Increase in the Extracellular Histamine Concentration in the Rat Striatum by μ-Opioid Receptor Activation

Abstract: The effects of morphine and selective ligands for μ-, κ-, and δ-opioid receptors on the extracellular histamine (HA) concentration in the striatum of freely moving rats were examined by in vivo microdialysis. On the day after implantation of the dialysis probe, the HA output per 30-min period was measured using HPLC-fluorometry. Morphine (3.8 mg/kg, s.c.) significantly increased the HA output by ∼200% 1–3 h after treatment. This effect was completely antagonized by naltrexone (1.6 mg/kg, s.c.). The HA output decreased to a level below 10% of the basal value by 4 h after treatment with ( S )-α-fluoromethylhistidine (77 mg/kg, s.c.). In such animals, morphine (3.8 mg/kg, s.c.) had no influence on the HA output. [ d -Ala²,MePhe⁴,Gly(ol)⁵]Enkephalin (DAGO; 0.2 µg, i.c.v.), a selective μ-agonist, significantly increased the HA output by ∼150% 0.5–1.5 h after treatment, and this effect was also completely blocked by naltrexone. A selective κ-agonist, U-50,488 (3.8 and 7.6 mg/kg, s.c.), and a selective δ-agonist, [ d -Pen², d -Pen⁵]enkephalin (0.5 and 2 µg, i.c.v.), had no effect on the HA output. These findings suggest that the stimulation of μ-opioid receptors by morphine and DAGO increases the extracellular HA concentration by accelerating HA release from nerve endings.     

On the Origin of Striatal Cholecystokinin Release: Studies with In Vivo Microdialysis

Abstract: In the present study, extracellular levels of the neuropeptide cholecystokinin (CCK), of the monoamine dopamine and its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and of the excitatory amino acids glutamate and aspartate were simultaneously monitored by microdialysis in the neostriatum of halothane-anesthetized rats under basal and K⁺-depolarizing conditions. Extracellular CCK and dopamine levels, but not glutamate and aspartate levels, were decreased by perfusion with a Ca²⁺-free medium, under both basal and K⁺-depolarizing conditions. HPLC revealed that the majority of the CCK-like immunoreactivity in the perfusates coeluted with CCK octapeptide. Striatal extracellular CCK levels were decreased by decortication plus callosotomy, with a parallel decrease in glutamate levels. Striatal extracellular levels of dopamine, DOPAC., and HVA were significantly decreased in animals treated previously with a unilateral 6-hydroxydopamine injection into the medial forebrain bundle. In these animals, however, the effect of decortication plus callosotomy on CCK and glutamate levels was not further augmented. Thus, this study supports the hypothesis of a neuronal origin of extracellular CCK and dopamine monitored with microdialysis in the striatum of the rat, and also supports the idea of a partly contralateral origin of corticostriatal CCK and glutamate inputs.     

Influence of Potassium Concentration in Microdialysis Perfusate on Basal and Stimulated Striatal Dopamine Release: Effect of Ceruletide, a Cholecystokinin-Related Peptide

Abstract: The in vivo microdialysis method was used to study the effect of the cholecystokinin-related peptide, ceruletide, on extracellular levels of dopamine (DA) in the striatum following perfusion with various K⁺ concentrations. Increasing the K⁺ concentration in the perfusate from 4 to 15 or 17.5 m M did not change basal DA release or release evoked by electrical stimulation of the medial forebrain bundle (MFB). However, when the perfusing solution contained 20 or 30 m M K⁺, dose-dependent reductions of both basal and MFB-stimulated DA release occurred. Subcutaneous administration of ceruletide at 160 μg/kg had no influence on the basal or MFB-stimulated DA release with 4 or 15 m M K⁺ in the perfusate. However, after perfusion with 17.5 m M K⁺, ceruletide significantly attenuated the basal and MFB-stimulated DA release. Carbachol (10 μ M ) locally applied via the dialysis probe also attenuated MFB-stimulated DA release after perfusion with 17.5 m M K⁺. From these results, we conclude that under appropriate depolarization of striatal DA terminals, ceruletide induces further depolarization and inactivation of nigrostriatal DA terminals. The present data suggest that this effect may be mediated via intrinsic cholinergic neurons in the striatum.     

