RFA-AA-00-001: TARGETED MUTATIONS TO STUDY ETHANOL-RELATED BEHAVIORS

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TARGETED MUTATIONS TO STUDY ETHANOL-RELATED BEHAVIORS Release Date: January 18, 2000 RFA: AA-00-001 National Institute on Alcohol Abuse and Alcoholism Letter of Intent Receipt Date: April 5, 2000 Application Receipt Date: May 5, 2000 THIS REQUEST FOR APPLICATIONS (RFA) USES THE MODULAR GRANT AND JUST-IN- TIME CONCEPTS. IT INCLUDES DETAILED MODIFICATIONS TO STANDARD APPLICATION INSTRUCTIONS THAT MUST BE USED WHEN PREPARING APPLICATIONS IN RESPONSE TO THIS RFA. PURPOSE The National Institute on Alcohol Abuse and Alcoholism (NIAAA) is soliciting proposals to use targeted gene disruption and overexpression techniques in mice to elucidate the roles of specific neuronal proteins in mediating the effects of ethanol on brain function and behavior. This manipulative approach offers the possibility of establishing a causal relationship between function of specific proteins and specific behaviors. The genetic approach complements the more traditional use of pharmacological agents to elucidate such causal relationships. It is applicable, in principle, to all neuronal proteins, including those for which pharmacological agents are as yet unavailable, and capable, in principle, of distinguishing roles of closely related proteins for which pharmacological agents of appropriate specificity are as yet unavailable. To be considered responsive to this RFA, applications must propose elucidation of the relationship between ethanol’s effects on a specific aspect of nervous system function and a specific behavioral effect of ethanol. NIAAA strongly encourages mouse geneticists with expertise in targeted mutagenesis techniques to seek collaborations with established alcohol researchers, in order to bring these powerful approaches to bear on the elucidation of the mechanisms of alcohol’s effects on behavior. Applications proposing creation of strains of mice bearing new targeted mutations or overexpressed transgenes, and applications proposing use of previously existing targeted mutations or overexpressed transgenes will all be considered responsive to this RFA. NIAAA hopes that research supported by this RFA will lead to more definitive validation of targets for future medications development. HEALTHY PEOPLE 2000 The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2000," a PHS- led national activity for setting priority areas. This Request for Applications (RFA), Targeted Mutations to Study Ethanol-Induced Behaviors, is related to the priority area of alcohol abuse and alcoholism. Potential applicants may obtain a copy of "Healthy People 2000" at http://odphp.osophs.dhhs.gov/pubs/hp2000 . ELIGIBILITY REQUIREMENTS Applications may be submitted by domestic and foreign, for-profit and non- profit organizations, public and private, such as universities, colleges, hospitals, laboratories, units of State and local governments, and eligible agencies of the Federal government. Racial/ethnic minority individuals, women, and persons with disabilities are encouraged to apply as Principal Investigators. MECHANISM OF SUPPORT This RFA will use the National Institutes of Health (NIH) research project grant (R01) and the NIAAA exploratory/developmental (R21) award mechanisms. Responsibility for the planning, direction, and execution of the proposed project will be solely that of the applicant. The total project period for an application submitted in response to this RFA may not exceed 5 years for the R01 mechanism, or 3 years for the R21 mechanism. Under the R21mechanism, direct costs are limited to $100,000 per year. (See Program Announcement PA- 99-131, NIAAA Exploratory/Developmental Grant Program, http://grants.nih.gov/grants/guide/pa-files/PA-99-131.html, for a complete description of the R21 mechanism.) This RFA is a one-time solicitation. Future unsolicited competing continuation applications will compete with all investigator-initiated applications and be reviewed according to the customary peer review procedures. The anticipated award date is September 29, 2000. Applicants may also submit applications for Investigator-Initiated Interactive Research Project Grants (IRPG). Interactive Research Project Grants require the coordinated submission of related research project grants (R01) from investigators who wish to collaborate on research, but do not require extensive shared physical resources. These applications must share a common theme and describe the objectives and scientific importance of the interchange of, for example, ideas, data, and materials among the collaborating investigators. A minimum of two independent investigators with related research objectives may submit concurrent, collaborative, cross- referenced individual R01 applications. Applicants may be from one or several institutions. Further information on these and other grant mechanisms may be obtained from the program staff listed in the INQUIRIES section of this RFA or from the NIAAA Web site http://www.niaaa.nih.gov/ under Research Programs/Program Announcements. FUNDS AVAILABLE The NIAAA intends to commit approximately $2 million in FY 2000 to fund six to eight new grants in response to this RFA. Because the nature and scope of the research proposed