E. Rosengren

Endogenous Release of Neuronal Serotonin and 5‐Hydroxyindoleacetic Acid in the Caudate‐Putamen of the Rat as Revealed by Intracerebral Dialysis Coupled to High‐Performance Liquid Chromatography with Fluorimetric Detection

Abstract: Extracellular levels of endogenous serotonin (5‐HT) and its major metabolite, 5‐hydroxyindoleacetic acid (5‐HIAA), were measured in the caudate‐putamen of anesthetized and awake rats using intracerebral microdialysis coupled to HPLC with fluorimetric detection. A dialysis probe (of the loop type) was perfused with Ringer solution at 2 μl/min, and samples collected every 30 or 60 min. Basal indole levels were followed for up to 4 days in both intact and 5,7‐dihydroxytryptamine (5,7‐DHT) lesioned animals. Immediately after the probe implantation, the striatal 5‐HT levels were about 10 times higher than the steady‐state levels that were reached after 7‐8 h of perfusion. The steady‐state baseline levels, which amounted to 22.5 fmol/30 min sampling time, remained stable for 4 days. In 5,7‐DHT‐denervated animals, the steady‐state levels of 5‐HT, measured during the second day after probe implantation, were below the limit of detection (<10 fmol/60 min). However, during the first 6h post‐implantation, the 5‐HT output was as high as in intact animals, which suggests that the high 5‐HT levels recovered in association with probe implantation were blood‐derived. As a consequence, all other experiments were started after a delay of at least 12 h after implantation of the dialysis probe. In awake, freely moving animals, the steady‐state 5‐HT levels were about 60% higher than in halothane‐anesthetized animals, whereas 5‐HIAA was unaffected by anesthesia. KCI (60 and 100 mM) added to the perfusion fluid produced a sharp increase in 5‐HT output that was eight‐fold at the 60 mM concentration and 21‐fold at the 100 mM concentration. In contrast, 5‐HIAA output dropped by 43 and 54%, respectively. In 5,7‐DHT‐lesioned animals, the KCl‐evoked (100 mM) release represented less than 5% of the peak values obtained for the intact striata. Omission of Ca2+ from the perfusion fluid resulted in a 70% reduction in baseline 5‐HT output, whereas the 5‐HIAA levels remained unchanged. High concentrations of tetrodotoxin (TTX) added to the perfusion medium (5‐50 μM) resulted in quite variable results. At a lower concentration (1 μM), however, TTX produceda 50% reduction in baseline 5‐HT release, whereas the 5‐HIAA output remained unchanged. The 5‐HT reuptake blocker, indalpine, increased the extracellular levels of 5‐HT sixfold when added to the perfusion medium (1 μM), and threefold when given intraperitoneally (5 mg/kg). By contrast, the 5‐HIAA level remained unaffected during indalpine infusion. Application of TTX (1 μM) under simultaneous 5‐HT uptake blockade induced a decrease in 5‐HT output by 62–71%. p‐Chloroamphetamine (2.5 mg/kg, i.p.) induced a 12‐fold increase in 5‐HT release and reduced the 5‐HIAA output by about 50%. The p‐chloroamphetamine‐induced increase in 5‐HT release was 10 times lower in the 5,7‐DHT‐denervated striatum. Pargyline (75 mg/kg, i.p.) increased the extracellular levels of 5‐HT 11‐fold within 6 h, and reduced the 5‐HIAA levels by 80%. The 5‐HT receptor agonist, 5‐methoxy‐N,N‐dimethyltryptamine (1 mg/kg, i.p.), produced an immediate reduction of about 50% in 5‐HT release and a small (11 %) decrease in 5‐HIAA output. It is concluded (1) that intracerebral microdialysis coupled to HPLC with fluorimetric detection provides a useful method for the study of extracellular 5‐HT and 5‐HIAA levels; (2) that steady‐state levels of 5‐HT and 5‐HIAA recovered in the dialysis perfusate are neuronally derived, but these steady‐state levels are reached only after a minimum of 7–8 h after probe implantation; (3) that changes in striatal extracellular levels of 5‐HT are closely related to changes in serotonergic synaptic activity; and (4) that extracellular levels of 5‐HIAA are a poor indicator of synaptic activity, and instead primarily reflect intraneuronal metabolism.

The macrophage migration inhibitory factor MIF is a phenylpyruvate tautomerase

A macrophage migration inhibitory factor (MIF), originally described as a product of activated lymphocytes, has been defined as a 12 kDa protein, expressed in a wide variety of tissues. Here MIF is identified as a phenylpyruvate tautomerase (EC 5.3.2.1) having p‐hydroxyphenylpyruvate and phenylpyruvate as its natural substrates. The definition of MIF as an enzyme may yield insight into the mechanism of action of this proinflammatory and immunomodulating cytokine.

