Differential Drug Responses on Native GABAA Receptors Revealing Heterogeneity in Extrasynaptic Populations in Cultured Hippocampal Neurons

Hippocampal pyramidal neurons potentially express multiple subtypes of GABA_A receptors at extrasynaptic locations that could therefore respond to different drugs. We activated extrasynaptic GABA_A receptors in cultured rat hippocampal pyramidal neurons and measured single-channel currents in order to compare the actions of two drugs that potentially target different GABA_A receptor subtypes. Despite the possible difference in receptor targets of etomidate and diazepam, the two drugs were similar in their actions on native extrasynaptic GABA_A receptors. Each drug produced three distinct responses that differed significantly in current magnitude, implying heterogeneous GABA_A receptor populations. In the majority of patches, drug application increased both the single-channel conductance (>40 pS) and the open probability of the channels. By contrast, in the minority of patches, drug application caused an increase in open probability only. In the third group high-conductance channels were observed upon GABA activation and drug application increased their open probability only. The currents potentiated by etomidate or diazepam were substantially larger in patches displaying high-conductance GABA channels compared to those displaying only low-conductance channels. Factors contributing to the large magnitude of these currents were the long mean open time of high-conductance channels and the presence of multiple channels in these patches. In conclusion, we suggest that the local density of extrasynaptic GABA_A receptors may influence their single-channel properties and may be an additional regulating factor for tonic inhibition and, importantly, differential drug modulation. This work is dedicated to the memory of Professor P. W. Gage.     

Homeostatic Competition between Phasic and Tonic Inhibition

The GABA_A receptors are the major inhibitory receptors in the brain and are localized at both synaptic and extrasynaptic membranes. Synaptic GABA_A receptors mediate phasic inhibition, whereas extrasynaptic GABA_A receptors mediate tonic inhibition. Both phasic and tonic inhibitions regulate neuronal activity, but whether they regulate each other is not very clear. Here, we investigated the functional interaction between synaptic and extrasynaptic GABA_A receptors through various molecular manipulations. Overexpression of extrasynaptic α6β3δ-GABA_A receptors in mouse hippocampal pyramidal neurons significantly increased tonic currents. Surprisingly, the increase of tonic inhibition was accompanied by a dramatic reduction of the phasic inhibition, suggesting a possible homeostatic regulation of the total inhibition. Overexpressing the α6 subunit alone induced an up-regulation of δ subunit expression and suppressed phasic inhibition similar to overexpressing the α6β3δ subunits. Interestingly, blocking all GABA_A receptors after overexpressing α6β3δ receptors could not restore the synaptic GABAergic transmission, suggesting that receptor activation is not required for the homeostatic interplay. Furthermore, insertion of a gephyrin-binding-site (GBS) into the α6 and δ subunits recruited α6_GBSβ3δ_GBS receptors to postsynaptic sites but failed to rescue synaptic GABAergic transmission. Thus, it is not the positional effect of extrasynaptic α6β3δ receptors that causes the down-regulation of phasic inhibition. Overexpressing α5β3γ2 subunits similarly reduced synaptic GABAergic transmission. We propose a working model that both synaptic and extrasynaptic GABA_A receptors may compete for limited receptor slots on the plasma membrane to maintain a homeostatic range of the total inhibition.     

Methyl CpG Binding Protein 2 Gene Disruption Augments Tonic Currents of γ-Aminobutyric Acid Receptors in Locus Coeruleus Neurons: IMPACT ON NEURONAL EXCITABILITY AND BREATHING*

