Monoamine Neurotransmitters and Their Metabolites in the Mature Rabbit Brain Following Induction of Hydrocephalus

Functional and behavioral disturbances associated with hydrocephalus may be due in part to altered neurotransmitter function in the brain. Hydrocephalus was induced in adult rabbits by injection of silicone oil into the cisterna magna. These and controls were killed 3 days, 1 and 4 weeks post-injection. Tissue concentrations of norepinephrine, epinephrine, serotonin, dopamine, and the metabolites 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and 3,4-dihydroxyphenylacetic acid (DOPAC) levels were determined in fifteen brain regions using HPLC. There were decreases in hypothalamic and medullary dopamine, transient decreases in basal ganglia serotonin, increases in thalamic noradrenaline, and increases in hypothalamic and thalamic epinephrine. Changes in the primary neurotransmitters may be attributable to damage of their axonal projection systems. Metabolite concentrations increased in the cerebrum. Reduced clearance of extracellular fluid which accompanies cerebrospinal fluid stasis may explain the accumulation of metabolites.     

THE CONCENTRATIONS OF FREE AMINO ACIDS AND OTHER ELECTROLYTES IN CEREBROSPINAL FLUID,IN VIVO DIALYSATE OF BRAIN,AND BLOOD PLASMA OF THE DOG*

The concentrations of free amino acids in plasma, CSF and in vivo dialysates of peripheral blood (neck sac fluid) and central nervous tissue (brain sac fluid) from each of five dogs (neck sac fluid from four of five dogs) were determined by ion-exchange chromatography. Dialysates were obtained by implanting small dialysis sacs filled with a dextran-saline solution into the subcutaneous tissue of the neck or the parenchyma of the brain at least 10 weeks before sample collection. The mean plasma concentration of most amino acids was within the range of values reported in the literature for human or dog plasma. The concentrations of most amino acids were higher in the neck sac fluid than in plasma; this discrepancy, however, was, for the most part, small and could most likely be accounted for by falling plasma free amino acid levels prior to sample taking. Previous conclusions that the CSF concentrations of most amino acids are lower than plasma concentrations are confirmed, although the present work indicates that there may be considerable individual variation in the CSF/plasma distribution ratio with respect to most amino acids. In the brain sac fluid the concentration of nearly every amino acid was consistently higher than that in CSF and lower than that in the neck sac fluid. The potassium concentration in the brain sac fluid was significantly higher than, and the total osmolality significantly lower than, those in the neck sac fluid. On the assumption that the brain sac fluid represents a dialysate of the brain extracellular fluid, these results contradict recent findings (Bito and Davson , 1965; 1966) indicating that the potassium concentration of the cortex extracellular fluid is lower than that of ventricular or cisterna magna CSF and certainly lower than that of plasma. Because of this and on the basis of consideration of the reaction of the brain to a foreign body, the possibility that the implanted brain sac lay on the‘blood side’of the bloodbrain barrier was suggested. Some implications of this possibility are discussed.     

Monoamines and their metabolites in somatosensory,visual, and cingulate cortices of adult rat: Differences in content and lack of sidedness

Small areas of somatosensory, visual and cingulate cortex were microdissected and assayed for their monoamine content by high-performance liquid chromatography with electrochemical detection. No differences were found between the right and the left hemisphere for any area nor for any of the monoamines. The values averaged from left and right hemispheres for the sensory areas were significantly different from the cingulate in the content of norepinephrine, 4-hydroxy-3-methoxyphenylglycol, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-hydroxyl-tryptophan, serotonin and 5-hydroxyindole-3-acetic acid. The two sensory cortices differed in their levels of norepinephrine, dopamine, 3–4-dihydroxyphenylacetic acid and homovanillic acid. In the latter comparison, the measured amounts were higher in somatosensory than in visual cortex. This biochemical heterogeneity in monoamine distribution may reflect specific innervation patterns for these compounds in these discrete cortical areas and allows differences in content to be related to functional specialization of the cerebral cortex.     

