Effects of Morris water maze testing on the neuroendocrine stress response and intrahypothalamic release of vasopressin and oxytocin in the rat

Adult male Wistar rats were trained in the Morris water maze (MWM) on 3 consecutive days to find a visible platform. Concomitantly, microdialysis samples from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei were collected in order to monitor local release of the neuropeptides vasopressin (AVP) and oxytocin (OXT), respectively, during controllable swim stress. Additionally, a separate set of animals was equipped with chronic jugular venous catheters to collect blood samples for analyzing plasma concentrations of corticotropin (ACTH) and corticosterone during training in the MWM. As measured by microdialysis, swimming in the MWM caused a significantly increased release of AVP within the PVN and of OXT within the SON on each of the 3 test sessions. In contrast to OXT in the SON, basal AVP concentrations in the PVN tended to rise from day to day. Plasma ACTH and corticosterone were found to be similarly elevated in response to MWM exposure on each of the test sessions. Taken together, these data demonstrate that testing in the MWM is not only associated with a significant activation of the hypothalamo-pituitary-adrenal axis but also with an intrahypothalamic release of AVP and OXT. If compared with findings using repeated forced swimming as an uncontrollable stressor (Wotjak, C.T., Ganster, J., Kohl, G., Holsboer, F., Landgraf, R., Engelmann, M., 1998. Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons. Neuroscience 85, 1209-1222), the present results suggest that (1) similarities in the release profiles of AVP in the PVN and plasma hormone levels are fairly independent from the controllability of the stressor and seem, thus, to primarily relate to the physical demands of the task, whereas (2) the different intra-SON OXT release profiles might be linked to the controllability of the stressor.     

Oxytocin, but not arginine vasopressin is involving in the antinociceptive role of hypothalamic supraoptic nucleus

Our pervious study has demonstrated that the hypothalamic supraoptic nucleus (SON) plays a role in pain modulation. Oxytocin (OXT) and arginine vasopressin (AVP) are the important hormones synthesized and secreted by the SON. The experiment was designed to investigate which hormone was relating with the antinociceptive role of the SON in the rat. The results showed that (1) microinjection of l-glutamate sodium into the SON increased OXT and AVP concentrations in the SON perfusion liquid, (2) pain stimulation induces OXT, but not AVP release in the SON, and (3) intraventricular injection (pre-treatment) with OXT antiserum could inhibit the pain threshold increase induced by SON injection of l-glutamate sodium, but administration of AVP antiserum did not influence the antinociceptive role of SON stimulation. The data suggested that the antinociceptive role of the SON relates to OXT rather than AVP.     

Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei

Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction.     

Oxytocin actions within the supraoptic and paraventricular nuclei: differential effects on peripheral and intranuclear vasopressin release

In response to forced swimming (FS), AVP is released somato-dendritically within the supraoptic nucleus (SON) and paraventricular nucleus (PVN), but not from neurohypophyseal terminals into blood. Together with AVP, oxytocin (OXT) is released within the SON and PVN. Here, we studied the role of intra-SON and intra-PVN OXT in the regulation of local AVP release and into the blood in male rats. Within the SON, bilateral retrodialysis of an OXT receptor antagonist (OXT-A) increased local AVP release in response to FS [60 s, 21 degrees C, vehicle twofold, not significant (ns); OXT-A: 15-fold increase, P < 0.05] without significantly affecting basal AVP release. In addition, local OXT-A elevated plasma AVP secretion under basal conditions (twofold increase, P < 0.05) without further elevation after FS. Within the PVN, exposure to FS elevated local AVP release, reaching significance only in the OXT-A group (vehicle: 1.4-fold, ns; OXT-A: 1.6-fold increase, P = 0.050). Bilateral OXT-A into the PVN did not affect peripheral AVP secretion either under basal or stress conditions. Basal ACTH concentrations tended to be elevated by local OXT-A within the PVN (1.7-fold increase, P = 0.076). In contrast, the swim-induced ACTH secretion was attenuated after retrodialysis of OXT-A within both the SON (at 5 min) and PVN (at 15 min) (P < 0.05 both) compared with vehicle. The results demonstrate a receptor-mediated effect of OXT within the SON and PVN on local and neurohypophyseal AVP release, which depends upon the activity conditions. Further, while exerting an inhibitory effect on hypothalamo-pituitary-adrenal axis activity under basal conditions, hypothalamic OXT is essential for an adequate acute ACTH response.     

