A procedure for detecting changes in the internal Ca2+ concentration in isolated nerve endings using the metallochromic dye arsenazo III

A method for detecting changes in the internal concentration of Ca2+ in synaptosomes from mouse brain is described. Synaptosomes were fused with phosphatidylcholine-phosphatidylserine unilamellar liposomes previously loaded with the metalochromic, Ca2+ -sensitive dye arsenazo III, in order to introduce the dye into the synaptosomes. The fusion was promoted by La3+. Changes in the differential absorption between 660 and 690 nm, which indicate changes in Ca2+ concentration, were followed in a double beam spectrophotometer. It was found that both in the dye-loaded liposomes and in the fused synaptosomes, the addition of the Ca2+ ionophore A23187 to the medium containing Ca2+ produced a notable change in the differential absorbance 660-690 nm. When the depolarizing alkaloid veratridine was added, there was no response in the liposomes, whereas a change in the differential absorbance 660-690 nm was detected in the fused synaptosomes containing arsenazo but only when Na+ was present in the medium. These fused synaptosomes were able to release labeled gamma-aminobutyric acid as a response to veratridine, in a Na+ -dependent manner, similarly to control non-fused synaptosomes. These results demonstrate the feasibility of fusion to introduce Ca2+ -sensitive dyes into isolated nerve endings from the mammalian brain and therefore to detect changes in their internal Ca2+ concentration.     

A comparison of the effects of acute and one year's continuous neuroleptic treatment on the release of [3H]glutamate and [3H]acetylcholine from rat striatal slices

The effect of neuroleptic drugs administered acutely or continuously for 1 year on the release of [³H]glutamate and [³H]acetylcholine from striatal slices in vitro has been compared.Acute in vivo administration of haloperidol, trifluoperazine and clozapine increased the potassium-evoked release of [³H]acetylcholine from striatal slices in a dose-dependent fashion, whereas sulpiride was without effect. None of the neuroleptics given acutely had any effect on the potassium-evoked striatal release of [³H]glutamate.Potassium-evoked striatal release of [³H]acetylcholine in animals receiving 1 year's continuous administration of haloperidol, trifluoperazine or sulpiride was no different from that in age-matched control animals, but was less than controls in animals receiving clozapine for 1 year. All drugs caused a decrease in potassium-evoked striatal [³H]glutamate release following drug administration for 1 year compared to age-matched controls.The reversal of the acute action of neuroleptic drugs on striatal [³H]acetylcholine and [³H]glutamate release is consistent with a functional increase in striatal dopamine transmission following long-term neuroleptic treatment.     

K+-stimulated release of labeled γ-aminobutyrate,glycine and taurine in slices of several regions of rat central nervous system

The effect of depolarization by high K⁺ concentration (68.5 mm) on the release of [³H] γ-aminobutyrate (GABA), [¹⁴C]glycine and [³⁵S]taurine was studied in superfused slices of rat cerebellum, striatum, hypothalamus, colliculi, cerebral cortex and ventral and dorsal halves of spinal cord. The release of [³H]GABA was notably stimulated by K⁺-depolarization in all regions studied, particularly in the cerebral cortex and the hypothalamus. The Ca²⁺-dependence of this phenomenon was studied in the cortex and ventral spinal cord; in both regions the K⁺-stimulated release was abolished when Ca²⁺ was omitted from the superfusing medium. The release of [¹⁴C]glycine was also stimulated in all regions, except the cerebellum, but to a lesser extent than that of GABA. This stimulation was Ca²⁺-dependent in the ventral spinal cord but not in the cerebral cortex. The release of [³⁵S]taurine was not affected by K⁺-depolarization in any of the regions studied.These results are consistent with a widely distributed neurotransmitter role for GABA. The Ca²⁺-dependence of glycine release in the spinal cord is in agreement with a role of this amino acid as a transmitter in this region. The finding that [³⁵S]taurine release was not stimulated by K⁺-depolarization in any of the regions studied, under experimental conditions identical to those resulting in an enhancement of [³H]GABA and [¹⁴C]glycine release, argues against a neurotransmitter role of this amino acid in brain and spinal cord.     

