An Analysis of Respondent Driven Sampling with Injection Drug Users (IDU) in Albania and the Russian Federation

Injection drug users in Tirana, Albania and St. Petersburg, Russia were recruited into a study assessing HIV-related behaviors and HIV serostatus using Respondent Driven Sampling (RDS), a peer-driven recruitment sampling strategy that results in a probability sample. (Salganik M, Heckathorn DD. Sampling and estimation in hidden populations using respondent-driven sampling. Sociol Method. 2004;34:193–239). This paper presents a comparison of RDS implementation, findings on network and recruitment characteristics, and lessons learned. Initiated with 13 to 15 seeds, approximately 200 IDUs were recruited within 8 weeks. Information resulting from RDS indicates that social network patterns from the two studies differ greatly. Female IDUs in Tirana had smaller network sizes than male IDUs, unlike in St. Petersburg where female IDUs had larger network sizes than male IDUs. Recruitment patterns in each country also differed by demographic categories. Recruitment analyses indicate that IDUs form socially distinct groups by sex in Tirana, whereas there was a greater degree of gender mixing patterns in St. Petersburg. RDS proved to be an effective means of surveying these hard-to-reach populations.
Introduction
Accessing hard-to-reach populations for HIV prevention and research activities has historically been challenging. Certain high-risk populations are fundamental in the proliferation of HIV, particularly in the early stages of a country’s epidemic. Owing to the stigmatized and often criminalized behaviors of injection drug users, for example, these populations avoid open contact with the rest of civil society. Respondent Driven Sampling (RDS) offers a solution to that dilemma, allowing researchers to access hard-to-reach populations through their social networks.2,3 A social network person includes other persons with whom he/she has a social relationship and who tend to engage in the same behaviors as the index person. This paper presents approaches used to implement RDS in Tirana, Albania and St. Petersburg, Russia and lessons learned in these two cities.
Materials and Methods
The studies in St. Petersburg and Tirana utilized RDS to recruit IDUs into an HIV-related behavioral survey. While the methodology behind RDS has been described elsewhere,3 in brief it is a referral-based system whereby participants refer a limited number of friends or acquaintances (who are also members of the target group) to participate. Study participants receive an incentive both for participating and successfully recruiting others. Data are analyzed using the recruiting linkages and yield a probability sample of the population in the target area.
For inclusion in either study, participants had to be at least 15 years old and report having injected drugs recreationally in the past 6 months. Data collection for both surveys included face-to-face interviews and encompassed a 7–8 week period at three sites in each city. In Tirana, data collection took place in offices of two non-governmental organizations (NGOs), conducting outreach to IDU and in one treatment hospital. Data collection in St. Petersburg was conducted in three sites in various parts of the city. One was a fixed site in the center of the city. The other two sites were located just outside the city center and open on weekend afternoons, operating from mobile vans at stable locations. One site was selected due to its proximity to an illegal drug market.
The data collection team at each site and shift consisted of two interviewers and one person serving as coupon manager, supervised by a study coordinator. The coupon manager recorded data for each participant on a form that included each individual’s self-reported network size, the coupon number brought to the interview, and the coupon numbers each was given for distribution. The Tirana sites also had a physician, laboratory technician and counselor as data collection included biological samples for HIV and other STIs.
Questionnaire topics included sociodemographic indicators, sexual and drug injection risk behaviors, knowledge of HIV and STIs, and previous HIV testing. Survey duration in Tirana was approximately 1.5 hour for both the behavioral and biological portions of the study, while in St. Petersburg data collection lasted approximately 45 minute. In accordance with RDS methodology, the initial participants of the study, known as “seeds”,4 were members of the target IDU population in their respective cities, and were selected to begin recruiting their peers into the study. In Tirana, 15 seeds launched the study, while in Russia recruitment was initiated with 11 seeds; 2 seeds were added later to generate more recruiting. The seeds selected in both cities were referred from local NGOs conducting outreach with the IDU population, as well as personal IDU contacts of study staff. Each participant in St. Petersburg received three coupons with an eight-week expiration date to recruit their peers while two coupons per participant were used in Tirana (no expiration dates). In St. Petersburg, three coupons continued to be distributed until approximately 200 participants were attained, after which participants were warned that the study would be ending within a few days. In Tirana, coupons were distributed until the desired sample size of 200 was reached; however, participants were warned from the beginning that once this was reached, no more coupons would be honored.
In Tirana, participants received cash incentives of 10 euros for participating and an additional 5 euros for each successful recruit. In St. Petersburg, use of monetary incentives was discouraged by all local stakeholders; therefore, participants were given a gift package consisting of a box of chocolates and personal hygiene products for completing the survey, and a box of chocolates or a can of coffee for each successful recruit. The gifts were equivalent to U.S. $10 for participating and U.S. $5 for recruiting.
Two hundred ten IDUs (not including seeds) were surveyed between July and August 2005 in Tirana, and 200 IDUs (not including seeds) were surveyed in St. Petersburg during the same time period.
Biometric measures were taken in Tirana to discourage study members from participating in multiple sites. Measurements consisting of wrist circumference multiplied by forearm length were taken and recorded. Participants were told that this was a unique measurement that could be used to determine if they attempted to return or access other data collection sites. Project staff also rotated between sites to decrease the likelihood of participants visiting multiple sites. In St. Petersburg, biometric measurements were not taken since local researchers thought it would induce suspicion among IDUs and consequently discourage participation. Recognition of potential duplicate participants depended upon study staff, who were outreach workers familiar with the target population and operated out of the same site for the entire 8 weeks of data collection.
The data were analyzed using RDS Analysis Tool version 5.4 (RDSAT). RDS population estimates and confidence intervals as calculated by RDSAT are reported. The homophily index describes the extent of in-group ties. A homophily index of H =  1.0 reflects perfect homophily, indicating that ties are formed with other members of the same group. In contrast, H =  −1.0 reflects perfect heterophily, indicating that ties are formed completely outside of the group.3 Both studies were approved by the FHI Protection of Human Subjects Committee and the Tirana study was also approved by the Medical Ethical Committee of Albania.
In both cities, information about whether anyone refused to accept coupons was elicited from those who returned to collect their compensation for successful recruiting. Approximately 20% (Tirana) and 5% (St. Petersburg) of those approached to participate refused to accept the coupon from participants. Of a total of 450 coupons distributed in Tirana, 210 were returned. In St. Petersburg, 200 out of a total of 629 coupons distributed were returned. Furthermore, in Tirana, only two potential participants were found to be ineligible for participation and once consent had been given, no participants withdrew from the process. This information is not yet available for St. Petersburg.
Results
Demographic Characteristics
Table 1 describes the characteristics of the study samples in Tirana and St. Petersburg. The sample in Tirana consists predominately of male IDUs, with only 14 female IDUs recruited for the survey. This contrasts substantially from the sample in St. Petersburg, where more females were represented compared to Tirana, with 36% being female and 64% being male.
Table 1Demographic information AlbaniaRussiaSex Males91% (86, 96)64% (55, 73) Females9% (4, 14)36% (27, 45)Education completed Did not attend school9% (5, 16)0% Primary17% (11, 26)0% Secondary/vocational67% (58, 76)84% (77, 92) University7% (3, 8)16% (8, 23)Age 15–22 years38% (33, 48)11% (7, 15) 23–27 years36% (27, 41)38% (29, 47) 28 years or more26% (19, 32)51% (42, 62)Current co-habitation situation Married living with spouse12% (6, 18)9% (4, 15) Married living with other sex partner0%4% (2, 9) Married not living with any partner3% (1, 6)4% (1, 7) Not married, living with sex partner20% (14, 26)33% (25, 42) Not married, not living with sex partner65% (58, 72)49% (39, 60)The numbers in parentheses represent the 95% confidence intervals as calculated by RDS.
In Tirana, the majority had completed at least middle school (eight classes), with a third of the sample completing middle school and a third completing high school (12 classes). Only 7% in Tirana had completed university. In St. Petersburg, the majority (84%) had completed either secondary or vocational education, and 16% had completed university. In Tirana, the sample was comprised of young IDUs with 38% between 15 and 22 years of age and 74% below 27 years of age. The St. Petersburg study included an older IDU population, with over half of participants 28 years of age or older and only 11% between 15 and 22 years of age. In St. Petersburg, males were slightly older than females; a much higher proportion of males were 28 years of age or older (66 vs 26% of females; data not shown).
The majorities of the samples are unmarried (85% in Tirana and 82% in St. Petersburg). In Tirana, the majority also did not live with a sex partner (65%), while another 20% were unmarried but living with a sexual partner. A higher proportion of IDUs in Russia reported being unmarried but living with a sex partner (33%).
Network Sizes
As a part of RDS methodology, respondents were asked about the size of their personal networks. Specifically, participants were asked how many people they know personally who used injecting drugs (i.e., “you know who they are, they know who you are, you have seen them in the last six months”).
In Tirana, male IDUs reported a larger average personal network size than female IDUs. In contrast, in St. Petersburg, females had a larger average personal network size than males (20 vs 17, respectively).
The pattern of network size by age is again very different between IDUs in Tirana and St. Petersburg. In Tirana, network size increases with age (from 16 among the youngest age group to 21 in the oldest age group), whereas in St. Petersburg, network size decreases with age (from 25 in the youngest age group to 15 in the oldest age group). The same trend and differences between the two cities is observed with regard to education, which is likely due to the collinearity between age and education.
Similarly, in Tirana, there was a trend of increasing network size with increasing duration of injection drug use. However, in St. Petersburg, the network size was larger for recent initiates and long-term injectors compared to those who injected for 1–5 years (Table 2).
Table 2History of injection drug useDuration of injection drug useAlbaniaRussia<1 year21% (11, 24)6% (3, 9)1–5 years72% (69, 83)43% (31, 51)>5 years6% (3, 11)52% (43, 63)
Recruiting
Table 3 represents the recruitment patterns of IDUs in Tirana and St. Petersburg with respect to sex, age and length of injection. RDS provides the opportunity to learn about the degree to which people tend to affiliate with others sharing their same characteristics. In Tirana, the gender affiliation patterns demonstrate a strong heterophily in women, whereby women recruited only men (H =  −1.00), while men recruited women 7% of the time (H =  0.16; this is probably due to the small number of women in the Tirana sample, with only 14 women participating). From a possible 28 coupons given to these women, 16 coupons were returned (57%). In contrast, the gender affiliation patterns of IDUs in St. Petersburg reflects neither strong homophily nor heterophily. Both males and females recruited males about the same percentage of times (63 and 57%, respectively). The heterophily index in males was −0.02 and homophily index was 0.12 for females.