might vary, it is anticipated that the size of awards will also vary. Although the financial plans of the NIAAA provide support for this program, awards pursuant to this RFA are contingent upon the availability of funds and the receipt of a sufficient number of applications of outstanding scientific and technical merit. RESEARCH OBJECTIVES Background Although the exact mechanism of interaction of ethanol with its neuronal molecular targets is not yet understood, there is evidence that ethanol interacts with specific hydrophobic domains of various neuronal membrane receptor proteins to alter their normal function. These interactions perturb the intra- and intercellular signaling systems in which those molecules function, thereby exerting diverse and profound effects on downstream neural responses, such as synaptic transmission. Examples of receptors for which some evidence supports a direct interaction with ethanol include ?- aminobutyric acid A (GABA-A), glutamate (both the N-methyl-D-aspartate (NMDA) and non-NMDA types), and several serotonin (5-HT) receptors. During chronic exposure to ethanol, cellular adaptations to the altered function of the pathways impacted acutely by ethanol occur, resulting in further changes in neuronal physiology, leading in turn to such neurobehavioral alterations as tolerance and dependence. The sheer multiplicity of changes in protein abundance, intracellular localization, covalent modification, and activity induced by ethanol greatly magnifies the challenge of determining which of these molecular changes are mechanistically responsible for ethanol’s effects on behavior. Pharmacological studies have implicated dopamine and serotonin receptors in ethanol consummatory behavior, GABA-A receptors in sedation and ethanol withdrawal phenomena, and NMDA receptors in ethanol-induced impairment of learning and memory, ethanol tolerance and withdrawal, and brain damage. Such studies, which involve deliberate perturbation of the activity of a protein of interest, can in principle establish a causal role for that protein in mediating a behavioral endpoint. Interpretation of such studies, however, is often complicated by drug effects on molecular targets besides the intended one. There is, moreover, a large body of studies which collectively catalogue molecular changes in neural tissue after ethanol treatment of cultured neurons, isolated brain slices, or whole animals. While such studies are useful for generating hypotheses, elucidation of mechanisms of ethanol’s effects on behavior requires targeted perturbation of activity of specific neuronal components. Newly developed methods for targeted alteration of gene function (gene knockouts and knockins ) offer the possibility of establishing a cause and effect relationship between an action of ethanol on a molecular target (along with attendant changes in the output of pathways downstream of this target) and an ethanol-induced behavioral change. In principle, if a behavioral effect of ethanol depends on the function of a protein, blocking that function by disrupting the corresponding gene would alter that behavioral effect. Whether ethanol’s behavioral effect would be diminished or enhanced would depend on whether ethanol normally stimulated or inhibited function of that protein. Methodological Considerations Many inbred strains of mice which an investigator might wish to use as a background strain for the development of a new gene knockout have sensory, motor, or other behavioral deficits which could confound the interpretation of knockout phenotypes . For example, it is easy to imagine how a background strain with taste deficits might interfere with measurements of conditioned taste aversion, how a hyperactive strain might interfere with measurements of conditioned place preference, or how a strain with impaired electrolyte balance might interfere with two-bottle choice drinking measurements. In view of these considerations, investigators clearly need to exercise care in the choice of genetic background strain for a knockout. Knockouts are typically generated by homologous recombination in embryonic stem cells derived from one of the substrains of mouse strain 129. For ease of detection of chimeric progeny, the targeted cells are injected into blastocysts of a contrasting genetic background. The chimeric progeny are then bred with mice of another genetic background (often the same as that of the recipient blastocysts, but in any case different from 129), and heterozygous mutant progeny are selected for further breeding. This breeding scheme results in mice in which the knockout mutation is carried on a mixed, segregating genetic background, i.e., one in which different individuals carry differing mixtures of the 129 and the other parental genetic backgrounds. Since 129 and the other parental strain may carry alleles with differing effects on the trait of interest, the variable genetic background of knockout and wild type control mice bred in this fashion may cause phenotypic variability which may obscure phenotypic differences arising from the knockout and wild type alleles of the targeted gene. For this and other compelling reasons, the 1997 Banbury Conference on Genetic Background in Mice has recommended that knockout mutations be maintained