GABAA Receptors In Hippocampal Neurons Research Articles

Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric potential and affect inhibitory GABAA receptor function in vitro.

Naturally occurring compounds such as sesquiterpenes and sesquiterpenoids (SQTs) have been shown to modulate GABAA receptors (GABAA Rs). In this study, the modulatory potential of 11 SQTs at GABAA Rs was analyzed to characterize their potential neurotropic activity. Transfected HEK293 cells and primary hippocampal neurons were functionally investigated using electrophysiological whole-cell recordings. Significantly different effects of β-caryophyllene and α-humulene, as well as their respective derivatives β-caryolanol and humulol, were observed in the HEK293 cell system. In neurons, the concomitant presence of phasic and tonic GABAA R configurations accounts for differences in receptor modulation by SQTs. The in vivo presence of the γ2 and δ subunits is important for SQT modulation. While phasic GABAA receptors in hippocampal neurons exhibited significantly altered GABA-evoked current amplitudes in the presence of humulol and guaiol, negative allosteric potential at recombinantly expressed α1 β2 γ2 receptors was only verified for humolol. Modeling and docking studies provided support for the binding of SQTs to the neurosteroid-binding site of the GABAA R localized between transmembrane segments 1 and 3 at the (+ α)-(- α) interface. In sum, differences in the modulation of GABAA R isoforms between SQTs were identified. Another finding is that our results provide an indication that nutritional digestion affects the neurotropic potential of natural compounds.

2-(4-methyl-thiazol-5-yl) ethyl nitrate maleate-potentiated GABAA receptor response in hippocampal neurons.

2-(4-methyl-thiazol-5-yl) ethyl nitrate maleate (NMZM), a derivative of clomethiazole (CMZ), had been investigated for the treatment of Alzheimer's disease (AD). The beneficial effects of NMZM in AD included reversing cognitive deficit, improving learning and memory as well as neuroprotection. The pharmacological effects of NMZM on GABAA receptors were reported previously; however, the mechanisms were unclear and were explored therefore. In this study, we demonstrated that NMZM improved learning and memory by alleviating scopolamine-induced long-term potentiation (LTP) suppression in the dentate gyrus of rats, indicating that NMZM had protective effects against scopolamine-induced depression of LTP. Next, we investigated the action of NMZM on GABAA receptors in hippocampal neurons and the binding site of NMZM on GABAA receptors. NMZM directly activated GABAA receptors in hippocampal neurons in a weak manner. However, NMZM could potentiate the response of GABAA receptors to GABA and NMZM positively modulated GABAA receptors with an EC50 value of 465μmol/L at 3μmol/L GABA while this potentiation at low concentration of GABA (1, 3μmol/L) was more significant than that at high concentration (10, 30μmol/L). In addition, NMZM could enhance GABA currents after using diazepam and pentobarbital, the positive modulators of GABAA receptors. NMZM could not affect the etomidate-potentiated GABAA current. It suggested that the binding site of NMZM on GABAA receptors is the same as etomidate. These results provided support for the neuroprotective effect of NMZM, which was partly dependent on the potentiation of GABAA receptors. The etomidate binding site might be a new target for neuronal protection and for drug development.

Regulation of dopamine D2 receptor-mediated extracellular signal-regulated kinase signaling and spine formation by GABAA receptors in hippocampal neurons

Dopamine (DA) signaling via DA receptors is known to control hippocampal activity that contributes to learning, memory, and synaptic plasticity. In primary hippocampal neuronal culture, we observed that dopamine D2 receptors (D2R) co-localized with certain subtypes of GABAA receptors, namely α1, β3, and γ2 subunits, as revealed by double immunofluorocytochemical analysis. Treatment with the D2R agonist, quinpirole, was shown to elicit an increase in phosphorylation of extracellular signal-regulated kinase (ERK) in hippocampal neurons. This phosphorylation was inhibited by pretreatment with the GABAA receptor agonist, muscimol. Furthermore, treatment of hippocampal neurons with quinpirole increased the dendritic spine density and this regulation was totally blocked by pretreatment with a MAP kinase kinase (MEK) inhibitor (PD98059), D2R antagonist (haloperidol), or by the GABAA receptor agonist, muscimol. These results suggest that D2R-mediated ERK phosphorylation can control spine formation and that the GABAA receptor negatively regulates the D2R-induced spine formation through ERK signaling in hippocampal neurons, thus indicating a potential role of D2R in the control of hippocampal neuronal excitability.

Cyclin-dependent kinase 5 is involved in the phosphorylation of gephyrin and clustering of GABAA receptors at inhibitory synapses of hippocampal neurons.

