microdialysis-excitatory-amino-acids-during-eeg-recordings-freely

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

University of Ferrara and National Institute of Neuroscience

1. We used electrophysiological and electron spin resonance (e.s.r.) techniques to study the mechanism of the protective effect of darodipine on rat isolated cardiomyocytes exposed to an exogenous source of oxygen free radicals (OFR). 2. The L-type calcium current (I(Ca,L)), action potential and cell shortening were measured in patch-clamped cells in the whole-cell configuration. I(Ca,L) blockade by darodipine was concentration-dependent, peak current being reduced by 20% with 50 nM and by 58% with 100 nM darodipine. The lowest concentration of darodipine did not affect action potential or twitch profile. 3. Exposure to OFR-generating solution (5 mM dihydroxyfumarate, DHF) caused the appearance of electrophysiological alterations and/or spontaneous activity in 73% of cells (n=26) within 5 min; action potential duration (APD) was prolonged (195+/-16 ms vs 140+/-6 ms in the control) and maximum diastolic potential (MDP) was reduced (-59.5+/-2.6 mV vs -69.8+/-0.8 mV in the control) (P<0.05, n=25). 4. A 2 min pretreatment with 50 nM darodipine significantly reduced the incidence of these arrhythmogenic events following a 5 min exposure to OFR (36% of cells, n=14; P<0.05 vs nonpretreated cells). Pretreatment with darodipine also prevented APD prolongation caused by OFR (137+/-12 ms after DHF vs 117+/-6 ms before DHF n=14, not significant) but not the decrease of MDP (-63.4+/-2.5 mV after DHF vs -70.9+/-1.0 mV before DHF, P<0.05). 5. The e.s.r. spectra obtained from the DHF-DMPO solution in the absence of darodipine demonstrated the presence of two components corresponding to two DMPO adducts. The addition of darodipine (50-500 nM) led to a concentration-dependent decrease in intensity of the signals, the intensity of the DMPO-COO.- adduct being decreased more than that of the DMPO-OH. adduct. 6. Our results demonstrate that darodipine dose-depentently blocks I(Ca,L) in rat isolated cardiomyocytes. Furthermore it exerts protective effects against free-radical-induced electrophysiological alterations independently of its calcium antagonistic properties; this effect is possibly due to trapping of specific radical species.

Protective effect of darodipine, a calcium antagonist, on rat cardiomyocytes against oxygen radical-mediated injury.

(1) Aim of this study was to gain insight into the mechanism of action of CHF3381, a novel putative antiepileptic and neuroprotective drug. (2) CHF3381 blocked NMDA currents in primary cultures of cortical neurons: maximal effect was nearly -80% of the NMDA-evoked current, with EC(50) of approximately 5 micro M. This effect was selective, reversible, use-dependent and elicited at the concentrations reached in the rodent brain after peripheral administration of therapeutic doses. (3) CHF3381 also inhibited voltage-gated Na(+) currents in an apparently voltage-dependent manner. However, this effect could be obtained only at relatively high concentrations (100 micro M). (4) Consistent with the mild effects on voltage-gated Na(+) channels, CHF3381 (100 micro M) failed to affect electrical stimulation-evoked glutamate overflow in hippocampal slices. In contrast, the anti-convulsant agent and Na(+) channel blocker lamotrigine (100 micro M) inhibited stimulation-evoked glutamate overflow by approximately 50%. (5) CHF3381 reduced kindled seizure-induced c-fos mRNA levels within the same brain regions, and to a similar level, as the selective NMDA receptor antagonist MK801, providing circumstantial evidence to the idea that CHF3381 blocks NMDA receptors in vivo. (6) The present mechanistic studies suggest that the primary mechanism of action of CHF3381 in the forebrain is blockade of NMDA receptors. On this basis, this compound may have a potential use in other diseases caused by or associated with a pathologically high level of NMDA receptor activation.

Mechanisms of action of CHF3381 in the forebrain.

The adult brain undergoes activity-dependent plastic modifications during pathologic processes that are reminiscent of those observed during development. For example, seizures induce neuronal loss, neurogenesis, axonal and dendritic sprouting, gliosis, and circuit remodeling. Neurotrophic factors and fibroblast growth factor-2 (FGF-2), in particular, are well-known mediators in each of these cellular events. The aim of this minireview is to summarize and discuss the data supporting the idea that FGF-2 may be involved in seizure generation and in their sequelae.

