Depolarization-induced Release Research Articles

A comparative study of the dopamine-acetylcholine interaction in telencephalic structures of the rat and of a reptile, the lizard Gekko gecko

The present study describes an investigation in which the dopamine-acetylcholine interaction in the caudate-putamen of the rat was compared with that in other telencephalic regions of the rat and in telencepalic regions of the Gekko gecko. For the rat these regions included the nucleus accumbens and the entorhinal and parietal cortices, for the gekko the striatum, the dorsal ventricular ridge and the cortex. All investigated brain regions in the rat and the gekko received dopaminergic projections from the ventral mesencephalon. The cholinergic fibers in the rat caudate-putamen and nucleus accumbens are mainly from intrinsic origin, whereas these fibers in the cortex of the rat and in the striatum and the cortex of the gekko predominantly originate from extrinsic sources. The dopamine-acetylcholine interaction was studied at the level of dopamine receptor-mediated inhibition of the depolarization-induced release of radiolabeled acetylcholine in vitro. It appeared that in the caudate-putamen and nucleus accumbers but not in the entorhinal and parietal cortices of the rat stimulation of D 2 receptors inhibits the release of acetylcholine. Although we could demonstrate the presence of D 2 dopamine receptor binding in all studied telencephalic structures of the gekko, D 2 receptor agonists were unable to inhibit the release of radiolabeled acetylcholine in these regions.

Depolarization-induced release of endogenous dopa from rat striatal slices

Depolarization-induced release of endogenous dopa from rat striatal slices

Depolarization-induced release of ATP from cholinergic synaptosomes is not blocked by botulinum toxin type A

We report here the effects of Botulinum Toxin type A on the release of ATP and Acetylcholine from Torpedo electric organ synaptosomes. Our results show that Botulinum Toxin type A inhibits specifically the K(+)-induced release of Acetylcholine from synaptosomes without affecting the release of ATP. Membrane potential and calcium uptake into cholinergic nerve terminals are not modified after Botulinum Toxin poisoning. It is suggested that either most of the ATP released during the depolarization of the cholinergic synaptosomes does not originate from cholinergic synaptic vesicles or that there are two populations of synaptic vesicles, Acetylcholine-enriched synaptic vesicles and ATP-enriched synaptic vesicles. However, the possibility that the ACh and ATP released could come from different intrasynaptosomal compartments cannot be excluded.

Neurochemical characterization of cysteine sulfinic acid, an excitatory amino acid, in hippocampus.

In this communication, I have summarized our studies on the possible roles of cysteine sulfinic acid (CSA) in the central nervous system (CNS), from these observations, CSA was suggested to be a neurotransmitter. We reported the presence of CSA in the CNS and subsequently characterized Na+-dependent high affinity uptake and depolarization-induced release of CSA. Depolarization-induced release of [14C]CSA from the preloaded hippocampal slices was specifically attenuated by benzodiazepines and GABA agonists. Synaptic membranes have a Na+-dependent specific binding site for cysteic acid, an analogue of CSA, which may be a possible binding site for CSA. This binding site seemed to be distinct from that for glutamate. To assess CSA as a physiologically active candidate which is distinct from glutamate, two neurochemical experiments were performed: one experiment determined the enhancement by excitatory amino acids of depolarization-induced release of [3H]GABA from the preloaded slices, and the other one monitored the cyclic AMP formation by excitatory amino acids in hippocampal slices. In both studies, differences in the responses to the various antagonists indicate that CSA receptors are distinct from glutamate receptors. Furthermore, we proposed that excitatory amino acid receptors which are subsequently linked to adenylate cyclase are functionally related to the Cl- channel.

