Measurement of Acetylcholine Release in Freely Moving Rats by Means of Automated Intracerebral Dialysis

The present study demonstrates the feasibility of measuring acetylcholine in perfusion samples collected by means of in vivo brain dialysis in the striata of freely moving rats. The output of the dialysis device was directly connected to an automated sample valve of a HPLC-assay system that comprises a cation exchanger, a post-column enzyme reactor, and an electrochemical detector. The presence of an acetylcholinesterase inhibitor (neostigmine) in the perfusion fluid was required for the detection of acetylcholine in the perfusate. Increasing concentrations of neostigmine induced increasing amounts of acetylcholine. Continuous perfusion with a fixed concentration (2 microM) of neostigmine resulted in gradually increasing amounts of collected acetylcholine over time although a considerable variation between successive samples exists. The brain dialysis technique was further validated by studying the effect of various drugs. Systemically administered atropine increased the output of acetylcholine, whereas the addition of tetrodotoxin to the perfusion fluid resulted in a complete disappearance of the neurotransmitter.     

On the Significance of Tyrosine for the Synthesis and Catabolism of Dopamine in Rat Brain: Evaluation by HPLC with Electrochemical Detection

Abstract: A chemical assay of tyrosine (Tyr) in nervous tissue is described. The method is based on a rapidly performed isolation of Tyr on small Sephadex G 10 columns, followed by reverse-phase HPLC in conjunction with amperometric detection. The method permitted the additional quantification of 3,4-dihydroxyphenylalanine, dopamine (DA), and its acidic metabolites. The method was applied to a study of the effects of γ-butyrolactone, haloperidol, haloperidol in combination with amfonelic acid, morphine, NSD 1015, and tyrosine methylester on the concentration of Tyr in the striatum, frontal cortex, hypothalamus, and cerebellum of rat brain. The effect of tyrosine methylester on DA and its acidic metabolites was investigated in the striatum and frontal cortex. Morphine and NSD 1015 were found to increase Tyr levels. γ-Butyrolactone, haloperidol, and haloperidol combined with amfonelic acid decreased the Tyr content in a manner related to their stimulatory effect on DA biosynthesis. These effects were restricted to DA-rich brain areas. It was concluded that during conditions of increased DA biosynthesis, the Tyr pool still possesses a considerable reserve capacity. The results bring into question the concept that brain Tyr is an important additional factor controlling catechol synthesis during increased tyrosine hydroxylase activity.     

In vivo dopamine receptor binding studies with a non-radioactively labeled agonist,dipropyl-5, 6-ADTN

Evidence is presented that a low dose of peripherally administered N, N-dipropylamino-5, 6-dihydroxytetralin (DiPr-5, 6-ADTN) specifically labels dopamine (DA) receptors in rat brain.Concentrations of this potent DA receptor agonist were determined by a highly selective method using reversed phase liquid chromatography and amperometric detection. The binding characteristics satisfy all criteria regarding saturability, stereospecificity, regional distribution and relation with pharmacological effects that are associated with DA receptor interactions. A rough estimation of the density of binding sites in the striatum resulted in values of 60–70 pmol/g. Lesioning the nigrostriatal pathway does not significantly alter the amount of ligand bound, nor do pretreatments with serotonergic, α-adrenergic or β-adrenergic antagonists. DiPr-5, 6-ADTN has thus been shown to be a useful ligand for labeling central DA receptors and a powerful tool in the study of DA-ergic mechanisms.     

Use of Microdialysis for Monitoring Tyrosine Hydroxylase Activity in the Brain of Conscious Rats

An on-line microdialysis system was developed which monitored the 3,4-dihydroxyphenylalanine (DOPA) formation in the striatum during infusion of a submicromolar concentration of an L-aromatic amino-acid decarboxylase inhibitor (NSD 1015). The absence of DOPA in dialysates of 6-hydroxydopamine-pretreated rats and the disappearance of DOPA after administration of alpha-methyl-p-tyrosine indicated that the dialyzed DOPA was derived from dopaminergic nerve terminals. Next we investigated whether the steady-state DOPA concentration in striatal dialysates could be considered as an index of tyrosine hydroxylase activity. The increase in DOPA output after intraperitoneal administration of haloperidol or gamma-butyrolactone and the decrease in DOPA output after intraperitoneal administration of apomorphine are in excellent agreement with results of postmortem studies, in which a decarboxylase inhibitor was used to measure the activity of tyrosine hydroxylase. The effect of haloperidol on DOPA formation was not visible when a U-shaped cannula (0.80 mm o.d.) was used. Some methodological problems related to microdialysis of the haloperidol-induced increase in DOPA formation are discussed. We concluded that the proposed model is a powerful and reliable in vivo method to monitor tyrosine hydroxylase activity in the brain. The method is of special interest for investigating the effect of compounds which are not able to pass the blood-brain barrier. As an application of the method in the latter situation, we report the effect of infusion the neurotoxin 1-methyl-4-phenylpyridinium ion (10 mmol/L infused over 20 min) on the activity of striatal tyrosine hydroxylase.     

