Transmitter Efflux Research Articles

The effects of optical, electrical and chemical stimulation on serotonin release from median raphe and hippocampus of mice

The present study has examined several characteristics of the release of [3H]5-HT from the median raphe nucleus (MRN) and hippocampus in terms of its dependence of nerve impulse. We used electrical stimulation and the sodium channel opener veratridine, which excite all of the neuronal processes in the stimulation field, and optogenetics to selectively stimulate those terminals which express channelrhodopsin-2 (ChR2) and compared 5-HT release evoked by electrical and chemical depolarization and by light. We injected an adeno-associated virus containing DNA construct encoding ChR2 into the MRN of mice and investigated [3H]5-HT release from MRN and hippocampal slices. Serotonergic nerve terminals was locally stimulated with 473 nm light (blue laser diode) and electrically by bipolar electrode and by veratridine and transmitter release was monitored by collecting the effluent in a fraction collector. Electrical filed stimulation and veratridine resulted in a significantly increase in the efflux of 5-HT, whereas optical stimulation of ChR2 expressing nerve terminals at various frequencies (10, 20, 50, 100 Hz) elicited only a negligible increase in 5-HT release either from the hippocampus or from the MRN itself. The electrically induced release of radioactive neurotransmitter was completely inhibited by perfusion with tetrodotoxin. We have also applied the 5-HT transporter inhibitor, fluoxetine and the GABAA blocker bicuculline to relieve released 5-HT from re-uptake and any endogenous inhibition. Nevertheless, the effect of optical stimulation remained closed to the detection limit under these condition. In conclusion, whereas our method is suitable to detect [3H]5-HT efflux in response to ongoing neuronal sodium channel activity its sensitivity is too low to detect transmitter efflux evoked by focal optogenetic stimulation. The most likely reason for the failure of detection of 5-HT efflux is that ChR2 is expressed only by a small subpopulation of nerve terminals.

Amphetamine makes caudate tissue more susceptible to oxygen and glucose deprivation

Amphetamine is being investigated to reduce morbidity following stroke. However, the medicinal use of amphetamine is complicated because this drug is addictive, cardiotoxic, and can be neurotoxic. Thus, further research into the safety of giving amphetamine to stroke patients is required. Here, we examine whether prior treatment with amphetamine has any effect on oxygen–glucose deprivation (OGD)-evoked transmitter efflux and mitochondrial function. To circumvent the well-documented cardiovascular effects of amphetamine we have used rat brain slices.Brain slices were exposed to 30μM of amphetamine for 10min 1h before being exposed to OGD. Using fast cyclic voltammetry in rat caudate, dopamine efflux induced by OGD was measured. The effect of amphetamine on mitochondrial function was examined using triphenyltetrazolium chloride (TTC) staining.Prior amphetamine exposure decreased the time to onset of OGD-evoked dopamine efflux (from 460 to 220s), suggesting that the caudate was more sensitive to OGD. This increased sensitivity to OGD was attenuated by pre-treatment with the dopamine transporter blocker GBR12909 (1μM). Pre-treatment with the dopamine D2 receptor antagonist metoclopramide (1μM) had no effect on the amphetamine-evoked sensitisation to OGD. Amphetamine decreased TTC staining in the caudate suggesting that amphetamine compromised the dopamine system by disrupting mitochondrial function.Amphetamine treatment may be harmful in stroke recovery by making the brain more vulnerable to ischaemia. These data also suggest that amphetamine abusers might be more susceptible to cerebral ischaemia.

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.

Effects of antiepileptic drugs on glutamate release from rat and human neocortical synaptosomes

