Most of our social scientists are not institution based… they are there for hire—Research consultancies and social science capacity for health research in East Africa

There is a serious shortage of senior African social scientists to lead health-related research in Africa. This is despite the existence of many African social science graduates, and decades of Northern funded research programmes intended to develop local capacity. To investigate the barriers to developing health social science research capacity in East Africa, 29 in-depth interviews, informal conversations and a group discussion were conducted with professionals in this field.
Introduction
There is a serious shortage of senior African social scientists to lead or manage health-related research in Africa (World Bank, 2000). This is despite the graduation of many African social scientists, and decades of Northern funded research programmes intended to develop local capacity (Nchinda, 2002; Simon, 2000). Whilst weak research capacity probably affects all areas of health, the HIV epidemic ‘offers a supreme test of how effectively African universities can respond to emerging challenges …’ (Zeleza, 2003, p. 84). Yet the shortage of senior social scientists is particularly apparent in sexual health. For instance, large-scale HIV/AIDS research programmes in both Tanzania and Uganda have been unable to recruit local social scientists to senior posts on international salaries, despite having trained local junior social scientists for over 10 years. Furthermore, international debates on social dimensions of sexual health, such as the hypothesis of permissive African sexuality (Caldwell, J, Caldwell, P, & Quiggin, 1989), are dominated by Northern academics, despite their sensitivity.
Health-related social science research capacity is the ability to investigate and define the social dimensions of health problems, set objectives, identify solutions and build sustainable institutions (cf. Sitthi-amorn & Somrongthong, 2000). Limited capacity is problematic at several levels. Most obviously, it requires non-local researchers generally unfamiliar with local life, reliant on interpreters, and prone to cultural misunderstandings with local fieldworkers. Service providers and policy makers have to base decisions on more superficial analyses, but ex-patriate-initiated research is less likely to have such practical application anyway (Costello & Zumla, 2000). At the broadest level, limited social science capacity restricts intellectual sovereignty (Zeleza, 2003), undermining political autonomy (RAWOO, 2002; Sitthi-amorn & Somrongthong, 2000).
The main explanations for limited research capacity have been identified in the literature as: inadequate resources for education at every level (Nchinda, 2002; Sall, 2003; Sitthi-amorn & Somrongthong, 2000); the drain of expertise to the North (Pang, Lansang, & Haines, 2002; Ramsay, 2002; Sall, 2003; Zeleza, 2003); dependence on Northern research funding (Jentsch & Pilley, 2003; Lansang & Dennis, 2004); inequitable access to the literature (Lansang & Dennis, 2004); unbalanced North–South research collaborations (Costello & Zumla, 2000; Jentsch & Pilley, 2003) and poor support from government (Nchinda, 2002; Sall, 2003; Sitthi-amorn & Somrongthong, 2000). Some see the perpetuation of inadequate research capacity as replicating the imbalance in global trade relationships (Zeleza, 2003) and essentially semi-colonial (e.g., Costello & Zumla, 2000). Others assume the good intentions of funders and research partners, but identify the perverse consequences of North–South collaborations (Edejer, 1999), such as poaching senior researchers from local institutions. Either way, limited research capacity in Africa should be an ethical issue for Northern researchers working there.
Whilst the main causes of weak research capacity are clearly macro-economic, the processes of conducting research might contribute to the problem and may be more readily modified (Green, 2003). Some reports have suggested that research consultancies, whilst augmenting meagre incomes, might marginalise teaching and research and undermine social science scholarship (Kwesiga, Mbago, & Chimanikire, 2000; Menken, Blanc, & Lloyd, 2002; Mkandawire, 1998; Sall, 2003). Being highly prescribed, consultancies have also been said to exacerbate the way African social science research is narrowly policy-bound (Allen, 1986; Sall, 2003), and increase Northern dominance of the research agenda (Mkandawire, 1998). ‘…consultants do not frequently choose to contradict the donor's agenda, … this would decrease their chances of … consultancies in the future.’ (Rossi, 2004, p. 27). Consultancies have been described as particularly problematic in East Africa (Sall, 2003), the University of Nairobi being called a ‘consultancy university’ (Allen, 1986, p. 25).
There have been many attempts to strengthen research capacity in developing countries, leading sponsors being the WHO and other UN agencies (e.g., the Special Programme for Research and Training in Tropical Diseases (TDR); UNDP/World Bank/WHO, 2003), national development agencies (e.g., Swiss, Canadian and Japanese), foundations such as Rockefeller and NGOs such as the Population Council. However, there is little evidence about the most effective approach (Simon, 2000), and debates continue over, for instance, investing in individuals or institutions (Costello & Zumla, 2000; Nchinda, 2002), whether post-graduate training in the North exacerbates the brain drain (Nchinda, 2002), and Southern control of research budgets (Lansang & Dennis, 2004; Nchinda, 2002). The role of consultancies, however, has received scant attention and almost exclusively in the grey literature.
In order to investigate the poor capacity for health-related social science research in East Africa, the processes perpetuating it and possible ways to improve it, a small-scale exploratory study was conducted in Tanzania, Kenya and Uganda. The general findings have been reported elsewhere (Wight, 2005). This short report focuses on the individualised nature of research activity and the role of individual research consultancies in shaping research capacity.
Methods
In 2003 and 2004, I conducted in-depth interviews with 29 leading professionals conducting, commissioning or supporting health-related social science research in East Africa (Table 1). The findings are biased towards Uganda, 18 interviewees being Ugandan, four Kenyan, three British, two North American, one Tanzanian and one Nigerian. This was primarily a snowball sample including seven senior social scientists from Makerere, the oldest university in East Africa with by far the largest research function in Uganda, and others from the London School of Hygiene and Tropical Medicine (three), the Universities of Nairobi (two) and Dar es Salaam (one) and from the leading independent research centres and research-supporting NGOs in Uganda and Kenya. None of the new universities were represented. To protect respondents’ anonymity small institutions are not named.
The interview schedule covered leadership of local social science research, training and career paths, ways of strengthening research capacity and barriers to this. The full schedule is available (as Appendix A).
Informal conversations were held with five senior and one junior researcher from the University of Dar es Salaam and National Institute for Medical Research, Tanzania, and nine junior researchers from Makerere and the MRC Programme on AIDS in Uganda. A group discussion was held with four of the Ugandans (three men, one woman), following the same schedule used for the in-depth interviews.
The interviews were summarised according to analytical themes. There was considerable concordance in accounts, which were not patterned by gender. Divergent opinions are considered in the Discussion. Interviewees were circulated the main report for comments and to confirm that their views were presented accurately. Four provided comments.
Findings
Severity of the problem
Nearly all those interviewed thought that there is a serious shortage of social science research capacity in East Africa, the few really good social scientists being overworked and overwhelmed with requests for collaboration. Most academic health-related social science research in Uganda was said to be run by Northerners, yet Uganda was thought to have stronger capacity than Kenya, with Tanzania coming third. Particular limitations identified were in qualitative research, analysis and writing skills, and health-related specialisms.
Interviewees stated that the vast bulk of social science research in East Africa is commissioned by NGOs or government departments, mostly funded from the North; consequently, it is highly applied and determined by external priorities. The few opportunities for academic research were said to come primarily from Northern researchers who win the funding, resulting in unbalanced collaborations.
Research processes: inter-collegiate support
Poor social science capacity was primarily related to under-development and global economic inequalities: very poor schooling, talented students choosing high status vocational courses, poor university facilities and teaching, research funded through Northern institutions, and the drain of senior researchers abroad (Wight, 2005). The problem has been exacerbated by the death of many junior and mid-level Researchers from AIDS (Pfau & Barton, 2004, RAWOO, 2002, Zeleza, 2003). However, the individualised nature of departments and lack of collegiate support were also identified as unhelpful. This was primarily attributed to lack of resources and staff's reliance on individual research consultancies, resulting in no writing skills training and limited publishing experience to share. A head of department observed that senior staff rarely co-author papers with junior colleagues, due to ‘the culture of individualism.’ In her department there was no formal system to support junior researchers, though she was planning a mentoring system. Staff rarely seem to comment on their colleagues’ draft papers; one senior respondent estimated that at Makerere only 1% of colleagues would have time.
Research processes: consultancies
Most of the research work conducted by social scientists in East Africa is in the form of consultancies. The proportion of academics’ time spent on them is unclear (Kwesiga et al., 2000), partly perhaps to disguise this from supervisors, but most estimates were around 50% of working time. Teaching takes up much of the rest, with very little for academic research.… in Makerere you can spend your entire time just working on very well paid, short-term consultancy studies for NGOs, … who want something done in three weeks, and will pay you very well … (Senior researcher, previously Uganda)
Extremely low university salaries create a powerful incentive for consultancies. A research associate's salary might be $250/month, while consultancies can pay $100–$250/day. In one research institute consultancies augment salaries from around $400/month to about $5000. A head of department explained: ‘… to rely on your salary would never make ends meet at all.’ Furthermore, in contrast to regular salaries, most researchers can avoid declaring consultancy fees for tax (30%).
Research commissioners, predominantly government departments or NGOs, usually seek a contract with individuals, or sometimes consultancy firms, but rarely with university departments. Private consultancy firms, often constituted for a particular brief, usually employ university staff to help with the bid and subsequent research.
Commissioning bodies are reportedly unwilling to pay overheads to institutions, and when they do, they are generally very low, e.g., 5–20% in Makerere departments, 20% at the University of Dar es Salaam, and a maximum of 15% at a Kampala independent research centre. The senior management at Makerere were said to encourage departments to become consulting firms and demand 30% overheads, but this leads university staff to work independently, undercutting university departments and earning more.
The predominance of research consultancies is critical for the development of research capacity. Financial insecurity leads researchers to take on any work available, and consequently:There are no research traditions being developed …. We are social scientists but very few are specialists … (Faculty dean)
Consultancy work also inevitably restricts academics’ time for teaching and supervision. At Dar es Salaam and Makerere consultancies should not interfere with normal academic work, but this is difficult to enforce:He will leave you. And who loses? This is the person you have trained up to PhD level, and now he is leaving you, and you have no one to teach … (Head of department)
Writing consultancy reports provides little incentive to develop analytical skills. Reports generally involve very tight timetables with little opportunity for peers’ critical input, are descriptive and have limited dissemination (sometimes for internal use only). Several researchers said they do not publish from consultancies because they need the funder's permission, but none knew of it being refused. More plausibly, there is rarely time for such writing. Consequently, the CVs of highly experienced researchers often list numerous consultancy reports but very few journal publications, jeopardising their applications for senior jobs.