CDK5 has been implicated in neural functions including growth, neuronal migration, synaptic transmission and plasticity of excitatory chemical synapses. Here we report robust effects of CDK5 on phosphorylation of the postsynaptic scaffold protein gephyrin and clustering of inhibitory GABAA receptors in hippocampal neurons. shRNA-mediated knockdown of CDK5 and pharmacological inhibition of cyclin-dependent kinases reduced phosphorylated gephyrin clusters and postsynaptic γ2-containing GABAA receptors. Phosphorylation of S270 is antagonized by PP1/PP2a phosphatase and site-directed mutagenesis and in vitro phosphorylation experiments indicate that S270 is a putative CDK5 phosphorylation site of gephyrin. Our data suggest that CDK5 plays an essential role for the stability of gephyrin-dependent GABAA receptor clusters in hippocampal neurons.

Activation of Brain Wnt signaling in vivo: Effect on LTP and Neurogenesis

The roles of the Wnt signaling pathway in several developmental processes, including synaptic differentiation, are well characterized. Wnts have a crucial role in synaptic physiology, as they modulate the synaptic vesicle cycle, the trafficking of neurotransmitter receptors and the interaction of these receptors with scaffold proteins in postsynaptic regions. Recent studies indicate that Wnts regulate synaptic plasticity and neurogenesis. We report here that the in vivo brain application of both agonists and modulators of Wnt receptors, results in improvement of several behavioral assays, concomitant with an increase in several pre and post‐synaptic proteins as well as in synaptic plasticity measured as long ‐ term potentiation (LTP) in hippocampal slices. These results were obtained with both canonical and non‐canonical Wnt modulators, including WASP‐1 and Foxy‐5. More recent studies, includes the effect of natural products as well as Wnts ligands on adult mammalian neurogenesis. In particular, we have examined the role of hypoxia on the activation of canonical Wnt signaling. Current studies are aim to ask whether non‐canonical Wnt signaling affects neurogenesis, since recent work indicated that Wnt‐5a modulates recycling of functional GABAA receptors in hippocampal neurons.

N-desmethylclozapine, a metabolite of the antipsychotic clozapine, modulates excitatory and inhibitory signals oppositely by acting on M1 muscarinic and GABAA receptors in hippocampal neurons

N-desmethylclozapine, a metabolite of the antipsychotic clozapine, modulates excitatory and inhibitory signals oppositely by acting on M1 muscarinic and GABAA receptors in hippocampal neurons

Flupirtine Modulates both KCNQ K+ Channels and GABAA Receptors in Hippocampal Neurons

KCNQ/M-channels are slowly gating and non-inactivating K+ channels. They are widely expressed in the nervous system and play major roles in regulation of neuronal excitability. Still controversial is the functional role of M-channels in neurotransmitter release. To clarify this issue, we sought to test drugs known to activate or inhibit M-channels for their effects on transmitter release or synaptic transmission, using hippocampal “micro-island” autapses or mass cultures. A possible confounding issue are effects of the drugs on excitatory or inhibitory inotropic receptors.

Constitutive Endocytosis of GABAAReceptors by an Association with the Adaptin AP2 Complex Modulates Inhibitory Synaptic Currents in Hippocampal Neurons

Type A GABA receptors (GABA(A)) mediate the majority of fast synaptic inhibition in the brain and are believed to be predominantly composed of alpha, beta, and gamma subunits. Although changes in cell surface GABA(A) receptor number have been postulated to be of importance in modulating inhibitory synaptic transmission, little is currently known on the mechanism used by neurons to modify surface receptor levels at inhibitory synapses. To address this issue, we have studied the cell surface expression and maintenance of GABA(A) receptors. Here we show that constitutive internalization of GABA(A) receptors in hippocampal neurons and recombinant receptors expressed in A293 cells is mediated by clathrin-dependent endocytosis. Furthermore, we identify an interaction between the GABA(A) receptor beta and gamma subunits with the adaptin complex AP2, which is critical for the recruitment of integral membrane proteins into clathrin-coated pits. GABA(A) receptors also colocalize with AP2 in cultured hippocampal neurons. Finally, blocking clathrin-dependant endocytosis with a peptide that disrupts the association between amphiphysin and dynamin causes a large sustained increase in the amplitude of miniature IPSCs in cultured hippocampal neurons. These results suggest that GABA(A) receptors cycle between the synaptic membrane and intracellular sites, and their association with AP2 followed by recruitment into clathrin-coated pits represents an important mechanism in the postsynaptic modulation of inhibitory synaptic transmission.