Stimulation-evoked Release Research Articles

Exercise increases caudate dopamine release and ventral striatal activation in Parkinson's disease.

The objective of this study was to examine the effects of aerobic exercise on evoked dopamine release and activity of the ventral striatum using positron emission tomography and functional magnetic resonance imaging in Parkinson's disease (PD). Thirty-five participants were randomly allocated to a 36-session aerobic exercise or control intervention. Each participant underwent an functional magnetic resonance imaging scan while playing a reward task before and after the intervention to determine the effect of exercise on the activity of the ventral striatum in anticipation of reward. A subset of participants (n = 25) completed [11 C] raclopride positron emission tomography scans to determine the effect of aerobic exercise on repetitive transcranial magnetic stimulation-evoked release of endogenous dopamine in the dorsal striatum. All participants completed motor (MDS-UPDRS part III, finger tapping, Timed-up-and-go) and nonmotor assessments (Starkstein Apathy Scale, Beck Depression Inventory, reaction time, Positive and Negative Affect Schedule, Trail Making Test [A and B], and Montreal Cognitive Assessment) before and after the interventions. The aerobic group exhibited increased activity in the ventral striatum during functional magnetic resonance imaging in anticipation of 75% probability of reward (P = 0.01). The aerobic group also demonstrated increased repetitive transcranial magnetic stimulation-evoked dopamine release in the caudate nucleus (P = 0.04) and increased baseline nondisplaceable binding potential in the posterior putamen of the less affected repetitive transcranial magnetic stimulation-stimulated hemisphere measured by position emission tomography (P = 0.03). Aerobic exercise alters the responsivity of the ventral striatum, likely related to changes to the mesolimbic dopaminergic pathway, and increases evoked dopamine release in the caudate nucleus. This suggests that the therapeutic benefits of exercise are in part related to corticostriatal plasticity and enhanced dopamine release. © 2019 International Parkinson and Movement Disorder Society.

Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy

The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl−-dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

3,4-Methylenedioxymethamphetamine, mephedrone, and β-phenylethylamine release dopamine from the cytoplasm by means of transporters and keep the concentration high and constant by blocking reuptake

The addiction-related behavioural effects of drugs of abuse are mediated by the mesocorticolimbic monoamine systems. We investigated the effects of 3,4-methylenedioxymethamphetamine (MDMA), mephedrone, β-phenylethylamine (β-PEA) methylphenidate (MPH) on dopamine release from mouse perfused nucleus accumbens and prefrontal cortex slices. The fractional release of [3H]-dopamine was measured at rest and in response to field stimulation. The distributions of [3H]-dopamine and its metabolites were determined using high-pressure liquid chromatography. The effect of drugs on [3H]-dopamine uptake was measured in synaptosomal P2 preparations from the frontal cortex and striatum. Similar to MDMA, mephedrone β-PEA increased the resting release of [3H]-dopamine from the nucleus accumbens and prefrontal cortex in a [Ca2+]o-independent manner, and the stimulation-evoked release was also augmented. In contrast, MPH failed to affect the resting release but potentiated the release in response to axonal activity. Similar to dopamine transporter antagonist GBR 12909, mephedrone, MDMA and MPH biphasically inhibited the [3H]-dopamine uptake. The administration of GBR 12909 and nisoxetine, or lowering the bath temperature prevented MDMA, mephedrone and β-PEA from enhancing the resting, cytoplasmic release of [3H]-dopamine, indicating the role of transporters in the release process. We conclude that amphetamine-like drugs of abuse and the trace amine β-PEA excessively increase the [Ca2+]o-independent, non-vesicular release of dopamine from the cytoplasm into the extrasynaptic space and inhibit the high-affinity transporters, thereby maintaining a high ambient, non-synaptic concentration of dopamine that may tonically control the activity of neurons equipped with dopamine receptors and is likely involved in the reinforcing effects and abusive potential of amphetamines.

Effects of articaine on [3H]noradrenaline release from cortical and spinal cord slices prepared from normal and streptozotocin-induced diabetic rats and compared to lidocaine.

