Presynaptic D2 dopaminergic receptors mediate inhibition of excitatory synaptic transmission in rat neostriatum

The effect of dopamine (DA) on excitatory synaptic transmission was studied in rat neostriatal neurons using intracellular- and whole-cell voltage clamp-recording methods. Depolarizing excitatory postsynaptic potentials (EPSPs) were evoked by cortical stimulation. Superfusion of DA (0.01–10 μM) reversibly decreases EPSP in a concentration-dependent manner and with a estimated IC₅ of 0.3 μM. In addition, the inhibitory effect induced by DA at a low concentratiion (0.1 μM) was antagonized by sulpiride (1–10 nM), a selective D₂ dopaminergic receptor antagonist. However, D₁ dopaminergic receptor antagonist SKF-83566 (1–5 μM) did not affect the blocking effect by DA 0.1 μM. Based on these findings, we conclude that DA at a low concentration ( 0.1 μM) reduced the excitatory response of neostriatal neurons following cortical stimulation via the activation of D₂, but not D₁ dopaminergic receptors, located on the terminals of corticostriatal neurons.     

High-sensitive liquid chromatographic method for determination of neuronal release of serotonin, noradrenaline and dopamine monitored by microdialysis in the rat prefrontal cortex

A high-sensitive liquid chromatographic method based on precolumn derivatization and fluorescence detection allowing simultaneous determination of serotonin (5-HT), noradrenaline (NA) and dopamine (DA) in brain microdialysis samples is described. 5-HT, NA and DA were derivatized with benzylamine and 1,2-diphenylethylenediamine in the presence of potassium hexacyanoferrate(III) and glycine, which yielded to highly fluorescent and stable benzoxazoles. The derivatized samples were separated on a microbore column (150 mm × 1.0 mm i.d., packed with C18 silica, 5 μm) within 60 min. The mobile phase consisted of acetonitrile–Briton–Robinson buffer (pH 7.2) (32:68, v/v) containing 5 mM Na₂EDTA and 5 mM octanesulfonic acid sodium salt. The detection limits (signal-to-noise ratio of 3) for 5-HT, NA and DA were 76, 42 and 95 amol/10 μl injected on-column, respectively. Microdialysis samples were collected at 10-min intervals from the probes implanted in the prefrontal cortex of awake rats. The basal levels of 5-HT, NA and DA were 7.3 ± 0.7, 5.3 ± 0.31 and 8.1 ± 0.47 fmol/5 μl (mean ± S.E.M., n = 5). Following 90-min perfusion with tetrodotoxin (1 μM) or calcium-free Ringer solution, the DA and NA levels were reduced to about 15 and 20%, respectively and the 5-HT levels to 45 and 60% of the basal levels, respectively. Reserpine, 12 h after a dose of 5 mg/kg i.p., reduced the extracellular 5-HT, NA and DA concentrations to about 34, 39 and 32% of the basal levels, respectively. In conclusion, the preset microdialysis/analytical method enables simultaneous monitoring of basal and pharmacologically reduced neuronal release of 5-HT, NA and DA in the rat brain.     

The in vivo microdialysis recovery of dopamine is altered independently of basal level by 6-hydroxydopamine lesions to the nucleus accumbens

