Depolarization-evoked Release Research Articles

Synaptotagmin-9 in mouse retina.

Synaptotagmin-9 (Syt9) is a Ca2+ sensor mediating fast synaptic release expressed in various parts of the brain. The presence and role of Syt9 in retina is unknown. We found evidence for Syt9 expression throughout the retina and created mice to conditionally eliminate Syt9 in a cre-dependent manner. We crossed Syt9fl/fl mice with Rho-iCre, HRGP-Cre, and CMV-Cre mice to generate mice in which Syt9 was eliminated from rods (rodSyt9CKO), cones (coneSyt9CKO), or whole animals (CMVSyt9). CMVSyt9 mice showed an increase in scotopic electroretinogram (ERG) b-waves evoked by bright flashes with no change in a-waves. Cone-driven photopic ERG b-waves were not significantly different in CMVSyt9 knockout mice and selective elimination of Syt9 from cones had no effect on ERGs. However, selective elimination from rods decreased scotopic and photopic b-waves as well as oscillatory potentials. These changes occurred only with bright flashes where cone responses contribute. Synaptic release was measured in individual rods by recording anion currents activated by glutamate binding to presynaptic glutamate transporters. Loss of Syt9 from rods had no effect on spontaneous or depolarization-evoked release. Our data show that Syt9 acts at multiple sites in the retina and suggest that it may play a role in regulating transmission of cone signals by rods.

Synaptotagmins 1 and 7 in vesicle release from rods of mouse retina.

Synaptotagmins are the primary Ca2+ sensors for synaptic exocytosis. Previous work suggested synaptotagmin-1 (Syt1) mediates evoked vesicle release from cone photoreceptor cells in the vertebrate retina whereas release from rods may involve another sensor in addition to Syt1. We found immunohistochemical evidence for syntaptotagmin-7 (Syt7) in mouse rod terminals and so performed electroretinograms (ERG) and single-cell recordings using mice in which Syt1 and/or Syt7 were conditionally removed from rods and/or cones. Synaptic release was measured in mouse rods by recording presynaptic anion currents activated during glutamate re-uptake and from exocytotic membrane capacitance changes. Deleting Syt1 from rods reduced glutamate release evoked by short depolarizing steps but not long steps whereas deleting Syt7 from rods reduced release evoked by long but not short steps. Deleting both sensors completely abolished depolarization-evoked release from rods. Effects of various intracellular Ca2+ buffers showed that Syt1-mediated release from rods involves vesicles close to ribbon-associated Ca2+ channels whereas Syt7-mediated release evoked by longer steps involves more distant release sites. Spontaneous release from rods was unaffected by eliminating Syt7. While whole animal knockout of Syt7 slightly reduced ERG b-waves and oscillatory potentials, selective elimination of Syt7 from rods had no effect on ERGs. Furthermore, eliminating Syt1 from rods and cones abolished ERG b-waves and additional elimination of Syt7 had no further effect. These results show that while Syt7 contributes to slow non-ribbon release from rods, Syt1 is the principal sensor shaping rod and cone inputs to bipolar cells in response to light flashes.

The Depolarization-Evoked, Ca2+-Dependent Release of Exosomes From Mouse Cortical Nerve Endings: New Insights Into Synaptic Transmission.

Whether exosomes can be actively released from presynaptic nerve terminals is a matter of debate. To address the point, mouse cortical synaptosomes were incubated under basal and depolarizing (25 mM KCl-enriched medium) conditions, and extracellular vesicles were isolated from the synaptosomal supernatants to be characterized by dynamic light scattering, transmission electron microscopy, Western blot, and flow cytometry analyses. The structural and biochemical analysis unveiled that supernatants contain vesicles that have the size and the shape of exosomes, which were immunopositive for the exosomal markers TSG101, flotillin-1, CD63, and CD9. The marker content increased upon the exposure of nerve terminals to the high-KCl stimulus, consistent with an active release of the exosomes from the depolarized synaptosomes. High KCl-induced depolarization elicits the Ca2+-dependent exocytosis of glutamate. Interestingly, the depolarization-evoked release of exosomes from cortical synaptosomes also occurred in a Ca2+-dependent fashion, since the TSG101, CD63, and CD9 contents in the exosomal fraction isolated from supernatants of depolarized synaptosomes were significantly reduced when omitting external Ca2+ ions. Differently, (±)-baclofen (10 µM), which significantly reduced the glutamate exocytosis, did not affect the amount of exosomal markers, suggesting that the GABAB-mediated mechanism does not control the exosome release. Our findings suggest that the exposure of synaptosomes to a depolarizing stimulus elicits a presynaptic release of exosomes that occurs in a Ca2+-dependent fashion. The insensitivity to the presynaptic GABAB receptors, however, leaves open the question on whether the release of exosomes could be a druggable target for new therapeutic intervention for the cure of synaptopathies.