Effect of chronic treatment with haloperidol on vasopressin release and behavioral changes by osmotic stimulation of the supraoptic nucleus.

Chronic treatment with dopamine D2 blockers in schizophrenic patients has been proposed as one of the causes of polydipsia and water intoxication, but this conclusion is still controversial. To investigate the relationship between dopamine D2 blockers and these syndromes, we designed a behavioral and neurochemical study using hyperosmotic stimulation in the supraoptic nucleus (SON) by microdialysis after chronic treatment with haloperidol in rats. Animals were injected with haloperidol decanoate (20 mg/kg, i.m.) or sesame oil at 2-week intervals for 8 successive weeks. During the 7th week, water-intake was increased 30-60 min after the hyperosmotic stimulation in both groups, but more so in haloperidol-treated animals compared to that in the control group. Moreover, arginine vasopressin (AVP) was released by the hyperosmotic stimulation in SON, but was not significantly different between groups. In addition, striatal dopamine levels 3-4 days after the microdialysis study showed a significant decrease in the haloperidol-treated animals. These results suggest that chronic treatment with haloperidol enhances water-intake produced by hyperosmotic stimulation in the SON but does not increase AVP levels in dialysates following hyperosmotic stimulation. Thus, these symptoms may be mediated by dopaminergic systems in brain.     

Activation of vasopressinergic and oxytocinergic neurons during aging in the Wistar rat

The activity of the hypothalamo-neurohypophyseal system (HNS) was determined in male Wistar rats from 3 to 32 months of age. Plasma levels of vasopressin (AVP) and oxytocin (OXT) were measured by means of a radioimmunoassay. In addition, the distribution of the Golgi apparatus marker enzyme thiamine-pyrophosphatase (TPP-ase) was measured as a parameter for neurosecretory activity in the hypothalamic supraoptic and paraventricular nuclei (SON and PVN). Plasma levels of radioimmunoassayable AVP were increased in the 32-month-old animals. Plasma levels of radioimmunoassayable OXT in 32-month-old animals did not differe from the levels found in the youngest group, but were higher than in 11-month-old animals. Neurosecretory activity in the SON was similar in 3- and 32-month-old animals, whereas in the PVN neurosecretory activity was increased in the 32-month-old animals. Urine excretion decreased between 6 and 11 months of age and remained on the same level until 32 months of age. In other words, instead of a loss of HNS function as has been suggested in the literature, an increased neurosecretory activity was observed in aged rats.     

Amino Acids in Rat Striatal Dialysates: Methodological Aspects and Changes After Electroconvulsive Shock

Extracellular levels of amino acids were estimated in dialysates of the rat striatum that were collected 1, 2, and/or more than 5 days after surgery, before (resting release) and during exposure to high K concentrations (50 mM) or electroconvulsive shocks. The resting release of several amino acids (Glu, Asn, Thr, Tau, Tyr, Gly, and Ala) was higher 9 days as compared to 1 day after surgery. In the 1-day preparation the resting release correlated highly with that observed with push-pull cannulas. The correlation with the tissue content of the amino acids was high only when they were divided into two groups (putative transmitters and metabolic intermediates). High K exposure produced increased output of Ala, ethanolamine (Eam), Asp, Glu, Tau, and Gly and a decrease in the egress of Gln 1 or 2 days after surgery. The effects on Asp and Glu had disappeared, and that on Gln reversed after 4-9 days. Electrically induced convulsions produced increased output of Ala, Gln, and Eam 1 or 2 days and 2 weeks after implantation of the probe. Changes were seen not only during but also (and some cases even more prominent) after the seizure. This study shows the usefulness of dialysis to monitor extracellular transmitter amino acids in the striatum of conscious rats (also bilateral dialysis was possible) for only a limited time after implantation of the probe. The dialysis method is suitable for longer time, when metabolic changes in amino acids are to be followed. In addition to transmitter release, glycolysis can be monitored by the measurement of Ala in the dialysate.     

