Promising Practices in Preconception Care for Women at Risk for Poor Health and Pregnancy Outcomes

Objectives: Two programs targeting urban African-American women are presented as promising models for preconception care, which includes interconception care. Methods: The Grady Memorial Hospital Interpregnancy Care (IPC) Program in Atlanta, Georgia, and the Magnolia Project in Jacksonville, Florida, are described. The IPC program aims to investigate whether IPC can improve the health status, pregnancy planning and child spacing of women at risk of recurrent very low birthweight (VLBW). The Magnolia Project aims to reduce key risks in women of childbearing age, such as lack of family planning and repeat sexually transmitted diseases (STDs), through its case management activities. Results: Seven out of 21 women in the IPC were identified as having a previously unrecognized or poorly managed chronic disease. 21/21 women developed a reproductive plan for themselves, and none of the 21 women became pregnant within nine months following the birth of their VLBW baby. The Magnolia Project had a success rate of greater than 70% in resolving the key risks (lack of family planning, repeat STDs) among case management participants. The black to white infant mortality (IM) ratio was better for the babies born to women managed in the Magnolia Project compared to the same ration for the United States. Conclusions: Preconception care targeted to African-American women at risk for poor birth outcomes appears to be effective when specific risk factors are identified and interventions are appropriate. Outreach to women at risk and case management can be effective in optimizing the woman's health and subsequent reproductive health outcomes.
Introduction
Current national level statistics demonstrate the potential for preconception care to improve the health of women and impact their reproductive health outcomes in terms of achieving optimally spaced, planned pregnancies and averting adverse birth outcomes. Each year in the United States, approximately 6 million pregnancies result in over 4 million live births. Almost half of all pregnancies are unintended, despite the fact that 62% of the 62 million women of reproductive age use a contraceptive method [1]. Chronic diseases and adverse health behavior known to affect pregnancy outcomes are prevalent among women of reproductive age (15–44 years). In 2002, 9.3% of women of reproductive age had diabetes, and 11.4% of pregnant women smoked tobacco and 10.1% of pregnant women consumed alcohol [2].
The U.S. Public Health Service has designated preconception care as a critical component of prenatal care [3]. Initiating interventions to address a woman's health risks upon her entry into prenatal care is often too late to impact the outcome of pregnancy. For example, if a woman has her first prenatal visit after the first month of pregnancy, it is too late to prescribe folic acid supplementation to help prevent neural tube defects as the neural tube closes by the 28th day of gestation. Likewise, women who are advised to discontinue their use of potentially teratogenic medications at the first prenatal visit may have already sustained damage to the fetus, as most organogenesis is complete by day 56 following conception [4].
Preconception care is the care of women of reproductive age before a first pregnancy or between pregnancies (commonly known as interconception care) to ensure that conditions and behaviors which may pose a risk to mothers and infants are identified and managed. A specific element of interconception care is the identification and reduction of risks indicated by a prior adverse pregnancy outcome. The major components of preconception care are risk assessment (identification of conditions that may be amenable to intervention that adversely affect reproductive health outcomes), education and health promotion, and medical and psychosocial interventions.
There are varying concepts of preconception care ranging from a pre-pregnancy planning visit in the months prior to conception to a component of each health encounter for women. Ideally, preconception care would be an integral component of primary and preventive care for women of reproductive age. Presently, the status of preconception care in the United States is far from ideal. Approximately one of six obstetrician/gynecologists or family physicians had provided preconception care to the majority of the women for whom they provided prenatal care [5].
Given the diversity of women of reproductive age, the numerous health and psychosocial issues that impact reproductive health outcomes and the varied settings for delivery of health care, a single ‘best’ model of preconception care for all women is unlikely. Rather, tailoring the delivery of preconception care interventions to women with specific circumstances and in specific care settings will be necessary. This paper describes two programs for delivering preconception care for women at-risk for poor health and pregnancy outcomes. One targets women whose risk status is defined by African-American ethnicity and delivery of a very low birth weight (VLBW; <1500 g) infant and, thus, specifically involves the provision of an interconception intervention. The other program targets women whose risk status is defined by African-American ethnicity and residence in Jacksonville-Duvall County, Florida, and involves the delivery of an intervention with preconception and interconception components.
The grady memorial hospital Interpregnancy Care (IPC) program
Background
Georgia ranks among the 10 states with the highest feto-infant mortality rate. The largest contributor to Georgia's comparatively high feto-infant mortality is the delivery of VLBW infants. VLBW delivery comprises only 2% of births in Georgia, yet accounts for approximately 50% of feto-infant mortality. African-American women in Georgia have 3–4 times the rate of VLBW delivery compared to whites. Approximately 66% of the observed racial disparity in feto-infant mortality rates between African-Americans and whites can be attributed to the higher rate of VLBW delivery among African-American women [6].
There is a close link between the poor health status of women and VLBW delivery [7–17]. The poor health status of women in Georgia is estimated to account for approximately 60% of Georgia's excessive infant mortality [18]. With each subsequent VLBW delivery, there is an increased chance that the woman's next pregnancy will result in recurrence of VLBW delivery [19]. In fact, the best predictor of whether a woman will have a VLBW delivery is her history of a previous VLBW delivery, with the rates of recurrence after a single VLBW delivery being approximately 1.5 times higher for African-American compared to white women (13.4% vs. 8.2%, respectively, for non-teen mothers; 26.8% vs. 16.4%, respectively, for teen mothers). The reason for recurrence of VLBW delivery is likely that aspects of the woman's pre-existing health status—including untreated or poorly managed medical problems and unaddressed nutritional, social, and behavioral risk factors—that may have contributed to delivery of the VLBW delivery persist after delivery and in subsequent pregnancies.
In 1998 the Georgia Task Force on Perinatal Care was convened to make recommendations for reducing Georgia's overall feto-infant mortality rate and racial disparities in feto-infant mortality. From its findings as outlined above, the Task Force specifically recommended that interpregnancy care be initiated and evaluated for women at risk for having recurrence of VLBW delivery. The Grady Memorial Hospital Interpregnancy Care (IPC) Program was initiated in response to the recommendation of the Task Force.
Target population
African-American women residing in Fulton or DeKalb counties, Georgia who qualify for county-supported indigent care services and who deliver liveborn or stillborn VLBW infants at Grady Memorial Hospital in Atlanta are considered eligible for the IPC program.
Program description
The Grady Memorial Hospital IPC program provides 24 months of integrated primary health care and dental services through enhanced nurse case management and community outreach via a Resource Mother. The Resource Mother is a layperson who is trained by the Fulton County Health Department using a specific set of objectives to facilitate life skills and health education acquisition to support high-risk women. The nurse case manager offers all women who deliver a VLBW (stillborn or liveborn) infant at Grady Memorial Hospital enrollment in the program during or soon after their delivery admission. Women who choose to enroll in the IPC program have their initial home visit with the Resource Mother within 1–2 weeks of discharge from the delivery admission; they are scheduled for their initial IPC clinical evaluation at a clinic within Grady Memorial Hospital at 4–6 weeks postpartum.
At the initial IPC clinical evaluation, the IPC program's family physician or nurse- midwife performs a comprehensive survey of medical, obstetrical, nutritional, psychological, and social issues (using standardized assessment tools); a thorough physical examination including pelvic exam; and laboratory evaluations to screen for anemia, nutritional deficiencies, sexually transmitted diseases (STDs) and reproductive tract infections. Standard postpartum care issues are addressed in the initial IPC clinical evaluation, thus eliminating a separate postpartum appointment.
As part of the evaluation, the participant and provider explicitly discuss a care plan for the 24-month period of the program. For each woman, the care plan addresses the following seven areas epidemiologically linked to LBW delivery: 1) Pregnancy intendedness and child-spacing through the provision of health education concerning the importance of achieving at least a 9-month (and preferably an 18-month) interpregnancy interval, assisting the woman to articulate her own reproductive plan and select a corresponding contraceptive method; 2) Management of chronic disease (if present) through the promotion of self-care and adherence to scheduled appointments in Grady Health System that are facilitated by nurse case management; 3) Screening and treatment for nutritional deficiencies; 4) Prevention, screening, and treatment for STDs and reproductive tract infections; 5) Treatment and referral for substance abuse (if present) including linkage with rehabilitation programs for illicit substance abuse, and support in and linkage with existing programs for tobacco and alcohol abuse; 6) Screening and treatment or support for depression, psychosocial stressors, and domestic violence; 7) Prevention, screening and treatment for periodontal disease.
