9,717 research outputs found
Meccanismi di vulnerabilità dei neuroni dopaminergici mesencefalici di ratto esposti a fattori neurotossici ambientali
No full textE’ sempre più accettata l’ipotesi secondo cui le malattie neurodegenerative come il morbo di Parkinson siano di origine multifattoriale (“multiple hit hypothesis”), cioè siano causate dalla concomitante o ripetitiva presenza di diversi fattori che cooperano alla morte cellulare. Tra questi quelli ambientali occupano un posto rilevante. Sebbene una grande varietà di processi neurologici potrebbero essere influenzati da neurotossine ambientali, il sistema dopaminergico sembra essere quello più colpito.
Negli ultimi 10 anni si è rivolta sempre maggiore attenzione alla L-BMAA (L-β-N-methylamino-L-alanine), un aminoacido non proteico trovato nei semi della Cycas micronesica, che sembra essere alla base dell’ “ALS-PDC complex”, una sindrome complessa caratterizzata da sintomatologie cliniche tipiche della sclerosi laterale amiotrofica (SLA), del morbo di Parkinson e dell’Alzheimer.
Recenti scoperte hanno dimostrato che questa tossina viene prodotta da una grande varietà di cianobatteri del genere Nostoc presenti in tutto il mondo. Pertanto, potenzialmente, tutta la popolazione umana potrebbe essere esposta a tale sostanza. Ciò potrebbe determinare il suo accumulo nell’organismo, sia in forma libera che legata alle proteine, da cui verrebbe poi rilasciata lentamente durante il catabolismo proteico.
Sulla base di queste considerazioni abbiamo voluto analizzare gli effetti di questo aminoacido sulle cellule dopaminergiche della SNc da un punto di vista elettrofisiologico, farmacologico, morfologico e tossicologico.
La BMAA (3 mM, 10 psi 1.0 s) causa una depolarizzazione dei neuroni dopaminergici della SNc inducendo una corrente entrante (media = 454.48 ± 34.65, n = 73) e aumenti transienti della concentrazione di calcio intracellulare (R medio = 0.368 ± 0.062, n = 13). Questi effetti sono mediati prevalentemente dall’attivazione dei recettori metabotropici del glutammato di gruppo I (mGluR1), in quanto vengono ridotti reversibilmente dall’antagonista selettivo, CPCCOEt (100 μM) (corrente: 41.56 ± 3.61 % del controllo, n = 24; calcio: 28.43 ± 5.96 % del controllo, n = 7). La corrente, ma non il calcio, indotta dalla BMAA è ridotta in piccola parte dal CNQX (10 μM) (corrente: 93,09 ± 1,97 % del controllo, n = 24; calcio: 100.17 ± 9.93 % del controllo, n=6), antagonista competitivo dei recettori AMPA.
Nelle cellule dopaminergiche della SNc gli aumenti di calcio indotti dall’attivazione dei recettori mGluR1 sono mediati dai canali SOCs/TRPC. Infatti, sia le correnti entranti che le variazioni di calcio intracellulare indotte dalla BMAA sono ridotte dagli antagonisti di tali canali, SKF 96365 (100 μM) (corrente: 42.125 ± 4.35 % del controllo, n = 8; calcio: 43.57 ± 7.9 % del controllo, n = 7) e Ruthenium Red (20 μM) (corrente: 27.05 ± 8.3 % del controllo, n = 6).
Inoltre, nonostante la BMAA in presenza di carbonato presenti una struttura chimica simile a quella dell’acido glutammico, essa non viene ricaptata dalla cellule dopaminergiche attraverso il trasportatore degli aminoacidi eccitatori EAAT.
E’ interessante notare che negli interneuroni GABAergici della SNc la BMAA attiva i recettori AMPA, ma non quelli metabotropici, senza indurre variazioni della concentrazione del calcio intracellulare. In queste cellule, tuttavia, l’agonista selettivo degli mGluR1, il DHPG (30 μM), evoca correnti entranti, a dimostrazione della presenza degli mGluR1 sugli interneuroni GABAergici.
A conferma della sua potenziale tossicità, esposizioni prolungate di fettine mesencefaliche (12, 20 e 30 minuti) alla BMAA inducono cambiamenti irreversibili su numerose proprietà cellulari. Tali modificazioni sono accompagnate dal rilascio massivo del citocromo C (Cyt C) nel citoplasma delle cellule dopaminergiche che viene completamente bloccato dall’aggiunta, nel mezzo di perfusione, di antagonisti dei recettori mGluR1 e AMPA.
