Effect of nootropic dipeptide noopept on CA1 pyramidal neurons involves α7AChRs on interneurons in hippocampal slices from rat.

Noopept (NP) is a proline-containing dipeptide with nootropic and neuroprotective properties. We have previously shown that NP significantly increased the frequency of spontaneous IPSCs in hippocampal CA1 pyramidal cells mediated by the activation of inhibitory interneurons in stratum radiatum. The cholinergic system plays an important role in the performance of cognitive functions, furthermore multiple behavioral and clinical facts link NP with the cholinergic system. The present study was undertaken to reveal the possible interaction of NP with neuronal nicotinic acetylcholine receptors (nAChRs). Currents were recorded from rat hippocampal neurons using the whole-cell, patch-clamp technique. NP (5 µM) increased the action potential firing frequency recorded from GABAergic interneurons in the stratum radiatum (SR) of CA1 region. This effect was almost completely abolished by the application of the α7 nAChR-selective antagonists α-bungarotoxin (α-BGT; 6 nM) and methyllycaconitine (MLA; 20 nM). The increase in the frequency of spontaneous IPSCs in CA1 pyramidal cells induced by NP was also eliminated by α7 nAChRs antagonists. These results imply the involvement of α7 nAChRs in the modulation of hippocampal neuronal activity caused by NP and indicate that a7 nAChRs are an important site of action of NP.

Synthesis and evaluation of avermectin-imidazo[1,2-a]pyridine hybrids as potent GABAA receptor modulators.

The γ-aminobutyric acid type A (GABAA) receptors are pentameric transmembrane protein complexes. They have attracted extensive attention from the scientific community due to their significant pharmacological potential. Here we report the first synthesis of avermectin-imidazo[1,2-a]pyridine hybrids promising as GABAA receptor positive allosteric modulators (PAMs). An efficient multi-step protocol was elaborated for the installation of the 6-methyl-2-(p-tolyl)imidazo[1,2-a]pyridine pendant to the Avermectin B1a and Ivermectin skeletons through a linker. A variety of linkers were used in order to study the effect of disturbances in the hybrid structure on the GABAA receptor affinity. In vitro experiments showed that the lead compounds exhibited high potency (IC50 = 207 and 359 nM) for binding at the benzodiazepine site of GABAA receptors. In silico studies suggest that the hybrids are able to bind at the Ivermectin binding site of the GABAA receptor. The functional properties of the highest-affinity hybrid (compound 15e) as GABAAR PAM were evaluated by patch-clamp electrophysiological recordings of GABA-mediated currents in rat cerebellar Purkinje neurons. The results obtained suggest that the potentiating effect of hybrid compound 15e is due to its interaction both with benzodiazepine- and Ivermectin-binding sites of GABAARs. Drug-induced behavioral responses in adult zebrafish for hybrids correlate with an alternative mode of action of avermectin and imidazo[1,2-a]pyridine pharmacophores. The investigation of avermectin-imidazo[1,2-a]pyridine hybrid molecules with activity as GABAA receptor modulators is important for the discovery of safe and effective drugs for the treatment of neurological disorders and pest control agents.

Development of 1,3-thiazole analogues of imidazopyridines as potent positive allosteric modulators of GABAA receptors.

Structure-activity relationship studies were conducted in the search for 1,3-thiazole isosteric analogs of imidazopyridine drugs (Zolpidem, Alpidem). Three series of novel γ-aminobutyric acid receptor (GABAAR) ligands belonging to imidazo[2,1-b]thiazoles, imidazo[2,1-b][1,3,4]thiadiazoles, and benzo[d]imidazo[2,1-b]thiazoles were synthesized and characterized as active agents against GABAAR benzodiazepine-binding site. In each of these series, potent compounds were discovered using a radioligand competition binding assay. The functional properties of highest-affinity compounds 28 and 37 as GABAAR positive allosteric modulators (PAMs) were determined by electrophysiological measurements. In vivo studies on zebrafish demonstrated their potential for the further development of anxiolytics. Using the OECD "Fish, Acute Toxicity Test" active compounds were found safe and non-toxic. Structural bases for activity of benzo[d]imidazo[2,1-b]thiazoles were proposed using molecular docking studies. The isosteric replacement of the pyridine nuclei by 1,3-thiazole, 1,3,4-thiadiazole, or 1,3-benzothiazole in the ring-fused imidazole class of GABAAR PAMs was shown to be promising for the development of novel hypnotics, anxiolytics, anticonvulsants, and sedatives drug-candidates.