A modulator of the low-voltage-activated T-type calcium channel that reverses HIV glycoprotein 120-, paclitaxel-, and spinal nerve ligation-induced peripheral neuropathies.

The voltage-gated calcium channels CaV3.1-3.3 constitute the T-type subfamily, whose dysfunctions are associated with epilepsy, psychiatric disorders, and chronic pain. The unique properties of low-voltage-activation, faster inactivation, and slower deactivation of these channels support their role in modulation of cellular excitability and low-threshold firing. Thus, selective T-type calcium channel antagonists are highly sought after. Here, we explored Ugi-azide multicomponent reaction products to identify compounds targeting T-type calcium channel. Of the 46 compounds tested, an analog of benzimidazolonepiperidine-5bk (1-{1-[(R)-{1-[(1S)-1-phenylethyl]-1H-1,2,3,4-tetrazol-5-yl}(thiophen-3-yl)methyl]piperidin-4-yl}-2,3-dihydro-1H-1,3-benzodiazol-2-one) modulated depolarization-induced calcium influx in rat sensory neurons. Modulation of T-type calcium channels by 5bk was further confirmed in whole-cell patch clamp assays in dorsal root ganglion (DRG) neurons, where pharmacological isolation of T-type currents led to a time- and concentration-dependent regulation with a low micromolar IC50. Lack of an acute effect of 5bk argues against a direct action on T-type channels. Genetic knockdown revealed CaV3.2 to be the isoform preferentially modulated by 5bk. High voltage-gated calcium, as well as tetrodotoxin-sensitive and -resistant sodium, channels were unaffected by 5bk. 5bk inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, 5bk did not bind human mu, delta, or kappa opioid receptors. 5bk reversed mechanical allodynia in rat models of HIV-associated neuropathy, chemotherapy-induced peripheral neuropathy, and spinal nerve ligation-induced neuropathy, without effects on locomotion or anxiety. Thus, 5bk represents a novel T-type modulator that could be used to develop nonaddictive pain therapeutics.

P.351 Omega-3 fatty acids for major depressive disorder with high inflammation: A personalized approach

Epilepsy is a chronic neurologic disorder which often induces numerous adverse long-term neurologic effects, such as behavioral and cognitive deficits, increased predisposition to additional seizures, and cell injury or death. Cognitive dysfunction, depression, anxiety and sleep disorders are some of the highly prevalent and most disabling complications of epilepsy. The mechanisms that lead to the generation of epileptic comorbidities are poorly understood. Treatment for epileptic complications still remains a challenge because of the poor adherence and drug interactions associated with multi drug prescriptions and also for the fear of worsening seizures by the individual medications for complications. Melatonin, an endogenous hormone secreted by pineal gland has a prominent role in epilepsy. Agomelatine is a novel antidepressant which acts as melatonin MT1 and MT2 receptor agonist and serotonin 5Ht2C receptor antagonist. The combined action at MT1/2 and 5HT2C receptors, reduction in the depolarization-evoked release of glutamate, strong neuroprotective action and possible antioxidant properties of agomelatine could make it a potential agent in the treatment of epilepsy. The effect of agomelatine on hippocampal neuronal cell survival and neurogenesis, neuroprotective effect in hippocampus and frontal cortex and the antioxidant potential may contribute to the protective action of agomelatine against epilepsy induced memory decline. Agomelatine is proven to be an antidepressant and it has relieved anxiety symptoms and improved the quality of sleep in patients with depressive disorder. The action of agomelatine as a melatonin agonist and the consequent circadian resynchronizing property as well as its action as 5-HT2C receptor antagonist, could possibly suggest an antidepressant and anxiolytic action of agomelatine in epilepsy induced depressive behavior and anxiety. Since one of the many causes of sleep disruption in epilepsy is circadian rhythm disturbances and sleep promoting and circadian effects of melatonin is attributed to the MT1and MT2 subtypes of human melatonin receptors, agomelatine may also have a promising effect on epilepsy induced sleep disruptions. Thus with all these potential pharmacological actions, agomelatine could be recommended as a potential drug to treat epilepsy and its complications.

