Jeremy J. Lambert

The 5-HT3B subunit is a major determinant of serotonin-receptor function

The neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) mediates rapid excitatory responses through ligand-gated channels (5-HT3 receptors). Recombinant expression of the only identified receptor subunit (5-HT3A) yields functional 5-HT3 receptors. However, the conductance of these homomeric receptors (sub-picosiemens) is too small to be resolved directly, and contrasts with a robust channel conductance displayed by neuronal 5-HT3 receptors (9–17 pS). Neuronal 5-HT3 receptors also display a permeability to calcium ions and a current–voltage relationship that differ from those of homomeric receptors,. Here we describe a new class of 5-HT3-receptor subunit (5-HT3B). Transcripts of this subunit are co-expressed with the 5-HT3A subunit in the amygdala, caudate and hippocampus. Heteromeric assemblies of 5-HT3A and 5-HT3B subunits display a large single-channel conductance (16 pS), low permeability to calcium ions, and a current–voltage relationship which resembles that of characterized neuronal 5-HT3 channels. The heteromeric receptors also display distinctive pharmacological properties. Surprisingly, the M2 region of the 5-HT3B subunit lacks any of the structural features that are known to promote the conductance of related receptors. In addition to providing a new target for therapeutic agents, the 5-HT3B subunit will be a valuable resource for defining the molecular mechanisms of ion-channel function.

The actions of propofol on inhibitory amino acid receptors of bovine adrenomedullary chromaffin cells and rodent central neurones

1 The interaction of the intravenous general anaesthetic propofol (2,6‐diisopropylphenol) with the GABAA receptor has been investigated in voltage‐clamped bovine chromaffin cells and rat cortical neurones in cell culture. Additionally, the effects of propofol on the glycine and GABAA receptors of murine spinal neurones were determined. 2 Propofol (1.7–16.8 μm) reversibly and dose‐dependently potentiated the amplitude of membrane currents elicited by GABA (100 μm) applied locally to bovine chromaffin cells. Intracellular application of propofol (16.8 μm) was ineffective. In rat cortical neurones and murine spinal neurones, extracellular application of 8.4 μm and 1.7–16.8 μm propofol respectively produced a potentiation of GABA‐evoked currents qualitatively similar to that seen in the bovine chromaffin cell. 3 The potentiation by propofol (1.7 μm) was not associated with a change in the reversal potential of the GABA‐evoked whole cell current. On outside‐out membrane patches isolated from bovine chromaffin cells, propofol (1.7 μm) had little or no effect on the GABA single channel conductances, but greatly increased the probability of the GABA‐gated channel being in the conducting state. 4 The potentiation of GABA‐evoked whole cell currents by propofol (1.7 μm) was not influenced by the benzodiazepine antagonist flumazenil (0.3 μm). A concentration of propofol (1.7 μm) that substantially potentiated GABA currents had little effect on currents induced by the activation of the GABAA receptor by pentobarbitone (1 mm). 5 Bath application of propofol (8.4–252 μm), to bovine chromaffin cells voltage clamped at −60 mV, induced an inward current associated with an increase in membrane current noise on all cells sensitive to GABA. Intracellular application of propofol (16.8 μm) was ineffective in this respect. Local application of propofol (600 μm) induced whole cell currents with a reversal potential dependent upon the Cl− gradient across the cell membrane. 6 On outside‐out membrane patches formed from bovine chromaffin cells, propofol (30 μm) induced single channels with mean chord conductances of 29 and 12 pS. The frequency of propofol channels was greatly reduced by coapplication of 1 μm bicuculline. Under identical ionic conditions, GABA (1 μm) activated single channels with mean chord conductances of 33, 16 and 10 pS. 7 Bath applied propofol (0.84–16.8 μm) dose‐dependently potentiated strychnine‐sensitive currents evoked by glycine (100 μm) in murine spinal neurones. 8 The relevance of the present results to the general anaesthetic action of propofol is discussed.