Subsequent visits to the IPC clinic are offered every 1–3 months, depending upon the extent and severity of the woman's health and social issues. Elements of the care plan are addressed at each subsequent visit to the IPC clinic. Peer group learning experiences are integrated with IPC health care visits and are modeled on the Centering Pregnancy concept of group prenatal care [20].
Resource Mother support services are focused on identification and management of psychosocial stressors, and life skills enhancement, including parenthood preparedness, safe housing, skills training, employment acquisition, and relationship issues. Resource Mother support is offered at least twice monthly in the form of home visits and telephone contact. Participants are able to contact the Resource Mother via her cell phone to request support, if needed.
Providers
Primary care and outreach services are delivered by a multidisciplinary team comprised of a family physician, an advanced practice nurse trained as both a nurse midwife and a family nurse practitioner, a periodontist, a nurse case manager, and a Resource Mother. Social workers affiliated with Grady Health System also support the IPC program and interface with the IPC team.
Outcome objectives
The purpose of the Grady Memorial Hospital IPC program is to investigate whether interpregnancy care can improve the health status, pregnancy planning and child spacing of women at risk of recurrent VLBW delivery. Findings will contribute to the field of primary health care of reproductive age women in several important ways: (a) the content of a successful IPC package for improving the health of high-risk women will be explored; (b) the concept of IPC will be tested as a means of improving attainment of desirable interpregnancy intervals and decreasing the occurrence of subsequent adverse pregnancy outcomes for high-risk women; (c) the cost of providing IPC to high-risk women will be studied.
Funding
The health care services rendered to IPC participants were provided through the services of the Grady Health System. Funding for the research and evaluation components of the IPC Program, including Vasser-Wooley Foundation, Healthcare Georgia Foundation, Centers for Disease Control and Prevention (CDC), Rockdale Foundation, and March of Dimes.
Results from the first year of the IPC program
Enrollment
During November 2003 through March 2004, the feasibility phase of the IPC program was initiated by enrolling the first sequential 29 eligible women who gave their informed consent for participation. During the enrollment period, a total of 47 women delivered VLBW infants at Grady Memorial Hospital. Of these 47 women, nine were not African-American or did not reside in Fulton or DeKalb counties, four declined enrollment, three who wished to be contacted afterdischarge from the hospital were unable to be contacted, and two were discharged from the hospital within 24 h and before being offered enrollment.
Participation
Twenty-one of 29 enrolled women (72.4%) successfully completed the initial 12 months of the planned 24 months of follow-up. During the first 12 months, eight women became disenrolled from the program two moved out of state (and informed us of their move), three electively disenrolled (two before prior to the initial home visit and the initial IPC clinical evaluation; one after a single IPC clinic visit), and three became lost to follow-up (two before the initial IPC clinical evaluation; one after a single IPC clinic visit and four home visits) all of whom who had noted problems with cocaine abuse in their medical records during their pregnancy.
Outcomes
A detailed program description and final results for all evaluated outcomes for the full 24 months of follow-up will be published when all follow-up and outcome ascertainment is complete (anticipated June 2006). Important findings related to the health status of women in the IPC intervention cohort who had at least two visits during the first 12 months of IPC are given below:Chronic diseases that were previously unrecognized or poorly managed were identified for 7/21 participants, and include valvular heart disease (1/21), sickle cell anemia (1/21), hypertension (2/21), diabetes (1/21), asthma (1/21), systemic lupus erythematosus (1/21), prolactinoma (1/21), panic attacks (1/21), and generalized anxiety disorder (1/21);Reproductive tract infections were diagnosed and treated for 15/21 participants;Iron-deficiency anemia was diagnosed and treated for 5/21 participants;Concerns about finances, employment, and needs of the child are almost universal stressors;Average annual outpatient charges per participant for IPC are $1,801 (average 4.6 visits, $389 per visit).
Important findings related to the reproductive outcomes during the first 12 months of IPC are as follows:With extensive case management and patient education, 21/21 women who had at least two visits during their first 12 months of IPC, stated a reproductive plan for themselves and initiated a contraceptive plan in accordance with their stated reproductive plan. Despite a stated desire to either avoid or delay a pregnancy (21/21 women), a significant number of barriers to effective contraception existed and were dealt with, including misinformation about contraceptive methods and reproduction, concerns about side effects of contraceptive methods, and perceptions of partners’ desires regarding child bearing and contraception.All of the women who had at least two visits during their first 12 months of IPC (21/21) achieved at least a nine-month interpregnancy interval (i.e., none have become pregnancy within nine months of the index VLBW delivery). For the 21 actively participating women, pregnancy ascertainment involved ongoing contact with the women and reviewing the Grady Healthy System medical record system for any evidence of health care visits or laboratory tests in which a pregnancy was diagnosed, incidentally noted, or treated. No pregnancies are known to have occurred within nine months of the index VLBW delivery for the eight women who became disenrolled, for which pregnancy ascertainment involved telephone follow-up, where possible (two women), and review of the Grady Health System medical record system. It is possible that those women with whom contact was lost have had a pregnancy for which no care was sought or care was sought outside of Grady Health System.
Barriers
In the United States, a major barrier to studying the impact of interpregnancy care on the health status and reproductive outcomes of high-risk women has been the lack of financial coverage for the health care component of the IPC intervention. For the IPC program in Atlanta, the Grady Health System has provided the two years of primary health care and treatment of all identified diseases for all enrollees.
Specific barriers exist for retaining participation in the IPC program. Women with substance abuse problems who do not enroll in formal substance abuse treatment programs are difficult to maintain in the IPC program. These women seem to have poor insight into the effects that substance abuse has on their repeated poor pregnancy outcomes. For some enrolled women, receiving health care services is less of a priority than securing employment, which negatively influences their health care seeking behaviors.
Next steps
Upon conclusion of the planned 24 months of follow-up, the final results for all evaluated outcome objectives from the feasibility phase will be reported. The IPC program is currently seeking grant support to conduct a randomized clinical trial to test the hypothesis of whether IPC can decrease the occurrence of subsequent adverse pregnancy outcomes for pregnancies conceived within 18 months of a VLBW delivery at Grady Memorial Hospital. The project plans to enhance participant retention by offering more opportunities for life skills enhancement, job training, and delivery of health care services via the community-based Grady Neighborhood Health Centers.
The magnolia project
Background
In 1998, infant mortality rates in Jacksonville-Duval County, Florida were increasing while statewide rates were declining. The Northeast Florida Healthy Start Coalition, Duval County Health Department and other community partners undertook a community assessment and identified racial disparities as a major factor contributing to the city's high infant mortality. The Coalition utilized the Perinatal Periods of Risk (PPOR) and findings from its Fetal & Infant Mortality Review (FIMR) project to analyze linked birth and death data. The results of this analysis indicated the need for interventions that focused on the health of women, particularly African-American women, before conception. The Coalition used this information to apply for federal Healthy Start funding under a 1999 initiative to address racial disparities in birth outcomes. This funding was used to establish The Magnolia Project, which adapts selected Healthy Start program models to at-risk African-American women of childbearing age who are not pregnant, but sexually active and likely to become pregnant. The project is implemented in a five-zipcode area of Jacksonville-Duval County that accounts for over half of all African-American infant mortality and about 30% of African-American births.
Target population
The Magnolia Project targets African-American women ages 15–44 living in a socioeconomic high-risk area of Jacksonville-Duval County. The project focuses on women of childbearing age who are likely to become pregnant and have some identified risk factor associated with poor birth outcomes. The project also maintains a small caseload of pregnant women (<10% of patients).
Program description
The Magnolia Project provides outreach, case management, risk reduction, support, well-woman prenatal care, health education, and community development. The project engages high-risk women through an empowerment model that promotes improved wellness and health, rather than future childbearing. Clinical services and health education are available to all eligible women in the target area. The project offers intensive case management to a subset of women who have specific risk factors identified through FIMR, including previous fetal or infant death or delivery of a LBW infant; repeated STDs; lack of family planning; substance abuse; first pregnancy before age 15; and lack of access to health care.