as standard inbred congenic lines (i.e., mutant and control lines identical at all loci in the genome, except for a small chromosomal segment containing the targeted gene), and should be analyzed either as such, or as F1 hybrids between strains of two different backgrounds . NIAAA encourages applicants to consider these recommendations seriously in the design of their proposals. Moreover, since nervous system function and behavior are the results of the combined actions of many gene products, it is to be expected that mice carrying the same mutation on different genetic backgrounds may display different phenotypes. This expectation has been abundantly realized, so that a full characterization of the effects of a knockout will require examination of its phenotype on several different genetic backgrounds.2, Germline (also referred to as traditional, classical, or constitutive) gene knockouts create animals in which function of the targeted gene is missing or altered from the time of conception, in all of the mutant individual’s cells. All knockout studies in the alcohol field to date have employed this approach. Although these studies (see Prior Use of Knockouts in Alcohol Research, below) have generated considerable excitement, interpretation of their results is complicated in several ways. Since, in germline knockouts, all of development takes place in the absence of normal function of the disrupted gene, it is difficult to distinguish whether an alteration in neural function or behavior observed in an adult is due to acute lack of gene product at the time of measurement of the phenotype, or whether these phenotypes are altered as a result of abnormal development occasioned by the lack of gene product. Moreover, if no phenotypic alteration is observed in mutant individuals, it is difficult to distinguish whether the targeted gene plays no role in the phenotype, or whether alterations in expression of other genes have occurred during development, compensating for deficiency of the targeted gene product. In addition, because function of the mutated gene is absent in all of the individual’s cells, germline knockouts often provide little information about the tissue or cell-type specificity of the targeted gene product’s role in influencing the phenotype (but see reference 3 for exceptions). Recently developed knockout methods circumvent these problems either by disrupting the gene only in a restricted set of tissues (rather than in the whole animal), by disrupting the gene (reversibly, in some cases) by means of a chemical inducer delivered according to a schedule decided by the investigator, or by overexpressing a transgene in a specifically chosen set of tissues.3, NIAAA strongly encourages applicants to incorporate these more sophisticated methods into the design of their projects. Prior Use of Knockouts in Alcohol Research Published reports have described neural and behavioral responses to ethanol in mice bearing germline knockouts of the following genes: 5HT1b receptor , , , GABA-A receptor subunits ?6 , and ?2L, dopamine receptors D1, D2, and D4, ?-endorphin, neuropeptide Y (NPY), fyn kinase, and protein kinase C (PKC) isoforms ? , and ? . The following examples of the use of knockouts to elucidate mechanisms of ethanol’s effects on nervous system function and behavior demonstrate collectively both the power and the limitations of this approach. Since previous pharmacological studies had indirectly implicated the ?6 subunit of the GABA-A receptor in mediating the intoxicating effects of ethanol, investigators used a germline knockout of the gene encoding this subunit to test this inferred role in an intact, behaving animal. Mutation of this gene had no effect on sensitivity to sedation by, acute functional or chronic tolerance to, or severity of withdrawal from ethanol.8,9 While a facile interpretation of these results might suggest no involvement of the ?6 subunit in any of these phenotypes, the investigators note that, since the withdrawal responses of the knockout were measured on a mixed genetic background of strains C57BL/6J and 129/SvJ, and since these two background strains differ in their withdrawal responses, it is possible that the 129- like withdrawal phenotype of both knockout and control mice may have resulted from alleles of other genes in the 129 genetic background, rather than the disrupted ?6 gene itself9. They also note that, since cerebellar GABA-A receptors in the knockout have reduced affinity for the GABA agonist muscimol, it is likely that the deficit of ?6 expression throughout development has been compensated by some process which has resulted in this change in the receptor’s pharmacological properties.8 It is thus unclear whether the ?6 subunit has no role in any of the measured responses to ethanol, or whether its true role has been masked by developmental compensation for the deficit of its expression. This uncertainty could be resolved by studies which knock out the gene in a restricted brain region, only after brain development has been completed. Because of inconsistent prior pharmacological evidence regarding the role of protein kinase C (PKC) in mediating ethanol’s effects on function of GABA-A receptors, investigators used a germline knockout of the ? isoform of this protein to test its role in receptor function and behavioral responses to ethanol.17 Since genetic disruption of the ? isoform is specific for this isoform alone, whereas pharmacological agents typically affect the activity of multiple isoforms, the genetic approach is better able to define a specific role for the ? isoform. Null mutants of ?