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INTRODUCTION The purpose of this competitive revision is to examine a novel means to reduce the severity of fetal alcohol effects. For over 20 years we have used a neonatal rat model to investigate the effects of late gestational alcohol exposure on brain and behavioral development. In the most recently funded period of the parent grant (5R01AA006902, Behavioral Effects of Neonatal Alcohol Exposure), we focused on one potential mechanism of alcohol-induced teratogenic effects and possible treatments/interventions related to this mechanism. Specifically, we have been investigating the hypothesis that NMDA receptor-mediated excitotoxicity occurs during periods of alcohol withdrawal, contributing to the adverse effects of alcohol on brain and behavioral development. This parent grant was funded as a revision to 5R01AA006902-19. In the original submission we had two distinct lines of investigation, 1) To test the potential of memantine administration during ethanol withdrawal to reduce the severity of fetal alcohol effects; and 2) To test the possibility that insulin like growth factor ( IGF-I), which enhances myelination, can mitigate the effects of prenatal alcohol exposure. That grant received a priority score of 195. The overall summary stated: “The application has a number of strengths that include very testable hypotheses and attention to detail with respect to both the study design and methodology. In addition, the study addresses an area of research that is of major importance. Despite all of these positive aspects there are a few concerns that dampened enthusiasm for the proposal. Most notable is that the specific aims are not integrated well. The proposal actually read as two separate proposals as the battery of tests are completely different between the specific aims. The second concern is that preliminary data examining memantine and IGF-I as potent therapeutic drugs is lacking. Overall this is a strong application and the resulting data should add to our understanding of the behavioral and neural effects of fetal alcohol exposure. With a few modifications it should be in the "outstanding" category.” – The Primary Reviewer stated “The only major concern is that the Specific Aims could be more integrated. They read almost as two separate research projects. For this reason, it seems reasonable to examine the effects of one system on the other to better integrate the Specific Aims.” The Second Reviewer stated “The only concern is the lack of preliminary data supporting memantine and especially IGF-I as potent therapeutic drugs, the use of different exposure paradigms, and lack of integration of the two aims.” The Third Reviewer added “This is a significant and innovative proposal; however enthusiasm is tempered slightly by the following concerns. This proposal is focused on treatment approaches for FAS, however the two specific aims aren’t tied in very well with respect to this issue and they read as two very separate goals.” Because there were few methodological concerns, in response to this critique we deleted the IGF studies because we were lacking pilot data (we did obtain pilot data on memantine) and submitted an application with only the aim of investigating the therapeutic effects of memantine, which was funded.

We have now collected data (funded through another source) and published one paper on the beneficial effects of IGF-I administration in animals exposed to alcohol during development. IGF-I attenuated motor deficits resulting from neonatal alcohol exposure, but did not significantly affect alcohol-related hyperactivity or spatial learning deficits. However, because of limited funding, we could not complete the studies in the original proposal examining myelination nor do a complete assessment of parameters that influence IGF-I effects on behavioral development. We are now requesting funds in this competitive revision to undertake such studies and revert to our two original specific aims in investigation of novel mechanisms and treatments for fetal alcohol spectrum disorders.

BACKGROUND AND SIGNIFICANCE The following Specific Aims in italics were taken from our original proposal. A. Specific Aims. For more than 20 years, we have been investigating the effects of 3rd trimester alcohol exposure using a neonatal exposure model in the rat. Recently, this work has concentrated on examining mechanisms of alcohol’s behavioral teratogenic effects. One hypothesis we have investigated is that NMDA receptor-mediated excitotoxicity occurs during periods of alcohol withdrawal, contributing to the adverse effects of alcohol on brain and behavioral development. One current aim is to continue pursuing this hypothesis and test the effects of memantine, a noncompetitive NMDA receptor antagonist, in blocking the behavioral and neuropathological teratogenic effects of alcohol [This aim was funded and is the basis of the parent grant]. Our second aim results from our human MRI studies where we reported a deficit in white matter and an increase in gray matter in certain brain regions. We also noticed a similarity between the behavioral effects of prenatal alcohol exposure and those that accompany disorders in which white matter deficits or degeneration play a major role. If the behavioral teratogenic effects of alcohol are related to deficits in the development or maintenance of myelination, it might be possible to mitigate ethanol’s effects with agents that affect white matter regeneration. Thus, our second aim is to investigate insulin like growth factor, IGF, as a potential agent for overcoming this white matter deficit. Besides providing evidence regarding the mechanisms by which prenatal alcohol exposure exerts its effects, this work has tremendous potential in providing information related to the treatment of fetal alcohol effects. Obviously, we have learned much about the effects of prenatal alcohol exposure since the identification of the fetal alcohol syndrome (FAS) 30 years ago, but one of the major problems facing the caregivers of affected children is that so little is known about effective treatments. There is no general standard of care for FAS, or a generally accepted medical or behavioral model upon which to base treatment. To date, while there has been much speculation about the mechanisms underlying the CNS anomalies common in children with FAS, very little has been published about ways to remediate or mitigate the damage. The purpose of this research is to continue examining the possibility that blocking NMDA receptors during ethanol withdrawal reduces fetal alcohol effects, and to test a novel treatment approach to one aspect of brain development, myelination, which is known to be affected by prenatal alcohol exposure. The specific aims are: 1) To test the potential of memantine administration during ethanol withdrawal to reduce the severity of fetal alcohol effects. 