The influence of subunit composition on the interaction of neurosteroids with GABAA receptors

The influence of the subunit composition of human GABA(A) receptors upon the GABA-modulatory properties of 5alpha-pregnan-3alpha-ol-20-one (5alpha,3alpha) has been examined using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. Steroid potency (EC(50)) is modestly influenced by the alpha-isoform (alpha(x)beta(1)gamma(2L); x=1-6). alpha(2)-, alpha(4)- and alpha(5)-containing receptors are significantly less sensitive to the action of low concentrations of 5alpha,3alpha (10-100 nM) when compared to alpha(1,3,6)beta(1)gamma(2L) receptors. Additionally, the maximal effect of the steroid is favoured at alpha(6)-containing receptors. The beta-isoform (alpha(1)beta(y)gamma(2L); y=1-3) has little influence on the GABA-modulatory effect of the neurosteroid. The EC(50) of 5alpha,3alpha is only modestly influenced by the omission of the gamma(2) subunit (alpha(1)beta(1)gamma(2L) vs alpha(1)beta(1)): while the maximal effect is favoured by the binary complex. However, the identity of the gamma subunit influences the GABA(A)-modulatory potency of 5alpha,3alpha with gamma(2)- and gamma(1)-containing receptors being the most and the least sensitive to 5alpha,3alpha, respectively. Finally, incorporation of the epsilon, or delta subunit dramatically reduces and augments the GABA-enhancing actions of the steroid, respectively. These findings provide evidence that 5alpha,3alpha discriminates amongst recombinant receptors of varied subunit composition. Furthermore, this selectivity may contribute to their neuronal specificity and behavioural profile.

Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids

1 The modulation of the γ‐aminobutyric acidA (GABAA) receptor by reduced metabolites of progesterone and deoxycorticosterone has been compared with that produced by depressant barbiturates in: (a) voltage‐clamp recordings from bovine enzymatically isolated chromaffin cells in cell culture, and (b) an assay of the specific binding of [3H]‐muscimol to a preparation of porcine brain membranes. 2 The progesterone metabolites 5α‐ and 5β‐pregnan‐3α‐ol‐20‐one (≥30 nm) reversibly and dosedependently enhanced the amplitude of membrane currents elicited by locally applied GABA (100 μm), and over the concentration range 30 nM‐100 μm stimulated the binding of [3H]‐muscimol. In contrast, 5α‐ and 5β‐pregnan‐3β‐ol‐20‐one (30 nM‐100 μm) had little effect in either assay, indicating a marked stereoselectivity of steroid action. 3 Scatchard analysis of the ligand binding data suggested an apparent increase in the number, rather than the affinity, of detectable [3H]‐muscimol binding sites as the principle action of the active steroid isomers. 4 GABA‐evoked currents were also potentiated by androsterone (1 μm) and the deoxycorticosterone metabolite 5α‐pregnane‐3α,21‐diol‐20‐one (100 nm). 5 Secobarbitone (10–100 μm), pentobarbitone (10–300 μm) and phenobarbitone (100–500 μm) reversibly and dose‐dependently potentiated the amplitude of GABA‐evoked currents in the absence of any change in their reversal potential. 6 At relatively high concentrations (≥ 30 μm) secobarbitone and pentobarbitone directly elicited a membrane current. It is concluded that such currents result from GABAA receptor‐channel activation since they share a common reversal potential with GABA‐evoked responses (≥0mV), are reversibly antagonized by bicuculline (3 μm), and potentiated by either diazepam (1 μm) or 5β‐pregnan‐3α‐ol‐20‐one (500 nm). 7 Secobarbitone (1 μm‐1 mm) dose‐dependently enhanced the binding of [3H]‐muscimol. In common with the active steroids, an increase in the apparent number of binding sites was responsible for this effect. 8 A saturating concentration (1 mm) of secobarbitone in the ligand binding assay did not suppress the degree of enhancement of control binding produced by 5β‐pregnan‐3α‐ol‐20‐one (30 nM‐100 μm). Similarly the steroid, at a concentration of 100 μm, did not influence the enhancement of [3H]‐muscimol binding by secobarbitone (1 μm‐1 mm). In all combinations of concentrations tested, the effects of secobarbitone and 5β‐pregnan‐3α‐ol‐20‐one on [3H]‐muscimol binding were additive. 9 In conjunction with previously published observations, the present data indicate close similarities in the GABA‐mimetic and potentiating actions of barbiturates and steroids. However, the results obtained with combinations of steroids and barbiturates in the ligand binding assay appear inconsistent with the two classes of compound interacting with a common site to modulate the GABAA receptor activity.

