Background: People with Huntington’s disease (HD) often require tailored healthcare and support packages that develop as the disease progresses. The Client Service Receipt Inventory (CSRI) gathers retrospective information on service utilization. This study investigated the use of formal services and informal care as measured by the CSRI and explored associations between informal care, disease severity and functional ability as measured by the Unified Huntington’s Disease Rating Scale Total Motor Score (UHDRS-TMS) and functional scales. Methods: All monitored longitudinal data from annual clinical assessments of UHDRS-TMS and functional assessments and CSRI collected under the auspices of the European Huntington’s Disease Network (EHDN) REGISTRY study between the years 2004 and 2009 were utilised in the analyses. Disease severity was reflected by UHDRS-TMS. Functional ability was measured using the UHDRS functional scales. CSRI data were analysed according to percentage use of individual formal services and total estimated hours per week of informal care. Regression analyses were conducted to identify any associations between disease severity, functional ability and hours of informal care.Results: 451 HD patients  completed one visit; 105 patients  completed two visits and 47 patients  completed three visits in total over the 5 year period. The mean time between visits was 1.2 years. At visit one, 74% of the participants reported being in receipt of at least one formal hospital-based service in the previous six months, and 89% reported receipt of formal primary and community care services. In contrast, at the third visit, 62% of people had used hospital based services and 94% formal community based services in the previous six months. Fifty % of individuals required some form of informal care in the home at visit 1; this increased to 68% at visits 2 and 3. The mean (SD) estimated weekly total informal care hours at visits 1, 2 and 3 were 32.8 (49.4); 21.6 (53.6) and 21.3 (62.4) respectively. Only the scores on the Functional Assessment Scale (FAS) accounted for the variance in the weekly total informal care hours at each visit. Conclusions: Although it must be acknowledged that service use is supply driven, most HD patients across Europe surveyed as part of this study were in receipt of formal primary and community care services and to a lesser extent formal hospital based services. There was however a large reliance on informal care in the home. The FAS appear to have predictive value on informal care requirements and may have utility in facilitating pro-active service provision and in particular when managing carer burden in this population. Huntington’s disease (HD) is an inherited, neurodegenerative disease resulting in clinical symptoms of progressive movement disorder, cognitive defects and behavioural changes that ultimately affects a person’s ability to participate independently in activities of daily living, work and community. With an approximately 20 year time course from symptom onset, death usually results from complications of falls, dysphagia, or aspiration Successful care will usually involve the efforts of a multi-disciplinary team working closely with the family to optimize quality of life over the life span of the disease Informal care is that which is delivered by non-expert carers who are usually family or friends, may range from full time care from person who lives with the HD patient to intermittent input from someone living elsewhere.  Informal care will vary according to the needs of the person with HD and may range from looking after all personal needs to help with shopping and care of home on less frequent basis. Informal carers are often required to provide both psychological and practical support over extended periods of time To be able to ensure that care needs are met, appropriate and informed service planning is required. The first step in this process is to quantify actual use of formal healthcare resources and the balance between formal care services and informal care use. http://www.ltnc.org.uk/research_files/RESULT_study.html) for this specific purpose but to date no data is available in published literature of specific service receipt in HD patients. Knowledge of such data has the potential to facilitate appropriate planning and resource allocation in line with standards of excellence for chronic conditions management. In the United Kingdom, a National Service Framework for people with Long Term Neurological Conditions (LTNCs) sets out quality requirements for care. These include prioritisation for personal care and support with appropriate support for family and carers and access to appropriate aids and home adaptations to promote living at home to facilitate living at home for as long as is able with as high a quality of life as possible The Client Service Receipt Inventory (CSRI) questionnaire was developed Disease severity is routinely quantified using the Unified Huntington’s Disease Rating Scales (UHDRS) The aim of this study was to investigate the use of formal and informal care services as recorded using retrospective CSRI data and to investigate any associations between use of care, disease severity and functional ability as measured by the UHDRS motor and functional scales. In these analyses, we specifically sought to identify, a) what care  HD patients currently use and b) whether there were any specific factors associated with the estimated amount of informal care required. To our knowledge such data has not previously been presented for a cohort of HD patients; we suggest that availability of such data in a cohort of HD patients from a cross-section of European countries is of use in quantifying and ultimately improving planning of service provision for HD patients.  Methods and materials:REGISTRY is a multi-centre, multi-national observational study with no experimental intervention. REGISTRY is managed by the European Huntington’s Disease Network (EHDN) and aims to build up an electronic database of patients with HD and their relatives. The main component of the ' REGISTRY ' study is the storage of anonymous medical data on an electronic database. One of the aims of REGISTRY is to obtain natural history data on an annual basis over an extended time period on a wide spectrum of individuals affected by HD to facilitate data mining studies. For the purposes of the analyses presented here, the monitored data of demography, CAG repeat, estimated age of onset, disease severity and functional ability as measured by the Unified Huntington’s Disease Rating Scale total motor score (UHDRS-TMS) and functional scales in combination with the CSRI has been utilised for data mining. Data was collected according to the standard REGISTRY protocol Data were analysed descriptively for demographics and according to percentage use of individual services and aggregates of service receipt namely 1) total number of individual formal services received 2) aggregates of hospital services (inpatient and outpatient) and primary and community care and 3) the carer or patient estimated total informal care hours  as recorded by the CSRI. As the focus on our investigations was to establish service use frequencies in the first instance and not costs per se, we do not report on investigations and diagnostic tests, average loss of income per week; journeys undertaken and occupation in these analyses.In order to identify specific factors associated with receipt of services at each visit, we utilised linear regression with estimated hours of informal care as the dependant variable and the measures of disease severity and functional ability as the independent variables.  All analyses were conducted using PASW version 18 Release 18.0.2 April 2010. CSRI data for 451 HD patients  was available for visit one (at enrolment to the study) of this observational study. Completed CSRI data for 105  and 47  HD patients was available for visits two and three respectively. The mean time between visits was 1.2 years. Countries represented were Austria (8.6%); Germany (33.7%); Italy (15.3%); Norway (3.3%); Poland (18.4%); Portugal (2.9%); Spain (10.6%); Switzerland (0.9%) and United Kingdom (6.2%). Mean (SD) estimated age of onset was 41.8 (12.8) years and age at visit 1 was 49.5 (13.1). Median (range) CAG repeat length was 45 (35-89). Median (range) scores at visit 1 on UHDRS-TMS, Functional assessment scale (FAS), Total Functional Capacity (TFC) and Independence Scale were 34 (0-106); 20 (0-25); 8 (0-13) and 80 (10-100) respectively. At Visit 2, median (range) scores on UHDRS-TMS, Functional assessment scale (FAS), Total Functional Capacity (TFC) and Independence Scale were 37 (0-111); 16 (0-25); 7 (0-13) and 75 (10-100) respectively.At Visit 3, median (range) scores on UHDRS-TMS, Functional assessment scale (FAS), Total Functional Capacity (TFC) and Independence Scale were 43 (0-97); 13 (0-25); 5 (0-13) and 70 (25-100) respectively. It is important to note the altered proportional representation of people with HD at each stage of the disease for each visit. At visit 1, most participants  were at Stage 1 of the disease; by visit 3 most participants  were at either stage 3 or 4 of the disease. Table 1 gives details of percentage of the sample at each stage of the disease for each visit. Table 1: Frequencies and valid percentages of HD patients at each visit stratified according to stage of disease (represented by TFC scores)  At visit 1, 335 of the 451 participants were in receipt of at least one formal hospital based service and 401 (89%) were in receipt of primary and community care services. In contrast, 94% of people used community based services at visit 3 and 62% used hospital based services. Fifty percent of individuals reported use of informal care in the home at visit 1; this increased to 68% at visits 2 and 3 (see Table 2).The increase in service use frequencies and care hours reported in visits 2 and 3 do however need to be considered in light of the greater proportion of people in stages 3 and 4 of the disease at visit 3 compared to those at visit 1 (see Table 1). Inpatient hospital service use increased dramatically over visits and outpatient service use decreased. Percentage of people in receipt of physiotherapy and speech and language therapy services increased at each visit; whilst social worker involvement remained relatively constant. Home help use increased over time. Notable is that occupational therapy, dentistry and other allied health professional services was not independently recorded in the version of CSRI used here and may well be under-reported in this situation. There was additionally no mention of key-worker involvement.     Table 2: Frequencies and valid percentages of HD patients receiving care services at each visit The use of wheelchairs increased over each visit, but all other appliance use remained fairly constant (see Table 3). Crutches and frames were used fairly infrequently whilst home adaptation  was a slightly more frequent occurrence.    