Claudia Michel

gamma-aminobutyric acid type B receptor splice variant proteins GBR1a and GBR1b are both associated with GBR2 in situ and display differential regional and subcellular distribution.

The subunit architecture of γ-aminobutyric acid, type B (GABAB), receptors in situis largely unknown. The GABAB receptor variants, characterized by the constituents GBR1a and GBR1b, were therefore analyzed with regard to their subunit composition as well as their regional and subcellular distribution in situ. The analysis was based on the use of antisera recognizing selectively GBR1a, GBR1b, and GBR2. Following their solubilization, GBR1a and GBR1b were both found by immunoprecipitation to occur as heterodimers associated with GBR2. Furthermore, monomers of GBR1a, GBR1b, or GBR2 were not detectable, suggesting that practically all GABAB receptors are heterodimers in situ. Finally, there was no evidence for an association of GBR1a with GBR1b indicating that these two constituents represent two different receptor populations. A size determination of solubilized GABAB receptors by sucrose density centrifugation revealed two distinct peaks of which one corresponded to dimeric receptors, and the higher molecular weight peak pointed to the presence of yet unknown receptor-associated proteins. The distribution and relative abundance of GBR2 immunoreactivity corresponded in all brain regions to that of the sum of GBR1a and GBR1b, supporting the view that most if not all GBR1 proteins are associated with GBR2. However, GBR1a was present preferentially at postsynaptic densities, whereas GBR1b may be mainly attributed to presynaptic or extrasynaptic sites. Thus, GBR1a and GBR1b are both associated with GBR2 to form heterodimers at mainly different subcellular locations where they are expected to subserve different functions.

GABAA receptor subtypes differentiated by their gamma-subunit variants: prevalence, pharmacology and subunit architecture.

Native GABAA receptors containing different gamma-subunit variants were distinguished immunobiochemically with antisera selectively recognizing the gamma 1-, gamma 2- and gamma 3-subunits. While GABAA receptors containing the gamma 2-subunits were confirmed to be rather ubiquitous in the adult brain, receptors characterized by the gamma 1- or gamma 3-subunit were of low abundance, as shown by immunoprecipitation. The three receptor populations differed strikingly in their benzodiazepine (BZ) site ligand binding profiles. The gamma 3-receptor population displayed reduced affinity for the full agonists clonazepam flunitrazepam and virtually lacked sensitivity to zolpidem. The gamma 1-receptor population displayed low affinity for all benzodiazepine site ligands tested, except for flunitrazepam, and could be differentiated from the gamma 2- and gamma 3-receptors by its low affinity for the inverse agonist beta CCM and its lack of affinity for the partial inverse agonist Ro 15-4513 and the antagonist flumazenil. Since flumazenil antagonizes all major effects of BZ agonists, gamma 1-receptors are not involved in mediating these actions in vivo. In immunopurified receptors, the gamma-subunit variants were found to be assembled with different variants of alpha- and beta-subunits, indicating that not only the gamma 2-subunit gamma 1- and gamma 3-subunits are part of various receptor subtypes. In addition, the gamma 2- and gamma 3-subunits can be co-assembled in native receptors, consistent with the subunit stoichiometry of two alpha-, one beta- and two gamma-subunits proposed previously for recombinant receptors.

GABAA receptors containing the α4-subunit : Prevalence, distribution, pharmacology, and subunit architecture in situ

Abstract: Recombinant GABAA receptors, expressed from α‐, β‐, and γ2‐subunits, are diazepam‐insensitive when the α‐subunit is either α4 or α6. In situ, diazepam‐insensitive receptors containing the α6‐subunit are almost exclusively expressed in the granule cell layer of the cerebellum. However, diazepam‐insensitive receptors are also expressed in forebrain areas. Here, we report on the presence of diazepam‐insensitive GABAA receptors in various brain areas containing the α4‐subunit. GABAA receptors immunoprecipitated with a newly developed α4‐subunit‐specific antiserum displayed a drug binding profile that was indistinguishable from those of α4β2γ2‐recombinant receptors and diazepam‐insensitive [3H]Ro 15‐4513 binding sites in rat brain membranes. In addition, α4‐subunit containing receptors and forebrain diazepam‐insensitive receptors are present at comparably low abundance in rat brain and exhibit virtually identical patterns of distribution. Analysis of the subunit architecture of α4‐subunit containing receptors revealed that the α4‐subunit contributes to several receptor subtypes. Depending on the brain region, the α4‐subunit can be coassembled with a second type of α4‐subunit variant being α1, α2, or α3. The data demonstrate that native receptors containing the α4‐subunit are structurally heterogeneous, expressed at very low abundance in the brain, and display the drug binding profile of diazepam‐insensitive [3H]Ro 15‐4513 binding sites. Pharmacologically, these receptors may contribute to the actions of nonclassical ligands such as Ro 15‐4513 and bretazenil.

