Reverse transport of glutamate during depolarization in immature hippocampal slices

We studied the source of extracellular glutamate released by hippocampal slices obtained from P14 or adult rats, during 50 mM K+ depolarization by using two potent inhibitors of Na+-dependent glutamate transport: l-trans-pyrrolidine-2,4-dicarboxylate (PDC), which is a relatively non-selective inhibitor of various glutamate transporter subtypes and dihydrokainic acid (DHK), a specific inhibitor of the glial transporter, GLT-1. Most depolarization-induced glutamate release was Ca2+-dependent in adults, while in P14 slices most glutamate release was Ca2+-independent. PDC decreased depolarization-induced glutamate release in P14 slices but not in adults. DHK increased glutamate release in adults but not in P14 slices. These data suggest that most depolarization-induced glutamate release in immature hippocampal slices is due to reversal of transport through a PDC-sensitive Na+-dependent glutamate transporter, presumably acting on presynaptic or cytoplasmic neuronal pools, and is not due to exocytosis from vesicular pools.     

Substrate specificity and developmental aspects of a presynaptic GABA receptor regulating glutamate release in the rat cerebellum

In order to better characterize the presynaptic GABA receptors regulating glutamate release in the cerebellum [Levi and Gallo, 1981], a number of GABA agonists and GABA transport inhibitors were tested for their ability to potentiate the depolarization-induced release of the glutamate analog D-[3H]aspartate from superfused cerebellar synaptosomes. Of all the compounds tested, only those which are known to interact specifically with GABA receptors were effective when tested on synaptosomal preparations. The order of effectiveness found was the following: muscimol congruent to 3-APS greater than or equal to P4S greater than isoguvacine greater than THIP. GABA uptake inhibitors were unable to enhance D-[3H]aspartate evoked release from synaptosomes, but were effective when tested in cerebellar slices; in the latter case, the activation of the GABA receptors may be achieved indirectly, through an increase of the extracellular GABA concentrations. The substrate specificity of the presynaptic GABA receptors regulating cerebellar acidic amino acid release appears to be similar to that reported for GABA receptors in radioligand binding studies and for GABA autoreceptors. Studies on synaptosomes from immature cerebella suggested that the presence of the potentiating effect on the acidic amino acid release by GABA agonists is correlated with the development of the parallel fiber terminals, which are believed to be the main site from which glutamate is released in the adult cerebellum.     

Enhancement by GABA of glutamate depolarization-induced release from cerebellar nerve endings

Cerebellar synaptosomes were superfused in the presence of D-[3H]aspartate (to label the glutamate 'reuptake pool') and [14C]glutamine (to label the 'new synthesis pool'). The depolarization-induced release of D-[3H]aspartate and of newly synthesized [14C]glutamate were potentiated by low concentrations of GABA (2--20 microM) or muscimol. The effect was probably mediated by the interaction of GABA with presynaptic GABA receptors localized in 'glutamergic' nerve endings, since it was antagonized by the GABA antagonists picrotoxin and bicuculline.     

A double-labeled preparation for simultaneous measurement of [3H]-noradrenaline and [14C]-glutamic acid exocytosis from streptolysin-O (SLO)-perforated synaptosomes

We have developed a novel method to examine [3H]-noradrenaline and [14C]-glutamate release from the same sample of streptolysin-O (SLO) perforated rat cortical synaptosomes. Ca2+ -dependent [3H]-noradrenaline and [14C]-glutamate release was examined at different temperatures and was found to be greater at 30 degrees C than at 25 degrees C. Ca2+ -dependent release of [3H]-noradrenaline is more ATP dependent than Ca2+ -dependent release of [14C]-glutamate. No significant reuptake of either neurotransmitter by the perforated synaptosomes was detected, indicating all the synaptosomes were indeed perforated. Incubations with 1 mM ouabain, a specific Na+,K+ -ATPase inhibitor, slightly increased Ca2+ -dependent release of both neurotransmitters. [3H]-noradrenaline is released from large dense-core vesicles and [14C]-glutamate is released from small clear synaptic vesicles, so one can directly compare and contrast neurotransmitter release mechanisms between large dense-core vesicles and small clear synaptic vesicles using this preparation.     