Pharmacological and ionic features of gamma-aminobutyric acid receptors influencing electrical properties of melanotrophs isolated from the rat pars intermedia

Characteristics of the gamma-aminobutyric acid receptors on melanotrophs of the rat pars intermedia were studied by intracellular recording. Muscimol and 3-amino-1-propanesulfonic acid, but not baclofen or glycine, mimicked the depolarization and conductance increase produced by gamma-aminobutyric acid on the melanotrophs. These effects appeared to be due to an increase in chloride ion conductance since the null potentials for all three drugs were the same and were affected by changes in external or internal chloride ion concentration but not by changes in the concentrations of other ions present in the recording solution or by the addition of the calcium-channel blocker cobalt. Bicuculline abolished the effect of muscimol. Picrotoxin reduced the effect of gamma-aminobutyric acid; so too did furosemide. Muscimol mimicked the ability of gamma-aminobutyric acid to reduce the depolarization produced by excess potassium and this effect was also blocked by bicuculline. Rat melanotrophs thus appear to possess gamma-aminobutyric acid receptor-ionophore complexes similar to the classical sort found in neurons in the mammalian central nervous system. Furthermore, the parallels between the electrical responses observed and secretory effects previously noted, reinforce the view that electrical activity may participate in stimulus-secretion coupling in melanotrophs.     

gamma-Aminobutyric acid and taurine release in the striatum of the rat during hypoglycemic coma, studied by microdialysis

Extracellular levels of striatal gamma-aminobutyric acid (GABA) and taurine were monitored during insulin-induced hypoglycemia using microdialysis. At the onset of isoelectricity in the electroencephalogram (EEG), a transient 5-fold increase in the levels of GABA occurred. Taurine levels increased 5 min following the onset of isoelectricity and continued to increase during the entire isoelectric period. The results demonstrate that events associated with the onset of isoelectricity during hypoglycemia trigger an increase in extracellular concentrations of GABA and taurine. The discrepancy in time-course of these changes may reflect differences in compartmentation, function and metabolism of the two amino acids.     

Effects of sodium-deficient media and of a calcium ionophore (A-23187) on the release of (3H)-noradrenaline, (14C)-α-aminoisobutyrate,and (3H)-γ-aminobutyrate from superfused slices of rat neocortex

The calcium ionophore A-23187, at a concentration of 250 μg/ml, had no significant effects on the efflux of (³H)-noradrenaline or of (¹⁴C)-α-aminoisobutyrate, in superfused slices from rat neocortex. Sodium-deficient solutions (sodium partly substituted by choline), however, were found to increase the efflux of (¹⁴C)-α-aminoisobutyrate and of (³H)-γ-aminobutyrate. The enhanced efflux of both these substances appeared with no latency, was maintained for long periods, and did not change appreciably when calcium was omitted. Media low in sodium enhanced noradrenaline efflux in a more complex manner. There was, initially, a small increase in efflux that was similar both in latency and lack of dependence upon calcium to that of the above amino acids. This was followed by a very large efflux peak of noradrenaline, with a change in the fractional rate constant of up to 0.06 min^?1. This large efflux peak did not occur when no calcium was added to the superfusing fluid. The efflux of noradrenaline occurred when calcium was replaced by strontium or barium ions, but the latter were less effective. Finally, when media free of added calcium were made sodium-deficient, there was a moderate increase in the efflux of noradrenaline that began after a delay of about 30 min.It is suggested that the effects of sodium-deficient media on the efflux of amino acids and transmitters are due to two different mechanisms. The first mechanism involves interference with sodium-dependent active transport processes that carry substances from the extracellular fluid across the plasma membrane; this could account for the increased efflux of α-aminoisobutyrate and of γ-aminobutyrate and for the first, small, increased efflux of noradrenaline, all of which are independent of external calcium concentration. The second mechanism is mediated by an increase in intracellular calcium, induced by low external sodium, which promotes secretion of noradrenaline present in synaptic vesicles. The large efflux of noradrenaline, that is calcium-dependent, might be the result of this mechanism. In addition, solutions low in both sodium and calcium induce non-specific increases in membrane permeability, that lead to the delayed release of noradrenaline.     

High affinity uptake and Ca2+-dependent release of glutamic acid in the developing cerebellum

This study was undertaken in order to evaluate the postulated role of glutamic acid as the neurotransmitter for the parallel fibers of the cerebellar cortex. We studied the Ca2+-dependent release and the high affinity uptake of glutamic acid in the developing cerebellum. The Ca2+-dependent release of glutamic acid from cerebellar molecular layer during development closely follows the time course of parallel fibers synaptogenesis. Little glutamic acid release was observed at 15 days, then it increased to the adult values at the 21st postnatal day. In the rat the bulk of synapses of the parallel fibers appear between the 15th and the 21st postnatal days, the time at which the nerve terminals of the climbing fibers, the other excitatory input to the Purkinje cells, are already developed. An enhanced Na+-dependent, high affinity uptake of glutamic acid was observed in the developing cerebellum relative to the adult rat. That this higher accumulation of glutamic acid is not related to a releasable pool is suggested by the fact that an enhanced glutamic acid, Ca2+-dependent release relative to the adult was not observed. These results support the view that glutamic acid is the transmitter for the cerebellar parallel fibers.     

