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Neurosteroid modulation of cortical network excitability
No full textNeurosteroids (NSs) such as allopregnanolone (ALLO) and tetrahydrodeoxy-corticosterone (THDOC ) are well known modulators of GABAA receptor function. While a large amount of data are available at the single cell level, their “global modulation” of network firing activity was not investigated in detail. NSs levels vary in the diverse brain regions and may change under different physiological and pathological conditions [1-3]. The NSs synthesizing enzymes, 5α-reductase type I and 3α-hydroxysteroid dehydrogenase have also different expression profiles, i.e. in the neocortex they co-localize in glutamatergic but not in GABAergic neurons [4]. Therefore these endogenous substances in excitatory neurons that co-express GABAA receptors and NSs synthetic enzymes can act in an autocrine manner [5] while in inhibitory cells their modulation is different.
The goal of our study was to investigate the effects of physiological NSs concentrations on the activity of networks formed by acutely dissociated mouse neocortical neurons. By using the multi-electrode arrays (MEA) technique the spontaneous reverberating activity of excitatory and inhibitory neurons was simultaneously recorded and analyzed in control and after perfusion with NSs. Their effects were analyzed, by using statistical methods [6], on physiological variables such as excitability (spikes-per neuron) and number of neurons engaged in bursts.
THDOC, at physiological concentrations, selectively decreased inhibitory interneuron activity, whereas at concentrations higher than 100 nM it inhibited both excitatory and inhibitory clusters. The analysis of the network activity after long time wash-out (8 hrs) highlight that the NS effect on inhibitory clusters persisted for hours producing a sort of long-term depression (LTD) in their excitability.
To further clarify this point we applied THDOC twice (after a 2h wash-out). The first application induced, as expected, a persistent depression of inhibitory neurons but after the second administration of THDOC no further LTD was observed. This experiment suggests that the first application of the NS produces some stable modifications, a sort of “memory” in the network that could be relevant also in vivo.
THDOC and ALLO, at low concentrations, are allosteric modulators of GABAergic neurotransmission but in the μM concentration range they also act as GABAA receptor agonists [7]. In agreement with these properties, our analysis of the global excitability of the network showed a sharp increase in NSs inhibitory effects at concentrations between 100 and 1000 nM, probably due to a direct agonistic activity at GABAA receptors.
Many single-cell studies of the effects of ALLO and THDOC have been performed, but no differences were reported between their effects. We show here that THDOC and ALLO, although consistently producing network inhibition at high concentrations, at low concentrations (10-100 nM) have different effects on excitatory and inhibitory clusters; moreover, the network recovered more easily from ALLO than from THDOC effect.
Previous studies suggested that tonic GABAergic currents are highly sensitive to NSs [8]. To investigate whether this happens also in our experimental mode we applied Gabazine (GBZ), a GABAA receptor antagonist, at concentrations that only block the phasic GABAergic current. GBZ 100 nM increased the activity of inhibitory neurons, leaving almost unaffected the excitatory neuron excitability suggesting that interneurons are controlling each other mainly through a phasic inhibition. In these conditions the excitability of the network was reduced but also the sensitivity to THDOC was unexpectedly decreased by approximately one order of magnitude compared to control.
A detailed analysis [6, 9] of the burst properties showed that in the presence of NSs, the neuronal activity changed and became heterogeneous because of the appearance of different states with occupancy probabilities strongly dependent on the drug concentration. In particular, we observed the random appearance of novel up-states, characterized by excitability features and engaged neurons different from those observed in control.
While the analysis of the “global network effect” of NSs provide information about the average changes in excitability of inhibitory and excitatory clusters, the “states analysis “ highlighted changes in the network connectivity: in the presence of neuromodulators different connectivity modes appear. What is the physiological significance of this effect?
Specific firing patterns recorded in selected neuronal populations encode information during physiological or pathological conditions [10] and changes in the connectivity induced by endogenous compounds are able to modify the response of the network.
Taken together, our results provide a new description of the mode of action of NSs and give some new insight in understanding the complexity of the network response to these endogenous modulators
"Glial-neuronal interactions: a crescendo in the degree of CNS complexity"
No full textCommentary on the importance of the interaction between neurons and glial cell
"Voltage-dependent calcium currents in trigeminal chick neurons."
