Decision Framework for the environmental management of explosive contaminated land.

The environmental risks from explosive manufacturing and testing activities are usually evaluated using a qualitative process such as environmental impact prioritisation as recommended by legislation and guidance. However, standard environmental management system (EMS) guidance rarely provides detailed information on how to objectively assess the significance of the environmental impacts based on a rational scientific evidence. Quantitative exposure and eco-toxicity assessments are frequently used in combination with environmental threshold limit guidelines, but these omit important environmental impacts such as physical damage to land, nuisance and contribution to climate change. These impacts are particularly relevant to the explosives industry where noise nuisance and physical damage are given high priority. In addition, contamination from explosive compositions may comprise mixtures of multiple legacy and new generation explosives such as 1,3,5-trinitro-1,3,5-triazinane (RDX), 2,4,6-trinitrotoluene (TNT), 5-nitro-1,2,4-triazol-3-one (NTO), 2,4-dinitroanisole (DNAN) and nitroguandine (NQ), which may have combined impacts not captured by conventional eco-toxicity assessments. Further, threshold limits for energetic materials in soil and water have not been established for most nations. Additionally, in the explosive industry wider concerns such as legislative compliance and stakeholder concerns may help to provide a more broadly applicable assessment of environmental impact. Therefore in this study a novel decision framework was developed to integrate empirical data with business risks to enable rational decision making for the environmental management of explosive manufacturing facilities. The application of the framework was illustrated using three case studies from the explosive manufacturing industry to demonstrate how the framework can be used to justify environmental management decision making. By linking the environmental impacts to business risks, we demonstrate that manufacturers are able to assess a wide spectrum of issues that might not be identified in the initial environmental assessment such as non-toxic pollution incidents, breaches in legislation and stakeholder perceptions.

GBA mutations in Parkinson disease: earlier death but similar neuropathological features.

BACKGROUND AND PURPOSE: Mutations in the glucocerebrosidase (GBA) gene are known to be a risk factor for Parkinson's disease (PD). Data on clinicopathological correlation are limited. The purpose of this study was to determine the clinicopathological findings that might distinguish PD cases with and without mutations in the GBA gene. METHODS: Data from the Arizona Study of Aging and Neurodegenerative Disorders were used to identify autopsied PD cases that did or did not have a GBA gene mutation. Clinical and neuropathological data were compared. RESULTS: Twelve PD cases had a GBA mutation and 102 did not. The GBA mutation cases died younger (76 vs. 81 years of age) but there was no difference in disease duration or clinical examination findings. No neuropathological differences were found in total or regional semi-quantitative scores for Lewy-type synucleinopathy, senile plaques, neurofibrillary tangles, white matter rarefaction or cerebral amyloid angiopathy scores. CONCLUSIONS: In longitudinally assessed, autopsied PD cases, those with GBA mutations had a younger age at death but there was no evidence for clinical or neuropathological differences compared to cases without GBA mutations. Due to the small GBA group size, small differences cannot be excluded.

Prognostic factors influencing fresh and frozen IVF outcomes: an analysis of the UK national database.

National registry data provides a valuable resource to quantify factors which influence the outcomes of IVF treatments. Multilevel logistic regression analyses for live birth and multiple births given a live birth were undertaken using data from the UK Human Fertilization and Embryology Authority's registry of treatments conducted between 2000 and 2005. This study analysed 119,930 fresh and 19,918 frozen transfers from 85,349 patients in 84 centres. As well as quantifying the effects of a range of previously identified prognostic factors, the analyses showed that embryo cryopreservation reduced the live birth rate substantially (odds ratio (OR) 0.60, 95% confidence interval (CI) 0.57-0.64) compared with typical fresh cycles. Prognostic factors for transfers following cryopreservation were largely similar to those for fresh cycles, with some evidence that female age is less important, and there were small differences in the effects of the numbers of embryos transferred and previous IVF attempts that can be ascribed to selection effects. No factors were identified which specifically predicted twin outcomes; patients with a high twin risk were those with a high chance of treatment success. After allowing for all prognostic factors, there remained clinically important variability between centres (median OR 1.4) and between patients (median OR 1.8). National registry data provides a valuable resource which can be used to determine to what extent clinical and patient characteristics influence the outcomes of IVF treatments. We fitted complex statistical models for live birth and multiple births to data from the UK national registry of treatments conducted between 2000 and 2005. The analysis included 119,930 fresh and 19,918 frozen transfers from 85,349 patients in 84 centres. As well as quantifying the effects of a range of previously identified prognostic factors, we were able to estimate the magnitude of the losses due to embryo freezing and thawing. The effects of clinical and patient characteristics on the outcomes for frozen transfers were largely similar to those for fresh cycles. No factors were identified which specifically predicted twin outcomes; patients with a high twin risk were those with a high chance of success. After allowing for all the identified prognostic factors, there remained clinically important variability between centres and between patients which may suggest that the patients and clinics differ in their chances of success due to characteristics which are not currently being measured.