2) To test the possibility that insulin like growth factor, which enhances myelination, can mitigate the effects of alcohol exposure To reiterate, this original submission (priority score of 195), was revised by deleting specific aim 2 and concentrating on the 1st specific aim because it was an extension of our previous work. Since our last competitive renewal much new has been learned about white matter changes in fetal alcohol spectrum disorders (FASD) and we have published data showing some beneficial effect of IGF-I on motor behavior deficits following developmental alcohol exposure. Thus we want to pursue our original aims of looking at IGF-I as a potential novel intervention for FASD. Following the identification of fetal alcohol syndrome (FAS) over 30 years ago, numerous clinical and preclinical studies have described the spectrum of adverse effects induced by alcohol exposure on the developing fetus. Heavy alcohol abuse during pregnancy not only increases the incidence of prenatal mortality, but produces a constellation of anomalies in the child, including facial dysmorphology, growth deficiencies, and central nervous system (CNS) dysfunction, the range of which is now referred to as fetal alcohol spectrum disorders (FASD) REF. Probably the most devastating effect is the prenatal alcohol-induced brain damage, which can be severe and long-lasting, and manifested as a range of intellectual, cognitive, and behavioral alterations. For example, prenatal alcohol exposure is associated with low IQ, motor dysfunction, altered social behavior, hyperactivity, executive function deficits, and deficits in learning and memory [see [4-6] and [7] for reviews]. Despite the known adverse effects of prenatal alcohol exposure, women continue to drink alcohol during pregnancy, at great personal and societal cost. Thus, it is imperative that we identify effective interventions and treatments to reduce the severity of alcohol-related neurodevelopmental disorders. To date, there have been few systematic investigations of treatment strategies for this population [2]. A number of experimental therapeutics reduces the severity of ethanol’s teratogenic effects. Some therapeutics are based on purported mechanisms of ethanol induced damage including neuroactive peptides [13], and antioxidants [14-17]. We have been examining the potential of NMDA receptor antagonists to mitigate ethanol’s teratogenic effects, based upon the hypothesis that NMDA receptor mediated excitotoxicity occurs during periods of withdrawal. Alcohol may also disrupt development by interfering with the production and/or response to neurotrophic factors. For example, the level of nerve growth factor (NGF) [2], the expression of NGF receptors [36] and NGF-mediated cell survival [84] are inhibited by pre- or perinatal ethanol treatment. Similar alcohol-related reductions have been reported for other neurotrophic factors, including brain derived neurotrophic factor (BDNF) [29, 50, 51, 57], basic fibroblast growth factor [41, 52, 61, 64, 81], glial derived neurotrophic factor (GDNF) [69, 70] and insulin-like growth factor (IGF) [20, 40, 89]. Conversely, administration of various trophic factors, including NGF, BDNF, GDNF and FGF can protect against some of alcohol’s teratogenic effects [9, 16, 19, 63, 70, 73, 93]. This proposal will focus on the neurotrophic factor IGF-I, which can protect against neuronal cell loss and enhance production of white matter. Overview of the effects of prenatal alcohol induced white matter deficits. Since 1992 we have conducted a series of investigations using magnetic resonance imaging (MRI) to study children with fetal alcohol spectrum disorders (FASD). One of our most interesting findings has been that the corpus callosum, the major fiber tract connecting the two hemispheres, appears to be especially sensitive to prenatal alcohol exposure. Our findings corroborated results from autopsy cases where there was either partial or complete absence of the corpus callosum [51, 52]. Although agenesis of the corpus callosum is not unique to FAS, it has been suggested that FAS might be a leading cause of this condition [57]. Agenesis of the corpus callosum may or may not be associated with mental retardation, but in any event, it is typically associated with subtle cognitive deficits and problems with motor and bimanual coordination. The majority of individuals with prenatal alcohol exposure do not have such severe alterations in the corpus callosum that can easily be seen in the MRI scans. However, a more in depth evaluation indicates changes in the size and shape of this structure. In one study, the midsagital section of the corpus callosum was divided into five equiangular regions. After controlling for overall reductions in brain size, areas corresponding to the most anterior and the two posterior regions of the corpus callosum were reduced in the children with histories of prenatal alcohol exposure [55]. While there is controversy over the behavioral implications of size differences in the various parts of the corpus callosum, similar differences in the size of the corpus callosum have been reported in cases of ADHD [58]. Other studies have used newer imaging techniques where the size, shape, and location of the corpus callosum were assessed. One study [59], compared the average location of the corpus callosum in FASD and control children after equalizing for overall brain size and the location of other structures. Striking differences were observed in the group average and corpus callosum displacement maps. These data not only confirmed the reduced size of the corpus callosum, specifically in the posterior regions, but demonstrated that it was also displaced in three-dimensional space. The posterior portion of the corpus callosum exhibited the largest displacement in both the inferior and anterior directions in alcohol-exposed children. A similar but less significant pattern of displacement was observed in the nondysmorphic FASD subjects, suggesting that neuroanatomical anomalies in the corpus callosum can occur without the characteristic FAS facial features. Importantly, this corpus callosum displacement was highly correlated with the alcohol-exposed children's performance on a verbal learning task, and the shape of the corpus callosum was, in fact, a better predictor of verbal learning deficits than verbal IQ. The Seattle group also has data indicating changes in callosal shape and correlations with behavioral outcomes [60, 61]. Given these changes in the corpus callosum, a major white matter tract, it seemed logical to examine white matter changes occurring elsewhere. We next conducted a series of studies utilizing whole-brain MRI analysis on a group exposed prenatally to alcohol using two distinct types of analyses. Both studies revealed that prenatal alcohol exposure is related to disproportionate reductions in cerebral white matter with relative sparing of cerebral gray matter. When overall microcephaly is taken into account, it appears that the parietal regions are particularly affected, with marked white matter volume reductions occurring in this area along with reductions in white matter density and increases in gray matter density (REF). Deficiencies in white matter may also contribute to the optic nerve damage commonly seen in FASD children [65-67]. Studies