Maria Alessandra Colivicchi

Neuro-inflammation induced in the hippocampus of 'binge drinking' rats may be mediated by elevated extracellular glutamate content

The neuropathological and immune changes induced in the brain by ‘binge drinking’ have been investigated in a rat model. Evidence of neuro‐inflammation was identified in the ‘binge drinking’ rat model of alcohol abuse after 3 weeks of administration of 2 or 3 g/kg ethanol (EtOH), three times per day for two consecutive days, followed by 5 days of abstinence: Firstly, alveolar macrophages, isolated from these animals, showed significant increases in inducible nitric oxide synthase, as assayed by nitrite release, both before and after lipopolysaccaharide stimulation. Secondly, significant numbers of activated microglia were present in the dentate gyrus region of the hippocampus of the ‘binge drinking’ model, after major histocompatibility complex class II staining, by comparison with the control. Microdialysis studies in the ventral hippocampus identified a significant increase in the basal extracellular concentration of glutamate, in both the 2 and 3 g/kg administered ‘binge drinking’ rats. In contrast, no changes in the hippocampal extracellular concentrations, of GABA and taurine, or the dopamine and serotonin metabolites were observed under basal conditions. A further dose of EtOH induced a significant decrease in the concentrations of both 3,4‐dihydroxyphenylacetic acid and 5‐hydroxyindoleacetic acid, whereas glutamate, taurine and GABA levels were unaffected. There was no evidence that EtOH preference was initiated by the ‘binge drinking’ regimen. Our results suggest that the possible toxicity associated with ‘binge drinking’ maybe directed by the elevated glutamate levels, which in turn, activate phagocytic cells to release their inflammatory cytokines and chemokines, ultimately leading to neuro‐inflammation.

Highly reactive oxygen species: detection, formation, and possible functions

The so-called reactive oxygen species (ROS) are defined as oxygen-containing species that are more reactive than O2 itself, which include hydrogen peroxide and superoxide. Although these are quite stable, they may be converted in the presence of transition metal ions, such as Fe(II), to the highly reactive oxygen species (hROS). hROS may exist as free hydroxyl radicals (HO·), as bound (“crypto”) radicals or as Fe(IV)-oxo (ferryl) species and the somewhat less reactive, non-radical species, singlet oxygen. This review outlines the processes by which hROS may be formed, their damaging potential, and the evidence that they might have signaling functions. Since our understanding of the formation and actions of hROS depends on reliable procedures for their detection, particular attention is given to procedures for hROS detection and quantitation and their applicability to in vivo studies.

Investigation on Acetylcholine, Aspartate, Glutamate and GABA Extracellular Levels from Ventral Hippocampus During Repeated Exploratory Activity in the Rat

The extracellular levels of aspartate, glutamate, γ-aminobutyric acid (GABA), and acetylcholine (ACh) were investigated by microdialysis, coupled with HPLC, in the ventral hippocampus of rats during two 30-min exploration periods. Motor activity was monitored. During exploration I, an increase in motor activity associated with a 315% increase in aspartate, 181% in glutamate, and 264% in ACh levels, occurred during the first 10 min. The increase in GABA level reached a maximum of 257% during the second 10 min. The neurotransmitter levels returned to basal values within 40 min. During exploration II, 1 h later, a smaller increase in neurotransmitter levels and motor activity was observed. In both explorations, the increase in neurotransmitter levels was completely abolished by 1 and 3 μM TTX. A statistically significant relationship was found between neurotransmitter extracellular levels and motor activity, for aspartate and glutamate in exploration I, and for ACh in exploration I and II. In conclusion, exploratory activity is associated with or depends on the activation of neuronal systems in the ventral hippocampus releasing aspartate, glutamate, GABA, and ACh. The activation is dampened by habituation.

Local GABAergic modulation of acetylcholine release from the cortex of freely moving rats.

