO-induced decrease in step-down latency in the passive avoidance test. The curve for these effects was bell-shaped; the effects were not observed at the dose of 1 mg/kg. Moreover, α-(-4-fluorophenyl-2-pyrimidinyl)-1-piperazine butanol (BMY 14802), a putative σ antagonist (1–10 mg/kg i.p), did not affect CO-induced amnesia, but when simultaneously administered with DTG, it completely prevented its effect in both tests. These findings indicate that σ₁ sites may mediate a complete but bell-shaped reversion of CO-induced amnesia. The exact mechanisms remain to be determined, but they may involve the modulation of both N-methyl-d-aspartate and cholinergic nicotinic systems.     

Dynorphin A-(1–13) attenuates basal forebrain-lesion-induced amnesia in rats

The effects of dynorphin A-(1–13), an endogenous κ opioid agonist, on basal forebrain (BF)-lesion-induced amnesia in rats were investigated using step-through-type passive avoidance task. The BF was lesioned by injecting the cholinergic neurotoxin ibotenic acid (6 μg/side). The number of rats achieving the cut-off time (600 s) of step-through latency (STL) in BF-lesioned group significantly decreased as compared with that in sham-operated group. Dynorphin A-(1–13) (0.3 μg) significantly increased the number of rats achieving the cut-off time of STL in BF-lesioned rats. These results suggest that dynorphins play an improving role in the impairment of memory processes in BF-lesioned rats.     

Dynorphin A (2-13) improves mecamylamine-induced learning impairment accompanied by reversal of reductions in acetylcholine release in rats

Accumulating evidence indicates that the endogenous opioid peptides dynorphin A (1-17) and synthetic dynorphin A (1-13) interact not only with opioid receptors but also with as yet poorly characterized non-opioid binding sites. Dynorphin A (1-13) improved impairments of learning and memory via not only kappa-opioid receptor-mediated, but also 'non-opioid' mechanisms. In the present study, the effects of des-tyrosine(1) dynorphin A (2-13) as a non-opioid metabolite of dynorphin A, and dynorphin A (1-13) on mecamylamine-induced impairment of the acquisition of learning in rats were investigated using a step-through type passive avoidance task. Further, hippocampal acetylcholine release was examined using in vivo microdialysis. Mecamylamine significantly shortened the step-through latency when given 30 min before the acquisition trial. Not only dynorphin A (1-13) but also dynorphin A (2-13) attenuated the mecamylamine-induced impairment of the acquisition of learning. The effect of dynorphin A (2-13) was not blocked by pre-treatment with nor-binaltorphimine (nor-BNI), a selective kappa-opioid receptor antagonist. Dynorphin A (2-13) completely abolished the decrease in the extracellular acetylcholine concentration induced by mecamylamine and this effect was not blocked by nor-BNI. Taken together with our previous findings, the present results may indicate that dynorphin A (2-13) improves impairment of learning and/or memory in 'non-opioid' mechanisms and dynorphin A (1-13) ameliorates impairment of the acquisition of learning via not only kappa-opioid receptor-mediated mechanisms but also 'non-opioid' mechanisms, by regulating the release of extracellular acetylcholine.     

Amnesia induced in mice by centrally administered β-amyloid peptides involves cholinergic dysfunction