Table 3: Frequencies and valid percentages of HD patients using aids and adaptations in the home at each visit The mean (SD) estimated weekly total informal care hours at visits 1, 2 and 3 were 32.8 (49.4); 21.6 (53.6) and 21.3 (62.4) respectively (see Table 3). Although the mean reported informal care hours were less at each visit; the mean number of informal care services used appeared to increase between visits 1 and 3. Mean number of formal primary and community services used  also appeared to increase over time whilst formal hospital based services reduced over time. This is consistent with what was seen in terms of percentages of HD patients using different services (see Table 2).  Table 4: Aggregates of service use for each visit2 8.6%; SE 59.9; F7.8 p<0.007) . At Visit 3, the Functional Assessment Scale accounted for 33 % of the variance in hours of informal care  .   Only the scores on the FAS accounted for the variance in the weekly total informal care hours at visit 1 (see Table 4). This remained a constant pattern over time. At Visit 2, the Functional Assessment Scale accounted for 9 % of the variance in hours of informal care  would be more appropriate in the context of an economic evaluation of a specific intervention Most HD patients surveyed as part of this study were in receipt of some formal primary and community care services and to a lesser extent hospital based services. Trends in service provision over time may be inferred from the longitudinal data presented but it is important to acknowledge that some bias may be introduced to the study simply as result of being a research participant in a large scale observational study such as REGISTRY. In addition, as the numbers of participants decrease with each visit, there is an associated change in proportional representation of people at each stage of the disease. We cannot infer that the cohort is deteriorating over time resulting in an increased service use; the changes in service use may simply be skewed by this proportional change in individuals at each stage of the disease. We therefore suggest that a general impression of service receipt is best judged at visit 1 (study entry) where any effect on reporting rates and the bias introduced by the annual visit or the altering proportional representation of the sample at each visit is limited. At enrolment to the REGISTRY study, 89% of HD patients assessed in this data mining study were in receipt of at least 1 form of primary or community care service. It is not possible to establish any measure of how integrated these services were and additionally there was no clear indication of key worker or occupational therapy involvement as this was not part of the standard questions employed in the REGISTRY CSRI. Social worker and nurse involvement was fairly minimal and use of formal home help was reported in only 11% of participants in this cohort. Approximately 1/5th of participants were in receipt of physiotherapy services at the first visit and the use of crutches and walking aids was infrequent. Such infrequent use of walking aids is congruent with suggestions in the literature that physiotherapists do not recommend routine use of aids as choreic movement and cognitive impairment may influence safe use of walking aids in HD patients When considering service use frequencies, it is useful to compare with other groups of patients with neurodegenerative diseases. A report (published in 2000) of community services for people with Multiple Sclerosis (MS) (n=150) living in the United Kingdom found that at that time of 45% of people with MS did not receive any form of community services This analysis is not without limitations. It is important to acknowledge that the descriptive data presented here is specific to this fairly small sample (to date REGISTRY has enrolled over 6000 participants so our data is only reflective of approximately 8 % of the HD population). Additionally, almost all of the participants enrolled in REGISTRY are able to reach a HD clinic for annual assessments and on review it was clear that only a small proportion of our sample were in the late stages of the disease.  It is therefore likely that the data presented here is not representative of the most severely affected HD patients.There will also be substantial differences across countries in terms of the support in the community that is available (i.e. service use is supply driven) and additionally there will clearly be inherent differences in health care systems between various European countries. The specific role that each member of the health care team may vary according to geographical region and this level of detail is not captured by the REGISTRY CSRI. Furthermore, there was no distinction between HD or non-HD related service use. It is difficult to say how much of the service use reported here is because of HD and there are many other factors which may lead a person to requiring formal health care services. Our data suggests a large reliance on informal care in the home in this group studied. At the first visit, half of all the HD patients in this study were in receipt of some manner of informal care in the home. It seems that informal carers will continue in this function up the very limits of their capacity and discussions with family members have highlighted some contributing factors. HD is seen as a family problem that ought to be managed by the involved family. Families trying to use available formal services encounter the problem of frequently changing care-givers and irregularity and restrictions in terms of the hours that the services can be provided. Such irregularity and frequent change is clearly not in agreement with the nature of HD and the person affected with HD. The HD patient is highly dependent on regularity and familiarity, making provision of community based care very challenging. Other studies of people with complex disability have suggested that where community care is limited, the burden of care often falls on the family and informal carers The data presented here is suggestive of the need for integrated and proactive care provision that addresses the requirements of the HD patients and their carers. It is clear that a method or a system to aid identification of factors indicative of the need for referral to formal services is required. Studies of the emotional experiences of family carers in HD and supportive care needs for people living with the disease highlight the high levels of emotional stress that can be associated with HD We would like to thank Dr Jonathan Gillard , Dr Lori Quinn , Dr Jenny Townhill (EHDN Language Co-ordination manager) and Professor David Cohen  for their helpful comments related to the manuscript presented here. Many thank to the contributors at the study sites that have diligently collected and entered data into the REGISTRY database (see Appendix 1).  Address for correspondenceMonica Bussebusseme@cardiff.ac.ukEmail: Department of Physiotherapy, School of Healthcare Studies, Cardiff University, Cardiff CF14 4XN, United Kingdom Competing InterestsThe authors have declared that no competing interests exist. Funding informationThere were no major sources of funding for this work. Each author is supported by their current individual institution.  Appendix 1Contributors  from the European Huntington’s disease Network to the data collected for analysis in this study are listed in alphabetical order by country below. AUSTRIAGraz : Raphael M. Bonelli; Brigitte Herranhof;  Anna Holl (formerly Hödl); Hans-Peter Kapfhammer; Michael Koppitz; Markus Magnet; Daniela Otti; Annamaria Painold; Karin Reisinger; Monika Scheibl; Karen Hecht; Sabine Lilek; Nicole Müller; Helmut Schöggl; Jasmin UllahInnsbruck : Florian Brugger; Caroline Hepperger; Anna Hotter; Philipp Mahlknecht; Michael Nocker; Klaus Seppi; Gregor Wenning; Lisa Buratti; Eva-Maria Hametner; Christiane Holas; Anna Hussl; Katharina Mair; Werner Poewe; Elisabeth Wolf;  Anja Zangerl; Eva-Maria Braunwarth   GERMANYAachen : Christoph Michael Kosinski; Daniela Probst; Christian Sass; Johannes  Schiefer; Christiane Schlangen; Cornelius J. WernerDinslaken (Reha Zentrum in Dinslaken im Gesundheitszentrums Lang): Herwig LangeDresden :  Andrea Friedrich; Ulrike Hunger; Matthias Löhle; Simone Schmidt;  Alexander Storch; Annett Wolz; Martin WolzFreiburg : Johann Lambeck, Birgit ZuckerHamburg : Kai Boelmans; Christos Ganos; Ute Hidding; Alexander Münchau; Michael Orth; Jenny Schmalfeld; Lars Stubbe; Simone ZittelHeiligenhafen (Psychatrium Heiligenhafen): Walburgis HeinickeMarburg KPP (Klinik für Psychiatrie und Psychotherapie Marburg-Süd): Bernhard LonginusMarburg Uni : Jens Carsten Möller; Ida RisslingMünchen (Huntington-Ambulanz im Neuro-Kopfzentrum - Klinikum rechts der Isar der Neurologischen Klinik und Poliklinik der Technischen Universität München): Alexander Peinemann; Michael Städtler; Adolf WeindlMünster : Natalie Bechtel; Heike Beckmann; Stefan Bohlen; Eva Hölzner; Herwig Lange; Ralf Reilmann; Stefanie Rohm, Silke Rumpf ; Sigrun Schepers Taufkirchen (Isar-Amper-Klinikum - Klinik Taufkirchen (Vils)): Antonie Beister; Matthias Dose; Kathrin Hammer; Janina Kieni; Gabriele Leythaeuser; Ralf Marquard; Tina Raab; Sven Richter; Amina Selimbegovic-Turkovic; Caroline Schrenk; Michele Schuierer; Alexandra Wiedemann Ulm : Andrea Buck; Julia Connemann; Daniel Ecker; Carolin Eschenbach; Christine Held; Bernhard Landwehrmeyer; Franziska Lezius; Solveig Nepper; Anke Niess; Michael Orth, Sigurd Süßmuth, Sonja Trautmann; Patrick Weydt ITALYBologna : Sabrina Capellari; Pietro Cortelli; Roberto Gallasi; Roberto Poda; Giovanni Rizzo; Cesa ScaglioneFlorence : Elisabetta Bertini; Elena Ghelli, Claudia Mechi; Marco Paganini; Silvia Piacentini; Silvia Pradella; Anna Maria Romoli; Sandro SorbiMilan :   Alberto Albanese; Stefano Di Donato; Cinzia Gellera; Silvia Genitrini; Caterina Mariotti; Daniela Monza; Lorenzo Nanetti; Domina Paridi; Paola Soliveri; Chiara TomaselloNaples : Giuseppe De Michele; Di Maio Luigi; Carlo Rinaldi; Cinzia Valeria Russo, Elena Salvatore; Tecla TucciPozzilli (Neurogenetics Unit – IRCCS Neuromed): Milena Canella; Valentina Codella, Francesca De Gregorio, Nunzia De Nicola, Tiziana Martino; Mariella Simonelli; Ferdinando Squitieri Rome : Anna Rita Bentivoglio; Alfonso Fasano; Marina Frontali; Arianna Guidubaldi; Tamara Ialongo; Gioia Jacopini; Giovanna Loria; Carla Piano; Silvia Romano; Francesco Soleti; Maria Spadaro; Paola Zinzi NORWAYOslo University Hospital : Arvid Heiberg; Marleen R van Walsem, Jan Frich; Raghild Wehus; Olaf Aaserud; Nancy BorgerødOslo University Hospital : Kathrine Bjørgo; Madelein Fannemel; Per Gørvell; Eirin Lorentzen; Susana Pro Koivisto; Lars Retterstøl; Torborg Overland; Bodil StokkeTrondheim : Inga Bjørnevoll; Sigrid Botne Sando POLANDGdansk : Emilia Sitek; Jaroslaw Slawek; Witold Soltan.Katowice : Magdalena Boczarska-Jedynak; Barbara Jasinska-Myga; Gregorz Opala.Krakow (Krakowska Akademia Neurologii): Krzysztof Banaszkiewicz; Andrzej Szczudlik; Monika Rudzińska; Magdalena Wójcik; Malgorzata Krawczyk.Poznan : Anna Bryl; Anna Ciesielska; Aneta Klimberg; Jerzy Marcinkowski; Justyna Sempołowicz; Daniel ZielonkaWarsaw-MU : Piotr Janik; Anna Kalbarczyk; Hubert Kwiecinski; Zygmunt Jamrozik.Warsaw-IPiN : Jakub Antczak; Katarzyna Jachinska; Maria Rakowicz; Przemyslaw Richter; Danuta Ryglewicz; Grzegorz Witkowski; Elzbieta Zdzienicka; Jacek Zaremba. PORTUGALLisbon-Santa Maria : Miguel Coelho; Leonor Guedes; Joaquim J Ferreira; Tiago Mendes; Tiago Mestre; Anabela Valadas.Lisbon-Fernando Fonseca : Cristina Costa;  Ângela Timóteo.Porto-Santo António : Sara Cavaco; Joana Damásio; Marina Magalhães Porto-São João : Miguel Gago; Carolina Garrett; Maria Rosália Guerra.  