Since a significant proportion of diabetic patients have clinical or subclinical neuropathy, there may be concerns about the use of local anaesthetics. The present study was designed to determine and compare the effects of articaine, a widely used anaesthetic in dental practice, and lidocaine on the resting and axonal stimulation-evoked release of [3H]noradrenaline ([3H]NA) in prefrontal cortex slices and the release of [3H]NA in spinal cord slices prepared from non-diabetic and streptozocin (STZ)-induced diabetic (glucose level=22.03±2.31mmol/l) rats. The peak of allodynia was achieved 9 weeks after STZ-treatment. Articaine and lidocaine inhibited the stimulation-evoked release in a concentration-dependent manner and increased the resting release by two to six times. These effects indicate an inhibitory action of these anaesthetics on Na+- and K+-channels. There was no difference in clinically important nerve conduction between non-diabetic and diabetic rats, as measured by the release of transmitter in response to axonal stimulation. The uptake and resting release of NA was significantly higher in the brain slices prepared from diabetic rats, but there were no differences in the spinal cord. For the adverse effects, the effects of articaine on K+ channels (resting release) are more pronounced compared to lidocaine. In this respect, articaine has a thiophene ring with high lipid solubility, which may present potential risks for some patients.

Correlation between the increased release of catecholamines evoked by local anesthetics and their analgesic and adverse effects: Role of K(+) channel inhibition.

Because local anesthetics are known to inhibit both sodium and potassium channels, and anesthetic properties have been attributed to the former effect, we compared their effects with those of tetrodotoxin (TTX), a selective Na(+) channel inhibitor with anesthetic activity, and 4-aminopyridine (4-AP), a selective potassium channel blocker with convulsive activity, on transmitter release during rest and in response to field (axonal) stimulation using the microvolume perfusion method and isolated prefrontal cortex and spinal cord slice preparations loaded with the radioactive transmitters [(3)H]dopamine ([(3)H]DA) and [(3)H]noradrenaline ([(3)H]NA). It is also known that local anesthetics may exert analgesic effect and, rarely, some adverse effects on the central nervous system (CNS). Neurochemical evidence demonstrated that local anesthetics administered at concentrations ranging from 0.5 to 5mM, which might have been intentionally or accidentally achieved in clinical practice (e.g., during spinal and epidural anesthesia or peripheral nerve block), led to presynaptic failures during neurochemical transmission, including inhibited transmitter release associated with axonal firing and markedly enhanced extraneuronal concentrations of transmitters due to increased resting, [Ca(2+)]o-independent release. Tetrodotoxin, a toxin with selective Na(+) channel-blocking properties, inhibited the stimulation-evoked release but failed to affect the resting release. In contrast, the potassium channel inhibitor 4-AP enhanced both the resting- and action potential-evoked transmitter releases. It is concluded that effects of local anesthetics on resting catecholamine release in the spinal cord may contribute to their action during neuropathic pain relief and spinal analgesia as well as to their side effects in the CNS.

Protective effect of rasagiline in aminoglycoside ototoxicity

Sensorineural hearing losses (SNHLs; e.g., ototoxicant- and noise-induced hearing loss or presbycusis) are among the most frequent sensory deficits, but they lack effective drug therapies. The majority of recent therapeutic approaches focused on the trials of antioxidants and reactive oxygen species (ROS) scavengers in SNHLs. The rationale for these studies was the prominent role of disturbed redox homeostasis and the consequent ROS elevation. Although the antioxidant therapies in several animal studies seemed to be promising, clinical trials have failed to fulfill expectations.We investigated the potential of rasagiline, an FDA-approved monomanine oxidase type B inhibitor (MAO-B) inhibitor type anti-parkinsonian drug, as an otoprotectant. We showed a dose-dependent alleviation of the kanamycin-induced threshold shifts measured by auditory brainstem response (ABR) in an ototoxicant aminoglycoside antibiotic-based hearing loss model in mice. This effect proved to be statistically significant at a 6-mg/kg (s.c.) dose. The most prominent effect appeared at 16kHz, which is the hearing sensitivity optimum for mice. The neuroprotective, antiapoptotic and antioxidant effects of rasagiline in animal models, all targeting a specific mechanism of aminoglycoside injury, may explain this otoprotection.The dopaminergic neurotransmission enhancer effect of rasagiline might also contribute to the protection. Dopamine (DA), released from lateral olivocochlear (LOC) fibers, was shown to exert a protective action against excitotoxicity, a pathological factor in the aminoglycoside-induced SNHL. We have shown that rasagiline enhanced the electric stimulation-evoked release of DA from an acute mouse cochlea preparation in a dose-dependent manner. Using inhibitors of voltage-gated Na+-, Ca2+ channels and DA transporters, we revealed that rasagiline potentiated the action potential-evoked release of DA by inhibiting the reuptake.The complex, multifactorial pathomechanism of SNHLs most likely requires drugs acting on multiple targets for effective therapy. Rasagiline, with its multi-target action and favorable adverse effects profile, might be a good candidate for a clinical trial testing the otoprotective indication.