Alterations in seizure susceptibility and in seizure-induced plasticity after pharmacologic and genetic manipulation of the fibroblast growth factor-2 system.

Fibroblast growth factor 2 (FGF-2) has multiple, pleiotropic effects on the nervous system that include neurogenesis, neuroprotection and neuroplasticity. Thus, alteration in FGF-2 expression patterns may have a profound impact in brain function, both in normal physiology and in pathology. Here, we used FGF-2 transgenic mice (TgFGF2) to study the effects of endogenous FGF-2 overexpression on susceptibility to seizures and to the pathological consequences of seizures. TgFGF2 mice display increased FGF-2 expression in hippocampal pyramidal neurons and dentate granule cells. Increased density of glutamatergic synaptic vesicles was observed in the hippocampus of TgFGF2 mice, and electrophysiological data (input/output curves and patch-clamp recordings in CA1) confirmed an increase in excitatory inputs in CA1, suggesting the presence of a latent hyperexcitability. Indeed, TgFGF2 mice displayed increased susceptibility to kainate-induced seizures compared with wild-type (WT) littermates, in that latency to generalized seizure onset was reduced, whereas behavioral seizure scores and lethality were increased. Finally, WT and TgFGF2 mice with similar seizure scores were used for examining seizure-induced cellular consequences. Neurogenesis and mossy fiber sprouting were not significantly different between the two groups. In contrast, cell damage (assessed with Fluoro-Jade B, silver impregnation and anti-caspase 3 immunohistochemistry) was significantly lower in TgFGF2 mice, especially in the areas of overexpression (CA1 and CA3), indicating reduction of seizure-induced necrosis and apoptosis. These data suggest that FGF-2 may be implicated in seizure susceptibility and in seizure-induced plasticity, exerting different, and apparently contrasting effects: favoring ictogenesis but reducing seizure-induced cell death.

Fgf-2 overexpression increases excitability and seizure susceptibility but decreases seizure-induced cell loss.

In the dorsal raphe nucleus (DRN) many inputs converge and interact to modulate serotonergic neuronal activity and the behavioral responses to stress. The effects exerted by two stress-related neuropeptides, corticotropin releasing factor (CRF) and nociceptin/orphaninFQ (N/OFQ), on the outflow of [(3)H]5- hydroxytryptamine were investigated in superfused rat dorsal raphe nucleus slices. Electrical stimulation (100 mA, 1 ms for 2 min) evoked a frequency-dependent peak of [(3)H]5- hydroxytryptamine outflow, which was sodium and calcium-dependent. Corticotropin releasing factor (1-100 nM), concentration-dependently inhibited the stimulation (3 Hz)-evoked [(3)H]5-hydroxytryptamine outflow; the inhibition by 30 nM corticotropin releasing factor (to 68 +/- 5.7%) was prevented both by the non selective CRF receptor antagonist alpha-helicalCRF(9-41) (alpha-HEL) (300 nM) and by the CRF(1) receptor antagonist antalarmin (ANT) (100 nM). The CRF(2) agonist urocortin II (10 nM) did not modify [(3)H]5- hydroxytryptamine outflow, ruling out the involvement of CRF(2) receptors. Bicuculline (BIC), a GABAA antagonist (10 microM), prevented the inhibitory effect of corticotropin releasing factor (30 nM), supporting the hypothesis that the inhibition was mediated by increased gamma-aminobutyric acid (GABA) release. Nociceptin/ orphaninFQ (1 nM-1 microM) exerted an antalarmin- and bicuculline-insensitive inhibition on [(3)H]5- hydroxytryptamine outflow, with the maximum at 100 nM (to 63+/- 4.2%), antagonized by the NOP receptor antagonist UFP-101 (1 microM). Dorsal raphe nucleus slices prepared from rats exposed to 15 min of forced swim stress displayed a reduced [(3)H]5-hydroxytryptamine outflow, in part reversed by antalarmin and further inhibited by nociceptin/orphaninFQ. These findings indicate that (i) both corticotropin releasing factor and nociceptin/orphaninFQ exert an inhibitory control on dorsal raphe nucleus serotonergic neurons; (ii) the inhibition by corticotropin releasing factor involves gamma-aminobutyric acid neurons; (iii) nociceptin/ orphaninFQ inhibits dorsal raphe nucleus serotonin system in a corticotropin releasing factor- and gamma-aminobutyric acid-independent manner; (iv) nociceptin/orphaninFQ modulation is still operant in slices prepared from stressed rats. The nociceptin/orphaninFQ-NOP receptor system could represent a new target for drugs effective in stress-related disorders.