Phosphorylated CRMP2 Regulates Spinal Nociceptive Neurotransmission.

The collapsin response mediator protein 2 (CRMP2) has emerged as a central node in assembling nociceptive signaling complexes involving voltage-gated ion channels. Concerted actions of post-translational modifications, phosphorylation and SUMOylation, of CRMP2 contribute to regulation of pathological pain states. In the present study, we demonstrate a novel role for CRMP2 in spinal nociceptive transmission. We found that, of six possible post-translational modifications, three phosphorylation sites on CRMP2 were critical for regulating calcium influx in dorsal root ganglion sensory neurons. Of these, only CRMP2 phosphorylated at serine 522 by cyclin-dependent kinase 5 (Cdk5) contributed to spinal neurotransmission in a bidirectional manner. Accordingly, expression of a non-phosphorylatable CRMP2 (S522A) decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), whereas expression of a constitutively phosphorylated CRMP2 (S522D) increased the frequency of sEPSCs. The presynaptic nature of CRMP2's actions was further confirmed by pharmacological antagonism of Cdk5-mediated CRMP2 phosphorylation with S-N-benzy-2-acetamido-3-methoxypropionamide ((S)-lacosamide; (S)-LCM) which (i) decreased sEPSC frequency, (ii) increased paired-pulse ratio, and (iii) reduced the presynaptic distribution of CaV2.2 and NaV1.7, two voltage-gated ion channels implicated in nociceptive signaling. (S)-LCM also inhibited depolarization-evoked release of the pro-nociceptive neurotransmitter calcitonin gene-related peptide (CGRP) in the spinal cord. Increased CRMP2 phosphorylation in rats with spared nerve injury (SNI) was decreased by intrathecal administration of (S)-LCM resulting in a loss of presynaptic localization of CaV2.2 and NaV1.7. Together, these findings indicate that CRMP2 regulates presynaptic excitatory neurotransmission in spinal cord and may play an important role in regulating pathological pain. Novel targeting strategies to inhibit CRMP2 phosphorylation by Cdk5 may have great potential for the treatment of chronic pain.

Betulinic acid, derived from the desert lavender Hyptis emoryi, attenuates paclitaxel-, HIV-, and nerve injury-associated peripheral sensory neuropathy via block of N- and T-type calcium channels.

The Federal Pain Research Strategy recommended development of nonopioid analgesics as a top priority in its strategic plan to address the significant public health crisis and individual burden of chronic pain faced by >100 million Americans. Motivated by this challenge, a natural product extracts library was screened and identified a plant extract that targets activity of voltage-gated calcium channels. This profile is of interest as a potential treatment for neuropathic pain. The active extract derived from the desert lavender plant native to southwestern United States, when subjected to bioassay-guided fractionation, afforded 3 compounds identified as pentacyclic triterpenoids, betulinic acid (BA), oleanolic acid, and ursolic acid. Betulinic acid inhibited depolarization-evoked calcium influx in dorsal root ganglion (DRG) neurons predominantly through targeting low-voltage-gated (Cav3 or T-type) and CaV2.2 (N-type) calcium channels. Voltage-clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after BA exposure. Betulinic acid inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, BA did not engage human mu, delta, or kappa opioid receptors. Intrathecal administration of BA reversed mechanical allodynia in rat models of chemotherapy-induced peripheral neuropathy and HIV-associated peripheral sensory neuropathy as well as a mouse model of partial sciatic nerve ligation without effects on locomotion. The broad-spectrum biological and medicinal properties reported, including anti-HIV and anticancer activities of BA and its derivatives, position this plant-derived small molecule natural product as a potential nonopioid therapy for management of chronic pain.