People with Rett syndrome and mouse models show autonomic dysfunction involving the brain stem locus coeruleus (LC). Neurons in the LC of Mecp2-null mice are overly excited, likely resulting from a defect in neuronal intrinsic membrane properties and a deficiency in GABA synaptic inhibition. In addition to the synaptic GABA receptors, there is a group of GABA_A receptors (GABA_ARs) that is located extrasynaptically and mediates tonic inhibition. Here we show evidence for augmentation of the extrasynaptic GABA_ARs in Mecp2-null mice. In brain slices, exposure of LC neurons to GABA_AR agonists increased tonic currents that were blocked by GABA_AR antagonists. With 10 μm GABA, the bicuculline-sensitive tonic currents were ∼4-fold larger in Mecp2-null LC neurons than in the WT. Single-cell PCR analysis showed that the δ subunit, the principal subunit of extrasynaptic GABA_ARs, was present in LC neurons. Expression levels of the δ subunit were ∼50% higher in Mecp2-null neurons than in the WT. Also increased in expression in Mecp2-null mice was another extrasynaptic GABA_AR subunit, α6, by ∼4-fold. The δ subunit-selective agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride and 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]]benzamide activated the tonic GABA_A currents in LC neurons and reduced neuronal excitability to a greater degree in Mecp2-null mice than in the WT. Consistent with these findings, in vivo application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride alleviated breathing abnormalities of conscious Mecp2-null mice. These results suggest that extrasynaptic GABA_ARs seem to be augmented with Mecp2 disruption, which may be a compensatory response to the deficiency in GABAergic synaptic inhibition and allows control of neuronal excitability and breathing abnormalities.     

γ-Hydroxybutyric Acid (GHB) Is Not an Agonist of Extrasynaptic GABAA Receptors

γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABA_B receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABA_A receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABA_A receptors, where GHB acts as an agonist with an EC₅₀ of 140 nM. We investigated three neuronal cell types that express a tonic GABA_A receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABA_A receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABA_B receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.     

Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors

GABA_A receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α₅ has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α₅ subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α₅ subunit-containing GABA_A receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α_4/6 subunit-containing GABA_A receptors, but is sensitive to L-655708, an antagonist of the GABA_A receptors containing α₅ subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α₅ subunit in the turtle spinal cord. Together, these results suggest that GABA_A receptors containing the α₅ subunit mediate the tonic inhibitory currents observed in VHIs.     

Extrasynaptic GABA(A) receptors and tonic inhibition in rat auditory thalamus

Background

Neural inhibition plays an important role in auditory processing and attentional gating. Extrasynaptic GABA_A receptors (GABA_AR), containing α₄and δ GABA_AR subunits, are thought to be activated by GABA spillover outside of the synapse following release resulting in a tonic inhibitory Cl⁻ current which could account for up to 90% of total inhibition in visual and somatosensory thalamus. However, the presence of this unique type of inhibition has not been identified in auditory thalamus.

Methodology/Principal Findings

The present study used gaboxadol, a partially selective potent agonist for δ-subunit containing GABA_A receptor constructs to elucidate the presence of extrasynaptic GABA_ARs using both a quantitative receptor binding assay and patch-clamp electrophysiology in thalamic brain slices. Intense [³H]gaboxadol binding was found to be localized to the MGB while whole cell recordings from MGB neurons in the presence of gaboxadol demonstrated the expression of δ-subunit containing GABA_ARs capable of mediating a tonic inhibitory Cl⁻ current.

Conclusions/Significance

Potent tonic inhibitory GABA_AR responses mediated by extrasynaptic receptors may be important in understanding how acoustic information is processed by auditory thalamic neurons as it ascends to auditory cortex. In addition to affecting cellular behavior and possibly neurotransmission, functional extrasynaptic δ-subunit containing GABA_ARs may represent a novel pharmacological target for the treatment of auditory pathologies including temporal processing disorders or tinnitus.     

Spontaneously Opening GABAA Channels in CA1 Pyramidal Neurones of Rat Hippocampus

Spontaneous, single channel, chloride currents were recorded in 48% of cell-attached patches on neurones in the CA1 region of rat hippocampal slices. In some patches, there was more than 1 channel active. They showed outward rectification: both channel conductance and open probability were greater at depolarized than at hyperpolarized potentials. Channels activated by γ-aminobutyric acid (GABA) in silent patches on the same neurones had similar conductance and outward rectification. The spontaneous currents were inhibited by bicuculline and potentiated by diazepam. It was concluded that the spontaneously opening channels were constitutively active, nonsynaptic GABA_A channels. Such spontaneously opening GABA_A channels may provide a tonic inhibitory mechanism in these cells and perhaps in other cells that have GABA_A receptors although not having a GABA_A synaptic input. They may also be a target for clinically useful drugs such as the benzodiazepines. Received: 31 August 1999/Revised: 2 November 1999     

