Marta Pazzagli

Effects of novelty and habituation on acetylcholine, GABA, and glutamate release from the frontal cortex and hippocampus of freely moving rats.

The involvement of the forebrain cholinergic system in arousal, learning and memory has been well established. Other neurotransmitters such as GABA and glutamate may be involved in the mechanisms of memory by modulating the forebrain cholinergic pathways. We studied the activity of cortical and hippocampal cholinergic, GABAergic and glutamatergic systems during novelty and habituation in the rat using microdialysis. After establishing basal release of the neurotransmitters, the animals were transferred to a novel environment and allowed to explore it twice consecutively for 30 min (60 min apart; exploration I and II). The motor activity was monitored. Samples were collected throughout the experiment and the release of acetylcholine (ACh), GABA and glutamate was measured. During the two consecutive explorations of the arena, cortical and hippocampal, ACh release showed a significant tetrodotoxin-dependent increase which was higher during exploration I than II. The effect was more pronounced and longer-lasting in the hippocampus than in the cortex. Cortical GABA release increased significantly only during exploration II, while hippocampal GABA release did not increase during either exploration. Motor activity was higher during the first 10 min of exploration I and II and then gradually decreased during the further 20 min. Both cortical and hippocampal ACh release were positively correlated with motor activity during exploration II, but not during I. During exploration II, cortical GABA release was inversely correlated, while hippocampal GABA release was positively correlated to motor activity. No change in cortical and hippocampal glutamate release was observed. In summary, ACh released by the animal placed in a novel environment seems to have two components, one related to motor activity and one related to attention, anxiety and fear. This second component disappears in the familiar environment, where ACh release is directly related to motor activity. The negative relationship between cortical GABA levels and motor activity may indicate that cortical GABAergic activity is involved in habituation.

Nerve growth factor increases extracellular acetylcholine levels in the parietal cortex and hippocampus of aged rats and restores object recognition

Male Wistar rats (3- and 20-month-old) were perfused i.c.v. with 1.5 micrograms of either nerve growth factor (NGF) or cytochrome C daily for 14 days. At the end of the infusion, the object-recognition test was carried out and extracellular acetylcholine levels (ACh) were measured in the cortex and hippocampus by transversal microdialysis technique. In 20-month-old control rats, the cortical and hippocampal ACh levels were 35 and 45% lower, respectively, than in 3-month-old rats and the ability to discriminate between a familiar and new object was impared. In the old rats treated with NGF, the ACh release as well as the behavioral performance showed no difference from those of young rats. These findings indicate that both ACh levels and memory impairment are improved in aged rats by NGF treatment and suggest that there is a relationship between object recognition and the activity of the forebrain cholinergic system.

A2 adenosine receptors: their presence and neuromodulatory role in the central nervous system

1. Adenosine is an endogenous neuromodulator that exerts its depressant effect on neurons by acting on the A1 adenosine receptor subtype. Excitatory actions of adenosine, mediated by the activation of the A2 adenosine receptor subtype, have also been shown in the central nervous system. 2. Adenosine A2a receptors are highly localized in the striatum, as demonstrated by the binding assay of the A2a selective agonist, CGS2680, and by analysis of the A2 receptor mRNA localization with in situ hybridization histochemistry. However, adenosine A2a, receptors, albeit at lower levels, are also localized in other brain regions, such as the cortex and the hippocampus. 3. In the striatum, adenosine A2a, receptors are implicated in the control of motor activity. Evidences exists of an antagonistic interaction between adenosine A2a and dopamine D2 receptors. 4. Utilizing selective agonists and antagonists for adenosine A2a receptors, their role in the modulation of the release of several neurotransmitters (acetylcholine, dopamine, glutamate, GABA) has been extensively studied in the brain (striatum, cortex, hippocampus). Controversial results have been obtained and, because the overall effect of endogenous adenosine in the brain is that of an inhibitory tonus, the physiological meaning of the excitatory A2 receptor remains to be clarified.