Effects and mechanism of action of isatin, a MAO inhibitor, on in vivo striatal dopamine release

Isatin is an endogenous indole that inhibits monoamine oxidase (MAO), being more selective for MAO-B than MAO-A isoform. By inhibiting MAO, isatin increases dopamine levels in the brain and, in animal models of Parkinson’s disease (PD) isatin is able to prevent dopamine depletion. Contradictorily, some studies indicate that isatin did not increase striatal dopamine levels, although it was able to improve the motor signs in PD model. Given these conflicting data, our aim was to study the effects and neurochemical mechanisms of action of isatin on in vivo dopamine release from rat dorsal striatum using brain microdialysis technique in conscious and freely moving animals. Our results showed that intrastriatal administration of 1, 5 or 10 mM isatin, for 1 h, significantly increased dopamine levels to 355 ± 104%, 700 ± 72%, and 1241 ± 146%, when compared with basal values, respectively. The highest concentration of isatin (10 mM) was used to investigate whether the dopamine overflow is due to an exocytotic release or due to a possible action on dopamine transporter (DAT). The removal of Ca++ from medium, administration of TTX (10 μM), or pretreatment with reserpine (10 mg/kg) significantly decreased by 90%, 83%, and 78%, respectively, the effect of isatin on dopamine levels. The blockade of DAT with nomifensine (50 μM) did not alter the effect of isatin; and isatin significantly increased the depolarization-evoked release of dopamine. These results suggest that isatin-induced dopamine release depends on vesicular dopamine content, and takes place due to a previous entry of Ca++ and terminal depolarization.

Effect of Two Disease-Causing Mutations on the Energetics and Kinetics of SNARE Assembly

Synaptic exocytosis relies on assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins into a four-helix bundle to drive membrane fusion. SNARE assembly occurs by zippering in a stepwise manner, beginning with slow N-terminal association and ending at the C-terminal with a rapid power stroke that lowers the barrier to membrane fusion. Imbalances in this process cause severe neuropathy. In particular, dominant missense mutations I67N and I67T in the neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25b) cause cortical hyperexcitability, myasthenia, ataxia, and intellectual disability. Recent electrophysiological measurements indicate that I67T mutation impairs spontaneous and evoked release in murine cortical slices. Similarly, transfection of bovine chromaffin cells with SNAP-25b I67N impairs depolarization-evoked release. However, the molecular mechanism underlying these phenotypes is unclear. Here, we used single-molecule optical tweezers to monitor in real-time the step-wise assembly of SNARE complex harboring SNAP-25b I67T/N. These measurements yielded the assembly energetics and kinetics of single SNARE complex molecules. Comparison between wild type and I67T or I67N mutant revealed that both disease-causing mutants significantly reduced the folding energy of C-terminal SNARE assembly by ∼10 kT, but did not affect N-terminal assembly. Such disruption of the C-terminal power stroke is expected to impair membrane fusion and is consistent with the corresponding phenotypes. In sum, our finding constitutes compelling evidence that SNAP-25b mutations cause neuropathy by greatly reducing the SNARE C-terminal folding energy and thus inhibiting neurotransmitter release.

Neuropeptide Y as a presynaptic modulator of norepinephrine release from the sympathetic nerve fibers in the pig pineal gland.

Norepinephrine (NE) released from the sympathetic nerve endings is the main neurotransmitter controlling melatonin synthesis in the mammalian pineal gland. Although neuropeptide Y (NPY) co-exists with NE in the pineal sympathetic nerve fibers it also occurs in a population of non-adrenergic nerve fibers located in this gland. The role of NPY in pineal physiology is still enigmatic. The present study characterizes the effect of NPY on the depolarization-evoked 3H-NE release from the pig pineal explants. The explants of the pig pineal gland were loaded with 3H-NE in the presence of pargyline and superfused with Tyrode medium. They were exposed twice to the modified Tyrode medium containing 60 mM of K+ to evoke the 3H-NE release via depolarization. NPY, specific agonists of Y1- and Y2- receptors and pharmacologically active ligands of α2-adrenoceptors were added to the medium before and during the second depolarization. The radioactivity was measured in medium fractions collected every 2 minutes during the superfusion. NPY (0.1-10 μM) significantly decreased the depolarization-induced 3H-NE release. Similar effect was observed after the treatment with Y2-agonist: NPY13-36, but not with Y1-agonist: [Leu31,Pro34]-NPY. The tritium overflow was lower in the explants exposed to the 5 μM NPY and 1 μM rauwolscine than to rauwolscine only. The effects of 5 μM NPY and 0.05 μM UK 14,304 on the depolarization-evoked 3H-NE release were additive. The results show that NPY is involved in the regulation of NE release from the sympathetic terminals in the pig pineal gland, inhibiting this process via Y2-receptors.