Reducing the incidence of twins from IVF treatments: predictive modelling from a retrospective cohort.

BACKGROUND: IVF treatments carry a high risk of twin pregnancy which confers a higher risk to the mother and child than singletons. Increased use of elective single embryo transfer (eSET) can reduce this twin rate. We aimed to utilize a previously published data set and statistical model based on routinely collected clinical data to predict the outcomes of policies that increase the proportion of eSET. METHODS: The models allow simultaneous prediction of outcomes from double embryo transfer (DET) and SET. These models were used to predict outcomes for different scenarios using SET in both the initial (fresh) transfer and over a complete cycle (transfer of all embryos created, with cryopreservation). A total of 16 096 cycles (12 487 fresh and 3609 frozen) from 9040 couples treated between 2000 and 2005 were included in the final analyses. RESULTS: For any transfer, SET has about a one-third lower live birth rate relative to DET: this can be partially mitigated by appropriate patient and treatment cycle selection, with several realistic policies performing similarly. However, if we consider complete cycles with embryo cryopreservation, it is possible for repeat SET to produce more live births per egg retrieval than repeat DET. CONCLUSIONS: All patients receiving SET would have a higher chance of successful treatment in that cycle if they received DET. The selection of appropriate patients for SET can partially ameliorate the overall loss. For complete cycles, repeat SET could produce more live births per egg retrieval than repeat DET. All treatments involving SET will increase the number of treatments required to achieve a successful outcome and this extra treatment burden will be a significant barrier to the implementation of such treatments.

Embryo and uterine influences on IVF outcomes: an analysis of a UK multi-centre cohort.

BACKGROUND: In order to optimize IVF strategies, particularly with the use of single embryo transfer, good predictive models are required. Here, we develop a model to allow such prediction, and the structure of the models point to more general conclusions about the mode of action of prognostic factors. METHODS: Anonymized data from consecutive embryo transfers in five IVF centres in the UK for the 2000-2005 period were extracted and the morphological grade based on common scoring criteria was included. There were 16 096 (12 487 fresh and 3609 frozen) transfers, for 8775 couples, available for analysis. Live birth data were fitted to a model with separate sub-models for embryo and recipient effects [the 'Embryo-Uterus' (EU) model]. All covariates were included, with sub-model selection using Akaike's information criterion. RESULTS: Age, number of embryos created, attempt number, previous history of pregnancy, duration of infertility, day of transfer and tubal diagnosis were all identified as significant prognostic factors, along with embryo grade and growth rate. Frozen transfers were substantially less likely to lead to a live birth with odds ratios of 1/3 to 1/2 compared with fresh transfers, with no evidence of differential loss for any particular patient group. Age acts predominantly through the embryo component with only a weak effect on the uterus. The embryo number, attempt number, previous pregnancies and duration of infertility act predominantly through the uterine environment. Both sub-models show significant heterogeneity between centres. CONCLUSIONS: The EU modelling framework has generated a model for predicting outcomes of embryo-transfer procedures, subject to the limitations of routinely collected data. With this large data set, the model allows identification of factors that act specifically on embryo viability or maternal receptivity. Variability in the two components between centres with similar overall outcomes suggests scope for further optimization of IVF treatment.

Are speed enforcement cameras more effective than other speed management measures? The impact of speed management schemes on 30 mph roads.

This paper presents the results of an evaluation of the impact of various types of speed management schemes on both traffic speeds and accidents. The study controls for general trends in accidents, regression-to-mean effects and migration, separately estimating the accident changes attributable to the impact of the schemes on traffic speed and on traffic volume. It was found that, when judged in absolute terms, all types of speed management scheme have remarkably similar effects on accidents, with an average fall in personal injury accidents of about 1 accident/km/year. In terms of the percentage accident reduction, however, engineering schemes incorporating vertical deflections (such as speed humps or cushions) offer the largest benefits: at 44%, the average reduction in personal injury accidents attributable to such schemes, is twice that at sites where safety cameras were used to control speeds (22%) and they were the only type of scheme to have a significant impact on fatal and serious accidents. Other types of engineering scheme (with a fall of 29% in personal injury accidents) were on average less effective in reducing accidents than schemes with vertical features but more effective than cameras. All types of scheme were generally effective in reducing speeds, with the largest reductions tending to be obtained with vertical deflections and the smallest with other types of engineering schemes.