The effects of hyperthermia on polyribosomes and amino acid levels in infant rat brain

Brain polyribosomes prepared from infant rats exposed to elevated ambient temperatures (39.5°C for 45 min) are highly disaggregated. Disaggregation is evident 15 min after the animals have entered the hot environment and is nearly maximal after 25 min. Substantial recovery of polyribosomes is seen 20 min after the hyperthermic rats are transferred from 39.5 to 33°C ambient temperature, indicating that heat-induced disaggregation is quickly reversible. During hyperthermia the levels of certain amino acids in the brain, particularly essential amino acids, rise markedly. The increases in amino acid levels are reversed within 1 h after the rats are transferred from the hot environment to 33°C ambient temperature. The levels of amino acids in blood plasma also rise during hyperthermia; however, the increases are relatively smaller and less selective than in the brain but are also reversed by returning the animals to 33°C ambient temperature.Injection of phenylalanine into infant rats causes hyperphenylalaninemia, elevated brain phenylalanine levels, and brain polyribosome disaggregation (Aoki & Siegel, 1970). The present observations show that elevated brain amino acid levels and polyribosome disaggregation are also linked in hyperthermic infant rats, but it is not yet clear if the two phenomena are causally related. Although the mechanisms of the increases of blood and brain amino acid levels remain to be elucidated, they may have a significant bearing on the functional status of the protein synthetic machinery of the brain, on the levels of neurotransmitter substances in the brain which are derived from amino acid precursors, and on the intermediary metabolism of the brain.     

Potassium effect on Na+, K+-ATPase activity of cultured newborn rat astroblasts during differentiation

High K⁺ concentration effect on Na⁺, K⁺-ATPase activity of cultured newborn rat astroblasts has been studied in conditions under which astrocytic differentiation could have taken place. In 14-day-old cultures, no potassium effect could be found reflecting the undifferentiated state of these cultures. After dibutyryl-cyclic AMP treatment, which induces a morphological transformation suggesting a differentiation, no K⁺ effect appears but the specific enzyme activities are higher than in control cells reflecting the development of numerous cellular processes. After 28 days of cultivation — a cultivation period which closely corresponds to the time of astrocytic differentiation in vivo — Na⁺,K⁺-ATPase activity at 25 and 50 mM K⁺ concentrations are higher than at 10 mM, suggesting that an astrocytic differentiation has taken place in vitro.     

Apical K+ channels of frog diluting segment: inhibition by acidification

The apical potassium conductance of the amphibian diluting segment is regulated by the intracellular pH. Alkalinisation of the cytosol, whether directly by bathing the cell in an alkaline medium, or following activation of an apical Na/H exchanger by aldosterone, results in an increase in the K conductance.Early distal tubules were isolated from slices of Frog kidneys and the apical membranes exposed by everting the tubule with the aid of microperfusion pipettes. Single channels in the apical membrane were studied in the cell-attached configuration while the tubules were bathed in Ringer with a pH of either 7.4 (control) or 6.6 (acid).Under control conditions single channel currents were readily seen at the resting membrane potential. Upon acidification of the superfusion solution the open probability of the channels was decreased from 0.76 to 0.15. Thus the reduction in apical conductance is brought about, at least in part, by a reduction in the open probability of the channels upon cellular acidification.     

Drug Modulation of Transductor Function of Na+,K+-ATPase

Experimental proof of the hypothesis on modulation of the transductor function of Na⁺,K⁺-ATPase by the ouabain—Ca²⁺ chelate complex was obtained by the method organotypic tissue culture. Quantum chemical estimations detected two principally different modes of Ca²⁺ ion chelation by ouabain molecule. It is hypothesized that the ouabain—Ca²⁺—Na⁺,K⁺-ATPase ligand-receptor complex is formed due to ion-ion bonds. The formation of the complex serves as the signal triggering the enzyme transductor function. It is experimentally proven that ouabain is incapable of inhibiting neurite growth in sensory neuron and heart tissue explants after removal of free calcium from the nutrient medium with EGTA. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 10, pp. 416–418, October, 2008     

The effect of some putative neurotransmitters on the release of 5-hydroxytryptamine and gamma-aminobutyric acid from slices of the rat midbrain raphe area.