Neurochemical Characterization of Cysteine Sulfinic Acid, an Excitatory Amino Acid, in Hippocampus

In this communication, I have summarized our studies on the possible roles of cysteine sulfinic acid (CSA) in the central nervous system (CNS), from these observations, CSA was suggested to be a neurotransmitter. We reported the presence of CSA in the CNS and subsequently characterized Na+-dependent high affinity uptake and depolarization-induced release of CSA. Depolarization-induced release of [14C]CSA from the preloaded hippocampal slices was specifically attenuated by benzodiazepines and GABA agonists. Synaptic membranes have a Na+-independent specific binding site for cysteic acid, an analogue of CSA, which may be a possible binding site for CSA. This binding site seemed to be distinct from that for glutamate. To assess CSA as a physiologically active candidate which is distinct from glutamate, two neurochemical experiments were performed: one experiment determined the enhancement by excitatory amino acids of depolarization-induced release of [3H]GABA from the preloaded slices, and the other one monitored the cyclic AMP formation by excitatory amino acids in hippocampal slices. In both studies, differences in the responses to the various antagonists indicate that CSA receptors are distinct from glutamate receptors. Furthermore, we proposed that excitatory amino acid receptors which are subsequently linked to adenylate cyclase are functionally related to the Cl- channel.

Storage and release of acetylcholine in rat cortical synaptosomes: Effects of d,l-2-(4-phenylpiperidino)cyclohexanol (AH5183)

A post-stimulation synthesis of acetylcholine (ACh), its incorporation into a ‘stable-bound’ (vesicular) compartment and subsequent release, were compared in K +-stimulated synaptosomes, in the absence and presence of 10 μM AH5183. The drug depressed by 16% the net intrasynaptosomal formation of ACh from 1 μM [ 3H]choline (Ch) in the medium, by competitively inhibiting ( K i⋍20μM ) the high-affinity Ch transport, but it had no direct effect on the intraterminal synthesis of ACh per se. The drug reduce of newly synthesized [ 3H]ACh into synaptic vesicles by 55% and subsequent K +-depolarization-induced release of [ 3H]ACh by 83%, although it had no effect on Ca 2+ influx into synaptosomes. These results are consistent with the hypothesis that AH5183 blocks cholinergic neurotransmission presynaptically by interfering with recharging of synaptic vesicles with ACh. Since the reduction of ACh release in the presence of AH5183 had no direct effect on ACh synthesis, these results also suggest that the transmitter release is not prerequisite for enhancement of Ch uptake and ACh synthesis in stimulated nerve terminals.

Insulin is released from rat brain neuronal cells in culture.

Depolarization of neuronal cells in primary culture from the rat brain by potassium ions in the presence of calcium or by veratridine caused a greater than three-fold stimulation of release of immunoreactive insulin. HPLC of the released insulin immunoreactivity from the neuronal cultures comigrated with the two rat insulins. The depolarization-induced release of insulin was inhibited by cycloheximide and was specific for neuronal cultures since potassium ions failed to cause the release in comparably prepared astrocytic glial cells from the rat brain. Prelabelling of neuronal cultures with [3H]leucine followed by depolarization resulted in the release of radioactivity that immunoprecipitated with insulin antibody. The release of [3H]insulin was biphasic. These observations suggest that neuronal cells from the brain have the capacity to synthesize insulin that could be released under depolarization conditions.

Dynorphin B-Containing Perivascular Axons and Sensory Neurotransmitter Mechanisms in Brain Blood Vessels

This is the first report demonstrating the existence of opiate-containing nerve fibers surrounding brain blood vessels. Dynorphin B, a tridecapeptide and potent opiate analgesic, was visualized by immunohistochemistry in guinea pig cerebral arteries comprising the circle of Willis and was measured by radioimmunoassay in canine middle cerebral arteries. This peptide, reportedly present in dorsal root ganglion cells, was observed by others to decrease the depolarization-induced release of substance P from primary sensory axons and, by so doing, to retard the development of neurogenic inflammation in target tissues. Consistent with an indirect action of dynorphin B, this peptide did not relax precontracted canine middle cerebral or basilar artery segments when added in vitro, nor did it modulate receptor-mediated relaxation on the addition of substance P. The presence of opiate-containing axons in or near trigeminovascular nerve fibers suggests novel mechanisms related to the modulation of pain possibly emanating from cerebral vessels.

