Ingrid Nylander

On the origin of extracellular glutamate levels monitored in the basal ganglia of the rat by in vivo microdialysis.

Abstract: Several putative neurotransmitters and metabolites were monitored simultaneously in the extracellular space of neostriatum, substantia nigra, and cortex and in subcutaneous tissue of the rat by in vivo microdialysis. Glutamate (Glu) and aspartate (Asp) were at submicromolar and γ‐aminobutyric acid (GABA) was at nanomolar concentrations in all brain regions. The highest concentration of dopamine (DA) was in the neostriatum. Dynorphin B (Dyn B) was in the picomolar range in all brain regions. Although no GABA, DA, or Dyn B could be detected in subcutaneous tissue, Glu and Asp levels were ≈5 and ≈0.4 µM, respectively. Lactate and pyruvate concentrations were ≈200 and ≈10 µM in all regions. The following criteria were applied to ascertain the neuronal origin of substances quantified by microdialysis: sensitivity to (a) K+ depolarization, (b) Na+ channel blockade, (c) removal of extracellular Ca2+, and (d) depletion of presynaptic vesicles by local administration of α‐latrotoxin. DA, Dyn B, and GABA largely satisfied all these criteria. In contrast, Glu and Asp levels were not greatly affected by K+ depolarization and were increased by perfusing with tetrodotoxin or with Ca2+‐free medium, arguing against a neuronal origin. However, Glu and Asp, as well as DA and GABA, levels were decreased under both basal and K+‐depolarizing conditions by α‐latrotoxin. Because the effect of K+ depolarization on Glu and Asp could be masked by reuptake into nerve terminals and glial cells, the reuptake blocker dihydrokainic acid (DHKA) or l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (PDC) was included in the microdialysis perfusion medium. The effect of K+ depolarization on Glu and Asp levels was increased by DHKA, but GABA levels were also affected. In contrast, PDC increased only Glu levels. It is concluded that there is a pool of releasable Glu and Asp in the rat brain. However, extracellular levels of amino acids monitored by in vivo microdialysis reflect the balance between neuronal release and reuptake into surrounding nerve terminals and glial elements.

The striatonigral dynorphin pathway of the rat studied with in vivo microdialysis--II. Effects of dopamine D1 and D2 receptor agonists.

In vivo microdialysis was used to study the effect of intracerebral administration of dopamine agonists on dynorphin B release in the striatum and substantia nigra of rats. The release of dopamine and GABA was also investigated. Administration of the dopamine D1 agonist SKF 38393 (10-100 microM) into the striatum increased extracellular dynorphin B and GABA levels in the ipsilateral substantia nigra, in a concentration-dependent manner. After a short-lasting increase, nigral dopamine levels were significantly decreased after the highest concentration of striatal SKF 38393. An increase in striatal dynorphin B, GABA and dopamine levels was also observed. When SKF 38393 (10 microM) was administered into the substantia nigra, nigral dynorphin B and GABA, but not dopamine levels increased. No significant effects were observed on striatal levels. Administration of the dopamine D2 agonist, quinpirole (100 microM), into the striatum decreased dopamine levels in both striatum and substantia nigra, while no effect was observed on striatal or nigral dynorphin B and GABA levels. Quinpirole (10-100 microM) given into the substantia nigra, decreased striatal dopamine levels in a concentration manner. In the nigra, a short-lasting increase in dopamine levels was observed following the highest concentration of nigral quinpirole (100 microM). The effect was followed by a decrease in dopamine levels. No significant effects were observed on striatal or nigral dynorphin B and GABA levels. The results show that stimulation of D1 receptors in striatum and substantia nigra leads to activation of the striatonigral dynorphin pathway. A parallel effect could also be seen on nigral GABA release.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain dynorphin and enkephalin systems in Fischer and Lewis rats: effects of morphine tolerance and withdrawal