Cellular Origins of Endogenous Amino Acids Released into the Extracellular Fluid of the Rat Striatum During Severe Insulin-Induced Hypoglycemia

The effect of severe insulin-induced hypoglycemia on the extracellular levels of endogenous amino acids in the rat striatum was examined using the brain microdialysis technique. A characteristic pattern of alterations consisting of a 9-12-fold increase in aspartate (Asp), and more moderate increases in glutamate (Glu), taurine (Tau), and gamma-aminobutyric acid (GABA), was noted following cessation of electroencephalographic activity (isoelectricity). Glutamine (Gln) levels were reduced both during and after the isoelectric period and there was a delayed increase in extracellular phosphoethanolamine (PEA) content. The effects of decortication and excitotoxin lesions on the severe hypoglycemia-evoked efflux of endogenous amino acids in the striatum were also examined. Decortication reduced the release of Glu and Asp both 1 week and 1 month post-lesion. The efflux of other neuroactive amino acids was not affected significantly. In contrast, GABA, Tau, and PEA efflux was attenuated in kainate-lesioned striata. Glu and Asp release was also reduced under these conditions, and a smaller decrease in extracellular Gln was noted. These data suggest that GABA, Glu, and Asp are released primarily from their transmitter pools during severe hypoglycemia. The releasable pools of Tau and PEA appear to be located in kainate-sensitive striatal neurons. The significance of these results is discussed with regard to the excitotoxic theory of hypoglycemic cell death.     

Somatostatin Potently Stimulates In Vivo Striatal Dopamine and γ-Aminobutyric Acid Release by a Glutamate-Dependent Action

Abstract: We have used in vivo microdialysis in anaesthetised rats to investigate whether somatostatin (SRIF) can play a neuromodulatory role in the striatum. When 100 n M SRIF was retrodialysed for 15 min, it increased concentrations of dopamine (DA) by 28-fold, γ-aminobutyric acid (GABA) by eightfold, and glutamate (Glu) by sixfold as well as those of aspartate (Asp) and taurine (Tau). These effects were both calcium- and tetrodotoxin-sensitive. Lower (10 or 50 n M ) and higher (1 µ M ) SRIF concentrations were less effective. Rapid sampling showed that whereas Asp and Glu concentrations were raised for 3 min at the start of 15-min SRIF infusions, those of DA were increased for 12 min. A second 15-min application of 100 n M SRIF given 135 min after the first application failed to increase transmitter release. An NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (200 µ M ), blocked SRIF (100 n M )-evoked Asp, Glu, Tau, and GABA release and reduced that of DA. An α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate antagonist, 6,7-dinitroquinoxaline-2,3-dione (100 µ M ), blocked SRIF-induced DA and Tau release and reduced that of Asp, Glu, and GABA. These results show that SRIF increases DA, Glu, Asp, GABA, and Tau release in the rat striatum and suggest that its actions on DA and GABA release are mainly mediated through increased excitatory amino acid release.     

Arginine vasopressin in hypothalamic paraventricular nucleus is transferred to the caudate nucleus to participate in pain modulation

Arginine vasopressin (AVP), which is synthesized and secreted in the hypothalamic paraventricular nucleus (PVN), is the most important bioactive substance in the pain modulation. Our pervious study had shown that AVP plays an important role in pain modulation in caudate nucleus (CdN). The experiment was designed to investigate the source of AVP in CdN by the nucleus push-pull perfusion and radioimmunoassay. The results showed that: (1) pain stimulation increased the AVP concentration in the CdN perfusion liquid, (2) PVN decreased the effect of pain stimulation which was stronger in both sides than in one side of PVN cauterization; and (3) L-glutamate sodium would excited the PVN neurons by the PVN microinjection that could increase the AVP concentration in the CdN perfusion liquid. The data suggested that AVP in the CdN might come from the PVN in the pain process, i.e., AVP in the PVN might be transferred to the CdN to participate in the pain modulation.     

Arginine vasopressin in hypothalamic paraventricular nucleus is transferred to the nucleus raphe magnus to participate in pain modulation

Hypothalamic paraventricular nucleus (PVN) is one of the main sources of arginine vasopressin (AVP) synthesis and secretion. AVP is the most important bioactive substance in PVN regulating pain process. Our pervious study has pointed that pain stimulation induced AVP increase in the nucleus raphe magnus (NRM), which plays a role in pain modulation. The present study was designed to investigate the source of AVP in the rat NRM during pain process using the methods of nucleus push–pull perfusion and radioimmunoassay. The results showed that pain stimulation increased the AVP concentration in the NRM perfusion liquid, PVN cauterization inhibited the role that pain stimulation induced the increase of AVP concentration in the NRM perfusion liquid, and PVN microinjection of l-glutamate sodium, which excited the PVN neurons, could increase the AVP concentration in the NRM perfusion liquid. The data suggested that AVP in the PVN might be transferred to the NRM to participate in pain modulation.     