Substantial evidences suggest that the increased cerebral deposition, and neurotoxic action of the β-amyloid peptide, the major constituent of senile plaques, may represent the underlying cause of the cognitive deficits observed in Alzheimer's disease. Herein, we attempted to verify this hypothesis by inducing a potential Alzheimer's-type amnesia after direct intracerebroventricular administration of aggregatedβ_25–35-amyloid peptide in mice. In this aim, mnesic capacities were evaluated after 6–13 days, using spontaneous alternation in the Y-maze, step-down type passive avoidance and place learning in a water-maze. Pretraining administration of aggregatedβ_25–35 peptide induced dose-dependent decreases in both alternation behaviour and passive avoidance, at doses of 3 and 9 nmol/mouse. A reduced but still significant impairment was observed when the peptide was not aggregated, or ‘aged’, by preincubation for 4 days at 37°C. Theβ_1–28 peptide, at 3 nmol/mouse, also induced a marked decrease in step-down latency. Posttraining, but not preretention, administration of_25–35 peptide also significantly impaired learning. The beneficial effects of cholinergic agents onβ_25–35-induced amnesia was examined using the cholinesterase inhibitor tacrine (THA, 1.3 and 4.3 μmol/kg i.p.) and the nicotinic receptor agonist (−)-nicotine (NIC, 0.06 and 0.2 μmol/kg i.p.). Both drugs induced a dose-dependent abrogation of theβ_25–35-induced decreases in alternation behaviour and passive avoidance. Furthermore, THA, at 1.3 μmol/kg, and NIC, at 0.2 μmol/kg, also reversed theβ_25–31-induced impairment of place learning and retention in the water-maze. Histological examination of Cresyl violet-stained brain sections indicated a moderate but significant cell loss within the frontoparietal cortex and the hippocampal formation of mice treated with agedβ_25–35 peptide (9 nmol). Examination of Congo red-stained sections in the same animals demonstrated the presence of numerous amyloid deposits throughout these brain areas. These results confirm that the deposition of β-amyloid peptide in the brain is in some way related to impairment of learning and cholinergic degeneration and suggest that the [25–35] fragment of the β-amyloid protein, sufficient to induce neuronal death in cultures, also induces an Alzheimer's-type amnesia in mice.     

Pharmacological characterization of the ameliorating effect on learning and memory impairment and antinociceptive effect of KT-95 in mice

3-Acetoxy-6beta-acetylthio-10-oxo-N-cyclopropylmethyl-dihydronormorphine (KT-95) is a synthesized compound that binds to mu-, delta- and kappa-opioid receptors in vitro. KT-95 induces analgesia and this effect is antagonized by nor-BNI, a selective kappa-opioid receptor antagonist. We have reported that kappa-opioid receptor agonists improve impairment of learning and memory in mice and/or rats. In this study, the effects of KT-95 were investigated in an acetic acid-induced writhing test and scopolamine-induced memory impairment test using spontaneous alternation performance in a Y-maze and a step-down type passive avoidance test. Male ddY mice were treated with KT-95 (0.24-2.35 micromol/kg, s.c.) 30 min before the behavioral test. In the writhing test, the antinociceptive effect of KT-95 (2.35 micromol/kg) was completely antagonized by nor-BNI (4.9 nmol/mouse, i.c.v.), but not by naloxone (3.05 micromol/kg, s.c.). KT-95 significantly improved the impairment of spontaneous alternation induced by scopolamine (1.65 micromol/kg, s.c.). The ameliorating effect of KT-95 was not antagonized by nor-BNI, but was almost completely antagonized by a selective sigma-receptor antagonist, N,N-dipropyl-2-[4-methoxy-3-(2-phenylenoxy)-phenyl]-ethylamine monohydrochloride (NE-100, 2.6 micromol/kg, i.p.). KT-95 also significantly improved scopolamine-induced learning and memory impairment in the passive avoidance test, although the effect was partial. Administration of KT-95 itself induced impairment of learning and memory. KT-95-induced impairment was not antagonized by naloxone, naltrindole, nor-BNI or NE-100 indicating that this impairment was not because of opioid receptor stimulation. These results suggested that although the KT-95-induced antinociceptive effect was mediated by kappa-opioid receptors, the KT-95-induced improvement in scopolamine-induced impairment of memory was mediated mainly via sigma-receptors and partially by kappa-opioid receptors.     