Aim of this study was to learn whether the antiepileptic drugs (AEDs) carbamazepine, lamotrigine, phenytoin, gabapentin, pregabalin, levetiracetam, and valproate (all at 100 μM) presynaptically influence the neurotransmission of the excitatory transmitter glutamate. The effects of these frequently used AEDs were examined on ³H-glutamate release from superfused synaptosomes of both rat and human neocortex. Release was evoked by elevation of buffer [K(+)] from 3 to 15 mM or by the Na(+) channel activator veratridine (1, 3.2, and 10 μM). Buffer [K(+)] elevation induced ³H-glutamate exocytosis, which was Ca(²+)-, but not Na(+)-, dependent and which was accompanied only in human tissue by release through transporter reversal. In rat tissue, release was diminished by the Na(+) channel inhibitors carbamazepine, lamotrigine, and phenytoin, which therefore may also affect presynaptic Ca(²+) channels. Interestingly, levetiracetam increased ³H-glutamate release. In contrast, the tested AEDs did not affect K(+)-evoked ³H-glutamate release in human tissue, neither when the transporters were operative nor when exocytosis was isolated by transporter blockade. Veratridine-evoked ³H-glutamate release was a Na(+)-dependent transmitter efflux through reversed transporters in both species which in human synaptosomes was accompanied by exocytosis. The latter depended on external Ca(²+). Carbamazepine, lamotrigine, and phenytoin reduced this release from both rat and human tissue. There is an obvious species difference in the effects of carbamazepine, lamotrigine, and phenytoin on K(+)-evoked ³H-glutamate release while their inhibitory effects on veratridine-evoked release were similar. Thus, the depression of ³H-glutamate release by carbamazepine, lamotrigine, and phenytoin may be due to inhibited synaptosomal Na(+) or Ca(²+) influx.

Hypoinsulinemia regulates amphetamine-induced reverse transport of dopamine.

The behavioral effects of psychomotor stimulants such as amphetamine (AMPH) arise from their ability to elicit increases in extracellular dopamine (DA). These AMPH-induced increases are achieved by DA transporter (DAT)-mediated transmitter efflux. Recently, we have shown that AMPH self-administration is reduced in rats that have been depleted of insulin with the diabetogenic agent streptozotocin (STZ). In vitro studies suggest that hypoinsulinemia may regulate the actions of AMPH by inhibiting the insulin downstream effectors phosphotidylinositol 3-kinase (PI3K) and protein kinase B (PKB, or Akt), which we have previously shown are able to fine-tune DAT cell-surface expression. Here, we demonstrate that striatal Akt function, as well as DAT cell-surface expression, are significantly reduced by STZ. In addition, our data show that the release of DA, determined by high-speed chronoamperometry (HSCA) in the striatum, in response to AMPH, is severely impaired in these insulin-deficient rats. Importantly, selective inhibition of PI3K with LY294002 within the striatum results in a profound reduction in the subsequent potential for AMPH to evoke DA efflux. Consistent with our biochemical and in vivo electrochemical data, findings from functional magnetic resonance imaging experiments reveal that the ability of AMPH to elicit positive blood oxygen level–dependent signal changes in the striatum is significantly blunted in STZ-treated rats. Finally, local infusion of insulin into the striatum of STZ-treated animals significantly recovers the ability of AMPH to stimulate DA release as measured by high-speed chronoamperometry. The present studies establish that PI3K signaling regulates the neurochemical actions of AMPH-like psychomotor stimulants. These data suggest that insulin signaling pathways may represent a novel mechanism for regulating DA transmission, one which may be targeted for the treatment of AMPH abuse and potentially other dopaminergic disorders.

Ectopic serotonin accumulation and efflux in rat mesencephalic slices after prior tissue ‘radiolabelling’

Radiolabelling of brain tissue has long been used to facilitate detection of transmitter efflux, on the assumption that egress of tritiated monoamines reflects that of the endogenous transmitter. The present study tested the hypothesis that the application of exogenous serotonin (5-HT) to mesencephalic slices, in the manner used during a typical radiolabelling protocol, leads to efflux of 5-HT from physiologically inappropriate loci such as other non-serotonergic neurones. We used fast cyclic voltammetry (FCV) to determine the effect of tissue pre-incubation with 5-HT on electrically-stimulated 5-HT efflux and reuptake in rat mesencephalic slices. Seven subregions were studied, including the dorsal raphe nucleus (DRN), dorsomedial periaqueductal grey (PAGdm) and the oral part of the pontine reticular nucleus (PnO). In control slices (pre-incubated without 5-HT), stimulated 5-HT efflux was only detectable in DRN, PAGdm and occasionally in PnO. In slices incubated in 5-HT (100 nM) for 30 min, stimulated 5-HT efflux was detected in all seven subregions studied. In such slices, citalopram (75 nM) increased efflux and reuptake t 1/2 in DRN to 201±21 and 487±117% of pre-drug values ( P<0.05) but had no significant effect on either measure in PnO. The 5-HT 1 autoreceptor agonist, 5-carboxamidotryptamine (5-CT, 100 nM) decreased efflux in DRN by 54±6% ( P<0.05), but was without effect (10±14%) in PnO. The present results show that pre-incubation in 5-HT allows stimulated 5-HT efflux from regions of the mesencephalon other than DRN and PAGdm. This stimulated 5-HT efflux is apparently not influenced by 5-HT transporters or 5-HT 1 autoreceptors, suggesting that efflux is ectopic, an artefact of the pre-incubation process. In summary, incubation of rat mesencephalic tissue in 5-HT, in the manner of a typical radiolabelling protocol, results in stimulated 5-HT efflux from non-physiological sites. The results of such transmitter efflux studies should thus be interpreted with caution.

Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor.

The trace amine para-tyramine is structurally and functionally related to the amphetamines and the biogenic amine neurotransmitters. It is currently thought that the biological activities elicited by trace amines such as p-tyramine and the psychostimulant amphetamines are manifestations of their ability to inhibit the clearance of extracellular transmitter and/or stimulate the efflux of transmitter from intracellular stores. Here we report the discovery and pharmacological characterization of a rat G protein-coupled receptor that stimulates the production of cAMP when exposed to the trace amines p-tyramine, beta-phenethylamine, tryptamine, and octopamine. An extensive pharmacological survey revealed that psychostimulant and hallucinogenic amphetamines, numerous ergoline derivatives, adrenergic ligands, and 3-methylated metabolites of the catecholamine neurotransmitters are also good agonists at the rat trace amine receptor 1 (rTAR1). These results suggest that the trace amines and catecholamine metabolites may serve as the endogenous ligands of a novel intercellular signaling system found widely throughout the vertebrate brain and periphery. Furthermore, the discovery that amphetamines, including 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy"), are potent rTAR1 agonists suggests that the effects of these widely used drugs may be mediated in part by this receptor as well as their previously characterized targets, the neurotransmitter transporter proteins.

α-Latrotoxin, Acting via Two Ca2+-dependent Pathways, Triggers Exocytosis of Two Pools of Synaptic Vesicles

alpha-Latrotoxin stimulates three types of [(3)H]gamma-aminobutyric acid and [(14)C]glutamate release from synaptosomes. The Ca(2+)-independent component (i) is insensitive to SNAP-25 cleavage or depletion of vesicle contents by bafilomycin A1 and represents transmitter efflux mediated by alpha-latrotoxin pores. Two other components of release are Ca(2+)-dependent and vesicular but rely on distinct mechanisms. The fast receptor-mediated pathway (ii) involves intracellular Ca(2+) stores and acts upon sucrose-sensitive readily releasable vesicles; this mechanism is insensitive to inhibition of phosphatidylinositol 4-kinase (PI 4-kinase). The delayed pore-dependent exocytotic component (iii) is stimulated by Ca(2+) entering through alpha-latrotoxin pores; it requires PI 4-kinase and occurs mainly from depot vesicles. Lanthanum perturbs alpha-latrotoxin pores and blocks the two pore-mediated components (i, iii) but not the receptor-mediated release (ii). alpha-Latrotoxin mutant (LTX(N4C)) cannot form pores and stimulates only the Ca(2+)-dependent receptor-mediated amino acid exocytosis (ii) (detectable biochemically and electrophysiologically). These findings explain experimental data obtained by different laboratories and implicate the toxin receptors in the regulation of the readily releasable pool of synaptic vesicles. Our results also suggest that, similar to noradrenergic vesicles, amino acid-containing vesicles at some point in their cycle require PI 4-kinase.

Substrate-induced regulation of gamma-aminobutyric acid transporter trafficking requires tyrosine phosphorylation.

Neurotransmitter transporters regulate synaptic transmitter levels and are themselves functionally regulated by a number of different signal transduction cascades. A common theme in transporter regulation is redistribution of transporter protein between intracellular stores and the plasma membrane. The triggers and mechanisms underlying this regulation are important in the control of extracellular transmitter concentrations and hence synaptic signaling. Previously, we demonstrated that the gamma-aminobutyric acid transporter GAT1 is regulated by direct tyrosine phosphorylation, resulting in an up-regulation of transporter expression on the plasma membrane. In the present report, we show that two tyrosine residues on GAT1 contribute to the phosphorylation and transporter redistribution. Tyrosine phosphorylation is concomitant with a decrease in the rate of transporter internalization from the plasma membrane. A decrease in GAT internalization rates also occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT1 surface expression. In support of this hypothesis, incubation of GAT1-expressing cells with transporter ligands alters the amount of GAT1 tyrosine phosphorylation, and substrate-induced surface expression is unchanged in a GAT1 mutant lacking tyrosine phosphorylation sites. These data suggest a model in which substrates permit the phosphorylation of GAT1 on tyrosine residues and that the phosphorylated state of the transporter is refractory for internalization.