No full textThe presence of action potentials, when sodium and potassium currents were blocked, has been investigated in trigeminal ganglion neurons, using the patch-clamp technique. In this conditions, inward currents, sensitive to the external application of cadmium, were detected. Activation and inactivation properties were investigated, as well as the behaviour of the current in the presence of extracellular Barium. The properties of these inward currents in trigeminal neurons are correlated to high threshold voltage-dependent calcium channels
Benzodiazepines outside the CNS
No full textCommentary on an article deling with the importance of benzodiazepines outside the CN
Non genomic modulation of ligand gated ionotropic receptors by thyroid hormones
No full textLigand gated ionotropic receptors are responsible for the fast neurotransmission in the brain and are the target of widely used drugs, such as anxiolytics, anticonvulsant and antidepressant, and of endogenously produced substances, e.g. hormones. In this review, the fast regulatory effects of thyroid hormones (THs, T3 and T4) on ligand gated ion channels will be analyzed and discussed. More precisely, the focus will be on receptors that mediate the majority of inhibitory and excitatory neurotransmission in the brain, respectively γ-aminobutyric acid (GABA) and glutamate ionotropic receptors.Brain is an important target of THs, as proved by profound alterations in its functionality related to hormonal imbalance. Indeed, dysthyroidism is often associated with several neuropsychiatric disorders that derive also from a dysfunction of GABAergic and glutamatergic neurotransmission. The molecular mechanisms responsible for these changes are not completely clarified yet. THs, through the binding of nuclear receptors, can modulate the expression of proteins directly or indirectly involved in neurotransmission; in addition, their non genomic fast modulation of ionotropic receptors mediating excitatory and inhibitory neurotransmission could also play an important role.THs modulate recombinant and native receptors activated by exogenous GABA or glutamate as well synaptic and extrasynaptic excitatory and inhibitory neurotransmission. These non genomic effects do not depend on protein phosphorylation or on the membrane integrin receptor αvβ3 activation.Keeping in mind that a local regulation of THs levels can occur in different brain regions the fast modulation of THs of synaptic activity could provide a rapid and efficacious control of the function of several brain circuitries
MODULATORY EFFECT OF THYROID HORMONES AND NEUROSTEROIDS OF GABA –EVOKED CURRENTS IN CULTURE DRGs.
No full textThyroid hormones (THs) and neurosteroids (NSs) are endogenous modulators of GABA-A receptors in the central and peripheral nervous system. Recent studies reported an increase in NSs levels after injury in rats ( Di Michele et al., 2000), and NSs administration reduces inflammatory and neuropathic pain (Charlet et al., 2008) suggesting their involvement in the physiology and pathology of pain transmission. Very little informations are available instead on the participation of THs in pain mechanisms (Bruno et al.2005). The aim of our study was to analyze the effect of THs (3-iodothyronine ,T3, thyroxine, T4) and of the NSs (Pregenolone Sulfate , PS, and allopregnenolone, ALLO) on GABA -evoked current in rat dorsal root ganglia cells in culture. The experiments were performed using the whole cell patch clamp technique. To highlight the nociceptors, at least one sub-population, we used capsaicin, a marker for pain cells expressing TRPV1, a specific nociceptive receptor. Our results show a reduction of GABA current in capsaicin positive cells after applications of THs (both at 10 μM ) and PS (10 uM) of 29,3% ± 8,2 for T3, 23,8%± 5,9 for T4 and 63,8%± 19,6 for PS. ALLO (10 μM) potentiated GABA current of 20 % ±3,5 and applied by itself elicited an inward current. Our results suggest a possible involvement of THs at the level of nociceptive peripheral transmission and open a new view on the complex influences existing among hormones and nociception
EFFECT OF NEUROSTEROIDS ON ARTIFICIAL AND NATIVE PLASMA MEMBRANES : role for the lipid bilayer in determining their mechanism of action.