Modulation of Tonic GABA Currents by Anion Channel and Connexin Hemichannel Antagonists

Anion channels and connexin hemichannels are permeable to amino acid neurotransmitters. It is hypothesized that these conductive pathways release GABA, thereby influencing ambient GABA levels and tonic GABAergic inhibition. To investigate this, we measured the effects of anion channel/hemichannel antagonists on tonic GABA currents of rat hippocampal neurons. In contrast to predictions, blockade of anion channels and hemichannels with NPPB potentiated tonic GABA currents of neurons in culture and acute hippocampal slices. In contrast, the anion channel/hemichannel antagonist carbenoxolone (CBX) inhibited tonic currents. These findings could result from alterations of ambient GABA concentration or direct effects on GABA_A receptors. To test for effects on GABA_A receptors, we measured currents evoked by exogenous GABA. Coapplication of NPPB with GABA potentiated GABA-evoked currents. CBX dose-dependently inhibited GABA-evoked currents. These results are consistent with direct effects of NPPB and CBX on GABA_A receptors. GABA release from hippocampal cell cultures was directly measured using HPLC. Inhibition of anion channels with NPPB or CBX did not affect GABA release from cultured hippocampal neurons. NPPB reduced GABA release from pure astrocytic cultures by 21%, but the total GABA release from astrocytes was small compared to that of mixed cultures. These data indicate that drugs commonly used to antagonize anion channels and connexin hemichannels may affect tonic currents via direct effects on GABA_A receptors and have negligible effects on ambient GABA concentrations. Interpretation of experiments using NPPB or CBX should include consideration of their effects on tonic GABA currents.     

Hyperpolarization-Activated Current (Ih) Is Reduced in Hippocampal Neurons from Gabra5?/? Mice

Changes in the expression of γ-aminobutyric acid type A (GABA_A) receptors can either drive or mediate homeostatic alterations in neuronal excitability. A homeostatic relationship between α5 subunit-containing GABA_A (α5GABA_A) receptors that generate a tonic inhibitory conductance, and HCN channels that generate a hyperpolarization-activated cation current (I_h) was recently described for cortical neurons, where a reduction in I_h was accompanied by a reciprocal increase in the expression of α5GABA_A receptors resulting in the preservation of dendritosomatic synaptic function. Here, we report that in mice that lack the α5 subunit gene (Gabra5−/−), cultured embryonic hippocampal pyramidal neurons and ex vivo CA1 hippocampal neurons unexpectedly exhibited a decrease in I_h current density (by 40% and 28%, respectively), compared with neurons from wild-type (WT) mice. The resting membrane potential and membrane hyperpolarization induced by blockade of I_h with ZD-7288 were similar in cultured WT and Gabra5−/− neurons. In contrast, membrane hyperpolarization measured after a train of action potentials was lower in Gabra5−/− neurons than in WT neurons. Also, membrane impedance measured in response to low frequency stimulation was greater in cultured Gabra5−/− neurons. Finally, the expression of HCN1 protein that generates I_h was reduced by 41% in the hippocampus of Gabra5−/− mice. These data indicate that loss of a tonic GABAergic inhibitory conductance was followed by a compensatory reduction in I_h. The results further suggest that the maintenance of resting membrane potential is preferentially maintained in mature and immature hippocampal neurons through the homeostatic co-regulation of structurally and biophysically distinct cation and anion channels.     

Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex: molecular mechanisms and functional implications

Serotonergic neurotransmission in prefrontal cortex (PFC) plays a key role in regulating emotion and cognition under normal and pathological conditios. Increasing evidence suggests that serotonin receptors are involved in the complex regulation of GABAergic inhibitory transmission in PFC. Activation of postsynaptic 5-HT₂ receptors in PFC pyramidal neurons inhibits GABA_A-receptor currents via phosphorylation of GABA_A receptor γ2 subunits by RACK1-anchored PKC. In contrast, activation of postsynaptic 5-HT₄ receptors produces an activity-dependent bi-directional regulation of GABA-evoked currents in PFC pyramidal neurons, which is mediated through phosphorylation of GABA_A-receptor β subunits by anchored PKA. On the presynaptic side, GABAergic inhibition is regulated by 5-HT through the activation of 5-HT₂, 5-HT₁, and 5-HT₃ receptors on GABAergic intereneurons. These data provide a molecular and cellular mechanism for serotonin to dynamically regulate synaptic transmission and neuronal excitability in the PFC network, which may underlie the actions of many antidepressant and antipsychotic drugs.     

Intracellular blockade of GABAA receptors in the rat hippocampal neurons

The intracellular blockade of GABA_A-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABA_A receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABA_A receptor channels substitute for Cl^? and in which Mg²⁺ and ATP are omitted to induce rundown of the GABA_A receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg²⁺- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABA_A-receptor-mediated synaptic currents and responses induced by the GABA_A receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg²⁺, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities.     

A Reinforcing Circuit Action of Extrasynaptic GABAA Receptor Modulators on Cerebellar Granule Cell Inhibition

GABA_A receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants.     

Waking Action of Ursodeoxycholic Acid (UDCA) Involves Histamine and GABA(A) Receptor Block

Since ancient times ursodeoxycholic acid (UDCA), a constituent of bile, is used against gallstone formation and cholestasis. A neuroprotective action of UDCA was demonstrated recently in models of Alzheimer''s disease and retinal degeneration. The mechanisms of UDCA action in the nervous system are poorly understood. We show now that UDCA promotes wakefulness during the active period of the day, lacking this activity in histamine-deficient mice. In cultured hypothalamic neurons UDCA did not affect firing rate but synchronized the firing, an effect abolished by the GABA_AR antagonist gabazine. In histaminergic neurons recorded in slices UDCA reduced amplitude and duration of spontaneous and evoked IPSCs. In acutely isolated histaminergic neurons UDCA inhibited GABA-evoked currents and sIPSCs starting at 10 µM (IC₅₀ = 70 µM) and did not affect NMDA- and AMPA-receptor mediated currents at 100 µM. Recombinant GABA_A receptors composed of α1, β1–3 and γ2L subunits expressed in HEK293 cells displayed a sensitivity to UDCA similar to that of native GABA_A receptors. The mutation α1V256S, known to reduce the inhibitory action of pregnenolone sulphate, reduced the potency of UDCA. The mutation α1Q241L, which abolishes GABA_AR potentiation by several neurosteroids, had no effect on GABA_AR inhibition by UDCA. In conclusion, UDCA enhances alertness through disinhibition, at least partially of the histaminergic system via GABA_A receptors.     

Characterization of Inhibitory GABA-A Receptor Activation during Spreading Depolarization in Brain Slice

Spreading depolarization (SD) is a slowly propagating wave of near complete depolarizations of neurons and glia. Previous studies have reported large GABA releases during SD, but there is limited understanding of how GABA release and receptor activation are regulated and influence the propagating SD wavefront, as well as an excitatory phase immediately following the passage of SD. The present study characterized GABA-A type receptor (GABA_AR) currents during SD generated by KCl microinjection in acute hippocampal slices from adult mice. Spontaneous GABA_AR-mediated currents (sIPSCs) were initially enhanced, and were followed by a large outward current at the wavefront. sIPSC were then transiently supressed during the late SD phase, resulting in a significant reduction of the sIPSC/sEPSC ratio. The large outward current generated during SD was eliminated by the GABA_AR antagonist gabazine, but the channel potentiator/agonist propofol failed to potentiate the current, likely because of a ceiling effect. Extracellular Cl⁻ decreases recorded during SD were reduced by the antagonist but were not increased by the potentiator. Together with effects of GABA_AR modulators on SD propagation rate, these results demonstrate a significant inhibitory role of the initial GABA_AR activation and suggest that intracellular Cl⁻ loading is insufficient to generate excitatory GABA_AR responses during SD propagation. These results provide a mechanistic explanation for facilitating effects of GABA_AR antagonists, and the lack of inhibitory effect of GABA_AR potentiators on SD propagation. In addition, selective suppression of GABA transmission in the late SD period and the lack of effect of GABA_A modulators on the duration of SD suggests that GABA modulation may not be effective approach to protect neurons during the vulnerable phase of SD.     