Cortical perfusion with GABA agonists and antagonists modulates the spontaneous release of cortical acetylcholine and GABA in freely moving rats. Twenty‐four hours after implantation of a dialysis fibre, cerebral cortex spontaneously released acetylcholine (3.8 ± 0.2 pmol/10 min) and GABA (6.6 ± 0.4 pmol/10 min) at a stable rate. Local administration of GABA (1 or 5 mm) or the GABAA agonist muscimol (25 or 50 μm) had no effect on the spontaneous release of acetylcholine. However, bicuculline (1–25 μm), a GABAA antagonist, added to the dialysis perfusate, elicited a concentration‐dependent increase of acetylcholine release to approximately double that of control. This effect of bicuculline (25 μm) was completely prevented by coperfusion with muscimol (50 μm). Local administration of the GABAB receptor agonist baclofen (10 or 50 μm) elicited a concentration‐dependent increase in spontaneous acetylcholine release with a maximal increase of about 60%. Intracortical administration of baclofen also decreased the spontaneous release of GABA. The GABAB receptor antagonist CGP 35348 (1 mm), administered alone for 20 min through the dialysis fibre, was without effect on spontaneous acetylcholine release; however, it completely blocked both the baclofen‐induced increase in acetylcholine release and the decrease in GABA release. These results suggest that cortically released GABA exerts a tonic influence on cholinergic activity.

α-synuclein and synapsin III cooperatively regulate synaptic function in dopamine neurons.

ABSTRACT The main neuropathological features of Parkinsons disease are dopaminergic nigrostriatal neuron degeneration, and intraneuronal and intraneuritic proteinaceous inclusions named Lewy bodies and Lewy neurites, respectively, which mainly contain α-synuclein (α-syn, also known as SNCA). The neuronal phosphoprotein synapsin III (also known as SYN3), is a pivotal regulator of dopamine neuron synaptic function. Here, we show that α-syn interacts with and modulates synapsin III. The absence of α-syn causes a selective increase and redistribution of synapsin III, and changes the organization of synaptic vesicle pools in dopamine neurons. In α-syn-null mice, the alterations of synapsin III induce an increased locomotor response to the stimulation of synapsin-dependent dopamine overflow, despite this, these mice show decreased basal and depolarization-dependent striatal dopamine release. Of note, synapsin III seems to be involved in α-syn aggregation, which also coaxes its increase and redistribution. Furthermore, synapsin III accumulates in the caudate and putamen of individuals with Parkinsons disease. These findings support a reciprocal modulatory interaction of α-syn and synapsin III in the regulation of dopamine neuron synaptic function. Summary: Absence or aggregation of α-synuclein induces a selective increase and redistribution of synapsin III, reorganizing synaptic vesicle pools, which could have implications for dopamine neuron degeneration in Parkinsons disease.

Differential regulation by N-methyl-d-aspartate and non-N-methyl-d-aspartate receptors of acetylcholine release from the rat striatum in vivo

The modulation of striatal cholinergic neurons by glutamatergic inputs was studied by monitoring the output of acetylcholine collected via a transversal microdialysis probe implanted into the striatum of freely moving rats. A transversal microdialysis membrane was inserted in the striatum and acetylcholine or GABA levels in the dialysate were measured. Acetylcholine levels in the dialysate were quantified by a high-performance liquid chromatography method with an electrochemical detector, while GABA levels were measured by a high-performance liquid chromatography method with a fluorescence detector. The dialysis membrane was perfused with Ringer solution containing 7 microM physostigmine sulphate and drugs, dissolved in the perfusion solution, were administered locally via the dialysis membrane. Local administration of the N-methyl-D-aspartate antagonist 3-[(RS)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (25-100 microM) brought about a decrease in striatal acetylcholine output which was dose-dependent, reversible and partially antagonized by 100 microM N-methyl-D-aspartate. On the other hand, local administration of the non-N-methyl-D-aspartate antagonist 2,3-dihydroxy-6-nitro-7-sulfamoil-benzo(F)quinoxaline was followed by an increase in acetylcholine output which reached a maximum of about +55% at 12.8 microM 2,3-dihydroxy-6-nitro-7-sulfamoil-benzo(F)quinoxaline and was readily reversed when the drug was withdrawn from the perfusion solution. Local administration of the non-N-methyl-D-aspartate receptor agonist (S)-alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (50 and 200 microM) decreased acetylcholine output and this effect was reversed by simultaneous perfusion with the GABA antagonist bicuculline (50 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

The release of amino acids from rat neostriatum and substantia nigra in vivo: a dual microdialysis probe analysis