Tyrosine-free amino acid mixtures reduce physiologically-evoked release of dopamine in a selective and activity-dependent manner

Depletion of the catecholamine precursor tyrosine using tyrosine-free amino acid mixtures is an important tool in neuropsychological studies, and often considered dopamine selective on the basis of neuropharmacological studies. However, little is known of the effects of tyrosine depletion when catecholamine neurons are activated physiologically. Here we investigated the effect of tyrosine-free amino acid mixtures on catecholamine release evoked in vivo using a stimulation paradigm aimed to approximate the phasic firing pattern of these neurons that accompanies cognitive and behavioural change. Dopamine and noradrenaline release was monitored by microdialysis in rat medial prefrontal cortex (mPFC) and striatum (chloral hydrate anaesthesia, perfusion medium containing 1 µM cocaine). Electrical stimulation of the medial forebrain bundle (MFB) caused a short-lasting, frequency-dependent increase in dopamine and noradrenaline. A full tyrosine-free amino acid mixture reduced the release of dopamine in mPFC and striatum, across a range of stimulation frequencies, and the effect was greater as stimulation frequency increased. Similar results were obtained using a smaller tyrosine-free amino acid mixture. In the same experiments showing decreased dopamine, neither tyrosine-free mixture of amino acids significantly altered stimulation-evoked release of noradrenaline. These results show that tyrosine depletion using tyrosine-free amino acid mixtures causes a selective, activity-dependent decrease in dopamine release when dopamine neurons are driven physiologically.

Influence of the hippocampus on amino acid utilizing and cholinergic neurons within the nucleus accumbens is promoted by histamine viaH1receptors

The influence of the neurotransmitter histamine on spontaneous and stimulation-evoked release of glutamate, aspartate, GABA and ACh in the nucleus accumbens (NAc) was investigated in vivo. Using the push-pull superfusion technique, histaminergic compounds were applied to the NAc and neurotransmitter release was assessed. In some experiments, the fornix/fimbria of the hippocampus was electrically stimulated by a microelectrode and evoked potentials were monitored in the NAc. Superfusion of the NAc with the H1 receptor antagonist triprolidine (50 μM) decreased spontaneous outflow of glutamate, aspartate and ACh, while release of GABA remained unaffected. Superfusion with histamine elevated release of ACh, without influencing that of the amino acids. Electrical stimulation of the fornix/fimbria enhanced the output of amino acids and ACh within the NAc. The evoked outflow of glutamate and ACh was diminished on superfusion with triprolidine, while release of aspartate and GABA was not affected. Superfusion of the NAc with histamine intensified the stimulation-evoked release of glutamate and Ach. Histamine also elevated the stimulation-induced release of aspartate, without influencing that of GABA. Presuperfusion with triprolidine abolished the reinforced effect of histamine on stimulation-evoked transmitter release within the NAc. Neuronal histamine activates H1 receptors and increases spontaneous release of glutamate, aspartate and ACh within the NAc. Stimulation of the hippocampal fornix/fimbria tract also enhances release of glutamate and ACh within the NAc and this effect is intensified by H1 receptor stimulation within the NAc. The latter effects, which are mediated by hippocampal afferences, might play an important role in mnemonic performance and in emotional processes such as anxiety and stress disorders.

Modulation of acetylcholine release by cholecystokinin in striatum – receptor specificity; role of dopaminergic neuronal activity

