Silencing of spontaneous activity at α4ß1/3δ GABAA receptors in hippocampal granule cells reveals different ligand pharmacology.

BACKGROUND AND PURPOSE: The δ-subunit-containing GABAA receptors, α4 ß1 δ and α4 ß3 δ, in dentate gyrus granule cells (DGGCs) are known to exhibit both spontaneous channel openings (i.e. constitutive activity) and agonist-induced current. The functional implications of spontaneous gating are unclear. In this study, we tested the hypothesis that constitutively active α4 ß1/3 δ receptors limit agonist efficacy. EXPERIMENTAL APPROACH: Whole-cell electrophysiological recordings of adult male rat and mouse hippocampal DGGCs were used to characterize known agonists and antagonists at δ-subunit-containing GABAA receptors. To separate constitutive and agonist-induced currents, different recording conditions were employed. KEY RESULTS: Recordings at either 24°C or 34°C, including the PKC autoinhibitory peptide (19-36) intracellularly, removed spontaneous gating by GABAA receptors. In the absence of spontaneous gating, DGGCs responded to the α4 ß1/3 δ orthosteric agonist Thio-THIP with a four-fold increased efficacy relative to recording conditions favouring constitutive activity. Surprisingly, the neutral antagonist gabazine was unable to antagonize the current by Thio-THIP. Furthermore, a current was elicited by gabazine alone only when the constitutive current was silenced (EC50 2.1 µM). The gabazine-induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ-/- mice and reduced in ß1 -/- mice, but could not be replicated in human α4 ß1/3 δ receptors expressed heterologously in HEK cells. CONCLUSION AND IMPLICATIONS: Kinase activity infers spontaneous gating in α4 ß1/3 δ receptors in DGGCs. This significantly limits the efficacy of GABAA agonists and has implications in pathologies involving aberrant excitability caused by phosphorylation (e.g. addiction and epilepsy). In such cases, the efficacy of δ-preferring GABAA ligands may be reduced.

GAT3 selective substrate l-isoserine upregulates GAT3 expression and increases functional recovery after a focal ischemic stroke in mice.

Ischemic stroke triggers an elevation in tonic GABA inhibition that impairs the ability of the brain to form new structural and functional cortical circuits required for recovery. This stroke-induced increase in tonic inhibition is caused by impaired GABA uptake via the glial GABA transporter GAT3, highlighting GAT3 as a novel target in stroke recovery. Using a photothrombotic stroke mouse model, we show that GAT3 protein levels are decreased in peri-infarct tissue from 6 h to 42 days post-stroke. Prior studies have shown that GAT substrates can increase GAT surface expression. Therefore, we aimed to assess whether the GAT3 substrate, L-isoserine, could increase post-stroke functional recovery. L-Isoserine (38 µM or 380 µM) administered directly into the infarct from day 5 to 32 post-stroke, significantly increased motor performance in the grid-walking and cylinder tasks in a concentration-dependent manner, without affecting infarct volumes. Additionally, L-isoserine induced a lasting increase in GAT3 expression in peri-infarct regions accompanied by a small decrease in GFAP expression. This study is the first to show that a GAT3 substrate can increase GAT3 expression and functional recovery after focal ischemic stroke following a delayed long-term treatment. We propose that enhancing GAT3-mediated uptake dampens tonic inhibition and promotes functional recovery after stroke.

Feasibility and resolution limits of opto-magnetic imaging of neural network activity in brain slices using color centers in diamond.

We suggest a novel approach for wide-field imaging of the neural network dynamics of brain slices that uses highly sensitivity magnetometry based on nitrogen-vacancy (NV) centers in diamond. In-vitro recordings in brain slices is a proven method for the characterization of electrical neural activity and has strongly contributed to our understanding of the mechanisms that govern neural information processing. However, this traditional approach only acquires signals from a few positions, which severely limits its ability to characterize the dynamics of the underlying neural networks. We suggest to extend its scope using NV magnetometry-based imaging of the neural magnetic fields across the slice. Employing comprehensive computational simulations and theoretical analyses, we determine the spatiotemporal characteristics of the neural fields and the required key performance parameters of an NV magnetometry-based imaging setup. We investigate how the technical parameters determine the achievable spatial resolution for an optimal 2D reconstruction of neural currents from the measured field distributions. Finally, we compare the imaging of neural slice activity with that of a single planar pyramidal cell. Our results suggest that imaging of slice activity will be possible with the upcoming generation of NV magnetic field sensors, while single-shot imaging of planar cell activity remains challenging.