SPAINBadajoz : Carmen Durán Herrera; Patrocinio Moreno GarciaGranada : Francisco Barrero; Blas Morales Burgos : Esther Cubo; Natividad Mariscal; Jesús SánchezMadrid-Clinico : Rocío García-Ramos García; Purificacion Pin Quiroga; Clara VillanuevaMadrid RYC : Mónica Bascuñana; Marta Fatás Ventura; Guillermo García Ribas; Justo García de Yébenes; José Luis López – Sendón Moreno, Patricia Trigo Cubillo Madrid FJD (Madrid-Fundación Jiménez Díaz): Pedro J García Ruíz, Asunción Martínez-Descals, María José Saiz Artiga; Vicenta SánchezBarcelona-Hospital Mútua de Terrassa : Miquel Aguilar Barbera; Dolors Badenes Guia; Laura Casas Hernanz ; Judit López Catena; Ana Rojo Sebastián, Pilar Quiléz Ferrer; Gemma Tome CarruescoBarcelona-Bellvitge : Jordi Bas; Núria Busquets; Matilde CalopaPalma : Aranzazú Gorospe; Inés Legarda; Penelope Navas Arques; María José Torres Rodríguez, Barbara VivesSevilla : Jose Manuel Garcia Moreno; José Chacón Peña; Luminita Dinca Avarvarei; Antonio Martín Bastida; Maria Fernández Recio; Luis Redondo Vergé; Violeta Sánchez Sánchez SWITZERLANDBern: Jean-Marc Burgunder; Yvonne Burgunder ; ; Alain Kaelin; Irene Romero; Michael Schüpbach; Sabine Weber Zaugg  UNITED KINGDOMAberdeen : Roisin Jack; Kirsty Matheson; Zosia Miedzybrodzka; Daniela Rae; Sheila Simpson; Fiona Summers; Alexandra UreCardiff : Monica Busse, Stephen Dunnett, Catherine Clenaghan, Ruth Fullham, Sarah Hunt, Lesley Jones, Una Jones, Hanan Khalil, Sara Minster, Michael Owen, Kathleen Price, Jenny Townshill, Anne Rosser. Edinburgh : Paul A. De Sousa; Maureen Edwards; Teresa Hughes (Scottish Huntington´s Association); Marie McGill; Pauline Pearson; Mary Porteous; Paul Smith (Scottish Huntington´s Association); Adam ZemanLeicester : Colin Bourne; Carole Clayton; Heather Dipple; Jackie Clapton, Janet Grant; Diana Gross; Caroline Hallam; Julia Middleton; Ann Murch, Dawn PatinoLondon : Michael Patton, Maria PattersonManchester : Natalie Arran; Jenny Callaghan; David Craufurd; Ruth Fullam; Liz Howard; Susan Huson; Emma Oughton; Lucy Partington-Jones;); Julie Snowden; Andrea Sollom; Cheryl Stopford; Jennifer Thompson; Iris Trender-Gerhad; Nichola Verstraelen (formerly Ritchie); Leann WestmorelandSheffield : Oliver Bandmann; Alyson Bradbury, Paul Gill, Helen Fairtlough, Kay Fillingham, Isabella Foustanos; Kirsty O’Donovan; Nadia Peppa, Katherine Tidswell, Oliver Quarrell


Nasonia vitripennis and identified 90 sequences encoding putative OBPs. This is the largest OBP family so far reported in insects. We report unique features of the N. vitripennis OBPs, including the presence and evolutionary origin of a new subfamily of double-domain OBPs (consisting of two concatenated OBP domains), the loss of conserved cysteine residues and the expression of pseudogenes. This study also demonstrates the extremely dynamic evolution of the insect OBP family: (i) the number of different OBPs can vary greatly between species; (ii) the sequences are highly diverse, sometimes as a result of positive selection pressure with even the canonical cysteines being lost; (iii) new lineage specific domain arrangements can arise, such as the double domain OBP subfamily of wasps and mosquitoes.Insects are the most diverse group of animals on the planet, comprising over 90% of all metazoan life forms, and have adapted to a wide diversity of ecosystems in nearly all environments. They have evolved highly sensitive chemical senses that are central to their interaction with their environment and to communication between individuals. Understanding the molecular bases of insect olfaction is therefore of great importance from both a basic and applied perspective. Odorant binding proteins (OBPs) are some of most abundant proteins found in insect olfactory organs, where they are the first component of the olfactory transduction cascade, carrying odorant molecules to the olfactory receptors. We carried out a search for OBPs in the genome of the parasitoid wasp  Nasonia vitripennis, commonly known as the jewel wasp. For instance, their courtship behaviour is guided by male Chemical senses are central to the life history of the parasitoid wasp, Nasonia genus is mainly comprised of three closely related species of wasps. N. vitripennis  is found throughout the world and is estimated to have diverged from N. giraulti and N. longicornis approximately 1.0 million years ago (Mya). The three species are normally genetically isolated as the result of Wolbachia-induced cytoplasmic incompatibility and laboratory strains, cured of Wolbachia are interfertile, provide a useful source of genetic and sequence variation for mapping studies. Consequently, Nasonia serves as a useful model system, particularly for the study of the genetics of complex traits and for comparative developmental genetics. The genomes of three Nasonia species, N. vitripennis, N. giraulti and N. longicornis have been sequenced Nasonia) approximately 200 Mya ago. The phylogenetic position of Nasonia is therefore very important for studies of sequence changes along the apocritan Hymenoptera lineage, which comprises both social and parasitoid groups.The Nasonia genome sequences to gain insight into the evolution and diversity of the odorant binding protein (OBP) family. Insect OBPs are small, water soluble proteins present at a very high concentration (up to 10 mM) in the chemosensillum lymph bathing the dendrites of olfactory nerve cells in antennae We searched the Drosophila melanogaster, transcripts encoding two OBP dimers (DmelOBP83cd and Dmel83OBPef) have been identified, with two complete OBP domains each with six conserved cysteines . Some OBPs are thought to form homo- and hetero-dimers in vivoOBPs typically contain six highly conserved cysteine residues, forming disulphide bonds that stabilise the 3-D structures Nasonia genome paper N. vitripennis genome as the supplementary material N. vitripennis and describe the unique features and evolutionary origin of the wasp OBPs.The N. vitripennis (Nvit) assembly v1.0 N. vitripennis ESTs and the raw genomic traces did not yield any additional sequences; it is therefore likely that we have identified the full complement of OBP-like sequences in N. vitripennis. This is the largest OBP family reported so far in any insect species Apis mellifera has only 21 OBP genes Solenopsis invicta only 12 Acyrthosiphon pisum only 15 Nasonia species, all 90 sequences identified in N. vitripennis have a clear ortholog in N. giraulti and N. longicornis. Interestingly, we did not find any “plus-C” OBPs Nasonia OBPs belonging to the “classic” or the “minus-C” OBP subfamilies N. vitripennis OBP genes using the “NvitOBP” prefix followed by a number incremented from the 5′ end of their scaffold to the 3′ end, in increasing scaffold order (Genome wide searches of the ld order . Of the ld order .p = 1.065779e−6), suggesting that the replacement of one cysteine (if such event is not deleterious) reduces the selective constraints acting on their former partner.The highly conserved pattern of cysteine residues in insect OBPs is very important for the disulphide bonding that stabilise the folded OBP structures N. vitripenis sequences encoding putative OBPs (OBP38-OBP46 and OBP48) with only little sequence similarity with the OBPs of other insect species. However, these 10 OBPs have significant similarity with those of the other wasp OBPs, which are in turn related to the classical OBPs of other insects. Moreover, the 10 N. vitripenis genes are found in the middle of a genomic cluster located on scaffold 9 containing predominantly OBP genes (D. melanogaster (Dmel), such as DmelOBP83cd and DmelOBP83ef, which have two complete OBP domains with no cysteine losses. This domain organization fits well with the functional hypothesis of OBP dimerization We have identified 10 unusual BP genes . Nine ofst domain from positions 25 to 186 and the 2nd domain from 187 to ∼240 of the multiple sequence alignment  that appear to have different evolutionary histories. The phylogenetic analysis suggests that NvitOBP47 is a classic OBP, with just one domain, whilst the NvitOBP43 sequence clusters with the 1ain OBPs . FurtherNasonia ESTs revealed a region on Scaffold 9 , within the double domain OBP gene cluster, containing several ESTs that partially overlap two pseudogenes (NvitOBP33 and NvitOBP34) and one gene (NvitOBP35). Two of these ESTs span across NvitOBP33 and NvitOBP34, and 11 ESTs cover exons of both NvitOBP34 and NvitOBP35  N. vitripennis, with a putative origin from a single duplication/fusion event, as shown on Phylogenetic analyses revealed that all double-domain equences . There aaffold 9 , suggestNasonia includes the double-domain genes  (DmelObp59a-AgamOBP29-TcasOBP45-NvitOBP64 and DmelObp73a-ApisOBP4) (We have found three orthologs of melOBP5)  and two pisOBP4) . This hiN. vitripennis. This is the largest OBP gene family so far reported in insects and has allowed us to identify some of unique features and to be able to study evolution origin of these features. This exceptionally high number of OBPs mirrors the large number of genes encoding olfactory receptors in this species (over 200 ORs) Nasonia OBPs are truly involved in olfaction, and whether the OBP expansion in Nasonia is necessarily related to any olfactory role. Further experiments are needed to determine which Nasonia OBPs are expressed in olfactory tissues, and what type of molecule they carry.Using the OBP sequence motif search Nasonia OBPs belong to the “classic” subfamily with no “Plus-C” OBPs but a large number which have lost at least one of the characteristic six conserved cysteines. Our most striking finding is a group of double-domain OBPs with little sequence similarity to OBPs of other species but with substantial similarity with the other NvitOBPs. These double-domain OBPs are thus different from the fusion of two classic OBP domains in that one of the two domains has undergone extensive loss of the canonical cysteines and become equivalent to “minus-C OBPs”. This is supported by the finding that the two domains of these NvitOBPs have the same gene structure, similar to the closely related classic OBPs, and are monophyletic. The results suggest that double-domain OBPs evolved from a single event. It is possible these functional double-domain OBP proteins may have acquired an equivalent function to that performed by two ‘classic’ OBPs acting as dimmers.All Taken together, the multiple occurrences of canonical cysteine losses, and the independent emergence of double domain OBPs in wasps, flies and mosquitos, represent a striking case of convergent evolution of protein structures. The main challenge remains to understand the functional significance of these structural changes.N. vitripennis genomic scaffolds