Cholecystokinin, a neuroactive peptide functioning as a neurotransmitter and neuromodulator in the central nervous system, mediates a number of processes and is implicated in neurological and psychiatric disorders such as Parkinson's disease, anxiety and schizophrenia. Striatum is one of the brain structures with the highest concentrations of CCK in the brain, rich in CCK receptors as well. The physiological effect of CCK on cholinergic interneurons, which are the major interneurons in striatum and the modulatory interactions which exist between dopamine, acetylcholine and cholecystokinin in this brain structure are still unclear. We studied the effect of cholecystokinin octapeptide (CCK-8) on the release of acetylcholine (ACh) from striatal slices of the rat brain. CCK-8 (0.01-0.1μM) showed no statistically significant effect on the basal but enhanced dose-dependently the electrically (2Hz)-evoked release of [(3)H]ACh. When slices were preperfused with 100μM sulpiride, a selective dopamine D(2) receptor antagonist, the CCK-8 (0.01μM) effect on electrically stimulated ACh release was increased nearly 2-fold. A similar increase was observed after depletion of endogenous dopamine (DA) from nigro-striatal dopaminergic neurons with 6-hydroxydopamine (6-OHDA) (2× 250μg/animal, i.c.v.). Furthermore in the presence of dopamine (100μM) or apomorphine (10μM), the prototypical DA receptor agonist, CCK-8 (0.01μM) failed to enhance the stimulation-evoked release of [(3)H]ACh. The D(2) receptor agonist quinpirol (1μM) abolished the CCK-8 effect on electrically stimulated ACh release as well. The increase in electrically induced [(3)H]ACh release produced by 0.01μM CCK-8 was antagonized by d,l loxiglumide (CR 1505), 10μM, a non-peptide CCK-A receptor antagonist and by Suc-Tyr-(OSO3)-Met-Gly-Trp-Met-Asp-β-phenethyl-amide (GE-410), 1μM, a peptide CCK-A receptor antagonist. The antagonistic effect of GE-410 on the CCK-8-potentiated, electrically induced release of [(3)H]ACh was studied in striatum for the first time. CAM 1028 (10μM), a CCK-B receptor antagonist, also prevented the potentiating effect of CCK-8 (0.01μM) on electrically stimulated release of [(3)H]ACh. The presented results indicate that (i) CCK-8 is capable of increasing ACh elicited by field electrical stimulation in striatum; (ii) CCK-8 is more effective in its ACh-stimulating effect when dopaminergic activity in striatum is blocked i.e. CCK-8-facilitated release of electrically induced ACh from cholinergic interneurons in the striatum is under the inhibitory control of the tonic activity of dopamine from the nigrostriatal pathway; (iii) the enhancing effect of CCK-8 on electrically evoked ACh release is mediated through both CCK-A and CCK-B cholecystokinin receptors located most likely on the cell bodies of cholinergic interneurons in striatum.

Transmitter releasing action of selegiline ((—)-deprenyl) from peripheral sympathetic nerves under different experimental conditions

A high concentration of selegiline ((-)-deprenyl; 10(-4) M) potentiated low frequency (2 Hz) nerve stimulation-evoked release of [3H]noradrenaline from the isolated main pulmonary artery of the rabbit in the presence of neuronal (cocaine, 3 X 10(-5) M) and extraneuronal (corticosterone, 5 X 10(-5) M) uptake blockers, and inhibited the postsynaptic response. The transmitter-releasing action of 10(-4) M selegiline was inhibited by a moderate increase of external K+ (23.6 mM). Excess K+ by itself abolished the nerve-evoked release of [3H]noradrenaline but did not increase the resting outflow of radioactivity. Excess Ca2+ (7.5 mM) increased the stimulation-evoked transmitter release. In the presence of excess Ca2+, selegiline (10(-4) M) was effective in increasing the [3H]noradrenaline release in response to nerve-stimulation. Excess Ca2+ partly antagonized the postsynaptic inhibitory action of selegiline. In Ca2+-free, 1 mM EGTA-containing Krebs solution both the nerve-evoked 3H release and the transmitter releasing action of selegiline were abolished in agreement with the 'Ca-hypothesis'. The voltage-dependent K+-channel blocker, 4-aminopyridine (10(-5) M), increased the nerve-stimulation-evoked release of tritium from arteries. If selegiline was also present in the perfusion medium the nerve-evoked transmitter release further increased. 4-Aminopyridine completely antagonized the inhibitory action of selegiline on the postsynaptic contraction.