P.1.h.025 Pharmacodynamic analyses of the anti-hyperalgesic and anti-allodynic effects of agomelatine in neuropathic rats

Epilepsy is a chronic neurologic disorder which often induces numerous adverse long-term neurologic effects, such as behavioral and cognitive deficits, increased predisposition to additional seizures, and cell injury or death. Cognitive dysfunction, depression, anxiety and sleep disorders are some of the highly prevalent and most disabling complications of epilepsy. The mechanisms that lead to the generation of epileptic comorbidities are poorly understood. Treatment for epileptic complications still remains a challenge because of the poor adherence and drug interactions associated with multi drug prescriptions and also for the fear of worsening seizures by the individual medications for complications. Melatonin, an endogenous hormone secreted by pineal gland has a prominent role in epilepsy. Agomelatine is a novel antidepressant which acts as melatonin MT1 and MT2 receptor agonist and serotonin 5Ht2C receptor antagonist. The combined action at MT1/2 and 5HT2C receptors, reduction in the depolarization-evoked release of glutamate, strong neuroprotective action and possible antioxidant properties of agomelatine could make it a potential agent in the treatment of epilepsy. The effect of agomelatine on hippocampal neuronal cell survival and neurogenesis, neuroprotective effect in hippocampus and frontal cortex and the antioxidant potential may contribute to the protective action of agomelatine against epilepsy induced memory decline. Agomelatine is proven to be an antidepressant and it has relieved anxiety symptoms and improved the quality of sleep in patients with depressive disorder. The action of agomelatine as a melatonin agonist and the consequent circadian resynchronizing property as well as its action as 5-HT2C receptor antagonist, could possibly suggest an antidepressant and anxiolytic action of agomelatine in epilepsy induced depressive behavior and anxiety. Since one of the many causes of sleep disruption in epilepsy is circadian rhythm disturbances and sleep promoting and circadian effects of melatonin is attributed to the MT1and MT2 subtypes of human melatonin receptors, agomelatine may also have a promising effect on epilepsy induced sleep disruptions. Thus with all these potential pharmacological actions, agomelatine could be recommended as a potential drug to treat epilepsy and its complications.

SY07-4 * DYSFUNCTION OF GABAERGIC SYSTEM IN THE INSULAR CORTEX CONTRIBUTES TO IMPAIRMENTS OF DECISION-MAKING IN METHAMPHETAMINE-DEPENDENT RATS

Introduction. Patients suffering from neuropsychiatric disorders such as substance-related and addictive disorders have impairments in decision-making, which may be associated with their behavioral abnormalities. However, the neuronal mechanisms underlying such impairments are largely unknown. To address this issue, we developed a gambling test for rodents using an 8-arm radial arm maze, and tested the effect of chronic methamphetamine (METH) treatment on decision-making in rats. Method. The c-fos staining was applied to determine the brain areas activated by the gambling test. Depolarization-evoked GABA release was measured by in vivo dialysis. Results. METH-dependent rats choose a high-risk/high-reward option more frequently, and assign higher subjective value to high returns, than control rats, suggesting that decision-making is impaired in the METH-dependent animals. Immunohistochemical analysis of c-fos following the gambling test revealed aberrant activation of the insular cortex (INS) and nucleus accumbens in METH-dependent animals. Pharmacological studies together with in vivo microdialysis showed that GABAergic neurotransmission in INS played a crucial role in decision-making. Conclusion. Our findings suggest that INS is a critical region involved in decision-making, and that insular GABAergic dysfunction in METH-dependent rats results in risk-taking behaviors associated with poor decision-making.