The effect of various putative transmitters has been studied on the efflux of [³H]5-hydroxytryptamine and [³H]γ-aminobutyric acid from small superfused slices of the midbrain raphe area of the rat. The slices apparently contained functionally intact terminals for these transmitters since a depolarizing stimulus (50 mM KCl) stimulated the rate of efflux of [³H]5-hydroxytryptamine and [³H]γ-aminobutyric acid in a calcium-dependent fashion. Furthermore, slices accumulated radioactivity with apparent high affinity mechanisms. γ-Aminobutyric acid (50 and 100 μ m) and substance P (100 and 500μ m) stimulated the efflux of [³H]5-hydroxytryptamine. The effect of γ-aminobutyrate was blocked by picrotoxin (50 μ m) but not by strychnine (1 μ m). Other inhibitory amino acids (β-alanine, taurine and glycine) were without effect on the release of [³H]5-hydroxytryptamine. l-Noradrenaline (0.2–1 mM) stimulated the efflux of [³H]γ-aminobutyric acid but not that of [3H]5-hydroxytryptamine. Phentolamine (10μ m) but not (±)-propranolol (10 μ m) abolished the effect of 1 mM noradrenaline. Neither dopamine nor 5-hydroxytryptamine influenced the efflux of [3H]γ-aminobutyric acid.It is possible that interactions in the midbrain raphe area proposed from other studies can be mediated through presynaptic influences on transmitter release.     

Effect of elevated extracellular potassium on the release of labelled noradrenaline,glutamate, glycine, β-alanine and other amino acids from rat brain cortex slices

The effect of an elevated concentration (46 mm) of extracellular potassium on the release, from superfused rat brain cortex slices, of labelled γ-aminobutyrate, glutamate, glycine, α- and β-alanine, aspartate, taurine and α-aminoisobutyrate has been studied; and a comparison made with the release of soluble (non-vesicular) noradrenaline (i.e. that transported into slices taken from the brains of animals pretreated with reserpine; the slices were incubated with nialamide). In all cases, with the possible exception of l-α-alanine, elevated potassium induced an increased efflux of these substances. Omission of calcium in the superfusing fluid markedly diminished the release of all the test substances, except, in part, that of β-alanine. It was also found that the time course of the induced efflux varied considerably for the different substances: substances such as noradrenaline and γ-aminobutyrate that are transported predominantly into axons and axon terminals, showed a ‘peak’ time course, with a maximal release within 2–4 min following potassium elevation, and the rate of release diminished rapidly in spite of the continued presence of high potassium. Such a decreased release was not due to exhaustion of the tissue stores of these substances. On the other hand, glutamate and glycine, substances that are thought to be transported predominantly into glial cells, attained their maximal release rates only 10 min after potassium was elevated, and such release was maintained without decrement, β-alanine showed a mixed type of release, with a small initial peak resembling that of the axonally located substances, and a delayed release similar to that of glycine and glutamate. The release of the rest of the amino acids also resembled that of glycine and glutamate. A correlation was found (r = 0.99, P < 0.01) between the area of the efflux curve that had a ‘peak’ shape, and the percentage of the substance that was transported into an axonal compartment.It is concluded that although elevated potassium concentrations can release substances from both axons and glia, and that a calcium dependency may exist in both cases, the time course of the induced efflux is markedly different when the substances are located in axons or in glial cells. Such a procedure may prove valuable for establishing grossly into which compartment a given substance is transported.     

Cellular localization of the uptake of [3H]taurine and [3H]β-alanine in cultures of the rat central nervous system

The cellular localization of the uptake of [³H]taurine and [³H]β-alanine was studied in cultures of rat central nervous system using autoradiography. In brain stem and spinal cord cultures, both amino acids were taken up by neurones and glial cells. In cerebellar cultures, [³H]taurine was accumulated by all glial cells and by a small number of neurones, whereas [³H]β-alanine was only taken up by glial elements. The uptake of both amino acids was sodium and temperature dependent, indicating an active transport mechanism.The results provide further support for the hypothesis that taurine and β-alanine are neurotransmitters in the mammalian central nervous system.     