of children with FASD illustrate a reduction in the size of the optic disc, indicating a reduction in the size of optic fibers. In addition, animal studies (see below) have shown that prenatal alcohol exposure reduces optic axon number and also alters myelination in the optic nerve. Diffusion tensor imaging (DTI) is a newer imaging technique that provides specialized information about the microstructure of white matter tissue within the brain. Specifically, DTI measures the diffusion of water molecules within white matter tissue, providing an estimate of the tissue’s overall maturity and organization. In general, increased water diffusion along a single direction (termed fractional anisotropy) and lower mean diffusion across all directions (mean diffusivity) signifies a more organized pattern of white matter, which implies more efficient neural processing. Whole fiber tractography DTI, a method that examines bundles of white matter fibers communicating across widespread brain regions, has recently been used to investigate white matter pathways in alcohol-exposed individuals. Evidence from these investigations, one of which is from my lab (Fryer, et al., 2008), suggests that several pathways display more disorganization in FASD, specifically those with connections to: 1) temporal (Lebel et al., 2008), 2) superior frontal (Fryer et al., 2008), and 3) occipital to inferior frontal and parietal regions (Fryer et al., 2008). DTI studies have also found microstructural damage and disorganization in the white matter tissue of the corpus callosum related to prenatal alcohol exposure, with abnormalities reported in posterior regions (splenium and isthmus) (Ma et al., 2005; Lebel et al., 2008; Sowell et al., 2008b; Wozniak et al., 2006) as well as anterior regions (the genu) (Ma et al., 2005; Lebel et al., 2008) and the overall body of the structure (Fryer et al., 2008). The finding of reduced white matter integrity along functional pathways in the brains of individuals with FASD complements earlier findings of decreased white matter density within specific regions of structural abnormality and provides further support for the idea that brain development and neuronal organization in FASD continues along an abnormal pathway long after the initial insult of prenatal alcohol exposure has passed. Evidence from animal studies also supports the adverse effects of prenatal alcohol on white matter. For example, in vivo studies have demonstrated that prenatal ethanol affects oligodendrocytes and delays myelin basic protein expression in various brain regions, including the cerebellum [86]. Such findings have been reported in rats, mice and guinea pigs. Similarly, in vitro studies demonstrate a delay in the development of myelin basic protein expression and altered oligodendrocyte morphology when exposed to alcohol [87]. Moreover, exposure limited to the 3rd trimester brain growth spurt reduces brain myelin accumulation and may mark a period of particular risk to myelin deficits [88]. Alcohol exposure during early postnatal development delays both acquisition and maturation of myelin and the oligodendroglia [89]. In addition, early postnatal exposure can lead to permanent alterations in the expression of mRNAs encoding myelin basic protein and myelin-associated glycoprotein [90]. This is not surprising, given that the 3rd trimester brain growth spurt is marked by rapid proliferation and maturation of oligodendrocyte precursors. Consistent with human studies, alterations in the corpus callosum and optic nerve have also been reported with various animal models. For example, several studies report that prenatal alcohol exposure reduces corpus callosum size [91, 92], although not all studies have found this [93]. Similarly, numerous studies found that early alcohol exposure induces optic nerve hypoplasia [94], delays the onset of myelination, reduces the number of myelinated axons, and reduces the thickness of the myelin sheath [66]. Prenatal alcohol delays the development of oligodendrocytes in the optic nerve and leads to aberrant myelin sheaths and myelin acquisition [66, 94]. Harris et al [95] reported that alcohol exposure limited to PD 4-9 leads to reductions in the proportion of myelinated axons in the optic nerve that persists at least until PD 30. With few exceptions [96], these findings are consistent with the clinical data. Finally, in addition to alcohol's effects on the CNS, alcohol exposure during development can alter Schwann cell proliferation and myelination [97]. Thus, ethanol's damaging effects on myelin production and associated glia occurs in both the CNS and PNS, a finding that has important implications for both cognitive functioning and control of peripheral muscles. In summary, data from several research groups indicate that white matter may be particularly affected by prenatal alcohol exposure. Consistent with the neuropathological profile, there are similarities between the behavioral problems seen in FASD children compared to individuals suffering from white matter dysfunction or deterioration, which lends further support to a relationship between white matter dysfunction and prenatal alcohol. Overview of the effects of IGF. Data from both clinical and animal studies indicate that a number of agents may successfully reduce the severity of myelin deficiencies. For example, several growth factors, including insulin-like growth factors (IGFs) have been shown to affect cells of the oligodendrocyte lineage. IGF-I belongs to a family of structurally related proteins including insulin and IGF-II. Insulin-like growth factors (IGF-I and IGF-II) are peptides that exert significant effects on cell metabolism and growth, and influence the development and differentiation of the CNS [99]. There is increasing evidence that IGF-I plays a role in oligodendrocyte development [100]. Oligodendrocytes possess receptors for IGFs and addition of IGF-I or IGF-II to developing brain cells in culture increases the number of oligodendrocytes, affecting proliferation, differentiation, and survival of cells [101,102]. Furthermore, IGF-I promotes the regeneration of oligodendrocytes, increases myelin gene expression, and increases myelin content both in vitro and in vivo, which has important implications for the proposed study. Thus, IGF-I may effectively serve as a treatment following a number of insults that affect white matter, even when administered after the insult [103]. For example, IGF-I increases both oligodendrocyte number and myelination following ischemia in the near-term fetal sheep [104]. It also reduces neuropathology in an experimental autoimmune encephalomyelitis, which serves as a model for demyelinating diseases [105]. Similarly, transgenic mice that overexpress IGF-I express greater myelin levels and are relatively protected against a reduction in myelin proteins resulting from undernutrition during the early neonatal period [106]. Moreover, IGF-I can promote myelination in the peripheral nervous system as well, by promoting proliferation, growth and survival of Schwann cells, and by both increasing myelination and inhibiting