Analysis of the presence and abundance of GABAA receptors containing two different types of alpha subunits in murine brain using point-mutated alpha subunits.

γ-Aminobutyric acid, type A (GABAA) receptors are pentameric proteins of which the majority is composed of two α subunits, two β subunits and one γ subunit. It is well documented that two different types of α subunits can exist in a singles GABAA receptor complex. However, information on the abundance of such GABAA receptors is rather limited. Here we tested whether mice containing the His to Arg point mutation in the α1, α2, or α3 subunit at positions 101, 101, and 126, respectively, which render the respective subunits insensitive to diazepam, would be suitable to analyze this issue. Immunodepletion studies indicated that the His to Arg point mutation solely rendered those GABAA receptors totally insensitive to diazepam binding that contain two mutated α subunits in the receptor complex, whereas receptors containing one mutated and one heterologous α subunit not carrying the mutation remained sensitive to diazepam binding. This feature permitted a quantitative analysis of native GABAA receptors containing heterologous α subunits by comparing the diazepam-insensitive binding sites in mutant mouse lines containing one mutated α subunit with those present in mouse lines containing two different mutated α subunits. The data indicate that the α1α1-containing receptors with 61% is the most abundant receptor subtype in brain, whereas the α1α2 (13%), α1α3 (15%), α2α2 (12%), α2α3 (2%), and α3α3 combinations (4%) are considerably less expressed. Only within the α1-containing receptor population does the combination of equal α subunits (84% α1α1, 7% α1α2, and 8% α1α3) prevail, whereas in the α2-containing receptor population (46% α2α2, 36% α2α1, and 19% α2α3) and particularly in the α3-containing receptor population (27% α3α3, 56% α3α1, and 19% α3α2), the receptors with two different types of α subunits predominate. This experimental approach provides the basis for a detailed analysis of the abundance of GABAA receptors containing heterologous α subunits on a brain regional level.

STRUCTURE OF GABAB RECEPTORS IN RAT RETINA

ABSTRACT In the search for yet unknown subtypes of GABAB receptors, the subunit architecture of GABAB receptors in the retina was analyzed using selective antisera. Immunopurification of the splice variants GABAB1a and GABAB1b demonstrated that both were associated with GABAB2. Quantitative immunoprecipitation experiments indicated that practical the entire GABAB receptor population in the retina consists of the receptor subtypes GABAB1a/GABAB2 and GABAB1b/GABAB2, although low levels of GABAB1c/GABAB2 cannot be excluded. The data rule out the existence of GABAB receptors containing the splice variants GABAB1d and GABAB1e. Moreover, no evidence for homomeric GABAB1 receptors was found. Among the splice variants, GABAB1a is by far the predominant one in neonatal and adult retina, whereas GABAB1b is expressed only late in postnatal development and in the adult retina. Since GABAB1a is expressed at high levels before functional synapses are formed, this specific receptor subtype might be involved in the maturation of the retina. Finally, subcellular fractionation demonstrated that GABAB1a, but not GABAB1b, is present in postsynaptic densities, suggesting a differential pre- and postsynaptic localisation of both splice variants.

On producing and sharing knowledge across boundaries: experiences from the interfaces of an international development research network

The number of large research networks and programmes engaging in knowledge production for development has grown over the past years. One of these programmes devoted to generating knowledge about and for development is National Centre of Competence in Research (NCCR) North–South, a cross-disciplinary, international development research network funded by the Swiss Agency for Development and Cooperation and the Swiss National Science Foundation. Producing relevant knowledge for development is a core goal of the programme and an important motivation for many of the participating researchers. Over the years, the researchers have made use of various spaces for exchange and instruments for co-production of knowledge by academic and non-academic development actors. In this article we explore the characteristics of co-producing and sharing knowledge in interfaces between development research, policy and NCCR North–South practice. We draw on empirical material of the NCCR North–South programme and its specific programme element of the Partnership Actions. Our goal is to make use of the concept of the interface to reflect critically about the pursued strategies and instruments applied in producing and sharing knowledge for development across boundaries.