Table 3.Recruitment patterns by age, sex, and duration of drug injection in Tirana and St. PetersburgRecruitersRecruiteesTiranaSt. PetersburgYoungest (15–22 years)Middle (23–27 years)Oldest (≥ 28 years)Youngest (15–22 years)Middle (23–27 years)Oldest (≥ 28 years)Age Youngest (15–22 years)42%35%23%10%54%36% Middle (23–27 years)33%38%29%19%36%45% Oldest (≥28 years)29%39%32%12%44%44%SexMaleFemaleMaleFemale Male93%7%63%37% Female100%0%57%43%Duration of drug injectionNew (<1 year)Mid-term (1–5 years)Long-term (>5 years)New (<1 year)Mid-term (1–5 years)Long-term (>5 years) New (<1 year)20%64%16%0%43%57% Mid-term (1–5 years)8%57%35%7%40%54% Long-term(>5 years)4%65%32%6%40%54%
The affiliation patterns of age recruiting relations in IDUs in Tirana reflects neither homophily nor heterophily. The homophily indices were 0.02 for the younger (15–22 years), 0.05 for the medium age (23–27 years) categories, and 0.09 for the older age group (≥28 years). As can be seen, both youngest and the oldest age categories recruited the medium age group approximately one-third of the time. Further, both the youngest and the medium age groups equally recruited the oldest age group (approximately 23–29% of the time). Lastly, the medium and the oldest age groups recruited the youngest group with equal vigor at about one-third of the time.
Among IDUs in St. Petersburg, the affiliation patterns of age relations reflect neither homophily nor heterophily. The homophily indices were −0.03 for both the younger (15–22 years) and the medium age (23–27 years) categories, and −0.15 for the older age group (≥28 years).
In Tirana, as with the relations by age, the affiliation patterns of relations based on duration of injection drug use reflects neither homophily nor heterophily. The homophily indices were 0.09 for new injectors (i.e., <1 year of injection experience), −0.10 for the mid-term users (1–5 years), and 0.09 for the longer-term injectors (>5 years). New users were recruited the least by the mid- and longer-term injectors (8 and 4%, respectively), and both the new and longer term injectors recruited mid-term injectors (64 and 65%, respectively).
In St. Petersburg, however, the affiliation patterns of relationships by duration of injection drug use differed between new injectors and longer-term injectors. The group of new injectors was completely heterophilous (H =  −1.0), reflecting a lack of in-group ties. However, both mid-term and longer-term injectors exhibited neither heterophily nor homophily (H =  −0.07 and H =  0.05, respectively).
Discussion
These two studies are among the first to report on the level of sexual and drug injection risk behaviors and knowledge about HIV prevention in a probability sample of IDUs in Tirana and St. Petersburg using RDS. The use of a systematic peer recruitment method afforded the possibility of capturing a wider range of IDUs than studies recruiting with outreach workers or from specific venues, such as STI clinics or needle exchange programs. Additionally, these two studies shed light on the social context by identifying social network relations among the IDUs.
The recruitment analysis suggests that IDUs in Tirana form socially distinct groups by sex. Female IDUs do not seem to interact with each other; no female IDU referred another woman. This suggests that intravenous drug use amongst females may be a highly stigmatized activity, or it may reflect their initiation into drug use by men.
In contrast, IDUs in St. Petersburg do not form socially distinct groups with regard to sex. The lack of strong ties within sex groups may be a reflection of injection behaviors in that they often inject in group settings which are likely to be of mixed sex and share needles with members of the opposite sex.
In Tirana, the lack of strong ties both within and between age groups most likely indicates that younger IDUs are probably not being initiated into injection drug use by injectors who are older in age. However, in St. Petersburg, there were no intra-group ties among younger females or among newer IDUs. All new IDUs appeared to be recruited only by long- and mid-term IDUs, and all younger female IDUs were primarily recruited by older male and female IDUs. This suggests that new IDUs, particularly female IDUs, have been initiated into injection drug use by older IDUs. The high prevalence of HIV and STIs5–7 in the IDU population in Russia places these new injectors at extremely high risk for HIV acquisition. The differences in average network sizes of male and female IDUs between Tirana and St. Petersburg may have implications for prevention programs. The larger network size of male IDUs compared to female IDUs in Tirana is not surprising given that the IDU population in Albania appears to be predominately male and clandestine for women. Therefore, it is apparently rare for female IDUs to know other female IDUs. In contrast, in St. Petersburg, female IDUs on average had a larger network size compared to male IDUs, which may be a reflection of the overlap of this population and the sex worker population. A sizable proportion are engaged in sex work so they likely know other sex workers from working in the same areas.
The younger age of IDUs in the Albanian sample indicates a potential for longer term drug use, allowing for greater transmission. Additionally, continued migration between borders with neighboring countries where prevalence amongst high-risk groups is higher (i.e., to Greece, Italy or Eastern European countries) may create a transmission–migration effect.8
The male-to-female ratio of almost 2:1 in St. Petersburg is consistent with the decreasing trend in the male-to-female ratio of IDUs previously reported, from 7:1 in 1991 to 5:1 in 2001.9 However, it is also possible that previous studies may have underestimated the proportion of female IDUs. RDS may reach female IDUs better than other sampling methodologies. This could be evidence that RDS can capture a more diverse and possibly a more representative sample of IDUs in St. Petersburg.
The Tirana sample is proportionately younger than the St. Petersburg sample, perhaps describing an epidemic that is newer or reflecting the population structure in Albania where 40% is under 25 years of age.10 Records indicate an increase in substance abuse treatment since 2000 and the first HIV case was detected in Albania in 1993—lending credence to the possibility of a younger epidemic.11
The co-habitation situation in Tirana differs from that in St. Petersburg, with a far greater proportion in St. Petersburg unmarried but living with a sex partner. It is possible that the more conservative nature of Albanian society and a holdover from the Albanian communist regime policies are reflected in this indicator.12 The finding in St. Petersburg that all new injectors were recruited only by long- and mid-term injectors is of concern. These social relations combined with the high prevalence of HIV in the IDU population in Russia place these new injectors at extremely high risk for HIV acquisition. This scenario is likely exacerbated by their large average network size (approximately 18).
The research conducted in Albania with IDUs is of vital importance. For the first time, policy makers and those involved with drug treatment and prevention of HIV and STIs have information to better plan and target interventions.
The pace of recruitment in both countries (approximately 8 weeks) seems to be fairly efficient. Differing numbers of coupons (two in Tirana and three in St. Petersburg) did not seem to affect overall recruitment with both studies reaching the desired sample size in approximately the same amount of time. In St. Petersburg, slow recruitment particularly at the fixed site at the city center was boosted by the addition of two seeds at week 6; participation of only 50 IDUs at week 6 increased to 200 by the eighth week. Over 200 IDUs were reached by the study in 7–8 weeks with 13–15 seeds in each city.
The body of literature regarding the use of RDS amongst hard-to-reach populations in developing countries is still in its nascence. Little is known about the effect of incentives on populations in developing countries or if random recruiting of peers (a fundamental assumption underlying the RDS methodology) is valid in countries where a large proportion of the population is poor. A greater body of research employing these methods in developing countries is needed.
Lessons learned from RDS methodology
Data collection during the summer was ideal in St. Petersburg given that some sites were mobile vans and participants had to wait outside. The season was also beneficial in Tirana, with data collection taking place before summer vacation. Given the large size of both cities, having three study sites in different parts of each city facilitated recruitment. Evening and Saturday hours were important to ensure that workers could participate. In St. Petersburg, upon recommendation from local NGOs, letters of support obtained from city police officials helped ensure that IDUs would not be harrassed at the study sites. In Tirana, biometrics were useful in creating a disincentive to participate multiple times. Involving NGO staff to recruit seeds and conduct data collection in the NGO offices enabled participants to feel comfortable about the process.
Limitations
Possible limitations to the study could have affected the results. In Tirana, the small numbers of women participating in the surveillance may indicate a strong desire to remain hidden, their limited numbers, or a reflection of poor recruiting. Because few women have been arrested or attended treatment facilities, there are no data regarding intravenous drug use amongst women in Tirana, or anywhere in Albania.11 In both cities, it is possible that RDS may have selectively attracted those IDUs desiring the incentives, which may have biased our sample. Formative research was conducted to assess if the amount would be coercive, so this is unlikely.
The major limitation of RDS is that it cannot control for non-response bias. Barriers to participation due to unknown characteristics of certain sub-groups or to factors beyond the control of the project could occur and are not measurable. Both studies attempted to determine the rate of non-response by asking participants returning to claim recruiting incentives about the number of attempts made before their coupons were accepted and the demographic characteristics of those who refused. They were also asked characteristics of those accepting the coupons. It is hoped that forthcoming analysis can assess if there is a difference between those who refuse, those who accept but do not show up, and those who accept and participate.
The coupon return rate in St. Petersburg was considerably lower than that of Tirana most likely due to the greater number of coupons given out by each participant in St. Petersburg (three coupons) than in Tirana (two coupons).
Conclusion
Because IDU participants were recruited through RDS, it is likely that the study sampled from a wide range of IDUs thereby increasing the sample representativeness in these two cities. Our study methodology likely drew a more diverse sample of IDUs compared to traditional recruitment by outreach workers or through needle exchange programs.
The studies in Tirana and St. Petersburg greatly enhance our understanding of IDUs and their network characteristics. They also provide evidence for the effectiveness of using RDS in recruiting hard-to-reach populations in environments where stigmatization and criminalization against their behaviors are commonplace.

injection drug users
albania
respondent driven sampling.


Transient severe metastatic calcification in acute renal failure

Metastatic calcification, a known complication of prolonged end-stage renal disease, is herein described for the first time in a 10-month-old boy with acute renal failure, manifesting as a painful and swollen arm. Imaging revealed diffuse calcification and technetium-99 methylene diphosphonate (99Tc-MDP) uptake around the humerus and axilla. Calcium and vitamin D restriction, followed by intravenous administration of sodium thiosulfate caused a full symptomatic, radio- and scintigraphic improvement.
Introduction
Vascular (“metastatic”) calcification (MC) (or calcific uremic arteriolopathy) is the most common type of extra-osseous calcification in end stage renal disease, manifesting as both medial and intimal calcification of large arteries [1]. It usually results from a deposition of calcified products in otherwise normal tissues as a result of hyperphosphatemia with or without hypercalcemia. It may affect the visceral organs, such as kidneys, lungs, and gastric mucosa, as well as joints, eyes, and skin [2]. The predisposing conditions of hyperphosphatemia and high (calcium×phosphorus) concentration products need to be persistent over weeks and months, since MC has been not been described in the setup of acute renal failure (even though a similar biochemical profile is seen there too).
We describe a case of MC in a 10-month-old child presenting with hemolytic uremic syndrome (HUS) and acute renal failure (ARF).