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system

Without medical progress, dementing diseases such as Alzheimers disease will become one of the main causes of disability. Preventing or delaying them has thus become a real challenge for biomedical research. Steroids offer interesting therapeutical opportunities for promoting successful aging because of their pleiotropic effects in the nervous system: they regulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination and influence cognitive processes, in particular learning and memory. Preclinical research has provided evidence that the normally aging nervous system maintains some capacity for regeneration and that age-dependent changes in the nervous system and cognitive dysfunctions can be reversed to some extent by the administration of steroids. The aging nervous system also remains sensitive to the neuroprotective effects of steroids. In contrast to the large number of studies documenting beneficial effects of steroids on the nervous system in young and aged animals, the results from hormone replacement studies in the elderly are so far not conclusive. There is also little information concerning changes of steroid levels in the aging human brain. As steroids present in nervous tissues originate from the endocrine glands (steroid hormones) and from local synthesis (neurosteroids), changes in blood levels of steroids with age do not necessarily reflect changes in their brain levels. There is indeed strong evidence that neurosteroids are also synthesized in human brain and peripheral nerves. The development of a very sensitive and precise method for the analysis of steroids by gas chromatography/mass spectrometry (GC/MS) offers new possibilities for the study of neurosteroids. The concentrations of a range of neurosteroids have recently been measured in various brain regions of aged Alzheimers disease patients and aged non-demented controls by GC/MS, providing reference values. In Alzheimers patients, there was a general trend toward lower levels of neurosteroids in different brain regions, and neurosteroid levels were negatively correlated with two biochemical markers of Alzheimers disease, the phosphorylated tau protein and the beta-amyloid peptides. The metabolism of dehydroepiandrosterone has also been analyzed for the first time in the aging brain from Alzheimer patients and non-demented controls. The conversion of dehydroepiandrosterone to Delta5-androstene-3beta,17beta-diol and to 7alpha-OH-dehydroepiandrosterone occurred in frontal cortex, hippocampus, amygdala, cerebellum and striatum of both Alzheimers patients and controls. The formation of these metabolites within distinct brain regions negatively correlated with the density of beta-amyloid deposits.

A cytoplasmic region determines single-channel conductance in 5-HT3 receptors.

5-Hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the ‘HA-stretch’) within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.

Extrasynaptic GABAA Receptors of Thalamocortical Neurons: A Molecular Target for Hypnotics

Among hypnotic agents that enhance GABAA receptor function, etomidate is unusual because it is selective for β2/β3 compared with β1 subunit-containing GABAA receptors. Mice incorporating an etomidate-insensitive β2 subunit (β2N265S) revealed that β2 subunit-containing receptors mediate the enhancement of slow-wave activity (SWA) by etomidate, are required for the sedative, and contribute to the hypnotic actions of this anesthetic. Although the anatomical location of the β2-containing receptors that mediate these actions is unknown, the thalamus is implicated. We have characterized GABAA receptor-mediated neurotransmission in thalamic nucleus reticularis (nRT) and ventrobasalis complex (VB) neurons of wild-type, β–/–2, and β2N265S mice. VB but not nRT neurons exhibit a large GABA-mediated tonic conductance that contributes ∼80% of the total GABAA receptor-mediated transmission. Consequently, although etomidate enhances inhibition in both neuronal types, the effect of this anesthetic on the tonic conductance of VB neurons is dominant. The GABA-enhancing actions of etomidate in VB but not nRT neurons are greatly suppressed by the β2N265S mutation. The hypnotic THIP (Gaboxadol) induces SWA and at low, clinically relevant concentrations (30 nm to 3 μm) increases the tonic conductance of VB neurons, with no effect on VB or nRT miniature IPSCs (mIPSCs) or on the holding current of nRT neurons. Zolpidem, which has no effect on SWA, prolongs VB mIPSCs but is ineffective on the phasic and tonic conductance of nRT and VB neurons, respectively. Collectively, these findings suggest that enhancement of extrasynaptic inhibition in the thalamus may contribute to the distinct sleep EEG profiles of etomidate and THIP compared with zolpidem.