The present study was designed to test the hypothesis that the active neurotransmitter processes of release and uptake affect the in vivo microdialysis recovery of dopamine (DA) in the nucleus accumbens (N ACC) of the rat. The in vivo recovery for DA was established for rats which had received either unilateral infusions of the neurotoxin 6-hydroxydopamine (6-OHDA, 8 μg) or vehicle (0.2 μg ascorbate). In the quantitative dialysis method used (point of no net flux method), DA is added to the perfusate at concentrations above and below the expected extracellular concentration (0, 5, 10 and 20 nM) and DA is measured in the dialysate from the brain to generate a series of points. A linear fit is performed, the slope of which is the in vivo recovery of the dialysis probe. The in vivo recovery of the 6-OHDA group was 30 ± 3% which was significantly lower (P < 0.002) than the in vivo recovery of the control group which was 60 ± 3% (mean ± SEM; n = 6/group). The zero intercept of this regression is the point of no net flux, which is the extracellular concentration of DA independent of the probe sampling characteristics. The extracellular DA concentration for the 6-OHDA group was 7.8 ± 1.1nM, which was not significantly different than the control group which was 6.9 ± 0.7nM. The tissue DOPAC/DA ratios of the 6-OHDA lesioned hemispheres were significantly higher than the contralateral hemispheres of the same animals (0.62 ± 0.1vs.0.27 ± 0.1; P < 0.02) while the DOPAC/DA ratios in the control group were not significantly different (0.24 ± 0.1vs.0.27 ± 0.1). The fractional DA efflux from the terminals in the 6-OHDA group was significantly higher than the fractional DA efflux of the control group (0.52 ± 0.08vs.0.03 ± 0.003; P < 0.0001), indicating that the remaining terminals have increased turnover of DA. Despite the increased turnover, however, the number of remaining release and uptake sites are not sufficient to maintain the high in vivo recovery observed in the control group.     

Effect of M1- and M2-muscarinic drugs on striatal dopamine release and metabolism: an in vivo microdialysis study comparing normal and 6-hydroxydopamine-lesioned rats

Microdialysis was used to study the effect of M1 and M2 selective agonists and antagonists on striatal dopamine release and metabolism. Microdialysis probes were implanted, under anesthesia, in the left and the right striatum of the normal rats and in the normal and denervated striatum of the nigral 6-hydroxydopamine-lesioned rats. Dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined by liquid chromatography and electrochemical detection. The different drugs were infused through the dialysis probe during 40 min. Pirenzepine (5 μM), a selective M1 antagonist, produced a significant decrease in DA release in the normal and the 6-hydrodopamine-lesioned rats, with no significant difference between both groups. Methoctramine, a selective M2 antagonist, produced a dose-dependent increase in DA release between 20 and 200 μM in the normal rats, with no significant effect on DOPAC and HVA. Infusing 75 μM methocramine produced a significant increase in DA release with a more pronounced effect in the intact animals compared to the 6-hydroxydopamine-lesioned animals. The non-selective agonist carbachol produced a decrease in dopamine release after infusion of 50 μM (M2 effect) and an increase in dopamine release after infusion of 50 mM (M1 effect) in the normal rats. Infusing 50 μM carbachol in the denervated striatum, produced a slight increase in DA release. Our data suggest that presynaptic M1-muscarinic receptors enhance and M2-muscarinic receptors inhibit DA release in the striatum of the rat; and that 3 weeks after 6-hydroxydopamine lesioning there may be a normalisation of the number of M1-receptors with a loss of M2-receptors at the denervated side.     

GABA, acting at both GABAA and GABAB receptors, inhibits the release of cholecystokinin-like material from the rat spinal cord in vitro.

Superfusion of slices of the dorsal zone of the lumbar enlargement of the rat spinal cord with an artificial cerebrospinal fluid allowed the collection of cholecystokinin-like material (CCKLM) whose Ca²⁺-dependent release could be evoked by tissue depolarization with 30 mM K⁺. Studies on the possible influence of GABA and related agonists on this process showed that the amino acid, the GABA_A agonist, muscimol, and the GABA_B agonist, baclofen, inhibited the K⁺-evoked release of CCKLM from the rat spinal cord in a concentration-dependent manner. Maximal inhibition did not exceed −40% with either agonist. Furthermore, the effects of GABA_A and GABA_B receptor stimulation were not additive. Whereas the effects of muscimol (10 μM) and baclofen (1 μM) could be completely antagonized by bicuculline (1 μM) and phaclofen (10 μM), respectively, complete blockade of the inhibition by GABA (1 μM) could only be achieved in the presence of both antagonists. These data indicate that both GABA_A and GABA_B receptors are involved in the negative influence of GABA onto CCK-containing neurones within the dorsal horn of the rat spinal cord. Apparently, these receptors are not located on CCK-containing neurones themselves, since the inhibitory effect of GABA on the K⁺-evoked release of CCKLM could be completely prevented by tetrodotoxin (1 μM). As CCK acts centrally as an endogenous opioid antagonist, such a GABA-inhibitory control of spinal CCK-containing neurones might participate in the analgesic action of the amino acid via the intrathecal route.     