The project's intensive case management is a collaborative team approach that supports participants based on a comprehensive assessment and a client care and goal plan. The plan is developed in collaboration with each participant and reflects her choices, preferences, and goals for coordination of services and activities that address identified risk factors associated with infant mortality. Case management services include:A comprehensive assessment of each participant's abilities and needs at program entry. This assessment includes completion of a problem checklist; screening for depression and stress; and evaluation of self-esteem. Participants are re-assessed periodically based on need and progress to determine resolution or recurrence of problems and risks, achievement of personal goals, and improvements in coping skills and self-esteem.Participant care and goal plan. This plan addresses the needs and preferences of each participant. The written plan is a collaborative process involving the participant and the case manager. It addresses health and social risks and outlines specific steps that the participant must follow to improve her health and well-being and to meet her goals for social, economic, and personal development.Ongoing monitoring and service coordination. The case management team monitors services to ensure that quality care is being provided, participants are addressing their care plans, and participants who are referred to another agency follow through and receive appropriate services. The risk profile (problem checklist) and other measures (stress, self-esteem) are updated at regular intervals (at 6 and 9 months).Anticipatory guidance, health education and advocacy. Case management staff addresses participant risk factors, ensures compliance with services, and promotes achievement of personal goals. Educational support groups are organized monthly. Women receive an incentive ($50 gift certificate) for each six months that they remain in the program, provided they have made progress in addressing their goals.
Providers
The clinical component of the program is staffed by a nurse-midwife and related nursing, lab and support staff. The program has a full-time health educator who provides a brief counseling intervention on key issues (e.g., use of multivitamins, nutrition, douching, and safe sex) to every woman who comes into the clinic. The clinic serves approximately 800 women annually. A team that includes a nurse, a social worker and two specially trained paraprofessionals provides intensive case management to about 75 women a year. Community development and outreach staff, as well as members of the Magnolia Project Community Council, who are indigenous neighborhood leaders, conduct outreach and community awareness activities. The project uses a collaborative, multi-agency staffing model. The Northeast Florida Healthy Start Coalition is the grantee and project administrator. Subcontractors include the Duval County Health Department (clinical care and case management) and Shands Jacksonville, a tertiary care and teaching hospital (case management and outreach). Staff is co-located at a storefront community site within walking distance of six public housing complexes. The University of North Florida Center for Community Initiatives serves as project evaluator.
Outcome objectives
The project addresses all the outcome objectives required by the Maternal and Child Health Bureau of the Health Resources and Services Administration (HRSA) regarding birth outcomes (LBW, VLBW, infant mortality, adequacy of prenatal care, etc.). However, because the project primarily focuses on women who are not pregnant, it has developed specific outcome objectives that examine its success in reducing key risks associated with poor outcomes through its case management activities. These include lack of family planning and STDs. Specific outcome objectives have been developed to measure the program's success in addressing participant risks identified at program entry and closure.
Funding
The Magnolia Project is primarily funded through a grant from the federal Healthy Start program ($925,000). Additional funding is provided through Medicaid and other third- party reimbursement for prenatal, family planning and STD services; state categorical funding through the health department (about $250,000 a year); and smaller community grants for special projects. Total 2004–2005 budget was just over $1 million.
Results to date
From 2001 to 2005, Magnolia Project clients were predominantly African-American (83%) and single (82%). The average age of participants was 25 years old. More than half (55%) had less than a high school education. Based on patient history and assessment, case management participants had an average of 7.9 risk factors or problems (current or history of) that could potentially impact a pregnancy. Seventy-two percent of all current problems were social problems (alcohol use, drug abuse, depression, family planning, housing, stress, and domestic violence). The remainder were medical problems (anemia, diabetes, hypertension, poor nutrition, previous poor outcome, STDs). The most common risk factors addressed in case management were family planning issues, education/training, bacterial vaginosis, repeated STDs, stress, and poor nutrition. Table 1 identifies frequency of participant risks/problems.Table 1Frequency of risks/problems, case management participants, The Magnolia Project, July 2001–May 2005 Risk factor/problemPercentage with history (n=1214)Percentage with current (n=1727)Social problems Family planning issues2643.5 Job placement8.233.0 Education/training11.327.3 Stress8.721.7 Housing4.115.7 Domestic violence7.211.3 Depression8.211.1 No source of care6.78.7 Lack of exercise7.08.7 Injury prevention/safety2.88.0 Drug abuse10.06.7 Transportation4.45.9 Sexual abuse7.04.4 Alcohol abuse6.44.1Medical problems Bacterial vaginosis12.930.2 Poor nutrition17.819.3 Repeated STDs12.416.5 Tobacco use13.215.5 Breast health3.911.3 Douching8.510.8 Abnormal pap9.59.5 Overweight11.69.5Source: Magnolia Project database; compiled by UNF Center for Community Initiatives, August 2005.
Two-hundred-forty-seven participants received at least three months of case management and were closed to care because they completed their care plans, voluntarily withdrew or were lost to service. For key risk factors, case management participants were most successful in resolving or managing issues related to domestic violence (68%) and poor nutrition (63%). The project was least successful in addressing substance abuse by participants (31%).
In October 2004, the HRSA Office of Performance Review conducted a site visit and assessment of Magnolia Project outcomes and activities. Its report noted that participants receiving case management services from 2001 to 2003 successfully addressed two priority risks: 86% of participants with family planning issues were consistently using a method at closure; 74% of participants with repeated STDs had no recurrent STDs at closure.
Barriers
Insufficient funding has limited expansion of the Magnolia Project. Existing funding streams focus primarily on pregnancy, not women's health. The Magnolia Project has successfully cobbled together categorical funding and Medicaid reimbursement for most clinical services, however, in Duval county, the delivery of most categorically funded services remains siloed (e.g., STDs, primary care, prenatal care).
Additionally, case management/risk assessment is not universally accepted or valued in the medical model of health care, even though many of the risk factors associated with poor outcomes are social, rather than medical, in nature. More longitudinal evaluations are needed to determine the impact of preconception intervention on pregnancy outcomes. Longitudinal evaluations are not easily incorporated into service delivery projects that rely on short-term outcomes for continued funding. Finally, although outcomes have improved in the target area since the implementation of the Magnolia Project, infant mortality rates in the overall community remain high. The project is unlikely to affect these rates because of its comparatively small reach.
Next steps
The Magnolia Project is aggressively seeking funding for a longitudinal evaluation. Current program evaluation is limited to an assessment of how well the project is achieving short-term objectives and objectives set by the funding agency (primarily focusing on birth outcomes for the small number of pregnant women served by the project). The CDC recently provided support to the Duval County Health Department for the initial design and piloting of tools for a longitudinal assessment of case management participants served by the project. Additionally, the project is working to interest local providers in replicating the intervention in other at-risk areas in Jacksonville-Duval County. Funding for replication is a significant challenge.
Discussion
The Grady IPC program and the Magnolia project use a classic public health model of care. They identify women at known risk for an adverse outcome and attempt to alter the woman's risk status to improve outcomes. These programs deliver aspects of preconception care to women at risk for poor health and pregnancy outcomes. The programs do, however, target different categories of high-risk African-American women and utilize different sites for contacting and interfacing with them. The IPC program identified women at risk based on race/ethnicity, qualification for charity care (based on financial status and geographic residence in two counties of metropolitan Atlanta) through Grady Memorial Hospital, and a prior poor birth outcome (VLBW delivery). The IPC program enrolled women soon after their VLBW delivery and provided interconception care in the clinical setting of the hospital (with community outreach). The Magnolia project serves women at risk based on race/ethnicity and residence in Jacksonville-Duval County, Florida. It provides preconception care in a community-based storefront setting with enhanced interconception care for women with a previous LBW delivery or a previous fetal or infant death. Access to the Magnolia project is enhanced by offering evening clinics and walk-in Wednesdays.
Both programs deliver services via a multidisciplinary team approach, though there are differences in the exact composition of the team. Inherent in the team approach of each program is attention to participants’ physical and psychological health and social well-being.
The programs emphasize several comparable intervention strategies, including community outreach via lay persons; psychosocial support; group education and health promotion modeled on the Centering Pregnancy philosophy of care; and provision of health services. Both programs also emphasize the provision of family planning services for helping women achieve intendedness of any subsequently conceived pregnancy.
Each program offers unique strategies as part of the interconception intervention. Because of the link between periodontal disease and preterm delivery (almost all VLBW deliveries are preterm), the IPC program incorporates the services of a periodontist to screen and treat women. To specifically enhance particpants’ life skills, the IPC program utilizes a layperson as a Resource Mother who has undergone a tailored training program through the local department of health. A unique feature of the Magnolia Project's approach is its reliance on indigenous neighborhood leaders (e.g., Magnolia Project Community Council) and community-based organizations to provide outreach, community awareness, and education. The Magnolia Project also has established a partnership with the Ryan White III program for STD/HIV/AIDS screening and treatment.