On preverbal requests

Release of both D-[3H]aspartate and endogenous amino acids uas measured in primary cultures of cerebellargranule cells.Tuo hour-pretreatment with the glycosphingolipids, GM1 or GT1b, attenuatedthestimulation of excitatory amino acid release induced by depolarizing concentrations of K− (50 mM). Gangliosides inhibited the phencyclidine (PCP)-sensitive component of depolarizationinduced release, i.e. the amplification of release that follous activation of NMDA receptorsby the endogenous glutamate.

In vivo release of corticosterone in discrete brain areas of rats. A push-pull perfusion study

The spontaneous and depolarization-induced release of corticosterone was studied in the medial hypothalamus (MH) and the central amygdala (AC) in vivo, using a push-pull perfusion system. Significant variations in the spontaneous release of corticosterone could be observed over time (3–8 h). Corticosterone in the brain superfusates did not seem to stem from adrenocortical sites since a continuous release of the glucocorticoid could also be followed in bilateral adrenalectomized rats. Superfusion of MH and AC with depolarizing stimuli led to an enhanced release of corticosterone. Of greater general significance is the first demonstration that the release of a steroid hormone can be measured in discrete brain areas under in vivo conditions.

Release of [ 3H]Norepinephrine: Alteration by early developmental exposure to diazepam

The effect of prenatal exposure to diazepam (over gestational days 13–20) on the release of tritiated norepinephrine ([ 3H]NE) from selected brain regions was analyzed to determine mechanisms whereby such exposure could disrupt functioning in specific NE neurons, as previously observed. Pregnant rats were administered diazepam (DZ) once daily at doses of 1.0, 2.5 or 10.0 mg/kg and the offspring studied as adults at 70–90 days of age and during development at 14, 21, 35 and 56 days of age. Release of [ 3H]NE was measured during in vitro incubation using 25 mM potassium as the depolarizing stimulus. As noted previously, prenatal exposure to DZ induced an effect only on NE neurons innervating the hypothalamus, sparing the NE innervation to the hippocampus and cerebellum. Prenatal exposure to DZ had no effect on the depolarized release of [ 3H]NE in the hypothalamus until after 35 days of age, a developmental pattern previously observed with respect to endogenous NE levels. In adult rat offspring, however, the depolarization-induced release of [ 3H]NE from the hypothalamus decreased 28%, 32% and 64% (relative to uninjected control values) in animals prenatally exposed to DZ at 1.0, 2.5 or 10 mg/kg/day respectively. Concurrent exposure of the pregnant dam to benzodiazepine antagonists (Ro 15-1788 or ethyl-β-carboline-3-carboxylate) prevented the effects of DZ (2.5 mg/kg/day) on [ 3H]NE release, demonstrating again the importance of the benzodiazepine binding site to the effects induced by the early DZ exposure. The initial accumulation of the [ 3H]NE was not altered by the prenatal exposure. The presence of an alpha-adrenergic antagonist, phentolamine, in the incubation medium reversed the decreased release of [ 3H]NE from the hypothalamus that was induced by prenatal exposure to DZ, suggesting that under basal conditions the hypothalamic NE neurons of the exposed animals may have been under greater modulation from the alpha-receptor. These data suggest that the NE innervation to the hypothalamus may have a unique relationship with the benzodiazepine binding site, compared with NE innervation to other regions, such that early exposure to benzodiazepines can alter the course of maturation of these neurons.

Correlation of rates of calcium entry and release of endogenous norepinephrine in rat brain region synaptosomes.

Voltage-dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were less than 15% of 0-1 s rates. Both resting (5 mM KCl) and depolarization-induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage-dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1- and 30-s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization-induced NE release was dependent on the presence of external calcium. These results show that calcium-dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.

Developmental features of rat cerebellar neural cells cultured in a chemically defined medium.

