Acetylcholine measurement of cerebrospinal fluid by in vivo microdialysis in freely moving rats.

Acetylcholine (ACh) and choline (Ch) levels in rat cerebrospinal fluid (CSF) were determined by in vivo microdialysis (CSF microdialysis) in both halothane-anesthetized and freely-moving rats. The Ch/ACh ratio in CSF perfused with Ringer's solution (30 microliters/30 min) containing 10(-5) M physostigmine, a centrally active cholinesterase inhibitor, was significantly lower than that in unprocessed CSF due to significantly higher ACh levels in the former. The successive measurement on the 2nd and 7th day after the guide cannula implantation demonstrated the feasibility of the CSF microdialysis method for repetitive monitoring of CSF ACh and Ch levels in freely moving rats without extensive tissue damage. Intraperitoneal administration of physostigmine caused an increase in CSF ACh levels, whereas administration of neostigmine, which cannot penetrate into the blood brain barrier, did not. Furthermore, a centrally active acetylcholinergic M1-receptor agonist, AF102B, produced an increase in CSF ACh and Ch levels. Thus, the present study demonstrates that CSF microdialysis is a useful method for evaluating overall central cholinergic activity and investigating the pharmacological effects of various drugs that act via the central cholinergic system.

The functional role of opioid receptors in acetylcholine release in the rat adrenal medulla.

The functional role of opioid receptors in acetylcholine release from splanchnic nerve terminals in the adrenal medulla was investigated in halothane-anesthetized rats. The extracellular acetylcholine level was measured by a newly developed in vivo adrenal microdialysis method. The potassium (K+)-evoked acetylcholine release from the splanchnic nerve terminals was inhibited by morphine (10 microM), a mu-opioid receptor agonist, and [D-Pen2,D-Pen5]enkephalin (DPDPE, 1 and 10 microM), a delta-opioid receptor agonist. These inhibitory effects of morphine and DPDPE were significantly abolished by naltrexone (9 mg/kg i.p.), a mu-opioid receptor antagonist, and naltrindole (9 mg/kg i.p.), a delta-opioid receptor antagonist, respectively. 5 alpha,7 alpha-beta-(-)- N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]benzene acetamide (U69593, 10 microM), a kappa-opioid receptor agonist, had no influence on the K(+)-evoked acetylcholine release. The findings suggest that both mu- and delta-opioid receptors might have a functional role in acetylcholine release from splanchnic nerve terminals in the adrenal medulla of the rat. The present study indicates that adrenal microdialysis is a useful method for studying the control mechanism of adrenomedullary function in the rat in vivo.

mu-Opioid receptors modulate noradrenaline release from the rat hippocampus as measured by brain microdialysis.

The modulation of noradrenaline (NA) release via presynaptic opioid receptors in the hippocampus of freely moving rats was studied by the use of brain microdialysis. Extracellular levels of NA were estimated by assaying its concentrations in the perfusion fluid using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). Spontaneous NA levels were reduced by tetrodotoxin (1 microM) co-perfusion and were increased by peripheral administration of desipramine (5 and 10 mg/kg, i.p.). Addition of potassium (K+, 60 and 120 mM) to the perfusion fluid evoked a concentration-dependent release of NA. K+ (120 mM)-evoked NA release was markedly reduced by removal of calcium (Ca2+) from the perfusion fluid. These results indicate that both the spontaneous and the K(+)-evoked NA release measured by the use of brain microdialysis coupled with HPLC-ECD can be used as indices of neuronal release from the noradrenergic nerve terminals. A mu-opioid receptor agonist, morphine (0.01-10 microM), when co-perfused with K+ (120 mM), produced a reduction of K(+)-evoked NA release in a concentration-dependent manner. Neither co-perfusion with a high concentration of [D-Pen2, D-Pen5]-enkephalin (DPDPE) (10 microM), an agonist selective for delta-opioid receptors, nor with U-69593 (10 microM), an agonist selective for kappa-opioid receptors, modified the K+ (120 mM)-evoked release of NA. Morphine-induced (1 microM) inhibition of NA release was blocked by a mu-opioid receptor antagonist, naltrexone (3 and 9 mg/kg, i.p). Naltrexone by itself did not alter the spontaneous NA levels or the K(+)-evoked NA release.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurochemical profiles in cerebrospinal fluid of stroke-prone spontaneously hypertensive rats.

The purpose of the present study was to evaluate stroke-prone spontaneously hypertensive rats (SHRSP) neurochemically by determining the cerebrospinal fluid (CSF) levels of acetylcholine (ACh), norepinephrine (NE) and serotonin (5-HT) as an index of central neuronal activity. The CSF ACh levels of 15- to 20-week-old SHRSP were significantly lower than those of age-matched Wistar Kyoto rats (WKY) both under the urethane/alpha-chloralose anesthesia and in freely moving conditions. The difference in the CSF ACh levels between SHRSP and WKY was more marked at 30-40 weeks. Sustained changes were not observed in the CSF NE and 5-HT levels. Thus, the progressive dysfunction in the central cholinergic system may characterize the pathophysiological state of this animal model with cerebral lesions caused by continuous high blood pressure.