Both programs have had success in accessing and providing services to the specific target group of women. Women who choose not to avail themselves of the services seem to have greater socioeconomic issues, such as substance abuse and lack of housing, jobs, and childcare. Due to the magnitude of some of the problems, maintaining contact is challenging and care often is not continuous.
Existing literature supports the strategy of addressing family planning to improve pregnancy outcomes. Preterm delivery, the underlying factor responsible for most LBW and VLBW deliveries, is the leading cause of neonatal mortality and among the leading causes of infant mortality. Haas and colleagues examined whether a woman's health status and risk factors before pregnancy are associated with her risk of preterm birth, independent of risk factors that occur during pregnancy. They found that, after adjusting for sociodemographic characteristics and prepregnancy and pregnancy risk factors, women who reported poor physical function during the month before conception were nearly twice as likely to deliver preterm. The authors concluded that a broader focus on the health of women before pregnancy might improve rates of preterm birth [21].
In a related editorial, Goldenberg suggests that reducing smoking, depression, and stress; treating periodontal disease and genital tract infections; and reaching a normal weight might all be necessary to substantially reduce the risk of preterm birth [22]. Goldenberg advocates for a Medicaid mandate that women who have had an adverse pregnancy outcome be eligible for continued medical coverage in the postpartum period to reduce risk factors and improve health status before the next pregnancy.
Data from the 1999 PRAMS survey indicate that the prevalence of unintended pregnancies in the United States ranges from 34 to 52% with 27 to 36% of these pregnancies being mistimed, and 6 to14% being unwanted [23]. In the United States, a clear causal association between unintended pregnancy and poor pregnancy outcomes has not been established. However, women with unwanted pregnancies are at greater risk for poor pregnancy outcomes than women with wanted pregnancies. Hogue asserts that one of the key approaches to prevention of high-risk pregnancy is assuring that the pregnancy is consciously desired, with adequate attention to preconception care [24].
Both programs demonstrate several components essential to any successful preconception care program: identification of risk factors amenable to change by the target population; tailored interventions for the target population; integration of preconception care into existing services; incorporation of family planning counseling and clinical services, health education, and community outreach. Preconception care aims to promote the health of women of reproductive age before conception and thereby improve pregnancy-related outcomes. Both the IPC program and the Magnolia Project show promise in achieving this goal for their target audiences.

preconception
interconception
family planning
birth outcomes
risk reduction


Structure and Function of the Hair Cell Ribbon Synapse

Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. While the electron microscopy hallmark of the hair cell afferent synapse — the electron-dense synaptic ribbon or synaptic body — has been recognized for decades, dissection of the synapse’s molecular make-up has only just begun. Recent cell physiology studies have added important insights into the synaptic mechanisms underlying fidelity and reliability of sound coding. The presence of the synaptic ribbon links afferent synapses of cochlear and vestibular hair cells to photoreceptors and bipolar neurons of the retina. This review focuses on major advances in understanding the hair cell afferent synapse molecular anatomy and function that have been achieved during the past years.
Introduction
Hearing relies on faithful synaptic transmission at the ribbon synapse of auditory hair cells. Figure 1 shows an electron micrograph of a typical hair cell ribbon synapse. The synapse is characterized by the presence of a large presynaptic organelle, the synaptic ribbon or dense body, that tethers synaptic vesicles and is itself anchored to the active zone. The performance of this synapse is remarkable in several aspects.
Figure 1Hair cell ribbon synapse. Electron micrograph of an inner hair cell afferent synapse of an 8-week-old mouse. The ribbon (rib) is anchored to the presynaptic plasma membrane and faces the postsynaptic density (PSD) of the auditory nerve fibers. The arrowhead and the star indicate a ribbon-associated synaptic vesicle and a free cytoplasmic coated vesicle, respectively.
PHASIC RELEASE PROPERTIES
The synapse’s precision to code the temporal fine structure of acoustic stimuli is unparalleled. For example, our capacity to locate sound sources in space depends on interaural time differences in the arrival of sound of only tens to hundreds of microseconds. Unlike a conventional synapse that is driven by action potentials, the hair cell afferent synapse responds to graded changes in membrane potential. While the action potential results in a strong all-or-none stimulus-secretion coupling, the hair cell afferent synapse is faced with the challenge of encoding sounds of extremely different intensities and, hence, must rely on mechanisms that allow for variable stimulus-secretion coupling. At the same time, the hair cell afferent synapse must also maintain temporal fidelity. For example, the firing of an auditory nerve fiber can follow at a constant phase relationship the periodicity of tonal stimuli into the low kHz range of frequencies, and we infer that the hair cell neurotransmitter release must comply with this extraordinary timing. Such temporal precision in synaptic transmission is even observed with sub-threshold stimuli, where the temporal pattern of spike firing regularizes already before there is an overall increase in the discharge rate.
TONIC RELEASE PROPERTIES
As we all can tell from our own experience, the cochlear hair cell afferent synapse must be able to encode prolonged, ever-present sounds. For example, despite adaptation, the constant background noise of the room’s air handling system is faithfully reported by the peripheral auditory system. To meet this requirement of audition, sustained neurotransmitter release must be maintained and implicates the need for extensive synaptic vesicle cycling (both, vesicle exo- and endocytosis). The hair cell afferent synapse’s capacity for sustained signal has often been attributed to the presence of the synaptic ribbon, and apparent depot of synaptic vesicles. However, recent morphological and physiological studies suggest additional/different roles for the hair cell synaptic ribbon, and have fuelled the structure-function debate at the ribbon synapse.
Molecular Anatomy and Physiology of the Hair Cell Synapse
We are still far from a comprehensive understanding of the molecular composition of the presynaptic active zone and the postsynaptic density of the hair cell afferent synapse. Homologies between retinal and inner ear sensory ribbon-type active zones are evident (Safieddine & Wenthold, 1999). Given the larger number of receptor cells in the retina (millions of photoreceptors vs. thousands of coding hair cells) and relative accessibility of the visual end organ, a more advanced molecular description is available presently for retinal ribbon synapses (e.g., tom Dieck et al., 2005) and promises to guide our understanding of auditory and vestibular ribbon synapses. For example, ribbons in both organs contain RIBEYE (Schmitz, Konigstorfer & Sudhof, 2000; Zenisek et al., 2003; Khimich et al., 2005), Bassoon and Piccolo (Khimich et al., 2005; tom Dieck et al., 2005). RIBEYE is thought to be a structural component of the ribbon but may also have enzymatic activity at the synapse (Schmitz et al., 2000). The photoreceptor ribbon also contains the close RIBEYE homologue and transcriptional co-repressor C-terminal binding protein 1 (tom Dieck et al., 2005). A putative role for this enzyme’s lysophosphatidic acid acyl-CoA transferase activity in membrane fission has been proposed (Valente et al., 2005, but see for an opposing view Gallop, Butler & McMahon, 2005). Some hint for a ribbon function in membrane fission may be seen in the accumulation of cisternal membrane profiles in ribbon-deficient inner hair cells (IHCs) (Khimich et al., 2005). However, any hypothesized enzymatic role for this ribbon constituent in synaptic transmission will require further investigation.
Bassoon, like Piccolo, is a large cytomatrix molecule found at the active zone, and most likely contributes to the anchoring of the ribbon at the active zone (Dick et al., 2001; Dick et al., 2003; Khimich et al., 2005). Deletion of a major part of the Bassoon gene (Altrock et al., 2003) results in the lack of synapse-anchored ribbons at most active zones of mouse IHCs. Moreover, the amplitude of a readily releasable pool of IHC synaptic vesicles is decreased, and both synchronous afferent synaptic transmission and hearing are impaired (Khimich et al., 2005). In addition, a minor reduction in the IHC Ca2+ current is observed, potentially reflecting impaired insertion of Ca2+ channels at the active zone due to the Bassoon and/or ribbon deficiency. Although less severe than the complete synaptic dysfunction resulting from the lack of CaV1.3 channels (Brandt, Striessnig & Moser, 2003), the loss of ribbons causes a synaptopathic hearing impairment with the previously described audiological signature of an auditory neuropathy (preserved cochlear amplification but impaired auditory evoked potentials). Interestingly, hearing was found intact in a Piccolo mutant (Sendin and Moser, unpublished data), but some immumoactivity was observed in the Synapses of the mutants. Perhaps the failure of the Piccolo mutation to impair hearing reflects a sufficient abundance of residual functional Piccolo protein in the mutant mouse or the action of some unknown compensatory mechanism.