Case report
A 10-month-old male infant was recovering from his first episode of atypical HUS. He belonged to a Bedouin tribe previously diagnosed by us [3] with recessive factor H deficiency. His clinical course was remarkable for microangiopathic hemolytic anemia, oliguric renal failure and hypertension with a depressed myocardial function, associated with resistant hypocalcemia (total calcium as low as 5.4 mg/dl) and hyperphosphatemia (as high as 13 mg/dl). The latter was eventually controlled with phosphor binders (aluminum hydroxide at first for several days and, later, calcium gluconate), intravenous administration of calcium (mainly due to an episode of cardiac arrest without hyperkalemia, as previously described for HUS [4]), low phosphorus diet and alphacalcidol, 0.25 μg q.d. (the latter only after serum phosphorus had been normalized). Medications were given through a right femoral vein catheter and peripheral veins in the right arm. Peritoneal dialysis was started, using regular calcium (3.5 meq/l) dialysate. Fresh frozen plasma (20 ml/kg) was given daily until there was evidence of HUS remission, and was then switched to 20 ml/kg twice a week, as previously done in homozygous factor H-deficient children [5].
Ten days after hospitalization, he was found to have a painful and mildly swollen left arm, mainly in the elbow area, with flexion limitation. There was no intravenous device in this arm. There were no high fever, local redness or swollen joints. The plain X-ray did not reveal any lesions or fractures. A week later the left shoulder and humeral area were more swollen and tender, with no specific nodule or bone deformity. On physical examination, the swelling was deeper than the dermis (mainly in the humeral area) and painful. The superficial skin did not show signs of ischemia, necrosis, ulceration or livedo reticularis. This time the plain X ray revealed diffuse calcification, involving mostly the arm muscles and the axilla (Fig. 1a). Calcium and phosphorus levels were 9.9 mg/dl and 6.6 mg/dl, respectively. Renal function remained stable but impaired (serum creatinine and urea concentrations were as low as 1.05 mg/dl and 90 mg/dl, respectively). Parathyroid hormone (PTH) levels were normal (25.7 pg/ml). Renal sonogram showed no evidence of nephrocalcinosis. A bone scan revealed significant technetium-99 methylene diphosphonate (99Tc-MDP) uptake along the areas that had seemed to be affected on the X-rays and physical examination (Fig. 1b). Treatment with peritoneal dialysis, alphacalcidol and calcium salts was stopped. Ketoconazole 1 mg/kg per day was given for 3 days, in addition to prednisolone 2 mg/kg per day for a week. Renal function improved, but the (Pi×Ca) products remained above 60 mg2/dl2. No improvement in the arm lesion was seen. On day 25 of hospitalization, intravenously administered sodium thiosulfate, 100 mg/kg q.d., was started for 12 days. A gradual symptomatic improvement in arm range of movement and resolution of local pain was seen. At follow-up 8 weeks afterwards, X- ray and bone scan revealed significant resolution of MC (Fig. 2). At long-term (>12 months) follow-up, no recurrence of MC was seen, in spite of recurrent events of HUS, eventually needing periods of peritoneal and hemodialysis.
Fig. 1Chest X-ray on day 18 (a) black arrow calcification on the axilla, arrowheads calcification lines along the humerus. Tc-99m MDP scintigraphy (b) shows increased abnormal uptake in the soft tissue of the left shoulder, humerus and left scapula. Kidneys are enlarged, with increased diffuse abnormal radiotracer activityFig. 2Repeated X-ray (a) and bone scan (b) after 1 month show no abnormal findings
Discussion
The unique MC features in our patient were its appearance early after the diagnosis of renal failure and its transient nature. Evidence for such early appearance of this complication comes only from an animal model of ARF (adenine-induced renal failure in rats), where microscopic metastatic calcification in aorta, coronary artery and other soft tissues were found after 4 weeks of uremia [6]. This was associated with hyperparathyroidism, hypocalcemia and hyperphosphatemia. In addition, a high index of suspicion may detect the MC early in the renal failure course, before it becomes ulcerated [7].
Current sophisticated imaging techniques suggest an incidence of subclinical or asymptomatic MC in 20–100% of patient with end-stage renal disease (ESRD) [8]. MC with soft tissue involvement in acute renal failure, to the best of our knowledge, has not been reported in children: using the PubMed and MESH search systems, and the terms “calcinosis” and “kidney failure, acute” (the term “calciphylaxis” is not an official indexing term), we found only three case reports of adults with such combination. There was a combination of risk factors for MC in our patient, including high dose parenteral calcium administration via both intravenous and peritoneal dialysis; high serum phosphorus levels due to renal failure and the hemolytic state; and the use of alphacalcidol [9]. These may have led to MC so early in the course of his disease. The possible correlation between renal failure due to HUS, a state where the basic damage is to the endothelium in small vessels, and MC is not supported by the literature search. Uremic calcification was thought to be a largely physico-chemical process facilitated by elevated Ca×P (i.e., “metastatic” calcification). Evidence now shows that vascular smooth muscle cells actively take up phosphate to form bio-apatite. This process is associated with a phenotypic transformation of vascular smooth muscle cells during which they express osteoblast markers [10].
Soft tissue accumulation of Tc99m MDP may be due to passive localization of tracer in slow fluid spaces (ascites, pleural effusion) when the blood concentration of tracer is high and these spaces do not clear as rapidly as the blood pool. Tracer may also bind in necrotic tissues (myositis, myonecrosis). Other causes of metastatic calcification include hypercalcemia from widespread destruction of bone (from metastatic cancer), parathyroid neoplasm and hypervitaminosis D. When the solubility product of calcium and phosphate is exceeded, there is precipitation of calcium in the extracellular space. Soft tissue calcification is, thus, another cause of soft tissue uptake of Tc99m MDP [11].
Reported therapeutic strategies for treating and preventing MC include: increasing dialysis dose, lowering serum calcium phosphorous and Ca×P solubility products, and avoiding calcium-based Pi binders and vitamin D analogs. Unfortunately, these methods have been ineffective in some patients with MC. Intravenously administered sodium thiosulfate (ST) increases the solubility of calcium deposits [12]. ST was shown to be successful in the treatment of both nephrolithiasis and tumoral calcinosis. ST has antioxidant effects on endothelial cells, but its exact mechanism of effect is unclear. It undergoes mainly renal clearance. Its half-life is increased from 15 min in the normal glomerular filtration rate (GFR) to 478 min in dialysis-dependent patients. The recommended adult dose is 12.5–25 g at the end of dialysis. The duration of this treatment is unclear. In our patient, a short ST course plus the withdrawal of enteral calcium and vitamin D were sufficient to resolve MC, which did not recur in the following events of HUS.

sodium thiosulfate
calcific uremic arteriolopathy
hemolytic uremic syndrome
peritoneal dialysis
radionuclide imaging
alfacalcidol


Measuring access to primary care appointments: a review of methods

Background Patient access to primary care appointments is not routinely measured despite the increasing interest in this aspect of practice activity. The generation of standardised data (or benchmarks) for access could inform developments within primary care organisations and act as a quality marker for clinical governance. Logically the setting of targets should be based on a sound system of measurement. The practicalities of developing appropriate measures need debate. Therefore we aimed to search for and compare methods that have been published or are being developed to measure patient access to primary care appointments, with particular focus on finding methods using appointment system data.
Background
Primary care is under scrutiny along with other public services to improve access to its users. Access in primary care is typically conceptualised as the achievable access to appointments with clinical professionals, although it is not routinely measured in most practices. It is widely recognised that it represents an important dimension in determining the quality of care [1,2]. Nevertheless, it is also known that waiting times can vary widely in differing localities and countries, from patients being seen the same day to a wait of several weeks. Most primary care organisations have no more than a perception of variable demand and no method of comparing fluctuating levels of access to appointments within or between practices. Measuring patient access could generate useful information for patients, clinicians and practice managers. Demand management initiatives and ways to optimise access could then be audited bearing in mind the impact of such initiatives on opportunity costs [3-5].
There are potential difficulties, recognised by a recent discussion document [6], that have been accentuated by policy and political influences. The NHS Plan in the UK [7] suggested that patients should have access to primary care services within 48 hours, but the concept lacked sufficient definition. The proposed new GMS contract includes an optional 48 hour target for access to GP appointments. But what exactly should we measure? Access can be measured at many different interfaces, from the wait for traditional services such as appointments with a clinician to the alternative solutions of nurse triage, nurse led clinics, telephone advice (including NHS Direct) or electronic mail responses. Differing interpretation of terms can also cause confusion. How soon for example should a problem that is defined by a patient as urgent be seen in general practice? The perception of urgent differs between patients themselves [8] and between doctors and patients. The concept of routine appointments is easier to define and quantify. A proposed measure is the waiting time for the next available routine appointment but this provokes debate about whether this should be practice-based or specified for individual clinicians. If the latter, factors such as part-time working, practitioner popularity, the creation of multiple review appointments are likely to rapidly diminish a clinician's accessibility. How transparent would a practice want to be about such data and how useful or acceptable would it be to publish information at the clinician level? [9]
Recognising the complex nature of this issue, we set out to review the literature. Our main aim was to search the international primary care literature for methods that had been, or were being developed to measure access to GP appointments, focusing on measures using appointment system data. Once identified, existing methods (tools, scales or other instruments) would be compared, with specific attention given to the type and levels of access they aimed to assess.
Method
Preliminary searches indicated that this area did not have an extensive or long-standing research literature; most articles had been published after 1998. A broad but systematic search process was designed to allow for a poorly indexed publication pool. Medline, PubMed, Clin Psyc and ASSIA were searched for relevant publications between 1990 and 2001. The following MeSH terms were used: family practice, health service accessibility (organisation and administration, statistics and numerical data, standards, trends, methods, manpower), appointments and schedules (waiting lists), research design, health service needs and demand, weights and measures, quality of healthcare, management audit, patient satisfaction, health service needs and demand. In addition the following keywords were used: general practice, access, appointments (same day, urgent, routine) appointment systems, measurement, measures, tools, scales, demand, availability, audit and waiting times. Terms were used both singly and in combination. Title searches were used to increase the sensitivity. All citations and abstracts were appraised for relevance and full articles selected for examination by two researchers independently (GE and WJ). Key authors were contacted directly [1,12,16,18,21,25,27] and searches conducted on their previous work. Departments of General Practice in Universities in the United Kingdom were also asked to send details of any relevant research. Conference literature [21] and non-peer reviewed literature obtained from websites was also appraised. Relevant websites were identified using  and the search terms health service delivery, access to primary care and general practice. The following sites were reviewed: the Royal College of General Practitioners [6], the National Primary Care Research and Development Centre [1], The National Primary Care Development Team [10] and the Centre for Innovation in Primary Care [11].
Studies or articles were included in the review if they described tools, scales, questionnaires or other methods of measuring actual patient access to appointments. We also included descriptions of methods that were currently being developed in this field, provided they had undertaken pilot studies and had completed one data collection exercise. Articles were excluded if they were purely editorial.