Effects of nicorandil on sympathetic neurotransmission in rat caudal artery

1. We examined the effects of nicorandil, an ATP-sensitive potassium (K(ATP)) channel opener and nitric oxide donor, on the release of noradrenaline from vascular sympathetic nerves. This effect was compared to the effect on vascular smooth muscle. 2. Caudal artery preparations from Wistar rats were electrically stimulated (1 Hz, 0.5-ms duration) and noradrenaline release in the artery was detected using an high-pressure liquid chromatography-electrochemical detection technique. The pharmacological properties of the prejunctional effect of nicorandil were determined using the nonselective K(ATP) channel blocker glibenclamide, the pancreatic beta-cell and brain-type K(ATP) channel blocker tolbutamide, and the smooth muscle-type K(ATP) channel blocker PNU-37883A. 3. Nicorandil inhibited the electrical stimulation-evoked noradrenaline release in a concentration-dependent manner. This inhibitory effect was abolished by 1 micromol/L glibenclamide and 10 micromol/L tolbutamide, but was not affected by 10 micromol/L PNU-37883A or 0.3 micromol/L ODQ. Nicorandil did not affect the noradrenaline transporter uptake 1 in the adrenergic nerve terminals. 4. Nicorandil produced a relaxation response in a concentration-dependent manner in the caudal artery pre-contracted with 0.3 micromol/L noradrenaline. This relaxation response was significantly diminished in the presence of 1 micromol/L glibenclamide, 10 micromol/L PNU-37883A and 0.3 micromol/L ODQ but not by 10 micromol/L tolbutamide. 5. These findings suggest that nicorandil inhibits noradrenaline release via the K(ATP) channels of sympathetic nerves. These channels may be pharmacologically different from those of vascular smooth muscle.

Effect of ω-Conotoxin GVIA on Noradrenaline Release from Postganglionic Sympathetic Neurones in Rabbit Aorta

The aim of the present work was to examine the effect of the selective N-type calcium blocking agent omega-conotoxin GVIA on stimulation-evoked release of noradrenaline from sympathetic nerves in rabbit isolated aorta with regard to stimulation frequency, extracellular Ca2+ concentration, and transmitter uptake. Rings of rabbit isolated aorta were preloaded with (-)-3H-noradrenaline and the fractional 3H-overflow evoked by electrical-field stimulation was determined by liquid scintillation spectrometry. Omega-conotoxin GVIA (3 x 10(-10)-3 x 10(-8) M) did not alter the spontaneous 3H-outflow. Omega-conotoxin GVIA (3 x 10(-10)-3 x 10(-8) M) caused a slowly developing reduction of stimulation-evoked 3H-overflow at 1 and 30 Hz. The Emax for the omega-conotoxin-induced inhibition was less (70%) at 30 Hz than that (96%) seen at 1 Hz. Short-term incubation with omega-conotoxin GVIA caused a subsequent steady-state inhibition. The inhibitory action of omega-conotoxin GVIA (3 x 10(-10)-3 x 10(-9) M) was inversely related to the extracellular Ca2+ concentration (6.5 x 10(-4)-2.7 x 10(-3) M). Cocaine (3 x 10(-5) M) plus corticosterone (4 x 10(-5) M), neuronal and extraneuronal uptake inhibitors, respectively, did not alter the inhibitory effect of omega-conotoxin GVIA (3 x 10(-9) M) on 3H-overflow evoked by stimulation at a frequency of either 1 or 30 Hz. It is concluded that omega-conotoxin GVIA acts on prejunctional N-type calcium channels to inhibit stimulation-evoked noradrenaline release from sympathetic neurone terminals in rabbit aorta. At a high frequency, another subtype calcium channel may possibly be involved. The action of omega-conotoxin GVIA is independent of neuronal and extraneuronal uptake mechanisms for noradrenaline, but dependent on the amount of Ca2+ to be transported across the neurilemma from the extracellular space into the neurone.

Involvement of Voltage-Dependent Ca2+ Channel β3 Subunit in the Autonomic Control of Heart Rate Variability

Noradrenaline release from sympathetic nerve terminals is dependent on Ca²⁺ entry through neuronal voltage-gated N-type Ca²⁺ channels. The accessory β₃ subunits of Ca²⁺ channels (Ca_Vβ₃) are preferentially associated with the α_1B subunit to form N-type Ca²⁺ channels, and are therefore expected to play a functional role in the stimulation-evoked release of noradrenaline. In this study, we employed Ca_Vβ₃-null, Ca_Vβ₃-overexpressing (Ca_Vβ₃-Tg), and wild-type (WT) mice to investigate the possible roles of Ca_Vβ₃ in the sympathetic regulation of heart rate in vivo. Telemetry was used to monitor the ECG and both time and frequency domain analyses were carried out to evaluate heart rate variability. In the frequency domain analysis, power spectral density of the RR interval series was computed using the fast Fourier transform algorithm. The resting heart rate was increased in Ca_Vβ₃-Tg mice compared with both Ca_Vβ₃-null and WT mice. Mice overexpressing Ca_Vβ₃ displayed decreased heart rate variability, which was measured by the time domain analysis of the standard deviation of RR intervals. In the frequency domain analysis, Ca_Vβ₃-Tg mice showed decreased spectral powers compared with WT and Ca_Vβ₃-null mice. Pharmacological blockade of β-adrenergic receptors with metoprolol decreased the heart rate in all genotypes, but the extent of the decrease was most obvious in Ca_Vβ₃-Tg mice. On the other hand, the spectral powers were decreased in response to parasympathetic blockade (atropine) in WT and Ca_Vβ₃-Tg mice. These results indicate the functional roles of Ca_Vβ₃ in regulating sympathetic nerve signaling.