A list of all described OBP-like protein sequences was built as described in Forêt and Maleszka (2006). The sequences were then used to build models for PSI-BLAST N. vitripennis were homogenised in 250 µL genomic DNA extraction buffer  in 1.5-mL eppendof tubes. The extraction mixture was heated at 70°C for 30 min, mixed with 35 µL of 8 M KAc solution, incubated on ice for 30 min. The supernatant containing DNA was obtained by centrifugation at 13,000×g for 10 min, and then extracted further with 280 µL chloroform∶phenol 1∶1. The DNA sample was treated with 2 µL RNase (10 mg/mL) at 37°C for 15 min, extracted again with 250 µL chloroform, and finally DNA was precipitated with 2.5 volume of 100% ethanol.Whole adult N. vitripennis adults was extracted using Trizol reagent (Invitrogen) according to the manufacturer's protocol. Reverse transcriptions were carried out using SuperScript II (Invitrogen). The cDNA for RT-PCR was prepared with an oligo(dT)15 primers (Promega) and SuperScript™ II Reverse Transcriptase RNAase H− (Invitrogen Life Technologies), and used as the PCR template.RNA from whole 2+, 0.4 µl dNTP mixture (10 mmol/L), 0.5 µl each of forward and reverse primers (25 mmol/L), and 0.1 µl HotStarTaq® DNA polymerase  and were done in a Hybaid thermocycler with thermo-cycling program: 15 min at 95°C followed by 35 cycles of 40 sec at 94°C, 40 sec at 53°C, and 40 sec at 72°C, with 7 min at 72°C after the last cycle. The PCR primers are Primer_F1 (5′-CCCAGGATAAGCAGTTTAGCTG-′3) on the second exon of the 3′-end of NvitOBP33, Primer_R2 (3′-TCATTTTCGACGAGCTGCTG-5′) on the first exon of the 5′-end of NvitOBP34 and Primer_R1 (3′-ATTTAGAGGATTACTGCGACGC-5′) on the last exon of NvitOBP35.The PCR amplifications contained 14.5 µl sterile water, 2 µl (10×) PCR buffer including 15 mmol/L Mghttp://tree.bio.ed.ac.uk/software/figtree/). Analyses of selective constraints were conducted using the codeml program from the PAML v4 software package Amino acid sequences were aligned with MAFFT v6 We inferred cysteine loss events by minimizing the number of events given the OBP phylogenetic gene tree (a maximum parsimony criteria). We tested if the cysteine loss events had occurred at random or had preferentially involved disulphide bond-forming cysteines. Assuming that each OBP has 6 cysteines, forming three disulphide bonds, after losing the first cysteine the probability of a second loss in a ‘bond’ or ‘non-bond’ cysteine is q = 1/5 and p = 4/5, respectively. We can thus use a binomial distribution to compute the probability that given a number of X events involving two cysteine replacements Y (or more) involve cysteines forming disulphide bonds by the binomial distribution.We delimited the OBP domains using sequence similarity, splicing pattern and cysteine profile information. We first determined the approximate boundary of the two domains by a dotplot analysis Click here for additional data file.Figure S2PCR products of the overlapping transcript within a genomic region containing three OBP genes from the cDNA (Lane 1 and 3) and gDNA (Lane 2 and 4) with the primer pair crossing whole region containing three OBP genes (Lane 1 and 2), and with the primer pair crossing the region containing NvitOBP33 and NvitOBP34 (Lane 3 and 4).(DOCX)Click here for additional data file.Figure S3The derived protein sequences of all 90 Nasonia vitripennis OBPs.(PDF)Click here for additional data file.Figure S4The Coding DNA sequences and EMBL entries of all 90 Nasonia vitripennis OBPs.(DOCX)Click here for additional data file.Table S1Accession numbers of protein sequences cited in the manuscript.(PDF)Click here for additional data file.


We found that (i) all five mutations were beneficial for the background on which they occurred; (ii) interactions between mutations drove a diminishing returns type epistasis, whereby epistasis became increasingly antagonistic as the expected fitness of a genotype increased; and (iii) the adaptive landscape revealed by the mutation combinations was smooth, having a single global fitness peak. Here we examine how the environment influences epistasis by determining the interactions between the same mutations in two alternative environments, selected from among 1,920 screened environments, that produced the largest increase or decrease in fitness of the most derived genotype. Some general features of the interactions were consistent: mutations tended to remain beneficial and the overall pattern of epistasis was of diminishing returns. Other features depended on the environment; in particular, several mutations were deleterious when added to specific genotypes, indicating the presence of antagonistic interactions that were absent in the original selection environment. Antagonism was not caused by consistent pleiotropic effects of individual mutations but rather by changing interactions between mutations. Our results demonstrate that understanding adaptation in changing environments will require consideration of the combined effect of epistasis and pleiotropy across environments.The fitness effect of mutations can be influenced by their interactions with the environment, other mutations, or both. Previously, we constructed 32 ( = 2 Escherichia coli to one simple environment are altered when the strain is grown in two novel environments. We found that fitness effects were greatly influenced by both the genetic and external environments. In several instances a change in environment reversed the effect of a mutation from beneficial to deleterious or caused combinations of beneficial mutations to become deleterious. Our results suggest that a complex or fluctuating environment may favor combinations of mutations whose interactions may be less sensitive to external conditions.The fitness effect of beneficial mutations can depend on how they interact with their genetic and external environment. The form of these interactions is important because it can alter adaptive outcomes, selecting for or against certain combinations of beneficial mutations. Here, we examine how interactions between beneficial mutations favored during adaptation of a lab strain of  The extent to which mutations interact with their genetic background (epistasis) and the role such interactions play in evolution is not well understood In addition to interactions within a genome, mutations can also interact with the external environment Relatively few studies have examined how epistasis and plasticity combine to influence mutational effects. One study that did found that all of 18 transposon insertion mutations were affected by either epistasis or plasticity, with half being affected by both Escherichia coli evolution experiment 5) strains comprising all mutation combinations in the two environments with the most extreme opposing plasticity. These measurements allowed us to isolate effects of epistasis (GxG), interactions between mutations and the environment (GxE), and interactions between epistasis and the environment (GxGxE) on the fitness of defined genotypes. More generally, we investigated how the adaptive landscape, and the indirect consequences of each mutational step, might change with the external environment.Here, we expand this investigation of how the external environment affects epistatic interactions between five beneficial mutations that fixed in one population of a long-term rbs, t = topA, s = spoT, g = glmUS and p = pykF) in 1,920 different environments. On average, rtsgp exhibited enhanced respiration over the ancestor (mean = 84.37±1.39 (SEM) compared to 72.45±1.14 (SEM); paired t-test, 1919t = 26.28, P = <0.0001) with significant differences in 203 environments  of the ancestor and the strain containing five beneficial mutations . By contrast, rtsgp was less fit in DM25 supplemented with guanazole than in the selection environment (DM25+guanazole: fitness of 1.116±0.029 (95% CI); DM25: 1.299±0.061 (95% CI), 9t = −5.779, P = 0.0003).We focused on two environments that revealed large differences in respiration between rtsgp and the ancestor to examine how genotype and environment interact to affect fitness. The largest relative increase in respiration was in the presence of EGTA, a Carbs: 2,11F = 1.100, P = 0.367; topA: 2,8F = 15.506, P = 0.002; spoT: 2,10F = 31.389, P<0.0001; glmUS: 2,10F = 3.513, P = 0.070; pykF: 2,10F = 149.730, P<0.0001)  . In summThe above results demonstrate that the effect of individual mutations depend on the environment . However, it is also possible that interactions between mutations depend on the environment , which would further influence the topology of the fitness landscape and make it much more difficult to predict the influence of environmental changes on evolutionary outcomes. To examine G×G×E we measured the fitness of all combinations of the five beneficial mutations in the two focal environments .Fitness of each of the 32 genotypes comprising each mutation combination was quantified in both novel environments and compared with prior findings in the original selection environment mε = −0.039±0.046 (95% C.I.), 25t = −1.740, P = 0.094) (mε = 0.057±0.022 (95% C.I.), 25t = 5.303, P<0.0001) . The opposite effect was seen in the guanazole environment (mean higher-order deviation = −0.247±0.196 (95% C.I.), 15t = 2.693, P = 0.017).To further examine the patterns of epistasis in the novel environments we focused on the effect of epistasis in determining the fitness of individual genotypes , S6. In = 0.094) . In the <0.0001) . In tota<0.0001) , S5. Botr = −0.748) (r = −0.281)   and S6.  −0.281) . We alsopykF mutation explained a significant portion of the variation in the relationship between epistasis and expected fitness in the guanazole environment  whereas pykF altered epistasis to generally increase genotype fitness . Genotypes lacking the rbs mutation again displayed a strong negative correlation between epistasis and expected fitness (r = −0.783), but adding the rbs mutation weakened the negative association between epistasis and expected genotype fitness without changing mean epistasis among these genotypes .In the EGTA environment, considering genotypes distinguished by the presence or absence of either pistasis . The topWe used a higher-throughput approach using overall population growth AUC, see  as a proE. coli to a constant laboratory environment   , but in iotropy) . Since tiotropy) , environiotropy) .These interactions may be especially important in determining evolutionary outcomes given initially rugged fitness landscapes In summary, the combination of phenotypic plasticity and epistasis can strongly influence how an organism adapts to a new environment. Although the five mutations examined here would not likely be the same favored in these new environments, our results demonstrate that epistatic interactions are not static and can determine which trajectories are selectively accessible during an adaptive walk in a fluctuating environment. As a result, the fate of a mutation depends on its individual effect, epistasis with preexisting mutations and on interactions with the prevailing environment. With growing opportunities to survey dynamics of many genotypes within evolving populations, studies of both inherent properties of individual alleles and effects of their interactions in multiple conditions would address how frequently pleiotropy and epistasis guide adaptive evolution.E. coli have been propagated for more than 50,000 generations in Davis Minimal (DM) medium supplemented with 25 µg/ml glucose (DM25) in a long-term evolution experiment studying the dynamics and genetic basis of adaptation E. coli strains were grown in rich Tsoy liquid media overnight from −80°C freezer stocks. Aliquots of overnight culture were transferred to 10 mL DM25 media to precondition the cultures for 24 hours prior to growth curves or fitness assays.Twelve populations of To identify external environments that interact with the five mutations , the resSince differences in respiration do not necessarily reflect differences in growth or fitness, growth rates and in some cases relative fitness (see below) of the rtsgp strain was compared with the ancestral strain in a variety of these external environments to confirm that respiration was representative of growth or fitness. These follow-up growth rate assays were confirmatory and qualitative, not quantitative .