Inhibition of serotonin outflow by nociceptin/orphaninFQ in dorsal raphe nucleus slices from normal and stressed rats: Role of corticotropin releasing factor.

The effects of nociceptin/orphanin FQ on putative serotonin (5HT) neurons of the dorsal raphe nucleus (DRN), known to modulate the behavioral responses to stress, were investigated in vivo and in vitro. In DRN slices from unstressed rats, nociceptin/orphanin FQ concentration-dependently inhibited the firing rate of putative 5HT neurons (EC(50) = 21.6 +/- 1.21 nM) and the selective NOP receptor antagonist UFP-101 shifted the concentration-response curve to the right (estimated pA(2) 6.86). Nociceptin/orphanin FQ potency was enhanced in slices prepared from rats previously subjected to a 15 min swim stress (EC(50) = 1.98 +/- 0.11 nM). Swim stress did not change the number or affinity of NOP receptors in DRN. Stress-elicited potentiation involved corticotropin-releasing factor (CRF)(1) receptors, GABA signaling and protein synthesis, being attenuated by pre-treatment with antalarmin (20 mg/kg, i.p.), diazepam (2.4 mg/kg, i.p.) and cycloheximide (2.5 mg/kg, i.p.), respectively. In anesthetized unstressed rats, locally applied nociceptin/orphanin FQ (0.03 and 0.1 ng/30 nl) inhibited the firing rate of DRN neurons (to 80 +/- 7 and 54 +/- 10% of baseline, respectively). Nociceptin/orphanin FQ inhibition was potentiated both 24 h after swim stress and 1 h after CRF (30 ng/30 nl intra-DRN). Stress-induced potentiation was prevented by the selective CRF(1) receptor antagonist, NBI 30755 (20 mg/kg, i.p.). In contrast, the inhibitory response of DRN neurons to the 5HT(1A) agonist, 8OH-DPAT (1 microg/1 microl, intra-DRN) was not potentiated by swim stress, ruling out a non-specific enhanced permeability of GIRK channel. Together, these findings suggest that CRF and the nociceptin/orphanin FQ/NOP system interact in the DRN during stress to control 5HT transmission; this may play a role in stress-related neuropsychopathologies.

Swim stress enhances nociceptin/orphanin FQ-induced inhibition of rat dorsal raphe nucleus activity in vivo and in vitro: role of corticotropin releasing factor.

Neurotrophic factors (NTFs) are involved in the regulation of neuronal survival and function and, thus, may be used to treat neurological diseases associated with neuronal death. A major hurdle for their clinical application is the delivery mode. We describe here a new strategy based on the use of progenitor cells called mesoangioblasts (MABs). MABs can be isolated from postnatal mesoderm tissues and, because of a high adhesin-dependent migratory capacity, can reach perivascular targets especially in damaged areas. We generated genetically modified MABs producing nerve growth factor (MABs-NGF) or brain-derived neurotrophic factor (MABs-BDNF) and assessed their bystander effects in vitro using PC12 cells, primary cultures, and organotypic cultures of adult hippocampal slices. MABs-NGF-conditioned medium induced differentiation of PC12 cells, while MABs-BDNF-conditioned medium increased viability of cultured neurons and slices. Slices cultured with MABs-BDNF medium also better retained their morphology and functional connections, and all these effects were abolished by the TrkB kinase blocker K252a or the BDNF scavenger TrkB-IgG. Interestingly, the amount of BDNF released by MABs-BDNF produced greater effects than an identical amount of recombinant BDNF, suggesting that other NTFs produced by MABs synergize with BDNF. Thus, MABs can be an effective vehicle for NTF delivery, promoting differentiation, survival, and functionality of neurons. In summary, MABs hold distinct advantages over other currently evaluated approaches for NTF delivery in the CNS, including synergy of MAB-produced NTF with the neurotrophins. Since MABs may be capable of homing into damaged brain areas, they represent a conceptually novel, promising therapeutic approach to treat neurodegenerative diseases.