Regarding the unitary theory of agonist and antagonist action at presynaptic adrenoceptors.

1. The linkage between potentiation of field stimulation-induced noradrenaline release and blockade of the presynaptic inhibitory effect of exogenous noradrenaline by a presynaptic antagonist was examined in superfused rabbit aorta preparations. 2. Rauwolscine clearly potentiated the release of noradrenaline in response to 100 pulses at 2 Hz but reduced the capacity of noradrenaline to inhibit transmitter release to a questionable extent, and then only when comparisons were made with untreated, rather then to rauwolscine-treated, controls. 3. Aortic preparations exposed for 60 min to rauwolscine followed by superfusion with antagonist-free Krebs for 60 min retained the potentiation of stimulation-induced transmitter release but no antagonism of the noradrenaline-induced inhibition could be detected at either of two noradrenaline concentrations when comparisons were made with rauwolscine treated controls. 4. Comparisons of the inhibitory effect of exogenous noradrenaline (1.8 x 10-6 M) on transmitter efflux in the presence and absence of rauwolscine pretreatment revealed that the antagonist enhanced rather than antagonized the presynaptic inhibition by noradrenaline. 5 It is concluded that the unitary hypothesis that asserts that antagonist enhancement of transmitter release and its blockade of noradrenaline induced inhibition are manifestations of a unitary event are not supportable.

Involvement of N- and P/Q- but not L- or T-type voltage-gated calcium channels in ischaemia-induced striatal dopamine release in vitro

Calcium influx and transmitter efflux are central events in the neuropathological cascade that occurs during and following cerebral ischaemia. This study explored the role of voltage-gated calcium channels (VGCCs) in ischaemia-induced striatal dopamine (DA) release in vitro. Slices (350 μm thickness) of rat neostriatum were superfused (400 ml/h) with an artificial cerebrospinal fluid (aCSF) at 34°C and subjected to episodes of `ischaemia' by reduction of the glucose concentration from 4 to 2 mM and gassing with 95% N 2/5% CO 2. DA release was monitored with fast cyclic voltammetry at implanted carbon fibre microelectrodes. The time to onset, time to peak, rate and magnitude of DA release were measured. Non-selective blockade of VGCCs with a high concentration of Ni 2+ (2.5 mM), markedly delayed ( P<0.01) and slowed ( P<0.05) DA release but preferential blockade of T-type VGCCs with a lower concentration (200 μM) had no effect. DA release was also unaffected by selective antagonism of L-type VGCCs with nimodipine and nicardipine (10 μM each). Selective blockade of N-type VGCCs with ω-conotoxin GVIA (100 nM) delayed DA release ( P<0.05) but did not affect its rate or magnitude. Blockade of P- and possibly Q-type VGCCs with ω-agatoxin IVA (up to 200 nM) both delayed ( P<0.05) and slowed ( P<0.05) DA release. Preferential blockade of P- type VGCCs with neomycin (500 μM) also delayed ( P<0.05) and slowed ( P<0.05) DA release. These findings suggest that N-, P- and possibly Q- but not L- or T-type VGCCs mediate ischaemia-induced DA release. Although it is not possible to say, on the basis of these results, that the effects are directly upon the dopamine terminals, these calcium channels nevertheless constitute promising targets for therapeutic intervention.