+ ancestral strain (REL607) that is able to utilize the sugar arabinose. The arabinose utilization phenotype was found to be neutral in each of these competitions but allowed for the two different cell types to be easily distinguishable on TA agar plates.The fitness of each constructed strain was determined relative to the ancestor by direct competitions as described previously Competitors were pre-conditioned in the medium used for the competition for 24 hours prior to all competitions. Each competitor was then standardized based on OD600 values and added to the competition environment. Competitions were typically carried out for three days with a 1∶100 mixture transferred to fresh media every 24 hours. Since the fitness effect of some mutations was small, multiday fitness assays were used to amplify subtle advantages. Mixtures of competing strains were plated on TA agar at the start and end of each competition to determine fitness. Relative fitness (w) was calculated as the ratio of natural logarithms of realized growth by each competitor over three days of competition. Assays were typically carried out with five-fold replication and no less than three-fold replication.F2,6 = 0.667, P = 0.244; sgp, F2,6 = 0.047, P = 0.258). Notwithstanding the difference, relative features of the fitness landscape do not seem to have changed   ). We notRelative fitness, w, was calculated as described above based on the change in the relative density of strains in direct competition with one another. The terms that we use to describe and quantify epistasis were adopted from da Silva et al. Genotypes consisting of more than two adaptive mutations were further analyzed for net higher-order epistatic interactions, defined as epistasis that occurs between three or more mutations that cannot be explained as the result of constituent lower-order interactions. As a result, net higher-order epistasis was calculated by subtracting the effect of lower-order interactions as shown in t-test with the t-statistic calculated as the ratio of the mean relative fitness to its standard deviation and the degrees of freedom based on the number of replicate assays to determine significance . The Standard Major Axis regression (SMA) was used in this analysis. Solid grey lines represent 95% confidence intervals. The dotted line signifies unity between observed and expected fitness  and represents the null model assuming no epistasis.(PDF)Click here for additional data file.Figure S2Model II regression of observed and expected fitness under the multiplicative null model in the EGTA environment (red). The Standard Major Axis regression (SMA) was used in this analysis. Solid grey lines represent 95% confidence intervals. The dotted line signifies unity between observed and expected fitness  and represents the null model assuming no epistasis.(PDF)Click here for additional data file.Figure S3Relationships between relative epistasis and expected fitness assuming no epistasis in the guanazole environment. Symbol color and shape defines the genotypes either containing  or lacking  the mutation of interest defined in the top right-hand corner. ‘-interaction’ and ‘-mutation’ P-values indicate the reduction in ANCOVA model fit when the mutation×independent variable interaction or mutation main effect terms are dropped from the full model.(PDF)Click here for additional data file.Figure S4Relationships between relative epistasis and expected fitness assuming no epistasis in the EGTA environment. Symbol color and shape defines the genotypes either containing  or lacking  the mutation of interest defined in the top right-hand corner. ‘-interaction’ and ‘-mutation’ P-values indicate the reduction in ANCOVA model fit when the mutation×independent variable interaction or mutation main effect terms are dropped from the full model.(PDF)Click here for additional data file.Table S1Biolog environments displaying differences in respiration between rtsgp and the ancestral strain.(DOCX)Click here for additional data file.Table S2External environments exhibiting differences in respiration and growth between ancestor and rtsgp genotypes.(DOCX)Click here for additional data file.Table S3Changes in fitness along each mutational trajectory in the EGTA environment.(DOCX)Click here for additional data file.Table S4Changes in fitness along each mutational trajectory in the guanazole environment.(DOCX)Click here for additional data file.Table S5Epistatic interactions in DM25+EGTA.(DOCX)Click here for additional data file.Table S6Epistatic interactions in DM25+guanazole(DOCX)Click here for additional data file.Table S7Significant interactions identified by six-way ANOVA.(DOCX)Click here for additional data file.Table S8Variance partitioning of interaction effects of six-way ANOVA.(DOCX)Click here for additional data file.Table S9Observed versus expected relative growth of the gp genotype in different external environments displaying epistatic interactions.(DOCX)Click here for additional data file.Table S10Observed versus expected relative growth of the rtsgp genotype in different external environments displaying epistatic interactions.(DOCX)Click here for additional data file.


Therefore miniaturized bioreactors inside Petri dishes (30 mm in diameter), containing porous PCL or poly-dimethylsiloxane (PDMS) membranes, were developed to allow the interactions of different cells with defined pores to be investigated in situ during both static and perfusion cultures. Investigation of two different cell types  and how they interact with a range of pores (100–350 μm in diameter) for up to 50 days indicated that the cells either ‘covered’ or ‘bridged’ the pores. Three distinct behaviors were observed in the way cortical astrocytes interacted with pores, while fibroblasts were able to quickly bridge the pores based on consistent “joint efforts”. Our studies demonstrate that the distinct pore sealing behaviors of both cell types were influenced by pore size, initial cell density and culture period, but not by medium perfusion within the range of shear forces investigated. These findings form important basic data about the usability of pores within scaffolds that could inform the design and fabrication of suitable scaffolds for various applications in tissue engineering.Open pores to maintain nutrient diffusion and waste removal after cell colonization are crucial for the successful application of constructs based on assembled membranes, in our case tubular scaffolds made of ɛ-polycaprolactone (PCL), for use in tissue engineering. Due to the complex three-dimensional structure and large size of such scaffolds needed for transplantable tissues, it is difficult to investigate the cell–pore interactions  Accordingly, considerable research has been undertaken to mimic three-dimensional (3D) ECM for tissue engineering scaffolds by generating well-defined architectures including porosity, surface topography and chemistry for optimizing cell and tissue growth The extracellular matrix (ECM) plays an important role in controlling cell behaviors Previously by combining prior knowledge of cell responses to chemical in vitro simulators to mimic the internal configuration of the membrane based porous 3D scaffolds and predict the cellular behaviors within, which is crucial for the optimization of scaffold design The success of tissue engineering is dependent not only on the migration of cells within the scaffolds but also on their survival by the maintenance of nutrient diffusion throughout the scaffold after cell colonization In order to increase the generality and transferability of this research, and also because of the potential applications of the Swiss rolls to various areas of tissue engineering, two different cell types were selected in this research. As the major supportive glial cell in the central nervous system 22.17 cells per T75 flask) and culturing the cells in poly-l-lysine coated T75 flask for 10–12 days. The cells were maintained in DMEM  supplemented with 10% fetal bovine serum (FBS)  and l-glutamine . Confluent flasks were shaken on a rotary platform overnight at 37 °C to remove contaminating oligodendrocyte precursor cells. The remaining cells after this procedure were 85–95% type 1 astrocytes as judged by labeling for glial fibrillary acidic protein (GFAP), a cell type specific marker for astrocytes. The other 5–15% were mainly oligodendrocyte precursor cells and type 2 astrocytes, which were identified with immuno-fluorescent microscopy in preliminary experiments (shown as supplementary data). These cells had bi-, tri- or multi- polar morphologies and were apparent only at an early culture stage (4–5 days). After that, type 1 astrocytes gradually dominated the culture. The astrocytes were passaged no more than 4 times and detached using trypsin/EDTA (0.02% solution) for experiments when almost 100% confluent. hTERT fibroblasts  were cultured using the same medium as astrocytes and detached for experiments when approximately 90% confluent.As described previously 2.2The poly-dimethylsiloxane (PDMS) membranes with defined pores were prepared through a number of steps. Initially, silicon wafers with micro-pillars of different sizes  were fabricated as previously reported 5 cells/ml). After static incubation (37 °C and 5% CO2) for 2 h to allow full cell attachment on the upper side (cell seeding side), the cell-loaded membranes were incorporated into one of the mini-bioreactors as described in the next section.Prior to experimentation both the PDMS and PCL membranes were sterilized with 70% ethanol for 24 h, rinsed thoroughly with sterilized RO water, dried, treated with a Harrick Plasma Cleaner  at Hi settings  for 1 and 5 min respectively, coated with collagen I solution , a gift from Devro Medical, Glasgow, UK) for 15 min and then washed thoroughly with phosphate-buffered saline (PBS). After that the membranes were fixed in 6-well tissue culture plates with a silicone ring , seeded with 2 ml of cell suspension (approximately 1 × 