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Thyroid hormones (THs) play a crucial role in the maturation and functioning of mammalian central nervous system. Thyroxine (T4) and 3, 3', 5-L-triiodothyronine (T3) are well known for their genomic effects, but recently attention has been focused on their non genomic actions as modulators of neuronal activity. In the present study we report that T4 and T3 reduce, in a non competitive manner, GABA-evoked currents in rat hippocampal cultures with IC₅₀s of 13±4μM and 12±3μM, respectively. The genomically inactive compound rev-T3 was also able to inhibit the currents elicited by GABA. Blocking PKC or PKA activity, chelating intracellular calcium, or antagonizing the integrin receptor αVβ3 with TETRAC did not affect THs modulation of GABA-evoked currents. THs affect also synaptic activity in hippocampal and cortical cultured neurons. T3 and T4 reduced to approximately 50% the amplitude and frequency of spontaneous inhibitory synaptic currents (sIPSCs), without altering their decay kinetic. Tonic currents evoked by low GABA concentrations were also reduced by T3 (40±5%, n=14), but not by T4. Similarly, T3 decreased currents elicited by low concentrations of THIP, a low affinity GABAA receptor agonist that preferentially activates extrasynaptic receptors, whereas T4 was ineffective. Thus, our data demonstrate that T3 and T4 selectively affect GABAergic phasic and tonic neurotransmission. Since THs concentrations can be regulated at the level of the synapses these data suggest that the network activity of the whole brain could be differently modulated depending on the relative amount of these two hormones. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Using the patch-clamp technique, we studied the effect of two novel adamantane derivatives, N-[2-(3,5-dimethyl-1-adamantyl)ethyl] guanidine (CR 3391) and N-[2-(3,5-dimethyl-1-adamantyl) ethyl]acetamidine (CR 3394), on NMDA receptors expressed in cortical neuron cultures. Our data show that CR 3391 and CR 3394 reduce NMDA-evoked currents (IC50 = 1.7 +/- 0.6 microM and 6.7 +/- 1.5 microM, respectively). This antagonism is non-competitive and is completely reversible. The effect of CR 3394, like that of memantine, was strongly voltage dependent. HEK293 cells expressing NR1a/NR2B recombinant NMDA receptors and immature neurons (DIV 8-9) were more sensitive to CR 3394 antagonism than NR1a/NR2A expressing cells and DIV 15 neurons. CR 3394 also reduced the duration and amplitude of miniature excitatory post-synaptic currents mediated exclusively by NMDA receptors (NMDA-mEPSCs). Both memantine and CR 3394 inhibited NMDA-evoked [3H]norepinephrine release from rat hippocampal slices in a concentration-dependent manner with similar potency. CR 3394, but not memantine, increased cathecholamine resting release at low micromolar concentrations. Moreover, in an in vitro model of neurotoxicity, CR 3394 strongly reduced glutamate- and NMDA-induced neuronal death. Taken together, our data highlight pharmacological features of CR 3394 in vitro that prompt us to further evaluate it as a candidate for the treatment of neurodegenerative disorders.

Using the patch-clamp technique, we studied the modulation of ionotropic gamma-aminobutyric acid (GABA) and glutamate neurotransmission by apigenin, a flavonoid with sedative and antidepressant activity. Apigenin reversibly reduced GABA-evoked currents mediated by alpha1beta2gamma2 receptors expressed in HEK293 cells. Amplitude and frequency of spontaneous postsynaptic inhibitory currents (sIPSCs) mediated by GABA(A) receptors were also decreased by apigenin in cultured cortical neurons. The flavonoid was almost inactive on alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) mediated currents while it reduced N-methyl-D-aspartate (NMDA) receptor mediated responses with a half maximal inhibiting concentration (IC50) of 10 microM. The flavonoid inhibited also peak amplitude and frequency of spontaneous postsynaptic excitatory currents (sEPSCs). Finally, apigenin is neuroprotective against glutamate-induced neurotoxicity in cerebellar and cortical neurons in culture. Our data reveal the antagonistic effect of apigenin on GABA and NMDA channels. While the inhibition on GABA receptor cannot explain the effects of the drug in vivo our data on NMDA channels reveal a new target of apigenin. A reduction of the network excitability could thus account for the sedative effects. Furthermore, our data suggest a potential neuroprotective activity of apigenin.

Exposure of cultured neurons to nanomolar concentrations of terfenadine prevented the NMDA receptor-mediated early appearance (30min.) of toxicity signs induced by the voltage sensitive sodium channel activator veratridine. Terfenadine also provided an histamine-insensitive protection against delayed neurotoxicity by veratridine (24h), occurring independently of NMDA receptor activation, while not protecting from excitotoxicity following direct exposure of neurons to glutamate. Terfenadine reduced tetrodotoxin-sensitive inward currents, and reduced intracellular cGMP formation following veratridine exposure. Our data suggest that nanomolar concentrations of TEF may reduce excitatory aminoacid release following neuronal depolarization via a presynaptic mechanism involving voltage sensitive sodium channels, and therefore may be considered as a prototype for therapeutic drugs in the treatment of diseases that involve excitatory aminoacid neurotransmission.