Serotonergic Facilitation of Acetylcholine Release In Vivo from Rat Dorsal Hippocampus via Serotonin 5-HT3 Receptors

Abstract: The serotonin (5-HT) releaser d -fenfluramine and its active metabolite d -norfenfluramine, or the 5-HT-uptake inhibitor citalopram, by increasing synaptic 5-HT availability, facilitated in vivo release of acetylcholine (ACh) from dorsal hippocampi of freely moving rats as determined by the microdialysis technique. The effects of d -norfenfluramine (7.5 mg/kg i.p.) and citalopram (10 μ M , applied by reverse dialysis) were prevented by a 14-day chemical lesion of the raphe nuclei, suggesting mediation by the 5-HT system in the cholinergic action of the drugs. The increase in extracellular ACh content induced by d -norfenfluramine (5 mg/kg i.p.) was antagonized by the 5-HT₃ receptor antagonists tropisetron (0.5 mg/kg i.p.) and DAU 6215 (60 μg/kg i.p.), but not by the mixed 5-HT₁ and 5-HT₂ receptor antagonist metergoline (2 mg/kg s.c.). In accordance with an involvement of the 5-HT₃ receptor in the ACh facilitation induced by d-norfenfluramine is the finding that the selective 5-HT₃ receptor agonist 2-methyl-serotonin (250 μg i.c.v., or 10 μ M applied by reverse dialysis) raised ACh release. The effect of the intracerebroventricular drug was prevented by the 5-HT₃ antagonists DAU 6215 (60 μg/kg i.p.) and ondansetron (60 μg/kg s.c.). These antagonists by themselves did not modify the basal ACh release, indicating that 5-HT does not tonically activate the 5-HT₃ receptors involved. In conclusion, the overall regulatory control exerted by 5-HT in vivo is to facilitate hippocampal ACh release. This is mediated by 5-HT₃ receptors probably located in the dorsal hippocampi.     

Extracellular N-Acetylaspartate in the Rat Brain: In Vivo Determination of Basal Levels and Changes Evoked by High K+

Abstract: The purpose of this study was to determine the extracellular concentrations of N -acetylaspartate (NAA) in the rat cerebral cortex, striatum, and hippocampus of halo-thane-anaesthetised rats by intracerebral microdialysis, and to examine the effects of high K⁺-induced local depolarisation, which provokes synchronous neurotransmitter release, cell swelling, and acid-base changes. Basal levels of NAA in the extracellular fluid (EOF) were determined by the zero net flux method. Tissue levels of NAA in the cortex, striatum, and hippocampus were 8.4, 5.7, and 7.2 mmol/kg, respectively. The corresponding extracellular concentrations of NAA were much lower (35.1, 83.7, and 23.0 tiM). High tissue/ECF concentration ratios may suggest little release or leakage of NAA under basal conditions, and potent reuptake mechanisms for NAA in the cellular membrane of CNS cells. There was no change in ECF NAA during K⁺-induced local depolarising stimuli produced in the striatum, but NAA levels consistently increased after the K⁺ stimuli, irrespective of whether or not Ca²⁺ was present in the perfusion medium. These data confirm that NAA is not a neurotransmitter and suggest strongly that NAA is not directly involved in the release and reuptake or metabolism of neuroactive compounds. The increase of NAA in the ECF immediately after K⁺ stimulation may reflect an involvement in brain osmoregulation and/or acid-base homeostasis.     