Hair cells express the SNARE proteins syntaxin 1, SNAP-25 and synaptobrevin 1 (Safieddine & Wenthold, 1999). These proteins are thought to build the key exocytic machinery, as their cleavage by clostridial neurotoxins shuts off exocytosis in other systems (e.g., Xu et al., 1998; Sakaba et al., 2005). The effects of clostridial neurotoxin action on hair cell afferent synaptic transmission remain to be investigated. Hair cell afferent synapses, like photoreceptor synapses, lack synapsins (Favre et al., 1986; Safieddine & Wenthold, 1999), a class of proteins that are involved in synaptic vesicle mobilization. Moreover, synaptotagmins 1 and 2, which are considered the calcium sensors of vesicle fusion in the central nervous system and neuroendocrine cells (Sudhof, 2004), have not been observed in hair cells (Safieddine & Wenthold, 1999). Genetic ablation of Synaptotagmin 7 in mice, which is expressed in IHCs (Safieddine & Wenthold, 1999) and involved in exocytosis of hippocampal neurons (Virmani et al., 2003), did not result in a hearing impairment (Nouvian and Moser, unpublished data). Thus, taken together, these findings suggest that either other synaptotagmin isoforms or other proteins altogether may act as the calcium sensor at the hair cell afferent synapse.
Despite some homologies, IHC ribbon synapses differ molecularly from retinal ribbon synapses. Most prominently, IHC transmitter release and sound coding are controlled by CaV1.3 Ca2+ channels (Platzer et al., 2000; Brandt et al., 2003; Brandt, Khimich & Moser, 2005), while visual signalling relies on stimulus-secretion coupling by retinal CaV1.4 channels (Bech-Hansen et al., 1998; Strom et al., 1998; Mansergh et al., 2005). Some vestibular hair cells, in addition, may involve non-L-type Ca2+ channels (Rodriguez-Contreras & Yamoah, 2001). Evidence obtained by various techniques in various species all suggests that CaV1.3 channels cluster at the ribbon-type active zones of the hair cell. These include loose-patch recordings (Roberts, Jacobs & Hudspeth, 1990), cell-attached recordings (Rodriguez-Contreras & Yamoah, 2001), Ca2+ imaging (e.g., Issa & Hudspeth, 1994; Tucker & Fettiplace, 1995; Zenisek et al., 2003) and immunohistochemistry (Sidi et al., 2004; Brandt et al., 2005). Estimates of the total number of Ca2+ channels per active zone have been obtained in frog saccular hair cells (∼90 channels, Roberts et al., 1990; but see alsoRodriguez-Contreras & Yamoah, 2001) and mouse cochlear IHCs (∼80, Brandt et al., 2005) by ensemble fluctuation analysis. This similarity in channel number is unexpected given the size differences between the active zones of the two species (see below and Table 1) and highlights how much more we have to learn about this intriguing synapse.
Table 1Size and kinetics of release components related to the synaptic ribbon and to morphological vessel pools associated from different species
Classical work by Robert’s and Fettiplace’s groups on intracellular calcium dynamics at the plasmalemma face of hair cells advocated the view that calcium domains were micromolar in concentration and micrometer in dimension. This work was based largely on computational models and studies of calcium-activated potassium channel gating, but was assumed to be also applicable to hair cell afferent synapse exocytosis. However, more recent experiments on the stimulus-secretion coupling in mouse IHCs suggested that exocytic fusion of a given readily releasable vesicle is controlled only by few nearby Ca2+ channels (Brandt et al., 2005). These authors argued that such a “nanodomain” control would support the temporal precision of coding also for weak sounds. Additional experiments using complimentary techniques are needed to further test this provocative hypothesis.
Rim binding protein, interacting with the active zone protein RIM and the CaV1.3 channel, is currently the best characterized candidate for a molecular link of channel and release site of a given vesicle (Hibino et al., 2002). However, functional evidence is still lacking and it remains possible that other proteins, e.g., scaffolding proteins of the cytomatrix of active zones like Bassoon or Piccolo, contribute. It is also possible that the synaptic ribbon itself is responsible for positioning of the CaV1.3 channel and readily releasable vesicles (Khimich et al., 2005). Clearly, molecular work, physiological analysis of mutants and particular immunoelectronmicroscopy are required to characterize the molecular interactions and the ultrastructural topography at the active zone. Figure 2 illustrates the subcellular localization of a few synaptic proteins.
Figure 2Molecular composition of the hair cell synapse. (A) Montage of a Nomarski image (note the hair cell bundles) and a confocal reconstruction of the mouse organ of Corti with immunolabeled ribbons (stained for RIBEYE/CtBP2, red) and postsynaptic transmitter receptor clusters (GluR2/3 glutamate receptor subtypes, green). Presumptive hair cell border (white) and postsynaptic fibers (orange) contacting an IHC were drawn illustration. (B) Previously identified molecular determinants of the hair cell ribbon synapse. 1Safieddine & Wenthold, 1999; 2Furness & Lawton, 2003; 3Eybalin et al., 2002; 4Schmitz et al., 2006; 5Khimich et al., 2005; 6Platzer et al., 2000; 7Brandt, Striessnig & Moser, 2003; 8Brandt, Khimich & Moser, 2005; 9Matsubara et al., 1996; 10Eybalin et al., 2004.
Besides a molecular coupling, other mechanisms contribute to localized Ca2+ signalling. The presence of the mobile, proteinaceous Ca2+ buffers calretinin, calbindin and parvalbumin has now been documented in a variety of cochlear and vestibular hair cells (e.g., Edmonds et al., 2000; Heller et al., 2002; Hackney et al., 2005). Their relevance for presynaptic Ca2+ signalling has been inferred from recordings of Ca2+-activated large-conductance K+ channels (Fettiplace, 1992; Roberts, 1993; Tucker & Fettiplace, 1996; Edmonds et al., 2000), simulations (Roberts, 1994) and capacitance measurements (Moser & Beutner, 2000; Spassova et al., 2004). Most likely they spatiotemporally restrict the presynaptic Ca2+ domains and, hence, improve the timing of synaptic transmission. However, discrepancies regarding both the amount and kinetic properties of buffers in different hair cells (Roberts, 1993; Edmonds et al., 2000 vs Moser & Beutner, 2000) suggest that our understanding of calcium buffers at the hair cell active zone synapse is far from complete. Examination of hair cells from mutant mice lacking the major proteinaceous Ca2+ buffers promises further insight into this important question.
In addition to synchronous synaptic transmission, the molecular machinery needs to be suited for long-lasting transmitter release. By yet unknown mechanisms CaV1.3 currents in hair cells are much less inactivating than in heterologous expression systems (Koschak et al., 2001). Most likely this is due to interactions with a special combination of auxiliary subunits and proteins, such as the class of Ca2+ binding proteins (Haeseleer et al., 2004). None of these auxiliary determinants of hair cell Ca2+ current are known so far.
Once the neurotransmitter is released into the synaptic cleft, it activates an AMPA-like receptor residing within a receptor cluster at the postsynaptic density of afferent auditory nerve fibers. In a classical study, Ottersen and his colleagues showed the abundance of the glutamate receptor subtypes (GluR) 2, 3 and 4 by immunogold labelling (Matsubara et al., 1996). Interestingly, the distribution of the glutamate receptor was not homogenous in the postsynaptic density (PSD): the receptor numbers increase at the border of the PSD. Using patch-clamp recordings from the postsynaptic endings contacting rat IHCs, Glowatzki & Fuchs (2002) showed that excitatory postsynaptic currents of spiral ganglion neuron afferent fibres are mediated by AMPA receptors. They failed to detect NMDA or Kainate currents. Recently, Eybalin and colleagues studied the developmental expression changes of postsynaptic glutamate receptors using western blotting, RT-PCR and immunohistochemistry (Eybalin et al., 2004). They showed that GluR2 becomes expressed only around the onset of hearing, while GluR3 and 4 are present before. Little is known about the other molecular determinants of the PSD. Davies et al. (2001) report the presence of PSD-95 and PSD-93 in the PSD of auditory nerve fibres.
To avoid saturation and excitotoxicity due to an excess of glutamate in the synaptic cleft, glutamate is taken up by glutamate transporters (EAAT) into supporting cell transporters (Furness & Lehre, 1997; Furness & Lawton, 2003; Rebillard et al., 2003). Interestingly, glutamate-aspartate transporters (GLA ST) are more abundant on the pillar IHC side, where high spontaneous rate fibers are located, compared to the modiolar cell side, where low spontaneous rate fibres are located (Liberman, 1982; Furness & Lawton, 2003).
