Hong-Jin Shu

Neurosteroid Access to the GABAA Receptor

GABAA receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABAA receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABAA receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.

Slow Actions of Neuroactive Steroids at GABAA Receptors

Neuroactive steroids are potent and efficacious modulators of GABAA receptor activity and are potent sedatives and anesthetics. These positive modulators of GABAA receptors both potentiate the actions of GABA at the receptor and, at higher concentrations, directly gate the channel. The contribution of direct gating to the cellular and behavioral effects of neuroactive steroids is considered of little significance because it has been generally found that concentrations well above those needed for anesthesia are required to gate channels. By studying solitary glutamatergic neurons devoid of synaptic GABA input, we show that direct gating occurs and significantly alters membrane excitability at concentrations ≤100 nm. We propose that the relevance of direct gating has been overlooked partly because of the extremely slow kinetics of receptor activation and deactivation. We show that slow deactivation of directly gated currents does not result from an inherently tight ligand-receptor interaction because the slow deactivation is markedly accelerated by γ-cyclodextrin application. We hypothesize that steroids access the relevant GABAA receptor site from a non-aqueous reservoir, likely the plasma membrane, and that it is slow reservoir accumulation and departure that accounts for the slow kinetics of receptor gating by neuroactive steroids.

The major brain cholesterol metabolite 24(S)-hydroxycholesterol is a potent allosteric modulator of N-methyl-D-aspartate receptors.

N-methyl-d-aspartate receptors (NMDARs) are glutamate-gated ion channels that are critical to the regulation of excitatory synaptic function in the CNS. NMDARs govern experience-dependent synaptic plasticity and have been implicated in the pathophysiology of various neuropsychiatric disorders including the cognitive deficits of schizophrenia and certain forms of autism. Certain neurosteroids modulate NMDARs experimentally but their low potency, poor selectivity, and very low brain concentrations make them poor candidates as endogenous ligands or therapeutic agents. Here we show that the major brain-derived cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC) is a very potent, direct, and selective positive allosteric modulator of NMDARs with a mechanism that does not overlap that of other allosteric modulators. At submicromolar concentrations 24(S)-HC potentiates NMDAR-mediated EPSCs in rat hippocampal neurons but fails to affect AMPAR or GABAA receptors (GABAARs)-mediated responses. Cholesterol itself and other naturally occurring oxysterols present in brain do not modulate NMDARs at concentrations ≤10 μm. In hippocampal slices, 24(S)-HC enhances the ability of subthreshold stimuli to induce long-term potentiation (LTP). 24(S)-HC also reverses hippocampal LTP deficits induced by the NMDAR channel blocker ketamine. Finally, we show that synthetic drug-like derivatives of 24(S)-HC, which potently enhance NMDAR-mediated EPSCs and LTP, restore behavioral and cognitive deficits in rodents treated with NMDAR channel blockers. Thus, 24(S)-HC may function as an endogenous modulator of NMDARs acting at a novel oxysterol modulatory site that also represents a target for therapeutic drug development.

Diverse voltage-sensitive dyes modulate GABAA receptor function

Voltage-sensitive dyes are important tools for assessing network and single-cell excitability, but an untested premise in most cases is that the dyes do not interfere with the parameters (membrane potential, excitability) that they are designed to measure. We found that popular members of several different families of voltage-sensitive dyes modulate GABAA receptor with maximum efficacy and potency similar to clinically used GABAA receptor modulators. Di-4-ANEPPS and DiBAC4(3) potentiated GABA function with micromolar and high nanomolar potency, respectively, and yielded strong maximum effects similar to barbiturates and neurosteroids. Newer blue oxonols had biphasic effects on GABAA receptor function at nanomolar and micromolar concentrations, with maximum potentiation comparable to that of saturating benzodiazepine effects. ANNINE-6 and ANNINE-6plus had no detectable effect on GABAA receptor function. Even dyes with no activity on GABAA receptors at baseline induced photodynamic enhancement of GABAA receptors. The basal effects of dyes were sufficient to prolong IPSCs and to dampen network activity in multielectrode array recordings. Therefore, the dual effects of voltage-sensitive dyes on GABAergic inhibition require caution in dye use for studies of excitability and network activity.

Characteristics of concatemeric GABAA receptors containing α4/δ subunits expressed in Xenopus oocytes

BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems.

