The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain

Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.     

Histochemical demonstration of transmitter release from noradrenaline,dopamine and 5-hydroxytryptamine nerve terminals in field stimulated rat brain slices

Summary The effect of electrical field stimulation on noradrenaline (NA), dopamine (DA) and 5-hydroxytryptamine (5-HT) nerve terminals in rat brain slicesin vitro was investigated. Slices prepared from the cerebral cortex or the neostriatum were incubated in physiologic buffer for 30 min and then superfused by buffer and stimulated by an electrical field (biphasic pulses, 10 Hz, 12 mA, 2 ms) for various time periods. The effect of the stimulation was studied at the cellular level with the histochemical fluorescence technique of Falck and Hillarp. The transmitter overflow into the superfusing buffer caused by the stimulation was studied with isotope technique. Cerebral Cortex NA Nerve Terminals. Stimulation caused release of NA from cortical NA nerve terminals. Already after 2 min stimulation a slight decrease of the fluorescence intensity of the nerve terminals could be found. Stimulation for 15 to 30 min greatly reduced the fluorescence intensity. In slices preincubated with³H-NA the stimulation-induced overflow of tritium during 2 min stimulation was about 15% (i.e. 15% of the tissue tritium content was overflowing into the superfusing buffer in response to stimulation for 2 min). During prolonged stimulation there was a considerable decline of the tritium efflux. Cerebral Cortex 5-HT Nerve Terminals. The 5-HT-analogue 6-hydroxytryptamine (6-HT) which is readily taken up into 5-HT nerve terminals was used to permit good visualization of these nerve terminals. Uptake of 6-HT into cortical NA nerve terminals was prevented by preincubation with 6-hydroxydopamine (6-OH-DA) or protriptyline. Stimulation for 15 to 30 min reduced the fluorescence intensity of the 5-HT nerve terminals. In slices preincubated with³H-5-HT the stimulation-induced overflow of tritium during 2 min stimulation was about 5%. The tritium efflux slowly decreased during continuous stimulation. Neostriatal DA Nerve Terminals. In slices frozen directly after preparation an intense diffuse fluorescence could be seen. After incubation in drug-free buffer at 37° C the fluorescence was localized in the varicosities of the DA nerve terminals. The central parts of the slices almost completely lacked specific fluorescence, while the outer zones were brightly fluorescent. No clear reduction of the fluorescence intensity of the DA nerve terminals in the outer zone could be observed after stimulation for 30 min. In slices preincubated with³H-DA the stimulation-induced overflow of tritium during 2 min stimulation was about 2%. The tritium efflux slowly decreased during continuous stimulation.It is suggested that the differences in release between the various nerve terminal systems foundin vitro reflect differences in transmitter release occurringin vivo. The comparably high release of NA per impulse from the cortical NA nerve terminals implicate that the discharge rate of these neuronsin vivo is very low.This investigation has been supported by grants from the Swedish Medical Research Council (B72-14X-2330-05A) and Magnus Bergwall's Foundation.The author is greatly indebted to Mrs. Annika Hamberger for her skillful technical assistance. For generous supplies of drugs thanks are due to Hässle, Göteborg, Sweden, through Dr. H. Corrodi (6-HT, 6-OH-DA and H44/68).     

Modulation of transmitter release by presynaptic resting potential and background calcium levels

Activation of presynaptic ion channels alters the membrane potential of nerve terminals, leading to changes in transmitter release. To study the relationship between resting potential and exocytosis, we combined pre- and postsynaptic electrophysiological recordings with presynaptic Ca(2+) measurements at the calyx of Held. Depolarization of the membrane potential to between -60 mV and -65 mV elicited P/Q-type Ca(2+) currents of < 1 pA and increased intraterminal Ca(2+) by < 100 nM. These small Ca(2+) elevations were sufficient to enhance the probability of transmitter release up to 2-fold, with no effect on the readily releasable pool of vesicles. Moreover, the effects of mild depolarization on release had slow kinetics and were abolished by 1 mM intraterminal EGTA, suggesting that Ca(2+) acted through a high-affinity binding site. Together, these studies suggest that control of resting potential is a powerful means for regulating synaptic function at mammalian synapses.     