Fragrant Dioxane Derivatives Identify β1-Subunit-containing GABAA Receptors

Nineteen GABA_A receptor (GABA_AR) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of β1-subunit-containing GABA_ARs is unknown. Here we report the discovery of a new structural class of GABA_AR positive modulators with unique β1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed α1βxγ2L (x-for 1,2,3) GABA_AR FDD were 6 times more potent at β1- versus β2- and β3-containing receptors. Serine at position 265 was essential for the high sensitivity of the β1-subunit to FDD and the β1N286W mutation nearly abolished modulation; vice versa the mutation β3N265S shifted FDD sensitivity toward the β1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to β1-negative cerebellar Purkinje neurons. Immunostaining for the β1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by β1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of β1-containing GABA_ARs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA_ARs.     

Neonatal estradiol exposure to female rats changes GABAA receptor expression and function,and spatial learning during adulthood

Exposure of female rats to estradiol during the perinatal period has profound effects on GABAergic neurotransmission that are crucial to establish sexually dimorphic brain characteristics. We previously showed that neonatal β-estradiol 3-benzoate (EB) treatment decreases brain concentrations of the neurosteroid allopregnanolone, a potent positive modulator of extrasynaptic GABA_A receptors (GABA_AR). We thus evaluated whether neonatal EB treatment affects GABA_AR expression and function in the hippocampus of adult female rats. Neonatal EB administration increased the expression of extrasynaptic α4/δ subunit-containing GABA_ARs and the modulatory action of THIP on tonic currents mediated by these receptors. The same treatment decreased the expression of synaptic α1/α4/γ2 subunit-containing receptors, as well as phasic currents. These effects of neonatal EB treatment are not related to ambient allopregnanolone concentrations per se, given that vehicle-treated rats in diestrus, which have opposite neurosteroid levels than EB-treated rats, show similar changes in GABA_ARs. Rather, these changes may represent a compensatory mechanism to counteract the long-term reduction in allopregnanolone concentrations, induced by neonatal EB. Given that both α4/δ receptors and allopregnanolone are involved in memory consolidation, we evaluated whether neonatal EB treatment alters performance in the Morris water maze test during adulthood. Neonatal EB treatment decreased the latency and the cumulative search error to reach the platform, as well as thigmotaxis, suggesting improved learning, and also enhanced memory performance during the probe trial. These enduring changes in GABA_AR plasticity may be relevant for the regulation of neuronal excitability in the hippocampus and for the etiology of psychiatric disorders that originate in development and show sex differences.     

Activation of functional α7-containing nAChRs in hippocampal CA1 pyramidal neurons by physiological levels of choline in the presence of PNU-120596

Background

The level of expression of functional α7-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal CA1 pyramidal neurons is believed to be very low compared to hippocampal CA1 interneurons, and for many years this expression was largely overlooked. However, high densities of expression of functional α7-containing nAChRs in CA1 pyramidal neurons may not be necessary for triggering important cellular and network functions, especially if activation of α7-containing nAChRs occurs in the presence of positive allosteric modulators such as PNU-120596.