The conflict between consultancies and academic publications reportedly generates a professional culture in which: ‘the point is to try and chase the quick money, and not take advantage of the chance of academic growth … people don’t value it very much.’ A faculty dean commented:Consultancies is not building the capacity of the person who is doing it. …. [Some] have even refused scholarships to do PhDs because they were busy doing consultancies.
Only two interviewees questioned the inevitability that consultancies detract from publications or teaching: ‘… consultancies can … be a source of writing … [and] training.’ (Director large research programme).
The high remuneration from consultancies, and tensions with teaching responsibilities, might encourage researchers to become full-time consultants. However, few do this because commissioners seek the ‘recognition and visibility’ of ‘high powered people’ in established university posts. Furthermore, it would be too insecure: ‘You would earn much more in the short-term, but then you would be unemployed in the long-term.’
Strengthening research capacity and likely barriers
Respondents proposed many ideas to strengthen health-related social science research capacity (see Wight, 2005). Here I focus on those to modify the individualism of research practice and consultancies.
Five senior interviewees identified the need to develop writing skills, for instance ‘to guide you through … the very complicated processes … and requirements’ to publish in international journals. Suggestions included experienced and in-experienced staff co-authoring, mentoring systems and support networks. The director of a research-facilitating NGO advocated posts dedicated to writing support, but with salaries adequate to prevent appointees taking on consultancies.
The potential advantages of institutional research consultancies were explored, and in particular establishing a norm of significant overheads, e.g., 30%. Everyone approved in principle. Overheads could be used for: libraries, computing and internet access; department-initiated research; disseminating reports; training staff and developing writing skills. Institutional consultancies might facilitate a more collective approach to research and assist management by departmental heads. Furthermore, paying overheads might benefit commissioners since they could require reports to be published, at least in an on-line journal.
However, several objections to institutional consultancies were also raised.The culture of institutionalising things is not there. Many think the institution is a barrier to them. And … the bureaucracy, you know, many people would prefer to have the money in their own accounts …. (Head of department)
Researchers anticipated the frustrations of inefficient institutional administrations, with long delays in finalising contracts or being paid. It was feared that, since some universities do not allow departmental bank accounts, the central administration might appropriate funds raised through departmental consultancies. Furthermore, fees would not only have to be shared with the institution, but would have to be declared for tax. Consequently, the director of a large programme thought: ‘… people will just try to get around it. They will get consultancies privately.’
Several interviewees said that commissioning agencies would not ‘… want to pay the institutional fee.’All the American universities [have] institutional overheads, but tell DfID that [they] have to be factored in [in Kampala] …: “Oh, no!” …. How am I supposed to run the project without institutional overheads? … they have the mentality that they can do it on the cheap. Africa is poor, but it is not cheap! (Director of large programme)
It was also argued that some agencies want to commission specific individual researchers, and that individual consultancies incentivise good work produced on time.
Some large donors practice their policies of strengthening institutional capacity by only contracting research through institutions, e.g., the Carnegie and Rockefeller Foundations, the World Bank, and the Swedish and Norwegian development agencies. However, some interviewees thought a concordat with all commissioning agencies to pay minimum overheads would be unrealistic, since East Africa is too dependent on donors.
Discussion
Although most respondents were Ugandan, the data from Kenya and Tanzania suggest that these findings apply across East Africa, while the broader literature (e.g., Carlsson & Wohlgemuth, 1996; Sall, 2003; Zeleza, 2003) and contacts with researchers elsewhere suggest they are relevant to much of sub-Saharan Africa. There is a serious shortage of health-related social science research capacity in this region, as evidenced by the Northern intellectual leadership of most academic research. This perpetuates ‘the international intellectual division of labour whereby African … social scientists … import appropriate … theory and, at best, export empirical data.’ (Zeleza, 2003, p. 111) African countries’ limited ability to define for themselves their problems and the solutions may have very practical consequences. For instance, Cleland and Watkins (2006, p. 2) argue that Africans’ frustratingly slow response to the HIV epidemic is because ‘the problem, and the remedies, were socially constructed in the West ….’
Like most previous studies, this one points to global economic inequalities as the primary cause of limited research capacity. However, unremarked in nearly all the published literature, these findings also suggest that the problem is perpetuated by the highly individualised character of research in East Africa, fuelled by the dominance of individually-contracted consultancies. ‘Most of our social scientists are not institution based, whether NGO or private. They are there for hire.’ (Faculty dean) Such consultancies seem to stunt research capacity: reports are generally not disseminated, thus not contributing to collective understandings, university departments are denied overheads, and staff are diverted from teaching, supporting colleagues, or publishing. Furthermore, consultancies exacerbate the narrow policy orientation of African social science research (Allen, 1986; Rossi, 2004; Sall, 2003).
Given their prominence, this study explored the potential for research consultancies to be used to strengthen research capacity, which to date has been largely ignored in the wider literature. The principle that consultancies should be contracted with institutions, rather than individuals, with overheads of around 30%, was widely accepted. This could fund many initiatives to strengthen research capacity, facilitate a more collective approach to research, and in the longer term might mean commissioning agencies get better value. Some African universities and research centres already regulate the division of consultancy fees between researchers and their institution, e.g., the University of KwaZulu-Natal and the REACH Trust, Malawi (Theobald & Nhlema, in press). However, established individual consultants would probably oppose institutionalisation, given very low university salaries and lack of confidence in departmental administration, due to experience of patronage, mismanagement and corruption (Zeleza, 2003). Furthermore, commissioning agencies were said to prefer individual consultancies as cheaper and more straightforward.
By and large, initiatives to strengthen research capacity do not address the issue of research consultancies, although in practice they are in competition for researchers’ commitment. This was clear in an academic research centre sponsored by an international NGO where researchers are prohibited from consultancy work. However, as noted above, some large donors further the development of institutional capacity by only contracting research through institutions, not individuals.
Unsurprisingly, respondents’ accounts were shaped by their professional and institutional positions, for instance leading them to defend their staff or externalise problems (see Wight, 2005). African interviewees gave more emphasis to economic factors, research commissioners’ restrictions and exclusion from Northern-dominated academic networks, while Northern interviewees were more likely to contrast East African with Northern professional cultures. However, these cultural differences were usually attributed to underlying structural/economic factors. Only one interviewee, an African, explicitly attributed inadequate research capacity to a global economy of academic research, in which Northern institutions actively maintain their dominance. Notably, the most critical reports of East African research came through informal conversations, rather than recorded interviews.
This has only been an exploratory study. Further research needs to clarify: the scale of individual consultancies across East Africa; whether revising commissioning practices would seriously contribute to research capacity; and, hardly represented here, the views of agencies commissioning consultancies.
While the underlying causes of poor research capacity require global economic reform, this study also points to the importance of individually contracted research consultancies in perpetuating the problem. Although they greatly augment meagre university salaries, they also seem to divert university staff from academic research and training the next generation of researchers, stunt the institutional capacity of university departments, restrict the sharing of research findings and perpetuate donors’ control of the research agenda. Commissioning bodies committed to strengthening research capacity should consider devising research contracts, and means to improve university administration, that ameliorate rather than exacerbate the problem.

research consultancies
health social sciences
research capacity
sub-saharan africa
knowledge economy
east african universities


A Kir6.2 mutation causing severe functional effects in vitro produces neonatal diabetes without the expected neurological complications

Aims/hypothesis Heterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects.
Introduction
ATP-sensitive potassium (KATP) channels link cellular metabolism to membrane electrical activity by regulating K+ fluxes across the plasma membrane [1]. They are found in multiple tissues but are of particular importance in regulating insulin secretion from pancreatic beta cells [1, 2]. At substimulatory glucose concentrations, KATP channels are open so that the membrane potential is hyperpolarised and Ca2+ influx and insulin secretion are prevented [3]. Glucose metabolism enhances ATP production, resulting in closure of KATP channels, stimulation of electrical activity, opening of voltage-gated Ca2+ channels and exocytosis of insulin granules.
KATP channels are hetero-octamers of Kir6.x and sulfonylurea receptor (SUR) subunits [4–6]. Four inwardly rectifying subunits (Kir6.2 in pancreatic beta cells) form the pore of the channel and four auxiliary SUR subunits (SUR1 in pancreatic beta cells) associate with the tetrameric pore and regulate its gating [7–9]. Binding and/or hydrolysis of Mg-nucleotides by the intracellular nucleotide-binding domains (NBDs) of SUR produces channel opening [10–13]. It is believed that reciprocal changes in the intracellular concentrations of ATP and MgADP are involved in the metabolic regulation of KATP channels.
Over the last 4 years, many different missense mutations in the genes encoding Kir6.2 (KCNJ11) and SUR1 (ABCC8) have been shown to cause permanent neonatal diabetes mellitus (PNDM). This is a rare disorder characterised by high blood glucose levels that manifests within the first 6 months of life. A subgroup of mutations were associated with a more severe clinical profile characterised by Delayed development of motor, intellectual and social skills, muscle weakness, Epilepsy, facial dysmorphism and Neonatal Diabetes (DEND syndrome) [14–16]. Mutations in Kir6.2 have also been found to cause a remitting relapsing form of neonatal diabetes that resembles transient neonatal diabetes mellitus [17, 18].
All Kir6.2 mutations analysed to date were heterozygous and most were de novo mutations [14, 19–21]. In most cases, the diabetes they caused could be successfully treated with sulfonylureas [14, 20, 22], which directly close KATP channels by binding to the SUR1 subunit of the channel [23]. All PNDM mutations result in a reduced KATP channel sensitivity to inhibition by MgATP in vitro [15, 24]. This is expected to cause an increased KATP current amplitude and reduced insulin secretion. Studies to date suggest that the severity of the clinical phenotype reflects the extent of the reduction of the channel ATP sensitivity. Thus, mutations that produce a small increase in KATP current in the presence of physiological concentrations of MgATP (1–5 mmol/l) lead to PNDM, whereas mutations that cause a larger increase in KATP current give rise to DEND syndrome [15].