Therapeutic potential of agomelatine in epilepsy and epileptic complications

Epilepsy is a chronic neurologic disorder which often induces numerous adverse long-term neurologic effects, such as behavioral and cognitive deficits, increased predisposition to additional seizures, and cell injury or death. Cognitive dysfunction, depression, anxiety and sleep disorders are some of the highly prevalent and most disabling complications of epilepsy. The mechanisms that lead to the generation of epileptic comorbidities are poorly understood. Treatment for epileptic complications still remains a challenge because of the poor adherence and drug interactions associated with multi drug prescriptions and also for the fear of worsening seizures by the individual medications for complications. Melatonin, an endogenous hormone secreted by pineal gland has a prominent role in epilepsy. Agomelatine is a novel antidepressant which acts as melatonin MT1 and MT2 receptor agonist and serotonin 5Ht2C receptor antagonist. The combined action at MT1/2 and 5HT2C receptors, reduction in the depolarization-evoked release of glutamate, strong neuroprotective action and possible antioxidant properties of agomelatine could make it a potential agent in the treatment of epilepsy. The effect of agomelatine on hippocampal neuronal cell survival and neurogenesis, neuroprotective effect in hippocampus and frontal cortex and the antioxidant potential may contribute to the protective action of agomelatine against epilepsy induced memory decline. Agomelatine is proven to be an antidepressant and it has relieved anxiety symptoms and improved the quality of sleep in patients with depressive disorder. The action of agomelatine as a melatonin agonist and the consequent circadian resynchronizing property as well as its action as 5-HT2C receptor antagonist, could possibly suggest an antidepressant and anxiolytic action of agomelatine in epilepsy induced depressive behavior and anxiety. Since one of the many causes of sleep disruption in epilepsy is circadian rhythm disturbances and sleep promoting and circadian effects of melatonin is attributed to the MT1and MT2 subtypes of human melatonin receptors, agomelatine may also have a promising effect on epilepsy induced sleep disruptions. Thus with all these potential pharmacological actions, agomelatine could be recommended as a potential drug to treat epilepsy and its complications.

Co-application of the GABAB receptor agonist, baclofen, and the mGlu receptor agonist, L-CCG-I, facilitates [3H]GABA release from rat cortical nerve endings

Interaction between different transmitter receptor systems is an emerging feature of neurotransmission at central synapses. G protein-coupled receptors' ability to form dimers or larger hetero-oligomers probably serves to facilitate the integration of diverse signals within the cell. We found that, in nerve terminals isolated from the cerebral cortices of rats, co-application of the GABAB agonist, baclofen, and of the non-selective mGlu agonist, L-CCG-I, potentiates the basal and depolarization-evoked release of [(3)H]GABA via a mechanism that involves mobilization of intracellular Ca(2+) ions. The effect of L-CCG-I+baclofen was abolished by the phospholipase C inhibitor U73122, reduced by Xestospongin C (an IP3 receptor blocker), and blocked by 2-APB, an IP3 receptor antagonist. Pretreatment of the synaptosomes with the lipid-soluble Ca(2+) chelator BAPTA-AM also inhibited the effects of L-CCG-I+baclofen. Subtype-selective non-competitive group I mGlu receptor antagonists, MPEP and CPCCOEt, had no effect on the release enhancement produced by baclofen+L-CCG-I. The enhancement was reversed by the GABAB receptor antagonist, CGP54626, and by the group I/group II mGlu receptor antagonist (R,S)-MCPG. The GABA release-enhancing effects of L-CCG-I+baclofen in our model might reflect the presence on cortical nerve endings of GABAB/group I mGlu receptor heteromers with pharmacological properties distinct from those of the component receptors. Activation of these heteromeric receptors might modify the function of the GABAB receptor in such a way that it facilitates GABAergic transmission, an effect that might be useful under conditions of excessive glutamatergic activity.

Intra-membrane Signaling Between the Voltage-Gated Ca2+-Channel and Cysteine Residues of Syntaxin 1A Coordinates Synchronous Release

The interaction of syntaxin 1A (Sx1A) with voltage-gated calcium channels (VGCC) is required for depolarization-evoked release. However, it is unclear how the signal is transferred from the channel to the exocytotic machinery and whether assembly of Sx1A and the calcium channel is conformationally linked to triggering synchronous release. Here we demonstrate that depolarization-evoked catecholamine release was decreased in chromaffin cells infected with semliki forest viral vectors encoding Sx1A mutants, Sx1AC271V, or Sx1AC272V, or by direct oxidation of these Sx1A transmembrane (TM) cysteine residues. Mutating or oxidizing these highly conserved Sx1A Cys271 and Cys272 equally disrupted the Sx1A interaction with the channel. The results highlight the functional link between the VGCC and the exocytotic machinery, and attribute the redox sensitivity of the release process to the Sx1A TM C271 and C272. This unique intra-membrane signal-transduction pathway enables fast signaling, and triggers synchronous release by conformational-coupling of the channel with Sx1A.