Involvement of Different Calcium Channels in the Depolarization‐Evoked Release of Noradrenaline from Sympathetic Neurones in Rabbit Carotid Artery*

The calcium channels coupled to noradrenaline release from sympathetic neurones in the rabbit isolated carotid artery were examined. Rings of carotid artery were preloaded with (-)-[(3)H]noradrenaline and the fractional (3)H overflow evoked by electrical-field stimulation was determined by liquid scintillation spectrometry. The N-type Ca(2+) channel blocking agent omega-conotoxin GVIA (3x10(-9)-6x10(-8) M) reduced the stimulation-evoked (3)H overflow. The maximal inhibition was seen with 3x10(-8) M. The maximal reduction was more marked at a low (2 Hz) stimulation frequency than at a high one (30 Hz). Mibefradil (10(-6) M) irreversibly reduced the (3)H overflow evoked by field stimulation (2 Hz). At 30 Hz, the reduction was more marked than at 2 Hz. Mibefradil (3x10(-6)-10(-5) M) enhanced the passive (3)H outflow. The reduction of the stimulation (30 Hz)-evoked (3)H overflow seen with omega-conotoxin GVIA (3x10(-8) M) was enhanced by mibefradil (10(-6) M) and unaffected by nimodipine (10(-5) M) and omega-agatoxin IVA (10(-8) M). We conclude that the stimulation-evoked release of noradrenaline from sympathetic neurones in rabbit carotid artery at a low frequency (2 Hz) is mediated mainly by the N-type calcium channels. At a high frequency (30 Hz), T-type Ca(2+) channels are also involved.

D 2 autoreceptor inhibition reveals oxygen-glucose deprivation-induced release of dopamine in guinea-pig cochlea

Dopamine (DA), released from the lateral olivocochlear (LOC) efferent terminals, the efferent arm of the short-loop feedback in the cochlea, is considered as a protective factor in the inner ear since it inhibits auditory nerve dendrite firing in ischemia- or noise-induced excitotoxicity leading to sensorineural hearing loss (SNHL). In the present study we investigated the effect of oxygen-glucose deprivation (OGD), an in vitro ischemia model, on guinea-pig cochlear [ 3H]DA release in a microvolume superfusion system. We found that OGD alone failed to induce a detectable elevation of [ 3H]DA level, but in the presence of specific D 2 receptor antagonists, sulpiride and L-741,626, it evoked a significant increase in the extracellular concentration of [ 3H]DA. D 2 negative feedback receptors are involved not exclusively in the regulation of synthesis and vesicular release of DA, but also in the activation of its reuptake. Thus, D 2 receptor antagonism interferes with the powerful reuptake of DA from the extracellular space. To explore the underlying mechanism of this DA-releasing effect we applied nomifensine and found that the effect of OGD on cochlear DA release in the presence of D 2 antagonists could be inhibited by this selective DA uptake inhibitor. This finding indicates that the OGD-evoked DA release was mainly mediated through the reverse operation of the DA transporter. The two structurally different D 2 antagonists also augmented the electrical field stimulation-evoked release of DA proving the presence of D 2 autoreceptors on dopaminergic LOC terminals. Our results confirm the presence and role of D 2 DA autoreceptors in the regulation of DA release from LOC efferents, and suggest a protective local mechanism during ischemia which involves the direct transporter-mediated release of DA. Increasing the release of the protective transmitter DA locally in the inner ear may form the basis of future new therapeutic strategies in patients suffering from SNHL.