In this paper, we identify a KCNJ11 mutation (L164P) that causes neonatal diabetes without obvious neurological complications. In functional studies, we show that the L164P mutation produces a large increase in the resting whole-cell current and a marked reduction in KATP channel sensitivity to inhibition by ATP. These effects are a secondary consequence of an increase in the channel open probability (Po) produced by the mutation. Surprisingly, other mutations that produce a similar increase in Po cause DEND syndrome. The L164P mutant channel was also far less blocked by the sulfonylurea tolbutamide, which explains why the patients were unable to transfer to glibenclamide therapy.
Methods
Participants Informed consent was obtained from all individuals investigated (or from their parents if they were children).
Molecular genetic analysis Genomic DNA was extracted from peripheral lymphocytes using standard procedures. The KCNJ11 gene was amplified and sequenced as described [25]. Other family members were also tested for the novel mutation. Family relationships were confirmed using a combination of six microsatellites on chromosome 11: D11S902, D11S419, D11S1397, D11S1901, D11S921 and D11S1888.
Oocyte preparation Female Xenopus laevis were anaesthetised with ethyl 3-aminobenzoate methanesulfonate salt (MS222; 2 g/l added to the water). One ovary was removed via a mini-laparotomy, the incision sutured and the animal allowed to recover. Subsequently, animals were operated on for a second time, but under terminal anaesthesia. Immature stage V–VI oocytes were incubated for 60 min with 1 mg/ml collagenase (Type V; Sigma, Poole, UK) and manually defolliculated. All procedures were carried out in accordance with UK Home Office Legislations and the University of Oxford ethical guidelines. Oocytes were coinjected with ∼0.8 ng wild-type or mutant Kir6.2 mRNA and ∼4 ng mRNA encoding SUR. The final injection volume was 50 nl per oocyte. Isolated oocytes were maintained in Barth’s solution and studied 1–4 days after injection.
Electrophysiology Wild-type or mutant Kir6.2 (GenBank D50581) were coexpressed with SUR1 in Xenopus oocytes (GenBank L40624) as described [26]. Because all the patients were heterozygous for the L164P mutation, their pancreatic beta cells will contain a mixture of wild-type and mutant Kir6.2 subunits. To simulate this heterozygosity, we coinjected Xenopus oocytes with a 1:1 mixture of mutant and wild-type Kir6.2 together with SUR1 mRNA. This is expected to give rise to a mixed population of channels composed of homomeric wild-type channels, homomeric mutant channels and heteromeric channels containing between one and three mutant subunits [21]. We refer to this global channel population as heterozygous channels.Whole-cell currents were recorded using a two-electrode voltage clamp in response to voltage steps of ±20 mV from a holding potential of −10 mV, filtered at 1 kHz and digitised at 4 kHz. Oocytes were perfused with a solution containing (in mmol/l): 90 KCl, 1 MgCl2, 1.8 CaCl2 and 5 HEPES (pH 7.4 with KOH). Metabolic inhibition was produced by 3 mmol/l sodium azide.Macroscopic currents were recorded from giant inside-out patches using an EPC10 amplifier (List Medical Electronics, Darmstadt, Germany) controlled with Pulse v8.74 software (Heka Electronik, Lambrecht, Germany). Macroscopic currents were elicited by 3 s voltage ramps from −110 to +100 mV (holding potential 0 mV), or recorded at a constant potential of −60 mV. They were filtered at 0.5 kHz and digitised at 1 kHz. The pipette solution contained (mmol/l): 140 KCl, 1.2 MgCl2, 2.6 CaCl2, 10 HEPES (pH 7.4 with KOH). The internal (bath) solution contained (mmol/l): 107 KCl, 1 K2SO4, 2 MgCl2, 10 EGTA, 10 HEPES (pH 7.2 with KOH) and Mg-nucleotides as indicated. Experiments were conducted at 20–22°C. Solutions were changed using a local perfusion system consisting of tubes of 200 μm diameter into which the tip of the patch pipette was inserted.Nucleotide concentration–inhibition curves were fit with the Hill equation:
where G is the KATP conductance in the presence of ATP, Gc is the KATP conductance in the absence of the nucleotide, [ATP] is the ATP concentration, IC50 is the nucleotide concentration at which inhibition is half maximal and h is the slope factor (Hill coefficient).Single-channel currents were measured at −60 mV, filtered at 5 kHz and digitised at 20 kHz. Unitary amplitude and Po were measured from the Gaussian fit to all-points amplitude histograms of tracts of current of 30–90 s duration.Data were analysed with in-house routines developed in the IgorPro platform (Wavematrics, Portland, OR, USA). Data are given as means ± SEM in the text and in the figures. Statistical significance was evaluated using a two-tailed Student t test and p < 0.05 taken to indicate a significant difference.
Results
Patient characteristics and genetics Three unrelated probands with permanent neonatal diabetes were shown to be heterozygous for the KCNJ11 gene mutation L164P (c.491T>C, p.Leu164Pro). All were female and had unaffected parents of different ethnic origin. Two patients have been reported previously [27, 28]. Mutation testing and microsatellite analysis of DNA from both parents and the child for two of the cases confirmed that the mutation had arisen de novo (the parents of the third case were not available for testing). None of the patients had any neurological complications or obvious developmental delay.The first patient is currently 8 years old. She was born in Singapore from Sri Lankan parents, at 38 weeks of gestation with a birthweight of 2.6 kg. She developed diabetes at 30 weeks of age. When she came to Australia at the age of 6.5 years, her HbA1c level was elevated (7.4%) and her diabetes required insulin treatment (0.45 U kg−1 day−1). Glibenclamide treatment (1.0 mg kg−1 day−1) was trialled for 2 months (at 7 years of age) but did not produce a decrease in her insulin requirement. She continues to require insulin (up to 0.7 U kg−1 day−1) with an HbA1c ranging from 7.2 to 8.2%. She has normal developmental milestones, her neurological examination is normal and she has no evidence of diabetic complications.The second patient, born in Australia of Afghan origin, is currently 2.7 years old. She was born at term following an uneventful pregnancy, with a birthweight of 2.7 kg. She presented at 8 weeks in diabetic ketoacidosis and was subsequently treated (at 2.5 months for 4 weeks, then at 5 months for 3 months) with glibenclamide (up to 1.1 mg kg−1 day−1, regimens of two times per day or three times per day), but had no reduction in insulin requirement (0.4 U kg−1 day−1) [28]. Her mean HbA1c since diagnosis has been 7.6% (range 7.2–8.7%). Assessment at 5 months by a neurologist was entirely normal, with a normal EEG. Subsequently, she has had normal developmental milestones and her neurological examinations have been normal at all follow up visits every 3 months. Diabetic complications have not been assessed due to the young age of the patient.The third patient is from Slovakia [27]. She was born at term with a birthweight of 2.6 kg, and was diagnosed with diabetes at 5 weeks of age. She is currently 20 years old and is treated with insulin (1.24 U kg−1 day−1). Glibenclamide transfer was not attempted because the patient also suffers from hepatitis C. She has poor glycaemic control, with an HbA1c level of 15.2%, and various diabetic complications including retinopathy and nephropathy. Psychomotoric development in childhood was normal. A neurological examination suggested the presence of diabetic motoric polyneuropathy in the lower extremities but there were no further neurological findings. Although her IQ was not tested, she successfully completed a specialised business school training suggesting normal mental development.
Functional analysis: effects on whole-cell KATP currents We examined the functional effects of the L164P mutation by heterologous expression in Xenopus oocytes. When wild-type Kir6.2/SUR1 channels are expressed in oocytes they are normally closed because they are inhibited by resting intracellular ATP concentrations ([ATP]i). They can be opened, however, by azide (3 mmol/l; Fig. 1), a metabolic inhibitor that lowers [ATP]i [29]. Heterozygous L164P (hetL164P) currents were about 18-fold larger at rest than wild-type currents, but increased twofold on metabolic inhibition indicating that the channel is only partially closed at resting ATP levels (Fig. 1). In contrast, homomeric L164P (homL164P) channels displayed a much greater resting current and were little affected by metabolic inhibition (Fig. 1).
Fig. 1Whole-cell KATP current. Mean steady state whole-cell currents evoked by voltage steps from −10 to −30 mV before (control, white bars) and after application of 3 mmol/l azide (grey bars) and in the presence of 3 mmol/l azide plus 0.5 mmol/l tolbutamide (black bars). The number of oocytes was five to seven in each case. G, KATP conductance; Gc, KATP conductance expressed relative to the conductance in the absence of the nucleotide. WT, wild-type; hetL164P and homL164P channels as indicated
The sulfonylurea tolbutamide (0.5 mmol/l) blocked whole-cell KATP currents by 98% but had no effect on homL164P currents (4% block). HetL164P channels were blocked by only 54% (Fig. 1).
Functional analysis: effects on KATP channel ATP sensitivity The increase in resting whole-cell KATP currents suggests that the L164P mutation may reduce the channel ATP sensitivity, as found for other PNDM mutations [21]. We first measured the ATP sensitivity of wild-type and mutant KATP currents in the absence of Mg2+, to isolate the effects of ATP on Kir6.2 (in the absence of Mg2+, ATP does not interact with SUR1 [11]). Both homL164P and hetL164P channels had severely impaired ATP sensitivity. HomL164P channels were not blocked at all by ATP, even at concentrations as high as 10 mmol/l. The concentration–inhibition curve for hetL164P showed a striking shift to higher ATP concentrations, with an IC50 of about 100 μmol/l, and a marked pedestal of unblocked current at very high ATP concentrations. The data were best fitted by assuming that in the heterozygous state about 20% of channels are never closed by ATP (Fig. 2a,b and Table 1).