Role of Adenylate Cyclase in the Modulation of the Release of Dopamine: A Microdialysis Study in the Striatum of the Rat

In the present study, we have applied the brain microdialysis technique to investigate the effect of the stimulation of adenylate cyclase on the extracellular levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in the striatum of freely moving rats. Infusion of 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP), 3-isobutyl-1-methylxanthine, or forskolin produced a significant increase in the release of DA. The effect of 8-Br-cAMP was tetrodotoxin, Ca2+, and dose dependent and was saturable. 8-Br-cAMP also caused an increase in the output of DOPAC and HVA. No effects were seen on the output of 5-HIAA, except at the highest 8-Br-cAMP concentration studied. Infusion of 8-Br-cAMP (25 microM, 1.0 mM, and 3.3 mM) together with infusion of (-)-sulpiride (1 microM) or systemic administration of (+/-)-sulpiride (55 mumol/kg i.p.) produced an additive effect on the release of DA. Infusion or peripheral administration of (-)-N-0437 (1 microM or 1 mumol/kg) both decreased the 8-Br-cAMP-induced increase in the release of DA. These results demonstrate that cyclic AMP may stimulate the release of DA, but it is unlikely that this second messenger is linked to presynaptic D2 receptors controlling the release of DA.     

Flexible life history responses to flower and rosette bud removal in three perennial herbs

In a garden experiment we investigated the response to continuous removal of either flower buds or rosette buds in three perennial grassland species ( Hypochaeris radicata , Succisa pratensis and Centaurea jacea ), which differ in longevity and flowering type. We distinguished two possible responses: compensation for lost buds by making more buds of the same type, and switching towards development of other life history functions. Both responses were demonstrated in our experiment, but bud removal had significantly different effects in each of the three species. The degree of compensation and the expression of trade-offs between life history functions differed markedly between species and seem related to longevity and developmental constraints. With respect to switching, our results suggest costs of reproduction and a trade-off between life history functions, at least for Hypochaeris and Succisa . For these species weight of new rosettes increased when resource allocation to flowering was inhibited. In Hypochaeris, we see that both compensation for lost flower buds and switching from lost rosette buds increased production of flower buds, underscoring the pivotal role of sexual reproduction in this short-lived species. The most prominent response seen in Centaurea is compensation for lost rosette buds, indicating that this long-lived species with monocarpic rosettes relies on rosette formation. Although Succisa does respond to bud removal, time is an important constraint in this species with long-lived rosettes and preformed flowering stalks. Trade-offs in Succisa seem to operate at a larger time scale, requiring long-lasting experiments to reveal them. We conclude that the response of these species to inflicted damage is likely to be linked to their longevity and developmental constraints.     

Discrimination between A-type and B-type conformations of double helical nucleic acid fragments in solution by means of two-dimensional nuclear Overhauser experiments

The solution structure of two double helical nucleic acid fragments, viz, r(CGCGCG) and d(CGCGCG), was probed by means of two-dimensional nuclear Overhauser effect spectroscopy. The two compounds were selected as models for the A-type and B-type double helical conformations, respectively, and it is shown that for each of the two model compounds the intensities of the NOE cross peaks between base- and H2' (deoxy)ribose proteins are qualitatively in correspondence with the relative NOE intensities expected on basis of the supposed duplex conformations. Thus our results indicate that NOE-data can be used to differentiate between A-and B-type double helical conformations in solution. Coupling constant data show that, except for G(6), all ribose rings in r(CGCGCG) adopt pure N (C3'-endo) conformations thereby manifesting that this molecule takes up a regular A-type double helical conformation in solution. In contrast, the deoxyribose rings in d(CGCGCG) retain conformational freedom in the duplex state, albeit that the N/S-equilibrium is biased towards the S (C2'-endo) sugar conformation. This finding indicates that in solution the B-DNA backbone is highly dynamic.     

