GRANT WRITING AND BUDGETING. Marc E. Freeman, Ph.D. Department of Biological Science Florida State University Tallahassee, FL 32306 850 644-3896. HOW TO WRITE YOUR FIRST GRANT. Nothing beats a good idea Be realistic Make the presentation clear and simple
Marc E. Freeman, Ph.D.
Department of Biological Science
Florida State University
Tallahassee, FL 32306
Prolactin (PRL) is one of the most versatile hormones of mammalian organisms. Besides its role in lactation, secretion of PRL contributes to a wide range of physiological functions, i.e. adaptation to new environment (22), immune functions (23) osmoregulation (24,25), reproduction (26) and behavior (27). PRL of anterior pituitary origin is secreted by lactotrophs (28). Lactotrophs have spontaneously high secretory activity, which is controlled primarily by tonic inhibitory input of hypothalamic origin (29), though they receive stimulatory input, as well (29-31).
The physiological PRL inhibiting factor is dopamine (DA) (32,33). DA is released from three, anatomically and functionally distinct hypothalamic neuroendocrine cell populations: I). periventricular hypothalamic dopaminergic (PHDA) neurons of the periventricular nucleus (A14), II) tuberohypophysial (THDA) and III) tuberoinfundibular (TIDA) neurons (34) of the arcuate nucleus (A12). PHDA and THDA neurons terminate in the intermediate and neural lobes of the pituitary gland, respectively (35,36). DA, released from THDA/PHDA terminals may reach the anterior lobe through the short portal vessels. On the other hand, TIDA neurons terminate in the external zone of the median eminence supplying the anterior pituitary gland with DA through the long portal vessels. Although the central role of TIDA neurons in hypothalamic control of PRL secretion is acknowledged, the relative contribution and importance of the distinct neuroendocrine DAergic neuron subpopulations to the regulation of PRL secretion is not understood.
To characterize the chronobiological and anatomical basis of regulatory mechanisms governing the rhythmic patterns of the DAergic input on PRL secretion. By conducting in vivo experiments in constant environments we will test whether the daily changes in the activities of neuroendocrine DAergic neuron populations are light entrained endogenous circadian rhythms. Tract tracing studies will be used to investigate the functional anatomical nature of the direct connection(s) between the circadian zeitgeber in the suprachiasmatic nucleus and the different neuroendocrine DAergic subpopulations. Antisense antagonism will be used to verify the functional significance of suprachiasmatic efferent fibers on the neuroendocrine DAergic neuron populations.
To characterize the feedback role of PRL in the daily activity of PHDA, THDA and TIDA neurons. The incidence of PRL-Rs in the THDA and TIDA neurons is higher than in PHDA neurons, although PHDA neurons also express PRL-R in ovarian steroid-replaced rats. We suggest that PRL feedback is inherently part of the THDA and TIDA neurons’ circadian regulation and it is inducible in PHDA neurons by ovarian steroids. In order to verify this hypothesis, we will compare the effects of PRL on the activity of PHDA, THDA and TIDA neuron populations in ovariectomized and ovarian steroid-replaced rats.
To identify the signal transduction mechanisms involved in the circadian regulation of the secretory activity of hypothalamic neuroendocrine DAergic neurons. We will identify the receptors, second messenger and cellular effector systems involved in mediating the effects of circadian input and PRL feedback on the neurosecretory activity of the PHDA, THDA and TIDA neuron subpopulations.
The neuroendocrine DAergic neuron populations and their feedback regulation (Fig. 1)
The hypothalamic A14 and A12 cell groups (Fig. 1), which are exclusively comprised of DAergic neurons (64,65) provide the pituitary gland with DA through two different routes: I) The tuberoinfundibular DAergic (TIDA) neurons send short projections to the external zone (EZ) of the median eminence. TIDA axons terminate on the basement membrane of the perivascular space surrounding the primary capillary loops of the portal system. From here the long portal vessels (LP) carry DA of TIDA origin to the anterior lobe (AL) of the pituitary gland. Perikarya of TIDA neurons show bimodal rostrocaudal distribution (66). The majority of TIDA neurons originate throughout the arcuate nucleus (ARN, A12), and a smaller
population arises from the periventricular nucleus (PeVN, A14) (66). II) The tuberohypophysial DAergic (THDA) neurons of the rostral ARN (A12) and the periventricular hypothalamic (PHDA) neurons of the PeVN (A14) have long axons, which course through the pituitary stalk (PS) and terminate in the neural (NL) and intermediate(IL) lobes of the pituitary gland, respectively (35). Short portal vessels(SP) provide communication between the neurointermediate and the AL (Figure 1).
Rats are sacrificed by an overdose of sodium pentobarbital, and perfused through a transcardial cannula with 50 ml of ice cold phosphate buffer (PBS, 0.1 M, pH 7.34, 295 mosm) immediately followed by 100 ml of ice cold fixative. The fixative applied usually is 4 % paraformaldehyde (PFA, in 0.1 M PBS, pH 7.5). The brains and pituitaries are postfixed for an hour in situ. After removal the brains and pituitaries are rinsed in PBS and immersed in 20% sucrose solution (in PBS, at 4ºC) until sinking. The tissue blocks are frozen on the freezing stage of a HM500OM cryostat (Zeiss, Germany). The brains are cut in 35-m coronal section between 300-4200 m post bregma and collected into 4 parallel series of free-floating sections. The sections are stored in cryoprotectant solution (133) until ICC is initiated. If the aforementioned tissue block does not contain an area with established immunoreactivity for the antigen in question, other areas of the brain (or other organs) are processed to provide positive and negative controls for the immunostaining. The pituitary glands are cut in 10-20 m coronal or horizontal sections, thaw-mounted on gelatin-subbed glass microscope slides and stored at -80ºC in closed tissue boxes containing Dryerite. Both the free floating brain sections and the mounted pituitary sections are thoroughly washed (5 X in PBS) prior to ICC. We have had ample experience with these techniques (21,37,38)
Basis of hypothesis A
Rationale for question
Experiment 1, 2, 3, 4……….
Rationale for question
Experiment 1, 2, 3, 4……….
Experimental results, interpretation, shortcomings, pitfalls
May consist of more than one hypothesis
Do not be afraid of redundancy with the Background
What is to be gained by asking the question?
Do NOT forget controls
Do NOT forget statistical analyses
Explain rationale for doses and times
Refer to previously described general methods
Describe methods unique to this experiment
What will you do if one of the unanticipated results emerge?
Be very careful that an unanticipated result does not doom the subsequent specific aims/questions
Why are you using the approach you are?
Why are you not using one of the alternatives?
What are the strengths and weaknesses of the approach you are using?
How will you deal with them?
Significance, Approach, Innovation, Investigator, Environment
Strengths and Weaknesses
Secure letters confirming their role
Antibodies, nucleotides, peptides
Obtain letters of collaboration
Request assignment to the Study Section you select
Not an integrated body of work
“OK, so you showed that there is message for the insulin receptor on the pancreas, now what?”
Ask SRA and Institute official
Obtain Summary Statement
Read Summary Statement thoroughly and often
Ask a mentor to read Summary Statement
Mentor and yourself should highlight issues for attention
Enumerating each criticism and the actions you propose to take
No finger pointing or accusations
Admit the reviewers were right
Reorganize and simplify presentation
Provide more detail
Provide preliminary data in response
Get a consultant
DIPLOMATICALLY point out reviewer error
AWAIT THE AWARD NOTICE!!!!!!!