-PKC show reduced sensitivity to ethanol-induced sedation and hypothermia, as well as reduced sensitivity of cerebellar GABA-A receptors to stimulation by ethanol. While developmental compensation for the deficit of activity of the knocked-out gene is always a concern with germline knockouts, this concern may be reduced in this case, since ?-PKC is not normally expressed during development until about one week postnatally. Tolerance to chronic ethanol exposure (as measured by sedative or hypothermic response to a challenge dose of ethanol) was either reduced in null mutants, or not, depending on the genetic background,18 demonstrating the interaction of multiple genes in the determination of this phenotype. Because of inferential evidence relating brain-regional NPY levels to alcohol preference in rats, investigators examined ethanol consumption and sedation sensitivity in mice bearing a germline knockout of the gene encoding this neuropeptide.15 Since pharmacological antagonists of NPY are not yet available, the genetic approach provides a practical method to examine the effects of a deficit of this peptide. Null mutant mice showed increased consumption of, and reduced sensitivity to sedation by ethanol. Transgenic mice overexpressing NPY in several brain regions consumed less ethanol, and were more sensitive to sedation by ethanol than the corresponding wild type. Because the transgenic mice did not overexpress NPY in the arcuate nucleus of the hypothalamus, it appears unlikely that ethanol and food consumption are regulated by a common mechanism in these mice. Although these mice overexpressed NPY in the amygdala, they (contrary to expectation) did not display lower basal anxiety than wild type mice. The reduced ethanol consumption of these mice is thus apparently not related to reduced anxiety. This study provides the first convincing evidence that NPY plays a role in the regulation of ethanol consumption and sedation. Further information on the role of NPY in regulation of these behaviors is likely to come from knockouts of the genes encoding the various NPY receptors. AREAS OF INTEREST General Considerations This RFA is soliciting applications to elucidate the neural mechanisms of ethanol’s effects on behavior and mechanisms of alcohol-seeking behavior. In order to be considered responsive to this RFA, applications must therefore propose to use gene knockouts to relate ethanol’s effects on specific domains of nervous system function to its effects on specific domains of behavior. Applicants are expected to propose use of current methodology designed to circumvent the difficulties of interpretation of results discussed earlier in this RFA (see Methodological Considerations, above). Since expertise in alcohol-related neuroscience and behavioral research and expertise in targeted mutagenesis in mice may not often coexist in the same laboratory, investigators are strongly encouraged to form collaborations, between institutions if necessary, to ensure the availability of the complete range of requisite expertise for design and execution of a proposed project. Sharing of Materials Generated Under this RFA Projects funded under this RFA are likely to lead to the creation of new gene-targeting constructs, congenic strains, and other research tools which will be of great value to the broader research community, beyond the laboratories which will create them. NIAAA strongly encourages the maximal dissemination of these tools to the broader research community, to ensure that they may be exploited to their full potential. NIAAA accordingly requires applicants who respond to this RFA to propose detailed plans for sharing the research resources generated through the grant. For this purpose, it is the opinion of NIAAA that dissemination of such resources through individual laboratories and websites is not sufficient, as it would force interested investigators to search numerous websites in order to gain access to research tools generated under this RFA. It is preferable that materials generated under this RFA should be placed in common, public repositories and databases that are widely accessible by investigators in the scientific community. It is expected that the investigator’s resource sharing plan will include a description of the mechanisms proposed for wide distribution of resources with investigators in the scientific community, and a timetable for distribution of resources. The reviewers will make an administrative comment on the adequacy of the proposed plan for resource sharing. (This comment will not affect the priority score of the proposal.) NIAAA program staff will consider the adequacy of the plan in determining whether to recommend an application for award. The sharing plan as approved, after negotiation with the applicant as necessary, shall become a condition of the grant award. Where appropriate, grantees may work with the private sector to make resources available