Involvement of GABAA receptors containing α6 subtypes in antisecretory factor activity on rat cerebellar granule cells studied by two-photon uncaging.

The antisecretory factor (AF) is an endogenous protein that counteracts intestinal hypersecretion and various inflammation conditions in vivo. It has been detected in many mammalian tissues and plasma, but its mechanisms of action are largely unknown. To study the pharmacological action of the AF on different GABAA receptor populations in cerebellar granule cells, we took advantage of the two-photon uncaging method as this technique allows to stimulate the cell locally in well-identified plasma membrane parts. We compared the electrophysiological response evoked by releasing a caged GABA compound on the soma, the axon initial segment and neurites before and after administering AF-16, a 16 amino acids long peptide obtained from the amino-terminal end of the AF protein. After the treatment with AF-16, we observed peak current increases of varying magnitude depending on the neuronal region. Thus, studying the effects of furosemide and AF-16 on the electrophysiological behaviour of cerebellar granules, we suggest that GABAA receptors, containing the α6 subunit, may be specifically involved in the increase of the peak current by AF, and different receptor subtype distribution may be responsible for differences in this increase on the cell.

Electrophysiological study of the effects of side products of RuBi-GABA uncaging on GABAA receptors in cerebellar granule cells.

The study of the GABAA receptor itself and its pharmacology is of paramount importance for shedding light on the role of this receptor in the central nervous system. Caged compounds have emerged as powerful tools to support research in this field, as they allow to control, in space and time, the release of neurotransmitters enabling, for example, to map receptors' distribution and dynamics. Here we focus on γ-aminobutyric acid (GABA)-caged compounds, particularly on a commercial complex called RuBi-GABA, which has high efficiency of uncaging upon irradiation at visible wavelengths. We characterized, by electrophysiological measurements, the effects of RuBi-GABA on GABAA receptors of rat cerebellar granule cells in vitro. In particular, we evaluated the effects of side products obtained after RuBi-GABA photolysis. For this purpose, we developed a procedure to separate the "RuBi-cage" from GABA after uncaging RuBi-GABA with a laser source; then, we compared electrophysiological measurements acquired with and without administering the RuBi-cage in the perfusing bath. In conclusion, to investigate the role of the "cage" molecules both near and far from the cell soma, we compared experiments performed changing the distance of the uncaging point from the cell.

Antisecretory Factor Modulates GABAA Receptor Activity in Neurons.

The antisecretory factor is an endogenous protein found in all mammalian tissues investigated so far. It acts by counteracting intestinal hypersecretion and various forms of inflammation, but the detailed mechanism of antisecretory factor (AF) action is unknown. We tested neuronal GABAA receptors by means of AF-16, a potent AF peptide derived from amino acids 36-51 from the NH2 part of AF. Cultured rat cerebellar granule cells were used, and the effects on the GABA-mediated chloride currents were determined by whole-cell patch clamp. Both the neurotransmitter GABA and AF-16 were added by perfusion of the experimental system. A 3-min AF-16 preincubation was more efficacious than 30 s in significantly elevating the rapidly desensitizing GABA-activated chloride current. No effect was found on the tonic, slowly desensitizing current. The GABA-activated current increase by AF-16 demonstrated a low k of 41 pM with a maximal increase of 37% persisting for some minutes after AF washout, independent from GABA concentration. This indicates an effect on the maximal stimulation (E%Max) excluding an altered affinity between GABA and its receptor. An immunocytochemical fluorescence approach with anti γ2 subunit antibodies demonstrated an increased expression of GABAA receptors. Thus, both the electrophysiological and the immunofluorescence approach indicate an increased appearance of GABAA receptors on the neuronal membrane. The rationale of the experiments was to test the effect of AF on a defined neuronal population of GABAA receptors. The implications of the results on the impact of AF on the enteric nervous system or on brain function are discussed.

Synthesis and pharmacological evaluation of functionalized isoindolinones on GABA-activated chloride currents in rat cerebellum granule cells in culture.

A focused N-substituted 3-(2-piperazin-1-yl-2-oxoethyl)-2-(pyridin-2-yl)iso-indolin-1-ones small library was synthesized for modulation of GABA-A receptor function and compared to Zopiclone for the ability to increase GABA-activated chloride currents. All compounds were tested for their effects on GABA-activated chloride currents in rat cerebellar granule cells by use of the whole-cell patch clamp technique. Electrophysiological studies on cultured cerebellar granule cells revealed 3-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2-(5-nitropyridin-2-yl)iso-indolin-1-one (Id) as a partial agonist displaying 34% increase of the 10µM GABA evoked peak chloride currents, antagonized by flumazenil. Moreover, a second group of compounds, with bulky functional groups at N-4 position of piperazine, have shown inverse agonist effects. The simple synthetic procedure and the possibility of modulating the efficacy of this class of ligands through additional structural modifications pave the way for further development of new molecules as a novel class of compounds able to interfere with benzodiazepine receptors.