Autoinhibition of transmitter release from PC12 cells and sympathetic neurons through a P2Y12 receptor‐mediated inhibition of voltage‐gated Ca2+ channels

Although feedback inhibition of noradrenaline release by coreleased nucleotides is a well known phenomenon, it remained unclear which P2 receptor subtypes and associated signalling cascades may be involved. In the rat pheochromocytoma cell line PC12, 2-methylthio-ADP reduced noradrenaline release triggered by K+ depolarization more potently than ADP and ATP, whereas UDP or UTP failed to do so. The inhibition by ADP was abolished by pertussis toxin and antagonized by reactive blue 2, 2-methylthio-AMP, and AR-C69931MX, but not by suramin. AR-C69931MX acted as a competitive antagonist with an apparent affinity of 2 nm, but did not alter noradrenaline release, when PC12 cells were continuously superfused. However, when the superfusion was halted during K+ depolarization, release was significantly reduced and this inhibition was attenuated by AR-C69931MX, thus revealing ongoing autoinhibition. Rises in cellular cyclic AMP did not alter depolarization-evoked release nor its reduction by ADP, even though the nucleotide did inhibit cyclic AMP accumulation. ADP and the direct Ca2+ channel blocker Cd2+ inhibited voltage-activated Ca2+ currents, but not ATP-induced currents, and both agents reduced K+-evoked, but not ATP-evoked, release. Hence, if voltage-gated Ca2+ channels do not contribute to stimulation-evoked release, ADP fails to exert its inhibitory action. In primary cultures of rat sympathetic neurons, ADP also reduced Ca2+ currents and K+-evoked noradrenaline release, and AR-C69931MX acted again as competitive antagonist with an apparent affinity of 3 nm. These results show that P2Y12 receptors mediate an autoinhibition of transmitter release from PC12 cells and sympathetic neurons through an inhibition of voltage-gated Ca2+ channels.

Presynaptic α 2-receptors regulate reverse Na +/Ca 2+-exchange and transmitter release in Na +-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [ 3 H ]noradrenaline ([ 3 H ]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3×10 −5 M; corticosterone, 5×10 −5 M). Substitution of most of the external Na + by Li + (113 mM; [Na +] 0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [ 3 H ]NA in a tetrodotoxin (TTX, 10 −7 M) sensitive manner. The reverse Na +/Ca 2+-exchange inhibitor KB-R7943 (3×10 −5 M) failed to inhibit the stimulation-evoked release of [ 3 H ]NA, but increased the resting outflow of neurotransmitter. The ‘N-type’ voltage-sensitive Ca 2+-channel (VSCC) blocker ω-conotoxin (ω-CgTx) GVIA (10 −8 M) significantly and irreversibly inhibited the release of [ 3 H ]NA on stimulation (∼60–70%). The ‘residual release’ of NA was abolished either by TTX or by reducing external Ca 2+ from 2.5 to 0.25 mM. The ‘residual release’ of NA was also blocked by the non-selective VSCC-blocker neomycin (3×10 −3 M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic α 2-receptor blocker yohimbine (3×10 −7 M). When the release of [ 3 H ]NA was blocked by ω-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na +-pump by removal of K + from the external medium increased both the resting and the axonal stimulation-evoked release of [ 3 H ]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after ω-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca 2+-removal (+1 mM EGTA). Similarly, external Li + (113 mM) or the reverse Na +/Ca 2+ exchange blocker KB-R7943 (3×10 −5 M) significantly inhibited the stimulation-induced transmitter release in ‘K +-free’ solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na +-pump was inhibited in the absence of functioning VSCCs, yohimbine (3×10 −7 M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10 −6 M), an agonist of presynaptic α 2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li +-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na +/Ca 2+-exchange and release neurotransmitter in Na +-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na +/Ca 2+-exchange may also be regulated by presynaptic α 2-receptors.

Distinct temperature-dependent dopamine-releasing effect of drugs of abuse in the olfactory bulb