Expression of fructose sensitive glucose transporter in the brains of fructose-fed rats

Glucose transporters play a critical role in mammalian brain energy metabolism because glucose is the principal brain energy source and these transporters promote glucose movement into neural cells. When glucose is unavailable, fructose can serve as an alternative energy source. Using real-time polymerase chain reaction and actin as a reference mRNA, we investigated the impact of fructose feeding on rat brain and other tissue mRNA expression of glucose transporter 5 which has high affinity for fructose. Brain mRNA levels of glucose transporter 5 increased 1.5-fold in 35-day old rats after 7 days of fructose feeding compared with controls, whereas it increased 2.5-fold in jejunum. Semi-quantitative analysis of protein expression by immunofluorescence of glucose transporter 5 in rat hippocampi indicated a 2.4-fold increase. We demonstrated the specificity of fructose feeding on glucose transporter 5 expression by showing that the expression of the neuronal glucose transporter 3 and insulin-regulated glucose transporter 4 were unaffected. In addition, the expression of glucose transporter 5 increased in fructose fed older adult rats (8-months and 12-months old) when compared with controls. These results suggest that short-term fructose feeding increases the expression of glucose transporter 5 in both young and aging adult rats. Increased brain expression of glucose transporter 5 is likely to be important in the role of fructose as an alternative energy source.

Cyclodextrins sequester neuroactive steroids and differentiate mechanisms that rate limit steroid actions.

Neuroactive steroids are potent modulators of GABAA receptors and are thus of interest for their sedative, anxiolytic, anticonvulsant and anaesthetic properties. Cyclodextrins may be useful tools to manipulate neuroactive effects of steroids on GABAA receptors because cyclodextrins form inclusion complexes with at least some steroids that are active at the GABAA receptor, such as (3α,5α)‐3‐hydroxypregnan‐20‐one (3α5αP, allopregnanolone).

γ-aminobutyric acid type A α4, β2, and δ subunits assemble to produce more than one functionally distinct receptor type.

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1–3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes.

Neurosteroid migration to intracellular compartments reduces steroid concentration in the membrane and diminishes GABA-A receptor potentiation

Neurosteroids are potent modulators of GABA‐A receptors. We have examined the time course of development of potentiation of α1β2γ2L GABA‐A receptors during coapplication of GABA and an endogenous neurosteroid (3α,5α)‐3‐hydroxypregnan‐20‐one (3α5αP). The simultaneous application of 3α5αP with 5 μm GABA resulted in a biphasic rising phase of current with time constants of 50–60 ms for the rapid phase and 0.3–3 s for the slow phase. The properties of the rapid phase were similar at all steroid concentrations but the time constant of the slower phase became successively shorter as the steroid concentration was increased. Potentiation developed very rapidly (τ= 130 ms) when cells were preincubated with 300 nm 3α5αP before application of GABA + 3α5αP, and in outside‐out patch recordings, suggesting that steroid diffusion to intracellular compartments competes with receptor potentiation by depleting the cell membrane of steroid. Very low steroid concentrations (3–5 nm) potentiated GABA responses but the effects took minutes to develop. Intracellular accumulation of a fluorescent steroid analogue followed a similar time course, suggesting that slow potentiation results from slow accumulation within plasma membrane rather than indirect effects, such as activation of second messenger systems. In cell‐attached single‐channel recordings, where 3α5αP is normally applied through the pipette solution, addition of steroid to the bath solution dramatically shifted the steroid potentiation concentration–effect curve to lower steroid concentrations. We propose that bath‐supplied steroid compensates for the diffusion of pipette‐supplied steroid out of the patch to the rest of the cell membrane and/or intracellular compartments. The findings suggest that previous studies overestimate the minimum concentration of steroid capable of potentiating GABA actions at GABA‐A receptors. The results have implications for the physiological role of endogenous neurosteroids.

Anticonvulsant and anesthetic effects of a fluorescent neurosteroid analog activated by visible light

Most photoactivatable compounds suffer from the limitations of the ultraviolet wavelengths that are required for activation. We synthesized a neuroactive steroid analog with a fluorescent (7-nitro-2,1,3-benzoxadiazol-4-yl) amino (NBD) group in the β configuration at the C2 position of (3α,5α)-3-hydroxypregnan-20-one (allopregnanolone, 3α5αP). Light wavelengths (480 nm) that excite compound fluorescence strongly potentiate GABAA receptor function. Potentiation is limited by photodepletion of the receptor-active species. Photopotentiation is long-lived and stereoselective and shows single-channel hallmarks similar to steroid potentiation. Other NBD-conjugated compounds also generate photopotentiation, albeit with lower potency. Thus, photopotentiation does not require a known ligand for neurosteroid potentiating sites on the GABAA receptor. Photoactivation of a membrane-impermeant, fluorescent steroid analog demonstrates that membrane localization is critical for activity. The photoactivatable steroid silences pathological spiking in cultured rat hippocampal neurons and anesthetizes tadpoles. Fluorescent steroids photoactivated by visible light may be useful for modulating GABAA receptor function in a spatiotemporally defined manner.