102.31/2″) were inserted through the plastic O-ring and the bottom silicone spacer into the rectangular perfusion chamber , and used as inlet and outlet respectively. The cannulae were connected with an Ismatec peristaltic pump , a medium reservoir (2.5 ml) and a 3-way valve by silicone tubes (Inside diameter: 1 mm). The rectangular chamber was subjected to medium perfusion with varying flow/shear rates  Prior to assembly, all the components of the mini-bioreactors (except the cell-loaded porous membranes) were sterilized in 70% (v/v) ethanol/30% (v/v) sterile water overnight, then washed thoroughly with PBS, sterilized RO water and dried. After the cell-loaded porous membranes were incorporated, all the chambers were filled with 2–3 ml of medium and placed in a larger sterilized Petri dish (9 cm in diameter) filled with 95% air/5% CO2.4® Red 580  stock solution (1 mm) was added into the culture medium in the module chamber to a final concentration of 100 nm. After 45 min incubation the cells were imaged using a fluorescence microscope.Cells cultured in the modules were also analysed using live cell fluorescent microscopy. Briefly, MitoTracker33.1Cortical astrocytes were loaded onto PCL membranes with defined pores (diameter: 300 μm) in static culture module I and monitored using time-lapse phase contrast microscope. Due to the translucent nature of the PCL membrane, cells on the non-pore areas were hard to observe, thus only the cells within the pores could be followed. Elongated single cells, large membranes of multiple cells or combination of both were found crossing 10–20% of the pores within 4 days culture as shown in 3.2In order to observe cells both on non-pore areas and within the pores, astrocytes were cultured on PDMS membranes with defined pore features in static culture modules I and II, monitored using time-lapse microscopy. Due to the transparency of PDMS membranes, the cells on the non-pore areas B–C, closFurther investigation indicated that two different approaches were employed by cells to interact with the pores: they either ‘covered’ the pores without migrating inside (Approach I) or ‘bridged’ the pores by migrating inside and attaching onto the inner aspects of the pore (Approach II) as illustrated in 3.3In static culture (module I and II) individual fibroblasts were observed to migrate inside the pores, remaining attached onto the edge with both ends (Approach I), spanning the empty space whilst actively sliding across the pores by elongation and contraction. Most of the elongated single fibroblasts “ripped” due to further stretching when PDMS membranes with larger pores (>150 μm in diameter) were used for culture. The “ripped” cells were observed to immediately re-attach onto the edges of the pores starting another round of sliding. As culture progressed, more and more cells migrated into the pores, attached to each other, and/or to the pore edge and formed a circular membrane along the edge. This membrane/cell cluster gradually increased in size and sealed the pores even those ≥300 μm in diameter within 2–4 weeks culture A. Quanti3.4Astrocytes and fibroblasts were seeded onto separate PDMS membranes with defined pores, statically cultured in the perfusion culture modules allowing cells to ‘cover’ or ‘bridge’ the pores, then perfused continuously with different flow/shear rates see  for vary3.55 cells/ml) were seeded separately inside silicone tubes  with different sizes (Inside diameter (I.D.): 0.57, 1.25, 2.06, 3, 4, 5, 9 mm, length: 3 mm) and cultured for 2 weeks by keeping the silicone tubes suspended horizontally in medium. The silicone tubes were then positioned vertically in the medium for live cell imaging. The different ‘bridging’ abilities of two types of cells were observed: fibroblasts were able to ‘bridge’ up to 3 mm wide tubes while astrocytes were able to ‘bridge’ tube with a diameter up to 2.06 mm as illustrated in To further investigate their bridging behaviors, fibroblasts and astrocytes  or ‘bridge’ the pores by migrating inside whilst attaching onto the edge (Approach II) as illustrated in In cultures of cortical astrocytes the interactions of cells with the pores could be classified to happen in three distinctive stages:Stage I: Individual oligodendrocyte precursor cells or type 2 astrocytes partly ‘covered’ or ‘bridged’ the pores using approaches I and II, but all these cells died off eventually.Stage II: Type 1-astrocyte colonies gradually dominated the culture and ‘covered’ some of the pores. However, before the cultures were confluent, the astrocyte sheets that partly covered the pores were always pulled away from the pores, or the freely suspended cell membrane was “torn apart” by the apparent pull of other astrocytes within the same colonies, but on non-porous areas, that migrated away from the pores.Stage III: When the cultures were confluent, astrocytes migrated into the pores, formed circular membranes with very regular and apparently tensed structures, and seemed to pull other astrocytes into the pores.Fibroblasts interacted with the pores in a way that was very different from that observed from cortical astrocyte cultures. First of all, fibroblasts mainly ‘bridged’ the pores after migrating inside these (approach II). Secondly, the fibroblasts were quicker to seal the pores, which was similar to previous observations using human dermal fibroblasts in vivo should remain open to allow nutrient diffusion as the neo-tissue forms One similarity between astrocytes and fibroblasts was that all the freely suspended multicellular structures that formed within the pores were robust. The structures either cell type formed were very resilient to medium perfusion. The formation of freely suspended cell-bridges was not obviously influenced, or even prevented by medium perfusion within the flow regime investigated. This cellular resilience was a surprise to us as perfusion was initially expected to be an efficient strategy to prevent the pores from being sealed by the ‘fragile’ cells. Ideally, some of the pores in 3D scaffold transplanted Fibroblasts are common cells in many tissues and are usually involved in various wound healing processes where they migrate into a wound, surround and even invade the grafted tissues The complex interactions of different cell types especially fibroblasts with defined pore features have been previously investigated using silicon nitride 5This research demonstrated that the two different cell types investigated showed time and density dependent variant pore sealing behaviors. These were influenced by pore size, initial cell density and culture period, but not obviously by medium perfusion within the range of shear forces investigated. Fibroblasts bridge pores given time - of almost any size, interact with the inner aspect of the pore and migrate across from one side of the membrane to the other. Astrocytes interact with the pores and bridge these as well, but they need to be almost confluent on the areas surrounding the pore before they start to seal pores smaller than about 100 μm as a sheet, that closes iris-like. These findings are important basic data about the usability of pores to keep mass transport routes open within scaffolds that could inform the design and fabrication of suitable scaffolds for various applications in tissue engineering. The methodology developed here, specifically the combining of a thin layer of a porous polymer membrane with the miniaturized static and perfusion culture modules that are accessible to various non-invasive microscopies could be further used to systematically investigate the interactions of other relevant cell types with defined pore features before a tissue specific scaffold is fabricated and thus speed up the engineering of designed scaffolds for tissue engineering.


Although chronic pain affects all age ranges, it is particularly common in the elderly. One potential explanation for the high prevalence of chronic pain in the older population is impaired functioning of the descending pain inhibitory system which can be studied in humans using conditioned pain modulation (CPM) paradigms. In this study we investigated (i) the influence of age on CPM and (ii) the role of expectations, depression and gender as potential modulating variables of an age-related change in CPM. 64 healthy volunteers of three different age groups  were studied using a classical CPM paradigm that combined moderate heat pain stimuli to the right forearm as test stimuli (TS) and immersion of the contralateral foot into ice water as the conditioning stimulus (CS). The CPM response showed an age-dependent decline with strong CPM responses in young adults but no significant CPM responses in middle-aged and older adults. These age-related changes in CPM responses could not be explained by expectations of pain relief or depression. Furthermore, changes in CPM responses did not differ between men and women. Our results strongly support the notion of a genuine deterioration of descending pain inhibitory mechanisms with age. Chronic pain represents one of the largest medical health problems in the developed world and affects about 19% of the adult population The descending pain modulatory system can selectively modulate pain by either inhibiting or facilitating nociceptive processing Given the high prevalence of pain among the elderly, surprisingly few studies have investigated age-dependent changes in endogenous pain control mechanisms so far. Previous CPM studies involving older participants support the notion that the capacity of endogenous pain control may decrease with age 78 potential participants of three different age groups  were recruited locally and enrolled in the study if they fulfilled the following inclusion criteria: (1) between the age of 18 and 80 years, (2) free from chronic pain, (3) not currently using any prescription analgesics, tranquilizers, antidepressants, anticonvulsants, (4) not pregnant. Of 78 potential participants, 14 were excluded from the final data analysis for the following reasons: 11 subjects did not finish the protocol (they withdrew from the experiment during the cold pressor task) and 3 subjects did not meet the inclusion criteria at the day of the experiment (HADS Anxiety ≥11). Thus data analyses were based on 64 complete data sets: 22 young adults , 17 middle-aged adults  and 25 older adults . The study was conducted in accordance with the Declaration of Helsinki and was approved by the local Ethics Committee of the Medical Council of Hamburg. All participants gave written informed consent and were free to withdraw from the study at any time.In this study we used a well-established CPM paradigm All participants were instructed using a standardized protocol. Participants were told that the purpose of the study was to characterize possible differences in the perception of two simultaneously applied painful stimuli comparing healthy participants of different age groups . First, participants were informed about the sequence of experimental procedures. After these general instructions, subjects filled in the HADS questionnaire and an assessment of acute pain was performed. If they fulfilled