Acetylcholine release in the rat hippocampus as studied by microdialysis is dependent on axonal impulse flow and increases during behavioural activation

Changes in extracellular levels of acetylcholine and choline in the hippocampal formation were measured using intracerebral microdialysis coupled to high performance liquid chromatography with post-column enzyme reaction and electrochemical detection. Various pharmacological and physiological manipulations were applied to awake unrestrained normal rats and rats subjected to a cholinergic denervation of the hippocampus by a complete fimbria-fornix lesion (1-2 weeks previously). Low baseline levels of acetylcholine (about 0.3 pmol/15 min sample) could be detected in the absence of acetylcholinesterase inhibition in all animals. However, in order to obtain stable and more readily detectable levels, the acetylcholinesterase inhibitor neostigmine was added to the perfusion medium at a concentration of 5 or 10 microM and was used during all subsequent manipulations. Addition of neostigmine increased acetylcholine levels approximately 10-fold (to 3.7 pmol 15 min) in the normal rats, which was about 4-fold higher than the levels recovered from the denervated hippocampi. Depolarization by adding KCl (100 mM) to the perfusion fluid produced a 3-fold increase in the extracellular acetylcholine levels, and the muscarinic antagonist atropine (3 microM) resulted in a 4-fold increase in the normal rats, whereas these drugs induced only small responses in the denervated rats. Neuronal impulse blockade by tetrodotoxin (1 microM) resulted, in normal rats, in a 70% reduction in extracellular acetylcholine levels. Sensory stimulation by handling increased acetylcholine levels by 94% in the normal rats, whereas this response was almost totally abolished in the denervated hippocampi. Behavioural activation by electrical stimulation of the lateral habenula resulted in a 4-fold increase in acetylcholine release in normal animals, and this response was totally blocked by a transection of the lateral habenular efferents running in the fasciculus retroflexus. The levels obtained by lateral habenula stimulation were reduced by about 95% in the rats with fimbria-fornix lesions. Following an acute knife transection of the fimbria-fornix performed during ongoing dialysis, acetylcholine levels dropped instantaneously by 70%, indicating that the extracellular acetylcholine levels in the hippocampus are maintained by a tonic impulse flow in the septohippocampal pathway. The extracellular levels of choline were reduced by about 30% after the addition of neostigmine in the normal rats, and increased by about 50% in both normal and denervated rats after addition of KCl to the perfusion fluid. No changes could be detected after atropine, handling, lateral habenula stimulation, or acute fimbria-fornix or fasciculus retroflexus transection.(ABSTRACT TRUNCATED AT 400 WORDS)

The apparent autoxidation rate of catechols in dopamine-rich regions of human brains increases with the degree of depigmentation of substantia nigra.

SummaryThe concentrations of the 5-S-cysteinyl adducts of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylalanine (DOPA) and the levels of noradrenaline (NA), DA, DOPAC and DOPA were determined in the putamen (PUT), caudate nucleus (CN) and substantia nigra (SN) of human post mortem brains with or without depigmentation and degeneration of the SN. The levels of DA, DOPAC and DOPA decreased with the degree of depigmentation and degeneration in the three brain regions while NA levels only decreased in SN and PUT. In general, the concentrations of the 5-S-cysteinyl adducts did not differ, but the ratios of 5-S-cysteinyl-DA/DA, 5-S-cysteinyl-DOPAC/DOPAC and 5-S-cysteinyl-DOPA/DOPA were higher in patients with a more depigmentated and degenerated SN, except for the 5-S-cysteinyl-DA/DA ratio in the PUT. Higher ratios were also found in the cell body areas compared to the neuron terminal areas.Thus depigmentation and degeneration of dopaminergic SN neurons, seem to be correlated to enhanced rates of autoxidation, possibly due to an impaired antioxidant capacity.

5-Hydroxytryptamine of the spinal cord normally and after transection

Der 5-Hydroxytryptamingehalt im Rückenmark des Kaninchens ist 0,26 µg/g. Querschnittsläsion durchs Rückenmark führt zu einer 85prozentigen Abnahme des 5-Hydroxytryptamingehaltes. Es wird darum angenommen, dass 5-Hydroxytryptamin in Nervenzellen des Zentralnervensystems lokalisiert ist.

Occurrence and distribution of 5-S-cysteinyl derivatives of dopamine, dopa and dopac in the brains of eight mammalian species

The 5-S-cysteinyl derivatives of dopamine, dopa (3,4-dihydroxyphenylalanine) and dopac (3,4-dihydroxyphenylacetic acid) were synthesized and used as reference compounds in high performance liquid chromatography analyses of extracts from various brain regions of eight mammalian species. All three metabolites were detected in the brains of all the species studied. The regional distribution of the metabolites was similar to that of dopamine; the metabolite concentrations ranged from less than 0.1 percent to more than 1 percent of the dopamine level, the highest ratios generally being found in substantia nigra. It is suggested that the 5-S-cysteinyl catechol metabolites have been formed after autoxidation of catechols to quinones and subsequent coupling to glutathione. The adduct thus formed is finally split by peptidases to yield the 5-S-cysteinyl derivatives.

