P. Migani

Kainic Acid Differentially Affects the Synaptosomal Release of Endogenous and Exogenous Amino Acidic Neurotransmitters

Abstract: Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome‐enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+‐dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40–80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D‐[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+‐stimulated release. Kainic acid at 0.1 mM had no clear‐cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier‐mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high‐affinity uptake of L‐glutamate and D‐aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed.

Protection from kainic acid neuropathological syndrome by NMDA receptor antagonists: Effect of MK-801 and CGP 39551 on neurotransmitter and glial markers

Systemic administration of kainic acid results in the development of a characteristic convulsive syndrome, accompanied by neuropathological alterations and loss of transmitter markers in some forebrain regions. Since some of these effects appear to involve the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors, the protection given by a non-competitive (MK-801) and a competitive (CGP 39551) NMDA receptor antagonist against the loss of glutamatergic and gamma-amino butyric acid (GABAergic) neurochemical markers was compared. Appropriate doses of both compounds (1 mg/kg MK-801 and 25 mg/kg CGP 39551) completely reversed the decrease of high affinity uptake of glutamate and activity of glutamate decarboxylase in the olfactory cortex, amygdala, hippocampus and lateral septum. In addition, they also essentially counteracted the increase of a glial marker, the enzyme glutamine synthetase, consequent to neuronal degeneration. The results confirmed that involvement of NMDA receptors is essential for the full expression of neuropathological effects of kainic acid. They also support the use of a competitive antagonist of the NMDA receptor, such as CGP 39551, to afford substantial protection against the excitotoxic damage, whilst giving fewer side effects and motor disturbances than MK-801.

Evidence of intrinsic cholinergic circuits in the optic tectum of teleosts

Choline acetyltransferase (CAT) was assayed in the optic tectum of 4 teleost species with different visual powers. The results showed a close relationship between the enzyme levels in the optic tectum and the development of the visual system. In the more visual species, the trout, CAT activity in the optic tectum was about 30-fold higher than in the catfish, whose visual system is much less developed. Two species with intermediate development of the visual system, the goldfish and the tench, showed intermediate levels of CAT activity. Kainic acid treatment caused a significant decrease in both CAT and acetylcholinesterase (AChE) in the goldfish optic tectum. Concomitant histological examination showed, among other effects, the disappearance of most neurons belonging to the pyramidal and fusiform type in the striatum fibrosum and griseum superficiale of the tectum. The comparative and experimental data therefore suggest that the relationship between cholinergic mechanisms and the visual function is, to a significant extent, connected with the presence of intrinsic cholinergic circuits in the optic tectum. The relevance of these findings, also in relation to the problem of the identification of the retino-tectal transmitter, is discussed.

Neurotoxic effect of kainic acid on ultrastructure and gabaergic parameters in the goldfish cerebellum

Kainic acid administration into the cerebellar dorsal lobe of the goldfish causes selective degeneration of some neuronal types. Stellate and Golgi neurons are very sensitive to the neurotoxin and undergo rapid degeneration. On the basis of their differential responses to kainic acid, Purkinje cells can be divided in two distinct sub-populations (i.e. sensitive and insensitive neurons). The degenerative changes of the Purkinje neurons are in addition remarkably slow in comparison with the same cells in mammals or with stellate and Golgi neurons in the goldfish. Granule cells, as well as the cerebellar afferent fiber system, are not significantly affected. Six days after kainic acid administration, the level of glutamate decarboxylase in the cerebellar dorsal lobe drops to about 40% of the control value. This result suggests that the neurons sensitive to kainic acid neurotoxicity are, at least in part, GABAergic. Light- and electron-microscopic autoradiography of cerebellar elements selectively accumulating [3H]GABA, supports this idea. Moderate decreases of acetylcholinesterase and protein content were also noticed in the kainic acid-treated cerebellar dorsal lobe.