Exposure of cultured cerebellar neurons to terfenadine prevented the N-methyl-D-aspartate (NMDA) receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. Delayed neurotoxicity by veratridine (24 h) occurring independently from NMDA receptor activation was also prevented by terfenadine. Terfenadine did not protect from excitotoxicity following direct exposure of neurons to glutamate. Our results suggest that terfenadine may modulate endogenous glutamate release following activation of VSSCs.

In rat cerebellar slices, we compared whole-cell recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) with Cl- currents resulting from pulses of GABA (1 mM, < 2 ms) to outside-out patches from Purkinje and granule neurons. sIPSCs in Purkinje cells decayed with a single fast exponential, as previously reported, whereas in granule cells sIPSC decay was best described by the sum of a fast and a slow exponential curve, with a variable contribution of the slow component to the peak current. GABA pulses to nucleated patches from granule cells elicited Cl- currents with decays similar to sIPSC decays, whereas in patches from Purkinje neurons GABA pulses produced Cl- currents decaying largely with a fast component, but often followed by a slower exponential. GABA concentration steps produced rapidly desensitizing currents in patches from both cerebellar neurons. In distinct cerebellar neurons, specific functional properties of GABAA receptors may relate to the presence of distinct receptor subtypes.

To assess the density and distribution of native and recombinant GABAA receptors we used label-fracture and fracture-flip technologies combined with immunocytochemistry using monoclonal and polyclonal Abs directed against the extracellular domain of the GABAA receptor protein located in the freeze-fracture replicas. In cortical neurons there is a high density of GABAA receptors on both soma and dendrites with some areas were the density of receptors is higher, but there are no well defined clusters. In cerebellar granule cells most of the receptors are distributed in round clusters both in neurites and soma. In astroglial cells the receptor density is lower than in neurons and only occasionally they appear in clusters. In cells transfected with cDNAs encoding for various molecular forms of GABAA receptor subunits, the receptor density is moderate when cDNAs for alpha, beta and gamma subunits are cotransfected; however, on cells cotransfected with cDNAs for beta and gamma subunits the receptor density is significantly lower. Recombinant receptors appear randomly distributed and occasionally they aggregate in small groups.

Benzodiazepine-induced modifications of GABA (gamma-aminobutyric acid) activated Cl- currents were studied in native GABAA receptors expressed in neonatal rat brain cortical neurons in primary cultures and in recombinant GABAA receptors expressed in transformed human embryonic kidney cells (293) after a transient transfection with cDNAs encoding for different molecular forms of alpha, beta, and gamma subunits of GABAA receptors. The efficacy of triazolam in cortical neurons was higher than that of diazepam. In transfected cells, triazolam showed a greater efficacy as a positive modulator of GABA-elicited Cl- currents in alpha 1 beta 1 gamma 1, alpha 1 beta 1 gamma 2, alpha 1 beta 1 gamma 3, alpha 6 beta 1 gamma 2 and alpha 1 beta 3 gamma 2 receptors than diazepam, except in alpha 3 beta 1 gamma 2 receptors where diazepam was more efficacious. When triazolam and diazepam were applied together to GABAA receptors assembled by transfecting cDNAs encoding for alpha 1 beta 1 gamma 1 subunits, the action of triazolam was curtailed in a manner related to the dose of diazepam. In recombinant receptors assembled with alpha 1 beta 1 gamma 1 receptors, maximally active doses of triazolam were more efficacious than those of clonazepam, alpidem, zolpidem, diazepam or bretazenil.

Using a competitive polymerase chain reaction assay, we have quantitated the absolute amounts of mRNA encoding 14 distinct subunits of the gamma-aminobutyric acid type A (GABAA) receptor in primary cultures of rat cerebellar granule neurons and cerebellar astrocytes. We found that the total amount of GABAA receptor subunit mRNA in astrocytes was 2 orders of magnitude lower than in neuronal cells. Furthermore, granule cell cultures expressed all 14 different GABAA subunit mRNAs, while the astroglial cultures contained detectable amounts of all the subunits expressed by granule cells except the alpha 6 and the gamma 2L subunits. Of the alpha subunit family members, the alpha 1, alpha 5, and alpha 6 mRNAs were prominent in granule cells, while the alpha 1 and alpha 2 mRNAs were abundant in astrocytes. Of the beta receptor subunit mRNAs, the beta 1 and beta 3 mRNAs were abundantly expressed in both cultures. The gamma 2S and gamma 2L mRNAs constituted the great majority of gamma subunit mRNAs in neurons, while the gamma 1 subunit mRNA was the most abundant gamma subunit mRNA in astrocytes. When various allosteric modulators of GABAA receptors were tested electrophysiologically, methyl 6,7-dimethoxy-4-ethyl-beta-carboline- 3-carboxylate (DMCM) was the only one to modulate chloride currents elicited by GABA in a significantly different manner in granule cells (negative modulation) compared with astrocytes (positive modulation). The latter effect was previously observed in transiently expressed recombinant GABAA receptors containing a gamma 1 instead of a gamma 2 subunit. Our quantitative mRNA results suggest that an important molecular determinant responsible for the DMCM-positive modulatory effect on astroglial native GABAA receptors is the presence of the gamma 1 subunit in the receptor assembly.