Methodology/Principal Findings

An approach previously developed for α7-containing nAChRs expressed in tuberomammillary neurons was applied to investigate functional CA1 pyramidal α7-containing nAChRs using rat coronal hippocampal slices and patch-clamp electrophysiology. The majority (∼71%) of tested CA1 pyramidal neurons expressed low densities of functional α7-containing nAChRs as evidenced by small whole-cell responses to choline, a selective endogenous agonist of α7 nAChRs. These responses were potentiated by PNU-120596, a novel positive allosteric modulator of α7 nAChRs. The density of functional α7-containing nAChRs expressed in CA1 pyramidal neurons (and thus, the normalized net effect of activation, i.e., response net charge per unit of membrane capacitance per unit of time) was estimated to be ∼5% of the density observed in CA1 interneurons. The results of this study demonstrate that despite low levels of expression of functional pyramidal α7-containing nAChRs, physiological levels of choline (∼10 µM) are sufficient to activate these receptors and transiently depolarize and even excite CA1 pyramidal neurons in the presence of PNU-120596. The observed effects are possible because in the presence of 10 µM choline and 1–5 µM PNU-120596, a single opening of an individual pyramidal α7-containing nAChR ion channel appears to transiently depolarize (∼4 mV) the entire pyramidal neuron and occasionally trigger action potentials.

Conclusions

1) The majority of hippocampal CA1 pyramidal neurons express functional α7-containing nAChRs. In the absence of PNU-120596, a positive allosteric modulator of α7 nAChRs, a lack of responsiveness of some hippocampal CA1 pyramidal neurons to focal application of 0.5–1 mM choline does not imply a lack of expression of functional α7-containing nAChRs in these neurons. Rather, it may indicate a lack of detection of α7-containing nAChR-mediated currents by patch-clamp electrophysiology. 2) PNU-120596 can serve as a powerful tool for detection and enhancement of responsiveness of low densities of functional α7-containing nAChRs such as those present in hippocampal CA1 pyramidal neurons. 3) In the presence of PNU-120596, physiological concentrations of choline activate functional CA1 pyramidal α7-containing nAChRs and produce step-like currents that cause repetitive step-like depolarizations, occasionally triggering bursts of action potentials in CA1 pyramidal neurons. Therefore, the results of this study suggest that in the presence of PNU-120596 and possibly other positive allosteric modulators, endogenous choline may persistently activate CA1 pyramidal α7-containing nAChRs, enhance the excitability of CA1 pyramidal neurons and thus act as a potent therapeutic agent with potential neuroprotective and cognition-enhancing properties.     

GABA(A) Receptor α Subunits Differentially Contribute to Diazepam Tolerance after Chronic Treatment

Background

Within the GABA_A-receptor field, two important questions are what molecular mechanisms underlie benzodiazepine tolerance, and whether tolerance can be ascribed to certain GABA_A-receptor subtypes.

Methods

We investigated tolerance to acute anxiolytic, hypothermic and sedative effects of diazepam in mice exposed for 28-days to non-selective/selective GABA_A-receptor positive allosteric modulators: diazepam (non-selective), bretazenil (partial non-selective), zolpidem (α₁ selective) and TPA023 (α_2/3 selective). In-vivo binding studies with [³H]flumazenil confirmed compounds occupied CNS GABA_A receptors.

Results

Chronic diazepam treatment resulted in tolerance to diazepam''s acute anxiolytic, hypothermic and sedative effects. In mice treated chronically with bretazenil, tolerance to diazepam''s anxiolytic and hypothermic, but not sedative, effects was seen. Chronic zolpidem treatment resulted in tolerance to diazepam''s hypothermic effect, but partial anxiolytic tolerance and no sedative tolerance. Chronic TPA023 treatment did not result in tolerance to diazepam''s hypothermic, anxiolytic or sedative effects.

Conclusions

Our data indicate that: (i) GABA_A-α₂/α₃ subtype selective drugs might not induce tolerance; (ii) in rodents quantitative and temporal variations in tolerance development occur dependent on the endpoint assessed, consistent with clinical experience with benzodiazepines (e.g., differential tolerance to antiepileptic and anxiolytic actions); (iii) tolerance to diazepam''s sedative actions needs concomitant activation of GABA_A-α₁/GABA_A-α₅ receptors. Regarding mechanism, in-situ hybridization studies indicated no gross changes in expression levels of GABA_A α₁, α₂ or α₅ subunit mRNA in hippocampus or cortex. Since selective chronic activation of either GABA_A α₂, or α₃ receptors does not engender tolerance development, subtype-selective GABA_A drugs might constitute a promising class of novel drugs.