Quinolinic acid is extruded from the brain by a probenecid-sensitive carrier system: a quantitative analysis

Although the neurotoxic tryptophan-kynurenine pathway metabolite quinolinic acid originates in brain by both local de novo synthesis and entry from blood, its concentrations in brain parenchyma, extracellular fluid, and CSF are normally below blood values. In the present study, an intraperitoneal injection of probenecid (400 mg/kg), an established inhibitor of acid metabolite transport in brain, into gerbils, increased quinolinic acid concentrations in striatal homogenates, CSF, serum, and homogenates of kidney and liver. Direct administration of probenecid (10 mM) into the brain compartment via an in vivo microdialysis probe implanted into the striatum also caused a progressive elevation in both quinolinic acid and homovanillic acid concentrations in the extracellular fluid compartment but was without effect on serum quinolinic acid levels. A model of microdialysis transport showed that the elevations in extracellular fluid quinolinic acid and homovanillic acid levels following intrastriatal application are consistent with probenecid block of a microvascular acid transport mechanism. We conclude that quinolinic acid in brain is maintained at concentrations below blood levels largely by active extrusion via a probenecid-sensitive carrier system.     

Haloperidol impairs auditory filial imprinting and modulates monoaminergic neurotransmission in an imprinting-relevant forebrain area of the domestic chick

In vivo microdialysis and behavioural studies in the domestic chick have shown that glutamatergic as well as monoaminergic neurotransmission in the medio-rostral neostriatum/hyperstriatum ventrale (MNH) is altered after auditory filial imprinting. In the present study, using pharmaco-behavioural and in vivo microdialysis approaches, the role of dopaminergic neurotransmission in this juvenile learning event was further evaluated. The results revealed that: (i) the systemic application of the potent dopamine receptor antagonist haloperidol (7.5 mg/kg) strongly impairs auditory filial imprinting; (ii) systemic haloperidol induces a tetrodotoxin-sensitive increase of extracellular levels of the dopamine metabolite, homovanillic acid, in the MNH, whereas the levels of glutamate, taurine and the serotonin metabolite, 5-hydroxyindole-3-acetic acid, remain unchanged; (iii) haloperidol (0.01, 0.1, 1 mm) infused locally into the MNH increases glutamate, taurine and 5- hydroxyindole-3-acetic acid levels in a dose-dependent manner, whereas homovanillic acid levels remain unchanged; (iv) systemic haloperidol infusion reinforces the N-methyl-d-aspartate receptor-mediated inhibitory modulation of the dopaminergic neurotransmission within the MNH. These results indicate that the modulation of dopaminergic function and its interaction with other neurotransmitter systems in a higher associative forebrain region of the juvenile avian brain displays similar neurochemical characteristics as the adult mammalian prefrontal cortex. Furthermore, we were able to show that the pharmacological manipulation of monoaminergic regulatory mechanisms interferes with learning and memory formation, events which in a similar fashion might occur in young or adult mammals.     

Phencyclidine (PCP) injected in the nucleus accumbens increases extracellular dopamine and serotonin as measured by microdialysis

Phencyclidine (PCP; 20 micrograms in 0.5 microliter) was tested by local brain injection for neurochemical effects in the nucleus accumbens and striatum of rats. Changes in dopamine turnover could not be detected in postmortem tissue assays. In contrast, extracellular levels of dopamine significantly increased as measured by microdialysis in freely moving animals. PCP also increased extracellular levels of serotonin and decreased 3,4-dihydroxyphenylacetic acid (DOPAC), but did not change homovanillic acid (HVA) or 5-hydroxyindoleacetic acid (5HIAA). Microdialysis suggests that PCP acts in some dopamine terminal regions to increase extracellular dopamine and serotonin.     

Significant correlation between cerebrospinal fluid and brain levels of norepinephrine, serotonin and acetylcholine in anesthetized rats

The present study was undertaken to determine cerebrospinal fluid (CSF) and brain levels of norepinephrine (NE), serotonin (5-HT) and their metabolites--3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenylacetic acid (HVA) and 5-hydroxyindole-3-acetic acid (5-HIAA)--in rats pretreated with 6-hydroxydopamine (6-OHDA) or 5,7-dihydroxytryptamine (5,7-DHT). In the 6-OHDA pretreated rats, both CSF and brain concentrations of NE, DOPAC and HVA sustained significant decreases as compared with those in non-treated rats. Positive and significant correlations between CSF and brain levels were observed in respect to NE, DOPAC and HVA. In 5,7-DHT pretreated rats, both CSF and brain concentrations of 5-HT and 5-HIAA were significantly decreased. A positive and significant correlation between CSF and brain levels in respect to 5-HT and 5-HIAA was observed. Further studies were carried out to determine ACh levels of both the CSF and the brain in microspheres (MS)-treated rats, which are used as a model of microembolization. The CSF ACh concentrations in MS-treated groups were significantly decreased as compared with those in non-treated rats. The brain ACh contents also tended to decrease in this group. A positive and significant correlation was observed between CSF and brain levels of ACh. These findings suggest that NE, 5-HT and ACh concentrations in the CSF are direct indications of central noradrenergic, serotonergic and cholinergic nerve activity, respectively.     