Are speed enforcement cameras more effective than other speed management measures? An evaluation of the relationship between speed and accident reductions.

In this paper, models are developed which enable a prediction of how the impact of speed management schemes on accidents varies both with speed changes and with site and scheme characteristics. It was found that, the impact of schemes with vertical deflections is independent of the change in mean speed: an accident reduction of 44% is predicted by the model irrespective of the impact on speed. For cameras and other types of engineering schemes, a simple relationship between the change in mean speed and the consequent change in accidents is available. For the range of mean speeds typically found on 30 mph roads, the percentage accident reduction per 1 mph speed reduction is around 4% for cameras and 7-8% for schemes with horizontal features. While larger percentage accident reductions are achieved per 1 mph speed reduction on lower speed roads, larger speed reductions and larger overall percentage accident reductions are obtained on roads with higher before mean speeds. It is possible to predict both changes in speeds and accidents before treatment using the models derived from this study and these models confirm that schemes with vertical deflections are most effective in reducing both speeds and accidents.

Sources of error in road safety scheme evaluation: a quantified comparison of current methods.

This paper considers the various factors that can have a confounding effect in the evaluation of road safety schemes and examines the extent to which current methods can effectively deal with these. A modification to current methods is proposed which allows the reduction in accidents attributable to risk and flow changes to be separately evaluated. Data are presented to demonstrate the relative magnitudes of the various sources of error. It is shown that a principal source of error is normally regression-to-mean (RTM) and a correction for this effect should always be applied. Changes in traffic flow can also result in substantial accident changes and it is important to establish whether flow changes have occurred and if they are attributable to the effect of the scheme.

Sources of error in road safety scheme evaluation: a method to deal with outdated accident prediction models.

This paper considers the errors that arise in using outdated accident prediction models in road safety scheme evaluation. Methods to correct for regression-to-mean (RTM) effects in scheme evaluation normally rely on the use of accident prediction models. However, because accident risk tends to decline over time, such models tend to become outdated and the estimated treatment effect is then exaggerated. A new correction procedure is described which can effectively eliminate such errors.

Differential involvement of 5-HT(1B/1D) and 5-HT6 receptors in cognitive and non-cognitive symptoms in Alzheimer's disease.

Growing evidence suggests that a compromised serotonergic system plays an important role in the pathophysiology of Alzheimer's disease (AD). We assessed the expression of 5-HT(1B/1D) and 5-HT(6) receptors and cholinacetyltransferase (ChAT) activity in post-mortem frontal and temporal cortex from AD patients who had been prospectively assessed for cognitive function using the Mini-Mental State Examination (MMSE) and behavioral changes using the Present Behavioral Examination (PBE). 5-HT(1B/1D) and 5-HT(6) receptor densities were significantly reduced in both cortical areas. 5-HT(1B/1D) receptor density was correlated to MMSE decline in the frontal cortex, supporting its implication in memory impairment. The best predictor for lowered 5-HT(6) receptor density in the temporal cortex was the PBE measure of overactivity. The 5-HT(6)/ChAT ratio was related to aggression both in the frontal and temporal cortex. Therefore, antagonists acting at 5-HT(6) receptors could be useful in the treatment of non-cognitive symptoms associated to AD.

A systematic compilation and classification of the literature on lameness in cattle.

This paper presents the first systematic review of the literature on lameness in cattle. It identifies, tabulates and classifies relevant published work and was conducted using electronic reference databases (BIDS ISI, BIOSIS, MEDLINE and the CAB Abstracts CD-ROM). A total of 1373 unique references were obtained from 1981 to 2000, of which 914 were written in the English language. A written search protocol was designed to ensure transparency and repeatability. Pilot studies were undertaken to create search terms that minimised bias and ensured relevance. Electronic files of the search terms allow the database to be updated in future. A further 93 references were included from the most recent international conference on lameness giving a total of 1007 English language references. The systematic review process is described, including a method of classifying papers according to their study design and statistical analysis, and it is hoped that other veterinary researchers will conduct similar reviews in their fields. The compiled and classified references are available as a searchable database through the web-sitehttp://cattle-lameness.dhs.org/. The review may be used in several ways; to identify practical interventions to reduce lameness in dairy cows and to use the resultant web-site as the basis of a decision support system for farmers, veterinarians and advisors.