Results
A total of 1763 citations were initially identified and 38 articles retrieved for detailed assessment from the Pubmed and the Medline searches. Clin Psych and ASSIA searches provided some overlap but no new relevant material. The most helpful pointers to relevant publications were obtained from the website searches and personal communications rather than the traditional search engines. Two broad approaches to the measurement of patient access were identified. Firstly, appointment systems in organisations were analysed in differing ways to provide numerical data and, secondly, patient perceptions (reports) of access were evaluated using survey techniques.
Methods using appointment system data
Table 1 summarises the six identified methods that were based on appointment system data.
Table 1
Comparison of methods based on appointments systems to measure access to primary care
Third appointment [21]
NEMAS [15]
Ledlow [14]
Access Response Index AROS [22]
Campbell [12]
Kendrick [13]
Measurement
The 3rd available routine appointment at 12 midday, one day per week, for every clinician
Date of patient call and appointment provided. GP requested and GP allocated.
Appointments demanded but not available in US style primary care clinics compared to community clinics. Demand versus availability gap coded into 4 categories.
Number of days until next available routine appointment (with any clinician) at 4 pm, every working day
Number of appointments provided at the beginning of the day and the number still available. Total number of patients seen during the day, noting the number of 'extras'
Number of appointments available at the start of the day and the number of patients seen as 'extras' at the end of the day
Frequency of data collection
Once a week
Continuous
Daily
Once a day
Twice a day
Twice a day
Weighted for part time staff
Yes
No
No
No
No
No
Named clinician access measured
Yes
Yes
No
No
No
No
Data analysis
Weekly median score and monthly average
Computerised
Demand versus availability gap
Computer to work out 5 day moving average
Data related to practice list size, with rates given per 1000 patients
Daily tally
Results
Weekly snapshot of patient access profile
Complete computerised analysis of practice appointment system
Feedback reports generated to clinic staff
Trends across weekly schedules.
Bar charts represent number of appointments offered versus number of patients seen. Start of day appointment availability categorised as low, medium and high
Graphical display of extras versus number of free appointments during the day
Extent of and reason for use
Primary Care Collaborative in England. To inform implementation of advanced access
145 teaching practices Audit
US Military Clinic Study
10 practices To inform improvement
19 practices Research Study
1 practice Research Study
Co-ordination
National Primary Care Development Team
Department of General Practice, University of Glasgow.
Healthcare Programs Central Michigan University.
University department of General Practice
University Department of General Practice, Edinburgh.
Department of Primary Health Care, University of Southampton.
Three methods determine appointment availability and/or the satisfaction of demand on a daily basis but do not measure the days wait for appointments. Campbell measured clinician availability by recording the number of provided appointments at the beginning of each day plus the number of these un-booked at this time [12]. At the end of the day numbers seen and numbers of extras were counted, and adjusted for practice list size. Kendrick and Kerry recorded the number of available appointments at the beginning of the day and the number of extras seen at the end [13]. Ledlow also suggested a categoriations system for recording differing levels of unmet daily demand in a military medical service [14]
Three methods measure access as days waited by representing appointment system data. A computerised package, NEMAS [15,16] enables practices to audit four areas: practice service provision (including 'appointment availability' and patient satisfaction), chronic disease management, drug monitoring and significant event analysis. The method calculates the mean time waited in days plus the minimum and maximum patient waits. Data can be presented for the whole practice or for individual GPs. Data entry involves using electronic forms to record the date of appointment request, the date the patient was seen, whether it was an elective, forced (i.e. next available clinician) or urgent appointment, the clinician requested and the clinician actually consulted. The costs of collecting data has varied considerably depending on which staff member is employed for the task [17]. Transfer of data from the practice system can be automatic with compatible systems (personal communication).
The National Primary Care Development Team is measuring access as part of the adoption of the system of 'Advanced Access', a system developed in the USA [18,19], as a response to patient waiting times of 4 – 6 weeks for routine appointments in primary care [20]. This method collects data on one day per week (which can vary) at 12 noon. The number of days to the third available routine appointment for each clinician is recorded and a median figure calculated to represent an access score for the specified week. Over a month, the average of four median values is taken to represent a monthly access score. The third appointment is chosen, rather than the first, in order to negate the effect of sudden cancellations which otherwise could give false impressions of availability if at the moment of measuring there is one sudden cancellation in a schedule that is otherwise booked for several days ahead. The third appointment has been found by trial and improvement to best represent the actual waits involved (personal communication). Embargoed appointments are not included. A system of weighting has been developed [21] for part time workers to enable their scores to be incorporated.
The Access Response Index (AROS) was developed as a rapidly calculable measure of organisational access [22]. This index is derived by counting the number of days until the next available routine appointment, with any clinician, once during every normal working day. The data is recorded at four pm – a time chosen to avoid the influence of embargoed appointments that many organisations use to maintain urgent same day availability. The results are plotted on a graph, the daily fluctuation represented by the raw data is smoothed to a demonstrate trends by calculating a 5-day moving average (a data point which is the mean of every successive 5-day group). An example of the data produced by this measure is shown in Figure 1.
Figure 1
AROS scores for routine appointment availability (data from 11 practices)
Methods using patient questionnaires
Four patient experience questionnaires were found that contained access assessments and the relevant items are outlined in Table 2. The General Practice Assessment Survey (GPAS) [23] was developed by the National Primary Care Research and Development Centre, by adapting the Family Practice Assessment Survey (FPAS)[24]. A second survey named Europep [25,26] has been validated in 10 European countries. Thirdly Baker describes the use of a validated surgery satisfaction questionnaire (SSQ) that he developed [27,28] and finally Grogan's patient Satisfaction Questionnaire contains two sections that ask about access and appointment availability[29].
Table 2
Patient survey instruments: items used to determine access perceptions
Survey items
Response ratings
GPAS [23]
6.
Thinking of times when you want to see a particular doctor:
a) 5 point scale, 1 = same day, 5 = more than 5 days
a) How quickly do you get an appointment?
b) How do you rate this?
b) Range from 1 = very poor, 6 = excellent
7.
Thinking of times when you are willing to see any doctor:
a) How quickly do you get an appointment?
8. Yes / No / Not applicable / Don't know
b). How do you rate this?
8.
If you need an urgent appointment to see your GP can you normally get one on the same day?
EUROPEP [25,26]
What is your opinion of the general practitioner and/or the practice over the past 12 months with respect to:
5 point scale (poor to excellent)
19)
Getting an appointment to suit you?
23)
Providing quick services for urgent health problems?
Baker [28]
10)
It can sometimes be difficult to get an appointment with my doctor at this surgery.
5 point agreement scale
14)
It can be hard to get an appointment for medical care right away.
Grogan [29]
33)
Getting an appointment at a convenient time is easy.
5 point agreement scale
34)
Appointments are easy to make whenever I need them.
35)
It is often difficult to get an appointment with a doctor.
36)
It is easy to see a doctor of my choice.
Discussion
Principal findings
This review of access measurement reveals the heterogeneous nature of the methods and the lack of any widely accepted conceptualisation of patient access. Identified measures are either practice centred using appointment data or patient orientated via surgery satisfaction questionnaires. It is clear that these two methods represent entirely different aims. It is not possible for episodic patient surveys to provide data that has enough currency or accuracy to inform organisational responses to patient demand.
Also demonstrated are inherent problems over definition of terms – defining what is to be measured and setting of targets. There appear to be three appointment categories over and above emergencies (which by definition cannot be given 'appointments'). Appointments for healthcare could be categorised into urgent, soon, and elective. Urgent appointments are typically seen as requests for same-day consultations. The soon category would fit problems that should be seen within two or three days to prevent escalation or symptom prolongation. Finally, routine or elective appointments suit individuals who value an agreed time window over other factors. The methods that represent patient waiting times for 'routine', i.e. elective appointments seem to hold promise as they avoid the definitional problems that surround 'urgent' appointments, and the different views that patients, clinicians and others have about 'urgency'. The task of deciding whether to represent access profiles for organisations or for individual clinicians also needs careful consideration. It may be more feasible (and less threatening) to routinely measure organisational access, especially if the data is to be used for benchmarking purposes.
To attempt to measure access means to obtain meaningful data from a dynamic system that is not always in equilibrium. The access experienced by an individual varies determined by demand, adequate appointment provision, sudden cancellations and block release of held appointments. We have distinguished two approaches to overcome this, either for a full statistical analysis using compatible systems or data retrieval software, or to use a simple snapshot method, deciding whether data-smoothing methods such as daily moving averages or aggregated weekly median scores are the best portrayal of overall access patterns.
Strengths and weaknesses of the study
Multiple search methods were used to ensure that the breadth of literature and online resources were examined as systematically as possible. The searches proved difficult and reflect the emergent status and the diversity of terms used in this area. We may have overlooked methods developed in other healthcare systems.
Implications of the findings to healthcare services and research
The lack of a widely agreed measurement method to represent patient access to primary care services will make it impossible for practices to compare their response to patient demand with any degree of certainty. The 'third appointment' system is the most widely used method and is currently supported by the National Primary Care Development Team in England (but has no equivalent support in Scotland, Wales or Northern Ireland). It is however a relatively complex manipulation of appointment system data, and incorporates individual clinician availability. It seems from first principles that the important requirements of a tool designed to measure a dynamic concept such as patient access is simplicity and ease of regular data collection, so that longitudinal data patterns capable of indicating trends in organisations can be generated rather than data on individual clinician availability.
A recent survey compared mechanisms used to manage requests for same day appointments[30]. Murray's proposal of doing today's work today (Advanced Access) eliminates appointment categories and the work involved in negotiating urgency by dealing with virtually all demand on the day it arises[31]. Access is an important determinant of healthcare quality but what are the implications of this approach to the balance of overall quality? Continuity of care, whilst not important for some problems is desirable for others [32,33]. Too drastic a shift in favour of access is likely to be at the cost of reduced continuity and a diminution of other services, such as screening and chronic disease management. Measures of organisational quality need to be aware of the dangers of focusing too much on one dimension, and should work towards the creation of measures that balance scores across internal and external requirements [34].
An effective consultation with a well trained clinician who knows the patient and who has access to a well structured longitudinal record will probably remain a gold standard and the issue of immediate 'access' should not be elevated above all the other components in this equation. Nevertheless, it seems prudent to develop measures that provide a better understanding of patient access to organisations with similar resources. Measuring the interval to the next available routine appointment, whilst measuring the measurable [35] will, if a consistent standard can be agreed, provide benchmark data, and mark an important step towards a compendium of methods to assess quality in primary care.
Conclusion
The two approaches of either using patient questionnaires or appointment system data to measure access are methods that represent entirely different aims. The latter method when used to represent patient waiting times for 'routine' elective appointments seems to hold promise as a useful tool and this avoids the definitional problems that surround 'urgent' appointments. The purpose for which the data is being collected needs to be borne in mind and will determine the chosen methods of data retrieval and representation
Competing Interests
The authors of this article have piloted the AROS Index.