Long-term Activation of Phosphoinositide Turnover Associated with Increased Release of Amino Acids in the Dentate Gyrus and Hippocampus Following Classical Conditioning in the Rat

The release of amino acids and the hydrolysis of inositol phospholipids were examined in parallel in three hippocampal areas following classical conditioning. Paired or unpaired tone(CS) - shock(US) presentations were given to animals engaged in a previously acquired food-motivated lever-pressing task. Conditioned suppression of lever-pressing was the behavioural measure of conditioning. Twenty-four hours after the last conditioning session, the dentate gyrus and areas CA3 and CA1 of the hippocampus were removed bilaterally from conditioned and pseudoconditioned animals, and slices cut and stored in liquid nitrogen for subsequent analysis. Crude synaptosomal pellets were prepared to investigate: (i) potassium-stimulated release of preloaded [3H]glutamate and [14C]aspartate in the presence and absence of extracellular Ca2+; (ii) [3H]inositol labelling of phosphoinositides and inositol phosphates; and (iii) [14C]arachidonic acid labelling of 1,2-diacylglycerol (1,2-DG). Potassium-stimulated, Ca2+-dependent release of [3H]glutamate in synaptosomes prepared from the dentate gyrus and area CA3 was significantly greater in conditioned animals than in pseudoconditioned animals. In area CA1, K+-stimulated, Ca2+-dependent release of [14C]aspartate was significantly increased in conditioned animals. These results confirm in synaptosomes, and extend to a period of 24 h our previous report of an increased release of transmitter in the dentate gyrus and hippocampus associated with classical conditioning. In parallel with the increased release of amino acids, learning was associated with a significant increase in labelling of phosphoinositides and inositol phosphates by [3H]inositol and a significant increase in labelling of 1,2-DG by [14C]arachidonic acid in the three hippocampal areas examined. It is suggested that a long-lasting presynaptic activation of inositol lipid metabolism may contribute to the learning-dependent increase in the capacity of hippocampal terminals to release transmitter and hence to the maintenance of a neurochemical trace which may, at least in part, underlie lasting changes in synaptic function built up during associative learning.     

Melittin enhances excitatory amino acid release and AMPA-stimulated 45Ca2+ influx in cultured neurons.

Melittin, a potent activator of phospholipase A2, enhanced both spontaneous and depolarization-induced release of D-[3H]aspartate in primary cultures of cerebellar granule cells. The action of melittin was concentration-dependent (EC50 value = 300 ng/ml) and did not require the presence of extracellular Ca2+. Melittin also stimulated the release of glutamate and aspartate, in addition to other endogenous amino acids (taurine, alanine and gamma-aminobutyric acid). These effects were accompanied by an enhanced influx of 45Ca2+, which was in part mediated by the activation of excitatory amino acid receptors by endogenous agonists. Low concentrations of melittin (50 ng/ml) potentiated the efficacy of AMPA in stimulating 45Ca2+ influx without affecting stimulation by kainate or by glutamate added in the absence of extracellular Mg2+ (a condition that favors the activation of NMDA receptors). These results indicate that activation of phospholipase A2 evokes both an enhanced glutamate release and an increased sensitivity of AMPA receptors, two events that may support synaptic facilitation and LTP formation.     