Generalised additive models and hierarchical logistic regression of lameness in dairy cows.

We examined the relationship between lameness (defined by locomotion score) and four time-related variables using data collected from a study of cattle lameness conducted in the UK from 1998 to 1992. The data were 19,667 locomotion scores for 1790 cows from 27 dairy herds; the four variables were time-from-calving, time of year, parity and time spent in the study. The shape of the relationships between calving and temporal variables and lameness were assessed using loess smoothed terms in a multivariable logistic generalised additive model (GAM). Polynomial relationships derived from the GAM then were included in a Bayesian hierarchical logistic-regression model incorporating between-herd, between-cow and within-cow random effects. The final hierarchical multivariable model showed that the most important variable influencing the probability of lameness was the time of scoring in the study; but, parity, time of year and time-from-calving also were significant. Between-herd and between-cow effects were of roughly equal importance.

A mixed-effects time-to-event analysis of the relationship between first-lactation lameness and subsequent lameness in dairy cows in the UK.

Data for 611 second-lactation and 251 third-lactation cows were examined using mixed-effects time-to-event models to determine the shape of the hazard, quantify relative risk and estimate herd- and sire-level variation in time to lameness. The semi-parametric Cox and fully parametric Weibull models were suggested from univariable Kaplan-Meier plots. Time to all-lameness, claw-horn lameness and skin lameness were modelled. Explanatory variables were season of current-lactation calving, age at first calving and first-lactation lameness history (whether all-lameness or claw-horn and skin lameness). In mixed-effects models of lactation-2 lameness, previously lame cows had a significantly increased hazard (hazard ratio (HR)=2.0 for all types of lameness and HR=3.2 for claw-horn lameness) compared to those not previously lame. These relationships were less marked in the third lactation. There was little evidence for an effect of age at first calving, whilst possible differences between calving seasons were observed. The hazard function suggested that the rate of lameness was roughly flat across each lactation. Herd-level variation was more evident for infectious foot diseases. The contribution of the sire increased with parity and might be important for sole ulcer and white-line disease.

Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.

GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.

Gabapentin is not a GABAB receptor agonist.

Recent experiments have demonstrated that formation of functional type B gamma-aminobutyric acid (GABA(B)) receptors requires co-expression of two receptor subunits, GABA(B1) and GABA(B2). Despite the identification of these subunits and a number of associated splice variants, there has been little convincing evidence of pharmacological diversity between GABA(B) receptors comprising different subunit combinations. However, Ng et al. [Mol. Pharmacol., 59 (2000) 144] have recently suggested a novel and important pharmacological difference between GABA(B) receptor heterodimers expressing the GABA(B1a) and GABA(B1b) receptor subunits. This study suggested that the antiepileptic GABA analogue gabapentin (Neurontin) is an agonist at GABA(B) receptors expressing the GABA(B1a) but not the GABA(B1b) receptor subunit. The importance of this finding with respect to identifying novel GABA(B) receptor subunit specific agonists prompted us to repeat these experiments in our own [35S]-GTPgammaS binding and second messenger assay systems. Here we report that gabapentin was completely inactive at recombinant GABA(B) heterodimers expressing either GABA(B1a) or GABA(B1b) receptor subunits in combination with GABA(B2) receptor subunits. In addition, in both CA1 and CA3 pyramidal neurones from rodent hippocampal slices we were unable to demonstrate any agonist-like effects of gabapentin at either pre- or post-synaptic GABA(B) receptors. In contrast, gabapentin activated a GABA(A) receptor mediated chloride conductance. Our data suggest that gabapentin is not a GABA(B)-receptor agonist let alone a GABA(B) receptor subunit selective agonist.

Microenvironment produced by acute myeloid leukemia cells prevents T cell activation and proliferation by inhibition of NF-kappaB, c-Myc, and pRb pathways.

