Excitatory amino acid receptors in the prefrontal cortex of aging mice.

The zones of the prefrontal cortex of Balb/c mice were tested for age-related changes of the ionotropic excitatory amino acid receptors density, together with zones of the dorsal cortex. Kainate, N-methyl-D-aspartate, and amino-3-hydroxy-5-methyloxazole-4-propionate sites were measured by slice receptor binding techniques in cortical zones from animals at the age of 6, 12, 18, and 24 months. An increase of the N-methyl-D-aspartate sites was detected in the medial prefrontal zone of mid-aged animals and was followed by a decrease at old age; a decrease of the N-methyl-D-aspartate and kainate sites was found for the medial dorsal (cingulate) cortex at old age. The age-related changes of receptor densities in the different cortical areas seem unrelated in origin. The sites decrease in the cingulate cortex could affect the transfer of the prefrontal cortex activity toward limbic structures.

Neurochemical parameters of the main neurotransmission systems in aging mice.

The present work was designed to study the effect of aging on some parameters of the glutamatergic, aminergic and cholinergic neurotransmission, in the main brain areas of mice of the long-surviving BALB/c-nu strain. We have assayed: (1) the density of three ionotropic receptors for excitatory aminoacids (EAA) which selectively bind kainic acid (KA), N-methyl-D-aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA); (2) the content of dopamine (DA), norepinephrine (NE) and serotonin (5-HT) and the levels of the DA metabolite dihydrophenylacetic acid (DOPAC) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA); (3) the level of the choline acetyltransferase (ChAT), the enzyme catalyzing the synthesis of acetylcholine. The parameters were measured in animals at the age of 6, 12, 18 and 24 months; the brain zones under test were the frontal cortex (FC), the corpus striatum (STR), the hippocampus (HIP), the medio-dorsal cortex (DC) and the cerebellum (CER). Significant age-related variations for the density of KA-type and NMDA-type receptors were found in STR and a decrease of the NMDA parameter was found in DC. Neither the monoamine and metabolite contents nor the ChAT levels showed any significant variation in all the tested areas. These findings suggest that an unbalance among different neurotransmission activities could take place with normal aging in rodents: it could be involved in the onset of the motor deficit which occurs in the elderly of these and other mammals.

Role of guanine nucleotides as endogenous ligands of a kainic acid binding site population in the mammalian cerebellum.

An endogenous inhibitor of the membrane binding of kainic acid was extracted from pig brain tissue and purified. The substance was identified as GMP by structural analysis: Most likely it corresponds to an inhibitor previously extracted from the rat brain. The nucleotide is active as an inhibitor for kainate binding on goldfish brain synaptosomes, probably owing to direct displacement on receptor sites; it is also active on a low-affinity kainate site population in membranes from rat cerebellum. The interaction of GMP with the latter sites leads to a concentration-dependent kainate binding increase or inhibition, thus demonstrating that these sites can bind the nucleotide and cooperatively increase their affinity. Other guanine nucleotides show interaction with these sites, by either an increase (GTP) or inhibition (cyclic GMP or GDP) of kainate binding. These findings support the view that a guanine nucleotide is the endogenous ligand of a receptor in the mammalian cerebellum similar to the kainate binding protein present with high density in the cerebellum of lower vertebrates, whose function is probably connected to the role of the glial cells in this zone.

An endogenous ligand for the kainate-type binding sites from rat brain.

Extracts from the rat brain were screened to identify a putative endogenous ligand for the binding sites of the neuroexcitant kainic acid (KA). The extracted substances were separated by chromatographic techniques and tested for their ability to inhibit KA binding to fish synaptosomes and to membranes from rat brain. A substance isolated in this way (rat kainate-binding inhibitor, RKBI) display a competitive interaction with KA for the low-affinity binding sites in rat brain membranes. According to the separation behavior in the purification step, RKBI is distinct from an inhibitor formerly isolated from fish nervous tissue (KBI). The substance exhibits positive co-operativity with KA for a very-low-affinity site population, particularly concentrated in the cerebellum, and could play a physiological role in this area.

Kainic acid neurotoxicity in the goldfish optic tectum. No protection by the NMDA receptor antagonist MK801 and partial protection by abolition of a tectal excitatory input.

Some features of the mechanism of kainic acid neurotoxicity were tested after the injection of this substance in the optic tectum of goldfish. A systemic treatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK801 did not influence the degree of toxicity, assessed by a decrement of enzymatic neuronal markers; instead, a significant neuronal rescue was obtained after the abolition of the excitatory input from the torus longitudinalis to the tectum.