Dynorphin A (1–13) Neurotoxicity in Vitro: Opioid and Non-Opioid Mechanisms in Mouse Spinal Cord Neurons

Dynorphin A is an endogenous opioid peptide that preferentially activates κ-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1–13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both κ-opioid and N-methyl- -aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through κ-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing κ-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both κ-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1–13) on intracellular calcium concentration ([Ca²⁺]_i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1–13) elevated [Ca²⁺]_i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 μM), 2-amino-5-phosphopentanoic acid (100 μM), or 7-chlorokynurenic acid (100 μM)—suggesting that dynorphin A (1–13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (−)-naloxone (3 μM), or the more selective κ-opioid receptor antagonist nor-binaltorphimine (3 μM), exacerbated dynorphin A (1–13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 μM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 μM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates κ-opioid receptors and suggests that κ receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1–13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.     

Pharmacological characterization of the ameliorating effect on short-term memory impairment and antinociceptive effect of KT-90 in mice

(−)-3-Acetyl-6β-acetylthio-N-cyclopropylmethyl-normorphine (KT-90) is a synthesized compound that binds to μ-, δ- and κ-opioid receptors in vitro. KT-90 induces analgesia in the tail-flick test and this effect is antagonized by nor-BNI, a selective κ-opioid receptor antagonist. However, lower doses of KT-90 antagonize morphine-induced analgesia. We reported that κ-opioid receptor agonists such as U-50,488H and dynorphin A (1-13), improved scopolamine-induced impairment of learning and memory in mice and/or rats. In this study, the effects of KT-90 were investigated in an acetic acid-induced writhing test and scopolamine-induced memory impairment test using spontaneous alternation performance in a Y-maze. Male ddY mice were treated with scopolamine (1.65 μmol/kg, s.c.) 30 min before the behavioral test. KT-90 (0.07–2.35 μmol/kg, s.c.) was injected 30 min before testing. In the writhing test, the antinociceptive effect of KT-90 (0.71 μmol/kg) was completely antagonized by a selective μ-opioid receptor antagonist, β-funaltrexamine (10.2 nmol/mouse, i.c.v.) and partially antagonized by nor-BNI (4.9 nmol/mouse, i.c.v.), but it was not antagonized by a selective δ-opioid receptor antagonist, naltrindole (9.1 pmol/mouse, i.c.v.). KT-90 significantly improved the impairment of spontaneous alternation induced by scopolamine. The ameliorating effect of KT-90 was not antagonized by nor-BNI, but was almost completely antagonized by a selective σ receptor antagonist, NE-100 (2.6 μmol/kg, i.p.). These results suggested that the KT-90-induced antinociceptive effect was mediated by μ- and partially by κ-opioid receptors, and the KT-90-induced improvement in scopolamine-induced impairment of spontaneous alternation was mediated mainly via σ receptors.     

Dynorphin A-induced rat hindlimb paralysis and spinal cord injury are not altered by the kappa opioid antagonist nor-binaltorphimine

The selective kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI) was used to distinguish a kappa opioid component in the mechanisms underlying the hindlimb paralysis, ischemia, and neuronal injury induced in the rat by the kappa opioid agonist dynorphin A. Spinal intrathecal (i.t.) injection of nor-BNI (20 nmol) either 15 min or immediately before i.t. injections of 5 or 20 nmol of dynorphin A failed to alter the dynorphin A-induced disruption of hindlimb motor function and nociceptive responsiveness. Nor-BNI also did not change the 3-fold increases in cerebrospinal fluid lactate concentrations produced by 20 nmol of dynorphin A. Neuroanatomical evaluations revealed that the cell loss, fiber degeneration, and central gray necrosis in lumbosacral spinal cords of rats treated with 20 nmol of dynorphin A were not altered by nor-BNI (20 nmol, i.t.). Thus, the spinal cord injury and associated neurological deficits resulting from i.t. injection of dynorphin A appear to be primarily, if not totally, attributable to its non-kappa opioid action(s).     