Processing of ARIA and release from isolated nerve terminals

The neuromuscular junction is a specialized synapse in that every action potential in the presynaptic nerve terminal results in an action potential in the postsynaptic membrane, unlike most interneuronal synapses where a single presynaptic input makes only a small contribution to the population postsynaptic response. The postsynaptic membrane at the neuromuscular junction contains a high density of neurotransmitter (acetylcholine) receptors and a high density of voltage-gated Na+ channels. Thus, the large acetylcholine activated current occurs at the same site where the threshold for action potential generation is low. Acetylcholine receptor inducing activity (ARIA), a 42 kD protein, that stimulates synthesis of acetylcholine receptors and voltage-gated Na+ channels in cultured myotubes, probably plays the same roles at developing and mature motor endplates in vivo. ARIA is synthesized as part of a larger, transmembrane, precursor protein called proARIA. Delivery of ARIA from motor neuron cell bodies in the spinal cord to the target endplates involves several steps, including proteolytic cleavage of proARIA. ARIA is also expressed in the central nervous system and it is abundant in the molecular layer of the cerebellum. In this paper we describe our first experiments on the processing and release of ARIA from subcellular fractions containing synaptosomes from the chick cerebellum as a model system.     

Calcium buffering in presynaptic nerve terminals. Free calcium levels measured with arsenazo III

The particulate fraction from osmotically shocked synaptosomes ('synaptosomal membrances') sequesters Ca when incubated with ATP]containing solutions. This net accumulation of Ca can reduce the free [Ca2+] of the bathing medium to sub-micromolar levels (measured with arsenazo III). Two distinct types of Ca sequestration site are responsible for the Ca2+ buffering. One site, presumed to be smooth endoplasmic reticulum, operates at low [Ca2+] (less than 1 microM), and has a relatively small capacity. Ca sequestration at this site is prevented by the Ca2+ ionophore, A-23187, but not by mitochondrial poisons. The secone (mitochondrial) site, in contrast, is blocked by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and oligomycin. Since the intraterminal organelles can buffer [Ca2+] to about 0.3-0.5 microM, this may be an upper limit to the normal resting level of [Ca2+]i in nerve terminals. In the steady state, total cell Ca and [Ca2+]i will be governed principally be Ca transport mechanisms in the plasmalemma; the intracellular organelle transport systems then operate in equilibrium with this [Ca2+]. During activity, however, Ca rapidly enters the terminals and [Ca2+]i rises. The intracellular buffering mechanisms then come into play and help to return [Ca2+]i toward the resting level; the non-mitochondrial Ca sequestration mechanism probably plays the major role in this Ca buffering.     

Calcium buffering in presynaptic nerve terminals. Free calcium levels measured with arsenazo III

The particulate fraction from osmotically shocked synaptosomes (‘synaptosomal membranes’) sequesters Ca when incubated with ATP-containing solutions. This net accumulation of Ca can reduce the free [Ca²⁺] of the bathing medium to sub-micromolar levels (measured with arsenazo III). Two distinct types of Ca sequestration site are responsible for the Ca²⁺ buffering. One site, presumed to be smooth endoplasmic reticulum, operates at low [Ca²⁺] (less than 1 μM), and has a relatively small capacity. Ca sequestration at this site is prevented by the Ca²⁺ ionophore, A-23187, but not by mitochondrial poisons. The second (mitochondrial) site, in contrast, is blocked by the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and oligomycin. Since the intraterminal organelles can buffer [Ca²⁺] to about 0.3–0.5 μM, this may be an upper limit to the normal resting level of [Ca²⁺]_i in nerve terminals. In the steady state, total cell Ca and [Ca²⁺]_i will be governed principally by Ca transport mechanisms in the plasmalemma; the intracellular organelle transport systems then operate in equilibrium with this [Ca²⁺]. During activity, however, Ca rapidly enters the terminals and [Ca²⁺]_i rises. The intracellular buffering mechanisms then come into play and help to return [Ca²⁺]_i toward the resting level; the non-mitochondrial Ca sequestration mechanism probably plays the major role in this Ca buffering.     