Pharmacological manipulation of the NMDA receptor differentially protects from systemic kainic acid neuropathology: evaluation through ornithine decarboxylase induction, morphology and GFAP immunohistochemistry.

The excitotoxic brain damage caused by systemic administration of kainic acid requires the activation of N-methyl-D-aspartate (NMDA) receptors in order to fully express its neurotoxic potency. We have tested the relative efficacy of different manipulations of the NMDA receptor on morphological, immunohistochemical and neurochemical parameters in this experimental model. A competitive (CGP 39551) and a non-competitive (MK 801) antagonist of the NMDA receptor, granted full protection against neuronal degeneration and consequent glial proliferation in the hippocampus and olfactory cortex, two regions severely affected by systemic administration of kainic acid. In addition, CGP 39551 completely counteracted the dramatic induction of the enzyme ornithine decarboxylase which occurs shortly after kainic acid administration. Systemic administration of high amounts of MgSO4 concomitantly and after kainic acid injection, appeared to partially prevent neuronal degeneration but had no clear effects on glial reaction and ornithine decarboxylase induction. Finally administration of an antagonist of the polyamine site present in the NMDA receptor (SL 82.0715), did not appear to have any protective effect at the dose used here. The present results help to better understand the ways by which it could be possible to counteract excitotoxic brain injuries.

Induction of brain ornithine decarboxylase after systemic or intrastriatal administration of kainic acid.

The activity of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, dramatically increases after different types of brain injuries. The role of this induction is still unclear. We report here data on the temporal pattern of ODC induction caused by the excitotoxin kainic acid. After systemic administration, ODC activity increases severalfold peaking at 8 h in the prefrontal cortex and at 16 h in the olfactory cortex and hippocampus. After intrastriatal injection, the peak of induction is reached at 32 h, while a smaller and more transient increase is also observed in the contralateral, saline-injected striatum. We suggest that ODC induction is initially linked to overactivation of neural circuits and, later on, to the development of widespread neural damage.

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.

Anatomical and neurochemical evidence for suicide transport of a toxic lectin, volkensin, injected in the rat dorsal hippocampus.

Volkensin, a ribosome-inactivating toxic lectin which has been proposed as a 'suicide transport' agent in the CNS, was unilaterally injected in the rat dorsal hippocampus at a dose of 1.2 ng. Three to 5 days after the injection, degenerating neurons were observed at the electron microscope in the medial septum-diagonal band area ipsilateral to the injection. Ten days after the injection, the number of pyramidal neurons in the CA3 region of the contralateral hippocampus, which are the major source of hippocampal commissural fibers, was obviously decreased. At the same survival time, the number of choline acetyltransferase (ChAT) immunoreactive neurons in the ipsilateral medial septum-diagonal band area was moderately but significantly decreased. These neurons are known to be the major source of the septohippocampal cholinergic projection. Concomitantly, microchemical assays of ChAT levels revealed a 25% decrease of enzyme activity in the medial septum-diagonal band area ipsilateral to the injection. This was accompanied by a 33% decrease of ChAT in the ipsilateral ventral hippocampus which was interpreted to be due, at least in part, to the degeneration of cholinergic septal neurons projecting to both the dorsal and the ventral hippocampus. Taken together, these results provide clear evidence that volkensin is taken up by nerve terminals in the injected area of the brain and retrogradely transported to the cell bodies originating the projection, which are killed by the toxin. The usefulness of the strategy of 'suicide transport' in the CNS is, therefore, confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of short- and long-term ganglioside treatment on the recovery of neurochemical markers in the ibotenic acid-lesioned rat striatum.

The striatum of adult rats was bilaterally lesioned with stereotaxic injections of ibotenic acid in a dose (16 nmoles) that resulted in subtotal lesions. Some rats received systemic ganglioside treatment starting the day before operation and lasting for 6 or 24 days after operation; they were compared with lesioned rat receiving systemic saline injections as well as with corresponding groups of sham-operated animals. Specific neurochemical markers for cholinergic neurons (choline acetyltransferase, ChAT), GABAergic neurons (glutamate decarboxylase; GAD), and astrocytes (glutamine synthetase; GS) were assayed to asses the neurochemical recovery promoted by ganglioside treatment. Twenty-four, but not six, days after operation a significant increase of ChAT and GAD was measured in the striatum of lesioned rats treated with gangliosides in comparison with the saline group. Furthermore, a significant increase of both enzymes occurred in the striatum of lesioned rats receiving ganglioside treatment for 24 days in comparison with rats receiving ganglioside treatment for 6 days only. A small but significant increase of ChAT was measured in the striatum of sham-operated rats after 24 days of ganglioside treatment in comparison with the corresponding saline group. Finally, the increase of GS caused by the glial reaction to the ibotenic acid lesion was not affected by ganglioside treatment. The results indicate that a relatively long-lasting ganglioside treatment stimulates the recovery of specific neuronal transmitter markers and that some effect is, in addition, exerted on unlesioned cholinergic striatal neurons.