Arginine vasopressin induces periaqueductal gray release of enkephalin and endorphin relating to pain modulation in the rat

Previous study has proven that microinjection of arginine vasopressin (AVP) into periaqueductal gray (PAG) raises the pain threshold, in which the antinociceptive effect of AVP can be reversed by PAG pretreatment with V2 rather than V1 or opiate receptor antagonist. The present work investigated the AVP effect on endogenous opiate peptides, oxytocin (OXT) and classical neurotransmitters in the rat PAG. The results showed that AVP elevated the concentrations of leucine-enkephalin (L-Ek), methionine-enkephalin (M-Ek) and beta-endorphin (beta-Ep), but did not change the concentrations of dynorphinA(1-13) (DynA(1-13)), OXT, classical neurotransmitters including achetylcholine (Ach), choline (Ch), serotonin (5-HT), gamma-aminobutyric acid (GABA), glutamate (Glu), dopamine (DA), norepinephrine (NE) and epinephrine (E), and their metabolic products in PAG perfusion liquid. Pain stimulation increased the concentrations of AVP, L-EK, M-Ek, beta-Ep, 5-HT and 5-HIAA (5-HT metabolic product), but did not influence the concentrations of DynA(1-13), OXT, the other classical neurotransmitters and their metabolic products. PAG pretreatment with naloxone - an opiate receptor antagonist completely attenuated the pain threshold increase induced by PAG administration of AVP, but local pretreatment of OXT or classical neurotransmitter receptor antagonist did not influence the pain threshold increase induced by PAG administration of AVP. The data suggested that AVP in PAG could induce the local release of enkephalin and endorphin rather than dynophin, OXT and classical neurotransmitters to participate in pain modulation.     

Centrally administered adrenomedullin 2 activates hypothalamic oxytocin-secreting neurons, causing elevated plasma oxytocin level in rats

We examined the effects of intracerebroventricular (i.c.v.) administration of adrenomedullin 2 (AM2) on plasma oxytocin (OXT) and arginine vasopressin (AVP) levels in conscious rats. Plasma OXT levels were markedly increased 5 min after i.c.v. administration of AM2 (1 nmol/rat) compared with vehicle and remained elevated in samples taken at 10, 15, 30, and 60 min. By contrast, plasma AVP levels were not significantly elevated in samples taken between 5 and 180 min after i.c.v. administration of AM2 except at the 30-min time point. Fos-like immunoreactivity (Fos-LI) was observed in various brain areas, including the paraventricular (PVN) and the supraoptic nuclei (SON) after i.c.v. administration of AM2 (2 nmol/rat) in conscious rats (measured at 90 min post-AM2 infusion). Dual immunostaining for OXT/Fos and AVP/Fos showed that OXT-LI neurons predominantly exhibited nuclear Fos-LI compared with AVP-LI neurons in the PVN and the SON. In situ hybridization histochemistry showed that i.c.v. administration of AM2 (0.2, 1, and 2 nmol/rat) caused marked induction of the expression of the c-fos gene in the PVN and the SON. This induction was significantly reduced by pretreatment with both the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37) (3 nmol/rat) and the AM receptor antagonist AM-(22-52) (27 nmol/rat). These results suggest that centrally administered AM2 mainly activates OXT-secreting neurons in the PVN and the SON, at least in part through the CGRP and/or AM receptors with marked elevation of plasma OXT levels in conscious rats.     

Catecholamine biosynthesis and vasopressin and oxytocin secretion in the spontaneously hypertensive rat: an in vitro study of localized brain regions

The in vitro synthesis of catecholamines and the secretion of vasopressin (AVP) and oxytocin (OT) was measured in localized regions of the hypothalamo-neurohypophyseal system in the spontaneously hypertensive rat (SHR). The posterior pituitary (PP), median eminence (ME) and supraoptic (SON) and paraventricular (PVN) nuclear regions were incubated in vitro in media containing 3H-tyrosine. Media and tissue levels of AVP and OT were measured as well as norepinephrine and dopamine content and biosynthesis. There were no differences in peptide release in either the PP, ME or SON. However, there was a marked increase in peptide release from the PVN of the SHR. Media AVP levels were 0.3 pg/ml/micrograms protein in the WKY as compared to 2.1 pg/ml/micrograms protein in the SHR. OT release was increased 2 fold, from 0.85 to 1.7 pg/ml/micrograms protein. PVN content of both AVP and OT was significantly lower in the SHR. ME and SON peptide levels were not changed, while neurohypophyseal AVP levels were increased in the SHR. With regard to the catecholamines appreciable norepinephrine synthesis was measured in the PVN and SON while there was little 3H-norepinephrine in the ME or PP. In the hypertensive rat, there was an increase in norepinephrine synthesis in the PVN with no change in the SON. These results provide further support for fundamental changes in the catecholaminergic and peptidergic systems of the hypothalamo-neurohypophyseal axis of the SHR.     