Structure-activity analysis of dynorphin A toxicity in spinal cord neurons: intrinsic neurotoxicity of dynorphin A and its carboxyl-terminal, nonopioid metabolites

Dynorphin A [dynorphin A (1-17)] is an endogenous opioid peptide that is antinociceptive at physiological concentrations. Levels of dynorphin A increase markedly following spinal cord trauma and may contribute to secondary neurodegeneration. Both kappa opioid and N-methyl-d-aspartate (NMDA) receptor antagonists can modulate the effects of dynorphin, suggesting that dynorphin is acting through kappa opioid and/or NMDA receptor types. Despite these findings, few studies have critically examined the mechanisms of dynorphin A neurotoxicity at the cellular level. To better understand how dynorphin affects cell viability, structure-activity studies were performed examining the effects of dynorphin A and dynorphin A-derived peptide fragments on the survival of mouse spinal cord neurons coexpressing kappa opioid and NMDA receptors in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A caused significant neuronal losses that were dependent on concentration (> or = 1 microM) and duration of exposure. Moreover, exposure to an equimolar concentration of dynorphin A fragments (100 microM) also caused a significant loss of neurons. The rank order of toxicity was dynorphin A (1-17) > dynorphin A (1-13) congruent with dynorphin A (2-13) congruent with dynorphin A (13-17) (least toxic) > dynorphin A (1-5) ([Leu(5)]-enkephalin) or dynorphin A (1-11). Dynorphin A (1-5) or dynorphin A (1-11) did not cause neuronal losses even following 96 h of continuous exposure, while dynorphin A (3-13), dynorphin A (6-17), and dynorphin A (13-17) were neurotoxic. The NMDA receptor antagonist MK-801 (dizocilpine) (10 microM) significantly attenuated the neurotoxic effects of dynorphin A and/or dynorphin-derived fragments except dynorphin A (13-17), suggesting that the neurotoxic effects of dynorphin were largely mediated by NMDA receptors. Thus, toxicity resides in the carboxyl-terminal portion of dynorphin A and this minimally includes dynorphin A (3-13) and (13-17). Our findings suggest that dynorphin A and/or its metabolites may contribute significantly to neurodegeneration during spinal cord injury and that alterations in dynorphin A biosynthesis, metabolism, and/or degradation may be important in determining injury outcome.     

Morphine and dynorphin(1–13) microinjected into the medial preoptic area and nucleus accumbens: effects on sexual behavior in male rats

The effects on sexual behavior of opiate receptor stimulation within A10 and A14 terminal areas were examined in the following experiments. Morphine (0.01–6 nmol) and dynorphin(1–13) (0.01–3 pmol) were microinjected into the medial preoptic area (MPOA). Morphine (10–100 pmol) and dynorphin (10–100 fmol) injected into the MPOA reduced both the latency to ejaculate and the number of intromissions triggering ejaculation. Morphine (6 nmol) produced a failure to resume copulating following the second ejaculation. Morphine (1–10 nmol) injected into the nucleus accumbens (ACC) shortened the latency to the first intromission and lengthened the second postejaculatory interval. Naloxone (3 mg/kg i.p.) reversed the effects of morphine on intromission latency and attenuated the lowering of ejaculatory threshold.     

Opioid-induced feeding: Localization of sensitive brain sites

These experiments were designed to identify brain sites at which opioids might act to influence ingestive behavior and to determine which opioid receptor types are involved. After food deprivation, rats were given microinjections of naloxone into several brain regions and food intake was measured. Injections into or near the paraventricular (PVN) or ventromedial (VMH) hypothalamic nuclei or the globus pallidus (GP) reduced food intake; injections into the striatum or lateral hypothalamus (LH) were ineffective. A second study examined the ingestive effects of roughly equimolar doses (1.43-1.75 nmol) of dynorphin A (DYN), beta-endorphin (beta-END), and D-Ala2,D-Leu5-enkephalin (DADLE) when injected into 4 different brain regions. Only DYN significantly increased food intake, and this effect was seen only with injections into the PVN and VMH. Beta-END stimulated water intake when injected into the PVN, VMH and GP but not the LH. Further studies indicated that with PVN injections, DYN was effective at a dose as low as 0.47 nmol, and that a higher dose of DADLE (4.39 nmol) did stimulate food intake. These studies support an important role for dynorphin and the kappa opioid receptor in the regulation of feeding and suggest that the opioid regulation of food and water intake can be differentiated both by sites of action and by effective agonists.     