Inhibitory effects of clonidine on serotonergic neuronal activity as measured by cerebrospinal fluid serotonin and its metabolite in anesthetized rats.

Clonidine-induced changes in the serotonergic neuronal activity of the central nervous system were estimated by measuring the concentrations of serotonin (5-HT) and its major metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), in the cerebrospinal fluid (CSF) of anesthetized rats. Clonidine (30 and 300 micrograms/kg, i.v.) led to 74% and 60% reductions in the concentration of 5-HT in the CSF 60 min after administration. CSF 5-HIAA concentrations were also decreased to 77% and 66%, respectively. Clonidine-induced (30 micrograms/kg, i.v.) decreases in CSF 5-HT and 5-HIAA concentrations were attenuated by pretreatment with idazoxan (5 mg/kg, i.p.). Idazoxan by itself did not alter the CSF 5-HT and 5-HIAA concentrations. Decreased CSF 5-HT and 5-HIAA concentrations after i.v. administration of clonidine (30 micrograms/kg) were abolished by noradrenergic denervation after pretreatment with 6-hydroxydopamine (200 micrograms/rat, i.c.v.). These results suggest the possibility that clonidine acts to inhibit the serotonergic neuronal activity, which is mediated via the alpha 2-adrenoceptors. It indicates, moreover, that noradrenergic nervous systems are involved in the clonidine-induced inhibition of serotonergic neuronal activity. Therefore, noradrenergic neurons play a significant role in mediating the actions of clonidine on serotonergic neuronal activity in the rat brain.

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.

Simultaneous high-performance liquid chromatographic determination of norepinephrine, serotonin, acetylcholine and their metabolites in the cerebrospinal fluid of anaesthetized normotensive rats.

A high-performance liquid chromatographic method with electrochemical detection was developed for the simultaneous determination of the levels of norepinephrine (NE), serotonin (5-HT), acetylcholine (ACh) and their metabolites in the cerebrospinal fluid (CSF) of anaesthetized rats. The response curve for each compound was linear for the concentration way of interest. The within- and between-day coefficients of variation (C.V.) for NE, 5-HT and their metabolites were less than 7.85% and 15.67%, respectively, and those for ACh and choline were less than 3.08% and 6.27%, respectively. This simultaneous determination should be useful for elucidating the noradrenergic, serotonergic and cholinergic nerve activity in the central nervous system.

Decreased monoamine oxidase activity in stroke-prone spontaneously hypertensive rat kidneys.

The present study was undertaken to measure kidney monoamine oxidase activity (MAO) isolated from three different rat groups: Wistar Kyoto rats (WKY), stroke-free cases of stroke-prone spontaneously hypertensive rats (SHRSP-control) and SHRSP-stroke cases (SHRSP-stroke). The SHRSP group was found to have less kidney MAO than the WKY group. Conversely, plasma norepinephrine concentration of SHRSP group was significantly higher than that of the WKY group (Jpn. Heart J., 25, 833-835, 1985). These findings suggest that the increased blood pressure in SHRSP may be related to increased plasma norepinephrine concentration caused by a decrease in degradation enzyme, MAO. An attempt was also made to determine the effects of clorgyline and deprenyl. No significant difference in MAO was observed between the kidneys isolated from the WKY, SHRSP-control and SHRSP-stroke groups. However, the inhibition curve of clorgyline reached a plateau after producing 33% inhibition of MAO. It was assumed that rat kidney mitochondrial MAO consists of 33% MAO-A type and 67% MAO-B type.

[Effect of ketanserin on blood pressure in rats].

Ketanserin, a selective serotonergic (5-HT2) antagonist, also has affinity for alpha 1-adrenoceptors. It is not clear whether the hypotensive mechanism of ketanserin is due to its antagonistic action to 5-HT2 receptor or to its affinity for alpha 1 adrenoceptors. The hypotensive mechanism of ketanserin was studied in both stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY). Anesthetized rats were used (alpha-chloralose + urethane, i.p.). Up to 3 ml of blood was drawn from each rat for analysis. Plasma norepinephrine (NE) was determined by radioenzymatic assay. Plasma serotonin (5-HT) was determined by HPLC-ECD. Adrenal nerve discharges were counted by a digital pulse counter. Ketanserin (0.5 mg/kg, 5.0 mg/kg, i.v.) produced a dose-dependent reduction of mean arterial pressure (MAP) in both SHRSP and WKY. MAP of SHRSP decreased significantly as compared with WKY. Both plasma NE and 5-HT showed a tendency to increase during ketanserin administration (5.0 mg/kg, i.v.). Ketanserin significantly antagonized the BP response induced by exogenously injected 5-HT (30 micrograms/kg) and NE (10 micrograms/kg). Adrenal nerve activity was reduced in parallel with the decrease in BP and HR. These findings suggest that ketanserin produced a decrease in BP via both peripheral and central action in rats.