Fig. 2ATP-inhibition of L164P channels is less that that of wild-type channels. a, c Currents recorded in inside-out patches excised from Xenopus oocytes expressing hetKir6.2-L164P/SUR1 (hetL164P) or homKir6.2-L164P/SUR1 (homL164P) channels, as indicated, in response to 3 s voltage ramps from −110 to +100 mV. ATP (10 mmol/l) was applied as indicated by the horizontal bars in the absence (a) or presence (c) of 2 mmol/l Mg2+. b, d, mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of the nucleotide (Gc), for wild-type (white circles, n = 9), hetL164P (white/black circles, n = 6) or homL164P (black circles, n = 4) channels. Experiments were carried out in the absence (b) or presence (d) of 2 mmol/l Mg2+. The continuous lines through the black circles were drawn by eye. The smooth curves are the best fit to the Hill equation with IC50 of 11 μmol/l (wild-type) and 100 μmol/l (hetL164P) (b, 0 mmol/l Mg2+) or IC50 of 16 μmol/l (wild-type) and 122 μmol/l (hetL164P) (d, 2 mmol/l Mg2+)Table 1ATP sensitivity of wild-type and mutant channels IC50 (Mg-free)h (Mg-free)IC50 (2 mmol/l Mg2+)h (2 mmol/l Mg2+)A (2 mmol/l Mg2+)%Imax (3 mmol/l MgATP)Wild-type9.6 ± 1.61.30 ± 0.0815.8 ± 3.00.99 ± 0.05n.a.0.01 ± 0.01hetL164P100 ± 8*1.10 ± 0.05118 ± 29*1.16 ± 0.340.29 ± 0.07*33.6 ± 3.4*homL164Pn.a.n.a.n.a.n.a.n.a.98 ± 1*Values are means ± SEM. The number of patches was four to nine in each case.A The fraction of unblocked current used to fit the ATP concentration–inhibition relationship; h Hill coefficient; IC50, ATP concentration (μmol/l) producing half-maximal inhibition; %Imax the per cent unblocked current in the presence of 3 mmol/l MgATP; n.a. not applicable (as no block).*p < 0.05 vs wild-type
Molecular mechanism of the reduced ATP sensitivity Mutations that reduce the ATP sensitivity of the KATP channel can act in several ways. They may prevent ATP binding directly. They may impair the mechanism by which nucleotide binding is coupled to channel gating. They may also stabilise the intrinsic open state of the channel (i.e. that in the absence of ATP), which shifts the gating equilibrium in the presence of ATP towards channel opening and thus indirectly reduces the channel ATP sensitivity (e.g. [15, 30, 31]).In a structural model of Kir6.2 [32], L164P lies within the channel pore, at a considerable distance from the ATP-binding site (Fig. 3). It is not predicted to interact directly with ATP. We therefore examined whether the L164P mutation alters intrinsic gating. Experiments were carried out in the absence of ATP, where intrinsic gating can be assessed. The L164P mutation had no effect on single-channel current amplitude (Table 2). However, the intrinsic Po was markedly increased, being 0.86 (n = 6) for homL164P compared with 0.4 (n = 4) for wild-type channels (Fig. 4 and Table 2). This suggests that, at least in part, the L164P mutation alters channel ATP sensitivity indirectly, via an increase in Po.
Fig. 3Homology model of Kir6.2 [32]. For clarity, only two subunits are shown. ATP (yellow) is shown in its binding site. Residue L164 is shown in redFig. 4The L164P mutation enhances single-channel activity. Representative single KATP channel currents recorded at −60 mV in inside-out patches from oocytes expressing wild-type or homL164P channels, as indicated. Currents were recorded in the absence of Mg2+ and nucleotidesTable 2Single-channel parameters for wild-type and homL164P channels Poi (pA)Wild-type0.39 ± 0.054.0 ± 0.1homL164P0.86 ± 0.01*4.1 ± 0.1Mean ± SEM values of intrinsic open probability (Po) and single-channel current (i) measured at −60 mVThe number of patches was five to six in each case* p < 0.05 vs wild-type
Effects on KATP channel ATP sensitivity in the presence of Mg2+ Previous studies have shown that Kir6.2 mutations associated with neonatal diabetes may not only decrease the sensitivity of Kir6.2 to ATP, but can also enhance channel activation by Mg-nucleotides [24]. We therefore explored the effect of the L164P mutation on the ATP sensitivity in the presence of 2 mmol/l Mg2+.Surprisingly, there was no difference in the effect of ATP in the presence and absence of Mg2+ on homL164P channels (Fig. 2c,d). This contrasts with what has been observed for mutations in the ATP-binding site that completely abolished ATP inhibition in the absence of Mg2+, such as R50P and G334D: channels containing these mutations were activated by MgATP [33, 34]. The IC50 for ATP inhibition of hetL164P channels was also not significantly affected by Mg2+, although the pedestal of unblocked current at high ATP concentrations was increased by about 50% (Table 1). At 3 mmol/l MgATP, a concentration within the physiological range, the unblocked current was 34%, substantially greater than that found for wild-type channels (<1%; Table 1).
Functional analysis: effects on MgADP sensitivity The lack of MgATP activation of hetL164P channels could be due to reduced functional coupling between SUR1 and Kir6.2-L164P, or to reduced MgATP binding/hydrolysis at the NBDs of SUR1. To explore the former possibility, we measured the ability of MgADP to activate hetL164P channels preblocked by 100 μmol/l MgATP. It was necessary to preblock the channels as the mutant channel has an open probability close to maximal and further activation is therefore not possible. Figure 5 shows that 30 μmol/l MgADP activates wild-type currents by 3.7 ± 1.8 fold (n = 3) and that 100 μmol/l MgADP did not produce any further increase (3.7 ± 1.2-fold, n = 3). In contrast, hetL164P channels were activated less: 1.4 ± 0.1 fold (n = 3) by 30 μmol/l MgADP and 1.5 ± 0.1 fold (n = 4) by 100 μmol/l MgADP.
Fig. 5Sensitivity to MgADP of wild-type and hetL164P channels. a Representative currents (I) recorded at −60 mV from inside-out excised membrane patches from Xenopus oocytes expressing wild-type or hetL164P channels, as indicated. Patches were exposed to 100 μmol/l ADP in the continuous presence of 100 μmol/l ATP: 2 mmol/l Mg2+ was present throughout. b Mean current in the presence of 100 μmol/l MgATP or 30 or 100 μmol/l MgADP plus 100 μmol/l MgATP, normalised to the current in the absence of nucleotides for wild-type (grey bars) and hetL164P (black bars) channels. Bars indicate means ± SEM. The number of patches was three to four in each case
Despite the fact that 100 μmol/l MgATP blocked mutant channels less, the lower extent of activation of hetL164P channels by 30 μmol/l MgADP is not due to the fact that channel activity is already maximal, as the amplitude is still significantly less than that in control solution (Fig. 5).
Discussion
We describe the clinical and functional effects of a KCNJ11 mutation, L164P, associated with permanent neonatal diabetes. This mutation causes a marked reduction in KATP channel inhibition by ATP primarily by stabilising the open state of the channel. This leads to an increase in the whole-cell KATP current, and in beta cells is expected to result in a reduction in insulin secretion. Surprisingly, unlike other mutations that reduced the channel ATP sensitivity by a similar amount, no motor or mental developmental delay was associated with the L164P mutation.
Structural considerations and molecular basis for reduced ATP sensitivity In a homology model of Kir6.2 [32], L164 lies partway along the permeation pathway, 35 Å away from the ATP-binding site (Fig. 3). It is therefore unlikely that it acts by reducing ATP binding directly. The side-chains of L164 point into the pore, forming a hydrophobic girdle that is narrow enough to prevent the passage of water and hydrated K+ ions [35], which suggests that L164 may form a hydrophobic gate within the pore. Functional studies support this idea. Following mutation of L164 to cysteine, cadmium ions were able to block the KATP channel with high affinity [36, 37], suggesting that the four cysteines (one on each subunit) come together close enough to form a high-affinity binding site for Cd2+. These data indicate that the side-chain of L164 must face into the pore, and that the pore is very narrow at this position. Substitution of a proline for L164 is expected to produce a kink in the α-helix [38] and disrupt the hydrophobic gate. It is therefore not surprising that the L164P mutation caused a dramatic effect on the Po. Mutation of L164 to cysteine, alanine, valine, threonine or glycine also produces a very large increase in Po [30, 36, 37, 39], consistent with the importance of this residue in channel gating.
The fact that the L164P mutation enhanced Po can explain, in part, the reduced ATP sensitivity of the channel. It is also possible that the mutation may have effects additional to stabilisation of the channel open state.
Effect of Mg2+ on Kir6.2-L164P/SUR1 channel ATP sensitivity In contrast to all PNDM mutations studied to date [24], MgATP was unable to enhance the activity of either homL164P or hetL164P channels. The lack of MgATP activation of homL164P channels may be due to the fact that Po is already very high and thus there is no scope for further activation. However, little MgATP activation was also observed for hetL164P channels. This is in marked contrast to other mutations that caused enhanced Po (I296L, [16], V59G [15]) where MgATP activated both homomeric and heterozygous channels. It is therefore possible that the L164P mutation reduces the efficacy of coupling between SUR1 and Kir6.2, and thereby decreases the ability of MgATP to stimulate channel activity. In support of this idea, MgADP activation of hetL164P channels was also reduced. This may explain the inability of Mg2+ to reduce ATP inhibition of hetL164P channels, since it is well established that MgATP must be hydrolysed to MgADP to stimulate channel activity.The reason for the lack of Mg-nucleotide activation of hetL164P channels is unclear. Because L164 lies within the pore, far away from the NBDs of SUR1, the effect must be mediated allosterically. It is possible that this is mediated by an interaction between the backbone of L164 itself, or transmembrane domain 2 (within which L164 lies), and the transmembrane domains of SUR1. In the absence of an atomic resolution structure of the KATP channel complex, however, this cannot be definitely determined.HomL164P channels were not blocked by tolbutamide, as expected because of their high Po [40]. The lower efficacy of tolbutamide on hetL164P channels (about 50% block) may reflect the enhanced Po of channels within the heterozygous population containing mutant subunits. It could also reflect impaired coupling between SUR1 and Kir6.2, such as that found for MgADP.
Clinical implications Previous studies of KCNJ11 mutations have suggested that there is a good correlation between the percentage of current that remains unblocked in the presence of 3 mmol/l MgATP and the clinical phenotype. Namely, currents that are >30% of maximal are associated with DEND syndrome, and those that lie between 5–10% of maximal with neonatal diabetes alone, compared with a current of <1% for wild-type channels (Fig. 6). The L164P mutation does not conform to this simple relationship between the functional effects of the mutation and the clinical phenotype. The magnitude of the hetL164P current in 3 mmol/l MgATP was 36%, yet none of the patients had extra-pancreatic symptoms.