Lewis rats are more likely to self-administer various drugs of abuse than Fischer rats. Here these two strains of rats were compared with regard to basal brain opioid peptide levels and the response to chronic morphine treatment and to naloxone-precipitated withdrawal. Lewis rats had lower basal dynorphin peptides in the substantia nigra, striatum (not Leu-enkephalinArg6) and VTA (not dynorphin B) and the pituitary gland. Leu-enkephalinArg6 levels were also lower in these structures (with the exception of striatum which had higher levels) and in the nucleus accumbens. There were also strain differences in the response to chronic morphine treatment; in the nucleus accumbens, morphine treatment increased dynorphin A levels in Fischer rats only, in the ventral tegmental area effects were opposite with increased dynorphin levels in Fischer and decreased levels in Lewis rats, in the hippocampus dynorphin levels were markedly reduced in Lewis rats only. In Fischer rats, chronic morphine strongly affected peptide levels in the substantia nigra and striatum, whereas Lewis rats responded less in these areas. Leu-enkephalin, which derives from both prodynorphin and proenkephalin, and Met-enkephalin, which derives from proenkephalin, were affected by chronic morphine mainly in Fischer rats, increasing levels in most of the brain areas examined. The results in this study show (1) strain differences in basal levels of prodynorphin-derived opioid peptides, (2) the prodynorphin system to be differently influenced by morphine in Lewis rats than in Fischer rats and 3) the proenkephalin system to be influenced by chronic morphine in brain areas related to reward processes only in Fischer rats.

Modulation of neurotransmitter release by cholecystokinin in the neostriatum and substantia nigra of the rat: regional and receptor specificity.

The effect of cholecystokinin peptides on the release of dynorphin B, aspartate, glutamate, dopamine and GABA in the neostriatum and substantia nigra of the rat was investigated using in vivo microdialysis. Sulphated cholecystokinin-8S in the dialysis perfusate (1-100 microM) induced a concentration-dependent increase in extracellular dynorphin B and aspartate levels, both in the neostriatum and substantia nigra. Striatal dopamine levels were only increased by 100 microM of cholecystokinin-8S, while in the substantia nigra they were increased by 10-100 microM of cholecystokinin-8S. Extracellular GABA and glutamate levels were increased following 100 microM of cholecystokinin-8S only. Striatal cholecystokinin-8S administration also produced a significant increase in nigral dynorphin B levels. Local cholecystokinin-4 (100 microM) produced a moderate, but significant, increase of extracellular dynorphin B and aspartate levels in the neostriatum and substantia nigra. No effect was observed on the other neurotransmitters investigated. A 6-hydroxydopamine lesion of the nigrostriatal dopamine pathway did not affect the increases in dynorphin B and aspartate levels produced by local administration of cholecystokinin-8S. Basal extracellular GABA levels were increased significantly in both the neostriatum and substantia nigra ipsilateral to the lesion. Nigral glutamate and aspartate levels were also increased in the lesioned substantia nigra, but in the lesioned neostriatum aspartate levels were decreased. The cholecystokinin-B antagonist L-365,260 (20 mg/kg, s.c.), but not the cholecystokinin-A antagonist L-364,718 (devazepide; 20 mg/kg, s.c.), significantly inhibited the effect of cholecystokinin-8S on striatal dynorphin B and aspartate levels. In the substantia nigra, however, the effect of cholecystokinin-8S on dynorphin B and aspartate levels was inhibited to a similar extent by both L-365,260 and L-364,718. Pretreatment with L-364,718, but not with L-365.260, prevented the increase in nigral dopamine levels produced by nigral cholecystokinin-8S administration. Taken together, these results suggest that cholecystokinin-8S modulates dynorphin B and aspartate release in the neostriatum and substantia nigra of the rat via different receptor mechanisms. In the neostriatum, the effect of cholecystokinin-8S on dynorphin B and aspartate release is mediated via the cholecystokinin-B receptor subtype, while in the substantia nigra, cholecystokinin-8S modulates dynorphin B and aspartate release via both cholecystokinin-A and cholecystokinin-B receptor subtypes. Cholecystokinin-8S modulates dopamine release mainly in the substantia nigra, via the cholecystokinin-A receptor subtype.