The sodium channel activator Lu AE98134 normalizes the altered firing properties of fast spiking interneurons in Dlx5/6+/- mice.

Mental disorders such as schizophrenia are associated with impaired firing properties of fast spiking inhibitory interneurons (FSINs) causing reduced task-evoked gamma-oscillation in prefrontal cortex. The voltage-gated sodium channel NaV1.1 is highly expressed in PV-positive interneurons, but only at low levels in principal cells. Positive modulators of Nav1.1 channels are for this reason considered potential candidates for the treatment of cognitive disorders. Here we examined the effect of the novel positive modulator of voltage-gated sodium channels Lu AE98134. We found that Lu AE98134 facilitated the sodium current mediated by NaV1.1 expressed in HEK cells by shifting its activation to more negative values, decreasing its inactivation kinetics and promoting a persistent inward current. In a slice preparation from the brain of adult mice, Lu AE98134 promoted the excitability of fast spiking interneurons by decreasing the threshold for action potentials. We then tested if Lu AE98134 could normalize the altered firing properties of FSINs in Dlx5/6+/- mutant mice. FSINs of this model for schizophrenia are characterized by broader action potentials and higher spike threshold. We found that in the presence of Lu AE98134, the firing frequency was increased while the spike duration and the threshold were decreased. Compounds with similar mode of action appear as promising candidates for restoring cognitive deficits present in schizophrenia.

Phencyclidine administration during neurodevelopment alters network activity in prefrontal cortex and hippocampus in adult rats.

Symptoms of schizophrenia have been linked to insults during neurodevelopment such as NMDA receptor (NMDAR) antagonist exposure. In animal models, this leads to schizophrenia-like behavioral symptoms as well as molecular and functional changes within hippocampal and prefrontal regions. The aim of this study was to determine how administration of the NMDAR antagonist phencyclidine (PCP) during neurodevelopment affects functional network activity within the hippocampus and medial prefrontal cortex (mPFC). We recorded field potentials in vivo after electrical brain stem stimulation and observed a suppression of evoked theta power in ventral hippocampus, while evoked gamma power in mPFC was enhanced in rats administered with PCP neonatally. In addition, increased gamma synchrony elicited by acute administration of the NMDAR antagonist MK-801 was exaggerated in neonatal PCP animals. These data suggest that NMDAR antagonist exposure during brain development alters functional networks within hippocampus and mPFC possibly contributing to the reported behavioral symptoms of this animal model of schizophrenia.NEW & NOTEWORTHY We show that insults with a NMDA receptor antagonist during neurodevelopment lead to suppressed evoked theta oscillations in ventral hippocampus in adult rats, while evoked gamma oscillations are enhanced and hypersensitive to an acute challenge with a NMDA receptor antagonist in prefrontal cortex. These observations reveal the significance of neurodevelopmental disturbances in the evolvement of schizophrenia-like symptoms and contribute to the understanding of the functional deficits underlying aberrant behavior in this disease.

Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission.

γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

Probing α4ßδ GABAA receptor heterogeneity: differential regional effects of a functionally selective α4ß1δ/α4ß3δ receptor agonist on tonic and phasic inhibition in rat brain.

In the present study, the orthosteric GABAA receptor (GABAAR) ligand 4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol (Thio-THIP) was found to possess a highly interesting functional profile at recombinant human GABAARs and native rat GABAARs. Whereas Thio-THIP displayed weak antagonist activity at α1,2,5ß2,3γ2S and ρ1 GABAARs and partial agonism at α6ß2,3δ GABAARs expressed in Xenopus oocytes, the pronounced agonism exhibited by the compound at α4ß1δ and α4ß3δ GABAARs was contrasted by its negligible activity at the α4ß2δ subtype. To elucidate to which extent this in vitro profile translated into functionality at native GABAARs, we assessed the effects of 100 µm Thio-THIP at synaptic and extrasynaptic receptors in principal cells of four different brain regions by slice electrophysiology. In concordance with its α6ß2,3δ agonism, Thio-THIP evoked robust currents through extrasynaptic GABAARs in cerebellar granule cells. In contrast, the compound did not elicit significant currents in dentate gyrus granule cells or in striatal medium spiny neurons (MSNs), indicating predominant expression of extrasynaptic α4ß2δ receptors in these cells. Interestingly, Thio-THIP evoked differential degrees of currents in ventrobasal thalamus neurons, a diversity that could arise from differential expression of extrasynaptic α4ßδ subtypes in the cells. Finally, whereas 100 µm Thio-THIP did not affect the synaptic currents in ventrobasal thalamus neurons or striatal MSNs, it reduced the current amplitudes recorded from dentate gyrus granule cells, most likely by targeting perisynaptic α4ßδ receptors expressed at distal dendrites of these cells. Being the first published ligand capable of discriminating between ß2- and ß3-containing receptor subtypes, Thio-THIP could be a valuable tool in explorations of native α4ßδ GABAARs.