Analgesic actions of dynorphin A(1–13) antiserum in the rat brain stem

This study was performed to evaluate the effects of dynorphin A(1–13) antiserum when microinjected into an active hyperalgesic region within the rat brain stem. When administered within the dorsal posterior mesencephalic tegmentum (DPMT) of intact conscious rats, dynorphin A(1–13) antiserum produced rapid onset and persistent prolongation of a low intensity thermally evoked tail avoidance response (LITETAR). These analgesic actions of the dynorphin A(1–13) antiserum appeared to be dose dependent. These studies support previous hypotheses about the existence of tonically active brain stem opioid hyperalgesic processes. Further, the results provide indirect evidence for a potential role of brain stem dynorphin(s) in facilitating pain.     

Dynorphin opioid inhibition of cocaine-induced,D1 dopamine receptor-mediated immediatem-early gene expression in the striatum

Neurons in the striatum that project to the substantia nigra contain the opioid peptide dynorphin. Stimulation of D1 dopamine receptors results in increased expression of mRNA encoding dynorphin as well as expression of immediate-early genes such as c-fos in these neurons. Levels of dynorphin vary in different regions of the normal rat striatum, being highest in ventral and medial striatum. In a prior study, we have shown that both regional and temporal patterns of c-fos induction following treatment with the indirect dopamine receptor agonist cocaine are inversely related to those of dynorphin expression. These results suggested that dynorphin is involved in regulating the responsiveness of these neurons to dopamine input. In the present experiments, we examined such a potential role for dynorphin by analyzing the influence of the dynorphin (kappa opioid receptor) agonist spiradoline on immediate-early gene induction by cocaine, and we determined that this immediate-early gene response is mediated by D1 dopamine receptors located in the striatum. As a marker of neuron activation, expression of c-fos and zif 268 immediate-early genes was assessed with quantitative in situ hybridization histochemistry. Results showed that (1) intrastriatal infusion of the D1 dopamine receptor antagonist SCH-23390 (2.5–250 pmol) resulted in a dose-dependent blockade of immediate-early gene induction by cocaine (30 mg/kg); (2) systemic administration of the kappa opioid receptor agonist spiradoline (0.5–10.0 mg/kg) decreased cocaine-induced expression of c-fos and zif 268 mRNAs in striatum in a dose-dependent manner; (3) intrastriatal infusion of spiradoline (1–50 nmol) also suppressed immediate-early gene induction by cocaine, demonstrating that kappa opioid receptors located in the striatum mediate such an effect; and (4) systemic and intrastriatal administration of spiradoline also affected immediate-early gene expression in cortex. These results demonstrate that, in striatum, immediate-early gene induction by cocaine is a D1 dopamine receptor-mediated process that is inhibited by activation of kappa opioid receptors. Therefore, these findings suggest that the striatal dynorphin opioid system acts directly and/or indirectly to inhibit dopamine input to striatonigral neurons through kappa opioid receptor-mediated processes in the striatum. © 1995 Wiley-Liss, Inc.     

Intrathecal administration of dynorphin A and its fragments increase heart rate and arterial pressure in the urethane anesthetized rat: mediation by a nonopioid mechanism.