Authors' Contributions
Peter Edwards, Melody Emmerson and Glyn Elwyn were involved in the development of the Aros index. Wendy Jones and Glyn Elwyn conducted the Aros pilot study and the literature review with contributions from Adrian Edwards and Richard Hibbs.
Pre-publication history
The pre-publication history for this paper can be accessed here:

measurement
access
primary care
appointments
waiting times.


Localization of metabotropic glutamate receptors in the outer plexiform layer of the goldfish retina

We studied the localization of metabotropic glutamate receptors (mGluRs) in the goldfish outer plexiform layer by light-and electron-microscopical immunohistochemistry. The mGluR1α antibody labeled putative ON-type bipolar cell dendrites and horizontal cell processes in both rod spherules and cone triads. Immunolabeling for mGluR2/3 was absent in the rod synaptic complex but was found at horizontal cell dendrites directly opposing the cone synaptic ribbon. The mGluR5 antibody labeled Müller cell processes wrapping rod terminals and horizontal cell somata. The mGluR7 antibody labeled mainly horizontal cell dendrites invaginating rods and cones and some putative bipolar cell dendrites in the cone synaptic complex. The finding of abundant expression of various mGluRs in bipolar and horizontal cell dendrites suggests multiple sites of glutamatergic modulation in the outer retina.
Introduction
Stimulation of vertebrate photoreceptors by light decreases the release of glutamate (Cervetto and MacNichol 1972; Murakami et al. 1972; Kaneko and Shimazaki 1976), which is sensed by glutamate receptors (GluRs) in the dendrites of second-order neurons. Glutamate receptors are roughly divided into ionotropic and metabotropic receptors (Eccles and McGeer 1979). Ionotropic receptors (iGluRs) form an integral ion channel, whereas metabotropic receptors (mGluRs) mediate responses through indirect mechanisms involving signal transduction cascades.
So far, eight different mGluRs have been cloned and classified into three groups based on their sequence similarities, second-messenger cascade, and pharmacology: group I comprises mGluR1 (Houamed et al. 1991; Masu et al. 1991) and mGluR5 (Abe et al. 1992) and their splice variants (1a–d and 5a–b); group II contains mGluR2 and mGluR3 (Tanabe et al. 1992; Tanabe et al. 1993); and group III includes mGluR4 (Tanabe et al. 1993), mGluR6 (Nakajima et al. 1993), mGluR7 (Okamoto et al. 1994; Saugstad et al. 1994), and mGluR8 (Duvoisin et al. 1995), and their splice variants (4a–b, 7a–b, 6a–b, and 8a–b). The lack of specific pharmacological tools to distinguish between mGluRs within a group makes it difficult to link a particular receptor type to a certain response property (for reviews, see Thoreson and Ulphani 1995; Schoepp et al. 1999).
In the retina, mGluRs play an important role in the transmission and modulation of visual signals. Light responses of ON-type bipolar cells (ON BCs) of a number of species, for instance, are sensitive to substances/drugs that target group III mGluRs (Shiells et al. 1981; Slaughter and Miller 1981; Thoreson and Miller 1993). The receptor activated by these drugs is most likely mGluR6 (Nakajima et al. 1993; Akazawa et al. 1994; Masu et al. 1995; Ueda et al. 1997; Vardi and Morigiwa 1997; Laurie et al. 1997; Vardi et al. 2000). In fish, amphibians, and mammals, group III mGluRs modulate the glutamate release of cones (Koulen et al. 1999; Hirasawa et al. 2002; Hosoi et al. 2005). Furthermore, pharmacological activation of both group I and group III mGluRs modulates horizontal cell (HC) responses (Nawy et al. 1989; Takahashi and Copenhagen 1992; Linn and Gafka 1999), suggesting that multiple mGluRs must be present in the fish outer plexiform layer (OPL).
At the moment, however, data available on the identity and distribution of the various mGluRs in the teleost outer retina are scarce (Yazulla et al. 2001; Klooster et al. 2001). We have therefore performed a light and electron microscopy study of the mGluRs present in the goldfish OPL. An antibody against the ON BC marker, protein kinase C (PKC; Negishi et al. 1988; Suzuki and Kaneko 1990; Yazulla and Studholme 1992), has been used in order to identify possible localization in mixed-input ON BCs (ON MBCs).
Materials and methods
Subjects
Goldfish (Carassius auratus), 12–15 cm standard body length, were obtained from a commercial supplier and maintained at 16°C in aerated tanks filled with tap water circulating through a biological filter system. The fish were fed and kept on a 12 h/12 h light-dark cycle. All animal experiments were approved by the ethical committee of the Royal Netherlands Academy of Arts and Sciences, acting in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Preparation
Dissections took place early in the morning (between 7 a.m. and 9 a.m.) under ambient light. The fish used in this study were therefore all in the light phase of their circadian regime. After cervical transection, the cornea and lens of light-adapted animals were removed, and the remaining eyecups were cut in half through the equator. Half-eyecups were placed vitreous-side-down on a Millipore filter (13 mm diameter, 8 μm pore size; Millipore, Amsterdam, The Netherlands) placed on a filter holder. Suction was applied to remove the vitreous; the sclera and retinal pigment epithelium were peeled away.
Light microscopy
Retinas were fixed at room temperature for 10 min in 0.1 M phosphate-buffered 4% paraformaldehyde (pH 6.5) and for another 10 min in 0.1 M sodium carbonate-buffered 4% paraformaldehyde (pH 10.4). These short fixation times were chosen to prevent loss of antigenicity. After being rinsed in 0.1 M phosphate buffer (PB, pH 7.4), the tissue was cryoprotected at room temperature for 30 min in PB containing 12.5% sucrose and for 1–2 h in PB containing 25% sucrose.
The pieces of retina, still attached to the filter, were embedded in Tissue Tek (Sakura Finetek Europe, Zouterwoude, The Netherlands) in an aluminum boat and frozen in liquid nitrogen. Sections (8–10 μm thick) were cut in a cryostat, mounted on poly-L-lysine-coated slides (Menzel-Gläser, Braunschweig, Germany), dried and stored in a non-frost-free freezer at −20°C until use.
Retinal sections were washed (2×, 10 min) in phosphate-buffered saline (PBS), blocked for 20 min in PBS with 2% normal goat serum (NGS; Jackson ImmunoResearch Lab, West Grove, USA), incubated for 18–48 h with the primary antibody (for dilutions, see Table 1) in PBS containing 0.3% Triton X-100 and 5% NGS at 4°C. After several washes (15 min, 3×) in PBS, sections were incubated in the secondary antibodies diluted in PBS containing 0.3% Triton X-100 for 30 min at 37°C (Cy3-conjugated antibody: 1:500, Alexa-conjugated antibody: 1:600).
Table 1Primary antibodies (LM light microscopy, EM electron microscopy, aas amino acids, PKC protein kinase C, mGluR metabotropic glutamate receptor)AntibodySourceCatalog numberLotTypeHostPurificationAntigenDilution for LMDilution for EMBlot (kDa)aPKCbSigma-Aldrich, St. Louis, USAP57040641H4847MonoclonalMouseProtein ABovine, within aas 296–317 of brain PKC1:5001:40080mGluR1αChemicon, Temecula, USAAB1551196011PolyclonalRabbitAffinityRat, C-terminal, aas 1180–1199 (PNVTYASVILRDYKQSSSTL)1:1001:100140mGluR2/3ChemiconAB1553196011PolyclonalRabbitAffinityRat, C-terminal, aas 860–872 (NGREVVDSTTSSL)1:1001:100100 and 190mGluR5ChemiconAB159624010139PolyclonalRabbitAffinityRat, C-terminal, aas 1159–1171 (LIIRDYTQSSSSL)1:1001:100116mGluR7Upstate, Lake Placid, USA07–23925162PolyclonalRabbitProtein AHuman, C-terminal, aas 899–912 (NSPAAKKKYVSYNN)1:5001:50097aWestern blots performed by external groups; relative mass values as reported in manufacturer’s data sheetsbProduced after Parker et al. 1984 and Young et al. 1988
Sections were cover-slipped with Vectashield (Vector Labs, Burlingame, USA) and stored at −20°C. Slides were observed on a Leica DMRD (Leica Microsystems, Wetzlar, Germany) fluorescence microscope equipped with filter sets that were designed for fluorescein isothiocyanate and Cy3. Sections from double-label experiments were observed on an inverted Zeiss Axiovert 100 M microscope equipped with the LSM 510 META laser scanning confocal module (Carl Zeiss Jena, Jena, Germany).
Two types of controls were performed. To control for unspecific labeling attributable to secondary antibodies, experiments were performed by omitting the primary antibody. This eliminated all labeling. To control for unspecific labeling attributable to the primary antibodies, preadsorption experiments were performed by mixing, in a 20-fold molar excess, a synthetic peptide having the amino acid sequence against which each mGluR antibody had been raised (see below) with the corresponding primary antibody overnight at 4°C. Incubation proceeded as previously described, with the same concentrations for the primary antisera. Labeling was also completely eliminated in these preadsorption controls.
Electron microscopy
The same fixation and cryoprotection procedures were used as described for light microscopy. The retinas were peeled off the filter and embedded in Tissue Tek (Sakura Finetek Europe, Zouterwoude, The Netherlands) in an aluminum boat. The boat was frozen in liquid nitrogen. Transverse frozen sections (30–40 μm thick) were obtained on a freezing microtome and collected in PB at room temperature (equivalent to freeze-thawing).
The retinal sections were incubated for 72 h with diluted antisera (for dilutions, see Table 1). After being rinsed, the tissue was incubated in a poly-horseradish-peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG (PowerVision, ImmunoVision Technologies, Daly City, USA). To visualize the peroxidase, the material was incubated in a TRIS-HCl diaminobenzidine (DAB, 0.05%) solution containing 0.03% H2O2. The DAB reaction product was subsequently intensified by the gold-substituted silver peroxidase method (Gorcs et al. 1986).
Sections were rinsed in sodium cacodylate buffer 0.1 M (pH 7.4) and post-fixed for 20 min in 1% OsO4 supplemented with 1.5 % potassium ferricyanide in sodium cacodylate buffer 0.1 M (pH 7.4). After being rinsed in sodium cacodylate buffer, the material was dehydrated and embedded in epoxy resin. Ultrathin sections were observed in a Philips EM201 electron microscope (Philips, Eindhoven, The Netherlands) and/or Technai (CM) 12 electron microscope (FEI Company, Eindhoven, The Netherlands). Sections examined with the Philips electron microscope were counter-stained with uranyl acetate and lead citrate.