The mechanisms of potentiation and inhibition of GABAA receptors by non-steroidal anti-inflammatory drugs, mefenamic and niflumic acids.

Fenamates mefanamic and niflumic acids (MFA and NFA) induced dual potentiating and inhibitory effects on GABA currents recorded in isolated cerebellar Purkinje cells using the whole-cell patch-clamp and fast-application techniques. Regardless of the concentration, both drugs induced a pronounced prolongation of the current response. We demonstrated that the same concentration of drugs can produce both potentiating and inhibitory effects, depending on the GABA concentration, which indicates that both processes take place simultaneously and the net effect depends on the concentrations of both the agonist and fenamate. We found that the NFA-induced block is strongly voltage-dependent. The Woodhull analysis of the block suggests that NFA has two binding sites in the pore - shallow and deep. We built a homology model of the open GABAAR based on the cryo-EM structure of the open α1 GlyR and applied Monte-Carlo energy minimization to optimize the ligand-receptor complexes. A systematic search for MFA/NFA binding sites in the GABAAR pore revealed the existence of two sites, the location of which coincides well with predictions of the Woodhull model. In silico docking suggests that two fenamate molecules are necessary to occlude the pore. We showed that MFA, acting as a PAM, competes with an intravenous anesthetic etomidate for a common binding site. We built structural models of MFA and NFA binding at the transmembrane ß(+)/α(-) intersubunit interface. We suggested a hypothesis on the molecular mechanism underlying the prolongation of the receptor lifetime in open state after MFA/NFA binding and ß subunit specificity of the fenamate potentiation.

Block of GABA(A) receptor ion channel by penicillin: electrophysiological and modeling insights toward the mechanism.

GABA(A) receptors (GABA(A)R) mainly mediate fast inhibitory neurotransmission in the central nervous system. Different classes of modulators target GABA(A)R properties. Penicillin G (PNG) belongs to the class of noncompetitive antagonists blocking the open GABA(A)R and is a prototype of ß-lactam antibiotics. In this study, we combined electrophysiological and modeling approaches to investigate the peculiarities of PNG blockade of GABA-activated currents recorded from isolated rat Purkinje cells and to predict the PNG binding site. Whole-cell patch-сlamp recording and fast application system was used in the electrophysiological experiments. PNG block developed after channel activation and increased with membrane depolarization suggesting that the ligand binds within the open channel pore. PNG blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC50 value of 1.12mM at -70mV. The termination of GABA and PNG co-application was followed by a transient tail current. Protection of the tail current from bicuculline block and dependence of its kinetic parameters on agonist affinity suggest that PNG acts as a sequential open channel blocker that prevents agonist dissociation while the channel remains blocked. We built the GABA(A)R models based on nAChR and GLIC structures and performed an unbiased systematic search of the PNG binding site. Monte-Carlo energy minimization was used to find the lowest energy binding modes. We have shown that PNG binds close to the intracellular vestibule. In both models the maximum contribution to the energy of ligand-receptor interactions revealed residues located on the level of 2', 6' and 9' rings formed by a bundle of M2 transmembrane segments, indicating that these residues most likely participate in PNG binding. The predicted structural models support the described mechanism of PNG block.

Modulation of ATP-induced currents by zinc in acutely isolated hypothalamic neurons of the rat.

1. Whole-cell patch-clamp and fast perfusion were used to study the effects of zinc on adenosine 5'-triphosphate (ATP)-induced responses of histaminergic neurons. 2. At 10-30 micro M ATP, Zn(2+) had biphasic effects on ATP responses. Zn(2+) at 3-100 micro M increased the ATP-induced currents, but inhibited them at higher concentrations. 3. At 300 micro M ATP, Zn(2+) predominantly but incompletely inhibited the currents. 4. At 5 and 50 micro M, Zn(2+) shifted to the left the concentration-response curve for ATP-induced currents, without changing the maximal response. At 1 mM, Zn(2+) inhibited ATP-induced currents in a noncompetitive way, reducing the maximal response by 58%. .Zn(2+) increased the decay time of ATP-evoked currents nine fold with an EC(50) of 63 micro M. Upon removal of high concentrations of Zn(2+), there was a rapid increase of the current followed by a slow decline towards the response amplitude seen with ATP alone. The appearance of a tail current is consistent with a Zn(2+)-induced increase of ATP affinity and an inhibition of its efficacy. 6. Thus, Zn(2+) acts as a bidirectional modulator of ATP receptor channels in tuberomamillary neurons, which possess functional P2X(2) receptors. The data are consistent with the existence of two distinct modulatory sites on the P2X receptor, which can be occupied by Zn(2+). 7. Our data suggest that zinc-induced potentiation of ATP-mediated currents is caused by the slowing of ATP dissociation from the receptor, while inhibition of ATP-induced currents is related to the suppression of ATP receptor gating.