Evidence for a neurotransmitter role of aspartate and/or glutamate in the projection from the torus longitudinalis to the optic tectum of the goldfish

Different experimental approaches have been used to demonstrate that aspartate and/or glutamate is a transmitter(s) in the projection from the torus longitudinalis to the marginal layer of the optic tectum in the goldfish. Slices of the optic tectum incubated in vitro in the presence of D-[3H]aspartate and processed for light microscopic autoradiography, demonstrated a preferential accumulation of the labeled compound in the marginal layer. Under the same experimental conditions several neurons in the central part of the torus longitudinalis selectively accumulated D-[3H]aspartate. Synaptosome-enriched preparations from the optic tectum showed high-affinity uptake for D-[3H]aspartate and the rate of the uptake was significantly decreased after disconnection from the ipsilateral torus longitudinalis. The same subcellular preparations showed Ca2+-dependent release of previously accumulated D-[3H]aspartate under high potassium stimulation. This release was significantly reduced in preparations from optic tecta 5 days after cutting their connection with the ipsilateral torus longitudinalis. Finally, D-[3H]aspartate injected in the optic tectum retrogradely labeled the fiber systems connecting the marginal layer with the ipsilateral torus longitudinalis as well as neuronal cell bodies in the torus longitudinalis itself. From autoradiographic experiments it was, in addition, noticed that several tectal neurons selectively accumulated D-[3H]aspartate in the cell bodies as well as in main dendritic trunks. This observation suggests tht aspartate and/or glutamate may be a transmitter(s) in some intrinsic circuits and extrinsic projections of the optic tectum.

Effect of kainic acid on ultrastructure and γ-aminobutyrate-related circuits in the optic tectum of the goldfish

Abstract Following unilateral microelectrophoretic delivery of kainic acid in the optic tectum of the goldfish, an ultrastructural and biochemical study was carried out. Kainic acid exerted a powerful neurotoxic effect against several types of tectal neurons, noticeably the periventricular neurons and the pyramidal and fusiform neurons of the stratum fibrosum et griseum superficiale. The neurotoxic effect of kainic acid was found to be highly selective. In fact, only some of the different neuronal populations underwent degenerative changes, while other neurons of the same type, and often in very close vicinity, were completely unaffected. Kainic acid neurotoxicity allows us therefore to discriminate between apparently homogeneous neuronal populations, probably on the basis of different neurochemical characteristics possessed by neurons of the same morphological type. The lack of neurotoxic effect against afferent fibres and axon terminals was assessed. Long-term observations of the affected optic tectum after kainic acid treatment demonstrated a remarkable level of structural rearrangement. A sharp decrease in the level of glutamate decarboxylase activity was noticed during the first six days after kainic acid treatment. This was followed by a partial recovery of enzyme activity, which, however, did not progress from 15 days to 2 months after operation. On the other hand no decrease of glutamate decarboxylase activity occurred in the left optic tectum six days and one month after surgical ablation of the right eye. These results suggest the presence of intrinsic γ-aminobutyrate-containing systems in the goldfish optic tectum. The existence of intrinsic neurons that take up γ-aminobutyrate was confirmed by light-microscopic autoradiography of the optic tectum of normal goldfish after local injection of [3H]γ-aminobutyrate.

Ultrastructural and neurochemical effects of the presumed cholinergic toxin AF64A in the rat interpeduncular nucleus

We have studied the effect of the presumptive cholinergic neurotoxin, ethylcholine mustard aziridinium ion (compound AF64A), on ultrastructure and neurochemical markers in the rat interpeduncular nucleus (IPN). Stereotaxic injections of 1 nmol of AF64A resulted in extensive degeneration of synaptic terminals within 40 h. Ultrastructural damage to neuronal cell bodies, dendrites and axons was also sometimes observed at this stage. Five days after the injection, more severe degenerative changes were observed in a larger number of neuronal cell bodies, axons and dendrites. High affinity uptake of [3H]choline, but not [3H]GABA, was significantly decreased 24 h after toxin injection. Five days after the injection, not only choline acetyltransferase but also glutamate decarboxylase levels were significantly decreased. Our results suggest that, in addition to presynaptic cholinergic neurotoxicity, AF64A also leads to degenerative alterations of non-cholinergic neurons. Our electron microscopic observations constitute the first ultrastructural report on neuropathological damage caused by AF64A.