No full textA Novel class of positive allosteric modulators of KA receptors
No full textKA-receptors (KARs) are widely expressed in the central nervous system and play an important role in short and long
term plasticity (Andrade-Talavera et al., 2012). Because KARs are involved in presynaptic modulation of neurotransmitter
release (Sihra and Moreno, 2013) and control both GABA and glutamate release (Jane et al., 2009) they represent a
potential target for cognition and memory enhancement drugs.
Compounds such as cyclothiazide and diazoxide, acting on AMPARs, potentiate glutamate currents through a removal of
receptors desensitization (Yamada and Tang, 2003). More recently benzothiazides derivatives, such as 7-chloro-3-
methyl-3, 4- dihydro
- 2H-1,2,4-benzothiadiazine 1,1-dioxide (IDRA21) and 8-chloro-2,3,5,6- tetrahydro
- 3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide (FUR) were studied because of their ability to cross
more efficiently the blood brain barrier and their capability to potentiate AMPA receptor activity (Bertolino et al.1993;
Battisti et al 2012). The modulation of these compounds of KAR activity was not investigated so far.
By using the patch clamp technique in the whole cell configuration we studied IDRA21 and FUR on primary culture of
cerebellum granule cells and in HEK293 transiently transfected with different subunits of KARs.
KA-evoked currents were potentiated by both IDRA 21 and FUR, even though the latter compound was more potent
(EC50IDRA21 191±58 μΜ; EC50RAC 7±2 μM).
Co-application of KA and GYKI 53655, an antagonist of AMPAR, elicits a fast desensitizing current of low amplitude that
was augmented in a dose-dependent manner by IDRA21 (EC50 = 538±240μM) and FUR (EC50 20±4μM).
To investigate whether a selectivity of the effect for certain KARs subunit exists, we analyzed the effect of both IDRA21
and FUR in HEK293 cells transiently transfected with GluR5, GluR6 and GluR5-KA1 subunits.
IDRA21 potentiates Glutamate-evoked currents in a dose-dependent fashion with EC50 of 595±167μM, 653±273μM and
668±271 in GluR5, GluR6 and GluR5-KA1, respectively. The potency of RAC was different in the diverse subunit
assembly being higher in GluR5 expressing cells ( EC50=113±50μM, 188±95μM and 550±145μM for GluR5, GluR6 and
GluR5-KA1, respectively). The efficacy of RAC was always higher than that of IDRA21 in all the subunit combination
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tested.
Our data evidentiate that IDRA21 and FUR not only modulate AMPAR but also KAR activity. Considered the importance
of KA receptors in memory procesess we think that these drugs exert their pharmacological effect also because of their
capability of modulate this receptor activity.
Neurosteroids modulation of mouse neocortical network: a multielectrode based study
No full textNeurosteroids (NSs) such as allopregnanolone (ALLO) and tetrahydrodeoxy-corticosterone (THDOC ) are well known modulators of GABAA receptor function. While a large amount of data are available at the single cell level, their “global modulation” of network firing activity was not investigated in detail. NSs levels vary in the diverse brain regions and may change under different physiological and pathological conditions [1-3]. The NSs synthesizing enzymes, 5α-reductase type I and 3α-hydroxysteroid dehydrogenase have also different expression profiles, i.e. in the neocortex they co-localize in glutamatergic but not in GABAergic neurons [4]. Therefore these endogenous substances in excitatory neurons that co-express GABAA receptors and NSs synthetic enzymes can act in an autocrine manner [5] while in inhibitory cells their modulation is different.
The goal of our study was to investigate the effects of physiological NSs concentrations on the activity of networks formed by acutely dissociated mouse neocortical neurons. By using the multi-electrode arrays (MEA) technique the spontaneous reverberating activity of excitatory and inhibitory neurons was simultaneously recorded and analyzed in control and after perfusion with NSs. Their effects were analyzed, by using statistical methods [6], on physiological variables such as excitability (spikes-per neuron) and number of neurons engaged in bursts.
THDOC, at physiological concentrations, selectively decreased inhibitory interneuron activity, whereas at concentrations higher than 100 nM it inhibited both excitatory and inhibitory clusters. The analysis of the network activity after long time wash-out (8 hrs) highlight that the NS effect on inhibitory clusters persisted for hours producing a sort of long-term depression (LTD) in their excitability.