Effect of K+ depolarization, tetrodotoxin, and NMDA receptor inhibition on extracellular adenosine levels in rat striatum

Extracellular adenosine in the striatum of adult male rats was measured by the use of a microdialysis fibre inserted transversely in the striatum. The adenosine concentration in samples of perfusate was determined by HPLC coupled to U.V. detection. The adenosine concentration (corrected for recovery) decreased after implantation of the probe. Two hours later it was 1.83 +/- 0.22 in anaesthetized rats, whereas it was 40% higher in rats in which anaesthesia had been discontinued. Twenty-four hours later the adenosine concentration was 0.124 +/- 0.09 microM; the addition of dipyridamole (100 microM), an adenosine uptake blocker, to the perfusate resulted in a 76% increase in adenosine concentration in the effluent, whereas addition of the adenosine deaminase inhibitor erythro-2-(hydroxy-3-nonyl) adenine (100 microM) caused a 260% increase. The addition of tetrodotoxin (1 microM) was followed by a decrease in basal adenosine concentration and a partial inhibition of the increase in adenosine evoked by K+ depolarization. The increase induced by high K+ was markedly inhibited by the NMDA receptor antagonist D(-)-amino-7-phosphoeptanoic acid (1 mM, D-AP7). These findings indicate that the extracellular adenosine level is influenced by neuronal activity, and that under strong depolarizing conditions the increase in adenosine level involves NMDA receptor activation.

Regulation of extracellular adenosine levels in the striatum of aging rats

Extracellular adenosine concentrations, evaluated by microdialysis in the striatum of young and aged rats, were 66.8 +/- 0.7 and 71.6 +/- 1.0 nM, respectively. The adenosine deaminase inhibitor EHNA (100 microM) increased the extracellular adenosine levels in young rats only. The adenosine kinase inhibitor iodotubercidin (10 microM) brought about the same increase in young and aged rats. In aged rats the resting adenosine outflow was reduced by the N-methyl-D-aspartate (NMDA) receptor antagonist D-(-)-2-amino-7-phophonoheptanoic acid (D-AP7) (1 mM). It is concluded that extracellular levels of adenosine in the striatum are not affected by age, irrespective of the differences in adenosine deaminase activity and that the release of excitatory amino acids is responsible for much of resting adenosine outflow in aged but not in young rats.

The glycopeptide CSF114(Glc) detects serum antibodies in multiple sclerosis

Synthetic glycopeptides have the potential to detect antibodies in multiple sclerosis (MS). In the present study, we analyzed the antibodies (IgM class, IgG class and IgG subclasses) to the synthetic glycopeptide CSF114(Glc) in the serum of 186 MS patients, 166 blood donors (BDs), 25 patients affected by meningitis/encephalitis, 41 affected by systemic lupus erythematosus (SLE) and 49 affected by rheumatoid arthritis (RA). The IgM antibody level to CSF114(Glc) was significantly increased in MS patients versus BDs (p<0.001) or versus other autoimmune diseases (SLE or RA, p<0.001). The IgG response was restricted to the subclass IgG2. IgM antibodies to CSF114(Glc) were found in 30% of relapsing/remitting MS patients and, at lower levels, in subjects affected by meningitis/encephalitis. The study of antibodies to CSF114(Glc) is a new, potential immunological marker of MS.

In vivo regulation of extracellular adenosine levels in the cerebral cortex by NMDA and muscarinic receptors

The adenosine concentration in samples of perfusate was determined 24 h after implantation of microdialysis fibre in the cortex. High performance liquid chromatography coupled with a fluorometric detector was used. K+ (100 mM) depolarization was followed by a 2- to 4-fold increase in adenosine efflux. The addition of tetrodotoxin (1 microM) to the perfusate was followed by a decrease in spontaneous and K(+)-evoked adenosine efflux. The increase induced by high K+ was markedly inhibited by the NMDA receptor antagonist, D(-)-2-amino-7-phosphonoheptanoic acid (1 mM, D-AP7), but not by the muscarinic receptor antagonist, atropine (1.5 microM). The acetylcholine esterase inhibitor, physostigmine (7 microM), and the muscarinic receptor agonist, oxotremorine (100 microM), significantly enhanced the K(+)-evoked increase in adenosine. The spontaneous efflux of adenosine was not modified by any of the drugs tested. A neurotoxic lesion of the cholinergic pathway innervating the cortex, although inducing a marked decrease in cortical choline acetyltransferase activity, did not significantly modify the cortical adenosine efflux. It is concluded that, under K(+)-depolarizing conditions, adenosine efflux is triggered by excitatory amino acids and enhanced by muscarinic activation.