Regional distribution of monoamines and monoamine metabolites in the brain of the crucian carp (Carassius carassius L.)

1. Monoamines (which all demand oxygen for their synthesis) and monoamine metabolites were quantified in 6 brain regions of the extremely anoxia tolerant crucian carp (Carassius carassius L.).2. The norepinephrine levels were generally twice as high as the dopamine levels. No epinephrine was found.3. The major dopamine metabolite seemed to be homovanillic acid. No 3,4-dihydroxyphenylacetic acid was found.4. Serotonin occurred at about the same levels as dopamine. The levels of the main serotonin metabolite, 5-hydroxyindole-3-acetic acid, were about 10% of that of serotonin.5. All three monoamines had a similar distribution, with the highest concentrations in hypothalamus and the lowest in cerebellum and vagal lobes.6. The distribution and levels of monoamines agreed with that of anamniote vertebrates in general, suggesting that the crucian carp has not adapted to anoxia by abandoning or minimizing the use of monoaminergic systems.     

Effect of probenecid on 5-hydroxyindoleacetic acid in cisternal cerebrospinal fluid of rats with portacaval anastomosis

Portal-systemic encephalopathy (PSE) is characterized by a neuropsychiatric disorder progressing through personality changes, to stupor and coma. Previous studies have revealed alterations of serotonin and of its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in brain tissue and CSF in experimental (rat) and human PSE. Increased brain 5-HIAA concentrations could result from its decreased removal rather than to increased serotonin metabolism. In order to evaluate this possibility, CSF 5-HIAA concentrations were measured using an indwelling cisterna magna catheter technique at various times following end-to-side portacaval anastomosis in rats (the most widely used animal model of PSE) treated with probenecid, a competitive inhibitor that blocks the active transport of acid metabolites out of the brain and CSF. Following portacaval anastomosis and probenecid treatment, CSF concentrations of 5-HIAA were increased to a greater extent than in sham-operated controls. When data were expressed as per-cent baseline values, the relative increase of CSF 5-HIAA in portacaval shunted rats following probenecid treatment was not significantly different from sham-operated controls. These findings confirm that increased 5-HIAA in the CNS in experimental PSE results from increased 5HT metabolism or turnover and that the probenecid-sensitive acid metabolite carrier is intact in PSE.     

Rat brain monoamine and serotonin S2 receptor changes during pregnancy

The concentrations of noradrenaline (NA), dopamine (DA), serotonin (5-HT), and their metabolites were determined in 5 brain areas of non-pregnant, 15 and 20 day pregnant and 4 day post-partum rats. Striatal 5-HT content was significantly lower in 15 and 20 day pregnant rats than in estrous controls. A significant decrease in striatal and frontal cortex 5-hydroxyindole-3-acetic acid (5-HIAA) concentration was observed in 15 day pregnant rats. Significant increases in hypothalamic and hippocampal NA levels were observed at 4 days post-partum. Frontal cortex serotonin S2 receptorKd was reduced in 4 day post-partum rats. There was no significant change in S2 receptorB _max during pregnancy. Levels of progesterone were negatively correlated with striatal DA, homovanillic acid (HVA), 5-HT, and 5-HIAA levels, hypothalamic DA, hippocampal 5-HT, and frontal cortex 5-HIAA values as well as striatal HVA to DA, and HVA to 3,4-dihydroxyphenylacetic acid (DOPAC) ratios and amygdaloid HVA to DOPAC ratios. The limbic neurotransmitter changes might possibly contribute to mood changes which occur during pregnancy and post-partum.     

High-performance liquid chromatography with electrochemical detection for the determination of levodopa, catecholamines and their metabolites in rat brain dialysates

A high-performance liquid chromatographic assay with electrochemical detection is described for the simultaneous determination of levodopa, 3-O-methyldopa, dopamine, dihydroxyphenylacetic acid, homovanillic acid, 3-methoxytyramine, noradrenaline, adrenaline, 3-methoxy-4-hydroxyphenylethylene glycol and 5-hydroxyindoleacetic acid in rat brain dialysates. Samples are obtained in vivo using the microdialysis technique. Microdialysis probes are placed in the brain area to be studied and neurochemicals are collected by perfusion of the probe with modified Ringer's solution. Direct injection of the dialysates allows rapid and reliable results to be obtained.     