We studied some aspects of the differentiation of rat cerebellar neural cells obtained from 8-day postnatal animals and cultured in a serum-free, chemically defined medium (CDM). The ability of the cells to take up radioactive transmitter amino acids was analyzed autoradiographically. The L-glutamate analogue 3H-D-aspartate was taken up by astroglial cells, but not by granule neurons, even in late cultures (20 days in vitro). This is in agreement with the lack of depolarization-induced release of 3H-D-aspartate previously observed in this type of culture. In contrast, 3H-(GABA) was scarcely accumulated by glial-fibrillary-acidic-protein (GFAP)-positive astrocytes, but taken up by glutamate-decarboxylase-positive inhibitory interneurons and was released in a Ca2+-dependent way upon depolarization: 3H-GABA evoked release progressively increased with time in culture. Interestingly, the expression of the vesicle-associated protein synapsin I was much reduced in granule cells cultured in CDM as compared to those maintained in the presence of serum. These data would indicate that in CDM the differentiation of granule neurons is not complete, while that of GABAergic neurons is not greatly affected. Whether the diminished differentiation of granule cells must be attributed only to serum deprivation or also to other differences in the composition of the culture medium remains to be established. 3H-GABA was avidly taken up also by a population of cells which were not recognized by antibodies raised against GFAP, glutamate decarboxylase, and microtubule-associated protein 2. These cells exhibited a stellate morphology, were stained by the monoclonal antibody A2B5, and have been characterized elsewhere [Levi et al, 1986] as bipotential precursors of oligodendrocytes and of a subpopulation of astrocytes bearing a stellate shape and capable of high-affinity 3H-GABA uptake.

Multiple opiate receptors may be involved in suppressing γ-aminobutyrate release in substantia nigra

Slices of rat substantia nigra were preloaded with tritiated γ-aminobutyrate (GABA) or dopamine (DA) and perfused with Krebs solution containing 5 μM aminooxyacetic acid or 10 μM nialamide to inhibit the catabolism of GABA and DA respectively. Repeated brief exposures to high potassium medium (+ 30 mM K + for 1 min) evoked a consistent pattern of calcium-dependent 3H efflux against which the effects of opiates (10–400 μM) were assessed. Opiate agonists inhibited K +-induced 3H-GABA efflux in the following decreasing order of potency : bremazocine > D-Ala 2-Met 5 -enkephalinamide (ENK) > SKF 10047 ⪢ morphine, consistent with the participation of κ,δ,σ and to a lesser extent μ opiate receptors respectively. Naloxone (1 μM) partially antagonised the response to morphine and ENK, while ICI 154129 attenuated ENK only. Save for a GABA-releasing action of SKF 10047 at high doses, none of the compounds altered basal outflow of 3H-GABA. Naloxone, in the dose range 10–400 μM, also significantly inhibited depolarisation-induced release of 3H-GABA. In parallel experiments none of the compounds tested were found to influence 3H-DA release in concentrations up to 40 μM, but thereafter suppressed K +-induced 3H-DA outflow indiscriminately. The results are discussed with reference to the possible mechanism(s) via which injected and endogenous opiates may affect motor performance by attenuating GABA transmission in the nigra.

Dopamine decreases cell excitability in rat striatal neurons by pre- and postsynaptic mechanisms

The mechanism by which dopamine (DA) decreases the amplitude of the EPSP-IPSP sequences evoked by cortical stimulation was investigated by means of electrophysiological and biochemical methods. Intracellular recordings indicate that DA decreases the amplitude of the excitatory and inhibitory events by reducing the increase in membrane conductance measured at the peaks of the EPSP-IPSP. The non-synaptic input resistance was not modified. In addition the catecholamine (+50/+200nA balanced current) was shown to decrease the action of glutamate (−30−80nA balanced current) and GABA (+40/+100nA balanced current) when iontophoretically applied. These observations suggest that DA interferes with the excitatory (glutamatergic) and inhibitory (GABAergic) transmission at the postsynaptic site in striatal neurons. However, the depression of cellular excitability elicited by DA could not be ascribed only to its interaction with synaptic transmission at the postsynaptic level. In fact the catecholamine also inhibited spike frequency driven by depolarizing pulses and decreased the depolarization-induced release of glutamate at the presynaptic site, as shown by biochemical experiments with striatal synaptosomal preparations. A neuromodulatory role of DA in the depression of the excitability of striatal neurons by presynaptic and postsynaptic mechanisms is suggested.