To further clarify this point we applied THDOC twice (after a 2h wash-out). The first application induced, as expected, a persistent depression of inhibitory neurons but after the second administration of THDOC no further LTD was observed. This experiment suggests that the first application of the NS produces some stable modifications, a sort of “memory” in the network that could be relevant also in vivo.
THDOC and ALLO, at low concentrations, are allosteric modulators of GABAergic neurotransmission but in the μM concentration range they also act as GABAA receptor agonists [7]. In agreement with these properties, our analysis of the global excitability of the network showed a sharp increase in NSs inhibitory effects at concentrations between 100 and 1000 nM, probably due to a direct agonistic activity at GABAA receptors.
Many single-cell studies of the effects of ALLO and THDOC have been performed, but no differences were reported between their effects. We show here that THDOC and ALLO, although consistently producing network inhibition at high concentrations, at low concentrations (10-100 nM) have different effects on excitatory and inhibitory clusters; moreover, the network recovered more easily from ALLO than from THDOC effect.
Previous studies suggested that tonic GABAergic currents are highly sensitive to NSs [8]. To investigate whether this happens also in our experimental mode we applied Gabazine (GBZ), a GABAA receptor antagonist, at concentrations that only block the phasic GABAergic current. GBZ 100 nM increased the activity of inhibitory neurons, leaving almost unaffected the excitatory neuron excitability suggesting that interneurons are controlling each other mainly through a phasic inhibition. In these conditions the excitability of the network was reduced but also the sensitivity to THDOC was unexpectedly decreased by approximately one order of magnitude compared to control.
A detailed analysis [6, 9] of the burst properties showed that in the presence of NSs, the neuronal activity changed and became heterogeneous because of the appearance of different states with occupancy probabilities strongly dependent on the drug concentration. In particular, we observed the random appearance of novel up-states, characterized by excitability features and engaged neurons different from those observed in control.
While the analysis of the “global network effect” of NSs provide information about the average changes in excitability of inhibitory and excitatory clusters, the “states analysis “ highlighted changes in the network connectivity: in the presence of neuromodulators different connectivity modes appear. What is the physiological significance of this effect?
Specific firing patterns recorded in selected neuronal populations encode information during physiological or pathological conditions [10] and changes in the connectivity induced by endogenous compounds are able to modify the response of the network.
Taken together, our results provide a new description of the mode of action of NSs and give some new insight in understanding the complexity of the network response to these endogenous modulators
NON GENOMIC EFFECTS OF THYROID HORMONES ON NICOTINIC NEUROTRANSMISSION
No full textThyroid hormones (THs) play a crucial role for the correct functioning of the mammalian central nervous system. Their genomic actions are well known and recently the attention has been focused on their novel non-genomic effects as modulators of several neurotransmitter systems (Losi et al. 2008, Puia et. al 2011).
Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the brain and play important roles in regulating neuronal activity in several brain areas.
By using the patch clamp technique in the whole cell configuration, we tested the effect of THs on nAChRs expressed in SHSY5Y neuroblastoma cells, in primary cultures of cortical neurons and in 293 HEK cell lines.
In SHSY5Y, THs showed a dose-dependent reduction of nicotine (NIC) (10μM) current ( IC50 T3 10+/- 2 μM; IC50 T4 7+/- 2.5 μM). Their effect was not competitive and more pronounced at high agonist concentrations. Similar results were obtained using Acetylcholine (Ach) as agonist. THs reduced NIC current in cortical neurons even though their modulatory effects were variable and less marked. To test possible subunit specificity for T3 and T4 modulation, we analyze their effect in HEK293 cells expressing α4β2 nAChRs. T3 (1μM) reduced Ach (1μM)-evoked current of 32 +/- 9 % and T4 of 26 +/- 6 %.
It's well known that several cognitive impairments have been resulted from THs deficiency and since “nicotinic circuits” are deeply implicated in learning and memory processes, is possible that also the reported non genomic effect of these hormones could regulate brain function
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