It was recently shown in the olfactory bulb (OB) that the response to olfactory stimulation might be related to local reinforcement mechanisms involved in discrimination of different odors. Therefore, it seemed interesting to study the effects of several drugs of abuse on the release of dopamine (DA) in the OB. Nicotine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), and cocaine at 37 °C increased the release of [ 3 H ] DA from olfactory bulb slice preparations of the rats. While nicotine, amphetamine, and MDMA directly evoked DA release, cocaine, by inhibiting the reuptake processes, enhanced the electrical stimulation-evoked release. At low temperature (17 °C), a condition in which the transmitter uptake carriers of the plasma membrane in both the normal and reverse mode of operation are inhibited, the nicotine-evoked [ 3 H ] DA release was potentiated, whereas those evoked by amphetamine and MDMA were inhibited. At low temperature the field stimulation-evoked [ 3 H ] DA release was potentiated, but under this condition cocaine failed to increase the release. Our results show that low temperature (a) increases the concentration of extracellular DA released by Ca 2+-dependent vesicular exocytosis elicited by nicotine, (b) inhibits the extracellular Ca 2+-independent amphetamine- and MDMA-induced release of DA that occurs by the reverse operation of membrane carriers transporting DA from the cytoplasm of presynaptic terminals to the extraneuronal space, and (c) does not alter the inhibitory effect of cocaine on DA uptake that increases the concentration of extracellular DA released by field stimulation. The findings that the drugs of abuse tested all enhanced the release of DA in the olfactory bulb suggest that local reinforcing mechanisms may also exist in this brain area. In addition, we also show that lowering the temperature in in vitro experiments is an easy and straightforward method for separating vesicular and cytoplasmic release of transmitters, and is suitable for studying the mechanism of catecholamine release evoked by drugs of abuse. This technique may be applicable in other neurochemical studies that need inhibition of the uptake carriers without the blockade of the ligand-gated ion channels caused by reuptake inhibitor drugs.

The effect of treatment with BRX-220, a co-inducer of heat shock proteins, on sensory fibers of the rat following peripheral nerve injury

In this study, we examined the effect BRX-220, a co-inducer of heat shock proteins, in injury-induced peripheral neuropathy. Following sciatic nerve injury in adult rats and treatment with BRX-220, the following features of the sensory system were studied: (a) expression of calcitonin gene-related peptide (CGRP); (b) binding of isolectin B4 (IB4) in dorsal root ganglia (DRG) and spinal cord; (c) stimulation-evoked release of substance P (SP) in an in vitro spinal cord preparation and (d) nociceptive responses of partially denervated rats. BRX-220 partially reverses axotomy-induced changes in the sensory system. In vehicle-treated rats there is a decrease in IB4 binding and CGRP expression in injured neurones, while in BRX-220-treated rats these markers were better preserved. Thus, 7.0 ± 0.6% of injured DRG neurones bound IB4 in vehicle-treated rats compared to 14.4 ± 0.9% in BRX-220-treated animals. Similarly, 4.5 ± 0.5% of DRG neurones expressed CGRP in the vehicle-treated group, whereas 9.0 ± 0.3% were positive in the BRX-220-treated group. BRX-220 also partially restored SP release from spinal cord sections to electrical stimulation of primary sensory neurones. Behavioural tests carried out on partially denervated animals showed that BRX-220 treatment did not prevent the emergence of mechanical or thermal hyperalgesia. However, oral treatment for 4 weeks lead to reduced pain-related behaviour suggesting either slowly developing analgesic actions or enhancement of recovery processes. Thus, the morphological improvement seen in sensory neurone markers was accompanied by restored functional activity. Therefore, treatment with BRX-220 promotes restoration of morphological and functional properties in the sensory system following peripheral nerve injury.

L-DOPA increases noradrenaline turnover in central and peripheral nervous systems

The ability of L-3,4-dihydroxyphenylalanine (L-DOPA) to release noradrenaline (NA) from peripheral and CNS neurons was studied using isolated rat vas deferens and in vivo frontal cortex microdialysis. Application of L-DOPA (30 μM) to vas deferens increased basal NA efflux but not electrical field stimulation-evoked release of NA when the tissue was pretreated with an inhibitor of MAO-B (clorgyline 1 μM) or an inhibitor of MAO-A and MOA-B (AGN-1133 1 μM). No effect on NA efflux was seen when the tissue was treated with rasagiline (0.005 μM; selective inhibitor of MAO-B), but rasagiline, AGN-1133 and clorgyline increased basal efflux of dopamine (DA) following L-DOPA. In microdialysis experiments, systemic administration of L-DOPA/carbidopa combination (50/12 mg.kg −1) increased the efflux of 3,4-dihydroxyphenylglycol and 3-methoxy,4-hydroxyphenylglycol but reduced that of NA. Microdialysate levels of NA, however were increased following L-DOPA/carbidopa when desipramine (1 μM) was infused locally via the probe, or following systemic administration of yohimbine (2 mg.kg −1). The data are consistent with the hypothesis that administration of L-DOPA is followed by increased axoplasmatic levels of DA which release NA from storage sites into the free cytoplasmatic pool.