I dati che ho riportato in questa tesi forniscono una chiara e plausibile dimostrazione di come la BMAA può essere tossica verso le cellule dopaminergiche della SNc, causando i sintomi neurologici della malattia di Parkinson.It is well known that several neurodegenerative diseases, such as Parkinson disease, have a multifactorial origin (multiple hit hypothesis), which suggests that neuronal loss is a result of multiple factors. Among them, environmental factors are the most important. Although a variety of neurological processes can be adversely affected, the dopaminergic system appears to be a major target for environmental neurotoxins. The hypothesis that L-BMAA (L-β-methylamino-L-alanine), a nonprotein amino acid found in the Cycas micronesica seeds in western pacific islands, is involved in the development of amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS-PDC complex) has risen and fallen since its initial proposal in 1987. In the last ten years the interest for this toxin has grown due to the discovery that it can be produced by many strains of Nostoc cyanobacterias, present throughout the world. Moreover L-BMAA can bind proteins. This bound form may function as an endogenous neurotoxic reservoir, accumulating and being released during protein catabolism.
In order to analyze the effects of this amino acid, we have performed electrophysiological, pharmacological, morphological and toxicological studies on dopaminergic neurons of SNc. In these neurons puff-application of L-BMAA (3 mM, 10 psi 1.0 s) causes an inward current (mean = 454.48 34.65, n = 73) and a transient increase of intracellular calcium (R mean = 0.368 ± 0.062, n = 13). These effects are mediated by the activation of group I metabotropic glutamate receptors (mGluR1) and they are reversibly blocked by the application of the antagonist CPCCOEt (100 μM) (current: 41.56 ± 3.61 % of control, n = 24; calcium: 28.43 ± 5.96 % of control, n = 7). Bath application of CNQX (10 μM), a competitive antagonist of AMPA receptors, partially inhibits the L-BMAA-induced current (current: 93,09 ± 1,97 % of control, n = 24) but it has no effect on the calcium concentration (100.17 ± 9.93 % of control, n = 6). SOCs/TRPC channels are present in the dopaminergic cells of SNc and they mediate the intracellular calcium increase due to the activation of mGluR1. Indeed SKF 96365 (100 μM) and Ruthenium Red (20 μM), two antagonists of TRPC channels, are able to reduce the L-BMAA-induced inward current (42.125 ± 4.35 % of control, n = 8 and 27.05 ± 8.3 % of control, n = 6 respectively). Moreover SKF 96365 (100 μM) reduces the intracellular calcium increase induced by L-BMAA (43.57 ± 7.9 % of control). It is known that L-BMAA, in the presence of carbonate, has a chemical structure similar to glutamic acid, however it is not re-uptaken by EAATs, the excitatory amino acid transporters. Interestingly, in GABAergic interneurons, L-BMAA activates AMPA receptors but not mGluR1, and this activation causes inward current without any change in intracellular calcium concentration. However mGluR1 are present in these neurons because application of DHPG (30 μM), the selective agonist, produces inward currents. In order to confirm the toxic effects of this amino acid we have treated midbrain slices with L-BMAA for 12, 20 and 30 minutes and we have seen irreversible modification of cellular properties (decrease in membrane resistance, inability to evoke firing, elevated intracellular calcium). As a consequence of the treatments, cytocrome C is released in the cytoplasm, but in the presence of AMPA and mGluR1 antagonists, this effect is blocked. In conclusion this study demonstrates that L-BMAA could be considered a possible toxic agent for the dopaminergic neurons and provides new insights into the role of this amino acid in the aetiology of Parkinson disease
Substantia nigra control of basal ganglia nuclei
No full textAbstract. The substantia nigra, located in the ventral mesencephalon,
is one of the five nuclei that constitute the basal
ganglia circuit, which controls voluntary movements.
It is divided into the pars compacta and the pars reticulata,
which mainly contain dopaminergic and GABAergic
cells respectively. Here we overview the electrophysiological
properties of these substantia nigra neurons in the pars
compacta and reticulata, together with their synaptic connections,
and discuss the functional effects of dopaminergic
and GABAergic inputs within the basal ganglia. We also
examine the phenomenon that when a deficiency of dopamine
(DA) occurs (e.g. in Parkinson’s disease), there is an
aberrant synaptic plasticity in the basal ganglia.
Moreover, we point out that the appearance of an altered
pattern of neuronal firing (beta-oscillations) and synchrony
among neurons in the subthalamic nucleus, the internal
globus pallidus, and the substantia nigra pars reticulata has
been related to motor symptoms and possibly, persistent
degeneration of DA-containing neurons.
Finally, we believe that, based on pathophysiological
data, new and significant targets for therapeutic intervention
can be identified and tested
The midbrain slice preparation. An in vitro model to select potential anti-parkinsonian drugs?