Bystander effect on brain tissue of mesoangioblasts producing neurotrophins.

Considerable evidence links dysfunction of serotonin (5-hydroxytryptamine, 5-HT) transmission to neurodevelopmental and psychiatric disorders characterized by compromised "social" cognition and emotion regulation. It is well established that the brain 5-HT system is under autoregulatory control by its principal transmitter 5-HT via its effects on activity and expression of 5-HT system-related proteins. To examine whether 5-HT itself also has a crucial role in the acquisition and maintenance of characteristic rhythmic firing of 5-HT neurons, we compared their intrinsic electrophysiological properties in mice lacking brain 5-HT, i.e. tryptophan hydroxylase-2 null mice (Tph2(-/-)) and their littermates, Tph2(+/-) and Tph2(+/+), by using whole-cell patch-clamp recordings in a brainstem slice preparation and single unit recording in anesthetized animals. We report that the active properties of dorsal raphe nucleus (DRN) 5-HT neurons in vivo (firing rate magnitude and variability; the presence of spike doublets) and in vitro (firing in response to depolarizing current pulses; action potential shape) as well as the resting membrane potential remained essentially unchanged across Tph2 genotypes. However, there were subtle differences in subthreshold properties, most notably, an approximately 25% higher input conductance in Tph2(-/-) mice compared with Tph2(+/-) and Tph2(+/+) littermates (p<0.0001). This difference may at least in part be a consequence of slightly bigger size of the DRN 5-HT neurons in Tph2(-/-) mice (approximately 10%, p<0.0001). Taken together, these findings show that 5-HT neurons acquire and maintain their signature firing properties independently of the presence of their principal neurotransmitter 5-HT, displaying an unexpected functional resilience to complete brain 5-HT deficiency.

Cellular resilience: 5-HT neurons in Tph2(-/-) mice retain normal firing behavior despite the lack of brain 5-HT.

New Psychoactive Substances (NPS) constitute a broad range of hundreds of natural and synthetic drugs, including synthetic opioids, synthetic cannabinoids, synthetic cathinones, and other NPS classes, which were not controlled from 1961 to 1971 by the United Nations drug control conventions. Among these, synthetic opioids represent a major threat to public health.

Article	Year
Protective effect of darodipine, a calcium antagonist, on rat cardiomyocytes against oxygen radical-mediated injury. British journal of pharmacology\| PubMed ID: 9421282	1997
Mechanisms of action of CHF3381 in the forebrain. British journal of pharmacology\| PubMed ID: 12890713	2003
Alterations in seizure susceptibility and in seizure-induced plasticity after pharmacologic and genetic manipulation of the fibroblast growth factor-2 system. Epilepsia\| PubMed ID: 15987254	2005
Fgf-2 overexpression increases excitability and seizure susceptibility but decreases seizure-induced cell loss. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 19052202	2008
Inhibition of serotonin outflow by nociceptin/orphaninFQ in dorsal raphe nucleus slices from normal and stressed rats: Role of corticotropin releasing factor. Neurochemistry international\| PubMed ID: 19418633	2009
Swim stress enhances nociceptin/orphanin FQ-induced inhibition of rat dorsal raphe nucleus activity in vivo and in vitro: role of corticotropin releasing factor. Neuropharmacology\| PubMed ID: 19747494	2010
Bystander effect on brain tissue of mesoangioblasts producing neurotrophins. Cell transplantation\| PubMed ID: 22525962	2012
Cellular resilience: 5-HT neurons in Tph2(-/-) mice retain normal firing behavior despite the lack of brain 5-HT. European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology\| PubMed ID: 26409296	2015
Novel Synthetic Opioids: The Pathologist's Point of View. Brain sciences\| PubMed ID: 30200549	2018

Mario Barbieri

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