Endogenous adenosine release from hippocampal slices : excitatory amino acid agonists stimulate release, antagonists reduce the electrically-evoked release

SummaryThe effect of excitatory amino acids and their antagonists on adenosine and inosine release has been investigated on unstimulated and electrically stimulated hippocampal slices.On unstimulated slices N-methyl-D-aspartate (NMDA), quisqualate and glutamate concentration-dependently evoked the release of adenosine and inosine. The effect of NMDA and quisqualate was antagonized by the NMDA receptor antagonist D(−)-2-amino-7-phosphonoheptanoic acid (D-AP7; 100 μmol/1) and the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 μmol/1) respectively. Glutamate (2 and 10 mmol/1)-evoked adenosine and inosine release was not antagonized by the NMDA and non-NMDA receptor antagonists indicating that the effect of glutamate is due to a metabolic rather than a receptor-mediated effect.Electrical field stimulation at 10 Hz also evoked a release of endogenous adenosine and inosine. Tetrodotoxin (0.5 gmol/1) abolished and absence of Ca2+ markedly reduced the electrically evoked release of adenosine and inosine. Adenosine and inosine release evoked by electrical stimulation at 20 Hz was significantly reduced in the presence of the NMDA receptor antagonist D-AP7, while at 10 Hz no consistent decrease was seen. In the presence of D-AP7 plus DNQX the 10 Hz-evoked adenosine and inosine release was reduced to about half. These data suggest that the electrically evoked release of adenosine and inosine is partly mediated by the release of excitatory amino acids which act at both non-NMDA and NMDA receptors.

Regional differences in the electrically stimulated release of endogenous and radioactive adenosine and purine derivatives from rat brain slices.

SummaryThe release of both radioactive and endogenous purines was investigated in rat brain cortical, hippocampal and striatal slices at rest and following stimulation with electrical fields.Purities were labelled by incubating the slices with 3H-adenine. The purine efflux at rest and that evoked by electrical stimulation (10 Hz, 5 min) was analyzed by HPLC with ultraviolet absorbance detection. Both radio-active and endogenous purines in the effluent consisted mainly of hypoxanthine, xanthine, inosine and adenosine. No qualitative differences in the composition of the released purines were found in the three areas investigated. Electrical stimulation evoked a net increase in both radioactive and endogenous purine release. However the increase in 3H-adenosine following electrical stimulation was twice as large as that of endogenous adenosine. The electrically evoked release of both radioactive and endogenous purines was greatest in hippocampal slices and progressively smaller in cortical and striatal slices. In the three areas the addition of 0.5 μM tetrodotoxin to the superfusing Krebs solution brought about a similar (83–100%) reduction in evoked 3H-purine and endogenous purine release. Superfusion of the slices with calcium-free Krebs solution containing 0.5 mM EGTA reduced evoked release of 3H-purines by 58–60% and that of endogenous purine components by 54–89%.The results demonstrate similar characteristics for both radioactive and endogenous purine release but indicate that the most recently synthetized adenosine is the most readily available for release. The features of the electrically evoked purine release support a neuronal origin of adenosine and derivatives and are consistent with the hypothesis of discrete regional differences in adenosine neuromodulation.

In Vivo Amino Acid Release From the Striatum of Aging Rats: Adenosine Modulation

The release of glutamate, aspartate, GABA, and taurine from the striatum of young (3 months), mature (12 months), and old (22 months), freely moving male rats was investigated by using a microdialysis fiber inserted transversally in the striatum. In old rats basal extracellular glutamate and aspartate levels were decreased vs. young rats (-38 and -49%, respectively). GABA and taurine levels were unmodified by age. In the presence of the adenosine receptor antagonist 8-phenyltheophilline (8-pT) at the concentration of 50 microM, both K(+)-evoked releases of glutamate and aspartate were more than doubled in young, but not in mature and old rats. 8-pT at the concentration of 500 microM significantly decreased glutamate basal levels and K(+)-evoked aspartate release in old rats only. GABA and taurine releases were not affected by 8-pT at either dose. Our findings indicate a modified adenosine modulation on glutamate and aspartate release in aged rats, that could result from a change in the balance between A1 and A2a adenosine receptor density or an alteration of A1 and A2a receptor-effector coupling.