demyelination. Consistent with this role of IGF-I, IGF-I null mutant mice exhibit increased neural cell density in the cortex and reduced myelin content [107]. In addition, IGF-I knockout mice exhibit reductions in oligodendrocyte number, as well as myelinated axons in the corpus callosum and anterior commissure [108]. Moreover, reductions in myelination may be independent of any changes in neuron or axonal number [100]. Although some have reported that myelin levels increase later in adulthood in IGF-I knockout mice, this may be due to overcompensation by IGF-II or other growth factors [100]. Importantly, this does not suggest that the effects of IGF-I are limited to glial cells and subsequent myelin formation, as IGF-I can also affect neuronal cells as well [109]. IGF-I can influence neuronal survival, dendritic growth and synaptogenesis. In fact, peripherally administered IGF-I can induce neurogenesis in the adult hippocampus [110]. So although we are interested in first examining white matter deficits, it is possible that IGF-I may also influence other components of neuronal development. IGF as a treatment for FASD. The possibility that IGF-I can reduce the severity of some of the neuropathology and behavioral alterations associated with developmental alcohol exposure is strengthened by the adverse effects of prenatal alcohol on IGF. Animal studies have shown that prenatal alcohol exposure reduces IGF-I expression in brain [112]. These findings are consistent with clinical reports of lower IGF-I and IGF binding proteins in FAS children [113]. Moreover, nutritional insufficiency lowers IGF-I levels without lowering other growth hormone levels, and undernutrition during the period of oligodendrocyte development and myelination causes severe, irreversible hypomyelination. So while animal studies indicate that alcohol has direct effects on IGF (and alters myelin production compared to nutritional controls), the combination of alcohol and undernutrition that is frequently observed in the clinical population (for example in South Africa, where rates of FAS may be as high as 80 per 1000) may be particularly devastating. The potential for IGF-I to serve as a possible treatment is further strengthened by its current clinical use. Although success with white matter disorders has not always been found (many of these conditions involve a chronic degenerative disease), IGF is well tolerated with few adverse side effects. For example, clinical trials have used IGF-I to promote growth in children with cystic fibrosis. Although they failed to find significant effects on growth, there were no adverse side effects of administering 80 μg twice a day [114]. IGF-I continues to be studied for a variety of clinical uses [115]. Although IGF-I may not be effective in the presence of alcohol [157,158], IGF-I might be used therapeutically during infancy or childhood in children exposed to alcohol prenatally (as modeled in the present studies). Animal Model Systems. Many of the important findings regarding the relationship between alcohol-related neuropathology and behavioral alterations, risk factors, mechanisms, and intervention strategies must rely on the use of animal model systems. Within human populations, accurate measures of dose, timing and pattern of alcohol use are difficult to obtain, and potential confounds of poor prenatal care and polydrug use may be present. These factors can be manipulated and controlled with animal model systems. The usefulness of these systems is strengthened by a robust concordance of alcohol’s effects between human and animal studies, with similarities in dysmorphia, growth, and brain and behavioral alterations [26-28]. When utilizing animal models, it is important to note that although all mammals undergo the same stages of brain development, the timing of development relative to birth differs among species. Rats are altricial and the brain development that occurs during gestation in the rat is equivalent to only the first two trimesters of brain development in the human [29]. During the human third trimester, the brain undergoes a period of rapid development commonly referred to as the “brain growth spurt” characterized by axonal growth, dendritic arborization, high rates of synaptogenesis, gliogenesis, myelination, and maturation of synaptic neurotransmission. It is now recognized that the brain is particularly vulnerable to alcohol-induced insults during this period [30]. The brain growth spurt begins at the end of the human second trimester and continues into the first two years of postnatal life. In the rat, the brain growth spurt begins after birth. Thus, to model the period of the human third trimester brain growth spurt, the rat must be exposed to alcohol postnatally. We will be examining IGF-I administration in subjects exposed to alcohol during the 3rd trimester equivalent brain growth spurt. CNS and behavioral development can be altered by alcohol exposure the neonatal brain growth spurt in the rat and we along with numerous other groups have successfully used this alcohol exposure model for many years. Moreover, although this exposure paradigm models late gestational alcohol exposure only, the consequences of alcohol exposure during this developmental period are concordant with those seen in humans and the model has proven extremely valuable in elucidating mechanisms, pathology, and treatment outcomes. We have been successfully using this model for almost 25 years.

A. SCOPE OF THE PROJECT. Most of the procedures outlined in the original application are to be utilized in this competitive revision, although some modifications have been made due to methodological improvements over the last several years. These will be discussed where appropriate. Preliminary Results. We have shown that administration of IGF-I after 3rd trimester equivalent alcohol can reduce the severity of some fetal alcohol effects, specifically alcohol-induced motor deficits. Subjects were exposed to 5.25 g/kg/day alcohol via intubation from PD 4-9, during the 3rd trimester equivalent brain growth spurt. This alcohol exposure produced binge-like patterns of alcohol exposure with peak blood alcohol levels of around 320 mg/dl. From PD 10-13, subjects received 10 μg/day IGF-I intranasally. This age of IGF administration was chosen as it is equivalent to early postnatal development in humans, mimicking a treatment that would be administered after a prenatally alcohol-exposed child was born. Subjects were then tested on a battery of tests to examine the potential therapeutic effects of IGF-I, including locomotor activity testing in an open field chamber (PD 18-21), parallel bar motor coordination (PD 30-32), and Morris spatial water maze (PD 45-52). Thus, all behavioral testing occurred when IGF-I would no longer have acute effects. As shown in Figures 1 and 2, ethanol exposure during development impaired motor coordination and IGF-I significantly attenuated ethanol-related deficits in motor performance. Interestingly, IGF-I did not significantly affect performance among controls. In contrast, IGF-I did not significantly affect ethanol-related alterations