Intrathecal administration of 6.50 nmol of dynorphin A (dyn A) (1-13) and (1-17) to the ninth thoracic (T9) spinal segment provoked a transient (5-10 min) increased in heart rate (40-60 beats per minute (bpm] and arterial pressure (20-25 mmHg). Intravenous administration and administration to the second thoracic (T2) segment failed to mimic the effect of T9 administration, suggesting that the cardiovascular effects of T9 administration did not occur via diffusion to the periphery or to the brainstem. The cardioacceleratory and hypertensive responses to T9 dyn A (1-13) administration were prevented by pretreatment with the nicotinic ganglion blocker hexamethonium (10 mg/kg), but were unaffected by bilateral adrenalectomy. These results suggest that the cardiovascular effects of dyn A were mediated predominantly via a sympathetic pathway that does not innervate the adrenal glands. The effects were not antagonized by pretreatment with the opiate receptor antagonist naloxone or by the specific kappa opiate receptor antagonist nor-binaltorphimine, suggesting that they were not mediated via activation of kappa opiate receptors. Further support for this conclusion was provided by experiments demonstrating that dyn A (3-13) (30 nmol), a dynorphin fragment which is devoid of kappa activity, mimicked the effect of dyn A (1-13), whereas administration of the synthetic kappa agonist U50, 488H (100 nmol), failed to elicit effects similar to those provoked by dyn A (1-13). It is concluded that the cardiovascular effects of intrathecal dyn A administration are mediated via a nonopioid mechanism.     

Intra-septal injections of glucose and glibenclamide attenuate galanin-induced spontaneous alternation performance deficits in the rat

Injection of the neuroactive peptide galanin into the rat hippocampus and medial septal area impairs spatial memory and cholinergic system activity. Conversely, injection of glucose into these same brain regions enhances spatial memory and cholinergic system activity. Glucose and galanin may both modulate neuronal activity via opposing actions at ATP-sensitive K⁺ (K-ATP) channels. The experiments described in this report tested the ability of glucose and the direct K-ATP channel blocker glibenclamide to attenuate galanin-induced impairments in spontaneous alternation performance in the rat. Intra-septal injection of galanin (2.5 μg), 30 min prior to plus-maze spontaneous alternation performance, significantly decreased alternation scores compared to those of rats receiving injections of vehicle solution. Co-injection of glucose (20 nmol) or the K-ATP channel blocker glibenclamide (5 nmol) attenuated the galanin-induced performance deficits. Glibenclamide produced an inverted-U dose–response curve in its interaction with galanin, with doses of 0.5 and 10 nmol having no effect on galanin-induced spontaneous alternation deficits. Drug treatments did not alter motor activity, as measured by overall number of arm entries during spontaneous alternation testing, relative to vehicle injected controls. These findings support the hypothesis that, in the septal region, galanin and glucose act via K-ATP channels to modulate neural function and behavior.     

Non-opioid dynorphin binding site on secretory vesicles of a pituitary-derived cell line

Accumulating evidence indicates that the endogenous opioid peptides dynorphinA-(1-17) and dynorphinA-(1-13) interact not only with opioid but also with yet poorly characterized non-opioid receptors. The latter have been implicated in a number of the effects of dynorphins including induction of ACTH release in sheep and in AtT 20 cells, a pituitary-derived mouse cell line. AtT 20 cells do not express opioid receptors and therefore are particularly suitable for search of non-opioid dynorphin receptors. We report here that ³H-dynorphinA-(1-13)-NH₂ associates specifically with AtT 20 cells, apparently through an uptake process and a binding site. Within the cell, it binds preferentially to fractions containing secretory vesicles, with a K_d of about 100 nM. DynorphinA-(1-17), and several non-opioid fragments of dynorphin, including A-(2-17), A-(2-16) and A-(2-13), compete with ³H-dynorphinA-(1-13)-NH₂ for that site with IC₅₀s ranging from 200 nM to 2 μM. ACTH(1-39) also competes with ³H-dynorphinA-(1-13)-NH₂ for the site with an IC₅₀ of about 300 nM. DynorphinA-(2-17) at μM concentrations stimulates release of ACTH from the isolated vesicles. The results indicate the presence of a non-opioid dynorphin binding site on the secretory vesicle fractions of AtT20 cells that might be involved in ACTH release. The ability of ACTH itself to compete for the binding sites associated with the vesicles suggest that those sites may be involved in an autocrine loop.     