Mucin release and calcium fluxes in isolated rat submandibular acini.

A method is described for preparing isolated rat submandibular acini by collagenase digestion followed by mechanical dispersion. As assessed by Trypan Blue exclusion, phase contrast microscopy, ATP content and release of mucins and lactate dehydrogenase, the acini are morphologically and functionally intact. Secretory function of isolated acini was similar to that of intact tissue in terms of time-course, dose dependence and degree of stimulation of mucin release by adrenergic secretagogues. Mucin release was increased to the same extent (approx. 3-4-fold) by either isoproterenol or noradrenaline at a maximally effective concentration (10 microM). Stimulation of mucin release by isoproterenol (10 microM), noradrenaline (10 microM) or adrenaline (10 microM) was inhibited by propranolol (30 microM) but not by phentolamine (30 microM). Isoproterenol (10 microM) increased both 45Ca2+ uptake and efflux from the acini, which was shown to represent a net release of calcium. However, there was a delay (approx. 10 min) in onset of stimulation of 45Ca2+ mobilization which was not apparent in isoproterenol stimulation of mucin release. Our results indicate that increases in intracellular calcium mobilization in response to a beta-adrenergic secretagogue do not trigger mucin secretion from rat submandibular acini.     

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.     

Removal of extracellular chloride suppresses transmitter release from photoreceptor terminals in the mudpuppy retina

Removal of extracellular Cl- has been shown to suppress light-evoked voltage responses of ON bipolar and horizontal cells, but not photoreceptors or OFF bipolar cells, in the amphibian retina. A substantial amount of experimental evidence has demonstrated that the photoreceptor transmitter, L-glutamate, activates cation, not Cl-, channels in these cells. The mechanism for Cl-free effects was therefore reexamined in a superfused retinal slice preparation from the mudpuppy (Necturus maculosus) using whole-cell voltage and current clamp techniques. In a Cl-free medium, light-evoked currents were maintained in rod and cone photoreceptors but suppressed in horizontal, ON bipolar, and OFF bipolar cells. Changes in input resistance and dark current in bipolar and horizontal cells were consistent with the hypothesis that removal of Cl- suppresses tonic glutamate release from photoreceptors. The persistence of light-evoked voltage responses in OFF bipolar cells, despite the suppression of light-evoked currents, is due to a compensatory increase in input resistance. Focal application of hyperosmotic sucrose to photoreceptor terminals produced currents in bipolar and horizontal cells arising from two sources: (a) evoked glutamate release and (b) direct actions of the hyperosmotic solution on postsynaptic neurons. The inward currents resulting from osmotically evoked release of glutamate in OFF bipolar and horizontal cells were suppressed in a Cl-free medium. For ON bipolar cells, both the direct and evoked components of the hyperosmotic response resulted in outward currents and were thus difficult to separate. However, in some cells, removal of extracellular Cl- suppressed the outward current consistent with a suppression of presynaptic glutamate release. The results of this study suggest that removal of extracellular Cl- suppresses glutamate release from photoreceptor terminals. Thus, it is possible that control of [Cl-] in and around photoreceptors may regulate glutamate release from these cells.     

Effect of carbachol on spontaneous transmitter release from rat motor nerve endings depending on extracellular potassium concentration

The effect of carbachol (10 µM) on the frequency of miniature end-plate potentials (MEPP) was studied in experiments on the Wistar rat soleus muscle during a change in extracellular potassium concentration from 2 to 15 mM. Between the range of potassium concentrations from 2 to 7.5 mM the cholinomimetic had no effect on spontaneous transmitter release. In higher potassium concentrations carbachol caused an increase in the frequency of MEPP. This facilitatory effect increased in strength with an increase in potassium concentration; at 15 mM the frequency of MEPP was increased up to 160%. The results confirmed the previous hypothesis that the action of the mimetic on spontaneous transmitter release, relaized through presynaptic acetylcholine receptors, depends on the initial level of polarization of nerve endings.S. V. Kurashov Kazan' State Medical Institute. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 470–475, July–August, 1984.     