Subunit-dependent interaction of the general anaesthetic etomidate with the γ-aminobutyric acid type A receptor

The GABA modulating and GABA‐mimetic actions of the general anaesthetic etomidate were examined in voltage‐clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant γ‐aminobutyric acidA (GABAA) receptor subunits. Currents mediated by recombinant receptors with the ternary subunit composition αxβyγ2L (where x=1,2,3 or 6 and y=1 or 2), in response to GABA applied at the appropriate EC10, were enhanced by etomidate in a manner that was dependent upon the identity of both the α and β subunit isoforms. For the β2‐subunit containing receptors tested, the EC50 for the potentiation of GABA‐evoked currents by etomidate (range 0.6 to 1.2 μm) was little affected by the nature of the α subunit present within the hetero‐oligomeric complex. However, replacement of the β2 by the β1 subunit produced a 9–12 fold increase in the etomidate EC50 (6 to 11 μm) for all α‐isoforms tested. For α1, α2 and α6, but not α3‐subunit containing receptors, the maximal potentiation of GABA‐evoked currents by etomidate was greater for β2‐ than for β1‐subunit containing receptors. This was most clearly exemplified by receptors composed of α6β1γ2L compared to α6β2γ2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC10 to 28±2% and 169±4% of the maximal GABA response, respectively. For α1 subunit‐containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the β subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5α‐pregnan‐3α‐ol‐20‐one was marginally higher for β1 rather than the β2 subunit‐containing receptor, although its maximal effect was similar at the two receptor isoforms. The GABA‐mimetic action of etomidate was supported by β2‐ but not β1‐subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either β isoform. For β2‐subunit containing receptors, both the agonist EC50 and the maximal current produced by etomidate were additionally influenced by the α isoform. It is concluded that the subtype of β‐subunit influences the potency with which etomidate potentiates GABA‐evoked currents and that the β isoform is a crucial determinant of the GABA‐mimetic activity of this compound. The nature of the α‐subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action.

Modulation of the GABAA receptor by progesterone metabolites

The naturally occurring progesterone metabolites 5β-pregnan-3α-ol-20- one and 5β-pregnane-3, 20-dione reversibly enhance membrane currents elicited by locally applied GABA in bovine adrenomedullary chromaffin cells. Such potentiation was not influenced by the benzodiazepine antagonist Ro 15-1788. At concentrations in excess of those necessary to evoke potentiation of GABA currents, 5β-pregnan-3α-ol-20-one and 5β-pregane-3, 20-dione directly activated a membrane conductance. The resulting currents were potentiated by phenobarbitone and diazepam, and abolished by the GABAA-receptor antagonist, bicuculline. On outside-out membrane patches, 5β-pregnan-3a-ol-20-one and 5β-pregnane-3, 20-dione activated single channel currents of similar amplitude to those evoked by GABA. The results suggest that certain naturally occurring steroids potentiate the actions of GABA and, additionally, directly activate the GABAA receptor.

How can drug discovery for psychiatric disorders be improved

Psychiatric disorders such as depression, anxiety and schizophrenia are leading causes of disability worldwide, and have a huge societal impact. However, despite the clear need for better therapies, and major advances in the understanding of the molecular basis of these disorders in recent years, efforts to discover and develop new drugs for neuropsychiatric disorders, particularly those that might revolutionize disease treatment, have been relatively unsuccessful. A multidisciplinary approach will be crucial in addressing this problem, and in the first Advances in Neuroscience for Medical Innovation symposium, experts in multiple areas of neuroscience considered key questions in the field, in particular those related to the importance of neuronal plasticity. The discussions were used as a basis to propose steps that can be taken to improve the effectiveness of drug discovery for psychiatric disorders.