Light-induced stimulation of retinal dopamine: a dose-response relationship

Light stimulates dopamine (DA) release in the retina. The purpose of this was to determine the threshold and dose-response relationship between ocular light exposure and retinal DA synthesis in vivo. Groups of dark-adapted rats were exposed to 0, 1, 3, 5, 10, 25, 50, 100 or 1000 microwatts per square centimeter (μW/cm² of white light for 15 min. Retinal DA and dihydroxyphenylalanine (DOPA) were subsequently quantified by liquid chromatography with electrochemical detection. Both the DA and DOPA data fit hyperbolic curves significantly (Pˇ0.01). Exposure to white light at 25 μW/cm² or greater appears to elicit the maximum response of these neurons. Threshold irradiance is calculated to be 3–5 μW/cm². These results indicate that retinal DA synthesis and presumably DA neuron activity have a graded response to increasing irradiances of white light.     

Effect of morphine on dynorphin B and GABA release in the basal ganglia of rats

In vivo microdialysis was used to study the effects of systemic, as well as intracerebral administration of morphine and naloxone on dynorphin B release in neostriatum and substantia nigra of rats. The release of dopamine (DA), γ-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) was also investigated. Systemic injection of morphine (1 mg/kg s.c.) induced long-lasting increases in extracellular dynorphin B and GABA levels in the substantia nigra, whereas DA, Glu and Asp levels, measured in the same region, were not significantly affected. No effect on striatal neurotransmitter levels was observed following systemic morphine administration. Local perfusion of the substantia nigra with morphine (100 μM) through the microdialysis probe also increased nigral dynorphin B and GABA levels. Perfusion of the neostriatum with morphine (100 μM) significantly increased GABA and dynorphin B levels in the ipsilateral substantia nigra, but no effect was observed locally. Naloxone blocked the effect of systemic morphine administration on nigral dynorphin B and GABA release, already at a dose of 0.2 mg/kg s.c. Naloxone alone, given either systemically (0.2–4 mg/kg s.c.) or intracerebrally (1–100 μM), did not affect dynorphin B or amino acid levels, either in neostriatum or in substantia nigra. However, naloxone produced a concentration-dependent increase in DA levels. The present results indicate that systemic morphine administration stimulates the release of dynorphin B in the substantia nigra, probably by activating the μ-subtype of opioid receptor, since the effect of morphine on nigral dynorphin B and GABA was antagonized by a low dose of naloxone. The increase in extracellular DA levels produced by high concentrations of naloxone, both in neostriatum and substantia nigra, indicates a disinhibitory effect of this drug on DA release, probably via a non-μ subtype of opioid receptors located on nigro-striatal DA neurones.     

Carbamazepine suppresses synchronized afterdischarging in disinhibited immature rat hippocampus in vitro

Bath application of therapeutic concentrations of the anticonvulsant carbamazepine suppressed penicillin-induced synchronized afterdischarging in immature rat CA₃ hippocampal pyramidal cells. Afterdischarging was completely abolished in all preparations at a concentration of 30 μM (IC₅₀ = 8.5 ± 1.4 μM; mean ± S.E.M.). The duration of the preceding epilptiform burst was not altered at this concentration and was diminished by only 24.4 ± 1.2% at a supratherapeutic concentration of 100 μM. These results suggest that a carbamazepine-sensitive neurophysiological mechanism distinct from those responsible for epileptiform burst generation plays a key role in the generation of afterdischarges in developing hippocampus.     