Relating Structural and Functional Vesicle Populations
MORPHOLOGY
Synaptic Ribbons
The active zone of the hair cell is characterized by an electron dense structure called synaptic body or ribbon. Most synaptic ribbons found in mature hair cells are anchored to the plasma membrane, one ribbon per active zone, and oppose a postsynaptic terminal. A small number of “floating” ribbons (<5 %) are observed, and probably reflect the turnover of these subcellular organelles (Zenisek et al., 2004; Khimich et al., 2005). Shape, size and number of the ribbons differ (see Table 1) between species as well as along the tonotopic axis, during development (Shnerson, Devigne & Pujol, 1981; Sobkowicz et al., 1982; Khimich et al., 2005) and even among different active zones within a single hair cell (Merchan-Perez & Liberman, 1996). For example, frog saccular hair cells display about 20 large, synaptic bodies irrespective of the cells’ location in the end organ (Roberts et al., 1990; Lenzi et al., 1999, 2002). Whereas in the chick cochlea, the number of synaptic bodies is constant in tall hair cells (functional homologue of the mammalian IHCs), the synaptic body diameter increases with increasing characteristic frequency (Martinez-Dunst, Michaels & Fuchs, 1997). Conversely, the number of ribbons varies along the tonotopic axis in many other species including turtle (plus small size differences: Sneary, 1988; Schnee et al., 2005), cat (Liberman, Dodds & Pierce, 1990), gerbil (Slepecky et al., 2000) and guinea pig (Hashimoto, Kimura & Takasaka, 1990).
Both the number and shape of the synaptic ribbons can also change with the onset of hearing. Mouse IHCs contain a large number (>20 in the apical cochlea, Nemzou et al., unpublished data) of small synaptic bodies (Shnerson et al., 1981; Sobkowicz et al., 1982) before the onset of hearing. After the onset of hearing only fewer but larger ellipsoid ribbons are observed (Shnerson et al., 1981; Sobkowicz et al., 1982; Francis et al., 2004; Khimich et al., 2005). An interesting feature described for cat IHCs is the spatial segregation of synapses with fibres of different spontaneous rates and thresholds. Thus, low-spontaneous-rate, high-threshold fibres contact the IHCs on their modiolar side, whereas high-spontaneous-rate, low-threshold fibres synapse on the pillar IHC side. EM analysis showed that the ribbon size and accordingly the number of ribbon-associated synaptic vesicles tend to increase with decreasing spontaneous rate of the contacted fibres (Liberman, 1980; Liberman et al., 1990; Merchan-Perez & Liberman, 1996).
Synaptic vesicles
Ribbons tether a monolayer of synaptic vesicles at a packing density that is ∼55% of maximum by 20 nm long filaments (Lenzi et al., 1999). These ribbon-associated vesicles represent only a tiny fraction of the huge number of synaptic vesicles contained in a hair cell. At least three vesicle populations can be morphologically distinguished at the hair cell ribbon synapse as illustrated by Figure 3:Docked vesicles (no discernible space between vesicle and plasma membranes)Ribbon-associated vesiclesFree cytosolic synaptic vesicles.
These vesicle populations have been quantified for hair cells of various species (Table 1). A variety of approaches have been used to assess the vesicle populations and accordingly, varying degrees of certainty surround these vesicle counts. The different approaches have included: precise three-dimensional EM counts based on few synapses (EM tomography: Fig 3, Lenzi et al., 1999; Lenzi et al., 2002; reconstructions based on ultrathin serial sections: Schnee et al., 2005); extrapolations from random ultrathin EM sections of tens of synapses (Khimich et al., 2005) and approximations based on ribbon surface areas and vesicle packing density estimates (Spassova et al., 2004; Khimich et al., 2005: more than 200 ribbons analyzed).
Figure 3Morphology of synaptic ribbons. (a, b and c) Electron micrographs of inner hair cell ribbon synapses of 8-week-old mouse. Ribbons are attached to the plasma membrane and surrounded by a monolayer of synaptic vesicles. Colored dots in (b) illustrate the morphologically defined classes of synaptic vesicles: red dots indicate the docked vesicle associated with the ribbon; yellow marks ribbon- associated SV which are not docked and green indicates outlying cytoplasmic vesicles. (d) Slice through an osmium-stained frog saccular hair cell synapse, reconstructed by electron tomography. Note the round shape of the synaptic body in contrast to the elipsoid one of the mouse. (e) Three-dimensional structure of presynaptic organelles from the same reconstruction shown in (d). The synaptic body (blue) is open and lies adjacent to the hair cell’s plasma membrane in red. Synaptic body-associated vesicles (yellow) surround the SB; outlying vesicles (green) lie further out in the cytoplasm. Also visible are coated vesicles (gold) and cisterns (purple).
Docked vesicles at a conventional synapse are viewed as a homogeneous anatomical pool. However, at the hair cell ribbon synapse docked vesicles are found in a variety of anatomical states; this includes underneath the ribbon (docked and ribbon-associated), at the active zone without contact to the ribbon, and outside the active zone. Lenzi et al., (1999) termed these last two classes of docked vesicles as “outlying”. The functional implications of the different classes of docked vesicles are poorly understood. Obviously, proximity to a calcium channel increases the likelihood that a docked vesicle is of functional significance. Accordingly, Lenzi et al., (1999) attempted to correlate the positions of docked vesicles and Ca2+ channels at the active zone. While the “outlying” vesicles are substantial in number, they are located further from the presumed calcium channel containing presynaptic densities than are other docked vesicles. “Outliers” has been alternatively used in evanescent wave microscopic studies (Zenisek, Steyer & Almers, 2000) to describe the “docking” and fusion of synaptic vesicles outside of functionally defined active zones.
PHYSIOLOGY
One fruitful way to investigate the compact afferent presynaptic terminal of the inner ear is to monitor exocytic fusion and endocytic retrieval of synaptic vesicle membrane as changes of the membrane capacitance (Cm) using the patch-clamp technique (for review seeNeher, 1998). During the fusion of the synaptic vesicle to the plasma membrane the cell surface and consequently Cm increase. This then is usually followed by slower fission accompanying Cm decrease. This technique has become increasingly popular to study the presynaptic function of hair cells in various species (Table 1). Upon depolarization, calcium influx, mainly through CaV1.3 L-type Ca2+ channels, triggers exocytosis of readily releasable synaptic vesicles. Depending on the stimulus intensity and duration, the resulting Cm increase may report exocytosis occurring at all active zones as well as the fusion of synaptic vesicles outside of active zones or even the fusion of non-synaptic membrane organelles (Coorssen, Schmitt & Almers, 1996). This lack of specificity associated with capacitance measurements can complicate its interpretation.
At least two kinetic components of exocytosis are commonly discriminated in the recordings of hair cell membrane capacitance following stimuli thought to trigger exocytosis. Such findings from a variety of species are also summarized in Table 1.
The first kinetic component of these membrane capacitance increases mediates exocytosis at extremely high rates but saturates within a few milliseconds. Hence, it is generally agreed that this component represents exocytosis of a small, finite pool of vesicles. Because it is the fastest discernible and exhaustible component of exocytosis several authors denominated it the readily releasable vesicle pool (RRP, Moser & Beutner, 2000; Spassova et al., 2004; Rutherford & Roberts, 2006) following the classical terminology of functional vesicle pools (Liley & North, 1953; Birks & MacIntosh 1961; Elmqvist & Quastel, 1965). Three major findings indicate that the first kinetic component is, indeed, involved in synaptic sound-coding by hair cells:Correlation between the RRP size and the number of afferent synapses made by a hair cell as it varies along the tonotopic axis of the cochlea (Schnee et al., 2005).Correlation between the RRP size and the number of synapse-anchored ribbons (Khimich et al., 2005).Correlation between the RRP size and the amplitude of the spiral ganglion compound action potential (reflecting synchronous activation of spiral ganglion neurons) (Khimich et al., 2005).