Synaptosomal release of newly-synthetized or recently accumulated amino acids. Differential effects of kainic acid on naturally occurring excitatory amino acids and on [d-3H]aspartate

Kainic acid, a powerful neuroexcitant and neurotoxin, stimulates the release of naturally occurring excitatory amino acids, l-glutamate and l-aspartate, from hippocampal synaptosomes. The release stimulation affects in a similar way both the general pool of the two amino acids and the fraction of l-glutamate and l-aspartate, newly-synthetized from precursors or recently accumulated through the high-affinity uptake mechanism. Kainic acid exerts its stimulatory action on the basal release of the two amino acids as well as on the high K(+)-stimulated release of l-glutamate. Kainic acid has, however, different effects on the release of exogenously accumulated [d-(3)H]aspartate. In particular, the high K(+)-stimulated release of this false transmitter is strongly inhibited by 1 mM kainic acid. The present data confirm the presynaptic action of kainic acid on the general as well as on the recently-formed pools of naturally occurring excitatory amino acids. At the same time, our results suggest that [d-(3)H]aspartate is not a reliable substitute for l-glutamate and l-aspartate, in release studies and that the radioactivity released after preloading with [d-(3)H]aspartate does not necessarily reflect the release of naturally occurring excitatory amino acids.

Kainic acid neurotoxicity does not depend on intact retinal input in the goldfish optic tectum

Kainic acid neurotoxicity has been studied in the optic tectum of the goldfish 4 weeks after eye enucleation. The effect of drug treatment has been tested with respect to both neurochemical and morphological parameters. The neurotransmitter-related enzymes, choline acetyltransferase, acetylcholinesterase and glutamate decarboxylase, show about 50% decrease in the deafferented tectum 6 days after kainic acid administration. Relevant morphological alterations of the tectal structure can also be noticed at the same stage. The neurotoxic effects of kainic acid in the deafferented optic tectum are therefore quite similar to the effects of previously noticed for the intact optic tectum of normal fish. Control experiments on the effect of optic nerve degeneration by itself on the levels of the neurotransmitter-related enzymes in the optic tectum, have shown no significant decrease in glutamate decarboxylase, a slight decrease in acetylcholinesterase and a more marked drop in choline acetyltransferase. The findings are discussed with reference to some of the hypotheses advanced in order to explain kainic acid neurotoxicity. It is proposed that the neurotoxic effect of kainic acid after removal of specific excitatory afferents, may vary in different nervous centers depending on differences of the remaining extrinsic connections and of the intrinsic neural circuits.

Cholinergic, GABAergic and excitatory amino acidic neurotransmission in the goldfish vagal lobe

SummarySome neurotransmitter systems operating in the goldfish vagal lobe, an hypertrophied gustatory center, have been studied by means of experimental (kainic acid injection and vagal rhizotomy), neurochemical and ultrastructural methods. The use of the neurotoxin, kainic acid, revealed the existence of cholinergic and GABAergic neurons in the vagal lobe. The results of histochemical observations and biochemical assays performed after rhizotomy of sensory and motor vagal roots, suggest that the motor neurons of the vagal motor layer are cholinergic. The same experiments also indicate that the primary gustatory afferents distributing to the sensory layer of the vagal lobe are, at least in part, cholinergic. By contrast, no decrease of excitatory amino acid uptake was demonstrated following the experimental lesions. GABA is likely to play an important role in the goldfish vagal lobe, particularly in the sensory layer, where the highest level of its synthetic enzyme, glutamate decarboxylase, is recorded. The significant decrease of glutamate decarboxylase in the sensory layer after vagal rhizotomy suggests that either GABAergic primary afferents reach the vagal lobe, or that deafferentation results in a decreased GABA synthesis in intrinsic GABAergic neurons.