on the activity level or Morris maze spatial learning tasks. Ethanol exposure significantly increased locomotor activity in the open field and this was not affected by IGF-I treatment. Similarly, ethanol exposure led to deficits in Morris maze spatial learning, as evidenced by increased heading angles during acquisition and impaired performance on the probe trial, an index of spatial memory; but IGF-I treatment did not alter ethanol-related deficits in spatial memory. Together, these data indicate that administration of IGF-I at this dose and during this developmental period targets motor areas of the CNS, such as the cerebellum, motor cortex, or striatum. Indeed, these are areas known to be adversely affected by developmental alcohol exposure and reductions in basic myelin protein have been reported in the cerebellum (REF). Research Design and Methods. Does IGF-I ameliorate the behavioral effects of neonatal alcohol exposure and does this correlate with white matter integrity? These experiments continue to examine possible treatments for FASD, a goal of the funded parent grant, and a specific aim in the original application (it was deleted, not based on scientific problems but based upon the suggestion from the reviewers that the two specific aims lacked sufficient integration.) IGF and myelin. As previously noted, both human and animal studies illustrate that prenatal alcohol exposure leads to a white matter deficit (REFS). The following experiments are tailored toward reducing the severity of white matter reductions by administering IGF-I, which has been shown to increase oligodendrocytes and enhance myelin expression. Rationale. Prenatal alcohol exposure can severely disrupt the development of white matter. In rodents, myelination begins in the first postnatal week and it is, incidentally, during this period that IGF-I expression in the brain peaks. When alcohol exposure is limited to the 3rd trimester equivalent, during this period of rapid myelin generation, alcohol leads to white matter reductions [95, 130]. One compound that has been shown to enhance oligodendrocyte development and increase myelin both in vivo and in vitro is insulin-like growth factor-I (IGF). Since acute ethanol exposure inhibits the glial cell proliferation response to IGF-I [131], it is uncertain if injections of IGF-I would be effective in stimulating myelin production in the presence of alcohol. However, our published data indicate that IGF-I is effective after the alcohol insult is complete. Importantly, this, in fact, would more closely model when a therapeutic intervention could be implemented in humans. Thus, the following study examines whether administration of IGF-I after alcohol exposure mitigates the adverse effects of ethanol on behavioral development when administered after ethanol treatment during the 3rd trimester equivalent. Our previous findings indicate that IGF-I attenuates ethanol’s adverse effects on motor behavior, but not on more cognitive tasks. This task specificity might be due to a dose effect, or be related to the developmental timing of IGF administration. The following studies study a broader range of doses and an earlier time of exposure, in order to answer these questions. Unlike many neurotrophic factors, IGF-I crosses the blood brain barrier [4, 77] and therefore has an effect on the CNS even when administered peripherally [77]. However, to reach the desired therapeutic dose in the CNS, higher IGF-I doses must be injected peripherally. Since IGF-I is a potent anti-apoptotic growth factor, it has been claimed that high doses of systemic IGF-I administration may have adverse side effects, including increased risk of tumorigenesis [101]. An alternate method for administering IGF-I into the CNS without presenting high doses peripherally is intranasal delivery, which has been shown to protect against neurological insults [58, 59]. This is the route we have decided on, and which we used in our published work. In the original application we had proposed icv administration. Experiment #1: Does IGF-I administration mitigate ethanol’s adverse effects on behavioral development? Protocol. On postnatal day (PD) 4, pups are randomly assigned to one of six treatment groups derived from the factorial combination of EtOH vs. SHAM and IGF (0, 10 or 15μg/10μl) (Peprotech, Rocky Hill, NJ). To control for potential litter effects, no more than one sex pair per litter will be assigned to any treatment group. On PD 4-9, EtOH subjects receive 5.25 g/kg/day (11.9% v/v) ethanol in a binge-like manner via intragastric intubation (27.5 ml/kg). Specifically, subjects receive 2.625 g/kg ethanol in a milk formula in each of two intubations, separated by two hours. Since alcohol causes reductions in suckling, EtOH subjects receive two additional intubations of a nutritionally balanced milk formula at 2-hour intervals following ethanol intubations [44]. During the SHAM intubation, the intubation tube is guided down the pup’s esophagus into stomach; however, no formula is administered. All subjects are returned to the dam between intubations. This is a fairly standard procedure and similar to what we have done previously and to what is used in various other labs [44, 106]. In the original protocol we proposed artificial rearing, but for the last several years have substituted oral gavage, as a less stressful, easier procedure. On PD 10-13, all subjects receive intranasal treatment under hypothermic anesthesia, with a third of the subjects receiving each dose. We will include 10 subjects per group. During the intranasal administration, all rat pups are protected with a latex glove and then placed into crushed ice/ice water for 6-8 minutes. After anaesthetized by hypothermia, subjects are placed on their backs and a total of 10 μl solution is administered in nose drops (1 μl/drop) over a 15-minute period, altering drops every 1.5 minute between the right and left nostril. The mouth and the opposite naris are closed during the administration so the drops could be naturally inhaled high into the nasal cavity. A second, third and fourth dose is given to each subjects 24, 48 and 72 hours after the first treatment, for a total of four doses of IGF-I or vehicle per rat. We have previously shown this method to serve as a successful means for delivering IGF-I to the CNS (REF). On PD 6, 20 μl of blood is collected from each pup’s tail 1.5 h after the second EtOH feed. Blood samples are centrifuged and supernatant collected. Samples are analyzed with the Analox Alcohol Analyzer (Model AM1, Lunenberg, MA) for blood ethanol content. On PD 10, subjects are coded via injections of India ink into paws so that each subject can be identified, but testers remain blind to treatment conditions. Subjects remain with the dam until weaning on PD 21. Following treatment, subjects will be tested on a series of behavioral tasks, to determine the functional significance of IGF-I treatment. The following behaviors, which we know to be sensitive to alcohol exposure during this period, will be tested: activity level (PD 18-21), parallel bar motor coordination (PD 30-32), Ledged tapered