Effects of 4-(2-Hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183) on rat cortical synaptosome choline uptake, acetylcholine storage and release

The cholinergic vesicular uptake blocker, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183), had several effects on presynaptic cholinergic function that depended on the duration of treatment and dose. The synthesis, storage and release of newly synthesized [3H]ACh were monitored because the vesicular uptake of this pool of transmitter may be preferentially affected by the drug. Initially, high concentrations of AH5183 (over 10 microM) increased the spontaneous release but decreased the K+ depolarization-induced release of newly synthesized transmitter. [3H]Choline efflux was not altered by the drug. High affinity choline uptake was slightly (10-20%) inhibited by AH5183 in an apparently competitive but time-dependent manner. In contrast to its initial effects on [3H]ACh release, AH5183 (50nM-100 microM) very potently inhibited both the spontaneous and K+-induced release of [3H]ACh but not of [3H]choline after a 60 min preincubation. [3H]ACh levels in cytoplasmic (S3) and crude membrane (P3) fractions were not affected by a 2-min incubation with 10 microM AH5183. After a 60-min preincubation with this drug dose, however, the P3 and S3 levels of newly synthesized transmitter were decreased and increased, respectively. Subsequent fractionation of synaptosomes by sucrose-density gradient centrifugation revealed that these reductions in P3 [3H]ACh-levels were referable to reductions in two subfractions D and H that have been reported to contain low density vesicles and denser vesicles associated with plasma membranes, respectively.

Calcium channel agonist and antagonist effects of the stereoisomers of the dihydropyridine 202–791

The effects of the pure stereoisomers of the novel dihydropyridine 202–791 on voltage sensitive calcium channels in nerve and cardiac muscle were examined. The (−)-isomer blocked depolarization-induced uptake of 45Ca 2+ into NG108-15 neuroblastoma x glioma cells, blocked the depolarization-induced release of [ 3H]-norepinephrine from PC12 cells and reduced the Vmax of the slow response action potential recorded from guinea pig papillary muscle. In contrast, the (+)-isomer enhanced these same processes. In papillary muscle, greater enhancement of the slow responses was observed at lower stimulation frequencies. Thus, the (−) and (+) stereoisomers of 202–791 can be shown to be calcium channel antagonist and agonist respectively.

Effects of anti-glutamate-binding protein antibodies on synaptic membrane ion flux, glutamate transport and release, and L-glutamate binding activities.

Antibodies (Abs) raised against the L-glutamate-binding protein (GBP) purified from bovine brain were used to define the possible physiologic activity of GBP in synaptic membranes. Three processes were examined for their sensitivity to the Abs: the excitatory amino acid stimulation of thiocyanate (SCN-) flux, the transport of L-glutamic acid across the synaptic membrane, and the depolarization-induced release of L-glutamate. Only the amino acid-induced changes in ion flux were inhibited by the anti-GBP Abs. The change in membrane potential produced by exposure of synaptic membranes to excitatory amino acids was measured as the increase in the uptake of the lipophilic anion SCN-. The L-glutamate-induced SCN- influx was 40 times more sensitive to inhibition by the anti-GBP Abs than the stimulation of ion flux by kainate, and 60 times more sensitive than that produced by quisqualate. The anti-GBP Abs did not inhibit the activation of ion flux produced by N-methyl-D-aspartate. The inhibition of glutamate-stimulated ion fluxes by the Abs was complete, whereas the inhibition of L-glutamate binding to either the rat or bovine brain GBP was not. The results obtained indicated that although the majority of the anti-GBP Abs were not directed against the glutamate recognition site of the GBP and of presumed synaptic membrane receptors, they were effective in blocking the activation of receptor-associated ion channels. Thus, the GBP may be considered a component of some excitatory amino acid receptor complexes.