Sampling and production of photomicrographs
At least 20 retinal sections obtained from a minimum of five animals were used for a given light microscopy or electron-microscopy experiment (i.e., single-immunolabeling at the light-microscopy level for mGluR1α). About five photomicrographs were taken from the most representative sections in each experiment.
Light micrographs of the single-label experiments were acquired as TIFF files directly from the Leica microscope by using a Leica 350 F digital B/W camera (1284×1028 pixels, 72 ppi). Light micrographs of the double-label experiments were acquired as TIFF files from the Zeiss LSM 510 META (1048×1024 pixels, 72 ppi).
For electron-microscopy experiments, the same number of retinal sections/animals as described above was examined at the light-microscopical level; labeled sections were chosen for ultra-thin sectioning. A minimum of 40 photoreceptor terminals were observed per ultra-thin section. Micrographs (n = 10–30) were taken from these sections, depending on the presence and amount of labeling.
Electron micrographs obtained with the FEI electron microscope were directly acquired as TIFF files (1024×1024 pixels, 72 ppi). Electron micrographs obtained from the Philips electron microscope were first printed from the negatives for analysis. The negatives of the prints that were selected for publication were scanned on a sprint scan 4000 scanner (Polaroid Nederland, Breda, The Netherlands) and acquired as TIFF files at 600 ppi.
All TIFF files were resampled at 400 ppi and subsequently re-sized and optimized for brightness and contrast by using Photoshop (Adobe Systems, San Jose, USA).
Immunochemicals
The primary antibodies used in this study were purchased from Chemicon International (Temecula, USA), Sigma-Aldrich (St. Louis, USA), and Upstate (Lake Placid, USA). Details of these antibodies are listed in Table 1. Goat anti-mouse Alexa was obtained from Molecular Probes (Breda, The Netherlands) and goat anti-rabbit Cy3 from Jackson ImmunoResearch Lab (West Grove, USA). For preadsorption controls, custom-made peptides were obtained from Eurogentec (Seraing, Belgium).
Western blotting
Goldfish retinas and brains were homogenized with a Teflon pestle in ice-cold PBS (pH 7.4) containing a tablet of protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany) per 50 ml PBS. After homogenization, a TRIS-buffered sample (pH 6.8) solution was added, with final concentrations of: 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.005% bromophenol blue. The samples were boiled for 2 min and centrifuged for 10 min at 14,600 g; supernatants were stored at −20°C until use. The diluted samples were boiled for 2 min (3×), cooled, and then spun in a microfuge at 14,600g.
For sodium dodecyl-sulfate polyacrylamide gel electrophoresis, samples were run through a polyacrylamide stacking gel at 20 mA and through a 13% polyacrylamide gel at 30 mA. Protein standards (Bio-Rad Laboratories, Veenendaal, The Netherlands) were run in adjacent lanes. Gels were electroblotted on a poly-vinylidene-di-fluoride blot membrane (Millipore) overnight at a constant current of 80 mA. After being rinsed with a 0.5 M TRIS buffer containing 1.5 M NaCl and 5% Tween 200, the membrane was blocked in the same buffer containing 2% dry milk for 1 h.
Subsequently, the membrane was incubated for 1 h in the primary antibodies (1:100), washed in the TRIS buffer, incubated in an HRP-conjugated goat anti-rabbit IgG (1:5000; Santa Cruz Biotechnology, Santa Cruz, USA), and washed in PBS. The immunoreaction was visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, USA) by using Kodak film. Exposures times were between 1 and 5 min.
Results
In this study, we aimed at a qualitative description of the mGluR composition in the goldfish OPL. To have a parameter for comparisons with regard to ON BC labeling with the antibodies against the various mGluRs, we first investigated the immunoreactivity pattern yielded by the PKC antibody, a widely used ON BC marker. Therefore, the labeling for PKC will be described first, followed by each of the mGluRs studied.
Immunolabeling for PKC
The PKC antibody was characterized by Western blot previously in a number of studies (Negishi et al. 1988; Suzuki and Kaneko 1990; Yazulla and Studholme 1992) and yielded similar results in various species (Table 1).
Light microscopy Immunolabeling for PKC in the goldfish retina at the light-microscopical level has been described in detail elsewhere (Suzuki and Kaneko 1990). Briefly, two populations of ON BCs were labeled: cells with small somata in the middle of the inner nuclear layer (INL) and small axonal terminations (cone-driven ON BCs), and cells with large flask-shaped somata and bulbous axon terminals (ON MBCs).
Electron microscopy: rod synapses In the rod synaptic complex, the antibody against PKC labeled invaginating dendrites at the central position and processes at some distance from the ribbon (Fig. 1a–c, arrowheads). The labeled structures were elongated (Fig. 1a), u-shaped (Fig. 1b), or round (Fig. 1c) and were found either directly opposite to the synaptic ribbon (Fig. 1a,b) or a little distance away from it (Fig. 1c). Dendrites with these characteristics and at these locations in the cyprinid retina were previously shown to belong to ON MBCs (Scholes 1975; Stell 1967, 1976, 1978; Saito et al. 1983, 1985; Klooster et al. 2001; Yazulla and Studholme 2001).
Fig. 1Ultrastructural localization of protein kinase C (PKC) in the outer plexiform layer. In the rod spherules (a–c), invaginating dendrites of putative mixed-input ON-type bipolar cells (ON MBCs) were positively labeled (arrowheads). Lateral elements of horizontal cells (HCs) were unlabeled (arrows), as were some invaginating processes that possibly belonged to OFF-type MBCs (asterisks). In the cone pedicles (d, e), PKC immunoreactivity was observed in small invaginating profiles at the base of the pedicles (arrowheads). These structures were never associated with the synaptic ribbons (SR). HC lateral elements were unlabeled (arrows). Bars 0.5 μmLateral processes immediately adjacent to the pre-synaptic ridge, previously classified as dendrites of rod-driven HCs (Stell 1967, 1976; Scholes 1975) were not labeled (Fig. 1a–c, arrows). Some invaginating processes were also not labeled (Fig. 1a,c, asterisks); their position and fine structure, in combination with the lack of labeling, suggested that they were either dendrites of OFF-type MBCs or rod-driven HCs (Stell 1967, 1976; Saito et al. 1983, 1985).
Electron microscopy: cone synapses PKC-immunoreactive structures were not abundant in the vicinity of the cone pedicles. When present, the labeled structures were never associated with the synaptic ribbon but were instead localized at the base of the cone pedicle (Fig. 1d,e, arrowheads). These were probably ON MBC processes approaching the cone pedicle. In the closely related carp, dendrites of horseradish peroxidase-injected ON MBCs have been found at similar sites in the cone synaptic complex (Saito et al. 1983, 1985). Lateral elements were unlabeled (Fig. 1d,e, arrows).
Immunolabeling for mGluR1α
Western blotting The mGluR1α antibody did not recognize any proteins in the goldfish retina, possibly because of the low concentration of the receptor in this tissue. The antibody produced a diffuse band at approximately 150 kDa in goldfish brain and rat samples (Fig. 2c), close to the predicted molecular mass of mGluR1α (Tables 1, 2). The immunoreactive protein found in the rat retina was faint, probably reflecting the restricted expression of this receptor in retinal tissue.
Fig. 2Immunolabeling for mGluR1α (ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer). a Immunoreactivity was concentrated at the OPL and outer half of the INL in which some BC somata were partially labeled (arrowheads). b Preadsorption with the control peptide (pread) abolished all labeling. c Western blot of total protein from goldfish retina (ret), goldfish brain (br), rat retina (ret), and rat cerebrum (cer). A protein at around 150 kDa, close to the predicted molecular mass of mGluR1α, was detected in goldfish brain, rat retina, and rat cerebrum. d, eRed and green channel confocal images of the same tissue section double-labeled with antibodies against mGluR1α (Cy3, d) and PKC (Alexa, e). Some structures were positive for mGluR1α but not for PKC (circles). Some small BCs were positive for the ON BC marker but not mGluR1α (asterisks). f Merged image of the double-labeling experiment. A band of mGluR1α immunoreactivity in the OPL partially overlapped with the dendritic processes of PKC-positive cells. Areas of overlap between the two proteins appear yellow. Bars 10 μmTable 2BLAST (basic local alignment search tool) results and relative molecular mass (Mr) of fish mGluRs (aa amino acids)Source proteinZebrafish matchesSequence comparisonLength sourceLength zebrafishMr sourceMr zebrafishThis studyRat mGluR1(NP_058707)Similar to bifunctional mGluR forextracellular Ca and Glu (previouslysimilar to mGluR1-XP_690446a)SourcePNVTYASVILRDYKQSSSTL1199 aa1158 aa133 kDa130 kDa150 kDaConsensusP+VTYASVILRDYKQSSSTLZebrafishPSVTYASVILRDYKQSSSTLSimilar to bifunctional mGluR forextracellular Ca and Glu, partial(XP_001346460a)SourcePNVTYASVILRDYKQSSSTL271 aa30 kDaConsensusP+VTYASVILRDYKQSSSTLZebrafishPSVTYASVILRDYKQSSSTLRat mGluR2(EDL77281)Hypothetical protein (previousmGluR2 precursor-XP_692887)SourceNGREVVDSTTSSL872 aa874 aa96 kDa97 kDa100 and230 kDaConsensusNGREVVDSTTSSLZebrafishNGREVVDSTTSSLHypothetical protein (XP_001341692)SourceNGREVVDSTTSSL754 aa85 kDaConsensusNGREVVDSTTSSLZebrafishNGREVVDSTTSSLNovel protein similar to vertebratemGluR3 (CAM56446)SourceNGREVVDSTTSSL884 aa98 kDaConsensusNGRE+VDSTTSSLZebrafishNGREIVDSTTSSLHuman mGluR7(AAB51763)Similar to GluR (previously similar tomGluR7-XP_697214)SourceNSPAAKKKYVSYNN915 aa668 aa102 kDa75 kDa50 kDaConsensusNSPAAKKKYVSYN+ZebrafishNSPAAKKKYVSYNDRat mGluR5(NP_058708)Similar to bifunctional mGluR forextracellular Ca and Glu (previouslysimilar to mGluR1-XP_690446a)SourceLIIRDYTQSSSSL1171 aa1158 aa128 kDa130 kDa60 and100 kDaConsensus+I+RDY QSSS+LZebrafishVILRDYKQSSSTLSimilar to bifunctional mGluR forextracellular Ca and Glu, partial(XP_001346460a)SourceLIIRDYTQSSSSL271 aa30 kDaConsensus+I+RDY QSSS+LZebrafishVILRDYKQSSSTLSimilar to mGluR5 (XP_696823)SourceLIIRDYTQSSSSL1173 aa130 kDaConsensusL++R Y+QSSSSLZebrafishLVLRHYSQSSSSLAccession numbers listed below protein names. Codes in parentheses indicate accession numbers in the NCBI protein database. a Proteins whose DNA coding sequences are almost identical (only 2 nucleotide substitutions).