Neuronal localization of dopamine and 5-Hydroxytryptamine in some mollusca

SummaryThe localization of biogenic monoamines in ganglionic tissues from Anodonta piscinalis, Helix pomatia, and Buccinum undatum has been studied by means of the histochemical fluorescence method of Falck and Hillarp.In cerebral, visceral, and pedal ganglia (besides nonfluorescent nerve cells) neurons emitting a green or yellow fluorescence were found. No other cell systems exhibiting a specific fluorescence were observed. An abundance of monoaminergic terminals were found in the central parts of these ganglia. Spectrophotofluorimetric determinations showed that there are large quantities of dopamine and 5-hydroxytryptamine in the tissues investigated. The amounts of dopamine and 5-hydroxytryptamine agree well with the distribution of green and yellow fluorescence, respectively, in the ganglia.There are many similarities between the vertebrate and the molluscan monoaminergic neurons. The morphology of the neurons is the same, the intraneuronal distribution of the monoamines is identical, depletion experiments with reserpine and denervation experiments give the same results, and the synaptic arrangement of monoaminergic fibres on non-adrenergic neurons has the same appearance. Apparently, however, dopamine and 5-hydroxytryptamine are the only monoamines acting as neuronal transmitters in the species investigated.

The neuromelanin of the human substantia nigra

The pigment of the human substantia nigra was isolated after extraction of lipids and proteins with 2% sodium cholate in 30% ethanol followed by 2% sodium dodecyl sulfate in 10% glycerol. The pigment was hydrolysed with HI or degraded by treatment with KMNO4 and the samples were examined for compounds known to derive from pheomelanin (4-amino-3-hydroxyphenylalanine, AHP and 4-amino-3-hydroxyphenylethylamine, AHPEA), or from eumelanin (pyrrole-2,3,5-tricarboxylic acid, PTCA). The HI hydrolysis yielded AHPEA in large quantities, indicating cysteinyldopamine as the main source of the pheomelanin moiety of the neuromelanin, but also trace amounts of AHP, derived from cysteinyldopa oxidation products. Dopamine and small quantities of dopa were also obtained by HI hydrolysis of the neuromelanin. The yield of PTCA was low, but the amounts observed show that part of the neuromelanin is of the eumelanin type, a fact compatible with an occasional exhaustion of the glutathione-cysteine reduction system at the site of neuromelanin formation.

Detection of 5-S-cysteinyldopamine in human brain

5-S-Cysteinyldopamine was synthesized and used as reference compound in HPLC analyses of extracts from various regions of human brain. The compound could be detected in brain regions rich in dopamine (caudate nucleus, putamen, globus pallidus and substantia nigra) but not in other regions (cerebellum, occipital cortex). The occurrence of 5-S-cysteinyldopamine in dopaminergic brain regions supports the hypothesis that dopamine in part undergoes autoxidation, leading to formation of highly reactive quinones. The newly discovered metabolite may prove useful in future studies of dopamine autoxidation and the possibly resultant cytotoxicity in aging and degenerative brain disorders.

Regulation of striatal serotonin release by the lateral habenula-dorsal raphe pathway in the rat as demonstrated by in vivo microdialysis : role of excitatory amino acids and GABA

Striatal extracellular levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were monitored with the microdialysis technique during electrical stimulation of the lateral habenula-dorsal raphe (LHb-NRD) pathway in halothane anaesthetized rats. A new double-loop probe, with an improved recovery factor, was implanted into the head of the caudate-putamen and perfused with Ringer solution containing 1 microM of the 5-HT uptake blocker indalpine. Samples were collected every 15 min and analyzed with HPLC coupled to fluorimetric detection. Low frequency stimulation of the LHb (1.5 and 3 Hz, 0.5 mA) produced no detectable changes in striatal indole levels, whereas 15 Hz stimulation induced a 70% increase in 5-HT release. This effect was most likely mediated by a direct LHb-NRD link, since it persisted after ibotenic acid lesions of the interpeduncular nucleus (which is the major projection area for the medial habenular nucleus), but was completely abolished after transection of the fasciculus retroflexus, which carries the axons of the LHb-NRD pathway. The possible identity of the transmitter operating in the LHb-NRD pathway was investigated by NRD injections of kynurenic acid, a potent blocker of excitatory amino acid transmission, and by NRD injections of the GABA antagonist bicuculline. Kynurenic acid (300 nl, 50 mM) did not by itself induce any detectable changes in spontaneous indole output, but completely blocked the effect of LHb stimulation. Injection of bicuculline (300 nl, 2 mM) increased the striatal 5-HT output by about 70%, and potentiated the effect of LHb stimulation by a further 50%. In none of the experiments performed in this study were there any significant changes in striatal 5-HIAA output. These data are compatible with the idea that excitatory amino acids in the LHb-NRD pathway are involved in the regulation of striatal 5-HT release, and that this influence is modulated by GABAergic synaptic activity at the level of the NRD.