No full textAbstract.
Most anti-parkinsonian drugs produce a dopamine-like effect on single dopaminergic neurons. Using electrophysiological recordings, this effect is observed as an inhibition of the spontaneous firing activity. The firing activity of dopaminergic cells throughout dopamine-mediated mechanisms is inhibited not only by dopamine direct agonists but also by substances that increase the release or synthesis, or block the uptake or reduce degradation of dopamine in the brain. Here we propose the electrophysiological approach on dopaminergic neurons in in vitro slice
preparation as a preliminary tool for selecting new potential anti-parkinsonian agents, before they are tested in more complex animal models
Memantine inhibits ATP-dependent K+ conductances in dopamine neurons of the rat substantia nigra pars compacta
No full text1-Amino-3,5-dimethyl-adamantane (memantine) is a noncompetitive
N-methyl-D-aspartate (NMDA) receptor antagonist
used in clinical practice to treat neurodegenerative disorders
that could be associated with excitotoxic cell death. Because
memantine reduces the loss of dopamine neurons of
the substantia nigra pars compacta (SNc) in animal models of
Parkinson’s disease, we examined the effects of this drug on
dopamine cells of the SNc. Besides inhibition of NMDA
receptor-mediated currents, memantine (30 and 100 M)
increased the spontaneous firing rate of whole-cell recorded
dopamine neurons in a midbrain slice preparation. Occasionally,
a bursting activity was observed. These effects were
independent from the block of NMDA receptors and were
prevented in neurons dialyzed with a high concentration of
ATP (10 mM). An increase in firing rate was also induced by
the ATP-sensitive potassium (KATP) channel antagonist tolbutamide
(300 M), and this increase occluded further effects
of memantine. In addition, KATP channel-mediated outward
currents, induced by hypoxia, were inhibited by
memantine (30 and 100 M) in the presence of the NMDA
receptor antagonist (5S,10R)-()-5-methyl-10,11-dihydro-
5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801)
(10 M). An increase in the spontaneous firing rate by memantine
was observed in dopamine neurons recorded with
extracellular planar 8 8 multielectrodes in conditions of
hypoglycemia. These results highlight KATP channels as possible
relevant targets of memantine effects in the brain.
Moreover, in view of a proposed role of KATP conductances
in dopamine neuron degeneration, they suggest another
mechanism of action underlying the protective role of memantine
in Parkinson’s disease
Metabotropic glutamate receptor 1 mediates the electrophysiological and toxic actions of the cycad derivative beta-N-Methylamino-L-alanine on substantia nigra pars compacta DAergic neurons
No full textAmyotrophic lateral sclerosis–Parkinson dementia complex (ALS-PDC) is a neurodegenerative disease with ALS, parkinsonism, and Alzheimer’s symptoms that is prevalent in theGuampopulation.-N-Methylamino alanine(BMAA)has been proposed as the toxic agent damaging several neuronal types in ALS-PDC, including substantia nigra pars compacta dopaminergic (SNpc DAergic) neurons. BMAA is a mixed glutamate receptor agonist, but the specific pathways activated in DAergic neurons are not yet known. We combined electrophysiology, microfluorometry, and confocal microscopy analysis to monitor membrane potential/current, cytosolic calcium concentration
([Ca2]i ) changes, cytochrome-c (cyt-c) immunoreactivity, and reactive oxygen species (ROS) production induced by BMAA. Rapid toxin applications caused reversible membrane depolarization/inward current and increase of firing rate and [Ca2]i in DAergic neurons. The inward current (IBMAA) was mainly mediated by activation of metabotropic glutamate receptor 1 (mGluR1), coupled to transient receptor potential (TRP) channels, and to a lesser extent,AMPAreceptors. Indeed, mGluR1 (CPCCOEt) and TRP channels (SKF 96365; Ruthenium Red) antagonists reduced IBMAA, and a small component of IBMAA was reduced by the AMPA receptor antagonist CNQX. Calcium accumulation was mediated by mGluR1 but not by AMPA receptors. Application of a low concentration of NMDA
potentiated the BMAA-mediated calcium increase. Prolonged exposure to BMAA caused significant modifications of membrane properties, calcium overload, cell shrinkage, massive cyt-c release into the cytosol and ROS production. In SNpc GABAergic neurons, BMAA activated only AMPA receptors. Our study identifies the mGluR1-activated mechanism induced by BMAA that may cause the neuronal degeneration and parkinsonian symptoms seen in ALS-PDC. Moreover, environmental exposure toBMAAmight possibly also contribute to idiopathic PD
Dual effects of L-DOPA on nigral dopaminergic neurons