Met-Enkephalin-Arg-Phe-like immunoreactive substances mediate electroacupuncture analgesia in the periaqueductal gray of the rabbit

The present study was undertaken to investigate whether the C-terminal extended Met-enkephalin heptapeptide (Met-enkephalin-Arg⁶-Phe⁷, MEAP) played a role in mediating the analgesic effect of electroacupuncture in rabbits. MEAP and its degrading enzyme inhibitor captopril as well as antiserum against MEAP were injected into the periaqueductal gray (PAG) via a previously implanted cannula. Their effects on nociception were tested by the escape response latency (ERL) elicited by radiant heat applied on the skin of the snout. (1) Microinjection of MEAP (30–240 nmol) into PAG produced a dose-dependent analgesic effect which was 2.5 times more potent than Met-enkephalin (MEK) and 3 times less potent than morphine. The complete reversal of the analgesia elicited by 240 nmol of MEAP by a small dose of naloxone (0.1 mg/kg, i.v.) indicates that the effect of MEAP is mediated by naloxone sensitive opioid receptors. (2) In rabbits, a dose-dependent analgesia was elicited by an intra-PAG injection of captopril (60–240 nmol). A single dose of 240 nmol captopril increased ERL by more than 100%. This effect could be reversed by 30 nmol of naloxone injected into the same site, or by antiserum recognizing MEAP (1 μL, titer 1:1500) but not by antiserum recognizing MEK (1 μl, 1:8000) suggesting that captopril was able to protect MEAP from degradation. (3) Intra-PAG injection of 60 nmol of captopril significantly potentiated the after effect of electroacupuncture (EA) induced analgesia. This effect could be blocked either by 30 nmol (but not 7.5 nmol) of naloxone, or by 1 μl (but not 0.1 μl) of MEAP antiserum. These results suggest that MEAP may cause analgesia by acting in PAG, and could be operative in EA analgesia.     

Upregulated dynorphin opioid peptides mediate alcohol-induced learning and memory impairment

The dynorphin opioid peptides control glutamate neurotransmission in the hippocampus. Alcohol-induced dysregulation of this circuit may lead to impairments in spatial learning and memory. This study examines whether changes in the hippocampal dynorphin and glutamate systems are related, and contribute to impairment of spatial learning and memory in a rat model of cognitive deficit associated with alcohol binge drinking. Hippocampal dynorphins (radioimmunoassay) and glutamate (in vivo microdialysis) were analyzed in Wistar rats exposed to repeated moderate-dose ethanol bouts that impair spatial learning and memory in the Water Maze Task (WMT). The highly selective, long-acting κ-opioid receptor (KOR) antagonist nor-binaltorphimine (nor-BNI) was administered systemically or into the hippocampal CA3 region to test a role of dynorphins in alcohol-induced dysregulations in glutamate neurotransmission and behavior in the WMT. The ethanol treatment impaired learning and memory, upregulated dynorphins and increased glutamate overflow in the CA3 region. Administration of nor-BNI after cessation of ethanol exposure reversed ethanol-induced changes in glutamate neurotransmission in animals exposed to ethanol and normalized their performance in the WMT. The findings suggest that impairments of spatial learning and memory by binge-like ethanol exposure are mediated through the KOR activation by upregulated dynorphins resulting in elevation in glutamate levels. Selective KOR antagonists may correct alcohol-induced pathological processes, thus representing a novel pharmacotherapy for treating of ethanol-related cognitive deficits.