A rapid and sensitive method for the analysis of brain monoamine neurotransmitters using ultra-fast liquid chromatography coupled to electrochemical detection

Electrochemical detection is often used to detect catecholamines and indolamines in brain samples that have been separated by conventional reverse-phase high performance liquid chromatography (HPLC). This paper presents the transfer of an existing chromatographic method for the determination of monoamines in brain tissues using 5 μm granulometry HPLC columns to columns with a particle diameter less than 3 μm. Several parameters (repeatability, linearity, accuracy, limit of detection, and stability of samples) for this new ultrafast high performance liquid chromatography (UHPLC) method were examined after optimization of the analytical conditions. The separation of seven compounds, noradrenaline, dopamine and three of its metabolites, dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine, and serotonin and its metabolite, 5-hydroxyindole-3-acetic acid was analyzed using this UHPLC-electrochemical detection method. The final method, which was applied to brain tissue extracts from mice, rats, and cats, decreased analysis time by a factor of 4 compared to HPLC, while guaranteeing good analytical performance.     

Enhancement of Brain Dopamine Metabolism by Tyrosine During Immobilisation: An In Vivo Study Using Repeated Cerebrospinal Fluid Sampling in Conscious Rats

Central dopamine (DA) and 5-hydroxytryptamine (5-HT) metabolism was monitored in conscious, freely moving rats by determination of levels of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) in CSF samples withdrawn repeatedly from the cisterna magna and treated with acid to hydrolyse DOPAC and HVA conjugates. The effect of tyrosine on DA metabolism was investigated. Time courses of metabolite concentrations in individual rats in a quiet room showed that tyrosine (20, 50, or 200 mg/kg i.p.) was without significant effect; brain changes were essentially in agreement. However, the increases of CSF DOPAC and HVA levels that occurred on immobilisation for 2 h were further enhanced by tyrosine (200 mg/kg). The associated increases of 5-HIAA level were unaffected. The corresponding increases of DA metabolite concentrations in the brains of immobilised rats given tyrosine were less marked than the CSF changes and only reached significance for "rest of brain" DOPAC. The CSF studies revealed large interindividual variation in the magnitude and duration of the effects of immobilisation on transmitter amine metabolism. These results may help toward the elucidation of possible relationships between the neurochemical and behavioural effects of stress.     

Effects ofp-chlorophenylalanine on cortical monoamines and on the activity of noradrenergic neurons

The catecholamines noradrenaline, dopamine, adrenaline, the indoleamine 5-hydroxy-tryptamine (5-HT; serotonin), and some of their major metabolites were assayed, using high performance liquid chromatography (HPLC), in the neocortex of normal rats as well as in animals in which 5-HT synthesis had been inhibited withp-chlorophenylalanine. Besides important depletions in serotonin and in 5-hydroxyindole-3-acetic acid, noradrenaline levels were significantly reduced, but the content in 3-methoxy-4-hydroxyphenylglycol was increased, indicating an augmented utilization of this amine. The levels of dopamine and 3-methoxytyramine were also reduced, although homovanillic acid and 3,4-dihydroxyphenylacetic acid levels remained constant. The spontaneous unitary activity of identified noradrenergic neurons in the Locus coeruleus was increased, indicating an hyperactivity of this system. These results can be interpreted in relation to functional interactions between the catecholamines and serotonin; i.e.: a decrease in endogenous serotonin results in the loss of a negative feedback control of noradrenaline release.Special Issue dedicated to Prof. Eduardo De Robertis.     

Effects of disulfiram and coprine on rat brain tryptophan hydroxylation in vivo

The effects of disulfiram and coprine on brain tryptophan hydroxylation, and on the brain-levels of serotonin and 5-hydroxyindole-3-acetic acid, were studied in 45 and 235 days old rats. Both drugs were found to affect the parameters measured. Disulfiram increased the rate of tryptophan hydroxylation and the serotonin level in young rats, while these parameters appeared to be unaffected in old disulfiram-treated rats. In contrast, coprine increased the rate of tryptophan hydroxylation and possibly also the serotonin level in old rats while no significant effects were seen in young coprine-treated rats. Regarding the 5-hydroxyindole-3-acetic acid concentration, this appeared to be increased by disulfiram in both age-groups, while no significant effects were found with coprine. The lack of similarity in the action of disulfiram and coprine, which are both potent aldehyde dehydrogenase inhibitors, suggests that the effects found were not caused by an impaired metabolism of monoamine-derived biogenic aldehydes.     