beam-traversal test: (PD 34-36), Morris Maze spatial learning (PD45-52) and, finally, paw laterality (PD 60). All procedures are described in the General Methods and all were proposed previously. In addition, given our previous motor findings we are including an addition task, the elevated beam task (Shallert, 2005) on PD 34-36. The developmental days chosen for testing are based on earlier findings from our lab and others indicating these are periods when such behavioral tasks are sensitive to early alcohol exposure. This series will allow us to determine if IGF-I is effective over a range of doses, behaviors and whether there is task specificity in the effects of IGF. Our previous work indicated task specificity, but IGF was only administered at a single dose. Interpretation and Significance of Results. If IGF-I is successful in reducing the severity of behavioral alterations associated with developmental alcohol treatment, it would suggest that this might serve as a potential therapeutic avenue for children exposed to alcohol prenatally. IGF has already been examined clinically in the potential treatment of cystic fibrosis, multiple sclerosis, and amyotrophic lateral sclerosis and may have potential for a number of neurodegenerative disorders. If IGF-I is unsuccessful or even exacerbates the adverse effects of early alcohol on behavioral development, then we will have to determine if it was successful in increasing white matter integrity (see Experiment #3). If beneficial effects are observed only on motor tasks, then it is likely that IGF-I targets the cerebellum, motor cortex, or striatum, areas known to be affected by early alcohol (REFS). If, however, beneficial effects are observed on other behaviors, such as the Morris water maze or open field activity, when doses are increased, it would suggest that the threshold for IGF-I effects is region-specific. Finally, paw laterality will be examined, as it is a task used to measure the functional integrity of the corpus callosum (REF). In sum, the pattern of behavioral effects will indicate the CNS regional specific effects of IGF-I and help to direct future studies on IGF-I and alcohol-induced neuropathology. Experiment #2: Does timing of IGF-I administration matter? The effects of IGF-I on motor performance, but not cognitive performance, as shown in the Preliminary Studies section, may be related to developmental differences in IGF among various CNS regions. The main effects of IGF-I on cell proliferation, survival and differentiation are mediated by IGF-I receptors, which are under critical spatial and temporal control. Although during PD 10-13, the expression of IGF-I receptors is high in the brain regions involved in neonatal ethanol induced behavioral impairment, there are some developmental differences. In the cerebellum, IGF-I mRNA is first detected on PD 0 and reaches its peak at about PD 12 [56]. Most cerebella Purkinje cells don’t express IGF-I immunoreactivity until PD 7 and start to lose their IGF-I immunoreactivity at PD 15 [87]. IGF-I receptors are not detectable until PD 4 and reach their maximum level at PD 14 [53]. However, in the hippocampus, the number of IGF-I receptors reaches its peak on embryonic day 18 and exhibits a reduction after birth [53]. IGF-I treatment in the present study occurred from PD 10-13, during the development period when IGF-I receptor expression is peak in the cerebellum. Thus, the timing of IGF treatment may explain the task specificity of IGF-I’s effects on ethanol-treated subjects. However, it is possible that IGF-I would have beneficial effects on other brain regions and corresponding behaviors if administered at a different developmental period. For example, even though levels are past their peak in the hippocampus, they are still high up until PD XXX. ADD SOME STUFF FROM THAT AUTORADIOGRAPHY PAPER. Sprague-Dawley rats will be randomly assigned to one of 4 postnatal treatment groups: ethanol-exposed+ IGF-I, ethanol exposed controls, saline exposed (pair fed) + IGF-I, and pair fed controls. Ethanol-exposed subjects will be exposed to binge-like ethanol (5.25 g/kg/day, producing peak blood alcohol levels (BALs) ~ 320 mg/dl) via oral gavage from postnatal days 4-6 (3rd trimester equivalents) in 2 intubations, as described in Experiment #1. We have data showing that while not as robust as the longer exposure, this period of time will induce significant behavioral changes on the tasks used in this study (REFS). Sham controls will be intubated without milk formula to control for any intubation-related stress. Ethanol-exposed and SHAM subjects will be randomly assigned within litter to one of 4 treatment groups using the optimal dose of IGF-I from Experiment #1: half of the subjects will receive IGF-I via nasal inhalation from PD 7-10, while the second group will receive saline vehicle. To determine peak BALs, 20 μl blood will be collected two hours after the alcohol intubation on PD 6 and analyzed with the Analox Alcohol Analyzer. Subjects (10 subjects/sex/group) will be tested on the same series of behavioral tasks, as described in Experiment #1. We hypothesize that administration of IGF during this developmental period may have a greater impact on brain regions involved in cognitive functioning. Experiment #3: Does IGF-I administration increase brain myelin integrity? Rationale. Administration of IGF-I can increase oligodendrocytes and myelin levels. To determine whether IGF-I increases myelin in animals exposed to alcohol during development, we originally proposed to measure levels of myelin basic protein (MBP) and proteolipid protein (PLP), the two major myelin-specific proteins (MSP). However, we are now proposing to use diffusion tensor imaging (DTI) to assess the integrity of white matter tracks. The reason for this is that we would like to obtain complementary information to our human DTI studies and we can obtain such information from our alcohol exposed animals not receiving IGF-I, as well as determine the integrity and extent of myelination. For example, recent work on comparing MRI to more basic histological techniques demonstrated that fractional anisotropy (FA) values in the corpus callosum mainly reflect changes in myelin structure Jito J; Fukami T; Nakasu S; Ito R; Morikawa S; Inusushi T; Nozaki K., 2009. Neurological Surgery [No Shinkei Geka], ISSN: 0301-2603, 2009 Feb; Vol. 37 (2), pp. 147-55; PMID: 19227156 Protocol. We will use one half of the subjects from the above experiments (probably all the animals from one sex, unless we find strong sex effects), contingent on the dose and timing factors that are effective. This study will be done in collaboration with Dr. Larry Frank from UCSD. We already have a subcontract with Dr. Frank for our human DTI studies, so this is an ongoing collaboration. After the conclusion of behavioral testing, brains will be fixed by intracardial perfusion of 1% parafolmadehyde/1.25 % glutaraldehyde. The fixed tissue will be stored in Fomblin solution and transported to the 7T Magnet Facility at UCSD for DTI scanning. All images will be acquired on the 7T Bruker Avance II at the UCSD CFMRI, a magnet