Presynaptic muscarinic modulation of nicotinic excitation in the rat neostriatum

In rat neostriatal slices, cholinergic agents were tested for their effects on endogenous ACh release and on electrical activity. ACh release was evoked by 25 mM K⁺ during two 5-min periods between which a slice was allowed to rest for 20 min; drugs were present during the second stimulation period. In the absence of a cholinesterase inhibitor, only Ch outflow was monitored. For the recording of electrical activity, intrastriatal stimulation evoked field potentials which were monitored in the absence and presence of drugs in the perfusate.Atropine (1–100 μM) increased endogeneous ACh release by 32–91% and effective doses were 10-fold lower in the presence of a cholinesterase inhibitor. Atropine also increased the amplitudes of synaptic population spikes in the field potentials.The muscarinic agonists muscarine (100 μM) and oxotremorine (25 and 100 μM) decreased endogenous ACh release. Atropine (10 μM) blocked the depressant effect of muscarine (100 μM). Muscarine (100 μM–1 mM) and oxotremorine (10–100 μM) decreased the electrically evoked excitation in the rat neostriatal slices, and their effects were reversed by atropine.Only higher concentrations of nicotine (1 and 5 mM) decreased the synaptic population spikes, but potassium-stimulated Ch outflow was not affected.It is concluded that in the neostriatum presynaptic muscarinic receptors modulate nicotinic excitation since potassium-stimulated ACh release and intrinsically evoked synaptic excitation are influenced by muscarinic drugs in the same way.     

Sumatriptan modifies cortical free radical release during cortical spreading depression. A novel antimigraine action for sumatriptan?

Increases in concentration of brain NO are proposed to initiate and mediate migraine headache. Triggered by focal depolarisation, spreading depression (SD) represents a suitable mechanism for eliciting widespread release of nitric oxide. The current study examines the effect of sumatriptan, a 5-HT_1B/1D agonist and effective antimigraine therapy, on free radical release (nitric oxide and superoxide) in SD in the simple and complex cortices of the rat and cat. Following initiation of SD, sumatriptan pretreatment (300 μg kg⁻¹ i.v., 15 min prior to SD) modulated all phases of nitric oxide release associated with each SD in both cats and rats. As a result, superoxide levels were observed to significantly (ANOVA, post hoc LSD) increase versus vehicle treated animals (saline 1 ml kg⁻¹ i.v. 15 min prior to SD) during specific phases of each SD depolarisation. Averaged over all SD depolarisations, mean peak SD nitric oxide levels per depolarisation were 0.73±0.23 μM (n=29) in cats, and 0.42±0.09 μM (n=34) in rats. Sumatriptan significantly (Students t-test, P<0.05, two tailed hypothesis, P<0.05) modulated this increase in cortical nitric oxide concentrations to 0.32±0.06 μM (n=25) and 0.22±0.07 μM (n=37) in cats and rats. Sumatriptan appears to decrease the amplitude of nitric oxide release but enhances extracellular superoxide concentrations in both lissencephalic and gyrencephalic cortices during SD.     

Dopaminergic regulation of serotonin release in the substantia nigra of the freely moving rat using microdialysis