Interactions Between Neuronal Histamine and Halothane Anesthesia in Rats

Abstract: Using an in vivo microdialysis method, we measured the release of histamine in the anterior hypothalamic area (AHy) of rats under several concentrations of halothane anesthesia (1, 0.5, and 0.2%). The release of histamine increased to 341 and 325% at halothane concentrations of 0.5 and 0.2%, compared with the basal level at anesthesia induced by 1% halothane. α-Fluoromethylhistidine (100 mg/kg i.v.), a specific and irreversible inhibitor of histidine decarboxylase, reduced the histamine release to <35% of the basal value at 1% halothane anesthesia in the AHy, and also decreased the anesthetic requirement for halothane, evaluated as the minimum alveolar concentration (MAC), by 26%. Furthermore, pyrilamine (20 mg/kg i.v.), a brain-penetrating H₁ antagonist, and zolantidine (20 mg/kg i.v.), a brain-penetrating H₂ antagonist, reduced the MAC for halothane by 28.5 and 16%, respectively. Although thioperamide (5 mg/kg i.v.), an antagonist of presynaptic H₃ autoreceptor, induced an approximate twofold increase in the level of histamine release in conscious freely moving rats, the same dose of thioperamide had little effect on the release of histamine under 1% halothane anesthesia in the AHy. Furthermore, thioperamide did not change the anesthetic requirement (MAC) for halothane. The present findings indicate that halothane anesthesia inhibits the release of neuronal histamine and that histaminergic neuron activities change the anesthetic requirement (MAC) for halothane through H₁ as well as H₂ receptors.     

Hypoxia-Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells

Abstract: Hypoxia (5% O₂) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca²⁺. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca²⁺]_i increase, showing that the Ca²⁺ influx was attributable to L- and N-type voltage-dependent Ca²⁺ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O₂). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K⁺ current. Among the K⁺ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K⁺ channels, inhibited the hypoxia-induced [Ca²⁺]_i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K⁺ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K⁺ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.     

Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Analysis of Its Action Site Using Sepiapterin

Abstract: Recently, we reported that 6 R - l - erythro -tetrahydrobiopterin (6 R -BH₄), a natural cofactor for hydroxylases of tyrosine and tryptophan, has a monoamine-releasing action independent of its cofactor activity. Here we attempted to determine whether 6 R -BH₄ acts inside the cell or from the outside of the cell by using brain microdialysis in the rat striatum. For this purpose, sepiapterin, an immediate precursor of 6 R -BH₄ in the salvage pathway, was used to selectively increase the intracellular 6 R -BH₄ levels. Dialytic perfusion of sepiapterin increased tissue levels of reduced biopterin (mainly 6 R -BH₄) but not the extracellular levels. Administration of sepiapterin increased the extracellular levels of 3,4-dihydroxyphenylalanine (DOPA) (an index of in vivo tyrosine hydroxylase activity) and of dopamine (DA) (an index of in vivo DA release). Either of the increases was eliminated after pretreatment with a tyrosine hydroxylase inhibitor α-methyl- p -tyrosine. Administration of 6 R -BH₄ increased extracellular levels of reduced biopterin, DOPA, and DA. After pretreatment with α-methyl- p -tyrosine, the increase in DOPA levels was abolished, but most of the increase in DA levels persisted. The increase in DA levels also persisted after pretreatment with nitric oxide synthase inhibitors. These data demonstrate that 6 R -BH₄ stimulates DA release directly, independent of its cofactor action for tyrosine hydroxylase and nitric oxide synthase, by acting from the outside of neurons.     

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.     