Altered function of hippocampal CA1 pyramidal neurons in the rTg4510 mouse model of tauopathy.

The formation of neurofibrillary tangles from the assembly of hyperphosphorylated tau leads to dendritic and axonal instability, synaptic degeneration, and neuronal loss. To understand the early physiological consequences of aberrant tau expression, we characterized the physiology of CA1 pyramidal neurons in rTg4510 female mice and non-transgenic (wt) littermate controls. We studied mice at the age of 10-12 weeks where only minimal hyperphosphorylated pretangle tau was present, and 22-24 weeks old mice with significant neurofibrillary tangle pathology. Our electrophysiological analysis included input-output relation, paired-pulse facilitation, and whole cell patch-clamp recordings of neurons to measure action potential threshold and action potential properties, chord-conductance, and characterization of AMPA receptor mediated synaptic transmission. We found that the input-output relation in field (excitatory postsynaptic potentials, EPSP) and whole cell recordings (excitatory postsynaptic currents, EPSC) were impaired in rTg4510 mice compared to wt controls at both ages. We measured a diminished tail current charge after depolarizing voltage input in rTg4510 mice compared to wt in both young and aged mice. Additionally, mini-EPSC properties (peak and decay time) were essentially similar between genotypes and age groups investigated. Surprisingly, in the 22-24 week old group, the mini-EPSC frequency was significantly increased (interevent interval 0.8 ± 0.1 in wt compared to 0.3 ± 0.1 in rTg4510 mice). These data indicate that the developmentally regulated expression of human P301L tau in CA1 pyramidal neurons coincide with changes in neuronal excitability but also that significant presynaptic changes occur late during the progression of tau pathology in this mouse model.

Impaired GABAergic inhibition in the prefrontal cortex of early postnatal phencyclidine (PCP)-treated rats.

A compromised γ-aminobutyric acid (GABA)ergic system is hypothesized to be part of the underlying pathophysiology of schizophrenia. N-methyl-D-aspartate (NMDA) receptor hypofunction during neurodevelopment is proposed to disrupt maturation of interneurons causing an impaired GABAergic transmission in adulthood. The present study examines prefrontal GABAergic transmission in adult rats administered with the NMDA receptor channel blocker, phencyclidine (PCP), for 3 days during the second postnatal week. Whole-cell patch-clamp recordings from pyramidal cells in PCP-treated rats showed a 22% reduction in the frequency of miniature inhibitory postsynaptic currents in layer II/III, but not in layer V pyramidal neurons of the prefrontal cortex. Furthermore, early postnatal PCP treatment caused insensitivity toward effects of the GABA transporter 1 (GAT-1) inhibitor, 1,2,5,6-tetrahydro-1-[2-[[(diphenyl-methylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid, and also diminished currents passed by δ-subunit-containing GABAA receptors in layer II/III pyramidal neurons. The observed impairments in GABAergic function are compatible with the alteration of GABAergic markers as well as cognitive dysfunction observed in early postnatal PCP-treated rats and support the hypothesis that PCP administration during neurodevelopment affects the functionality of interneurons in later life.

Repeated potentiation of the metabotropic glutamate receptor 5 and the alpha 7 nicotinic acetylcholine receptor modulates behavioural and GABAergic deficits induced by early postnatal phencyclidine (PCP) treatment.

The underlying mechanism of the GABAergic deficits observed in schizophrenia has been proposed to involve NMDA receptor hypofunction. An emerging treatment strategy therefore aims at enhancing GABAergic signalling by increasing the excitatory transmission onto interneurons. We wanted to determine whether behavioural and GABAergic functional deficits induced by the NMDA receptor channel blocker, phencyclidine (PCP), could be reversed by repeated administration of two drugs known to enhance GABAergic transmission: the positive allosteric modulator (PAM) of the metabotropic glutamate receptor 5 (mGluR5), ADX47273, and the partial agonist of the α7 nicotinic acetylcholine receptor (α7 nAChR), SSR180711. Adolescent rats (4-5 weeks) subjected to PCP treatment during the second postnatal week displayed a consistent deficit in prepulse inhibition (PPI), which was reversed by a one-week treatment with ADX47273 or SSR180711. We examined GABAergic transmission by whole cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSC) in pyramidal neurons in layer II/III of prefrontal cortex (PFC) and by activation of extrasynaptic δ-containing GABAA receptors by THIP. Following PCP treatment, pyramidal neurons displayed a reduced mIPSC frequency and up-regulation of extrasynaptic THIP-induced current. ADX47273 treatment restored this up-regulation of THIP-induced current. Reduced receptor function seems to be the underlying cause of the reported changes, since repeated treatment with ADX47273 and SSR180711 decreased the induction of spontaneous inhibitory current caused by acute and direct agonism of mGluR5s and α7 nAChRs in slices. These results show that repeated administration of ADX47273 or SSR180711 reverses certain behavioural and functional deficits induced by PCP, likely through down-regulation or desensitisation of mGluR5s and α7 nAChRs, respectively.