Several other lines of evidence support the notion that the first kinetic component of exocytosis represents a synaptic relative pool of vesicles. Resistance of the readily releasable pool to inhibition by exogenous slow calcium buffers infers a co-localization of this functional pool with Ca2+ channels (Moser & Beutner, 2000; Spassova et al., 2004). Calcium channels are widely believed to be concentrated at hair cell active zones (e.g., Roberts et al., 1990; Issa & Hudspeth, 1994; Tucker & Fettiplace, 1995; Zenisek et al., 2003; Sidi et al., 2004; Brandt et al., 2005). Nanodomain control of synaptic vesicle exocytosis (Brandt et al., 2005) requires an intimate spatial relationship between the readily releasable pool and calcium channels. There is also good correspondence between the quantal content predicted by measurement of action potential-driven exocytic Cm changes in immature IHCs (Beutner & Moser, 2001; Johnson, Marcotti & Kros, 2005) and direct measures of postsynaptic currents from the afferent fiber (Glowatzki & Fuchs, 2002). Finally, additional supportive evidence comes from comparison of the timing of peripheral auditory adaptation and the kinetics of RRP depletion and recovery.
Adaptation is a common property of sensory systems. It reduces encoding redundancy by decreasing the sensitivity to constant stimulation, is proposed to optimize a sensory system’s operating range (Koutalos & Yau, 1996) and maximize its information transfer (Brenner, Bialek & de Ruyter van Steveninck, 2000). In the peripheral auditory system, adaptation is likely localized to the cochlear hair cell afferent synapse because hair cell receptor potentials do not adapt to maintained acoustic stimulation (Holton & Weiss, 1983; Russell & Sellick, 1978). Previous work (Furukawa & Matsuura, 1978; Moser & Beutner, 2000) suggested that the exhaustion and replenishment of the readily releasable pool of vesicles in the hair cell contribute to a fast form of adaptation and recovery from it in the auditory nerve (Westerman & Smith, 1984; Yates, Robertson & Johnstone, 1985).
Parsons and colleagues (Spassova et al., 2004) employed both in vitro patch-clamp recordings of hair cell synaptic vesicle fusion and in vivo single-unit recording of cochlear nerve activity at the same synapse to further examine this hypothesis and probe the possible auditory significance of the hair cell ribbon synapse structure-function relationship. Similar to other hair cell preparations, exocytosis of the chick cochlear tall cell’s readily releasable pool is fast, saturating in < 50 ms, and its recovery is also rapid, regaining 95% of its initial amplitude following a 200 ms period of repolarization. Interestingly, sound-evoked afferent synaptic activity also adapted and recovered with similar time courses as readily releasable pool exhaustion and recovery. This work supports the view that the fast exocytic component is of synaptic relevance by providing evidence that exhaustion and replenishment of the readily releasable pool mediates behaviorally relevant phenomena — namely short-term auditory nerve adaptation and its recovery from adaptation.
In an elegant two-photon imaging study Ashmore and colleagues utilized the turnover of the membrane dye FM1-43 to track the exocytosis of synaptic vesicles at hair cell active zones. Their findings on the readily releasable pool of the adult guinea pig IHCs were similar to descriptions provided in other species by capacitance measurements. The significance of these findings is that the imaging experiments, unlike the capacitance measurements, are spatially resolved and lend credence to previous interpretations of the first exocytic component. Optically recorded exocytosis was extremely fast at the onset of a depolarization (initial rate of 3,000 vesicles per second) and saturated with a time constant of 30 ms in mature guinea pig hair cells (Griesinger, Richards & Ashmore, 2005). This intial rate is comparable to the initial rate of exocytosis evoked at one ribbon synapse of mature mouse IHCs by maximal stimulation amounting to ∼6,000 vesicles per second (after Cm conversion into vesicle numbers using 28 aF/vesicle). Moreover, estimates of the time course of recovery from depletion obtained by the two methods were quite similar, too (Table 1).
Both the amplitude and kinetics of the first kinetic component in mature mouse and guinea-pig hair cells were found to depend on the stimulus strength (Brandt et al., 2005; Griesinger et al., 2005). This is in contrast to a study on frog saccular hair cell exocytosis where the kinetics, but not amplitude of exocytosis, varied with stimulus amplitude (Edmonds, Gregory & Schweizer, 2004). The reasons for this discrepancy among the different experiments are currently unclear. Moser and colleagues have suggested the Ca2+ nanodomain control of RRP exocytosis in mouse IHCs (Brandt et al., 2005). This hypothesis predicts that an increase in intensity of stimuli would recruit more calcium channel-release site units, presumably increasing the amplitude of the RRP without major changes of release kinetics. Classical quantal analysis at the goldfish hair cell synapse (Furukawa, Kuno & Matsuura, 1982) also revealed intensity-dependent increases in the number of available vesicles (as opposed to changes in the release probability). While the synaptic relevance of the first kinetic component is indisputable, clearly a detailed mechanistic understanding of it awaits further experimentation.
Our current knowledge about the further kinetic component (s) is much more limited. This is mainly due to the lack of spatial resolution of Cm recordings of exocytosis, which report fusion to the plasma membrane no matter whether it happens at the synapse or elsewhere. The slower kinetic components are typically triggered by prolonged, strong depolarizing steps (tens of ms to s in duration) and have the potential to drive calcium levels within the cell to levels that could not be achieved with more physiological time-varying, smaller-amplitude stimuli. The relative resistance of the CaV1.3 calcium channel to inactivation allows for the influx of large numbers of calcium ions during prolonged depolarizations that may be sufficient to saturate cellular calcium buffers. Clearly, aphysiologic calcium gradients within the hair cell promise to further compound the limitations of membrane capacitance measurements and cloud the interpretation of secondary, slower exocytic components.
Since the first Cm measurements of hair cell exocytosis (Parsons et al., 1994), we have been astonished and puzzled by the huge amount of membrane turnover during prolonged depolarization. During the first second of depolarization thousands of vesicles are released (see Table 1). A membrane turnover of more than one equivalent of the initial hair cell’s surface has been reported for maximal stimulation over several seconds (Schnee et al., 2005). In this respect, the hair cell’s exocytic capacity exceeds that of retinal photoreceptors and bipolar neurons. This difference between retinal cells and hair cells is most dramatic when examining the fast exocytosis evoked by Ca2+ uncaging. A uniform step increase in calcium concentration through the cell’s cytoplasm triggers the exocytosis of all fusion-competent vesicles. Flash photolysis of caged calcium in the goldfish bipolar nerve terminal results in an average capacitance increase of ∼150 fF (Heidelberger et al., 1994), whereas a similar stimulus in the mouse IHC drives a 10-fold larger increase in membrane capacitance (Beutner et al., 2001). This discrepancy in the amount of exocytosis is even more remarkable given that a hair cell contains nearly 4 times fewer, but similar-sized and vesicle-populated synaptic ribbon active zones as the retinal bipolar terminal (von Gersdorff et al., 1996; Khimich et al., 2005). Hence we have to either assume that the turnover at a hair cell ribbon synapse is 40 times faster than that of a bipolar neuron or to conclude that hair cells contain an extremely large pool of fusion-competent vesicles that reside outside of the active zone. Spatially-resolved exocytic measurements, such as imaging or conventional post-synaptic recordings, will be required to ascertain the contribution of the hair cell’s large pool of fusigenic vesicles to synaptic relevant exocytosis.
Is it Possible at all to Relate Anatomically and Physiologically Defined Pools of Synaptic Vesicles?
It would be a significant breakthrough in our understanding of the hair cell ribbon synapse if we managed to arrive at a joint anatomical and physiological definition of synaptic vesicle populations. This goal requires relating physiologically derived capacitance or fluorescence changes to anatomically defined synaptic vesicle numbers. Unfortunately, several technical issues limit the accuracy and certainty of this pursuit. Estimations of single-vesicle capacitance are required to determine the equivalent number of vesicles associated with a given Cm increase. Surface calculations based on the vesicle diameter in fixed tissue (possibly correcting for a potential shrinkage bias) and an assumption of the specific membrane capacitance (usually 10 μF/μm2, Breckenridge & Almers, 1987) are commonly employed. Both are prone to errors complicating this structure-function approach. The inherent problems with the fixation artefacts are highlighted by the factor of 2 discrepancy between “in vitro” single-vesicle capacitance values derived from anatomical studies (37 aF, Lenzi et al., 1999 and 28 aF, Khimich et al., 2005) and the more direct “in vivo” estimates obtained by electrophysiology in other systems (∼50 aF, Klyachko & Jackson, 2002 and 65 aF, Sun, Wu & Wu, 2002).