Rat-1 Fibroblasts Engineered with GAD65 and GAD67 cDNAs in Retroviral Vectors Produce and Release GABA

Abstract: The effects of the adenosine A₁ agonist N ₆-cyclohexyladenosine (CHA) on MPTP-induced dopamine (DA) depletion in the striatum of C57BL/6 mice were studied. Twenty hours after a single injection of MPTP (30 mg/kg, s.c.), the toxin caused 62% depletion of striatal DA. CHA (0.2–3 mg/kg, s.c.), when given together with MPTP, prevented the toxin-induced DA depletion in a dose-dependent manner. This protective action was apparently mediated by the A¹ receptors, because this effect was selectively antagonized by pretreating the animals with the A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine (25 mg/kg, i.p.) but not with the A₂ antagonist 1,3-dipropyl-7-methylxanthine (25 mg/kg, i.p.). When CHA (3 mg/kg) was injected 5 h after MPTP administration, at which point striatal DA levels were already reduced significantly, a rapid and complete recovery of the striatal DA levels occurred. These neurochemical data suggest that the A₁ agonist CHA is potentially useful as a neuroprotective agent against MPTP-induced toxicity.     

Muscarinic Receptor-Mediated Increase in Extracellular Inositol 1,4,5-Trisphosphate Levels in the Rat Hippocampus: An In Vivo Microdialysis Study

Abstract: The extracellular concentration of inositol 1,4,5-trisphosphate (IP₃) has been monitored in the ventral hippocampus of the anesthetized rat by using a microdialysis technique coupled to a radioreceptor assay. Three hours after the implantation of the cannula, basal extracellular concentration of IP₃ (corrected for a 9% recovery) was 71 n M (0.39 pmol/60-µl fraction) and remained stable for at least 5 h. Local infusion of carbachol for 60 min caused a significant concentration-related increase in extracellular IP₃ levels (0, 24, and 57% at 1, 50, and 100 µ M , respectively). Acetylcholine (100 µ M ) and muscarine (100 µ M ) increased IP₃ outflow by 40 and 42%, respectively. The effect of carbachol was fully prevented by coinfusion of 10 µ M pirenzepine and reduced by 1 µ M tetrodotoxin indicating that the carbachol response is mediated by neuronal muscarinic receptors. These data demonstrate the feasibility of using microdialysis and a radioreceptor assay to measure IP₃ in the extracellular space. This approach could prove useful for the study of the in vivo operation of muscarinic and, by extension, a number of receptors coupled to phosphoinositide turnover.     

Studies of Inositol Phosphate Export from Neuronal Tissue In Vitro

Abstract: Recent in vivo microdialysis studies have demonstrated the presence of extracellular levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] that can be increased in a concentration-dependent manner by muscarinic receptor activation. The aim of the present study was to determine whether extracellular levels of Ins(1,4,5)P₃ could be measured in vitro. Despite rapid increases in internal Ins(1,4,5)P₃ levels after stimulation with 1 m M carbachol, there was no change in external levels in both rat brain cortical slices and human neuroblastoma SH-SY5Y cells. Suprafusion of myo -[³H]inositol-prelabelled hippocampal slices with 1 m M carbachol caused an increase in ³H-inositol phosphates over basal levels in the perfusate after 10 min, reaching a peak (223 ± 56% of basal) 20 min after suprafusion with carbachol was started. This response to carbachol was potentiated in the presence of 30 m M K⁺. Analysis of the individual ³H-inositol phosphates in the perfusate revealed that levels of [³H]inositol monophosphate, [³H]inositol bisphosphate, [³H]inositol trisphosphate, and [³H]inositol tetrakisphosphate were all significantly increased. A similar increase in extracellular ³H-inositol phosphates was demonstrated in SH-SY5Y cells incubated with 1 m M carbachol for 30 min. This response was again enhanced by 30 m M K⁺, although the intracellular response was not potentiated. Possible roles for extracellular inositol phosphates are discussed.     