dedicated to animal imaging. Dr. Frank is also applying for a challenge grant for DTI work in animals, and is well recognized as a leader in DTI. The following section was drafted by Dr. Frank in terms of the DTI work that they will be doing on rat brain to optimize DTI imaging. DTI of the contrast-enhanced perfusion-fixed brains will be performed 14-21 days postmortem. Sample temperature is maintained using calibrated, thermostatically controlled airflow at 10.0 degrees C throughout data acquisition. All MR imaging data are acquired using an 11.7 Tesla Bruker Avance II imaging system equipped with 1500mT/m Micro2.5 microimaging gradients, and a customized 3D diffusion weighted RARE (DW-RARE) sequence (Pipe et al., 2002) with double-echo diffusion preparation (Reese et al., 2003) and image-based phase correction (Tyszka and Frank, 2008). The acquisition has an isotropic spatial resolution of 100 μm3 with 32 diffusion encoding directions and 4 reference (b0) volumes. The diffusion directions were chosen using an approximate solution to the Thomson electrostatic repulsion problem (Jones et al., 1999). The following sequence parameters for DW-RARE acquisition will be used: TR/TE: 125/19.8ms, isotropic voxel size: 100 μm3, matrix size =256x160x128, echo train length: 8, nominal b-value: 1500 s/mm2, total imaging time: 3 hours 12 minutes. An accurate b-matrix for each image will be calculated by numerical simulation of the pulse sequence gradient waveforms and used for diffusion tensor reconstruction. Interpretation and Significance of Results: These data will allow us to determine how early alcohol exposure affects myelin integrity and whether IGF-I can affect the development of myelin and white matter tracks in alcohol-exposed subjects. Such data will provide critical information regarding the mechanisms by which IGF-I may be effective. There are two limitations to this study. First, although these data will provide a general look at overall myelin, they will not elucidate whether changes in myelin reflect increased number of myelinated axons, thicker myelin sheaths, or whether the distribution of myelinated axons is altered. Secondly, our results will not discount the possibility that IGF-I is also affecting neuronal development, in addition to myelin. Nevertheless, if IGF-I does affect myelin levels in alcohol-exposed subjects, these data would have important implications for FASD treatments. Finally, if we find that there are changes in myelin levels within a region, we will follow-up with a study to evaluate neuronal cell number via stereological analyses to determine if the changes in myelin are related to changes in neuronal survival. General Methods. All of these procedures are well established in the laboratory. Breeding. Multiparous Sprague-Dawley (S-D) dams are bred at the Animal Care Facilities in the CBT. Subjects are mated overnight and the presence of a sperm plug the following morning is used to determine gestational day (GD) 0. Pregnant animals are then singly housed in a temperature and humidity controlled nursery with food and water ad libidum on a 12:12 hour light/dark schedule. One day following birth, which usually occurs on GD 22, litters are randomly culled to 10 pups with the exception that 5 female and 5 males are kept when possible. Neonatal Alcohol Treatment: Gavage. In these studies, a procedure previously used by our group and others [41] will be utilized. The gavage tube is Polyethylene 10 tubing (11 cm in length), held by flexible Tygon tubing. Ethanol-treated groups are intubated with a nutritionally-balanced milk diet containing 2.625 g/kg ethanol (XXX v/v) ethanol (EtOH) at approximately 10:00 hr and again, two hours later, at 12:00. Milk only will be intubated at 2:00 and 4:00, since ethanol-exposed subjects will not suckle during intoxication. Sham control groups are intubated, but no milk formula is injected. To provide nutrition during the ethanol treatment, subjects are gavaged with two additional milk-only feedings (2-hr interval). During these feedings, control subjects are gavaged but do not receive additional milk. Between feedings, subjects are returned to the dam and monitored throughout the day. Several labs have used this treatment regimen successfully. Blood Alcohol Concentrations. Blood samples are collected from all subjects in all studies. Mean peak blood alcohol levels (BALs) could be obtained from a subset of non-experimental subjects, however, measurements from individual experimental subjects both confirms treatment and allows for the ability to correlate individual performance data with BAL. Moreover, previous studies indicate that the stress associated with drawing a small volume of blood has no significant effect on behavioral or brain development (e.g., see ref. [138]). Twenty microliters of blood are drawn from the tail of each pup collected into micropipettes 1.5 hours after the start of the last alcohol intubation on PD 6 to determine peak BAL. Blood samples are analyzed enzymatically with an Analox Alcohol Analyzer (Analox, USA). Behavioral Methods. Activity Assessment. Consistent with clinical studies of children prenatally exposed to alcohol, exposure to alcohol during the neonatal brain growth spurt produces increases in activity level (REFS). Activity level will be monitored in Plexiglas activity cages (40 x 40 x 30.5 cm) within an optical beam activity monitor (Hamilton-Kinder, San Diego, CA). The monitors and cages are located in enclosed chambers, equipped with fans providing airflow and masking noise. The chambers are cleaned prior to each testing session to remove any odor cues. Each subject is placed in the center of the chamber and interruptions of the infrared beams are recorded electronically. A variety of activity measures are automatically collected every 5 minutes during a 60-min test. Subjects are tested for four consecutive days, during the dark cycle. Total distance traveled, time in motion, rearing and thigmotaxis (wall hugging) will be examined. This will provide measures of overall activity as well as emotionality. Parallel Bar Motor Task. Both autopsy and MRI studies report that the cerebellum is one brain region particularly sensitive to alcohol-induced volumetric reductions. Numerous studies have demonstrated that 3rd trimester equivalent alcohol exposure produces severe cerebellar damage and deficits in balance and fine motor coordination. The parallel bar task is a test of motor coordination, shown to be sensitive to both early alcohol exposure [139] and cerebellar damage [140]. In fact, the severity of alcohol-induced motor deficits is significantly correlated with the severity of cerebellar Purkinje cell loss [141]. The parallel bar apparatus consists of two parallel steel rods (0.5-cm diameter each, 91 cm long) held between two end platforms (15.3 x 17.8 cm). The rods are fastened with screws onto a rack of 28 grooved slots (0.5 cm apart) on each platform, thereby allowing the distance between the rods to be manipulated. The platforms and rods stand 63 cm above a floor of wood chip bedding. On the first day of testing, the subject is initially placed