Tumors produce a variety of immunosuppressive factors which can prevent the proliferation and maturation of a number of normal hemopoietic cell types. We have investigated whether primary acute myeloid leukemia (AML) cells have an effect on normal T cell function and signaling. Tumor cell supernatant (TSN) from AML cells inhibited T cell activation and Th1 cytokine production and also prevented activated T cells from entering the cell cycle. These effects occurred in the absence of AML cell-T cell contact. We have demonstrated that AML TSN contained none of the immunosuppressors described to date, namely gangliosides, nitric oxide, TGF-beta, IL-10, vascular endothelial growth factor, or PGs. Furthermore, IL-2 did not overcome the block, despite normal IL-2R expression. However, the effect was overcome by preincubation with inhibitors of protein secretion and abolished by trypsinization, indicating that the active substance includes one or more proteins. To determine the mechanism of inhibition, we have studied many of the major pathways involved in T cell activation and proliferation. We show that nuclear translocation of NFATc and NF-kappaB are markedly reduced in T cells activated in the presence of primary AML cells. In contrast, calcium mobilization and activation of other signal transduction pathways, namely extracellular signal-regulated kinase1/2, p38, and STAT5 were unaffected, but activation of c-Jun N-terminal kinase 1/2 was delayed. Phosphorylation of pRb by cyclin-dependent kinase 6/4-cyclin D and of p130 did not occur and c-Myc, cyclin D3, and p107 were not induced, consistent with cell cycle inhibition early during the transition from G(0) to G(1). Our data indicate that TSN generated by AML cells induces T cell immunosuppression and provides a mechanism by which the leukemic clone could evade T cell-mediated killing.

GABA(B2) is essential for g-protein coupling of the GABA(B) receptor heterodimer.

GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.

Epileptogenesis and enhanced prepulse inhibition in GABA(B1)-deficient mice.

The recent cloning of two GABA(B) receptor subunits, GABA(B1) and GABA(B2), has raised the possibility that differences in GABA(B) receptor subunit composition may give rise to pharmacologically or functionally distinct receptors. If present, such molecular diversity could permit the selective targeting of GABA(B) receptor subtypes specifically involved in pathologies such as drug addiction, spasticity, pain, and epilepsy. To address these issues we have developed a GABA(B1) subunit knockout mouse using gene targeting techniques. In the brains of GABA(B1) null mice, all pre- and postsynaptic GABA(B) receptor function was absent demonstrating that the GABA(B1) subunit is essential for all GABA(B) receptor-mediated mechanisms. Despite this, GABA(B1) null mice appeared normal at birth, although by postnatal week four their growth was retarded and they developed a generalized epilepsy that resulted in premature death. In addition, GABA(B1) heterozygote animals showed enhanced prepulse inhibition responses compared to littermate controls, suggesting that GABA(B1) deficient mice exhibit increased sensorimotor gating mechanisms. These data suggest that GABA(B) receptor antagonists may be of benefit in the treatment of psychiatric and neurological disorders in which attentional processing is impaired.

Cortical spreading depression produces increased cGMP levels in cortex and brain stem that is inhibited by tonabersat (SB-220453) but not sumatriptan.

Migraine headache is proposed to be mediated by nitric oxide (NO). Suitable mechanisms for eliciting increases in brain NO concentration in migraineurs have not yet been identified, although, animal models highlight cortical spreading depression (CSD) as a potential candidate. These studies have focused on CSD-associated NO release at highly acute time points (min-hours) and have not employed markers of NO metabolism with direct clinical application e.g. cGMP. The current study evaluated changes in plasma cGMP concentrations 3 h, 24 h and 3 days post-CSD and compared these to cortical and brainstem cGMP concentrations at 3 days. Moreover, this study also examined the effect of sumatriptan, a clinically effective antimigraine agent, and tonabersat (SB-220453) a potential novel antimigraine agent, on any observed changes in cGMP. Following pre-treatment with vehicle (n=3), sumatriptan (300 microg kg(-1) i.v, n=3) or tonabersat (SB-220453 10 mg kg(-1) i.p., n=3), CSD was evoked in anaesthetised rats by a 6-min KCl application to the parietal cortex. In the vehicle-treated group a median of eight depolarisations, were observed. Sumatriptan had no effect on the number of depolarisations, whereas tonabersat significantly reduced the number of events (median=2). No depolarisation events were observed throughout the recording period in the sham group. Following KCl application plasma cGMP concentrations were reduced up to 24 h post-CSD, but not significantly different from sham animals at 3 days. CSD in vehicle-treated animals produced a highly significant elevation in cGMP concentration in the brain stem 3 days after application of KCl. cGMP concentration increased 2.3-fold from 68+/-8 fmol/mg in sham animals (n=3) to 158+/-28 fmol/mg in the vehicle group. This increase in brain stem cGMP was abolished by tonabersat pre-treatment but not by sumatriptan.

The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.