Regulation of Serotonin Release in the Frontal Cortex and Ventral Hippocampus of Homozygous Mice Lacking 5-HT1B Receptors: In Vivo Microdialysis Studies

Abstract: To assess the involvement of the serotonin receptor subtype 5-HT_1B as terminal autoreceptor regulating 5-HT release in mice, we compared basal values and potassium-evoked changes of extracellular 5-HT levels obtained by in vivo microdialysis in two serotoninergic terminal projection areas of conscious wild-type mice with those measured in homozygous mutant mice lacking the gene encoding the 5-HT_1B receptor. In the frontal cortex and ventral hippocampus, basal and K⁺-evoked 5-HT release did not differ between the two strains of mice studied. The infusion via reverse microdialysis of the selective 5-HT_1B receptor agonist CP-93,129 (500 n M ) decreased significantly K⁺-evoked 5-HT release in the frontal cortex (by −44%) and ventral hippocampus (by −32%) of wild-type mice but had no effect in mutants. In a similar manner, the mixed 5-HT_1B-5-HT_1D receptor agonist sumatriptan (800 n M ) decreased significantly K⁺-evoked 5-HT release in the frontal cortex (by −46%) of wild-type mice but had no effect in mutants. These results demonstrated that 5-HT_1B knockout mice are not as sensitive to full (CP-93,129) and mixed (sumatriptan) 5-HT_1B receptor agonists as are wild-type mice. These data provide in vivo evidence that, in mice, 5-HT_1B, but not 5-HT_1D, autoreceptors inhibit 5-HT release at nerve terminals located in the frontal cortex and ventral hippocampus.     

Acetylcholine Release in the Rat Prefrontal Cortex In Vivo: Modulation by α2-Adrenoceptor Agonists and Antagonists

Abstract: We have previously shown that the release of acetylcholine (ACh) in the medial prefrontal cortex of the conscious rat, as measured by microdialysis, is increased following intraperitoneal injection of the selective α₂-adrenoceptor antagonist (+)-efaroxan. To characterize further the receptor pharmacology of this response, the effects of other selective α₂-adrenoceptor ligands were examined. The α₂-adrenoceptor antagonists idazoxan (2.5 and 20 mg/kg), atipamezole (2.5 mg/kg), and fluparoxan (10 mg/kg) increased ACh outflow by up to 250–325% of basal levels over a 3-h period following intraperitoneal injection. The α₂-adrenoceptor agonists UK-14304 (2.5 mg/kg) and guanabenz (2.5 mg/kg) reduced ACh outflow by 80 and 60%, respectively. Clonidine (0.00063–0.16 mg/kg) had no significant depressant effect and at 2.5 mg/kg increased ACh outflow to 233% of basal levels. These results indicate a modulatory role for α₂-adrenoceptors on the release of ACh in the rat prefrontal cortex in vivo. Based on the facilitatory effects produced by the antagonists alone, this α₂-adrenoceptor modulation appears to be tonic and inhibitory. The ability of α₂-adrenoceptor antagonists to enhance ACh outflow suggests a therapeutic usefulness in disorders where cortical ACh release deficits have been implicated.     

Dopaminergic Properties of Cultured Rat Carotid Body Chemoreceptors Grown in Normoxic and Hypoxic Environments

Abstract: Using dissociated carotid body (CB) cultures prepared from neonatal (postnatal days 5–7; P7) or juvenile (postnatal day 19–20; P20) rats, we compared catecholaminergic properties and mechanisms of O₂ sensing in glomus cells grown in normoxic (Nox; 20% O₂) and chronically hypoxic (CHox; 6% O₂) environments for up to 2 weeks. In Nox cultures, basal dopamine (DA) release, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was similar for P7 and P20 cultures (∼0.3 pmol/1,000 cells/15 min) and was unaffected by culture duration (2 vs. 12 days). Acute hypoxia (5 and 10% O₂) caused a dose-dependent stimulation (6× and 3× basal, respectively) in DA release, that was inhibited by nifedipine (10 µ M ). DA release was also stimulated by high extracellular K⁺ (30 m M ) and iberiotoxin (200 n M ), a selective blocker of P o ₂-regulated, Ca-dependent K⁺ channel in glomus cells. The stimulatory effect of iberiotoxin was similar to 5% O₂ in P20 cultures, but substantially less (about one-half) in P7 cultures. In contrast, in CHox cultures, basal DA release was substantially elevated, ∼8× Nox levels, although this did not correlate with significant differences in stores. Further, whereas acute hypoxia (5% O₂) and high K⁺ also stimulated DA release in CHox cultures (∼2× and ∼3× basal), iberiotoxin (200 n M ) did not. Thus, after chronic hypoxia in vitro, there is an enhanced basal catecholamine release and an apparent down-regulation of functional Ca-dependent K⁺ channels in CB chemoreceptors. These cellular adaptations may relate to changes in CB chemosensitivity during chronic hypoxemia.     