Light microscopy mGluR1α immunoreactivity in the goldfish retina was concentrated in the OPL (Fig. 2a) and outer half of the INL in which the somata of some bipolar cells were labeled (Fig. 2a, arrowheads). A confocal examination of the immunofluorescence pattern yielded by this antibody revealed diffuse labeling in the INL, inner plexiform layer (IPL), and ganglion cell layer (GCL; Fig. 2d). Red and green images of a confocal view were taken from a retinal slice double-labeled for mGluR1α (Fig. 2d) and PKC (Fig. 2e); the yellow areas in the merged image (Fig. 2f) indicated the strong colocalization in ON BC processes in the OPL. However, mGluR1α immunoreactivity in this layer was not restricted to PKC-positive processes. Furthermore, the small PKC-positive ON BCs were not positive for mGluR1α (Fig. 2d,e, asterisks). Conversely, the mGluR1α-antibody labeled structures (presumably amacrine cell somata) in the inner border of the INL that were not PKC-positive (Fig. 2d,e, circles).
Electron microscopy: rod synapses In the rod spherule, mGluR1α immunoreactivity was present at invaginating dendrites in close apposition to the synaptic ribbons (Fig. 3a–d, arrowheads). These processes were elongated (Fig. 3a), ellipsoidal (Fig. 3b), goblet-shaped (Fig. 3c), or round (Fig. 3d), consistent with ON MBC dendrites. Roughly two invaginating dendrites were stained per rod spherule; the remaining invaginating dendrites (Fig. 3a,b, asterisks) were not labeled. These were possibly OFF MBCs, rod-driven HCs, or a subtype of ON MBC negative for mGluR1α.
Fig. 3Ultrastructural localization of mGluR1α in the OPL. In the rod terminals (a–d), ON MBC invaginating processes at the summit of the synaptic ribbon (SR) were positively labeled (arrowheads). Some invaginating processes were not labeled (asterisks); these might belong to OFF MBCs, to rod-driven HCs, or to a subtype of ON MBC negative for mGluR1α. Occasionally, a laterally positioned dendrite showed mGluR1α immunoreactivity (thick arrow in d). In the cone pedicles (e, f), mGluR1α immunoreactivity was found in small invaginating profiles that lay at the base of the pedicles or close to the ribbon (arrowheads) and that might have originated in BCs. In addition, a HC dendrite occupying the lateral position of the triads was positively labeled (thick arrows in e), whereas the other lateral element was unlabeled (arrows). The antibody also labeled HC spinules in the cone terminals (thick arrows in f). Bar 0.25 μmThe antibody also labeled single lateral elements facing the synaptic ribbon (Fig. 3d, thick arrow). Whenever a lateral element was labeled, the other one was unlabeled (Fig. 3d, arrow). Although processes in the lateral position are classically attributed as belonging to HCs (Stell 1967, 1976), we could not fully exclude the possibility that some of the labeled processes were aberrant ON MBC dendrites.
Electron microscopy: cone synapses In the cone pedicles, various types of post-synaptic structures were labeled. In addition to small invaginating processes (Fig. 3e,f, arrowheads), which could belong to BCs, mGluR1α immunoreactivity was also present at one lateral dendrite of some cone triads (Fig. 3e, thick arrows). The position and fine structure of these dendrites were consistent with those of cone-driven HCs (Stell 1967, 1976; Sakai and Naka 1983). When one lateral HC dendrite in a triad was positive for mGluR1α, the opposite HC dendrite was not (Fig. 3e, thick and thin arrows, respectively). A few invaginations in the cone pedicle containing discontinuous patches of electron-dense material in the membrane and resembling HC spinules (Wagner 1980) exhibited mGluR1α immunoreactivity (Fig. 3f, thick arrows).
Immunolabeling for mGluR2/3
Western blotting The antibody against mGluR2/3 reacted with proteins of approximately 100 kDa in goldfish brain, rat retina, and rat cerebrum (Fig. 4c). This is the predicted molecular mass of mGluR2 and mGluR3 (Tables 1, 2). In the goldfish retina, only a protein at about 200 kDa was detected, which also appeared in the other three tissue samples. This probably represents receptor dimers (Poli et al. 1999), which occur naturally in many G-protein-coupled receptors, including mGluRs (Bouvier 2001; Kubo and Tateyama 2005; Pin et al. 2005; Poli et al. 2003; Beraudi et al. 2007). In goldfish and rat retinas, a smaller band at around 40 kDa was detected.
Fig. 4Immunolabeling for mGluR2/3 (ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer). a Immunoreactivity was seen as tiny bands in the OPL (thick arrows), irregular patches in the INL, and diffuse labeling in the IPL. b Preadsorption with the control peptide (pread) abolished all labeling. c Western blot of total protein from goldfish retina (ret), goldfish brain (br), rat retina (ret), and rat cerebrum (cer). A protein at around 100 kDa, close to the predicted molecular mass of mGluR2 and mGluR3, was detected in goldfish brain, rat retina, and rat cerebrum. The antibody also detected a protein at 200 kDa (probably receptor dimers) in all four tissues. d, eRed and green channel confocal images of the same tissue section double-labeled with antibodies against mGluR2/3 (Cy3, d) and PKC (Alexa, e). f Merged image of the double-labeling experiment. No colocalization with ON BCs was found. Bars 10 μm. g, h In a few cone pedicles, mGluR2/3 immunoreactivity was found in HC central and lateral elements of the cone triad (thick arrows). Note the unlabeled lateral processes (thin arrows) and synaptic ribbons (SR). Bars 0.5 μm
Light microscopy Immunocytochemistry with the mGluR2/3 antibody revealed signal in the OPL and INL and strong labeling in the IPL. Tiny discontinuous immunoreactive bands were visible in the OPL (Fig. 4a, thick arrows). Confocal images showed a similar pattern, with a slightly stronger OPL labeling (Fig. 4d, thick arrows). Clearly labeled somata could not be distinguished, although immunolabeling in the INL was concentrated in patches. Immunoreactivity for PKC (Fig. 4e) did not colocalize with mGluR2/3 (Fig. 4d), as can be seen by the absence of yellow areas in the merged confocal image (Fig. 4f).
Electron microscopy: rod synapses No immunoreactive processes could be identified in the rod synaptic terminals.
Electron microscopy: cone synapses HC dendrites occupying both the lateral and central position were positive for mGluR2/3 in only a few cone pedicles (Fig. 4g–h, thick arrows). The fine structure of the central processes was consistent with that of HC dendrites because of their large dimensions compared with BC dendrites, cytoplasm devoid of organelles, some electron-dense amorphous material applied to the cytoplasmic surface, and concavity of the surface facing the synaptic ribbon (Stell 1967, 1976; Scholes 1975). No mGluR2/3 immunoreactivity was seen in structures resembling BC dendrites, in agreement with our light-microscopical findings.
Immunolabeling for mGluR5
Western blotting Immunoblots for mGluR5 revealed a protein at around 100 kDa in goldfish retina and brain and in rat cerebrum (Fig. 5c). An additional band at 60 kDa was detected in the goldfish samples and in the rat retina. A heavier protein of around 145 kDa was detected in rat retina and cerebrum, roughly corresponding to the deduced molecular mass of rat mGluR5 (Koulen et al. 1997).
Fig. 5Immunolabeling for mGluR5 (ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer). a Immunoreactivity was concentrated in the ONL (open arrows), with some granular labeling in the OPL, INL, and IPL (thick arrow labeled HC body). b Preadsorption with the control peptide (pread) abolished all labeling. c Western blot of total protein from goldfish retina (ret), goldfish brain (br), rat retina (ret), and rat cerebrum (cer). A protein around 100 kDa was detected in the goldfish tissue and rat brain. The antibody also a smaller band (60 kDa) in the goldfish samples and in the rat retina, and a heavier band (145 kDa) in the rat retina and cerebrum. d, eRed and green channel confocal images of the same tissue section double-labeled with antibodies against mGluR5 (Cy3, d) and PKC (Alexa, e). f Merged image of the double-labeling experiment, showing no colocalization. Bars 10 μm. g Labeling is restricted to processes ensheathing photoreceptor terminals in the ONL (open arrows). h A similar labeling pattern (open arrows) is obtained with the monoclonal antibody against glial fibrilary acidic protein (GFAP; clone G-A-5, Sigma-Aldrich; 1:400), which labels exclusively Müller cells (SR synaptic ribbon). Bars 0.5 μm
Light microscopy The mGluR5 antibody labeled vertical bands at the outer nuclear layer (ONL) in both conventional (Fig. 5a, open arrows) and confocal microscopy (Fig. 5d, open arrows) and yielded granular labeling in the OPL, INL, and IPL. Some HC somata were positively labeled at the outer margin of the INL (Fig. 5a,d, thick arrows). There was no colocalization with PKC (Fig. 5d–f).
Electron microscopy Ultrastructurally, mGluR5 immunoreactivity was seen in longitudinally oriented structures between photoreceptor terminals (Fig. 5g, open arrows). This pattern was consistent with previous reports concerning the localization and ultrastructural appearance of Müller cell processes (Villegas 1960; Lasansky 1961; Pedler 1963; Miller and Dowling 1970). Furthermore, it resembled closely that of immunolabeling for glial fibrilary acidic protein (Fig. 5h, open arrows), which is exclusively expressed by glial cells (Bignami and Dahl 1977; Liepe et al. 1994).
Immunolabeling for mGluR7
Western blotting A protein of approximately 50 kDa was labeled in goldfish and rat samples, with an additional band of around 75 kDa in the goldfish brain. In the rat retina, a protein of 200 kDa was also recognized. A clear band could not be distinguished in rat cerebrum, although there was a larger precipitation around 50 kDa (Fig. 6c), at some distance from the value reported by the producer (Table 1).
Fig. 6Immunolabeling for mGluR7 (ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer). a Immunoreactivity was concentrated in the OPL (arrowheads), with some granular labeling in the INL, IPL, and GCL. b Preadsorption with the control peptide (pread) abolished all labeling. c Western blot of total protein from goldfish retina (ret), goldfish brain (br), rat retina (ret), and rat cerebrum (cer). A pronounced band at around 50 kDa was produced in all four samples. Additional immunoreactive proteins were found at 75 kDa in the goldfish tissue and at 200 kDa in the rat retina. d, eRed and green channel confocal images of the same tissue section double-labeled with antibodies against mGluR7 (Cy3, d) and PKC (Alexa, e). A BC soma (arrowheads) and an HC soma (thick arrows) were positive for mGluR7 but negative for PKC. f Merged image of the double-labeling experiment, showing no colocalization in ON BCs. g In rod spherules, lateral elements were labeled (thick arrows). h In the cone pedicles, small profiles not related to the synaptic ribbons (SR) were positive (arrowheads), as were lateral elements of the triads (thick arrow). Note the unlabeled lateral elements (thin arrows). Bars 0.25 μm
Light microscopy The antibody against mGluR7 immunoreactivity yielded strong labeling in the OPL (Fig. 6a, arrowheads) and granular labeling at the INL, IPL, and GCL. Occasionally, positive amacrine and ganglion cell somata were seen. Confocal images of double-labeling with PKC (Fig. 6d–f) did not show colocalization: mGluR7-positive areas were concentrated close to the outer border of the INL, immediately below the band of PKC-positive ON BC dendrites. Some mGluR7-positive somata that were not labeled for PKC could be identified in the outer half of the INL; their form and position was consistent with that of HCs (Fig. 6d,e, thick arrow) and OFF BCs (Fig. 6d,e, arrowhead).