Astrocyte origin of activity‐dependent release of ATP and glutamate in hippocampal slices: real‐time measurement utilizing microelectrode biosensors

It is well known that astrocytic and neuronal transmitter release processes are important for signalling, and that activity-dependent release of adenosine nucleotides and transmitters occurs after stimulation. Neurons and astrocytes can account for the source of ATP efflux. In this issue of the BJP, Heinrich et al. characterized K(+) depolarization-evoked release of ATP, adenosine and glutamate in hippocampal slices, utilizing microelectrode biosensors for simultaneous real-time recordings of multiple transmitter effluxes. They demonstrated efflux of ATP, adenosine and glutamate from hippocampus slices, in response to K(+) -depolarization, with distinct kinetics and mechanisms, suggesting a coordinated pattern of transmitter release. Surprisingly, it turned out that a considerable amount of the transmitter efflux measured under these conditions had a glial origin. For a long time, it was believed that the glial cell did not play a major role in neurotransmission, but the latter results somewhat change this view. The release of ATP and glutamate from glial cells under these conditions involved P2X7 receptors, and a source of adenosine accumulation independent of the metabolism of extracellular ATP was identified. This study also highlighted a novel use of multi-enzymatic microelectrode biosensors, which enabled a better characterization of transmitter release processes with higher temporal and spatial resolution than obtained previously. This technique was originally developed and used for the detection of purine release. In the present study, it was modified to identify the interplay between different transmitters, measured simultaneously in hippocampal slices.

Clozapine decreases exploratory activity and increases anxiety-like behaviour in the Wistar–Kyoto rat but not the spontaneously hypertensive rat model of attention-deficit/hyperactivity disorder

The spontaneously hypertensive rat (SHR) is the most widely used animal model of ADHD. SHR has been found to have increased glutamate-stimulated noradrenaline release from varicosities in several brain areas. Besides its effects on dopamine D4 receptors, clozapine, an atypical antipsychotic with antagonistic effects on α1-adrenoceptors, may reduce activation of α1-adrenoceptors in SHR and thereby attenuate their hyperactivity. The aims of the study were to determine the effect of clozapine (post-natal day (P) 21–P35, 10mg/kg/day) on SHR and Wistar-Kyoto (WKY), SHR's normotensive control, and a standard laboratory strain, Sprague Dawley (SD). Rat behaviour was assessed in the open field (P32), novel object (P33) and elevated plus maze (P34) tests that measured locomotor and anxiety-related behaviour. An in vitro superfusion technique was used to measure [3H]noradrenaline release in prefrontal cortex (PFC) and hippocampal slices (P35 or P36). Clozapine decreased exploratory activity in WKY, consistent with antagonism of dopamine D4 and α1-adrenoceptors reducing the behavioural response to novelty. Clozapine also increased anxiety-related behaviour of WKY. However, clozapine did not affect SHR, suggesting that genetic predisposition may play a role in determining clozapine's behavioural effects. WKY have been shown to have higher levels of dopamine D4 receptor expression in the PFC than SHR, which may be a reason for their elevated response to clozapine. SHR released more [3H]noradrenaline from PFC and hippocampal slices in response to glutamate- and elevated potassium-stimulation, compared to WKY and SD rats. However clozapine treatment did not affect glutamate-, GABA- or depolarization-evoked release of [3H]noradrenaline.

Involvement of the cGMP pathway in the osthole‐facilitated glutamate release in rat hippocampal nerve endings

Osthole, an active constituent isolated from Cnidium monnieri (L.) Cusson, has previously been shown to have the capacity to increase depolarization-evoked glutamate release in rat hippocampal nerve terminals. As cGMP-dependent signaling cascade has been found to modulate glutamate release at the presynaptic level, the aim of this study was to further examine the role of cGMP signaling pathway in the regulation of osthole on glutamate release in hippocampal synaptosomes. Results showed that osthole dose-dependently increased intrasynaptosomal cGMP levels. The elevation of cGMP levels by osthole was prevented by the phosphodiesterase 5 inhibitor sildenafil but was insensitive to the guanylyl cyclase inhibitor ODQ. In addition, osthole-induced facilitation of 4-aminopyridine (4-AP)-evoked glutamate release was completely prevented by the cGMP-dependent protein kinase (PKG) inhibitors, KT5823, and Rp-8-Br-PET-cGMPS. Direct activation of PKG with 8-Br-cGMP or 8-pCPT-cGMP also occluded the osthole-mediated facilitation of 4-AP-evoked glutamate release. Furthermore, sildenafil exhibited a dose-dependent facilitation of 4-AP-evoked release of glutamate and occluded the effect of osthole on the 4-AP-evoked glutamate release. Collectively, our findings suggest that osthole-mediated facilitation of glutamate release involves the activation of cGMP/PKG-dependent pathway.