Effects of K-stimulation and precursor loading on the in vivo release of dopamine, serotonin and their metabolites in the nucleus accumbens of the rat

The efflux of endogenous 3,4-dihydroxyphenylethylamine (DA) 5-hydroxytryptamine (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the nucleus accumbens of the anesthetized rat was studied using a push-pull cannula. Local perfusion for 10 minutes with 35 mM K⁺ significantly (P<0.01) increased the release of DA and 5-HT, but not their metabolites, from their respective control levels of 0.95 and 0.04 pmol/15 min to 2.5 and 0.23 pmol/15 min. Exposure to 35 mM K⁺ a second and third time resulted in a decrement in the amount of stimulated release for both DA and 5-HT. This decrease was prevented by local perfusion for 10 minutes with 50 uM L-tyrosine and -tryptophan starting 30 minutes before each episode of depolarization. The baseline amounts of DOPAC, HVA and 5-HIAA observed in the perfusates were several fold higher than the basal levels found for 5-HT and Da. In the absence of precursors, the efflux of DOPAC, HVA and 5-HIAA decreased approximately 60, 40 and 25%, respectively, from the first to the last baseline fraction collected. Addition of precursors prevented the decrease for DOPAC and 5-HIAA but not for HVA. The data indicated that (a) the release of DA and 5-HT, along with their metabolites, could be simultaneously measured with the present procedure, and (b) when using the push-pull cannula, local perfusion with precursors may be necessary following periods of sustained and/or repeated stimulation in order to replenish the monoamine transmitter pools.     

Transient Elevation of Interstitial N-Acetylaspartate in Reversible Global Brain Ischemia

Abstract: We evaluated the changes of interstitial N -acetylaspartate (NAA) concentration ([NAA]_e) in rat striatum by microdialysis following transient global ischemia and depolarization. The dialysate NAA concentration ([NAA]_d) values were corrected for the in vivo recovery to obtain [NAA]_e, by the use of [³H]mannitol in the perfusion fluid. During global ischemia the relative loss (RL) of [³H]mannitol decreased to 40% of preischemic values, reflecting the decrease in extracellular volume fraction. During reperfusion RL of [³H]mannitol quickly normalized. The [NAA]_d doubled during transient ischemia, which, after correction for in vivo recovery, corresponds to a fivefold increase in [NAA]_e ( p < 0.05). Reperfusion induced a >10-fold increase of [NAA]_e ( p < 0.01) with subsequent normalization after 45 min. KCI at 100 m M caused a reversible 50% reduction in RL of [³H]mannitol and a three times increase in [NAA]_e ( p < 0.05) but no further increase when normal perfusate was reintroduced. The mechanisms of NAA release from neurons are unknown but may involve the activation of unknown channels/carriers—possibly in relation to a volume regulatory response. The present study shows that the distribution of NAA in brain is dynamically regulated in acute ischemia and suggests that changes of NAA levels could be caused by other means than neuronal loss.     