Electron microscopy: rod synapses Lateral elements of the rod triads were labeled, consistent with the labeling of putative HCs reported above (Fig. 6g, thick arrows). We found no evidence of MBC labeling in rod spherules.
Electron microscopy: cone synapses A few small processes away from the ribbons were labeled by the mGluR7 antibody (Fig. 6h, arrowheads). Their small profiles resembled those of BCs. This observation, added to the finding that the antibody labeled PKC-negative BC somata (Fig. 6d,e, arrow), suggested that these were OFF BC profiles. Lateral elements (Fig. 6h, thick arrow) and spinules were also labeled, consistent with HC labeling at the light-microscopical level (Fig. 6d,e, thick arrows). These labeled HC processes were however rare; most of the lateral elements were not positive (Fig. 6h, thin arrows).
Discussion
Summarizing scheme
This study shows that mGluRs of all three groups can be found in the goldfish OPL. The main findings are summarized in Fig. 7. Cell types have been identified based on the results obtained at both light-microscopy and electron-microscopy levels.
Fig. 7Representation of a rod spherule, a Müller cell process, and a cone pedicle, depicting the localization of PKC and the patterns yielded by the various mGluR antibodies (red group I mGluRs, green group II mGluRs, blue group III mGluRs, SR synaptic ribbon)
In the rod spherule, PKC labels invaginating dendrites of putative ON MBCs. The mGluR1α antibody labels similar structures and some lateral elements. Lateral elements of rod triads are also positive for mGluR7. Müller cell processes between the rod spherules are positive for mGluR5. We have found no evidence for mGluR2/3 in the rod synaptic complex.
In the cone pedicles, PKC labels fine dendrites at the base of the terminal or invaginating structures away from the ribbons. Elements at similar positions and with similar fine structure are positive for mGluR1α and mGluR7. BC central processes in the cone triads are positive for mGluR1α. Spinules are positive for mGluR1α and mGluR7, and lateral elements are labeled by the mGluR1α, mGluR2/3, and mGluR7 antibodies. Finally, HC central elements are positive for mGluR2/3.
Specificity of the antibodies
No data are available on the amino acid and/or nucleotide sequences of goldfish mGluRs. The closest available information is from the zebrafish. This database is however not fully completed, and the various zebrafish mGluRs have not been unambiguously identified (Table 2). The finding of candidates for zebrafish mGluR2 and mGluR6 has been reported, but the actual nucleotide and amino acid sequences have not been disclosed (Bjarnadottir et al. 2005).
We therefore used multiple data sources to infer the specificity of the antibodies used in this study: Western blots and BLAST searches in the zebrafish genome for the amino acid sequences in Table 1 (at http://www.ncbi.nlm.nih.gov/BLAST/), the sequence alignments (with http://www.ch.embnet.org/software/LALIGN_form.html and http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html), and the molecular mass calculations (at http://www.basic.northwestern.edu/biotools/proteincalc.html). We also “BLASTed” the proteins retrieved from the zebrafish database against all other species available, in order to check for protein similarities among species.
To choose good candidates for fish mGluRs among the BLAST hits, we relied on the scores and E-values; the cutoff was E<0.01. We adopted a stringent significance level, because the chance of obtaining random matches with short amino acid sequences was relatively large. All scores used for these comparisons were higher than 1,000. For the C-terminal mGluR5 peptide, the cutoff was lowered to E=0.35, since no candidates could be found with the same stringency as for the remainder of the peptide sequences.
As summarized in Table 2, all of the anti-mGluR antibodies used in this study were raised against polypeptides that yielded mGluR-like hits when “BLASTed” against the zebrafish database. Two homologs were found for mGluR1, but one of these (XP_001346460) was an incomplete sequence that might have corresponded to a fragment of the other (XP_690446), since they only differed by one amino acid and their respective coding sequences by two nucleotides. For mGluR2/3, three putative mGluRs were identified (Table 2), but one of them had a slightly smaller molecular mass than the other two (85 kDa). We did not however detect multiple proteins around 80–100 kDa in our immunoblots, which suggested that XP_001341692 was not present in the goldfish retina.
According to the database search, the C-terminal peptides of mGluR5 and mGluR7 have one fish homolog each. The mGluR5 candidate shows lowest amino acid sequence similarity at the C-terminus, but the lack of overlapping expression patterns between mGluR1 and mGluR5 in the OPL of the goldfish retina excludes cross-reactivity of anti-mGluR5 and mGluR1.
In summary, we feel confident about the specificity of the commercially available anti-mGluR peptide antibodies, since the carboxytermini of mammalian and fish mGluR homologs are nearly identical, and the molecular masses of fish and mammalian homologs are similar (Table 2).
mGluRs of Müller cells
The finding of mGluR5 in Müller cells is in agreement with two previous studies of isolated Müller cells from the salamander; these studies have shown that activation of group I mGluRs has two major effects. First, it leads to the closure of the K+ conductance of these cells, which is by far the largest in Müller cells (Schwartz 1993). Since their glutamate uptake is both voltage-dependent (Brew and Attwell 1987; Schwartz and Tachibana 1990) and regulated by the internal K+ concentration (Barbour et al. 1988), one can imagine that mGluR5, by modulating this K+ conductance, indirectly modulates glutamate uptake by changing the membrane potential and/or internal K+ concentration.
The second effect of group I mGluR activation in Müller cells is the generation of Ca2+ waves by triggering the release of Ca2+ from internal stores (Keirstead and Miller 1997). The physiological consequences of this effect are not completely clear, but they could contribute to lateral signaling among Müller cells, as in astrocytes (Kim et al. 1994).
mGluRs of HCs
Here, we show that rod and cone-driven HCs of the goldfish express mGluRs from all three groups, although the exact mGluR composition of these cells differs slightly from reports in other species (i.e., no mGluR1 in cat HCs: Cai and Pourcho 1999; no mGluR2/3 in catfish HCs: Gafka et al. 1999).
We have found that lateral elements in the rod triads are positively labeled for mGluR1α and mGluR7. Since the labeling for mGluR1α is rare, we cannot at this point positively identify these processes as belonging to either rod-driven HCs or MBCs. In the cone pedicle, mGluRs seem to be expressed in a highly organized manner. mGluR1α and mGluR7 tend to be localized to spinules and to dendrites occupying the lateral position in the cone triad, whereas mGluR2/3 has been found at both lateral and central processes directly opposing the synaptic ribbon.
This extremely ordered pattern indicates that either a single cone-driven HC type expresses distinct mGluRs in different dendrites, or that the expression of a given mGluR is restricted to a certain subpopulation of HCs (i.e., the distribution is cell-type-specific). In cyprinid retinae, three physiological and morphological HC types have been described (MacNichol and Svaetichin 1958; Norton et al. 1968; Stell et al. 1982); one could easily imagine that each HC class expresses only one or two mGluRs at their dendritic tips.
The light responses of HCs are mediated by AMPA (2-amino-4-phosphonobutyric acid)/kainic acid receptors (Lasater and Dowling 1982; Slaughter and Miller 1983; Zhou et al. 1993) and are not abolished by mGluR agonists or antagonists (Yang and Wu 1989; Luo and Liang 2003), showing that mGluRs are not implicated in the direct generation of light responses. They are most likely involved in the modulation of the light-driven conductance of these cells. In the fish retina, for instance, mGluR agonists have been shown to modify both light-modulated (Nawy et al. 1989; Yasui et al. 1990; Takahashi and Copenhagen 1992) and voltage-gated conductances of cone-driven HCs (Dixon and Copenhagen 1997; Linn and Gafka 1999; Gafka et al. 1999).
mGluRs of ON MBCs
The labeling of ON MBC processes, in the rod spherule, with the antibody against mGluR1α corroborates previous studies in fish (Klooster et al. 2001; Yazulla and Studholme 2001; Yazulla et al. 2001) and mammalian (Koulen et al. 1997) species, although it differs from the situation in the cat retina, in which mGluR1α is pre-synaptic to rod-driven BCs (Cai and Pourcho 1999). Since no evidence has as yet been presented for the participation of group I mGluRs in the generation of the light responses of ON BCs, one can only speculate that this protein plays a modulatory role in the first synapse. In many parts of the central nervous system, group I mGluRs are involved in controlling the intracellular calcium concentration and membrane channel activity (for a review, see Fagni et al. 2000). In the spinal cord neurons of the lamprey, for instance, mGluR1 has been shown to regulate the resting membrane potential and thus the excitability of the cells by controlling a leak current (Kettunen et al. 2003).
Although there is physiological evidence for the existence of a group III mGluR in fish ON MBCs (Shiells et al. 1981; Nawy and Copenhagen 1987; Kamermans et al. 2004), we have not been successful in obtaining specific immunoreactivity with the antibodies commercially available against (mammalian) mGluR4, mGluR6, and mGluR8. Furthermore, there is as yet no candidate for mGluR6 in the zebrafish database, which makes the production of custom-made antibodies against this protein difficult. One would however expect these cells to express an mGluR6-like receptor, given the many physiological similarities between the rod-driven conductance in fish and mammalian ON BCs.
Whether and in what manner this mGluR6-like receptor and the mGluR1α-like protein interact in ON MBCs is still to be resolved. G-protein-coupled receptors can interact at many levels. For instance, gamma-aminobutyric acidB receptors form complexes with mGluR1α dimers and thereby regulate the sensitivity of the latter to glutamate (for a review, see Kubo and Tateyama 2005). Interactions between mGluRs, however, have so far not been described. Another level of interaction lies within the second-messenger cascade. Until recently, only one G-protein (Goα) had been described in ON BCs (Vardi et al. 1993), which suggests that mGluR1α and mGluR6 share (part of) the same intracellular machinery. Nonetheless, a second G-protein (Gi2α) has been shown to coexist with Goα in rat ON BCs and not to couple to mGluR6 (Tian and Kammermeier 2006), providing an independent G-protein signaling pathway within these cells; this pathway might involve mGluR1α.

metabotropic glutamate receptor
retina
immunohistochemistry
bipolar cell
horizontal cell
electron microscopy
goldfish, carassius auratus (teleostei)