A metabotropic glutamate receptor regulates transmitter release from cone presynaptic terminals in carp retinal slices.

The role of group III metabotropic glutamate receptors (mGluRs) in photoreceptor-H1 horizontal cell (HC) synaptic transmission was investigated by analyzing the rate of occurrence and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in H1 HCs uncoupled by dopamine in carp retinal slices. Red light steps or the application of 100 microM cobalt reduced the sEPSC rate without affecting their peak amplitude, which is consistent with hyperpolarization or the suppression of Ca(2+) entry into cone synaptic terminals reducing vesicular transmitter release. Conversely, postsynaptic blockade of H1 HC AMPA receptors by 500 nM CNQX reduced the amplitude of sEPSCs without affecting their rate. This analysis of sEPSCs represents a novel methodology for distinguishing between presynaptic and postsynaptic sites of action. The selective agonist for group III mGluRs, l-2-amino-4-phosphonobutyrate (L-APB or L-AP4; 20 microM), reduced the sEPSC rate with a slight reduction in amplitude, which is consistent with a presynaptic action on cone synaptic terminals to reduce transmitter release. During L-APB application, recovery of sEPSC rate occurred with 500 microM (s)-2-methyl-2-amino-4-phosphonobutyrate (MAP4), a selective antagonist of group III mGluR, and with 200 microM 4-aminopyridine (4-AP), a blocker of voltage-dependent potassium channels. Whole-cell recordings from cones in the retinal slice showed no effect of L-APB on voltage-activated Ca(2+) conductance. These results suggest that the activation of group III mGluRs suppresses transmitter release from cone presynaptic terminals via a 4-AP-sensitive pathway. Negative feedback, operating via mGluR autoreceptors, may limit excessive glutamate release from cone synaptic terminals.     

Differential release of amino acids, neuropeptides, and catecholamines from isolated nerve terminals

We have investigated transmitter release from small and large dense-core vesicles in nerve terminals isolated from guinea pig hippocampus. Small vesicles are found in clusters near the active zone, and large dense-core vesicles are located at ectopic sites. The abilities of Ca2+ channel activation and uniform elevation of Ca2+ concentration (with ionophores) to evoke secretion of representative amino acids, catecholamines, and neuropeptides were compared. For a given increase in Ca2+ concentration, ionophore was less effective than Ca2+ channel activation in releasing amino acids, but not in releasing cholecystokinin-8. Titration of the average Ca2+ concentration showed that the Ca2+ affinity for cholecystokinin-8 secretion was higher than that for amino acids. Catecholamine release showed intermediate behavior. It is concluded that neuropeptide release is triggered by small elevations in the Ca2+ concentration in the bulk cytoplasm, whereas secretion of amino acids requires higher elevations, as produced in the vicinity of Ca2+ channels.     

Reactive oxygen species induced by presynaptic glutamate receptor activation is involved in [H]GABA release from rat brain cortical nerve terminals