Autoregulation of histamine release in brain by presynaptic H 3-receptors

Regulation of histamine release was studied mainly on brain slices prelabeled with l-[ 3-H]histidine and depolarized by increased extracellular K + concentration or veratridine in a non-superfused system. The released 3H-labeled amines, isolated by ion-exchange chromatography from a large excess of 3H-labeled precursor consisted by more than 95% of unchanged [ 3H]histamine. Exogenous histamine reduced the release of neosynthesized [ 3H]histamine via stimulation of previously characterized H 3-receptors whereas it did not modify the 3H-labeled amine release from slices prelabeled with preformed [ 3H]histamine. The maximal inhibitory effect of exogenous histamine progressively diminished as the strength of the depolarizing stimulus or the external Ca 2+ concentration were elevated. On the contrary H 3-receptor antagonists like impromidine or burimamide enhanced the depolarization-induced release of [ 3H]histamine, an effect which was particularly marked when slices were loaded with histamine by preincubation with [ 3H]histidine in high concentration. These results suggest that the inhibition of [ 3H]histamine release by exogenous histamine acting via H 3-receptor stimulation is mediated by a restricted access of Ca 2+ and that its extent is influenced by the degree of autostimulation by endogenous histamine as well as, possibly, by actual internal Ca 2+ concentration. In addition the decrease in external Ca 2+ concentration shifted rightwards the concentration-response curve to histamine. The autoinhibitory effect of exogenous histamine was found on slices from various regions, known from lesion studies to contain terminals of extrinsic histaminergic neurons. It did not apparently involve interneurones, not being prevented in slices in which the traffic of action potentials was blocked by tetrodotoxin. It also remained unaffected in striatal slices in which the neuronal cell-bodies were selectively destroyed by prior local infusion of kainic acid. Finally exogenous histamine inhibited [ 3H]histamine release from depolarized synaptosomes of rat cerebral cortex, with anEC 50 value similar to that found with slices and was antagonised by impromidine with an apparentK i value similar to that displayed at H 3-receptors. It is concluded that histamine modulates its own release from cerebral neurones by interacting with H 3-presynaptic autoreceptors and via mechanisms similar to those previously evidenced on other aminergic systems.

Release of LHRH in vitro and anterior pituitary responsiveness to LHRH in vivo during sexual maturation in pullets (Gallus domesticus).

An in-vitro superfusion technique was used to study basal and depolarization-induced (32 mmol K+/l) release of LHRH from the mediobasal hypothalamus (MBH) of pullets at 8-25 weeks of age. Plasma LH concentrations and the incremental change (delta LH) after an i.v. injection of 1 or 15 micrograms synthetic ovine LHRH/kg body weight were also determined. Between 8 and 25 weeks of age, significant (P less than 0.01) increases in basal and depolarization-induced release of LHRH (93 and 330%, respectively) were accompanied by a significant (P less than 0.01) rise in the residual LHRH content of MBH tissue (152%), observations which suggest that the ability of the hypothalamus to synthesize and secrete LHRH increases as sexual maturation proceeds. However, plasma LH, which reached a maximum concentration of 2.05 +/- 0.43 micrograms/l at 15 weeks, fell significantly (P less than 0.05) to 1.14 +/- 0.05 micrograms/l at 25 weeks. Since delta LH in response to exogenous LHRH showed a marked and progressive decline between 12 and 20 weeks of age, the low plasma concentration of LH typical of the mature hen is probably attributable to a direct negative-feedback action of ovarian steroids on the anterior pituitary gland rather than to an impaired secretion of LHRH from the median eminence. It is suggested that a dramatic increase in the responsiveness of LHRH nerve terminals in the MBH to depolarization by 32 mmol K+/l between 20 and 25 weeks of age (mean age at onset of lay 21.9 weeks; range 19-25 weeks) may reflect the development of hypothalamic responsiveness to the positive feedback action of progesterone.