Looking back: plausible links between a research intervention and the course of development in the Tajik Pamirs

Promoting sustainable development in the mountainous region of the Tajik Pamirs is socially, economically, and ecologically challenging. This region of Tajikistan was always considered of high geostrategic importance by the Soviet Union because it shares borders with China and the Indian subcontinent (i.e. with Pakistan and Afghanistan). The breakdown of the Soviet Union led to the disintegration of Central Asian states and vital subsidies that had become the backbone of mountain economies in these states were abruptly cut (Figure 13). In Tajikistan, the political transition after independence caused impoverishment, economic slowdown, and environmental degradation throughout the whole country and particularly in the Pamirs. A decline in living conditions and outmigration were the consequence. At the same time, refugee infl ux due to the civil war raging in other regions of Tajikistan aggravated the situation, bringing the region close to a humanitarian catastrophe. With the support of development assistance from the international community, the situation stabilised in the mid1990s but remained critical, with continued dependence on foreign aid.

Maximising the impact of research: the NCCR North-South approach: Fourth NCCR North-South Report on Effectiveness

Interest is growing in the impact that science can have on reducing poverty in the global South. If we understand impact as the “demonstrable contribution that excellent research makes to society and the economy”, the concept encompasses a variety of contributions of research-related knowledge and skills that benefit people and the environment. One reason for the growing interest in impact in this context is research councils’ increasing focus on documenting the social and environmental benefits of science, as indicated by the above quotation from the British research councils. Another reason is that research funding agencies from the private and public sectors are now more interested in social innovations for solving problems on the ground. Research can indeed influence policymakers’ views, policy development, funding patterns, and implementation or practice. This is promising for those who would like to improve – and prove – the influence research can have on policy and practice. It is also of importance for better understanding the intended and unintended effects of research. This report presents the NCCR North-South approach to increasing the impact of development-oriented research. It explains how we can maximise our impact and how we can assess whether our efforts have worked, based on six case studies from around the world. The report is of interest to all researchers who wish to respond to policy and practice from their point of view and who are keen on publicising their evidence. It is also relevant to those who teach how to maximise research impact.

Partnership Actions for Mitigating Syndromes (PAMS): Experience with a transdisciplinary tool in the NCCR North-South programme

Partnership Actions for Mitigating Syndromes (PAMS) are small transdisciplinary projects which bring scientific research insights from the NCCR North-South into policy and practice. They are implemented by researchers from different disciplines in collaboration with non-scientific actors. PAMS aim to implement and test approaches, methods and tools developed in research, in order to identify promising strategies and potentials for sustainable development. In this sense, they are solution-oriented. This paper will provide insights into our experience with PAMS, with a special focus on the implementation of transdisciplinarity and its outcomes. From 2001 to 2010, 77 PAMS were implemented in Africa, Asia and Latin America. An internal evaluation of the first 55 projects was conducted in 2006. Results of this evaluation led to a refinement and improvement of the tool. A second internal evaluation is currently underway in the NCCR North-South. This evaluation will provide an overview of 22 new PAMS. We will look at partners involved, project beneficiaries, activities implemented, outcomes achieved, and lessons learnt. In the first evaluation, transdisciplinarity was considered as “a form of collaboration within scientific fields … and as a form of continuous dialogue between research and society” (Messerli et al., 2007). The evaluation report concluded that this understanding of transdisciplinarity was not satisfactorily applied in the 55 projects. Only about half of the PAMS addressed mutual exchange between researchers and society. Some involved only one specific field of research and clearly lacked interdisciplinary co-operation, and most often knowledge was transferred mainly unilaterally from the scientific community to society, without society having any effect on science. It was therefore recommended to address transdisciplinarity more carefully in Phase 2 PAMS. The second evaluation, which is currently under way, is analysing whether and how this recommendation has been met, based on criteria defined in the NCCR North-South’s Outcome Monitoring Strategy. The analysis is focusing on partners with whom researchers interact and investigating whether practices have changed both in research and society. We are also exploring the role of researchers in PAMS. Preliminary results show that researchers can assume different roles, from direct implementation, mediation, and promotion of social learning between different actors, to giving advice as neutral outsiders.