to the wider research community at a reasonable cost. Applicants may request funds to defray the costs of sharing resources, with adequate justification. For more detailed guidance on NIH’s policies on resource sharing, applicants are referred to Principles and Guidelines for Recipients of NIH Research Grants and Contracts on Obtaining and Disseminating Biomedical Research Resources, http://www.ott.nih.gov/policy/rt_guide_final.html . Research Questions The following examples of possible uses of targeted mutagenesis are for illustration only, and are not exclusive. What are the neuronal gene products, biochemical and electrophysiological pathways, brain regions, and neural circuits mediating o consumption of ethanol? o sensitivity to ethanol-induced ataxia or sedation? o stimulant properties of ethanol? o acute functional tolerance to ethanol? o anxiolytic properties of ethanol? o tolerance resulting from chronic ethanol exposure? o withdrawal from chronic ethanol? o rewarding and reinforcing properties of ethanol? o neurotoxicity and cognitive deficits resulting from chronic ethanol exposure? What is the precise mechanistic relationship between the rewarding and reinforcing properties of ethanol and o sensitivity to ethanol-induced ataxia or sedation? o stimulant properties of ethanol? o acute functional tolerance to ethanol? o anxiolytic properties of ethanol? o tolerance resulting from chronic ethanol exposure? o withdrawal from chronic ethanol? LETTER OF INTENT Prospective applicants are asked to submit a letter of intent that includes a descriptive title of the proposed research, the name, address, and telephone number of the Principal Investigator, the identities of other key personnel and participating institutions, and the number and title of the RFA in response to which the application may be submitted. Although a letter of intent is not required, is not binding, and does not enter into the review of a subsequent application, the information that it contains allows Institute staff to estimate the potential review workload and avoid conflict of interest in the review. The letter of intent is to be sent to RFA-AA-00-001, Extramural Project Review Branch, NIAAA 6000 Executive Boulevard, Suite 409, MSC 7003, Bethesda, MD 20892-7003 by the letter of intent receipt date listed in the heading of this RFA. APPLICATION PROCEDURES The research grant application form PHS 398 (rev. 4/98) is to be used in applying for these grants. These forms are available at most institutional offices of sponsored research and from the Division of Extramural Outreach and Information Resources, National Institutes of Health, 6701 Rockledge Drive, MSC 7910, Bethesda, MD 20892-7910, telephone 301/710-0267, email: GrantsInfo@nih.gov. SPECIFIC APPLICATION INSTRUCTIONS FOR MODULAR GRANTS The modular grant concept establishes specific modules in which direct costs may be requested, as well as a maximum level for requested budgets. Only limited budgetary information is required under this approach. The just-in-time concept allows applicants to submit certain information only when there is a possibility for an award. It is anticipated that these changes will reduce the administrative burden for the applicants, reviewers, and Institute staff. The research grant application form PHS 398 (rev. 4/98) is to be used in applying for these grants, with the modifications noted below. BUDGET INSTRUCTIONS Modular Grant applications will request direct costs in $25,000 modules, up to a total direct cost request of $250,000 per year. (Applications that request more than $250,000 direct costs in any year must follow the traditional PHS 398 application instructions.) The total direct costs must be requested in accordance with the program guidelines and the modifications made to the standard PHS 398 application instructions described below: PHS 398 FACE PAGE - Items 7a and 7b should be completed, indicating Direct Costs (in $25,000 increments up to a maximum of $250,000) and Total Costs [Modular Total Direct plus Facilities and Administrative (F&A) costs] for the initial budget period. Items 8a and 8b should be completed indicating the Direct and Total Costs for the entire proposed period of support. DETAILED BUDGET FOR THE INITIAL BUDGET PERIOD - Do not complete Form Page 4 of the PHS 398. It is not required and will not be accepted with the application. BUDGET FOR THE ENTIRE PROPOSED PERIOD OF SUPPORT - Do not complete the categorical budget table on Form Page 5 of the PHS 398. It is not required and will not be accepted with the application. NARRATIVE BUDGET JUSTIFICATION - Prepare a Modular Grant Budget Narrative page. (See http://grants.nih.gov/grants/funding/modular/modular.htm . for sample pages.) At the top of the page, enter the total Direct Costs requested for each year. This is not a Form page. Under Personnel, list key project personnel, including their names, percent of effort, and roles on the project. No individual salary information should be provided. However, the applicant should use the NIH appropriation language salary cap and the NIH policy for graduate student compensation in developing the budget request. For Consortium/Contractual costs, provide an estimate of total costs (Direct plus F&A) for each year, each rounded to the nearest $1,000. List the individuals/organizations with whom consortium or contractual arrangements have been made, the percent effort of key personnel, and the role on the project. Indicate whether the collaborating institution is foreign or domestic. The total cost for a consortium/contractual arrangement is included in the overall requested Modular Direct Cost amount. Include the letter of intent to establish a consortium. Provide an additional narrative budget justification for any variation in the number of modules requested. BIOGRAPHICAL SKETCH - The Biographical Sketch provides information used by reviewers in the assessment of each individual"s qualifications for a specific role in the proposed project, as well as to evaluate the overall qualifications of the research team. A biographical sketch is required for all key personnel, following the instructions below. No more than three pages may be used for each person. A sample biographical sketch may be viewed at: http://grants.nih.gov/grants/funding/modular/modular.htm . - Complete the educational block at the top of the Form page, - List position(s) and any honors, - Provide information, including overall goals and responsibilities, on research projects ongoing or completed during the last three years, and - List selected peer-reviewed publications, with full citations. CHECKLIST - This page should be completed and submitted with the application. If the F&A rate agreement has been established, indicate the type of agreement and the date. All appropriate exclusions must be applied in the calculation of the F&A costs for the initial budget period and all future budget years. The applicant should provide the name and phone number of the individual to contact concerning fiscal and administrative issues if additional information is necessary following the initial review. The RFA label available in the PHS 398 (rev. 4/98) application form must be affixed to the bottom of the face page of the application. Failure to use this label could result in delayed processing of the application such that it may not reach the review committee in time for review. In addition, the RFA title and number must be typed on line 2 of the face page of the application form and the YES box must be marked. The sample RFA label available at: http://grants.nih.gov/grants/funding/phs398/label-bk.pdf has been modified to allow for this change. Please note this is in pdf format. Submit a signed, typewritten original of the application, including the Checklist, and three signed, photocopies in one package to: CENTER FOR SCIENTIFIC REVIEW NATIONAL INSTITUTES OF HEALTH 6701 ROCKLEDGE DRIVE, ROOM 1040, MSC 7710 BETHESDA, MD 20892-7710 BETHESDA, MD 20817 (for express/courier service) At the time of submission, two additional copies of the application must be sent to: RFA :AA-00-001 Extramural Project Review Branch National Institute on Alcohol Abuse and Alcoholism 6000 Executive Boulevard, Suite 409, MSC 7003 Bethesda, MD 20892-7003 Rockville, MD 20852 (for express/courier service) Applications must be received by the application receipt date listed in the heading of this RFA. If an application is received after that date, it will be returned to the applicant without review. The Center for Scientific Review (CSR) will not accept any application in response to this RFA that is essentially the same as one currently pending initial review, unless the applicant withdraws the pending application. The CSR will not accept any application that is essentially the same as one already reviewed. This does not preclude the submission of substantial revisions of applications already reviewed, but such applications must include an introduction addressing the previous critique. REVIEW CONSIDERATIONS Upon receipt, applications will be reviewed for completeness by the CSR and responsiveness by the NIAAA. If the application is not responsive to the RFA, CSR staff may contact the applicant to determine whether to return the application to the applicant or submit it for review in competition with unsolicited applications at the next review cycle. Applications that are complete and responsive to the RFA will be evaluated for scientific and technical merit by an appropriate peer review group convened by the NIAAA in accordance with the review criteria stated below. As part of the initial merit review, a process will be used by the initial review group in which applications receive a written critique and undergo a process in which only those applications deemed to have the highest scientific merit, generally the top half of the applications under review, will be discussed, assigned a priority score, and receive a second level review by the National Advisory Council on Alcohol Abuse and Alcoholism. Review Criteria The goals of NIH-supported research are to advance our understanding of biological systems, improve the control of disease, and enhance health. In the written comments reviewers will be asked to discuss the following aspects of the application in order to judge the likelihood that the proposed research will have a substantial impact on the pursuit of these goals. Each of these criteria will be addressed and considered in assigning the overall score, weighting them as appropriate for each application. Note that the