Histamine H 3 receptors modulate depolarization-evoked [ 3H]-noradrenaline release from rat olfactory bulb slices

We have studied the effect of histamine H 3 receptor (H 3R) activation on the depolarization-evoked release of labeled neurotransmitters from slices of the rat olfactory bulb (rOB). The presence of pre-synaptic H 3Rs was evidenced by the specific binding of the H 3R ligand N-α-[methyl- 3H]histamine to membranes from rOB synaptosomes (maximum binding, B max, 106 ± 19 fmol/mg protein; dissociation constant, K d, 0.68 ± 0.11 nM) which was inhibited by selective H 3R ligands (immepip, (R)(−)-α-methylhistamine (RAMH) and clobenpropit) with affinities similar to those previously reported for H 3Rs expressed in other rat brain areas. Perfusion of rOB slices with the selective H 3R agonist RAMH (0.1 and 1 μM) had no effect on the release of [ 3H]-γ-aminobutyric acid ([ 3H]-GABA), [ 3H]- d-aspartate, [ 3H]-dopamine or [ 3H]-5-hydroxytryptamine ([ 3H]-5-HT) evoked by depolarization with high K + (20 or 40 mM). [ 3H]-Noradrenaline release induced by 20 mM K + was reduced in a modest but significant manner by RAMH (94.9 ± 1.7% and 83.1 ± 2.1% of control release at 0.1 and 1 μM, respectively). The effect of 1 μM RAMH was blocked by the selective H 3R antagonist/inverse agonist clobenpropit (5 μM). When tested alone clobenpropit and a second H 3R antagonist/inverse agonist, ciproxifan (both at 1 μM) significantly increased K +-evoked [ 3H]-noradrenaline release to 119.4 ± 4.2% and 120.0 ± 3.7% of K + alone, respectively. Ciproxifan (1 μM) had no effect on the depolarization-evoked release of the other labeled neurotransmitters. These data indicate that H 3Rs with constitutive activity modulate noradrenaline release in rOB, presumably through a pre-synaptic action. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’.

Chronic stress enhances calcium mobilization and glutamate exocytosis in cerebrocortical synaptosomes from mice

Objectives: Our previous study showed that acute restraint stress enhances depolarization-induced increases in intrasynaptosomal free calcium (Ca2+) concentration ([Ca2+]i) and Ca2+-dependent glutamate release in mouse cerebrocortical nerve terminals (synaptosomes). In the present study, we investigated the effects of chronic stress on [Ca2+]i and glutamate release in cerebrocortical synaptosomes from mice.Methods: Male ddY strain mice were randomly assigned to one of two experimental groups: control group and chronic stressed group. Mice in the chronic stressed group were subjected to immobilization stress for 2 hours daily for a period of 21 days. [Ca2+]i and glutamate release in cerebrocortical synaptosomes isolated from the mice were determined by fura-2 fluorescence assay and enzyme-linked fluorometric assay, respectively.Results: Chronic stress caused a significant increase in resting [Ca2+]i and significantly enhanced the ability of the depolarizing agents K+ and 4-aminopyridine (4-AP) to increase [Ca2+]i. It also brought about a significant increase in spontaneous (unstimulated) glutamate release and significantly enhanced K+- and 4-AP-evoked Ca2+-dependent glutamate release. Synaptosomes were more sensitive to the depolarizing agents at lower concentrations following chronic stress than after acute stress. The pretreatment of synaptosomes with a combination of omega-agatoxin IVA (a P-type Ca2+ channel blocker) and omega-conotoxin GVIA (an N-type Ca2+ channel blocker) completely suppressed the enhancements of [Ca2+]i and Ca2+-dependent glutamate release in chronic stressed mice.Discussion: These results indicate that chronic stress enhances depolarization-evoked glutamate release by increasing [Ca2+]i via stimulation of Ca2+ entry through P- and N-type Ca2+ channels, and that chronic stress increases the sensitivity to depolarizing agents.