No full textL-DOPA (Levodopa) remains the gold standard for the treatment of motor symptoms of Parkinson's disease (PD), despite indications that the drug may have detrimental effects in cell culture. Classically, l-DOPA increases the production of dopamine (DA) in nigral dopaminergic neurons, while paradoxically inhibiting the firing of these neurons due to activation of D2 autoreceptors by extracellularly released DA. Using a combination of electrophysiology and calcium microfluorometry in brain slices, we have identified a novel effect of L-DOPA on dopaminergic neurons when D2 receptors were blocked. Under these conditions, L-DOPA (0.03-3 mM) evoked an excitatory effect consisting of two components. The 'early' component observed during and immediately after application of the drug, was associated with increased firing, membrane depolarization and inward current. This excitatory response was strongly attenuated by CNQX (10 μM), pointing to the involvement of TOPA quinone, an auto-oxidation product of L-DOPA and a potent activator of AMPA/kainate receptors. The 'late' phase of excitation persisted >30 min after brief L-DOPA application and was not mediated by ionotropic glutamate receptors, nor by D1, α1-adrenergic, mGluR1 or GABAB receptors. It was eliminated by carbidopa, demonstrating its dependence on conversion of L-DOPA to DA. Exogenous DA (50 μM) also evoked a glutamate-receptor independent increase in firing and an inward current when D2 receptors were blocked. In voltage-clamped neurons, both L-DOPA and DA produced a long-lasting increase in [Ca(2+)]i which was unaffected by block of ionotropic glutamate receptors. These results demonstrate that L-DOPA has dual, inhibitory and excitatory, effects on nigral dopaminergic neurons, and suggest that the excitation and calcium rise may have long-lasting consequences for the activity and survival of these neurons when the expression or function of D2 receptors is impaired
N-Glycans mutations rule oligomeric assembly and functional expression of P2X(3) receptor for extracellular ATP
No full textProperties of dopaminergic neurons in organotypic mesencephalic-striatal co-cultures - evidence for a facilitatory effect of dopamine on the glutamatergic input mediated by α-1 adrenergic receptors
No full textOrganotypic cultures (OCs) have been widely used to investigate the midbrain dopaminergic system, but only a few studies focused on the functional properties of dopaminergic neurons and their synaptic inputs from dopaminergic and non-dopaminergic neurons also contained in such cultures. In addition, it is not clear whether the culturing process affects the intrinsic neuronal properties and the expression of specific receptors and transporters. We performed patch-clamp recordings from dopaminergic neurons in mesencephalic–striatal co-cultures obtained from transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase promoter. Some (10 ⁄ 44) GFP+ neurons displayed a bursting activity that renders the firing of these cells similar to that of the dopaminergic neurons in vivo. The culturing process reduced the hyperpolarization-activated current (Ih) and the expression of D2 receptors. Downregulation of D2 receptor mRNA and protein was confirmed with reverse transcriptase polymerase chain reaction and Western blotting. Immunocytochemistry revealed that many synaptic terminals, most likely originating from dopaminergic neurons, co-expressed the dopamine (DA) transporter and the vesicular glutamate transporter-2, suggesting a co-release of DA and glutamate. Interestingly, exogenous DA decreased glutamate release in young cultures [days in vitro (DIV) 26) DA increased glutamate release by acting on a-1 adrenoreceptors. The facilitatory effect of DA on glutamatergic transmission to midbrain dopaminergic neurons may be important in conditions when the expression of D2 receptors is compromised, such as long-term treatment with antipsychotic drugs. Our data show that midbrain OCs at DIV > 26 may provide a suitable model of such conditions
Metadata Representations for Queryable ML Model Zoos
No full textMachine learning (ML) practitioners and organizations are building model zoos of pre-trained models, containing metadata describing properties of the ML models and datasets that are useful for reporting, auditing, reproducibility, and interpretability purposes. The metatada is currently not standardised; its expressivity is limited; and there is no interoperable way to store and query it. Consequently, model search, reuse, comparison, and composition are hindered. In this paper, we advocate for standardized ML model metadata representation and management, proposing a toolkit supported to help practitioners manage and query that metadata.Web Information SystemsHuman-Centred Artificial Intelligenc
A Manifesto of Nodalism
No full textThis paper proposes the notion of Nodalism as a means describing contemporary culture and of understanding my own creative practice in electronic music composition. It draws on theories and ideas from Kirby, Bauman, Bourriaud, Deleuze, Guatarri, and Gochenour, to demonstrate how networks of ideas or connectionist neural models of cognitive behaviour can be used to contextualize, understand and become a creative tool for the creation of contemporary electronic music
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