It has previously been demonstrated, in dual probe microdialysis studies, that stimulation of the neostriatum with kainic acid causes the release of GABA both locally within the neostriatum and distally in the substantia nigra, observations that are consistent with the known anatomy of the basal ganglia. The object of the present study was to further examine the characteristics of GABA release and to determine whether taurine, which has been proposed to be present in striatonigral neurons, has similar characteristics of release, and to examine the release of excitatory amino acids under the same conditions. To this end, dual probe microdialysis studies were carried out on freely-moving rats. The application of kainic acid to neostriatum enhanced the release of GABA, taurine, aspartate and glutamate locally in the neostriatum and distally in the substantia nigra. The distal release of each amino acid in the substantia nigra was sensitive to the administration of 6,7-dinitroquinoxaline-2,3-dione and tetrodotoxin to the neostriatum. Similarly the local release of GABA, aspartate and glutamate but not taurine was sensitive to the intrastriatal application of 6,7-dinitroquinoxaline-2,3-dione or tetrodotoxin. It is concluded that the release of taurine from the substantia nigra has similar characteristics to that of GABA and may be released from the terminals of striatonigral neurons following the stimulation of their cell bodies in the neostriatum. The release of taurine in the neostriatum however, is likely to be mediated mainly by different mechanisms and not related to neuronal activity. The release of excitatory amino acids is likely to involve indirect effects in the neostriatum and polysynaptic pathways in the substantia nigra.

Validation of a robust and sensitive method for detecting hydroxyl radical formation together with evoked neurotransmitter release in brain microdialysis

Sodium terephthalate was shown to be a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), which lacks the drawbacks of the salicylic acid method. Reaction of the almost non‐fluorescent terephthalate (TA2−) with hydroxyl radicals or ferryl‐oxo species resulted in the stoichiometric formation of the brilliant fluorophor, 2‐hydroxyterephthalate (OH‐TA). Neither hydrogen peroxide nor superoxide reacts in this system. This procedure was validated for determining hROS formation during microdialysis under in vivo conditions as well as by in vitro studies. The detection limit of OH‐TA in microdialysis samples was 0.5 fmol/μL. Derivatization of samples with o‐phthalaldehyde, for amino acid detection, had no effect on OH‐TA fluorescence, which could easily be resolved from the amino acid derivatives by HPLC, allowing determination in a single chromatogram. Use of terephthalate in microdialysis experiments showed the neurotoxin kainate to evoke hROS formation in a dose‐dependent manner. The presence of TA2− in the perfusion fluid did not affect basal or evoked release of aspartate, glutamate, taurine and GABA. Assessment of cell death ‘ex vivo’ showed TA2− to be non‐toxic at concentrations up to 1 mM. The in vitro results in the Fenton system (Fe2+ + H2O2) indicate a mechanism whereby TA2− forms a primary complex with Fe2+ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer.

Extracellular levels of brain aspartate, glutamate and GABA during an inhibitory avoidance response in the rat

The extracellular levels of aspartate, glutamate and GABA were measured by microdialysis, coupled with an HPLC method, in rat prefrontal cortex (mPFC) and ventral hippocampus (VH) before and during the performance of a step‐down inhibitory task. The basal levels of glutamate were about 50% higher than those of aspartate, and GABA levels were about 20‐folds smaller than those of the excitatory amino acids. There were no significant differences in the basal levels of any of the three amino acids between the two brain regions. The extracellular levels of aspartate increased during acquisition and recall trials in both VH and mPFC, whereas those of glutamate increased in the VH during acquisition only. A significant increase in GABA levels was also detected during acquisition but only in the mPFC. The neuronal origin of the increased extracellular levels of aspartate, glutamate and GABA was demonstrated by administering tetrodotoxin directly into the mPFC or VH by reverse dialysis. These findings, together with previous evidence from our and other laboratories, indicate a differential release of aspartate and glutamate from excitatory neurons during the performance of behavioral responses, and therefore, distinct roles for the two excitatory amino acids should be envisaged.

Species‐specific chemosignals evoke delayed excitation of the vomeronasal amygdala in freely‐moving female rats

Male rat chemosignals attract females and influence their reproductive status. Through the accessory olfactory bulb and its projection target, the posteromedial cortical nucleus of the amygdala (PMCo), species‐specific chemosignals detected by the vomeronasal organ (VNO) may reach the hypothalamus. To test this hypothesis in vivo, behavioural activation and neurotransmitter release in the PMCo were simultaneously monitored in freely moving female oestrus rats exposed to either rat or mouse urinary stimuli, or to odorants. Plasma levels of the luteinizing hormone were subsequently monitored. All stimuli induced an immediate behavioural activation, but only species‐specific chemosignals led to a delayed behavioural activation. This biphasic behavioural activation was accompanied by a VNO‐mediated release of the excitatory amino acids, aspartate and glutamate, in the PMCo. The late behavioural and neurochemical activation was followed by an increase in the levels of circulating luteinizing hormone. In conclusion, these data show that only species‐specific chemosignals induce a delayed behavioural activation and excitatory activation of the PMCo, which is dependent on an intact VNO.