Release of Neuropeptide FF, an Anti-Opioid Peptide, in Rat Spinal Cord Slices Is Voltage- and Ca2+-Sensitive: Possible Involvement of P-Type Ca2+ Channels

Abstract: Neuropeptide FF (NPFF), an FMRFamide-like peptide with antiopioid properties, inhibits morphine-induced analgesia but also produces hyperalgesia. In the present study, the mechanisms of NPFF release were investigated in an in vitro superfusion system with rat spinal cord slices. The opening of voltage-sensitive Na⁺ channels with veratridine (20 µ M ) induced calcium-dependent NPFF release, which was abolished by tetrodotoxin (1 µ M ), suggesting that NPFF release depends on nerve impulse activity. We also showed that NPFF release was a function of the extent of depolarization and was calcium dependent. The 30 m M K⁺-induced release was blocked by Co²⁺ or Ni²⁺ (2.5 m M ) but was unaffected by Ca²⁺ channel blockers of the L type—Cd²⁺ (100 µ M ), nifedipine or nimodipine (10 µ M ), diltiazem (20 µ M ), or verapamil (50 µ M )—or the N type—ω-conotoxin GVIA (1 µ M ). In contrast, ω-agatoxin IVA (1 µ M ) led to a 65% reduction in NPFF release, suggesting that P-type Ca²⁺ channels play a prominent role. The 35% remaining release resulted from activation of an unknown subtype. The NPFF-like material in superfusates recognized spinal NPFF receptors, suggesting that NPFF release in the spinal cord has a physiological role.     

Extracellular release of photosynthetic products by freshwater phytoplankton populations, with special reference to the algal species involved

SUMMARY. Over three successive years, depth profiles of C-fixation and excretion, chlorophyll- a concentrations, phytoplankton species composition and bacterial numbers were determined in Lake Vechten, a slightly eutrophic lake in The Netherlands. Special attention was given to the method used to measure extracellular release.
Excretion of dissolved organic ¹⁴C depended largely upon the photo-synthetic activity of the phytoplankton, ranging from 0–2.5 mg m^-1 h^-1, representing a percentage extracellular release (PER) of 0–25%.
During a period in August, however, a subsurface chlorophyll- a maximum at 5–7 m depth coincided with high excretion rates of up to 10 mg Cm^-3 h^-1 (PER = 55%). Phytoplankton analysis revealed a stratification in numbers of Mallomonas caudata with a maximum at 5–7 m depth.
The results suggest that in these water layers bacterial populations grew at the expense of the dissolved organic carbon compounds excreted by Mallomonas caudata. This means that extracellular release can temporarily function as an important nutrient source for the heterotrophie community in addition to the more or less constant dissolved organic carbon pool.     

Neuronal Release of Serotonin in the Cerebellum of Behaving Rats: An In Vivo Microdialysis Study

Abstract: Release of endogenous serotonin [5-hydroxy-tryptamine (5-HT)] in the cerebellum of awake rats was characterized using in vivo microdialysis. 5-HT output was increased (∼70%) by local application of KCl (100 m M ) and was reduced (∼60%) by both tetrodotoxin (0.5 µ M ) and omission of Ca²⁺ from the perfusion fluid. 5-HT release was decreased (∼70%) by the selective 5-HT_1A agonist 8-hydroxy-2-(di- n -propylamino)tetralin (0.25 mg/kg, s.c.), and this effect was rapidly reversed by a selective 5-HT_1A antagonist, N -[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N -(2-pyridinyl)cyclohexane-carboxamide trihydrochloride (WAY-100635; 0.1 mg/kg, i.p.). These results indicate that a large portion of the measurable 5-HT output in the cerebellum is of neuronal origin, is dependent on impulse flow, and is sensitive to 5-HT_1A autoreceptor activation. Further studies examined the relationship between 5-HT levels and general activity of the animals across the light-dark transition and during behavioral manipulations. Both 5-HT levels and behavioral activity were significantly elevated during the dark period, with changes in 5-HT efflux closely paralleling changes in activity. Similar increases (∼40%) in 5-HT output were observed during both feeding and feeding in the presence of a stressor (tail pinch). These findings suggest that behavioral state is an important factor determining neuronal 5-HT release in cerebellum under physiological conditions.