Anticonvulsive Activity in Audiogenic DBA/2 Mice of 1,4-Benzodiazepines and 1,5-Benzodiazepines with Different Activities at Cerebellar Granule Cell GABAA Receptors.

Herein, we tested in a model of generalized reflex epilepsy in mice different 1,4-benzodiazepines and 1,5-benzodiazepines with agonistic activity at the GABAA receptor population contributing to the peak component of the chloride current elicited by GABA in cerebellar granule cells (CGCs) in culture. The substances have all higher lipophilia than clobazam, an antiepileptic drug well known and used in human therapy. This ensures that they all can pass relatively easily the blood-brain barrier (BBB). The benzodiazepines were administered intraperitoneally (i.p.) and tested for their activity against sound-induced tonic and clonic seizures in a genetic model of experimental epilepsy, the DBA/2 mouse. Our data demonstrates an interesting inverse correlation between the ED50s and the efficacy (E %) of the drugs in increasing the peak chloride current elicited by GABA in cerebellar granule cells in culture. There is indication of the existence of a threshold of E % above which the increase of ED50 with increasing E % becomes linear. This is statistically significant for the clonic phase, whereas it is at the limit of significance for the tonic one. A possible interpretation of these results is that in this epilepsy model, projections from the cerebellum exert a convulsion prevention activity.

Study of the Interaction of 1,4- and 1,5-Benzodiazepines with GABAA Receptors of Rat Cerebellum Granule Cells in Culture.

The effects of a classical 1,4-benzodiazepine agonist, such as diazepam, its catabolite N-desmethyl-diazepam (nordiazepam), and 1,5-benzodiazepines such as clobazam and RL 214 (a triazolobenzodiazepine previously synthesized in our labs) were evaluated on native GABAA receptors of cerebellar granule cells in culture. The parameter studied was the increase of GABA-activated chloride currents caused by these substances. The contributions of α6 ß2/3 γ2 and α1 α6 ß2/3 γ2 receptor subtypes to the increase of GABA-activated chloride current were investigated by comparing the effects of such substances in the presence vs. the absence of furosemide. Furosemide is in fact able to block such receptors. It was found that the percent enhancement of peak GABA-activated current doubled for diazepam, clobazam, and RL 214. However, it did not change for N-desmethyl-diazepam. These results indicate that diazepam, clobazam, and RL 214 interact exclusively with α1 ß2/3 γ2 receptors, while N-desmethyl-diazepam seems to interact with not only α1- but also α6-containing receptors.

GABAA Receptors of Cerebellar Granule Cells in Culture: Interaction with Benzodiazepines.

GABAA receptor mediated inhibition plays an important role in modulating the input/output dynamics of cerebellum. A characteristic of cerebellar GABAA receptors is the presence in cerebellar granule cells of subunits such as α6 and δ which give insensitivity to classical benzodiazepines. In fact, cerebellar GABAA receptors have generally been considered a poor model for testing drugs which potentially are active at the benzodiazepine site. In this overview we show how rat cerebellar granule cells in culture may be a useful model for studying new benzodiazepine site agonists. This is based on the pharmacological separation of diazepam-sensitive α1 ß2/3 γ2 receptors from those which are diazepam-insensitive and contain the α6 subunit. This is achieved by utilizing furosemide/Zn2+ which block α6 containing and incomplete receptors.

Catatonic features in major depression relieved by electroconvulsive treatment: parallel evaluation of the status of platelet serotonin transporter.

The aim of this research was to follow parallelly the clinical status of a patient and the dynamics of the serotonin transporter (SERT), a likely player in the effect of electroconvulsive treatment (ECT), a powerful tool against deep depression. A patient affected by major depression with catatonic features, not responding to pharmacological therapy, underwent ECT. Evaluations of the binding of labelled paroxetine to venous blood platelet SERT were parallel to the assessments of clinical improvements. The density of platelet SERT, starting from a low level before ECT, displayed an initial steep increase peaking the day after the third electroconvulsive session (5 days after the start of ECT). This was followed by a rapid decrease, which seemed to precede the process of clinical recovery. These results were found in a case of unavoidable ECT treatment. If generalizable, they suggest interesting ideas about the still mysterious mechanism of ECT antidepressant action.

Epileptic seizures but not pseudoseizures are associated with decreased density of the serotonin transporter in blood platelet membranes.