Fig. 6Mean KATP current (expressed as a % of maximum) measured in the presence of 3 mmol/l MgATP from inside-out patches expressing heterozygous mutant channels as indicated. The dashed line indicates the maximal current amplitude normally associated with PNDM. iDEND, intermediate DEND syndrome (i.e. neonatal diabetes with developmental delay [21]). Data for wild-type (WT) and Kir6.2-R201H channels are from [14], for Y330C and F333I from [26], for Q52R, V59G and R201C from [15], for V59M from [24], for I296L from [16] and for G334D from [34]The reason for this anomaly is not clear, particularly as the resting whole-cell current was also very large. Previous studies have also indicated that such large whole-cell currents are invariably associated with more severe clinical symptoms. The lack of responsiveness of the patient to sulfonylureas is consistent with the very large increase in Po and the resting whole-cell current. It is therefore likely that hetL164P channels expressed in Xenopus oocytes are a reasonable model for the pancreatic beta cell KATP channels of the patients. We are therefore forced to conclude that compensatory mechanisms must ameliorate the extra-pancreatic effects of this mutation. The fact that the patients had very different ethnic origins suggests the compensatory mechanism is unlikely to reflect a shared genetic background: however, it does appear to be specific to the L164P mutation, which is unique (to date) in having a marked functional effect in vitro despite causing neonatal diabetes without neurological complications.Importantly, a maximally effective concentration of tolbutamide only blocked hetL164P channels by 50%. All patients to date whose channels are blocked by <65% have not been able to transfer from insulin treatment to sulfonylurea therapy. This suggested that our patients would be unable to transfer to sulfonylurea therapy, as indeed was found to be the case. This is likely to be due to the fact that L164P destabilises the long closed state of the channel, to which sulfonylureas preferentially bind, and which is rarely entered in channels with enhanced Po. Our results further suggest that not all patients with permanent neonatal diabetes will necessarily respond to sulfonylurea treatment, and indicates that knowledge of the functional effect of the mutation is helpful for predicting the drug response in patients.


Effect of disconnecting the orbital prefrontal cortex from the nucleus accumbens core on inter-temporal choice behaviour: A quantitative analysis

Previous experiments showed that destruction of the orbital prefrontal cortex (OPFC) or the nucleus accumbens core (AcbC) in rats altered choice between two delayed food reinforcers. Application of a quantitative model of inter-temporal choice suggested that lesions of either structure increased the delay-dependent degradation of reinforcer value (delay discounting); destruction of the OPFC (but not the AcbC) also increased the relative value of the larger reinforcer. This experiment examined the effect of disconnecting the OPFC from the AcbC on inter-temporal choice. Rats received excitotoxin-induced contralateral lesions of the OPFC and AcbC (disconnection), severing of the anterior corpus callosum (callosotomy), a combined lesion (disconnection + callosotomy) or sham lesions. They were trained in a discrete-trials progressive delay schedule to press levers A and B for a sucrose solution. Responses on A delivered 50 μl of the solution after a delay dA; responses on B delivered 100 μl after a delay dB. dB increased across blocks of trials; dA was manipulated across phases of the experiment. Indifference delay, dB(50) (value of dB corresponding to 50% choice of B), was estimated for each rat in each phase, and linear indifference functions (dB(50)vs. dA) were derived. The disconnection + callosotomy group showed a lower intercept of the indifference function (implying a higher rate of delay discounting) than the sham-lesioned group; the disconnection group showed a similar but less robust effect, whereas the callosotomy group did not differ significantly from the sham-lesioned group. The results suggest that OPFC–AcbC connections are involved in delay discounting of food reinforcers, but provide no evidence for an involvement of OPFC–AcbC connections in regulating sensitivity to reinforcer size.
1
Introduction
It is widely accepted that the capacity of a reinforcer to exert control over operant behaviour is a direct function of its size and an inverse function of the delay that precedes its delivery. These two principles are placed in mutual opposition in inter-temporal choice schedules, in which the subject is required to choose between two reinforcers that differ along both dimensions. For example, the subject may be confronted with a choice between a smaller reinforcer, A, of size qA, delivered after a short delay, dA, and a larger reinforcer, B, of size qB, delivered after a longer delay, dB.
Inter-temporal choice schedules have provided valuable insights into the behavioural and neurobiological bases of ‘delay discounting’, the hypothetical process whereby the efficacy or ‘value’ of a reinforcer decays as a function of delay. Recent work has implicated the core of the nucleus accumbens (AcbC) [1,2,6,8] and the orbital region of the prefrontal cortex (OPFC) [23,26,42] in inter-temporal choice behaviour. However, the interpretation of results obtained using these schedules is complicated by the fact that magnitude and delay of reinforcement are generally manipulated simultaneously. Therefore it is often unclear whether the effect of an intervention on inter-temporal choice behaviour has been brought about by a change in the rate of delay discounting, by a change in the organism's sensitivity to relative reinforcer size, or both [8,19,31]. One approach to overcoming this problem is the application of null equations derived from quantitative models of inter-temporal choice [19,29–31]. For example, according to one such model [19], the overall value of a reinforcer, V, is determined by the multiplicative combination of two hyperbolic expressions that define the effects of delay and magnitude upon reinforcer value:where K is the delay-discounting parameter [29] and Q is a parameter expressing sensitivity to reinforcer size [19]. Faced with a choice between two reinforcers, A and B, the organism is presumed to select the reinforcer with the higher value. However, by experimentally manipulating the sizes and delays of the two reinforcers, it is possible to establish a state of ‘indifference’, in which A and B are selected with equal frequency. Indifference between A and B is taken to imply equality of value, i.e., VA = VB. Using Eq. (1) to define VA and VB and solving for dB(50) (the delay to reinforcer B at the point of indifference), yields the following null equation:in which dB(50) is linearly related to dA. By examining the effect of an intervention on this relation, it is possible to deduce whether the intervention has affected the organism's rate of delay discounting, its sensitivity to reinforcer size, or both. Since K makes no contribution to the slope of the function, a change in slope implies a change in sensitivity to reinforcer size, whereas a change in intercept in the absence of a concomitant change in slope implies a change in the rate of delay discounting [19].
Destruction of the OPFC was found to increase the slope of the linear indifference function, indicating that the lesion affected sensitivity to reinforcer size; the lack of a concomitant increase in the intercept suggested that the rate of delay discounting had also been increased [26] (see Section 4 for further explanation). In contrast, lesions of AcbC reduced the intercept without significantly altering the slope of the function, implying a selective effect on delay discounting [2]. While these observations implicate both the OPFC and the AcbC in delay discounting, they leave unanswered the question of whether the two structures play independent roles in delay discounting, or whether they contribute to an integrated mechanism regulating inter-temporal choice behaviour. The present experiment was intended to address this question: the effect of functional disconnection of the OPFC and AcbC on inter-temporal choice behaviour was examined using the linear indifference relation epitomised by Eq. (2).
The principal anatomical link between the OPFC and the AcbC is an ipsilateral excitatory glutamatergic corticofugal pathway, which is believed to comprise one link in a cortico-striato-thalamo-cortical circuit. Inhibitory γ-aminobutyric acid (GABA)ergic efferents from the AcbC project to the internal pallidum and substantia nigra pars reticulata. These structures in turn send inhibitory projections to diencephalic structures, whose excitatory corticopetal projections complete the circuit. In order to effect functional disconnection of the OPFC and AcbC without totally ablating either structure, a ‘disconnection lesion’ [12,14] was employed, which consisted of unilateral excitotoxic destruction of the OPFC in one hemisphere and of the AcbC in the other hemisphere. As recent evidence indicates that transcallosal fibres may make a significant functional contribution to cortico-striatal connections [9], the effects of callosotomy alone, and callosotomy combined with the disconnection lesion were also examined.
2
Methods
The experiment was carried out in accordance with UK Home Office regulations governing experiments on living animals.
2.1
Subjects
Sixty experimentally naive female Wistar rats (Charles River UK) approximately 4 months old and weighing 250–300 g at the start of the experiment were used. They were housed individually under a constant cycle of 12 h light and 12 h darkness (light on 06:00–18:00 h), and were maintained at 80% of their initial free-feeding body weights throughout the experiment by providing a limited amount of standard rodent diet after each experimental session. Tap water was freely available in the home cages.
2.2
Surgery
Anaesthesia was induced with halothane (4% in oxygen), and the rat positioned in a stereotaxic apparatus (David Kopf), with the upper incisor bar set 3.3 mm below the inter-aural line. Anaesthesia was maintained with 2% halothane in oxygen during surgery. Holes were drilled in the skull for introduction of a microinjection cannula or leucotome (see below). Disconnection: sixteen rats received unilateral lesions of the OPFC and the contralateral AcbC (the sides being counterbalanced across rats). The following coordinates (mm, measured from bregma) were used to locate the OPFC: site (i): AP +3.7, L ±1.2, DV −4.8; site (ii): AP +3.7, L ±2.8, DV −4.4. The coordinates for the AcbC were: AP +1.2, L ±1.8, V −7.1. Injections were given via a 0.3-mm diameter cannula connected by a polyethylene tube to a 10-μl Hamilton syringe. In each site, 0.5 μl of a 0.1-M solution of quinolinic acid (2,3-pyridinedicarboxylic acid) in phosphate-buffered 0.9% NaC1 (pH 7.0) was injected at a rate of 0.1 μl per 15 s. The cannula was left in position for 3 min after completion of the injection. Callosotomy: 15 rats underwent a midline leucotomy in order to sever the anterior corpus callosum. The leucotome, similar to that described by Gold et al. [17], was constructed from a 1-μl Hamilton microsyringe. A curved wire could be extruded from the tip of the syringe needle; when fully extended, the wire projected approximately 3 mm in the AP plane (i.e. at right angles to the needle). Two midline cuts were made: (i) the tip of the needle was positioned at AP 0.0, DV −5.0, the wire extruded in a rostral direction, and the tip slowly raised to DV −1.0; the wire was then retracted into the needle, and the needle was withdrawn from the brain; (ii) the tip of the needle was repositioned at AP +2.0, DV −4.0 and the procedure repeated. Disconnection + callosotomy: 15 rats underwent both the disconnection and callosotomy procedures described above. Sham lesion: fourteen rats underwent the same surgical procedures as the disconnection-lesioned group, except that the vehicle solution alone was injected into the target sites.
