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 301/435-0714, 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.

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phenotypes of inbred mouse strains: implications and recommendations for molecular studies.  
Psychopharmacology 132:107-124.
  Banbury Conference on Genetic Background in Mice (1997) Mutant mice and neuroscience:  
recommendations concerning genetic background.  Neuron 19:755-759.
  Piccioto MR (1999) Knock-out mouse models used to study neurobiological systems.  Crit Rev 
Neurobiol 13:103-149.
  Picciotto MR, Wickman K (1998) Using knockout and transgenic mice to study 
neurophysiology and behavior.  Physiol Rev 78:1131-1163.
  Crabbe JC, Phillips TJ, Feller DJ, Hen R, Wenger CD, Lessov CN, Schafer GL (1996) Elevated 
alcohol consumption in null mutant mice lacking 5-HT1B serotonin receptors.  Nature Genetics 
14:98-101.
  Risinger FO, Bormann NM, Oakes RA (1996) Reduced sensitivity to ethanol reward, but not 
ethanol aversion, in mice lacking 5-HT1B receptors.  Alcoholism Clin Exp Res 20:1401-1405.
  Risinger FO, Doan AM, Vickrey AC (1999) Oral operant ethanol self-administration in 5-
HT1b knockout mice.  Behav Brain Res 102:211-215.
  Homanics GE, Ferguson C, Quinlan JJ, Daggett J, Snyder K, Lagenaur C, Mi ZP, Wang XH, 
Grayson DR, Firestone LE (1997) Gene knockout of the ?6 subunit of the ?-aminobutyric acid 
type A receptor:  lack of effect on responses to ethanol, pentobarbital, and general anesthetics.  
Mol Pharmacology 51:588-596.
  Homanics GE, Le NQ, Kist F, Mihalek R, Hart AR, Quinlan JJ (1998) Ethanol tolerance and 
withdrawal responses in GABAA receptor alpha 6 subunit null allele mice and in inbred 
C57BL/6J and Strain 129/SvJ mice.  Alcoholism Clin Exp Res 22:259-265.
  Homanics GE, Harrison NL, Quinlan JJ, Krasowski MD, Rick CEM, de Blas AL, Mehta AK, 
Kist F, Mihalek RM, Aul JJ, Firestone LL (1999) Normal electrophysiological and behavioral 
responses to ethanol in mice lacking the long splice variant of the ?2 subunit of the ?-
aminobutyrate type A receptor.  Neuropharmacology 38:253-265.
  El-Ghundi M, George SR, Drago J, FletcherPJ, Fan T, Nguyen T, Liu C, Sibley DR, Westphal 
H, O’Dowd BF (1998) Disruption of dopamine D1 receptor gene expression attenuates alcohol-
seeking behavior.  Eur J Pharmacology 353:149-158.
  Phillips TJ, Brown KJ, Burkhart-Kasch S, Wenger CD, Kelly MA, Rubinstein M, Grandy DK, 
Low MJ (1998) Alcohol preference and sensitivity are markedly reduced in mice lacking 
dopamine D2 receptors.  Nature Neurosci 1:610-615.
  Rubinstein M, Phillips TJ, Bunzow JR, Falzone TL, Dziewczapolski G, Zhang G, Fang Y, 
Larson JL, McDougall JA, Chester JA, Saez C, Pugsley TA, Gershanik O, Low MJ, Grandy DK 
(1997) Mice lacking Dopamine D4 receptors are supersensitive to ethanol, cocaine, and 
methamphetamine.  Cell 90:991-1001.
  Grisel JE, Mogil JS, Grahame NJ, Rubinstein M, Belknap JK, Crabbe JC, Low MJ (1999) 
Ethanol oral self-administration is increased in mutant mice with decreased ?-endorphin 
expression.  Brain Res 835:62-67.
  Thiele TE, Marsh DJ, Ste Marie L, Bernstein IL, Palmiter RD (1998) Ethanol consumption 
and resistance are inversely related to neuropeptide Y levels.  Nature 396:366-369.
  Miyakawa T, Yagi T, Kitazawa H, Yasuda M, Kawai N, Tsuboi K, Niki H (1997) Fyn-kinase 
as a determinant of ethanol sensitivity:  relation to NMDA-receptor function.  Science 278:698-
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  Harris RA, McQuilkin SJ, Paylor, Abeliovich, Tonegawa S, Wehner JM (1995) Mutant mice 
lacking the ? isoform of protein kinase C show decreased behavioral actions of ethanol and 
altered function of ?-aminobutyrate receptors.  Proc Natl Acad Sci USA 92:3658-3662.
  Bowers BJ, Owen EH, Collins AC, Abeliovich A, Tonegawa S, Wehner JM (1999) Decreased 
ethanol sensitivity and tolerance development in ?-protein kinase C null mutant mice is 
dependent on genetic background.  Alcoholism Clin Exp Res 23:387-397.
  Hodge CW, Mehmert KK, Kelley SP, McMahon T, Haywood A, Foster Olive M, Wang D, 
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modulators and alcohol in mice lacking PKC?.  Nature Neurosci 2:997-1002.



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