all inclusion criteria (see 2.1) a calibration procedure was performed to determine the individual temperatures corresponding to a pain level of 50–60 on a 0–100 visual analogue scale . To this end, we pseudo-randomly applied six stimuli á six seconds each with different intensities ranging from 45.5–49.5°C to the right volar forearm, every temperature was presented once. Participants were asked to rate the intensity of each stimulus on a VAS which was presented on a computer screen in front of the subjects and ranged from 0 = “no sensation” to 100 = “most intense pain imaginable”. Two vertical white lines represented the two endpoints 0 and 100 of the VAS, a third white line was set at 25 labeled as “pain threshold”. Subjects indicated the pain intensity of each heat pain stimulus by moving a red bar between the two endpoints using two buttons of a computer mouse. The maximum stimulation temperature was restricted to 49.5°C in order to avoid any tissue damage. This calibration procedure ensured that all participants perceived the phasic heat pain stimuli  as equally painful (VAS 50–60).www.neurobs.com).The application of the thermal stimuli, the presentation of the VAS and the recording of behavioral data was performed using the software Presentation  and the last six stimuli  were applied without any other concomitant procedures. During the application of test stimuli seven to twelve ( = block II), the conditioning stimulus was applied.We used phasic heat pain stimuli as test stimuli (TS). The test stimuli were applied to the right volar forearm  of the participants using a 30×30 mm Peltier-Thermode . Each stimulus had a duration of six seconds . Pain ratings on the VAS were obtained immediately after each stimulus. A total of 18 test stimuli were applied. The first ( = block I), a message on the computer screen prompted the participants to immerse their left foot into a bath with ice water (∼0°C). The intensity of the conditioning stimulus was rated once in the middle of the cold pressor task  using a VAS presented on a computer screen with the same endpoint labels 0 = “no sensation” and 100 = “most intense pain imaginable” and a third white line set at 25 labeled as “pain threshold”. At the end of block II another message on the computer screen instructed the participants to take their foot out of the ice water. Prior to the experiment subjects were asked to focus their attention on the heat stimuli applied to the arm while having their foot immersed into the ice water and it was pointed out again that they could withdraw from the experiment at any time by telling the supervising experimenter. Finally heat pain stimuli 13–18 (block III) were applied without concomitant painful stimulation to the foot.A cold pressor task was used as the conditioning stimulus (CS). After completion of the first block of six heat pain stimuli , 50 = “no change” ( = no change of heat pain at the arm during the cold pressor task), and 100 = “maximum pain” ( = pain applied to the arm would get worse during the cold pressor task and when marking 100 the experiment would not be continued).The Hospital Anxiety and Depression Scale (HADS) As in previous studies To test if there was a significant CPM response in each of the three different age groups, we conducted three separate one sample t-tests for every single age group. Group differences of CPM responses between the three age groups were analyzed using non-parametric Kruskal-Wallis tests as variables in the different age groups were not normally distributed. Significant main effects were followed by post-hoc Mann-Whitney U tests.P<0.05 (two-tailed) was considered statistically significant. Post-hoc Mann-Whitney U tests and t-tests for applied stimulation temperatures, depression scores and CPM responses were conducted using Bonferroni adjusted alpha levels of 0.017 per test (0.05/3) accounting for every independent test within in each age group.One way analysis of variance (ANOVA) or the appropriate non-parametric tests  for non-normally distributed variables were used to test for potential differences in pain intensity ratings of the test (block I) and conditioning stimuli (cold pressor task) across groups. The effect of age on CPM response was assessed using a linear regression model with CPM response as dependent variable and age as independent variable. To test for further modulatory influences on the CPM response, we used stepwise multiple regression analysis with CPM response as dependent variable and age, expectation, depression and gender as independent variables ( = predictors). Relationships between the different variables were explored using Pearson product moment correlations and hierarchical regression analyses. Prior to the hypothesis-driven analyses of age-dependent differences of the CPM response we tested for potential differences in mean pain ratings of test stimuli in block I, of the conditioning stimulus and of the applied stimulation temperatures between the three different groups which could account for possible differences in CPM responses between the three groups. There were no group differences in mean pain ratings of test stimuli in block I  = 1.559, p = 0.219) or in pain ratings for the conditioning stimuli  = 0.327, p = 0.722). In line with the known age-dependent increase in pain thresholds Also, expectation regarding the effect of the conditioning stimulus on the test stimuli did not differ between the different age groups (H(2) = 3.3 p = 0.188).Depression scores differed significantly between the three age groups (H(2) = 12.7, p = 0.002). Post hoc Mann-Whitney U Tests showed significant lower depression scores for young participants compared to middle-aged  and older adults  whereas no differences between middle-aged and older adults were found .As hypothesized CPM responses differed significantly between the three age groups (H(2) = 11.4, p = 0.003, see Post-hoc Mann-Whitney U tests revealed significant differences between young and middle-aged adults  and between young and older adults , whereas no group differences between middle-aged and older adults could be detected .Looking at the group-specific CPM responses in more detail, we found that young adults showed a highly significant CPM response  = 4.3, p<0.001, one-sample t-test), whereas middle-aged  and older adults  = 0.8, p = 0.411, one-sample t-test) did not show significant CPM responses.Additionally we tested whether the decline in CPM response could be predicted from age using a linear regression analysis. This analysis revealed a significant age-dependent reduction of CPM responses: β = −0.406, t(61) = 4.796, p<0.001. Thus, age explained a significant proportion of variance in CPM responses, R2 = 0.165, F = 12.231, p = 0.001 see .When using another strategy of calculating the CPM response, i.e. comparing the mean pain ratings of block I and block II, very similar results were observed: Here again CPM responses differed significantly between the three age groups  = 8.706, p<0.001). Post hoc t-tests showed significant differences between young and middle-aged adults (t(37) = 3.823, p<0.001) and between young and older adults (t(45) = 3.198, p = 0.003), whereas no differences between middle-aged and older adults could be detected (t(40) = −0.743, p = 0.462).Moreover, young adults showed a highly significant CPM response  = 4.56, p<0.001, one-sample t-test) whereas middle-aged  = −0.726, p = 0.48) and older adults  = 0.39, p = 0.70) did not.To identify potential modulating factors regarding the CPM response in more detail, we conducted correlation analyses for CPM response and expectation, gender and depression. We solely found a significant relationship between CPM responses and depression . However, the stepwise multiple regression analysis (predictors tested: age and depression) revealed that depression ( = HADS depression score) had no additional influence on CPM responses over and above the influence of age. This might be due to the significant positive relationship between age and depression .The other correlations did not reach statistical significance  and were therefore not included in the above mentioned multiple regression analysis.In this study we investigated the influence of age on CPM responses and the role of potentially modulating variables, including expectation, depression and gender. Using a well-established CPM-paradigm in three age groups of healthy participants we showed a significant decrease of CPM responses on suprathreshold thermal pain stimuli with age. Importantly, this age-dependent decline in endogenous pain modulatory capacity could not be explained by other factors such as expectation, depression or gender.Our results of an age-dependent decline in CPM responses confirm previous observations showing an age-dependent reduction of CPM responses Previous studies have used CPM responses as a valuable marker of endogenous pain modulatory capacity. For instance, variations in CPM responses have been shown to predict the risk for developing chronic pain after certain operations The DNIC response that has been characterized in animals represents a basal physiological response that occurs even without the contribution of cortical brain areas However, these observations do not allow for conclusions regarding the role of implicit, not manipulated expectations the participants or patients may hold regarding the CPM procedure. To address this issue, we obtained individual expectancy ratings of how the cold pressor task may influence the perception of the test stimulus prior to the CPM procedure. Our findings show that the expectation does not significantly influence CPM responses and that CPM-related expectations do not change with age. These findings are at least partly in line with a previous study by Larivière et al. who also concluded from their data that the age-dependent reduction of CPM responses cannot be explained by changes in expectation A second factor that is known to substantially modulate pain perception is depression In addition to cognitive-affective factors, CPM responses might be gender-sensitive. Numerous studies have shown that the prevalence of chronic pain is higher in women than in men The underlying mechanisms in the generation of CPM responses in humans include spinal There is ample evidence for a decrease in brain volume with age Another important factor might be the reduction in neurotransmitter availability in the elderly. The literature supports a role for both opoidergic In summary, we show that CPM responses which are indicative of endogenous pain modulation, decline with increasing age. This effect could not be explained by expectation, depression or gender. Our data therefore provide further evidence for a genuine age-dependent change of the descending pain modulatory system. In combination with insights into altered neurotransmitter availability in the elderly, our data could allow for a better understanding of neurophysiological mechanisms underlying pain in the elderly and help the development of improved treatment options for the growing number of older individuals in our population to counteract its detrimental consequences.