The density of the serotonin transporter in the plasma membranes of blood platelets was evaluated by labelled paroxetine binding in three different groups. These groups were: normal controls, epileptic patients having undergone a recent seizure (less than 4 days before) and patients who equally recently presented psychogenic non-epileptic seizures (pseudoseizures). Real seizures resulted in a significant decrease of membrane serotonin transporter density. In the instances of pseudoseizures, its membrane density was undistinguishable from that of normal controls. These data lend further support to the idea that down regulation of serotonin transporter may play a homeostatic role in the cessation of epileptic seizures.

Protective effects of some creatine derivatives in brain tissue anoxia.

Some derivatives more lipophylic than creatine, thus theoretically being capable to better cross the blood-brain barrier, were studied for their protective effect in mouse hippocampal slices. We found that N-amidino-piperidine is harmful to brain tissue, and that phosphocreatine is ineffective. Creatine, creatine-Mg-complex (acetate) and phosphocreatine-Mg-complex (acetate) increased the latency to population spike disappearance during anoxia. Creatine and creatine-Mg-complex (acetate) also increased the latency of anoxic depolarization, while the delay induced by phosphocreatine-Mg-complex (acetate) was of borderline significance (P = 0.056). Phosphocreatine-Mg-complex (acetate) significantly reduced neuronal hyperexcitability during anoxia, an effect that no other compound (including creatine itself) showed. For all parameters except reduced hyperexcitability the effects statistically correlated with tissue levels of creatine or phosphocreatine. Summing up, exogenous phosphocreatine and N-amidino piperidine are not useful for brain protection, while chelates of both creatine and phosphocreatine do replicate some of the known protective effects of creatine. In addition, phosphocreatine-Mg-complex (acetate) also reduced neuronal hyperexcitability during anoxia.

A quantum-dot nanocrystal study of GABAA receptor subunits in living cerebellar granule cells in culture.

Quantum dots (QDs) are semiconductor nanocrystals emerging as a new class of fluorescent labels with large brightness, multi color fluorescence emission and resistance against photobleaching. Here we have used QDs as biological markers in an immunofluorescence approach. In this work GABA(A )receptors of rat cerebellar granule cells have been studied and in particular we have visualized the beta(2/3) and delta subunits in live cells. The results obtained were compared to those gathered with conventional probes. The images of the delta subunit in living cells appear to correspond to those expected for a subunit part of GABA(A )receptors mediating tonic inhibition in the granules cell bodies.

Is the platelet serotonin transporter different in venous vs. arterial blood?

The binding of labelled paroxetine to the serotonin transporter (SERT) of platelet membranes has been studied in both venous and mixed venous/arterial blood of the rat. In addition, we studied the inhibition of paroxetine binding to SERT by quipazine and N-methyl-quipazine (NMQ). The results indicate differences in affinity for the two test drugs, quipazine and NMQ, in venous vs. mixed venous/arterial blood. This suggests different post-translational modifications of SERT in platelets of arterial vs. venous blood.

Decrease of serotonin transporters in blood platelets after epileptic seizures.

Serotonin transporter (SERT) was studied by [3H]-paroxetine binding in blood platelets from controls and epileptic patients with generalized convulsive seizures. The average KD and BMax were not different in the two cases. However, a significant decrease was found in the serotonin transporter density in the platelet membranes from patients having undergone an epileptic seizure less than 4 days before. This circumstance may indicate a homeostatic reaction to the epileptic attack.

Different chloride electrochemical gradients across the plasma membrane in subcellular compartments of rat cerebellum granules.

The effects of GABA on intracellular Ca2+ have been studied in neonatal rat cerebellum granule cells (CGC) in culture by Oregon Green and two-photon excitation fluorescence microscopy. This technique allowed the study of [Ca2+]i both in cell bodies and neurites. Working with a perfusion chloride concentration corresponding to the average extracellular level, we found that GABA induced an increase in [Ca2+]i in the cell bodies in many of the cells studied with a maximum at day 4 in vitro. This effect disappeared after day 6. However, no increase in [Ca2+]i was ever found in neurites at standard [Cl-]e. On the other hand, an increase of [Ca2+]i was found also in neurites when [Cl-]e was close to zero. The [Ca2+]i increases were blocked by both bicuculline methiodide and nimodipine. The results indicate the presence of an outward directed electrochemical gradient for chloride in the cell bodies which results in depolarization by GABA via GABA(A) receptor activation. Calcium ion influx ensues due to activation of voltage-gated calcium channels (VGCC). This phenomenon may mediate the well-known trophic effect of GABA on these cells at this developmental stage, via an action of [Ca2+]i on the transcriptional activity of the nucleus. No calcium accumulation takes place in neurites due to either no or a reverse (hyperpolarizing) electrochemical gradient for chloride ions. Such a circumstance in later developmental stages may be of importance for the phasic component of GABA-mediated inhibition.

Structure-affinity relationship studies on arylpiperazine derivatives related to quipazine as serotonin transporter ligands. Molecular basis of the selectivity SERT/5HT3 receptor.