A critical assessment of electrochemical procedures applied to the measurement of dopamine and its metabolites during drug-induced and species-typical behaviours

Monoamine neurotransmitters and their metabolites are electro-oxidizable at the surface of electrodes implanted in brain, and this has raised the expectation that in-vivo electrochemical procedures may be used routinely to monitor dynamic changes in dopamine (DA), noradrenaline and serotonin during species-typical and drug-induced behaviours. This expectation is slowly becoming a reality as various laboratories refine electrochemical procedures and design recording electrodes that are selective for specific neurotransmitters. The most important step is to conduct rigorous assessments of these procedures in-vivo to confirm that they are indeed valid and reliable bioprobes for a given neurochemical species. The present review describes the different electrochemical procedures and recording electrode designs currently used to measure monoamines in freely moving animals. Emphasis is placed on recording electrodes used to monitor DA in the extracellular compartment of the brain. In-vitro and in-vivo validation studies, demonstrating the selectivity and stability of these electrodes, are discussed in detail with respect to five criteria: (a) voltammograms recorded in-vivo are identical to those recorded in-vitro; (b) independent confirmation of the species detected and agreement of concentration estimates with other neurochemical methods; (c) sufficient electrode sensitivity to measure impulse-flow dependent transmitter efflux; (d) the dependency of the electrochemical signal on the presence of an intact neuronal system; and (e) predictable and reproducible effects of drugs or other manipulations on the electrochemical signal. The final section discusses recent applications of in-vivo electrochemical procedures for the monitoring of DA neurotransmission in terminal regions of the mesotelencephalic DA system during: (a) operant behaviour reinforced by brain-stimulation reward or drugs of abuse; (b) species-typical motivated behaviours, as exemplified by feeding and sexual behaviour, and (c) in response to environmental stressors.

Regulation of stimulus-dependent hippocampal acetylcholine release by okadaic acid-sensitive phosphoprotein phosphatases

Isolated nerve endings (synaptosomes) from rat hippocampas were used to characterize the influence by serine/threonine-specific phosphoprotein phosphatase (PP) inhibitors on acetylcholine release. Brief exposure to low concentrations of selective PP inhibitors (okadaic acid and calyculin A) caused a concentration-dependent attenuation of stimulus-dependent (calcium-evoked or potassium-evoked) [ 3H]acetylcholine ([ 3H]ACh) release, while having no effect on the rate of basal transmitter efflux. In view of the observed potencies for okadaic acid and calyculin A (pseudo-IC 50) values near 3 nM), these data indicate that Type 1 (PP1) or Type 2A (MA) enzymes play a permissive role in exocytotic [ 3H]ACh release. In contrast, the absence of any measurable effect by sodium ortho-vanadate argues against a similar influence by tyrosine-specific phosphoprotein phosphatases. While the neuronal substrate(s) responsible for PP regulation of [ 3H]ACh release are unknown, the underlying mechanism clearly differs from that through which muscarinic autoreceptors act since inhibition by okadaic acid and oxotremorine (an autoreceptor agonist) are additive and the former is not blocked by the muscarinic receptor antagonist atropine. Based upon these results, we conclude that dephosphorylation steps catalyzed by okadaic acid-sensitive PP represent an important regulatory mechanism for stimulus-dependent transmitter release in septo-hippocampal cholinergic neurons.

Blockade of striatal 5-hydroxytryptamine2 receptors reduces the increase in extracellular concentrations of dopamine produced by the amphetamine analogue 3,4-methylenedioxymethamphetamine.

5-Hydroxytryptamine2 (5-HT2) receptor antagonists have been shown to interfere with the stimulation of striatal dopamine synthesis and release produced by the amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA). To localize the receptors responsible for the attenuation of MDMA-induced release, 5-HT2 receptor antagonists were infused via the microdialysis probe directly into the brains of awake, freely moving rats before the systemic administration of MDMA. Intrastriatal infusions of the selective 5-HT2 antagonist MDL 100,907 produced a concentration-dependent inhibition of MDMA-induced dopamine release. Similar results were observed with intrastriatal infusions of the 5-HT2 antagonist amperozide. In contrast, infusion of MDL 100,907 into the mid-brain region near the dopaminergic cell bodies was without effect on the MDMA-induced elevation of extracellular dopamine in the ipsilateral striatum. Neither antagonist attenuated basal transmitter efflux nor the MDMA-stimulated release of [3H]dopamine from striatal slices in vitro indicating that the in vivo effect of the antagonists was not due to inhibition of the dopamine uptake carrier. Intrastriatal infusion of tetrodotoxin reduced both basal and MDMA-stimulated dopamine efflux and eliminated the effect of intrastriatal MDL 100,907. The results indicate that 5-HT2 receptors located in the striatum augment the release of dopamine produced by high doses of MDMA. Furthermore, these 5-HT2 receptors appear to be located on nondopaminergic elements of the striatum.