We previously demonstrated that aged ovariectomized rats that had received prior estradiol treatment in middle-age exhibited increased levels of estrogen receptor alpha (ERα) in the hippocampus as well as enhanced hippocampal dependent memory as compared to aged rats that had not received mid-life estradiol treatment. These effects persisted long after the estradiol treatment had been terminated. The goal of the current experiment was to determine if increased expression of ERα in the hippocampus, in the absence of exogenously administered estrogens, can impact the hippocampus and cognitive function in aging ovariectomized rats. Middle-aged rats were trained for 24 days on an eight-arm radial maze spatial memory task. All rats were then ovariectomized. Forty days later, rats received either lentiviral delivery to the hippocampus of the gene encoding ERα (lenti-ERα) or a control virus. Rats were tested on delay trials in the radial-maze in which delays of varying lengths were imposed between the fourth and fifth arm choices. Following behavior testing, hippocampi were immunostained using western blotting for ERα, the ERα-regulated protein choline acetyltransferase, and phosphorylation of the ERα-regulated kinases, ERK/MAPK and Akt. Results revealed that aging ovariectomized rats that received delivery of lenti-ERα to the hippocampus exhibited enhanced spatial memory as indicated by increased arm-choice accuracy across delays as compared to ovariectomized rats that received control virus. Western blot data revealed that lenti-ERα delivery significantly increased levels of ERα and phosphorylated ERK/MAPK and had no impact on levels of ChAT or phosphorylation of Akt. Results indicate that increasing hippocampal levels of ERα in aging females in the absence of ovarian or exogenously administered estrogens leads to increases in phosphorylation of ERK/MAPK as well as in enhanced memory. Results of basic research conducted over the last two decades support a role for estrogens and estrogen receptors in the modulation of learning and memory Results of our investigation into the long-term impact on the brain and memory of short-term exposure to estrogens in middle-age are consistent with a role for ERα in female cognitive aging. Aged ovariectomized rats that previously had been treated with estradiol for 40 days during middle-age displayed enhanced memory performance as compared to aged ovariectomized rats that had no estradiol treatment The hypothesis that hippocampal ERα can positively impact cognition in the absence of ovarian or endogenously administered estrogens is supported by data in which viral-vector delivery of ERα to the hippocampus of young adult ovariectomized ERα knockout mice improved performance on a task of spatial learning and memory 6 transducing units of virus in 0.5 µl) were delivered per hippocampus. Strong expression was observed within 9 days after injection. We confirmed that transfection efficacy in rats was similar to what we previously reported for transduction of ERα in ERαKO mice Before using our lenti-viral vectors to test our hypotheses, we examined their expression in rat hippocampus to determine if the transfection efficacy in rats was similar to what we previously reported for transduction of ERα in ERαKO mice After confirming efficacy of our viral vectors, we purchased animals that were used to test our hypotheses. Fourteen middle-aged female Long-Evans hooded rats, retired breeders (∼11 months of age), were purchased from Harlan Sprague Dawley Inc. . All animals used in the study had experienced multiple pregnancies. Whether results of the present study would generalize to results obtained using virgin rats remains to be determined because reproductive histories of rats affect the hippocampus and hippocampus-dependent behavior This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Procedures were approved by the Institutional Animal Care and Use Committee of Tulane University (Permit Number: 0393). All surgery was performed under anesthesia induced by ketamine and xylazine and all efforts were made to minimize suffering.One week after arrival, rats were placed on diets to maintain body weights at 85–90% of pre-surgery weights and were trained to obtain food rewards  from the arms of an elevated eight-arm radial maze purchased from Lafayette Instruments . The maze consisted of black metal floors and clear acrylic walls with arms (10 cm wide × 70 cm long × 20 cm high) extending out from an octagonal center (33 cm across). The maze was located in the center of a 3×5 m room and raised approximately 1 m from the floor. Several extramaze cues, including overhead fluorescent lights, desk, chairs, sink, and door, were visible from the maze.To begin a trial, a rat was placed in the center compartment in a pseudorandom orientation and had access to all eight arms. Arm choices were recorded by an observer seated in a fixed location approximately 1 m away from the maze. An arm choice was scored if the rat traversed half the length of an arm. Food rewards, located in recessed cups at the end of each arm, were not visible until the rat reached the end of an arm. To control for possible odor cues, each cup contained froot loop residue that was present throughout the experiment. An arm choice was scored if the rat traversed half the length of an arm. Rats were allowed to choose arms in any order until all arms had been visited or 5 min had elapsed. Errors were reentries into previously visited arms. Performance was assessed by the number of errors of the first eight arm choices. Each animal received one trial per day across 24 days of acquisition.Following radial maze acquisition, rats were ovariectomized while under anesthesia induced by injection of ketamine  and xylazine . Rats were trained on the radial maze acquisition task once per week to retain performance levels until testing (see below).), the current infusion sites were chosen to provide intense coverage in CA1 and dentate gyrus of the dorsal hippocampus, with some expression extending into CA3. Rats were given two weeks to recover and to allow for optimal expression of the virus before behavioral testing.Forty days after ovariectomy, a time period of ovarian hormone deprivation that we previously showed significantly decreases hippocampal levels of ERα Rats were re-trained in the maze for two days using the same acquisition protocol as described above. Performance of all rats was at pre-surgery levels as measured by number of errors of the first 8 choices. Additionally, the rate at which rats completed the maze task was also at pre-surgery levels, indicating that viral infusions did not impact motor or other non-mnemonic functions. Behavioral testing consisted of daily delay trials in the radial maze during which various delays (1 minute –6 hours) were imposed between the fourth and fifth arm choices to increase memory load Approximately two days following behavior testing, all behaviorally tested animals were killed and the dorsal hippocampus from each hemisphere of each rat was dissected on ice, quick-frozen on dry ice, and stored at −80°C until processing. Tissue was homogenized in 15 µl/mg lysis buffer containing 1 mM EGTA, 1 mM EDTA, 20 mM Tris, 1 mM sodium pyrophosphate tetrabasic decahydrate, 4 mM 4-nitrophenyl phosphate disodium salt hexahydrate, 0.1 µM microcystin, and 1% protease inhibitor cocktail (Sigma-Aldrich). Samples were centrifuged for 15 min at 1000×g at 4°C, protein concentration of supernatants was determined , and each sample was diluted 1∶1 with Laemmli Sample Buffer  mixed with 350 mM D,L-dithiothreitol, boiled for 5 min, and stored at −80°C.For each sample obtained from each rat, 35 µg of total protein were loaded and separated at 200 V on 7.5%  or 10%  SDS-PAGE gels (Bio-Rad) for 60 min. Molecular weight markers  were included with each run. The specificity of the ERα antibody used was previously determined Two procedures were conducted to confirm endocrine status. First, daily vaginal smears were collected by lavage during the final 2 weeks before stereotaxic surgery. Slides were stained with 0.2% toluidine blue, and microscopically examined. Smears of all ovariectomized rats were characterized by a predominance of leukocytes. Second, at the time the rats were killed, 1-cm-long sections of the right uterine horn (cut at the base) were extracted and weighed. All rats presented with atrophied uterine horns, confirming success of ovariectomies.Arm-choice accuracy data  from each delay were averaged across the two days of testing and analyzed by two-ANOVA (treatment x delay) with treatment as the between subjects factor and repeated measures on delay. Western blotting data were analyzed by independent sample t-tests with treatment as the factor. = 3.885, P = .077) indicating greater accuracy in the Lenti-ERα rats  = 2.665, P = .033) on arm-choice accuracy indicating that increasing the delay negatively impacted accuracy  = 5.136, P = .047) indicating significantly greater accuracy in the Lenti-ERα rats for information that had to be remembered across delays (As illustrated in s delays . There ws delays . There w(10) = 2.519, P = 0.03) for protein levels of ERα, indicating that rats that received lenti-ERα infusions had significantly increased levels of ERα in the hippocampus as compared to rats that received control lenti-cherry infusions. There was no significant effect of treatment on protein levels of β-actin.As illustrated in As illustrated in (10) = 2.321, P = 0.043) for protein levels of phosphorylated p42 MAPK, indicating that rats that received lenti-ERα infusions had significantly increased levels of phosphorylated p42 MAPK in the hippocampus compared to rats that received control lenti-cherry infusions (As illustrated in nfusions . There wWestern blots revealed bands of phosphorylated and total Akt-like immunoreactivity at approximately 60 kDa. There was no significant effect of treatment for protein levels of phosphorylated or total Akt .The results of the current experiment demonstrate for the first time that increasing levels of ERα in the hippocampus of aging females in the absence of ovarian or endogenously administered estrogens increases activation an ERα-regulated kinase and enhances cognitive function. Lenti-viral delivery of the gene encoding ERα to the hippocampus of middle-aged ovariectomized rats resulted in significantly improved ability to remember information across time delays in a hippocampus-dependent radial-arm maze task as compared to hippocampal delivery of a control virus. In addition to impacting cognition, increasing hippocampal levels of ERα resulted in increased levels of phosphorylated ERK/MAPK, regulation of which is mediated by ERα signaling. These data provide support for the hypothesis that treatments that increase or maintain levels of ERα in the hippocampus in aging females are beneficial to the hippocampus and hippocampus-dependent behavior. Importantly, they demonstrate that cognitive benefits exerted by increased levels of ERα present following the cessation of ovarian function do not require administration of estrogens to be expressed.The current data demonstrate that increasing levels of hippocampal ERα in ovariectomized rats can impact learning and memory in a similar manner as does exogenous administration of estrogens. For example, like the effects of viral-vector delivery of ERα to aging ovariectomized rats in the current experiment, estradiol administration to young adult In the absence of ovarian or exogenously administered estrogens, ERα may be activated by novel mechanisms involving ligand-dependent and ligand-independent actions. For example, ERα may be activated via estradiol synthesized locally in the hippocampus In addition to enhancing cognition, increasing hippocampal levels of ERα via viral vector delivery increased phosphorylation of ERK/MAPK in the hippocampus, results consistent with the possibility of novel mechanisms involving ligand-dependent and/or ligand-independent actions at ERα. For example, hippocampus-derived estradiol is hypothesized to act locally at membrane estrogen receptors, including ERα In the current study, we saw no effect of viral vector delivery of ERα to the hippocampus on levels of ChAT. These results are in contrast to our previous results in which in parallel to increases in ERα induced by a previous short-term exposure to estradiol, aging ovariectomized rats also displayed increases in levels of ChAT Although the mechanisms are as not yet determined, there is mounting evidence that the expression or function of ERα in the hippocampus is selectively decreased during aging. In rats, aged females had a 50% reduction in the number of synapses in CA1 of the hippocampus that contained ERα immunoreactivity as compared to young females and the ERα levels were responsive to estradiol treatment in young, but not old rats A decline in ERα expression or function may mediate the “critical period” of estrogen effects In conclusion, these data demonstrate that increasing levels of ERα in the hippocampus following the cessation of ovarian function enhances hippocampus-dependent memory and increases phosphorylation of ERK/MAPK. Further, they represent the first demonstration that estrogen receptor can impact the hippocampus and cognition in aging females in the absence of ovarian steroids or exogenously administered estrogens. The current data add to a growing body of evidence that changes in levels of ERα that occur following the cessation of ovarian function in the aging female contribute to age-related cognitive decline.