Effects of Prostaglandin E2 and Capsaicin on Behavior and Cerebrospinal Fluid Amino Acid Concentrations of Unanesthetized Rats: A Microdialysis Study

Abstract: Prostaglandin E₂ (PGE₂) delivered to the spinal cord produces an increased sensitivity to noxious (hyperalgesia) and innocuous (allodynia) stimuli. The mechanisms that underlie this effect remain unknown, but a PGE₂-evoked enhancement of spinal neurotransmitter release may be involved. To address this hypothesis, we examined the effect of PGE₂ on CSF concentrations of amino acids and also the modulatory effect of PGE₂ on capsaicin-evoked changes of spinal amino acid concentrations using a microdialysis probe placed in the lumbar subarachnoid space. Amino acids were quantified using HPLC with fluorescence detection. Addition of 1 mM, but not 10 or 100 µM, PGE₂ to the perfusate for a 10-min period (flow rate, 5 µl/min) evoked an immediate increase (80–100%) in glutamate (Glu), aspartate (Asp), taurine (Tau), glycine (Gly), and γ-aminobutyric acid (GABA) concentrations. Similarly, capsaicin infusion (0.1–10 µM) induced a dose-dependent increase in Glu, Asp, Tau, Gly, GABA, and ethanolamine levels. Significant increases in amino acid levels evoked by PGE₂ or capsaicin were associated with a touch-evoked allodynia. The combination of PGE₂ (10 µM) and capsaicin (0.1 or 1.0 µM) at concentrations that individually had no effect together evoked a significant increase (60–100%) in Glu, Asp, Tau, Gly, and GABA concentrations and produced tactile allodynia. These data demonstrate that spinally delivered PGE₂ or capsaicin substantially elevates CSF concentrations of both excitatory and inhibitory amino acids. The capacity of PGE₂ to enhance and prolong capsaicin-evoked amino acid concentrations may be one of the mechanisms by which spinal PGE₂ produces hyperalgesia and allodynia.     

Locus Coeruleus Lesions Decrease Oxytocin and Vasopressin Release Induced by Hemorrhage

The role of the noradrenergic nucleus Locus Coeruleus (LC) on hemorrhage-induced vasopressin (AVP) and oxytocin (OT) secretion was examined. Rats with LC lesion were submitted to three 1-min hemorrhage sessions at 5-min intervals; 15% of the total blood volume was withdrawn in each session. OT and AVP were measured in plasma, paraventricular (PVN) and supraoptic (SON) nuclei and in posterior pituitary (PP). LC Lesion did not affect basal plasma AVP or OT levels, but partly blocked the increase in plasma AVP and OT induced by hemorrhage. Hemorrhage produced decreases in content of AVP and OT in the PVN and SON and increased levels in the PP. These responses were attenuated in the lesioned group, but only in the PVN and PP. Data suggest a stimulatory role of the inputs from LC to PVN neurons on hemorrhage-induced OT and AVP secretion and that, this pathway is critical in the hypo-volemic neuroendocrine reflex.Special Issue Dedicated to Miklós Palkovits.     

Time-Related Cortical Amino Acid Changes After Basal Forebrain Lesion: A Microdialysis Study