CORRELATIONS BETWEEN A FLUORIMETRIC and MASS FRAGMENTOGRAPHIC METHOD FOR THE DETERMINATION OF 3-METHOXY-4- HYDROXYPHENYLACETIC ACID and TWO MASS FRAGMENTOGRAPHIC METHODS FOR THE DETERMINATION OF 3-METHOXY-4- HYDROXYPHENYLETHYLENE GLYCOL IN CEREBROSPINAL FLUID

One-hundred and twenty-two lumbar cerebrospinal fluid (CSF) samples were assayed for 3-methoxy-4-hydroxyphenylacetic acid (HVA) by both a fluorimetric and mass fragmentographic method. The correlation coefficient (cc) and residual standard deviation (Syx) of the results were calculated as 0.966 and 23.3 ng/ml, respectively. If only samples containing less than 100ng/ml of HVA were considered, somewhat different values for cc and Syx were found (0.854 and 10.0 ng/ml, respectively). The data obtained by the fluorimetric method were consistently 17% lower than those obtained by the mass fragmentographic method. Spiking experiments resulted in 96.5 ± 7.8% recovery for the fluorimetric method, whereas the use of a deuterated internal standard was found to compensate completely for losses in the mass fragmentographic method. In addition the correlation between two different mass fragmentographic methods for the simultaneous determination of HVA and 3-methoxy-4-hyd-roxyphenylethylene glycol (MOPEG) in CSF was studied. The measurements were performed in different laboratories and resulted in correlation coefficients of 0.941 and 0.940 and residual standard deviations of 7.6 and 1.0 ng/ml for HVA and MOPEG, respectively. From all data we conclude that mass fragmentographic methods for the determination of catecholamine metabolites in CSF are superior to fluorimetric methods because of their selectivity, reproducibility and accuracy.     

Norepinephrine Release in the Rat Pineal Gland: The Input from the Biological Clock Measured by In Vivo Microdialysis

Abstract: The sympathetic innervation of the rat pineal gland was investigated, measuring the norepinephrine (NE) release by on-line in vivo microdialysis. NE was assayed using an HPLC method with precolumn derivatization and fluorescence detection. Its high sensitivity and reliability made it very suitable to monitor the low levels of NE in the dialysates (12.5 fmol during nighttime, 3 fmol during daytime). To increase NE levels, the monoamine reuptake inhibitor cocaine was added to Ringer's solution at concentrations of 10⁻⁶ and 10⁻⁵ M . This resulted in increases of neurotransmitter output of 167 and 219%, respectively, but did not change the qualitative and/or quantitative outcome of other experiments. Perfusion with 10⁻⁶ M tetrodotoxin for 1 h resulted in a decrease of the NE release by >80%, whereas perfusion with the α₂-receptor antagonist yohimbine caused a twofold increase. These results indicate that the NE release in the rat pineal was of neuronal origin and regulated by a negative feedback mechanism involving inhibitory presynaptic α₂-receptors. Long-term (i.e., 16 h) measurements are described, showing the circadian properties of NE release. A pronounced rhythm is reported, showing extremely sharp transitions between low daytime and high nighttime values. Increases and decreases are reported to occur within the duration of collecting one sample (20 min). For comparison, the rhythm of melatonin release was also recorded. The on and off switches of the sympathetic input correlated well with the circadian rhythm of melatonin release and can thus be considered as the primary clock signal, inducing the nightly production of melatonin.     

NMDA Receptor Antagonist Ketamine Impairs Feature Integration in Visual Perception

Recurrent interactions between neurons in the visual cortex are crucial for the integration of image elements into coherent objects, such as in figure-ground segregation of textured images. Blocking N-methyl-D-aspartate (NMDA) receptors in monkeys can abolish neural signals related to figure-ground segregation and feature integration. However, it is unknown whether this also affects perceptual integration itself. Therefore, we tested whether ketamine, a non-competitive NMDA receptor antagonist, reduces feature integration in humans. We administered a subanesthetic dose of ketamine to healthy subjects who performed a texture discrimination task in a placebo-controlled double blind within-subject design. We found that ketamine significantly impaired performance on the texture discrimination task compared to the placebo condition, while performance on a control fixation task was much less impaired. This effect is not merely due to task difficulty or a difference in sedation levels. We are the first to show a behavioral effect on feature integration by manipulating the NMDA receptor in humans.