A competitive inhibitor of kainic acid binding from the goldfish nervous tissue.

An inhibitor of the receptor binding of the neuroexcitant kainic acid was extracted from the nervous tissue of the goldfish and purified. The substance acts as a competitive inhibitor (displacer) on the kainate binding sites in membranes from the fish nervous system; this action is selective since the substance does not affect the membrane binding of glutamate, the common ligand for the excitatory amino acid binding sites. The interaction of the substance with the fish kainate binding sites displays a positive cooperativity, similar to that measured for kainic acid itself. Thus the endogenous kainate binding inhibitor (KBI) can be assumed as a candidate for the role of physiological ligand of receptors for kainic acid in the fish. The substance, at the tested concentration, does not significantly affect the binding of kainic acid in membranes from rat brain while it is active on the sites from the pigeon cerebellum. The relevance of these findings for the understanding of the functional heterogeneity of the kainate receptors in different species is discussed.

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.

Distribution of kainic acid receptor binding sites in the goldfish CNS: evidence for the existence of intracellular sites.

The density of binding sites for kainic acid was measured both in fresh slices and in the particulate fraction of the homogenate from various zones of the CNS of goldfish. Binding in the homogenate fraction was always found higher than in the corresponding slices, which should be representative of receptors located on the outer surface of the cellular membrane. The hypothesis is discussed that the sites demonstrated by homogenization are located in the intracellular compartment.

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

Some 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.

Synaptosomal release of newly-synthetized or recently accumulated amino acids. Differential effects of kainic acid on naturally occurring excitatory amino acids and on [d-(3)H]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 differentially affects the synaptosomal release of endogenous and exogenous amino acidic neurotransmitters.

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 of 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.

[3H] kainic acid binding sites in the synaptosomal-mitochondrial (P2) fraction from goldfish brain.

Binding of [3H]kainic acid to the synaptosomal-mitochondrial fraction (P2) of the goldfish brain was studied. Specific binding to this fraction represents about half of the total binding capability of the homogenate particulate material and is enriched in synaptic membranes; it is greater by about two orders of magnitude than those given for rat brain and pigeon optic tectum membranes. Association of the ligand-site complex has a time constant lower than 1 min and the same is true for the main component of the dissociation process. The binding equilibrium is apparently not affected by substances contained in the fraction material. The analysis of the dose-response data showed a main receptor population (B max = 139 pmol/mg protein) which displayed positive cooperativity (nH = 1.29). The same behaviour was shown by washed membranes from the same fraction but, in this case, the affinity for the ligand was lower (apparent affinity constants: K'D = 0.28 nM for the intact fraction and K'D = 0.38 nM for membranes). A smaller population of sites with higher affinity was also detected both in the intact fraction and in membranes. Among the substances tested as displacers of kainic acid from the synaptosomal sites, the most effective were quisqualate and L-glutamate. Folic acid and its dihydro and tetrahydro derivatives were half as potent as glutamate whereas methyltetrahydrofolic acid and folinic acid had a very weak action. The difference between these sites and those detected on rat brain membrane preparations is discussed.

Neurotransmitter-related markers in the normal and experimentally lesioned telencephalon of the goldfish.

The telencephalon of teleost fish shows high affinity uptake for D-[3H]aspartate, intermediate levels of GABAergic markers and low levels of cholinergic enzymes. Experimental results (resection of the olfactory tracts or unilateral kainic acid administration in the telencephalon) suggest that: the projection from the olfactory bulbs to telencephalic targets is mediated by aspartate and/or glutamate; and a population of GABAergic neurons is present in a telencephalic area which is considered homologous to part of the striatal complex of land vertebrates. From the present results, it appears that the neurochemical approach can be used, in conjunction with neuroanatomical methods, to study evolutionary problems of telencephalic function.

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.