We investigated the production of reactive oxygen species (ROS) as a response to presynaptic glutamate receptor activation, and the role of ROS in neurotransmitter (GABA) release. Experiments were performed with rat brain cortical synaptosomes using glutamate, NMDA and kainate as agonists of glutamate receptors. ROS production was evaluated with the fluorogenic compound dichlorodihydrofluorescein diacetate (H₂DCF-DA), and GABA release was studied using synaptosomes loaded with [³H]GABA. All agonists were found to stimulate ROS production, and specific antagonists of NMDA and kainate/AMPA receptors, dizocilpine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-done (CNQX), significantly inhibited the ROS increase. Spontaneous as well as agonist-evoked ROS production was effectively attenuated by diphenyleneiodonium (DPI), a commonly used potent inhibitor of NADPH oxidase activity, that suggests a high contribution of NADPH-oxidase to this process. The replacement of glucose with pyruvate or the simultaneous presence of both substrates in the medium led to the decrease in spontaneous and NMDA-evoked ROS production, but to the increase in ROS production induced by kainate. Scavenging of agonist-evoked ROS production by a potent antioxidant N-acetylcysteine was tightly correlated with the inhibition of agonist-evoked GABA release. Together, these findings show that the activation of presynaptic glutamate receptors induces an increase in ROS production, and there is a tight correlation between ROS production and GABA secretion. The pivotal role of kainate/AMPA receptors in ROS production is under discussion.     

Acetylcholine--inhibition of transmitter release from adrenergic nerve terminals mediated by muscarinic receptors.

The evidence is reviewed for the presence of muscarinic receptors on the sympathetic nerves to blood vessels. Activation of these receptors by acetylcholine in doses that are too small to affect the smooth muscle cells directly inhibits the release of norepinephrine evoked by electric impulses or potassium ions. This inhibitory action of acetylcholine is prevented by muscarinic blocking agents and is probably due to hyperpolarization of the adrenergic nerve terminals.     

Leptinotarsin-D, a neurotoxic protein, evokes neurotransmitter release from, and calcium flux into, isolated electric organ nerve terminals

Previous work has demonstrated that the neurotoxin leptinotarsin elicits release of neurotransmitter from mammalian nerve terminals, and it has been suggested that the toxin may act either as a direct agonist of voltage-sensitive calcium channels in these terminals (Crosland et al., 1984) or as a calcium ionophore (Madeddu et al., 1985a,b). Preliminary studies (Yeager et al., 1987) demonstrated that leptinotarsin also evokes transmitter release from isolated elasmobranch electric organ nerve terminals. We now report further investigations of the effects of leptinotarsin in this system. The action of the toxin is saturable, releasing about the same small fraction of total transmitter as that released by depolarization. An upper limit for the concentration for half maximal release is estimated to be 4 nM. Leptinotarsin-evoked transmitter release exhibits behavior very similar to depolarization-evoked release with respect to dependence on Ca2+, Ba2+, and Sr2+ and blockade by Co2+, Cd2+, and trifluoperazine. Leptinotarsin also promotes the uptake of calcium into synaptosomes to a degree similar to that caused by depolarization by K+. The binding of leptinotarsin to nerve terminals is probably Ca2+ dependent and receptor mediated. Taken together with the behavior of leptinotarsin-evoked release in other preparations, these results are consistent with the hypothesis that this toxin acts by opening a presynaptic calcium channel. However, the possibility that leptinotarsin is a calcium ionophore cannot be excluded.     

Opioid inhibition of GABA release from presynaptic terminals of rat hippocampal interneurons.

Opiates and the opioid peptide enkephalin can cause indirect excitation of principal cortical neurons by reducing inhibitory synaptic transmission mediated by GABAergic interneurons. The mechanism by which opioids mediate these effects on interneurons is unknown, but enkephalin hyperpolarizes the somatic membrane potential of a variety of neurons in the brain, including hippocampal interneurons. We now report a new, more direct mechanism for the opioid-mediated reduction in synaptic inhibition. The enkephalin analog D-Ala2-Met5-enkephalinamide (DALA) decreases the frequency of miniature, action potential-independent, spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) without causing a change in their amplitude. Thus, we conclude that DALA inhibits the action potential-independent release of GABA through a direct action on interneuronal synaptic terminals. In contrast, DALA reduces the amplitude of action potential-evoked, GABA-mediated IPSCs, as well as decreases their frequency. This suggests that the opioid-mediated inhibition of non-action potential-dependent GABA release reveals a mechanism that contributes to reducing action potential-evoked GABA release, thereby decreasing synaptic inhibition.