The effects of morphine treatment and morphine withdrawal on the dynorphin and enkephalin systems in sprague-dawley rats

The effect of morphine tolerance and withdrawal on prodynorphin peptides was studied in relevant brain areas and in the pituitary gland of male Sprague-Dawley rats, and compared with effects on the proenkephalin-derived peptide Met-enkephalin. After 8 days of morphine injections (twice daily), dynorphin A and B levels increased in the nucleus accumbens and dynorphin A levels increased also in the striatum. Morphine treatment increased striatal Met-enkephalin. Leu-enkephalinArg6 levels were reduced in the ventral tegmental area (VTA). Morphine-treated rats had very low Leu-enkephalinArg6 levels in the hippocampus as compared to saline control rats. Comparison of the relative amounts of dynorphin peptides and the shorter prodynorphin-derived peptides, Leu-enkephalinArg6 and Leu-enkephalin, revealed a relative increase in dynorphin peptides versus shorter fragments in the nucleus accumbens, VTA and hippocampus. Morphine-tolerant rats had lower levels of dynorphin A in both lobes of the pituitary gland, whereas hypothalamic dynorphin levels were unaffected by morphine. Leu-enkephalinArg6 levels were reduced in the hypothalamus, but not changed in the pituitary gland. Naloxone-precipitated withdrawal accentuated the increase in dynorphin A and B levels in the accumbens and dynorphin A levels in the striatum, while inducing an increase in enkephalin levels in the accumbens and Met-enkephalin in the VTA. In the hippocampus, Leu-enkephalinArg6 levels remained low in the withdrawal state. The low dynorphin levels in the anterior part of the pituitary gland were reversed by naloxone, whereas the low dynorphin A levels in the neurointer-mediate lobe were even lower in the withdrawal state. In conclusion, morphine tolerance and withdrawal affected prodynorphin-derived peptides in areas related to central reward mechanisms, and in the pituitary gland. The dynorphin peptides and the LeuenkephalinArg6 fragment were not affected similarly, indicating an effect also on metabolic interconversion.

Differential dopaminergic regulation of the neurotensin striatonigral and striatopallidal pathways in the rat

Recently the existence of a neurotensin striatonigral pathway strongly up-regulated by methamphetamine has been demonstrated. The aim of the present study was to investigate, using immunohistochemistry and radioimmunoassay, the modulation of this pathway by dopamine antagonists. Rats were injected either with methamphetamine alone or together with the dopamine D1 receptor antagonist, SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-be nzapine hydrochloride), or with the dopamine D2 receptor antagonist, sulpiride. Both techniques showed that this neurotensin striatonigral pathway is regulated by dopamine D1 receptors, since SCH 23390 totally prevented the methamphetamine-induced increase in neurotensin-like immunoreactivity, both in the striatum and in the substantia nigra pars reticulata. Conversely, sulpiride was unable to counteract the effect of methamphetamine in these two areas, suggesting that dopamine D2 receptors are not involved in the regulation of this neurotensin pathway. On the other hand, neurotensin-like immunoreactivity was markedly increased in striatal cell bodies and in the globus pallidus after treatment with sulpiride, indicating that this pathway is mainly regulated by dopamine D2 receptors.

Processing of prodynorphin-derived peptides in striatal extracts. Identification by electrospray ionization mass spectrometry linked to size-exclusion chromatography

Proteolytic processing of prodynorphin-derived peptides in rat brain was studied with the help of high performance size exclusion chromatography (SEC) connected to electrospray ionization mass spectrometry. Extracts from rat striatum were incubated with individual synthetic dynorphin peptides. Dynorphin A was the most resistant to proteolytic cleavage, converting slowly to Leu-enkephalin (0.3 pmol/min), whereas dynorphin B was processed to this pentapeptide at a 10(4)-fold higher rate. Minor cleavage was also observed between Arg6-Arg7. Alphaneoendorphin was also rapidly metabolized to Leu-enkephalin (6 nmol/min) and, to a lesser extent, to Leu-enkephalinArg6. This new strategy for studying peptidases can easily be adapted to identification of components present in body fluids.