2.3
Apparatus
The rats were trained in standard operant conditioning chambers (CeNeS Ltd., Cambridge, UK) of internal dimensions 25 cm × 25 cm × 22 cm. One wall of the chamber contained a recess into which a peristaltic pump could deliver a 0.6 M sucrose solution. Two apertures situated 5 cm above and 2.5 cm to either side of the recess, through which motor-operated retractable levers could be inserted into the chamber. The levers could be depressed by a force of approximately 0.2 N. A 2.8-W lamp was mounted 2.5 cm above each lever; a third lamp was mounted 10 cm above the central recess. Six red light-emitting diodes were mounted in a row, 4 cm apart, 5 cm above the levers. The operant chamber was enclosed in a sound-attenuating chest; masking noise was generated by a rotary fan. An Acorn microcomputer programmed in Arachnid BASIC (CeNeS Ltd., Cambridge, UK), located in an adjoining room, controlled the schedules and recorded the behavioural data.
2.4
Behavioural training
Two weeks after surgery, the food-deprivation regimen was introduced and the rats were gradually reduced to 80% of their free-feeding body weights. They were then trained to press two levers (A and B) for sucrose reinforcement, and were exposed to a discrete-trials continuous reinforcement schedule in which the two levers were presented in random sequence for three sessions. After this initial training, they underwent daily training sessions under the discrete-trials delayed reinforcement schedule for the remainder of the experiment. Each experimental session consisted of six blocks of six trials, except in phases 4 and 5 when sessions consisted of five blocks. The trials were 90 s in duration, with the exception of phase 5, in which the duration was increased to 120 s in order to accommodate the long delay to reinforcement (see below). The six blocks were signalled by illumination of the six light-emitting diodes: in block 1 the first (left-most) diode was illuminated, in block 2 the first and second diodes were illuminated, and so on. The first two trials of each block were forced-choice trials in which each lever was presented alone in random sequence. The other four trials were free-choice trials in which both levers were presented. The beginning of each trial was signalled by illumination of the central light above the reinforcer recess. After 2.5 s the lever or levers (depending on the type of trial) were inserted into the chamber. When a lever-press occurred, the lever(s) were withdrawn, the central light was extinguished, and the light located above the lever that had been depressed was illuminated. This light remained illuminated until the delivery of the reinforcer, and was then extinguished. The chamber remained in darkness until the start of the following trial. If no lever-press occurred within 5 s of the lever(s) being inserted, the lever(s) were retracted and the central light extinguished. (This seldom happened except during the first few training sessions.) A response on lever A initiated a fixed delay dA, following which 50 μl of the 0.6 M sucrose solution was delivered. A response on lever B initiated a variable delay dB, after which 100 μl of the same sucrose solution was delivered. The positions of levers A and B (left vs. right) were counterbalanced across subjects.
The experiment consisted of six phases, in which the value of dA was set at 1, 2, 4, 8, 12 and 0.5 s, respectively. In each phase, the value of dA was held constant. In each session the value of dB was set equal to dA in the first block of trials. In subsequent blocks dB was increased in increments of 75%. In phases 4 and 5, when dA was 8 and 12 s, respectively, computing five increments of 75% would have generated a value of dB that was longer than the duration of a trial in the sixth block of trials; therefore the number of blocks was limited to five in these phases. The first phase continued for 100 sessions, phase 2 for 50 sessions, and the remaining phases for 40 sessions.
Experimental sessions were carried out 7 days a week, at the same time each day, during the light phase of the daily cycle (between 08:00 and 14:00 h).
2.5
Histology
At the end of the behavioural experiment, the rats were deeply anaesthetised with sodium pentobarbitone, and perfused transcardially with 0.9% sodium chloride, followed by 10% formol saline. The brains were removed from the skulls and fixed in formol saline for 1 week. Forty micrometer coronal sections were taken through the regions of the OPFC and AcbC (approximately from AP +5.0 to AP 0.0) using a freezing microtome.
2.5.1
Cresyl violet staining
The procedure was similar to that described previously [26]. Alternate sections were mounted on chrome-gelatine-coated slides and air dried, hydrated by successive immersion in 95, 70 and 50% ethanol, stained in 0.25% cresyl violet for 2 min at room temperature, dehydrated by successive immersion in 50, 70, 95, and 100% ethanol and xylene, and mounted with DPX.
2.5.2
Immunocytochemistry
In the other sections neurone-specific nuclear protein (NeuN) was labelled as described by Jongen-Relo and Feldon [21]. Our protocol has been described elsewhere [2]. Briefly, freshly sliced sections were rinsed in 0.1 M phosphate buffered saline (PBS) and placed in 0.5% H2O2 in PBS for 30 min. After twice rinsing in PBS, they were placed for 1 h in a blocking solution (10% normal horse serum [Vector Laboratories, Peterborough, UK], 1% bovine serum albumin [BSA, Sigma–Aldrich, Gillingham, UK] and 0.3% Triton X-100 [Sigma–Aldrich] in PBS). They were incubated for 48 h at 4 °C with the primary antibody (monoclonal mouse anti-NeuN serum [1:5000, Chemicon, Chandlers Ford, UK] in 1% normal horse serum, 1% BSA and 0.3% Triton X-100 in PBS), washed twice in PBS, and incubated for 2 h at room temperature in biotinylated horse antimouse serum (Vector Laboratories) (1:1000 in 1% BSA and 0.3% Triton X-100 in PBS). After further rinsing in PBS, they were placed for 2 h in avidin–biotin–horseradish peroxidase complex (1:200, ABC-Elite, Vector Laboratories) in PBS. After two further rinses in PBS, they were placed in a chromagen solution (0.05% diaminobenzidine [Sigma–Aldrich] and 0.01% H2O2 [Sigma–Aldrich]) for 5 min. The reaction was observed visually and stopped by rinsing in PBS. The sections were floated on to chrome-gelatine-coated slides and mounted with DPX.
An investigator who was blind to the behavioural results performed the microscopic examination. Drawings of the area of the lesions were superimposed on the appropriate coronal sections in the stereotaxic atlas of Paxinos and Watson [34].
2.6
Data analysis
Data from 5 of the 60 rats were discarded. Histological examination revealed two rats with misplaced excitotoxin-induced lesions; one rat in the disconnection + callosotomy group was discarded because of a failed callosotomy. Data from two further rats were discarded because they showed persistent exclusive responding on one lever. This left 13 rats in the sham-lesioned group, 14 in the disconnection group, 15 in the callosotomy group and 13 in the disconnection + callosotomy group.
2.6.1
Preference functions and linear indifference functions
For each rat, the percentage choice of lever B in the free-choice trials (%B) was computed for each block of trials from the pooled data from the last 10 sessions of each phase of the experiment. Plots of %B vs. dB were derived for each rat, and the indifference delay (dB(50): the value of dB corresponding to %B = 50%) was estimated by linear interpolation between the two delays which fell on either side of %B = 50% (i and j) using the formula: dB(50) = dB(i) + ([dB(j)-dB(i)].[%Bi − 50]/[%Bi − %Bj]) [39]. Plots of dB(50)
vs. dA were obtained for each rat, and linear functions were fitted by the method of least squares; goodness of fit was expressed as r2, the proportion of the data variance accounted for by the fitted function. The slope and intercept of the linear indifference functions were analysed by two-factor ANOVA (presence/absence of disconnection × presence/absence of callosotomy) followed by multiple comparisons of the lesioned groups with the sham-lesioned group using Dunnett's test. Linear indifference functions (dB(50)
vs. dA) were also derived for the group mean data. The slopes and elevations of these functions were analysed by one-factor ANOVA (group) followed by multiple comparisons of the lesioned groups with the sham-lesioned group using Dunnett's test, as described by Zar [43]: the slopes were first analysed, and in the absence of significant between-group variation in slope, a common weighted slope value was adopted in order to make comparisons among the elevations. (‘Elevation’ refers to the y-axis location of the function taking the range of observed data into account, whereas ‘intercept’ refers to the intersection of the function with the y-axis location [43].)
2.6.2
Psychophysical analysis of preference functions
Logistic functions were fitted to the group mean %B data and the %B data from each rat in each phase of the experiment: %B = 100/(1 + [dB/dB(50)]ɛ). This function defines a descending sigmoid curve which is symmetrical in semi-logarithmic co-ordinates; dB(50) and ɛ are parameters, dB(50) being the point of intersection of the logistic curve with the indifference line, and ɛ being the slope of the function. These parameters were used to derive the limen ([dB(25) − dB(75)]/2, where dB(25) and dB(75) are the estimated values of dB corresponding to %B = 25 and %B = 75, respectively), and the index of relative precision, the Weber fraction, was defined as limen/dB(50). The Weber fraction was subjected to repeated-measures ANOVA (phase); as no significant effect of phase was revealed, the Weber fractions were averaged across phases and subjected to two-factor ANOVA (presence/absence of disconnection × presence/absence of callosotomy), as described above.
3
Results
3.1
Behavioural data
3.1.1
Preference functions and linear indifference functions
Preference functions (%B vs. dB) derived for the four groups in all six phases of the experiment are shown in Fig. 1 (left-hand graphs). In all four groups, preference for lever B declined as a function of the delay to reinforcer B (dB). The horizontal lines in the graphs show the indifference level (i.e., %B = 50); the value of dB at which the preference function crossed this level (i.e. dB(50)) increased as a function of increasing values of dA, reflecting a progressive rightward displacement of the curve.