Positive modulation of δ-subunit containing GABA(A) receptors in mouse neurons.

δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) receptors in mouse neurons in vitro and in vivo. Whole-cell patch-clamp recordings were carried out in the dentate gyrus in mouse brain slices. In granule cells, AA29504 (1 µM) caused a 4.2-fold potentiation of a tonic current induced by THIP (1 µM), while interneurons showed a potentiation of 2.6-fold. Moreover, AA29504 (1 µM) increased the amplitude and prolonged the decay of miniature inhibitory postsynaptic currents (mIPSCs) in granule cells, and this effect was abolished by Zn²âº (15 µM). AA29504 (1 µM) also induced a small tonic current (12.7 ± 3.2 pA) per se, and when evaluated in a nominally GABA-free environment using Ca²âº imaging in cultured neurons, AA29504 showed GABA(A) receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo. We propose that AA29504 potentiates δ-containing GABA(A) receptors to enhance tonic inhibition, and possibly recruits perisynaptic δ-containing receptors to participate in synaptic phasic inhibition in dentate gyrus.

Inhibition of gamma-aminobutyric acid uptake: anatomy, physiology and effects against epileptic seizures.

The transport of gamma-aminobutyric (GABA) limits the overspill from the synaptic cleft and serves to maintain a constant extracellular level of GABA. Two transporters, GABA transporter-1 (GAT-1) and GAT-3, are the most likely candidates for regulating GABA transport in the brain. Drugs acting either selectively or nonselectively at GATs exert distinct anticonvulsant effects, presumably because of distinct regions of action. Here I shall give a brief review of the localization and physiology of GATs and describe effects of selective and nonselective inhibitors thereof in different animal models of epilepsy.

Activation of NMDA receptors in rat dentate gyrus granule cells by spontaneous and evoked transmitter release.

Activation of N-methyl-D-aspartate (NMDA) receptors by synaptically released glutamate in the nervous system is usually studied using evoked events mediated by a complex mixture of AMPA, kainate, and NMDA receptors. Here we have characterized pharmacologically isolated spontaneous NMDA receptor-mediated synaptic events and compared them to stimulus evoked excitatory postsynaptic currents (EPSCs) in the same cell to distinguish between various modes of activation of NMDA receptors. Spontaneous NMDA receptor-mediated EPSCs recorded at 34 degrees C in dentate gyrus granule cells (DGGC) have a frequency of 2.5 +/- 0.3 Hz and an average peak amplitude of 13.2 +/- 0.8 pA, a 10-90% rise time of 5.4 +/- 0.3 ms, and a decay time constant of 42.1 +/- 2.1 ms. The single-channel conductance estimated by nonstationary fluctuation analysis was 60 +/- 5 pS. The amplitudes (46.5 +/- 6.4 pA) and 10-90% rise times (18 +/- 2.3 ms) of EPSCs evoked from the entorhinal cortex/subiculum border are significantly larger than the same parameters for spontaneous events (paired t-test, P < 0.05, n = 17). Perfusion of 50 microM D(-)-2-amino-5-phosphonopentanoic acid blocked all spontaneous activity and caused a significant baseline current shift of 18.8 +/- 3.0 pA, thus identifying a tonic conductance mediated by NMDA receptors. The NR2B antagonist ifenprodil (10 microM) significantly reduced the frequency of spontaneous events but had no effect on their kinetics or on the baseline current or variance. At the same time, the peak current and charge of stimulus-evoked events were significantly diminished by ifenprodil. Thus spontaneous NMDA receptor-mediated events in DGGC are predominantly mediated by NR2A or possibly NR2A/NR2B receptors while the activation of NR2B receptors reduces the excitability of entorhinal afferents either directly or through an effect on the entorhinal cells.