Abstract: Cholinergic basal forebrain (BF) lesions in experimental animals have been used as a potential model for cholinergic deficits in cortex and hippocampus that occur in normal aging and Alzheimer's disease (AD). Glutamatergic cortical neurons are also affected in AD and could be part of the neurodegenerative process. In the present study, the effect of bilateral BF lesion with ibotenic acid microinjection on cortical extracellular amino acid levels was determined. Samples were collected every 20 min with microdialysis probes in awake, freely moving rats under basal and potassium stimulation conditions and measured by HPLC with fluorescence detection. Microdialysis experiments were performed 13 days, 21 days, and 30 days after BF lesion. The effectiveness of the lesion was shown by a significant 30% depletion in acetyl-CoA:choline O -acetyltransferase (EC 2.3.1.6) activity in the frontal cortex. Under basal conditions at 13 days only extracellular levels of taurine (Tau) and Glu were significantly reduced. Tau and Glu levels were recovered after 21 days and 30 days, respectively. In contrast, increase in Gly levels reaches its significance only at 30 days after lesion. Significant increases of Gln levels were observed at 21 days and 30 days. Asp and Ser levels remained constant throughout the period studied. Potassium stimulation led to increased Asp, Glu, Gly, and Tau levels, whereas Gln content decreased and Ser remained unaltered. As Ser is not believed to be a neurotransmitter, its lack of variation in any of the experimental conditions studied supports specific neuronal changes of the other amino acids. Results are discussed with reference to data observed in AD patients and possible mechanisms underlying the changes are suggested.     

Extracellular Concentrations of Taurine,Glutamate, and Aspartate in the Cerebral Cortex of Rats at the Asymptomatic Stage of Thioacetamide-Induced Hepatic Failure: Modulation by Ketamine Anesthesia

Subclinical hepatic encephalopathy (SHE) was produced in rats by two intraperitoneal injections of TAA at 24 h intervals and the animals were examined 21 days later. Concentrations of the neuroactive amino acids taurine (Tau), glutamate (Glu) and aspartate (Asp), were measured in the cerebral cortical microdialysates of thioacetamide (TAA)-treated and untreated control rats. During microdialysis some animals were awake while others were anesthetized with ketamine plus xylazine. There was no difference in the water content of cerebral cortical slices isolated from control and SHE rats, indicating a recovery from cerebral cortical edema that accompanies the acute, clinical phase of hepatic encephalopathy in this model. When microdialysis was carried out in awake rats, dialysate concentrations of all the three amino acids were 30% to 50% higher in SHE rats than in control rats. Ketamine anesthesia caused a 2.2% increase of water content of cerebral cortical slices and increased Asp, Glu, and Tau concentration in microdialysates of control rats. In SHE rats, ketamine anesthesia produced a similar degree of cerebral edema, however, it did not alter Asp and Glu concentrations in the microdialysates. These data may reflect on one hand a neuropathological process of excitotoxic neuronal damage related to increased Glu and Asp, on the other hand neuroprotection from neuronal swelling indicated by Tau redistribution in the cerebral cortex. The reduction of the effects of SHE on Glu and Asp content in ketamine-anesthesized rats is likely to be due to interference of ketamine with the NMDA receptor-mediated component of the SHE-evoked excitatory neurotransmitter efflux and/or reuptake of the two amino acids. By contrast, the SHE-related increase of Tau content was not affected by ketamine anesthesia, indicating that the mechanism(s) underlying SHE-evoked accumulation of Tau must be different from the mechanism causing release of excitatory amino acids. The results with ketamine advocate caution when using this anesthetic in studies employing the cerebral microdialysis technique for measurement of extracellular amino acids.     

Enhancement of Dopamine Release In Vivo from the Rat Striatum by Dialytic Perfusion of 6R-l-erythro-5,6,7,8-Tetrahydrobiopterin

We have previously reported that intracerebroventricular administration of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4), a cofactor for tyrosine hydroxylase, enhances biosynthesis of 3,4-dihydroxyphenylethylamine (dopamine) in the rat brain. In the present study, we have more precisely examined the effects of 6R-BH4 on dopamine release in vivo from the rat striatum using brain microdialysis. The amount of dopamine collected in striatal dialysates was determined using HPLC with electrochemical detection after purification with an alumina batch method. When the striatum was dialyzed with Ringer solution containing various concentrations of 6R-BH4 (0.25, 0.5, and 1.0 mM), dopamine levels in striatal dialysates increased in a concentration-dependent manner. Biopterin had little effect on dopamine levels in dialysates. The 6R-BH4-induced increase in dopamine levels in dialysates was abolished after pretreatment with tetrodotoxin (50 microM) added to the perfusion fluid, but after pretreatment with nomifensine (100 mg/kg, intraperitoneal injection), an inhibitor of dopamine uptake mechanism, a larger increase was observed. After inhibition of tyrosine hydroxylase by pretreatment with alpha-methyl-p-tyrosine (250 mg/kg, intraperitoneal injection), most of the increase persisted. These results suggest that 6R-BH4 has a dopamine-releasing action, which is not dependent on biosynthesis of dopamine.