In central nervous system gamma-aminobutyric acid (GABA) inhibits neuronal activity by acting on GABA type A (GABAA) receptors. These heterooligomeric integral membrane proteins include a GABA-gated Cl- channel and various allosteric modulatory sites where endogenous modulators and anxiolytic drugs act to regulate GABA action. In vivo, various anxiolytic drugs exhibit a wide range of variability in their modulatory efficacy and potency of GABA action. For instance, bretazenil modulatory efficacy is much lower than that of diazepam. Such low efficacy could be due either to a preferential modulation of specific GABAA receptor subtypes or to a low modulatory efficacy at every GABAA receptor subtype. To address these questions we studied drug-induced modifications of GABA-activated Cl- currents in native GABAA receptors of cortical neurons in primary cultures and in recombinant GABAA receptors transiently expressed in transformed human embryonic kidney cells (293) after transfection with cDNAs encoding different molecular forms of alpha, beta, and gamma subunits of GABAA receptors. In cortical neurons the efficacy of bretazenil was lower than that of diazepam, whereas the potency of the two drugs was similar. In cells transfected with gamma 2 subunits and various molecular forms of alpha and beta subunits bretazenil efficacy was always lower than that of diazepam. However, in cells transfected with gamma 1 or gamma 3 subunits and various forms of alpha and beta subunits the efficacy of both diazepam and bretazenil was lower and always of similar magnitude. When bretazenil and diazepam were applied together to GABAA receptors including a gamma 2 subunit, the action of diazepam was curtailed in a manner related to the dose of bretazenil.

The 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.

Cl- currents elicited by gamma-aminobutyric acid (GABA) application were recorded with the whole-cell tight-seal technique from voltage-clamped cortical neurons of neonatal rats in primary culture. The peripheral benzodiazepine recognition site ligand 4'-chlorodiazepam [Ro 5-4864; 7-chloro-1,3-dihydro-1-methyl-5-(4-chlorophenyl)-2H-[1,4]-benzodiazep in-2- one] inhibited the GABA-generated currents in a dose-dependent manner. Also, a beta-carboline (DMCM; 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester), acting as a negative allosteric modulator of GABAA receptors, reduced the intensity of GABA-generated currents with similar efficacy but greater potency. Flumazenil (Ro 15-1788; 8-fluro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-[1,5-a] [1,4]-benzodiazepine-3-carboxylate ethyl ester) antagonized DMCM inhibition but not that elicited by 4'-chlorodiazepam. The isoquinoline carboxamide PK 11195, an antagonist of 4'-chlorodiazepam effects in other systems, failed to antagonize the action of 4'-chlorodiazepam. The transient expression of various molecular forms of GABAA receptors in the human embryonic kidney cell line 293 allowed a study of the minimal structural requirements for the inhibition of GABA-induced Cl- currents by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. GABA-elicited Cl- currents in cells coexpressing alpha 1 and beta 1 subunits of GABAA receptors were inhibited by bicuculline and picrotoxin, but not by DMCM or 4'-chlorodiazepam. Conversely, the GABA currents in cells coexpressing alpha 1 beta 1 and gamma 2 subunits were inhibited by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. Since the Cl- currents generated by GABA in some molecular forms of GABAA receptors are inhibited by bicuculline and picrotoxin only, 4'-chlorodiazepam cannot be acting isosterically with picrotoxin.

Abstract: Thyroid hormones (THs) are well known for their genomic effects but recently attention has focused also on their nongenomic actions as rapid modulators of membrane receptors. Here we show that thyroxine (T4) and 3,3′,5′-l-triiodothyronine (T3) rapidly decrease N-methyl-d-aspartate (NMDA)-evoked currents in rat hippocampal cultures with potency in the micromolar range. The effect is not mediated by glutamate or glycine binding sites as an increase in agonist or glycine concentration does not alter TH potencies. Furthermore THs’ effect on NMDA receptors is independent of voltage and of subunit composition. The mechanism of THs’ antagonistic effect does not involve PKC phosphorylation of NMDA receptors since neither blocking nor stimulating PKC changed THs’ modulation. T3, but not T4, inhibits also kainate-evoked currents in hippocampal neurons in culture. In hippocampal pyramidal neurons in slice, T3, but not T4, significantly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting their amplitude and decay. In cultured rat cortical neurons THs prevented glutamate-induced neuronal death at concentrations similar to those effective on glutamatergic receptors. Taken together our data show for the first time that THs can rapidly affect ionotropic glutamatergic receptors in hippocampal neurons, an effect that could have an important role in their modulation of brain function in physiological and pathological states. [Copyright &y& Elsevier]