The physiological approach to pool definition also faces technical limitations. The rate of the first or fastest kinetic component is limited by the bandwidth and signal-to-noise ratio of the recording equipment. Much confusion in the retinal bipolar terminal literature stems from the initial definition of a readily releasable pool with a time constant of ∼100 ms (von Gersdorff & Matthews, 1994) prior to the subsequent discovery of a 5-fold smaller, but nearly 50-fold faster kinetic component of exocytosis (Mennerick & Matthews, 1996; Neves & Lagnado, 1999). At the chick hair cell synapse, which features some of the smallest-numbered and -sized synaptic ribbons, the total number of docked vesicles is ∼100. Exocytosis of these vesicles would generate a capacitance increase perhaps as small as 3 fF, at or below the resolution of whole-cell capacitance measures and likely obscuring the observation of this pool that is readily measured in cells with large ribbon structures.
What Is the Anatomical Substrate of the Fast Kinetic Component in Hair Cell Exocytosis?
Several caveats described above constrain our ability to answer this question. However, recognizing these limitations and uncertainties, it remains an important and interesting question underlying our understanding of the hair cell afferent synapse. The recent literature provides two alternative hypotheses: it may preferentially draw on readily releasable vesicles docked at the plasma membrane of the active zone (Moser & Beutner, 2000; Khimich et al., 2005; Schnee et al., 2005; Rutherford & Roberts, 2006) or involve release of all ribbon-associated vesicles (Edmonds et al., 2004; Spassova et al., 2004). Table 1 highlights these views. The first hypothesis is consistent with independent fusion of the individual vesicles, similar to what is expected for docked vesicles at the active zone of conventional synapses. The second hypothesis requires the rapid recruitment and fusion of non-docked ribbon-associated vesicles. As active vesicle transport seems too slow (see discussion in Edmonds et al., 2004), different modes of exocytosis, such as compound and “piggy back” (cumulative) fusion, have been considered (Parsons & Sterling, 2003; Edmonds et al., 2004; Spassova et al., 2004). Functional evidence for multivesicular release at the hair cell ribbon synapse was first obtained by Glowatzki and Fuchs (2002). Using direct post-synaptic recordings at rat afferent synapses, they observed large variations in the amplitude of kinetically quite homogeneous postsynaptic currents, as well as clear composite events characterized by multiple quanta (Glowatzki & Fuchs, 2002). Clearly, the exact presynaptic release mechanism of vesicle release is likely to have great impact on the transfer characteristics of the hair cell synapse and remains an extremely important problem for continued study.
This review cannot solve the debate over the anatomical basis of the hair cell fast exocytic component. However, we aim to fuel the on-going discussion with some selected comparisons of data complied from various studies and summarized in Table 1. Any considerations of structure-function comparisons are complicated by the fact that the physiological pool size estimates obtained by different groups can vary substantially even for hair cells of the same species (Edmonds et al., 2004; Rutherford & Roberts, 2006, see discussion in Rutherford’ paper). Moreover, the morphological vesicle counts or estimations vary dramatically in their degree of precision (from direct counts in electron microscopical 3-D reconstructions to approximations based on ribbon size, see Morphology section above and Table 1). In addition, we need to remember that our capacitance-based approximations probably overestimate the number of vesicles due to underestimation of single-vesicle capacitances (due to the shrinkage during chemical fixation, see above). With these caveats in mind, studies on hair cells of the frog (work from the Roberts lab) and the mouse (work from the Moser lab) provide the most compelling structure-function correlations, in part because both the anatomy and physiology were carried out in the same laboratory. In each case, their observations support the hypothesis that for these particular hair cells RRP is defined by the fusion of docked vesicles (Table 1). Correcting the conversion factor for shrinkage would tend to still improve the match between RRP size and docked vesicle number.
The question still lingers as to whether non-docked vesicles can be released rapidly at ribbon synapses. Zenisek et al. (2000), using evanescent wave microscopy of single FM1-43 stained vesicles in retinal bipolar nerve terminals, provide some of the most direct evidence addressing this question. They reported that the first component of exocytosis was dominated by “resident” (probably docked) vesicles at the hot-spots of exocytosis, whereas arrivals or “newcomers” contributed only marginally within the first milliseconds. This argues against a major contribution of non-docked, ribbon-associated vesicles. On the other hand, Griesinger et al. (2005) concluded from two-photon imaging of basolateral hot-spots of FM1-43 fluorescence that rapid exocytosis of non-docked vesicles does, indeed, occur at hair cell ribbon synapses. A certain degree of caution, however, is required in interpreting either of these studies, as the best resolution of an optical microscope (200 nm) is comparable to the size of most synaptic ribbons.
The apparent differences between the behavior of ribbon-tethered vesicles in these two types of ribbon synapses highlight the fact that it could be an oversimplification to assume that a common anatomical substrate underlies the first/fastest recorded kinetic component of hair cell exocytosis. The physical dimensions of hair cell active zones, shapes of synaptic ribbons, best stimulation frequency, and possibly endogenous calcium buffering vary dramatically across end organ, species and in some cases even within the same end organ of a given species. Thus, subsequent studies may reveal that different hair cells use different anatomical substrates for different functional pools of synaptic vesicles to meet their respective synaptic demands.
What about the Anatomical Substrate of Subsequent, Slower Kinetic Components of Exocytosis?
Things are still more complicated for the further kinetic component(s) of exocytosis reported by capacitance measurements. Most studies concur that replenishment of readily releasable vesicles occurs at high rates, exceeding those of conventional synapses and even retinal ribbon synapses (Moser & Beutner, 2000; Spassova et al., 2004; Edmonds et al., 2004; Griesinger et al., 2005). However, whether the slow kinetic components of exocytosis simply represent the serial refilling of the readily releasable pool and or parallel exocytosis at “ectopic” fusion sites is debatable. Schnee et al. (2005) proposed a purely serial and synaptic model to explain the three kinetic components of the depolarization-induced exocytic Cm changes in turtle hair cells. They inferred that the second component reflected exocytic turnover of the ribbon-associated vesicle population, while vesicle recruitment to the ribbon would become rate-limiting thereafter. Not surprisingly, a similar model was appropriate to describe destaining of FM1-43 hotspots and recovery of staining in the study of Griesinger and colleagues. However, these optical studies differ from the capacitance recordings in the turtle hair cell (Schnee et al., 2005), as they are spatially resolved and focus on membrane turnover at individual active zones.
Findings that argue for a significant contribution of ribbon-independent and at least in part parallel extrasynaptic exocytosis to sustained exocytic Cm changes include:Robust sustained exocytosis in hair cells that lack 90% of their synapse-anchored ribbons (Khimich et al., 2005)Presence of docked synaptic vesicles at extrasynaptic stretches of the plasmamembrane (Lenzi et al., 1999, 2002)The extensive fast exocytosis during flash photollysis, which by far exceeds fusion of the readily releasable pool (Beutner et al., 2001), potentially arguing for the presence of many fusion-competent vesicles outside the active zone.
In addition, exocytosis of such “outlying” vesicles has been described in retinal bipolar nerve terminals by evanescent microscopy (Zenisek et al., 2000). Hence, taken together, present evidence supports the interpretation that sustained hair cell exocytosis reported by Cm tracking represents both the serial re-supply of vesicles to the active zones and the parallel extrasynaptic turnover of synaptic vesicles. However, the quantitative contribution of the slow exocytic components to synaptically relevant neurotransmitter release remains to be determined.
Conclusion and Outlook
The multidisciplinary approach to the hair cell ribbon synapse has for the first time quantitatively described important aspects of the synapse’s structure and function. Most importantly, despite all limiting uncertainty, we begin to relate molecules, structure and function. Although still debated, the concept of a readily releasable pool has been substantiated for the ribbon synapse of several hair cells. We have gained a few insights into the mechanisms underlying the incredible temporal precision of synapses that participate in the coding of sound. Moreover, in addition to learning about the molecular physiology, the availability of mouse mutants with defined synaptic lesions provided the possibility to study the properties of synaptopathic hearing impairment in greater detail. However, we are far from a comprehensive molecularly defined model of ribbon structure and function. The molecular dissection of the hair cell synapse is technically challenging due to the low amount of tissue and will require much more time and effort. Ear-specific genetic deletion also will be helpful to investigate synaptic protein function in hair cell sound coding. A more precise and direct biophysical analysis of single hair cell synapses will require combined pre- and postsynaptic recordings as well as optical measurements, such as evanescent wave microscopy and confocal techniques. The optical approach will be strongly facilitated by the generation of genetically targeted fluorescent vesicle tags.

hair cell
hair cell
ribbon synapse
synaptic ribbon
exocytosis
calcium
synaptic protein
synaptic vesicle pool