Fig. 2 shows indifference functions (dB(50)
vs. dA) for the group mean data. In each group, the linear function accounted for more than 96% of the variance of the group mean data (r2 > 0.96). Comparisons were made between the function derived for each of the lesioned groups and the function derived for the sham-lesioned group [43]. An initial test on the homogeneity of the slopes indicated that there was no significant effect of group upon the slope [F(3,20) = 1.8, P > 0.05], and the common (weighted) slope (3.16) was therefore adopted in statistical comparisons of the elevations of the functions. There was a significant effect of group on the elevation [F(3,23) = 24.4, P < 0.01]. Multiple comparisons (Dunnett's test) showed that the elevations of the functions derived for the disconnection and disconnection + callosotomy groups differed significantly from that of the sham-lesioned group [t(9) = 4.0, P < 0.05, and t(9) = 5.2, P < 0.01, respectively], whereas there was no significant difference between the elevations of the callosotomy and sham-lesioned groups [t(9) = 0.2, P > 0.3].
Linear indifference functions were also fitted to the data from the individual rats. The group mean values (+S.E.M.) of the slope and intercept of the function are shown in Fig. 3. A two-factor analysis of variance (callosotomy × disconnection) showed no significant effect of either factor nor any significant interaction on the slope [all Fs < 1]. There was a significant effect of the disconnection lesion on the intercept [F(1,52) = 8.7, P < 0.005], but no significant effect of the callosotomy [F < 1] and no significant interaction [F < 1]. Multiple comparisons with the sham-lesioned group (Dunnett's test) showed that only the disconnection + callosotomy group differed significantly from the sham-lesioned group. The goodness of fit of the linear function did not vary significantly among the groups [F < 1]; the function accounted for >86% of the data variance for individual rats in all groups [r2 = 0.865 ± 0.022].
3.1.2
Psychophysical analysis of preference functions
The logistic psychometric functions derived for the group mean data (Fig. 1, right-hand panels) accounted for 95% of the data variance (r2 > 0.95) in all cases. The logistic function could be fitted to 311 of the 330 preference functions obtained for the individual rats in the six phases of the experiment (94.2%); functions could be fitted to the data from all six phases in 48 of the 55 rats. Analysis of variance (disconnection × callosotomy) indicated that the goodness of fit was not significantly affected by either the callosotomy [F < 1] or the disconnection lesion [F(1,51) = 1.2, P > 0.2], and there was no significant interaction [F < 1]. The overall mean (±S.E.M.) value of r2 derived from all rats in all phases of the experiment was 0.966 ± 0.005. There was good agreement between the values of dB(50) derived from the logistic functions (dB(50)logist.) and those derived by linear interpolation (dB(50)interp.). The slope of the regression of dB(50)logist.
vs. dB(50)interp. (1.02 ± 0.01) did not deviate significantly from unity, and the intercept (−0.16 ± 0.31) did not deviate significantly from zero; the correlation (r) between the two estimates was 0.971.
The Weber fraction derived from the logistic function was not systematically related to the value of dA. A single-factor analysis of variance with repeated measures (incorporating data from the 48 rats that generated Weber fractions from all 6 phases) showed no significant effect of phase [F(5,235) = 1.9, P > 0.05]. The Weber fraction was therefore averaged across phases for each rat; the group mean values (+S.E.M.) are shown in Fig. 4 (left-hand histogram). Analysis of variance (callosotomy × disconnection) revealed a significant main effect of the disconnection lesion [F(1,51) = 6.6, P < 0.05], but no significant effect of the callosotomy [F(1,51) = 2.7, P > 0.1] and no significant interaction [F < 1]. Multiple comparisons (Dunnett's test) did not reveal any significant differences between the lesioned groups and the sham-lesioned group.
The group mean values of the slope of the logistic functions (ɛ) (+S.E.M.) are shown in Fig. 4 (right-hand histogram). Analysis of variance (callosotomy × disconnection) showed a significant main effect of the disconnection lesion [F(1,51) = 7.2, P < 0.05], but no significant main effect of the callosotomy [F < 1] and no significant interaction [F < 1]. None of the lesioned groups differed significantly from the sham-lesioned group (Dunnett's test).
3.2
Histology
Examples of the lesions are shown in Fig. 5. OPFC: injection of quinolinic acid into the OPFC resulted in gliosis and atrophy of the ventral and lateral orbital regions. There was some damage to the medial prefrontal cortex (medial orbital, infralimbic and prelimbic cortices) in some rats. The AP extent of the lesion was from about +3.2 to +4.5; in no case did the lesion extend caudally as far as the anterior margin of the nucleus accumbens. AcbC: coronal sections showed ventricular dilatation and atrophy in the ventral striatal area. The NeuN labelled sections showed that there was extensive neuronal loss in the area of the AcbC of all lesioned animals, with some neuronal loss in the ventral and medial portions of the caudate-putamen in some animals; the shell region of the nucleus accumbens was essentially spared. Callosotomy: the corpus callosum was completely severed between AP +0.5 to +1.6 in most animals. There was some sparing of the corpus callosum posteriorly (caudal to AP +0.5), possibly reflecting the curvature of the leucotome. Destruction of the callosum was generally accompanied by some ventricular dilatation in the vicinity of the lesion, and the mesial surfaces of the cortex overlying the lesion showed some damage.
4
Discussion
Injections of quinolinic acid into the OPFC and AcbC produced lesions of similar extent to those seen in previous experiments in which excitotoxins have been used to lesion these structures [OPFC: 13, 23, 25, 26, and 42; AcbC: 1, 2, 3, 5, 6, and 35]. In the case of the OPFC lesion, the main area of damage included the ventral and lateral orbital (VO and LO) regions. There was also some intrusion into the medial orbital (MO), infralimbic (IL) and prelimbic (PrL) regions in some animals. According to the subregional classification recommended by Uylings and van Eden [40] and Kesner [22], the lesion embraced the ventral (VO) and lateral OPFC (LO), with some involvement of the medial PFC (MO, IL and PrL). In the case of the AcbC lesion, the area of destruction was mainly restricted to the target structure. Some additional damage was inflicted to the ventral portion of the caudate-putamen in some rats; and in some cases NeuN staining revealed a band of neuronal loss in the medial caudate-putamen adjacent to the lateral ventricle. The mesial shell region of the nucleus accumbens was spared. The callosotomy lesion was generally successful in severing the corpus callosum anterior to the caudal margin of the AcbC.
The discrete-trials schedule used in this study was an adaptation of the progressive delay schedule developed by Evenden and Ryan [10]. The rats in all four groups showed a progressive shift in preference from the larger to the smaller reinforcer as the delay to the larger of the two reinforcers (dB) was progressively increased across successive blocks of trials. This is consistent with previous studies that have used this schedule [2,6–8,10,11,23,24,26,33,42]. As in previous experiments [2,23,24,26], we used a geometric progression to determine the values of dB in successive blocks of trials, thereby allowing the range of values of dB to be adapted to the value of dA, which was systematically manipulated across the six phases of the experiment. The resulting preference functions (relation between %B and dB; see Fig. 1) were used to compute the indifference delays to the larger reinforcer (dB(50)) in each phase. This measure formed the basis of the linear indifference functions (see below).
As in previous studies employing Evenden and Ryan's [11] protocol [2,6–8,10,11,23,24,26,33,42], the preference functions seen in this experiment were characterized by a gradual reduction of %B as a function of dB. As noted by Bezzina et al. [2], this is apparently inconsistent with models of inter-temporal choice that are based on the computation of hypothetical ‘values’ of reinforcers. Such models generally assume that organisms should invariably select the more highly valued of two mutually exclusive reinforcers [18,19], leading to the prediction that the ideal preference function should be a step function, %B falling precipitously from near 100% to near 0% around the point at which VA = VB. Bezzina et al. [2] proposed that the gradual decline in preference generated by progressive delay schedules may represent a discrimination gradient for reinforcer value, and adopted a standard psychophysical approach to analyse the preference functions, an analytical approach that was also used in the present study. As in Bezzina et al.'s [2] experiment, a two-parameter logistic function adequately described the group mean preference functions (Fig. 1, right-hand panels) and more than 90% of the preference functions obtained from individual rats. The two parameters of this function define its slope (ɛ) and its locus on the abscissa (dB(50)). Combination of ɛ and dB(50) allows computation of the Weber fraction, the traditional measure of the relative precision of discrimination [15,16,20,28]. The data shown in Fig. 4 indicate that ɛ tended to be lower and the Weber fraction higher in the disconnection-lesioned and disconnection + callosotomy groups than in the sham-lesioned group. This resembles Bezzina et al.'s [2] finding with bilateral AcbC lesions, which induced a robust increase in the Weber fraction, consistent with an impairment of discriminative precision. It appears that functional disconnection of the OPFC from the AcbC had a similar effect on value discrimination as destruction of the AcbC.
The locus of the preference function is defined by dB(50), the delay to reinforcer B corresponding to indifference between the two reinforcers. According to hyperbolic models of inter-temporal choice [19,29], indifference implies equality of the values of the two reinforcers, which provides the basis for deriving the linear function expressed by Eq. (2). This equation offers a means of distinguishing between changes in inter-temporal choice behaviour brought about by effects on the hypothetical processes of delay discounting (K) and magnitude discounting (Q). The slope of the linear function reflects the physical magnitudes of the two reinforcers (qA and qB) and Q; a change in slope therefore implies a change in Q. The intercept of the function is influenced jointly by Q and K. Therefore, while an increase in Q causes an increase in both the slope and the intercept, an increase in K simply diminishes the intercept. If both parameters are increased, Q's effect on the intercept is countered by the change in K, whereas its impact on the slope remains unaltered [19].
In the present experiment, the indifference functions of the four groups did not differ significantly in terms of slope; however, there were significant between-group differences in the intercept. The disconnection-lesioned group showed a somewhat lower intercept than the sham-lesioned group. This effect was statistically significant when the functions fitted to the group mean data were compared; however it failed to reach significance when the functions fitted to the data from individual rats in the two groups were compared. A more robust effect on the intercept was seen in the case of the disconnection + callosotomy group, whose intercept was significantly lower than that of the sham-lesioned group, in the case of the functions fitted to the group mean data and also the functions fitted to the individual-subject data. The callosotomy had no significant effect on the intercept. These results indicate that functional disconnection of the AcbC from the OPFC selectively altered the delay discounting parameter, K. The lower intercept seen in the disconnection + callosotomy group implies a higher value of K, in other words, a higher rate of delay discounting, than that of the sham-lesioned group. The fact that callosotomy alone had no discernable effect on performance suggests that ipsilateral cortifugal fibres may be mainly responsible for the functional connection between the OPFC and AcbC; however, the fact that the combined disconnection + callosotomy lesion had a more robust effect on inter-temporal choice than the conventional lesion suggests that inter-hemispheric connections may also make a subsidiary contribution. The existence of inter-hemispheric cortico-striatal connections has been known for some time [32], although their functional importance has only recently been established. Dunnett and his colleagues [9,41] recently found that a combined disconnection + callosotomy lesion was needed to effect complete functional disconnection of the prefrontal cortex and dorsal striatum in the case of delayed alternation performance.