The 5-HT 2 receptor antagonist, MDL 28,133A, disrupts the serotonergic-dopaminergic interaction mediating the neurochemical effects of 3,4-methylenedioxymethamphetamine

The selective 5-HT 2 receptor antagonist MDL 28,133A dose dependently blocked the long-term deficits in rat brain 5-HT concentrations produced by the substituted amphetamine analogue 3,4-methylenedioxymethamphctamine (MDMA). This protective effect of MDL 28,133A could be abolished by coadministration of the dopamine precursor, L-dihydroxyphenylalanine (L-DOPA). Electrophysiological experiments demonstrated that the ability of MDL 28,133A to block the MDMA-induced slowing of A 9 dopaminergic neurons was also sensitive to L-DOPA administration. Both sets of experiments suggest an interaction of MDL 28,133A at the level of dopamine synthesis. Consistent with this explanation, MDL 28,133A antagonized the MDMA-induced stimulation of dopamine synthesis in vivo. MDMA-induced 5-HT release did not reduce the firing rate of dopaminergic neurons as assessed by dopamine depletion following synthesis inhibition with α-mcthyl-p-tyrosinc (α-MPT). This indicates that the effect of 5-HT 2 receptor antagonists on MDMA-induced dopamine synthesis is not due simply to the removal of an inhibitory serotonergic input followed by an increase in dopamine cell firing and autoreceptor activation. MDL 28,133A was also shown to be without effect on the sensitivity of terminal dopamine autoreceptors. The results are consistent with the hypothesis that 5-HT 2 receptors are permissive for the stimulation of dopamine synthesis necessary to support MDMA-induced transmitter efflux.

Adrenergic presynaptic antagonists and their mechanism of action in smooth muscle.

The effects of veratridine and of yohimbine on the efflux of norepinephrine from guinea pig ureters was examined to gain insight into presynaptic receptor function. Ureter segments were stimulated at 1 and 2 Hz with 100 pulses in the absence and presence of yohimbine, veratridine, or the combination. Veratridine (4.5 or 6 x 10(-7) M) increased transmitter release. The enhancements of release by the adrenergic antagonist yohimbine and by veratridine were not additive, suggesting a common site of action. The lack of additivity was not linked to a ceiling effect since tetraethylammonium, which increases release by block of potassium channels, had an additive effect with veratridine. Protection experiments, done with veratridine as the protecting agent against phenoxybenzamine blockade, supported the interpretation that adrenergic antagonists and veratridine act at a common locus to enhance transmitter efflux. Although veratridine enhanced transmitter efflux like yohimbine, it only slightly reduced the inhibitory capacity of norepinephrine, confirming the likelihood of discrete sites of agonist and antagonist action. Veratridine and alpha-receptor antagonists might combine presynaptically with sites on the sodium channels of sympathetic nerve terminals to alter channel gating, namely the shifting of sodium channel activation to more negative potentials, and in this way increase the liberation of norepinephrine.

Mechanism of Neurotransmitter Release Elicited by the Preferential ?1-Adrenergic Receptor Agonist Phenylephrine in Superfused Superior Cervical Ganglion Cells in Culture

Phenylephrine increased [3H]norepinephrine efflux and accumulation of cyclic AMP in cultured rat superior cervical ganglion cells superfused with Tyrode's solution. The purpose of this study was to determine the mechanism and relationship between these two events. Electrical stimulation (1-2 Hz), potassium chloride (50 mM), and the preferential alpha 1-adrenergic receptor agonist phenylephrine (1-100 microM) increased fractional tritium efflux, whereas methoxamine, cirazoline, and amidephrine were relatively ineffective. Phenylephrine, but not methoxamine and cirazoline, also increased cyclic AMP accumulation. Phenylephrine-induced tritium efflux was not altered by alpha- and beta-adrenergic receptor antagonists or by removal of extracellular calcium. Phenylephrine-induced cyclic AMP accumulation was blocked by the beta-adrenergic receptor antagonists propranolol and atenolol. Forskolin (10 microM) and the nonhydrolyzable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (100 microM) had minimal effect on tritium efflux. However, phenylephrine-evoked increase in tritium efflux was dose dependently attenuated by the neuronal uptake blocker cocaine, and phenylephrine dose-dependently inhibited the incorporation of [3H]norepinephrine into neuronal stores. We conclude that the increase in tritium efflux induced by phenylephrine is independent of cyclic AMP accumulation and appears to be mediated by uptake of phenylephrine via the neuronal carrier-mediated amine transport process, which in turn promotes efflux of the adrenergic transmitter from its storage sites.