The functional regulation by dopamine (DA) receptors of serotonin (5-HT) release from the rat substantia nigra (SN) was investigated using in vivo microdialysis. A D₁- and D₂-receptor-mediated inhibition of nigral 5-HT release was demonstrated in this study. Continuous administration of the D₁-receptor agonist CY 208243 (10 μM) through the probe did not alter extracellular DA nor 5-HT from the SN, whereas intranigral administration of the D₁-receptor antagonist SCH-23390 HCl (10 μM) significantly increased both DA (to 214%) and 5-HT release (to 168%) from the SN. Co-perfusion of the D₁-receptor agonist and antagonist did not change nigral DA nor 5-HT release compared to perfusion of the antagonist alone. The continuous intranigral perfusion of the D₂-receptor agonist, (−)-quinpirole HCl (1 μM) significantly decreased both DA and 5-HT release to 71% and 78%, respectively. These decreases were abolished when the D₂-receptor antagonist S(−)-sulpiride (10 μM) and the D₂-receptor agonist (−)-quinpirole HCl (1 μM) were co-perfused. In contrast, the intranigral perfusion of the DA precursor, -DOPA (5 μM; 1 h), significantly increased nigral and striatal 5-HT release to 202% and 155%, respectively. This enhanced nigral 5-HT release might not be receptor-mediated. The results of the present study suggest a D₁ and D₂ regulation of nigral 5-HT release, either directly mediated by DA receptors on nigral 5-HT terminals or indirectly by nigral GABA, Glu or Asp. Alternatively, the observed DA–5HT-interaction in the SN might not reflect a local interaction but might involve an interaction at the level of the serotonin cell body region, the dorsal raphe nuclei (DRN).     

Stimulation by interleukin-1 (IL-1) of the release of rat corticotropin-releasing factor (CRF), which is independent of the cholinergic mechanism, from superfused rat hypothalamo-neurohypophysial complexes

The effects of interleukin-1 (IL-1) and interferon-γ (Ifn-γ) on the release of corticotropin-releasing factor (CRF) from superfused hypothalamo-neurohypophysial complexes (HNC) of rats were examined in the present study. In this in vitro system, the release of CRF from HNC was not affected by any dose of human recombinant Ifn-γ tested (0.1, 1 and 10 nM). In contrast, a rapid increase of CRF from HNC was elicited in a dose-dependent manner by human recombinant IL-1 and -1β in concentrations of 0.1–10 nM. The involvement of the cholinergic system in the mediation of the stimulatory effect of IL-1 on CRF release was evaluated. Acetylcholine in concentrations of 1–100 nM also elicited a rapid increase of CRF. The increase in CRF release induced by 10 nM of acetylcholine was completely suppressed in the presence of both hexamethonium (10 μM) and atropine (50 μM), a nicotinic and a muscarinic receptor antagonist, respectively. On the other hand, the increase in CRF release induced by 10 nM IL-1 or -1β was not affected by these two antagonists. These results indicate that IL-1 stimulates of CRF release through an action on the hypothalamo-neurohypophysial system, most likely on the hypothalamus, and that the stimulatory effect of IL-1 is probably independent of the cholinergic system.     

Beta-agkistrodotoxin inhibits large-conductance calcium-activated potassium channels in rat hippocampal CA1 pyramidal neurons

Effect of β-agkistrodotoxin (β-AgTx), a presynaptic neurotoxin purified from snake venom, on large-conductance calcium-activated potassium channels (BK_Ca) was studied in rat hippocampal CA1 pyramidal neurons using inside-out configuration of patch-clamp technique. The results showed that in equimolar K⁺ (150 mM) and 1 μM intracellular Ca²⁺ conditions, internal application of β-AgTx inhibited the activity of BK_Ca by reducing open probability (P_o) of the channels in a concentration-dependent manner. High concentration (74 nM) of β-AgTx completely eliminated opening of the channels. However, 37 nM β-AgTx (at −40 mV) decreased P_o from 0.49±0.07 to 0.03±0.03, switched two open time constants (0.51±0.32 and 8.77±1.63 ms) to be a single time constant of 0.46±0.40 ms. The results indicate that inhibition of BK_Ca by β-AgTx may account for the facilitatory phase of the toxin on acetylcholine release from nerve terminals.     