HOMOVANILLIC ACID AND 3-METHOXY-4- HYDROXYPHENYLETHYLENEGLYCOL PRODUCTION BY THE MONKEY SPINAL CORD

The release of homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) into CSF by the monkey spinal cord was investigated with spinal subarachnoid perfusion of 20 rhesus monkeys. The preperfusion concentration of HVA in lumbar CSF was 365 ng/ml and in cisternal CSF was 365 ng/ml, while the concentrations of MHPG were 28.3 and 40.4 ng/ml respectively. HVA originating from the spinal cord appeared in the perfusate at a rate of 2.4 and MHPG at 1.4 ng/min. Treatment with probenecid either intraperitoneally or intrathecally did not alter the rate of release into CSF of these metabolites by the spinal cord but did significantly increase the rate of appearance in the cisterna magna of HVA originating from the brain. MHPG and HVA in lumbar CSF are therefore derived in part from spinal cord metabolism.     

Morphine tolerance in arthritic rats and serotonergic system

To understand whether chronic inflammation alters the development of morphine tolerance, the tail-flick test was used to evaluate the analgesic effect of morphine (75 mg tablet, s.c.) in the arthritic rats at the day 9-12 after the inoculation with Freund's adjuvant. Spinal cord monoamines and amino acid neurotransmitters were concomitantly measured. Chronic inflammation attenuated the antinociceptive effect of morphine as tolerance developed faster in the arthritic rats compared to the vehicle-treated controls. In addition, ratio of 5-hydroxyindole-3-acetic acid/5-hydroxytryptamine (5-HIAA/5-HT) increased in the lumbar spinal cord of arthritic rats without any change in the concentrations of norepinephrine, glutamate, aspartate or GABA. Interestingly, increased serotonin turnover in the spinal cord was observed in both control and arthritic rats 24 hours after morphine treatment. Overall, the results suggest a significant role of serotonin up-regulation in the spinal cord during chronic pain and the development of morphine tolerance.     

Intracerebrally administered (6r)-l-erythro-tetrahydrobiopterin does not affect extracellular levels of dopamine and serotonin metabolites in rat striatum in vivo during measurement by brain micro-dialysis system

By the use of the brain micro-dialysis technique combined with HPLC, the changes in the extracellular levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and a serotonin(5-HT) metabolite, 5-hydroxyindoleacetic acid (5-HIAA) were examined in the rat striatum before and after intracerebral injection of a vehicle or (6R)-l-erythro-tetrahydrobiopterin (6R-BH₄), the natural form of the cofactor for the tryrosine hydroxylase and tryptophan hydroxylase. No apparent change after the 6R-BH, treatment was found in the levels of DA, DOPAC, HVA and 5-HIAA in the striatal dialysate. In contrast, the levels of total biopterin in both the operated (dialysis probe-implanted) and unoperated striatum of 6R-BH₄-treated rats increased by 23- and 93-fold, respectively, when compared with those of the control, vehicle-treated rats. The results indicate that increased levels of the tetrahydrobiopterin cofactor may not affect the release of DA and the extracellular level of DA and 5-HT metabolites in the physiologically normal brain.     

Increased Cerebrospinal Fluid Dopamine and 3,4-Dihydroxyphenylacetic Acid Levels in Huntington''s Disease: Evidence for an Overactive Dopaminergic Brain Transmission

Levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), 3-methoxy-4-hydroxyphenylglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) in the CSF of patients with Huntington's disease (HD) were measured by HPLC. CSF DA, DOPAC, and MHPG levels were found to be increased in HD patients. Levels of HVA, 5-HIAA, and NA in the CSF of HD patients did not differ from those of controls. Changes in CSF DA and DOPAC levels were consistent with previous findings of increased DA tissue content in some brain areas of patients with HD. These results suggest that CSF DOPAC levels could be a more reliable index of overactive dopaminergic brain systems in HD than CSF HVA levels.     

FMRF-NH2-like mammalian octapeptide: possible role in opiate dependence and abstinence

Yang et al. have isolated from bovine brain an octapeptide, FLFQPQRF-NH2 (F-8-F-NH2), with certain antiopiate properties. Malin et al. previously found that ICV injection of this peptide could precipitate an opiate abstinence syndrome in dependent rats. RIA revealed significantly higher levels of F-8-F-NH2 immunoreactivity in CSF withdrawn from the cisterna magna of morphine-dependent rats as opposed to CSF withdrawn from sham-implanted controls. ICV infusion of IgG from antiserum against F-8-F-NH2 significantly reduced the number of abstinence signs subsequently precipitated by naloxone in morphine-dependent rats.