Neural elements containing glutamic acid decarboxylase (GAD) in the dorsal lateral geniculate nucleus of the rat; immunohistochemical studies by light and electron microscopy

Immunoreactive constituents of the dorsal lateral geniculate nucleus of adult albino rats were examined by light- and electron-microscopy, using the unlabelled antibody enzyme method, following treatment of brain slices with a purified antibody to glutamic acid decarboxylase. The neuropil of the dorsal lateral geniculate nucleus displayed a conspicuous granular immunoreactivity. In addition, the antibody was bound to a class of small neurons of characteristic morphology. These cells possessed few (commonly 2-4) sparsely branched, long dendrites from some of which immunoreactive appendages were traced. Many cells were bipolar in form, and the dendrites of some appeared to be preferentially orientated. The immunoreactive cells closely resembled intrinsic interneurons characterized in previous Golgi studies of this nucleus. By electron-microscopy, immunoreactive presynaptic elements were present both in the extraglomerular neuropil and in the synaptic glomeruli. The former were axon terminals containing flattened synaptic vesicles and making Gray type II axo-dendritic synaptic contact; they appeared to correspond to axon terminals whose origin in the thalamic reticular nucleus has been established in previous studies, but it is possible that some were axon terminals of intrinsic interneurons. The immunoreactive glomerular components also contained flattened vesicles, were presynaptic to presumptive projection cell dendrites, postsynaptic to retinal axon terminals, and participated in triplet (triadic) and other complex synaptic arrangements. They corresponded in all respects to the synaptic portions of the complex dendritic appendages of intrinsic interneurons, identified and characterized in previous studies. The finding that there are high levels of glutamic acid decarboxylase in the cell bodies, dendritic shafts and dendritic appendages of intrinsic interneurons in the dorsal lateral geniculate nucleus of the rat, and in the axon terminals of fibres projecting to this site from the thalamic reticular nucleus, allows us to conclude that the inhibitory inputs to the geniculo-cortical projection cells from both of these sources are probably mediated by gamma-aminobutyric acid.     

Selective uptake of [3H]glutamine and [3H]glutamate into neurons and satellite cells of dorsal root ganglia in vitro

The uptake of [³H]glutamate and [³H]glutamine into rat dorsal root ganglia has been examined by autoradiography and thin-layer chromatography. [³H]glutamate was selectively accumulated by satellite glial cells and after 10 min, 53% of this had been converted to [³H]glutamine. [³H]glutamine, on the other hand, entered neuronal perikarya and 40% was converted to [³H]glutamate. It is suggested that these selective uptake processes provide supporting evidence for the existence of a neuronal-glial glutamine cycle in dorsal root ganglia. Small dark (B) cells accumulated 6 times as much [³H]glutamine as did large light (A) cells. The reasons for this marked difference in the metabolism of the two main types of dorsal root ganglion neurone are discussed.     

Ultrastructural, functional and biochemical characteristics of mouse and human neuroblastoma cell lines

Three commercially available neuroblastoma cell lines were analyzed to determine their ultrastructural, functional and neurochemical characteristics. The cell lines were Neuro-2a and NB41A3, derived from mouse neuroblastoma, and IMR-32, derived from a human neuroblastoma.Ultrastructurally, the neuroblastoma exhibited two major differences. Both mouse neuroblastoma cell lines contained virus-like particles, whereas the human neuroblastoma was free of virus particles. Scanning electron-microscopy revealed that the external morphology in the mouse cell lines was heterogeneous, suggesting the presence of more than one cell type, while the external morphology of the human cell line was homogeneous, suggesting the presence of a single cell type.Neurochemically, choline acetyltransferase, tyrosine hydroxylase and glutamate decarboxylase, synthetic enzymes for acetylcholine, catecholamines and γ-aminobutyric acid, respectively, were present in each cell line. Mean choline acetyltransferase activity was highest in NB41A3, but differences in this activity among the three lines were not statistically significant. Tyrosine hydroxylase activity in IMR-32 was significantly higher than in the mouse neuroblastoma cells. Glutamate decarboxylase activity was equivalent in all three cell lines.Functionally, each cell line was tested to determine its ability to form cholinergic synapses on cultured striated muscle cells. Only the IMR-32 cells formed appreciable numbers of synapses with muscle cells as detected by electrophysicological recording.The delineation of several characteristics of these three neuroblastoma cell lines will allow other investigators to choose the most appropriate of these cell lines for their studies. The heterogeneity of cell types of the mouse neuroblastoma may compromise biochemical measurements as each cell type may have different characteristics. Since only the IMR-32 form cholinergic synapses and have no more choline acetyltransferase activity than the other two cell lines, the presence of this enzyme is not a reliable marker for the ability to form synapses.