The striatonigral dynorphin pathway of the rat studied with In vivo microdialysis—I. Effects of K+-depolarization, lesions and peptidase inhibition

Extracellular levels of dynorphin B were analysed with in vivo microdialysis in the neostriatum and substantia nigra of halothane-anaesthetized rats. Dopamine and its metabolites, 3,4-dihydroxyphenyl-acetic acid and homovanillic acid, as well as GABA were simultaneously monitored. Chromatographic analysis revealed that the dynorphin B-like immunoreactivity measured in perfusates collected under basal and K(+)-depolarizing conditions co-eluted with synthetic dynorphin B. Dynorphin B, GABA and dopamine levels were Ca(2+)-dependently increased by K(+)-depolarization, while 3,4-dihydroxyphenylacetic acid and homovanillic acid levels were decreased. Dopamine and its metabolites, but not dynorphin B or GABA levels, were significantly decreased after a unilateral 6-hydroxydopamine injection into the left medial forebrain bundle. In contrast, following a unilateral injection of ibotenic acid into the striatum, dynorphin B and GABA levels were decreased by > 50% in striatum and substantia nigra on the lesioned side, whereas no significant changes were observed in basal dopamine levels. The inclusion of the peptidase inhibitor captopril (50-500 microM) into the nigral perfusion medium produced a concentration-dependent increase in nigral extracellular levels of dynorphin B. In the striatum, a delayed increase in dynorphin B and GABA levels could be observed following the nigral captopril administration, but this effect was not concentration-dependent. Thus, we demonstrate that extracellular levels of dynorphin B, dopamine and GABA can simultaneously be monitored with in vivo microdialysis. Extracellular dynorphin B appears to originate from neurons, since the levels were (i) increased in a Ca(2+)-dependent manner by K(+)-depolarization, and (ii) decreased by a selective lesion of the striatum, known to contain cell bodies of dynorphin neurons in the striatonigral pathway. Furthermore, (iii) the increase in nigral dynorphin B levels by peptidase inhibition suggests the presence of clearance mechanisms for the released dynorphin peptides.

Effect of morphine on dynorphin B and GABA release in the basal ganglia of rats.

In vivo microdialysis was used to study the effects of systemic, as well as intracerebral administration of morphine and naloxone on dynorphin B release in neostriatum and substantia nigra of rats. The release of dopamine (DA), gamma-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) was also investigated. Systemic injection of morphine (1 mg/kg s.c.) induced long-lasting increases in extracellular dynorphin B and GABA levels in the substantia nigra, whereas DA, Glu and Asp levels, measured in the same region, were not significantly affected. No effect on striatal neurotransmitter levels was observed following systemic morphine administration. Local perfusion of the substantia nigra with morphine (100 microM) through the microdialysis probe also increased nigral dynorphin B and GABA levels. Perfusion of the neostriatum with morphine (100 microM) significantly increased GABA and dynorphin B levels in the ipsilateral substantia nigra, but no effect was observed locally. Naloxone blocked the effect of systemic morphine administration on nigral dynorphin B and GABA release, already at a dose of 0.2 mg/kg s.c. Naloxone alone, given either systemically (0.2-4 mg/kg s.c.) or intracerebrally (1-100 microM), did not affect dynorphin B or amino acid levels, either in neostriatum or in substantia nigra. However, naloxone produced a concentration-dependent increase in DA levels. The present results indicate that systemic morphine administration stimulates the release of dynorphin B in the substantia nigra, probably by activating the mu-subtype of opioid receptor, since the effect of morphine on nigral dynorphin B and GABA was antagonized by a low dose of naloxone. The increase in extracellular DA levels produced by high concentrations of naloxone, both in neostriatum and substantia nigra, indicates a disinhibitory effect of this drug on DA release, probably via a non-mu subtype of opioid receptors located on nigro-striatal DA neurones.

Levels of dynorphin peptides, substance P and CGRP in the spinal cord after subchronic administration of morphine in the rat

Rats were rendered dependent on morphine by repeated injections of morphine, in increasing doses for 14 days and sacrificed. Levels of peptides in the dorsal spinal cord and dorsal root ganglia were analyzed in rats decapitated 2 hr, 24 hr (acute abstinent) or 7 days (late abstinent) respectively, after the last injection of drug. Dynorphin A was significantly decreased in rats abstinent for 24 hr, while dynorphin B remained unaffected. Substance P and CGRP, both putative transmitters in nociceptive primary afferent neurones, and partly existing together in the same neurone, were affected differently. Significantly less substance P but unchanged levels of CGRP were detected in rats abstinent for 24 hr, while on the other hand, CGRP but not levels of substance P, were increased 2 hr after the final injection. In dorsal root ganglia, levels of substance P were lower at 2 hr, while levels of CGRP were unaffected. In late (7 days) abstinence, no effect of opiate on any peptide was detected.