Evidence for 5-HT4 receptor involvement in the enhancement of acetylcholine release by p-chloroamphetamine in rat frontal cortex

The roles of endogenous serotonin (5-HT) and 5-HT receptor subtypes in regulation of acetylcholine (ACh) release in frontal cortex of conscious rats were examined using a microdialysis technique. Systemic administration (1 and 3 mg/kg, i.p.) of the 5-HT-releasing agent p-chloroamphetamine (PCA) elevated ACh output in a dose-dependent manner. Depletion of endogenous 5-HT by p-chlorophenylalanine significantly attenuated the facilitatory effect of PCA on ACh release. The PCA (3 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT₄ receptor antagonists RS23597 (50 μM) and GR113803 (1 μM), while the 5-HT_1A antagonist WAY-100135 (10 mg/kg, i.p.; 100 μM), 5-HT_1A/1B/β-adrenoceptor antagonists (−)-pindolol (8 mg/kg, i.p.) and (−)-propranolol (150 μM), 5-HT_2A/2C antagonist ritanserin (1 mg/kg, i.p.; 10 μM) and 5-HT₃ antagonist ondansetron (1 mg/kg, i.p.; 10 μM) failed to significantly modify the effect of PCA. These results suggest that PCA-induced enhancement of 5-HT transmission facilitates ACh release from rat frontal cortex at least in part through 5-HT₄ receptors.     

Inhibition by the neurosteroid allopregnanolone of basal and stress-induced acetylcholine release in the brain of freely moving rats

The neurosteroid allopregnanolone is a potent and efficacious modulator of γ-aminobutyric acid (GABA) type A receptors. The effects of intracerebroventricular injection of allopregnanolone (5 to 15 μg/5 μ1) on basal and stress-induced release of acetylcholine were investigated in various regions of the brain areas of freely moving rats and compared with those of the benzodiazepine midazolam (1 to 10 μg/5 μ1). Allopregnanolone inhibited (20–55%) basal acetylcholine release from the prefrontal cortex and hippocampus, but not from the striatum, in a dose-dependent manner. At a dose of 10 μg, allopregnanolone also completely prevented the increase in hippocampul acetylcholine release induced by foot-shock stress. Midazolam, inhibited basal acetylcholine release in all three brain regions as well as stress-induced acetylcholine release in the hippocampus, and showed a greater potency in these effects than allopregnanolone. These results suggest that endogenous neurosteroids may participate in the GABAergic modulation of central cholinergic function during basal conditions as well as after stress.     

Methods for isolation and characterization of intracerebral arterioles in the C57/BL6 wild-type mouse

Vascular control mechanisms have been studied extensively in mice. However, an in vitro characterization of penetrating intracerebral arterioles has not been reported. We describe methods for isolation and cannulation for mouse intracerebral arterioles. This technique allows analysis of mouse cerebral arteriolar physiology and pharmacology without the confounding influences of the surrounding brain elements. Penetrating intracerebral arterioles from adult C57/BL6 wild-type (WT) mice were isolated at 4 °C, transferred to an inverted microscope and cannulated at both ends using a dual glass micropipette system, wherein intraluminal flow (0.2 μl/min) and pressure (60 mmHg) were maintained. The arterioles developed spontaneous tone when the chamber was warmed to 37 °C, with the resulting diameter reaching 68.4±0.9% of passive diameter (29.8±1.1 μm). After the development of spontaneous tone, incremental changes in luminal pressure from 20 to 140 mmHg induced myogenic responses. Acidosis (pH 6.8) and alkalosis (pH 7.6) caused dilation (20.0±1.4%) and constriction (17.2±1.4%), respectively. Extraluminal adenosine (ADO (10 μM); 24.3±3.6%) and sodium nitroprusside (SNP (10 μM); 28.6±4.1%) and intraluminal adenosine 5′-triphosphate (ATP (10 μM); 20.0±3.9%) resulted in vasodilation similar in magnitude to that observed in rat arterioles. This information provides a foundation for elucidating cerebral vascular control mechanisms in genetically engineered mice.     