Culture of mature hippocampus slices for 4 days in a newly developed medium: preservation of transmitter release and leucine incorporation into protein

A procedure and a medium for sustaining mature hippocampus slices in vitro for 4 days are described. The ionic composition of the medium, in which the slices were incubated for 1 h of recovery following preparation, strongly affected their ability, 4 days later, to take up and to release D-[3H]aspartate and [14C]GABA. A medium deficient in Na+ and Ca2+ proved best for recovery of the fresh slices prior to transfer to culture medium. The newly developed CSF-like culture medium was the best among several media tested in maintaining the potential of the slices for uptake and for induced release of D-[3H]aspartate and [14C]GABA. Glutamine, present in most culture media, appeared to be particularly toxic. Relative to fresh slices, the slices after 4 days in culture maintained 118% and 97% of the uptake of D-[3H]aspartate and of [14C]GABA respectively. K(+)-induced release of D-[3H]aspartate and of [14C]GABA was 104% and 82% of the respective values in fresh slices. Under the optimal culture conditions worked out, the slices also regained a considerable capacity for incorporation of labelled leucine into protein, which was low in fresh slices.     

Long-term potentiation is associated with an increase in calcium-dependent, potassium-stimulated release of [14C]glutamate from hippocampal slices: an ex vivo study in the rat

Long-term potentiation (LTP) was induced in the commissural pathway to CA3 in vivo. Tissue slices were prepared from potentiated and control hippocampi and preloaded with [14C]glutamate ([14C]glu). K+-stimulated release of [14C]glu from these slices was compared in the absence and presence of extracellular Ca2+. High-affinity uptake and Ca2+-independent release of [14C]glu were not affected by the induction of LTP, but Ca2+-dependent release from potentiated tissue was significantly greater than that from control tissue. These results demonstrate that LTP induced in vivo in the commissural pathway to CA3 is associated with enhanced release of preloaded glutamate in vitro.     

D-[3H]aspartate release from hippocampus slices studied in a multiwell system: controlling factors and postnatal development of release

Medium components and various factors were tested to define optimal conditions for D-[3H]aspartate release. Isolation of the hippocampus and preparation of the slices in a medium without Ca2+ increased the release of D-[3H]aspartate in response to veratridine when subsequently tested in a regular Ca2+ containing medium. Apparently, the absence of Ca2+ during preparation of the slices reduced irreversible damage due to hypoxic conditions which prevail throughout the interval between killing the animal and immersion of the slices in a well oxygenated medium. Substitution of 10 mM Mg2+ for Ca2+ was an efficient procedure to test for Ca2+ dependence of D-[3H]aspartate release induced by veratridine. The inhibition was readily reversible when Ca2+ was readded. Veratridine (50 microM) was superior to high K+ (45 mM) in inducing D-[3H]aspartate release under all conditions tested in slices of mature animals. Furthermore, veratridine-induced release could be completely blocked by tetrodotoxin while K+-induced release was essentially unaffected by this toxin. Postnatal development of the D-[3H]aspartate release induced by veratridine was found to require 40-45 days, whereas release induced by K+ reached about 80% of maximum at postnatal day 22. K+-induced release appears to reach maturation when most hippocampal cells have been formed while veratridine-induced release probably requires completion of the neural circuit, involving also extensive sodium channel formation. These investigations were conveniently performed using a modified plastic culture box in which 24 slice systems can be studied simultaneously.     

beta-Agkistrodotoxin inhibition of Ca2+-dependent release of glutamate,aspartate, glycine and gamma-aminobutyric acid from cerebrocortical synaptosomes following its binding to synaptic membrane

The binding properties of beta-AgTX, a snake pre-synaptic toxin, membranes and its effect on transmitter release from cerebrocortical synaptosomes were investigated. Assay of [125I]-beta-AgTX binding to rat synaptic membrane revealed a high affinity binding site for the toxin within the synaptic membrane. Preincubation with beta-AgTX inhibited K+-evoked Ca2+-dependent glutamate release from synaptosomes in a concentration-dependent manner, as determined by an on-line enzyme-linked fluorometric assay. The toxin also blocked the Ca2+-dependent release of other transmitters, aspartate, glycine, and GABA induced by K+-depolarization. However, Ca2+-ionophore, ionomycin-stimulated Ca2+-dependent transmitter release was not significantly affected by beta-AgTX, indicating that the toxin inhibits transmitter release by reducing the entry of Ca2+ into cytoplasm. It is suggested that beta-AgTX-binding site in synaptic membrane is related to the release of a variety of transmitters.