A series of quipazine derivatives, previously synthesized to probe the 5-HT(3) receptor, was evaluated for its potential interaction with serotonin transporter (SERT). Some of them show nanomolar affinity for the rodent SERT comparable to or slightly higher than quipazine or N-methylquipazine. Subsequently a candidate was selected on the basis of its SERT affinity and submitted to a molecular manipulation of the basic moiety. The structure-affinity relationships obtained provided information on the role of the fused benzene ring of quipazine in the interaction with the SERT binding site and on the stereoelectronic requirements for the interaction of both the heteroaromatic component and the basic moiety. Moreover, the comparison of the structure-affinity relationships obtained in the present work with those concerning the interaction of these heteroarylpiperazine derivatives with 5-HT3 receptor suggested some molecular determinants of the selectivity SERT/5HT3 receptor.

Subcellular distribution of alpha1 and beta2/3 GABA(A) receptor subunits in sensory neurons of the bovine trigeminal mesencephalic nucleus: evidence suggesting their axoplasmic transport.

Neurons were free-hand isolated under the stereomicroscope from the bovine trigeminal mesencephalic nucleus. These neurons were challenged with monoclonal antibodies against the alpha1 and the beta2/3 subunits of the GABA(A) receptor. The neurons showed a strong reaction to both antibodies. The reaction was mainly intracellular in the round cell bodies and in the axoplasm of the axon emerging from these pseudounipolar cells. This suggests the synthesis of these subunits in the cell body and their transport along the axon.

Two-photon imaging of calcium accumulation in rat cerebellar granule cells.

Topical accumulation of calcium ions in neurites and cell bodies of rat cerebellar granule cells was studied by two-photon microscopy in neurons loaded with the Ca-sensitive fluorescent indicator Oregon Green 488 Bapta. High potassium caused a rapid surge of internal calcium ([Ca2+]i) in the cell body, followed by a plateau. In neurites, [Ca2+]i reached a peak and then decreased back to the control level. In contrast, in neurons stimulated by NMDA, [Ca2+]i reached a steady level and remained constant as long as the agonist was present in the bath, either in the cell bodies or in neurites. In the latter, the response to NMDA treatment was smaller and heterogeneous, and [Ca2+]i increased in certain segments of the neurite, but not in others.

The combined disruption of microfilaments and microtubules affects the distribution and function of GABAA receptors in rat cerebellum granule cells in culture.

The role of the microfilaments and microtubules cytoskeleton in the stability of the subcellular distribution and function of GABAA receptors has been studied in rat cerebellar granule cells in culture. The disruption of either the microfilaments or the microtubules structures did not result in detectable changes in the receptors distribution, as assessed by immunocytochemistry, or in their function, as assessed by the whole-cell patch-clamp approach. A distinct disruption of both the subcellular distribution and the function of the GABAA receptors was found only if both microfilaments and microtubules were destroyed. The results suggest that, in the short term, the plasma membrane localization/stabilization and function of these receptors in granule cells are largely independent from microfilaments and microtubules individually, although they obviously depend on the presence of an organized cellular framework.

A non-peptide NK1 receptor agonist showing subpicomolar affinity.

3-Quinolinecarboxamides have been synthesized and evaluated for their binding to the human NK(1) receptor. Several secondary amide derivatives show NK(1) receptor affinity in the picomolar range. The most active compound, hydroxymethylcarboxamide 3h showing an IC(50) value in the subpicomolar range, behaved as an agonist of NK(1) receptor in endothelial cell proliferation, inositol phosphate turnover, and NO-mediated cyclic GMP accumulation, thus proving it to be the first non-peptide NK(1) receptor agonist showing very high potency.

Immunocytochemical study of alpha 1 and beta 2/3 subunits of GABAA receptors in freehand isolated vestibular Deiters' neurons.

Vestibular Deiters' neurons have been isolated from bovine brain by the Hydén's freehand dissection technique and challenged with monoclonal antibodies directed toward the alpha 1 and beta 2/3 subunits of the GABAA receptors. Subsequent challenge with fluorescent secondary antibodies and confocal microscopy allowed the study of the cellular distribution of such subunits. In Deiters' neurons the beta 2/3 subunit displayed a clear presence all along the cell body profile and the initial parts of the dendrites. The alpha 1 subunit was found highly present all over the cell interior except the nuclear profiles. The strong presence inside the cells possibly masked its presence on the plasma membrane. However, in part of the cells studied a distinct presence on the plasma membrane was evident. This subunit was visualized also all along the long dendrites of these neurons. The approach we describe here, involving freehand isolated mature neurons from adult animals, may allow a better characterization of the tridimensional distribution of different types of neuronal GABAA receptors in the respect of the approach with brain slices.