Previous experiments have examined the effects of selective lesions of the OPFC and AcbC on inter-temporal choice behaviour. Kheramin et al. [23,26] found that bilateral destruction of the OPFC increased the slope of the linear indifference function without altering the intercept, an effect that was attributed to an increase in the rate of delay discounting combined with increased sensitivity to relative reinforcer size (see also [25]). Cardinal et al. [6,8] first reported that bilateral lesions of the AcbC rendered rats more ‘intolerant’ to delay of reinforcement. This finding was recently confirmed and extended by Bezzina et al. [2], using the indifference-equation approach; these authors reported that destruction of the AcbC lowered the intercept of the linear indifference function without significantly altering the slope, implying an increase in the rate of delay discounting. These findings suggest that both the OPFC and AcbC may contribute to delay discounting. The present results further suggest that delay discounting may be regulated by an integrated mechanism that involves both these structures, and that the integrity of both structures and their connecting fibres may be important for the effective control of behaviour by delayed reinforcers. Interestingly, the present results provide no evidence for an involvement of OPFC–AcbC connections in regulating sensitivity to reinforcer size. This suggests that while the OPFC may integrate information on multiple features of reinforcers, including both size and delay [37,38], its role in delay discounting may be more specifically related to its connections with the AcbC. Data consistent with this notion have recently been obtained using the progressive ratio schedule. Mathematical analysis of performance on this schedule [27] yields a quantitative index of instantaneous reinforcer value that is sensitive to variations of reinforcer size [4,36]. This index has been found to be reduced by lesions of the OPFC [25] but not by lesions of the AcbC [3], suggesting that the OPFC, but not the AcbC, may be involved in determining sensitivity to reinforcer size.

orbital prefrontal cortex
nucleus accumbens core
inter-temporal choice
rat
delay discounting
disconnection lesion


How are podocytes affected in nail–patella syndrome?

Nail–patella syndrome is an autosomal-dominant hereditary disease named for dysplastic fingernails and toenails and hypoplastic or absent kneecaps evident in patients with the syndrome. Prognosis is determined by the nephropathy that develops in many such patients. Besides podocyte foot-process effacement, pathognomonic changes in the kidney comprise electron-lucent areas and fibrillar inclusions in the glomerular basement membrane. These characteristic symptoms are caused by mutations in the gene encoding the transcription factor LMX1B, a member of the LIM-homeodomain gene family. Comparable with the human syndrome, homozygous Lmx1b knockout mice lack patellae and suffer from severe podocyte damage. In contrast, however, podocin and the α3 and α4 chains of collagen IV are absent in the glomeruli of Lmx1b knockout mice. Further studies with podocyte-specific Lmx1b knockout mice have confirmed the importance of LMX1B in podocytes, as these mice apparently develop foot processes initially but lose them later on. We therefore conclude that LMX1B is essential for the development of metanephric precursor cells into podocytes and possibly also for maintaining the differentiation status of podocytes. LMX1B can serve as a model system to elucidate a genetic program in podocytes.
For many years, the mesangial cell took the forefront of glomerular research (see, for example, [1, 2]), but its pedestal was first slowly shaken by the painstaking morphological investigations of Wilhelm Kriz (confer [3, 4] for an early and late review, respectively) and finally abruptly toppled by irrefutable genetic evidence pointing towards the podocyte as a crucial cell in the glomerulus [5]. Meanwhile, the podocyte is firmly rooted in pathogenetic models of glomerular diseases and it is hard to imagine that it will leave again. Although mutations in WT1, a gene encoding a transcription factor of the Cys2His2-zinc finger family, had been found responsible for the podocytopathies Denys-Drash syndrome [6], WAGR syndrome [7] and Frasier syndrome [8], the article by Karl Tryggvason’s group on the identification of mutations in NPHS1 added another dimension [5]. NPHS1 is mutated in patients suffering from congenital nephrotic syndrome of the Finnish type. It codes for nephrin, a component of the slit diaphragm, and therefore is an essential part of the glomerular filtration barrier [9–11]. Since then several other genes have been cloned that when mutated lead to glomerular disease and which in the kidney are (almost) specifically expressed in podocytes. They are LMX1B [12–14], NPHS2, the gene encoding podocin [15]; ACTN4, the gene encoding α-actinin-4 [16]; CD2AP [17, 18] and TRPC6 [19, 20]. In the following years, an increasing amount of evidence has accumulated on the specific role of these proteins in the podocyte. Nephrin and podocin participate in the formation of the slit diaphragm complex, α-actinin-4 crosslinks actin filaments in podocytes, and TRPC6 belongs to a special class of cation channels. Very little, however, is known about how the podocyte-specific expression of these genes is achieved. The sparse evidence that has been published concerns WT1, which binds to sequences in the promoter regions of the Podxl gene (encoding podocalyxin) [21] and of the NPHS1/Nphs1gene [22, 23]. Although WT1 activates the respective reporter constructs, the induction of the endogenous NPHS1/Nphs1 gene by WT1 was described by one group [23] but not another [22].
Nail–patella syndrome has been known for many decades as a hereditary disease and was one of the first genetic disorders for which linkage was established. In addition to the obvious limb abnormalities, nephrologic symptoms develop in ∼40% of these patients over the course of several decades. On an ultrastructural level, the moth-eaten appearance of the glomerular basement membrane together with fibrillar deposits is considered typical of nail–patella syndrome. In addition, podocytes lose their foot processes (for references, see [24]). In 1998, not only were the first mutations in the LMX1B gene published for patients suffering from nail–patella syndrome [12–14], but a report also appeared on the first characterisation of the Lmx1b knock-out mouse [25]. A more careful analysis of the kidney phenotype in the Lmx1b knock-out mouse revealed pronounced retardation in the development of podocytes that did not elaborate foot processes and slit diaphragms. Consistent with this finding was the splitting of the glomerular basement membrane and the reduced number of endothelial fenestrations, because podocytes synthesise proteins of the glomerular basement membrane and control differentiation of glomerular endothelial cells [26, 27]. Attractive explanations for these morphological defects have come from the observations that the α3 and α4 chains of collagen IV are no longer detected in the glomerular basement membrane ([28] and personal observations), that the Nphs2 gene is no longer expressed in podocytes of homozygous Lmx1b knock-out mice, and that podocytes in homozygous Lmx1b knock-out mice produce less vascular endothelial growth factor (VEGF) [26, 27]. Further molecular analysis demonstrated that LMX1B bound to AT-rich sequences in the first intron of the COL4A4 gene [28] and in the promoter region of the NPHS2 gene [26, 27].
Although the model that LMX1B activates the expression of COL4A4 and NPHS2, and that inactivating mutations in the LMX1B gene secondarily lead to the loss of collagen IV and podocin and therefore to the characteristic alterations in nail–patella syndrome patients certainly is an attractive one, several caveats have to be mentioned as well. Firstly, we could not demonstrate activation of a reporter construct with 4.4 kbp of the NPHS2 promoter by LMX1B [27], although another group showed an approximate twofold activation of the reporter gene controlled by four concatemerised LMX1B binding sites from the NPHS2 promoter [26]. Secondly, when we stably transfected a human cervical carcinoma cell line HeLa cells (which admittedly bears only a very remote similarity to podocytes) with an LMX1B cDNA, we found no upregulation of the endogenous NPHS2 gene [27]. Thirdly, podocin and the α3 and α4 chains of collagen IV were still present in glomeruli from patients with nail–patella syndrome [29]. And fourthly, the constitutive podocyte-specific inactivation of Lmx1b in the mouse does not lead to the loss of podocin or collagen IV [30]. What do these apparently discrepant results mean? If LMX1B already acts at a very early stage of podocyte development, specifically before the NPHS2, COL4A3 and COL4A4 genes are turned on (by other transcription factors?), the podocyte will just not have reached an advanced enough stage of development to produce podocin and collagen IV. In other words, LMX1B may rather exert a permissive influence and, for example, initiate the spreading of the foot processes upon which podocin would be produced and slit diaphragms be elaborated.
The constitutive podocyte-specific inactivation of Lmx1b represents a more comparable model for human nephropathy, but the mice only survive for ∼2 weeks after birth [30], again limiting their usefulness. In those animals, podocin and collagen IV α3/α4 are still present, and it appears as if foot processes and slit diaphragms are first elaborated and then lost secondarily (Fig. 1). Does LMX1B therefore play a role not only for the initial development of podocytes but also in the maintenance of their differentiation status? Clearly, more elaborate mouse models with an inducible inactivation of Lmx1b in adult animals are needed to answer this question. Such mice will also have the additional advantage of permitting the isolation of sufficient amounts of glomeruli, which can be used for DNA microarrays and identification of LMX1B target genes. Mouse genetics has already provided us with the verification of LDB1 as an interaction partner of LMX1B [30], and it may in the end help us to identify a genetic hierarchy acting in podocytes by telling us what factors control the expression of LMX1B in podocytes, what other proteins LMX1B interacts with and what genes are regulated by LMX1B in this peculiar cell type.
Fig. 1a–f Ultrastructural and immunohistochemical characterisation of mice with podocyte-specific inactivation of Lmx1b. In 11-day-old mice, the podocyte-specific inactivation of Lmx1b leads to the loss of foot processes and to a thickened glomerular basement membrane (arrows in b). However, despite the inactivation of Lmx1b, podocin and the α4 chain of collagen IV are still produced (d, f). +/lox control mice with one wild-type and one floxed Lmx1b allele; lox/lox mice with two floxed Lmx1b alleles; Cre presence of the Cre transgene under control of the human NPHS2 promoter. Bars: 5 μm (a, b), 20 μm (c–f). With permission from [30]

lmx1b
slit diaphragm
mouse model
target genes
podocyte foot process
promotor analysis