Expression and function of P2X purinoceptors in rat histaminergic neurons.

(1) The pharmacology of ATP responses and the expression pattern of seven known subunits of the P2X receptor were investigated in individual histaminergic neurons of the tuberomamillary nucleus (TM). (2) ATP (3-1000 micro M) evoked fast non-desensitizing inward currents in TM neurons. 2-methylthioATP (2MeSATP) displayed the same efficacy but a lower potency, EC(50)s 84 micro M versus 48 micro M, when compared with ATP. Adenosine-diphosphate (ADP), uridine-triphosphate (UTP) and alpha beta methylene-ATP (alphabeta-meATP) were inactive. (3) ATP-mediated whole cell currents were potentiated by acidification of the recording solution (pH 7.5 and 6.6 were compared). (4) Single-cell RT-PCR (scRT-PCR) analysis revealed that the P2X(2) receptor is expressed in all PCR-positive neurons. Each of the P2X(1), P2X(3), P2X(4), P2X(5) and P2X(6) mRNAs were detected in less than 35% of the cells. (5) Suramin antagonized ATP responses with an IC(50) of 4.2 micro M and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 1 micro M) reduced ATP responses to 43% of control, when antagonists were pre-applied 90s before the agonist. Cibacron blue (3 micro M) given together with ATP potentiated control responses by 67%, but inhibited it to 10% after pre-application. (6) 2',3'-O-(2,4,6-Trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) antagonized ATP responses with an IC(50) of 7 micro M. (7) Pharmacological properties of ATP responses together with scRT-PCR data suggest that P2X(2) is the major purinoceptor on the soma of TM neurons, however the presence of heteromeric P2X(2/5) receptors in some neurons cannot be excluded.

GABA(A) receptor heterogeneity in histaminergic neurons.

Histaminergic neurons of the tuberomamillary nucleus display pacemaker properties; their firing rate is regulated according to behavioural state by gabaergic inhibition. Whole-cell recordings and single-cell RT-PCR from acutely isolated rat tuberomamillary neurons were used to characterize GABA -evoked currents and to correlate them with the expression pattern of 12 GABAA receptor subunits. We report differences in sensitivity to GABA and zinc as well as in the modulation of IPSC-decay times by zolpidem in histaminergic neurons expressing gamma-subunits at different levels. Immunocytochemistry and pharmacological analysis of whole-cell GABA-currents in these neurons revealed that all carry the gamma2-subunit protein and that all receptors contain at least one gamma-subunit. Neurons with different expression levels of gamma-subunits displayed a difference in cooperativity of GABA and zolpidem binding which we explain by the presence of one vs. two gamma-subunits in one receptor. Thus, we describe here native GABAA receptor function in relation to its stoichiometry.

Mechanisms of GABA(A) receptor blockade by millimolar concentrations of furosemide in isolated rat Purkinje cells.

The action of diuretic furosemide on the GABA(A) receptor was studied in acutely isolated Purkinje cells using the whole-cell recording and fast application system. Furosemide blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC(50) value > 5 mM at -70 mV. The inhibition was rapid in the onset, fully reversible and did not require drug pre-perfusion. The termination of GABA and furosemide co-application was followed by transient increase in the inward current 'tail' current, which was not observed when furosemide was continuously present in the solution. The degree of furosemide block did not depend on GABA concentration. Furosemide block increased with membrane depolarization. Five millimolar furosemide depressed GABA currents by 32.4+/-1.3% at -70 mV and by 76.7+/-5.0% at +70 mV. Analysis of the voltage dependence of the block suggests that furosemide binds at the site located within GABA(A) channel pore with a dissociation constant of 5.3+/-0.5 mM at 0 mV and electric distance of 0.27. Our results provide evidence that furosemide interacts with Purkinje cell GABA(A) receptors (most probably composed of alpha1beta2/3gamma2 subunits) through a low affinity site located in channel pore and suggest that furosemide acts as a sequential open channel blocker, which prevents the dissociation of agonist while the channel is blocked.