application does not need to be strong in all categories to be judged likely to have major scientific impact and thus deserve a high priority score. For example, an investigator may propose to carry out important work that by its nature is not innovative but is essential to move a field forward. (1) Significance: Does this study address an important problem? If the aims of the application are achieved, how will scientific knowledge be advanced? What will be the effect of these studies on the concepts or methods that drive this field? Is the proposed study likely to clarify the mechanistic relationship between ethanol’s effects on a specific domain of neural function and its effects on a specific domain of behavior? (2) Approach: Are the conceptual framework, design, methods, and analyses adequately developed, well integrated, and appropriate to the aims of the project? Does the applicant acknowledge potential problem areas and consider alternative tactics? (3) Innovation: Does the project employ novel concepts, approaches, or methods? Are the aims original and innovative? Does the project challenge existing paradigms or develop new methodologies or technologies? If the development of new gene knockouts, congenic strains, or other research tools is proposed, are they significantly different or improved from tools already available to the research community? (4) Investigator: Is the investigator appropriately trained and well suited to carry out this work? Is the work proposed appropriate to the experience level of the principal investigator and other researchers (if any)? Will the team of investigators and collaborators receive sufficient participation or guidance from individuals with documented expertise in targeted mutagenesis in mice to make successful creation of the proposed mutants highly likely? (5) Environment: Does the scientific environment in which the work will be done contribute to the probability of success? Do the proposed experiments take advantage of unique features of the scientific environment or employ useful collaborative arrangements? Is there evidence of institutional support? In addition to the above criteria, in accordance with NIH policy, all applications will also be reviewed with respect to the following: o the reasonableness of the proposed budget and duration in relation to the proposed research, and o the adequacy of the proposed protection for animals or the environment, to the extent they may be adversely affected by the project proposed in the application. Reviewers should also comment in an Administrative Note on the adequacy of plans for sharing of research tools developed with support from this RFA. These comments will be advisory to program staff and should not affect the priority score. Schedule Letter of Intent Receipt Date: April 5, 2000 Application Receipt Date: May 5, 2000 Peer Review Date: July, 2000 Council Review: September 13, 2000 Earliest Anticipated Start Date: September 29, 2000 AWARD CRITERIA Award criteria that will be used to make award decisions include: o scientific merit (as determined by peer review), o availability of funds, o programmatic priorities, o adequacy of protection for animal subjects, and o adequacy of plans for sharing research tools developed with support from this RFA. INQUIRIES Inquiries concerning this RFA are encouraged. The opportunity to clarify any issues or questions from potential applicants is welcome. Direct inquiries regarding programmatic issues to: Robert W. Karp, Ph.D. Division of Basic Research National Institute on Alcohol Abuse and Alcoholism 6000 Executive Boulevard, Suite 402, MSC 7003 Bethesda, MD 20892-7003 Telephone: (301) 443-2239 FAX: (301) 594-0673 Email: rkarp@willco.niaaa.nih.gov Direct inquiries regarding fiscal matters to: Ms. Linda Hilley Office of Planning and Resource Management National Institute on Alcohol Abuse and Alcoholism 6000 Executive Boulevard, Suite 504, MSC 7003 Bethesda, MD 20892-7003 Telephone: (301) 443-4703 FAX: (301) 443-3891 Email: lhilley@willco.niaaa.nih.gov AUTHORITY AND REGULATIONS This program is described in the Catalog of Federal Domestic Assistance No. 93.273. Awards are made under authorization of the Public Health Service Act, Title IV, Part A (Public Law 78-410, as amended by Public Law 99-158, 42 USC 241 and 285) and administered under NIH grants policies and Federal Regulations 42 CFR 52 and 45 CFR Part 74. This program is not subject to the intergovernmental review requirements of Executive Order 12372 or Health Systems Agency review. The PHS strongly encourages all grant recipients to provide a smoke-free workplace and promote the non-use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, any portion of a facility) in which regular or routine education, library, day care, health care, or early childhood development services are provided to children. This is consistent with the PHS mission to protect and advance the physical and mental health of the American people. REFERENCES Crawley JN, Belknap JK, Collins A, Crabbe JC, Frankel W, Henderson N, Hitzemann RJ, Maxson SC, Miner LC, Silva AJ, Wehner JM, Wynshaw-Boris A, Paylor R (1997) Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies. Psychopharmacology 132:107-124. 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