Response to cyanide of two types of glomoid cells in mature rat carotid body

Cells belonging to glomoids of mature rat carotid bodies were studied using the whole-cell patch clamp technique following acute dissociation. The recorded population encompassed two subtypes: one type (n=202), termed G(out), was characterized by a small voltage-dependent inward current (43±9pA, mean ±S.E.M.), large outward current (671±31 pA@⁺40 mV), high membrane resistance (1910 ± 110M Ω) and low capacitance (5.1 ± 0.1pF). A second subtype (n=56), termed G(in), had significantly lower membrane resistance (177 ± 35 MΩ), membrane capacitance (15.0 ± 1.0 pF) and little voltage-dependent current. Neither subtype supported generation of multiple action potentials during depolarization in the current clamp mode. Intracellular staining of the recorded cells by Lucifer yellow showed co-localization of both subtypes to clusters of cells which stained positively for catecholamines. Somal diameter was slightly, but significantly, larger for G(in) cells 8.7 ± 0.4 μM, n=7) compared to G(out) cells (7.8±0.2 μM, n=31) and all cells had fine cytoplasmic process s extending around neighboring cells. During recordings using the perforated patch technique, histotoxic hypoxia significantly decreased a voltage-dependent outward current in G(out) cells by 113±60pA (n=13), and decreased the holding current by 10±4pA (n=13) from a control value of −32±6pA. In G(in) cells, cyanide significant decreased membrane resistance and decreased holding current by 55±28pA from a control value of +120±42pA (n=7), but caused no significant change in outward current. These results show that glomoids of mature rat carotid bodies contain at least two types of cells which differ in their morphologic and electrophysiologic characteristics. The subtypes rapidly respond to histotoxic hypoxia and thus may mediate separate roles in the organ response to chemostimuli.     

Effects of veratridine and high potassium on micro-opioid receptor internalization in the rat spinal cord: stimulation of opioid release versus inhibition of internalization

Veratridine and high K⁺-induced μ-opioid receptor (MOR) internalization in rat spinal cord slices by evoking opioid release. Veratridine induced up to 75% MOR internalization but showed an atypical concentration–response: its effect increased steeply from 5 μM to 10 μM, and declined thereafter to disappear at 100 μM. At 100 μM, veratridine also inhibited of MOR internalization induced by exogenous endomorphin-2. This inhibition was caused by Na⁺ entry, since the Na⁺ ionophore monensin (50 μM) also inhibited endomorphin-induced MOR internalization. In contrast, veratridine induced neurokinin 1 receptor internalization (by evoking substance P release) without any inhibition at high concentrations. KCl evoked up to 80% MOR internalization, which disappeared in the presence of lidocaine or in the absence of peptidase inhibitors, indicating that it involved neuronal firing and peptide release. Unlike veratridine, KCl did not inhibit MOR internalization at high concentrations. However, both KCl and veratridine evoked more MOR internalization when applied for 2 min than for 20 min because of a direct inhibition of MOR internalization with the longer incubation times. These results show that short incubations with 20 μM veratridine or 30 mM KCl are optimal stimuli to evoke opioid release and MOR internalization in the spinal cord.     

Effects of taurine on depolarization-evoked release of amino acids from rat cortical synaptosomes

Effects of taurine on endogenous aspartic acid (Asp), glutamic acid (Glu) and γ-aminobutyric acid (GABA) release has been investigated using synaptosomes prepared from rat cerebral cortex. Although basal release of these amino acids was not affected, taurine inhibited KCl (30 mM)-evoked overflow of Asp, Glu and GABA in a concentration-dependent manner with potencies (IC₅₀) of 1 μM, 0.8 μM and 5 nM, respectively. Taurine (10 μM) maximally inhibited K⁺-evoked Asp, Glu and GABA overflow by 28, 37 and 65%, respectively. Phaclofen (10 μM, a GABA_B receptor antagonist), but not bicuculline (10 μM, a GABA_A receptor antagonist), counteracted the inhibition of GABA overflow, although the inhibition of Asp and Glu overflow was not attenuated. These data suggest that taurine may inhibit GABA release through the activation of presynaptic GABA_B autoreceptors and, at high concentration, also act on Asp- and Glu-nerve terminals to regulate release of excitatory amino acids in rat cortex.