β‐adrenoceptor‐mediated facilitation of [3H]‐noradrenaline release from the intramural nerves of bovine mesenteric lymphatic vessels

1. Isolated segments of bovine mesenteric lymphatic vessels were loaded with [3H]-noradrenaline and its efflux in response to field stimulation at 2 Hz (0.3 ms pulses, 1 min train) examined. 2. Isoprenaline (10(-6) M) increased evoked fractional 3H efflux (3H released as a percentage of total tissue 3H at the onset of stimulation) from its control value of 0.67 +/- 0.09 to 1.2 +/- 0.18% (s.e.mean; n = 5). 3. Propranolol (10(-6) M) alone had no effect on evoked fractional 3H efflux but blocked the increase in transmitter overflow induced by isoprenaline (10(-6) M). 4. In the presence of the alpha-adrenoceptor antagonist phentolamine (10(-6) M) the facilitatory effect of isoprenaline (10(-6) M) on transmitter efflux was enhanced. In 5 experiments isoprenaline increased evoked fractional 3H efflux from its control value of 1.07 +/- 0.17 to 2.5 +/- 0.37% when phentolamine was present. 5. Adrenaline (10(-8) M) increased evoked fractional 3H efflux from its control value of 0.76 +/- 0.09 to 1.13 +/- 0.17% (n = 7). 6. These findings are consistent with the presence of presynaptic beta-adrenoceptors in this preparation. There is no evidence that these receptors play any role in feedback regulation of transmitter release.

Cellular and molecular correlates of aging in the nervous system

Three characteristic features of aging in the nervous system are reviewed: deficits in the regulation of nerve-cell calcium levels, increased leakage of synaptic transmitters and changes in neuronal arborization. In hippocampal cells and motor nerve terminals, the rate of calcium clearance from the immediate vicinity of the membrane decreases with age. There is further evidence of decreased rates of transmembrane Ca 2+ flux in synaptosomal preparations from aged animals. Stimulation-evoked transmitter release, which is calcium dependent, appearsto increase at some neuromuscular junctions during aging; in contrast, high-K +-induced release in brain tissue appears to decline. A much more consitent age-related increase in “basal” transmitter efflux, under nonstimulated conditions, has been observed in both the peripheral and the central nervous system. This may be related to increased nerve-terminal arborization, which occurs in actively used muscles during aging. Likewise, dendritic branching becomes more extensive with moderate age; at advanced age, though, branching decreases. Furthermore, motor nerve terminal branching also decreases with age in muscles subject to disuse. This variability among morphologic features during aging illustrates the need to standardize ages and preparations when comparing these types of data.

Clonidine and presynaptic adrenoceptor theory

The effects of clonidine, a presumed selective presynaptic alpha 2-adrenoceptor agonist or partial agonist, were examined in guinea-pig atria. Split left atrial preparations were stimulated transmurally at 2 Hz with 100 pulses of 0.5 ms duration and the efflux of 3H-transmitter determined. Clonidine inhibited efflux at 3 X 10(-8)M to 3 X 10(-7)M by about 30%. Yohimbine, at a concentration (10(-6)M) which caused a 3 fold increase in the release of 3H-transmitter during field stimulation, did not alter the ability of clonidine to inhibit transmitter efflux. At 10(-6)M clonidine alone had no significant effect on the stimulation-induced efflux of 3H-transmitter, but in the presence of yohimbine (10(-6)M), inhibited efflux by over 50%. The inhibitory effect of noradrenaline (10(-6)M) on 3H-transmitter efflux was antagonized by clonidine at 10(-6)M but not at 10(-8)M, although neither concentration of clonidine alone inhibited transmitter efflux. The present findings indicate that the effects of clonidine on the efflux of noradrenaline from sympathetic nerves cannot be accommodated within the currently held view that the compound is an agonist or partial agonist on presynaptic alpha 2-adrenoceptors. It appears that clonidine has multiple sites of action few of which are antagonized by a concentration of the prototypical presynaptic antagonist yohimbine, which enhances efflux 3 fold.