on each platform for 30 sec. Then, the subject is carefully placed on the rods halfway between the platforms, with both left paws on one bar and both right paws on the other bar. Subjects usually move toward the escape platform, given their position. Four successive alternating steps with the hind legs on the rods constitute a successful traversal. If the subject places two hind paws on one rod, falls or swings under the rods, the trial is unsuccessful. If the subject fails to move after 5 min, it is removed, placed in a holding cage for 1 min and replaced onto the rods. The initial distance between the rods is set at 3.0 cm. Subjects are tested for up to five consecutive trials at a given width between rods, with an intertrial interval (ITI) of 5 to 10 sec. Once successful at a given width, the subject is placed in a holding cage while the distance between the rods is increased at 0.5-cm increments. If unsuccessful after five consecutive trials, testing for the day is terminated. Subjects are tested a maximum of 15 trials a day, for 3 consecutive days. Each day, the distance between rods is set at the last successful distance for each individual animal. The maximum distance successfully traversed as well as the ratio of successful to total traversals serve as measures of performance. Ledged Beam Test. Ledge tapered beam-traversal test: On PD 34, rats will be tested on the ledge tapered beam-traversal test, a task that is particularly sensitive to motor impairments (Schallert et al., 2002, Fleming et al., 2004) and can be used to assess the extent to which therapeutic interventions can alleviate those impairments (Schallert 2006). The ledged tapered beam is 165cm in total length and is split into 3 distinct “bins.” Each of these bins contains a 1.5cm wide raised ledge that is raised above a 2-cm step-down surface. On the first training day, rats are placed at one edge of the beam, under a bright light to initiate movement, and are encouraged to walk down the tapered beam towards their home-cage. The home-cage is covered with a dark cloth and placed at the end of the beam to promote traversal. During the first trials, until a successful traversal is made, rats are gently assisted towards the end of the beam by tapping on the beam, lifting the tail, or pushing their hind limbs towards the home-cage. On the first day of training, rats must achieve 5 successful traversals without experimenter assistance. These 5 traversals serve as learning trials and are not scored for errors. On the following day, rats are placed on the beam and their movement recorded for a total of 5 trials. Recordings will be rated by experimenter’s blind to the experimental groups and scored for errors. An error is counted if any limb slips from the tapered ledge and contacts the step-down surface. Task difficulty can be increased with the addition of a mesh-grid surface, placed over the tapered portion of the beam, with errors being counted whenever a limb slips from the grid and contacts the tapered ledge surface. This increase in difficulty necessitates the addition of a second training day, during which the rats learn to traverse the mesh-grid surface with experimenter assistance. Morris Water Maze. The Morris spatial learning water maze is a task that relies on the functional integrity of the hippocampus (REF), a brain area known to be damaged by neonatal alcohol exposure (REF). Previous studies from our group and others have found this task to be sensitive to neonatal alcohol exposure (REF). This task utilizes a circular water tank (121 cm diameter) filled with water (26° C) made opaque with the addition of powdered milk. The tank is housed in a room filled with spatial cues (e.g. sink, lights, posters, and the experimenter, who is always in the same location). An escape platform (13 x 13 cm) is hidden 1.5 cm below the surface and cannot be seen by the subject because of the opacity of the water. Each subject is marked with nontoxic ink that allows a video tracking system to observe the rat within the white pool. A trial is initiated with the placement of the rat at one of 12 starting positions along the perimeter of the tank. The starting position differs for each trial and follows a pseudorandom sequence, with the condition that the subject must start once in each quadrant of the tank each day. During each trial, the subject is placed in the tank, facing the rim, and must swim to the location of the escape platform. If the subject fails to escape within 70 sec, the subject is manually placed on the platform. The subject remains on the platform for 10 sec before being removed. Between trials, subjects are kept in a heated environment (31° C) to prevent any hypothermia. Subjects are tested for four trials each day with an ITI of 3-5 min. Subjects are tested for 6 consecutive days, beginning on PD 45. After the last acquisition trial, the platform is removed for a 60-sec probe trial. On PD 51-52, subjects are tested with a visible platform, to determine if any deficits are related to performance measures, like vision, swimming ability, or motivation. On these days, white sheets surround the pool, removing spatial cues, and the platform is set above water level, made visible with a brightly colored hue. The location of the platform changes on each trial, thus subjects must learn to swim to the platform, regardless of spatial location. Subjects are tested for 4 trials on each of 2 days. The platform location is in each of the quadrants per day. Ideally, separate subjects would be tested on a visible platform task in a manner identical to the spatial condition. However, that would require doubling the number of subjects, which would not be practical. We have found that alcohol-exposed subjects exhibit spatial learning deficits with no differences in visible platform learning using the current protocol. All data are collected via a video camera interfaced with a PC and software (HVS Image). Latency to escape, path length, percentage of time in each quadrant, and initial heading angle are measured with trial and day as within-subject factors. On the probe trial, the area within 26 cm around where the platform should have been is identified and the number of passes through and the amount of time spent in this area serve as measures of search pattern. Paw laterality. Paw laterality will also be examined, as reduced laterality has been associated with corpus callosum damage and is also known to be affected by prenatal alcohol exposure [143, 144]. Testing sessions take place in a reaching apparatus made of three contiguous plastic cubicles, each 24 cm x 8 cm x 16 cm, separated by opaque walls. A cylindrical feeding tube (11 mm) is placed in the face wall of the cubicles, projecting at chest high when the rat stands on its hind legs. Each rat participates in three videotaped testing sessions. Rats are initially 24 hours food deprived and then placed into one of the cubicles of the reaching apparatus, and remain in there until they have either reached 50 times for food (Fruit Loops) or one hour has passed. A single Fruit Loop is placed in the reaching tube for each trial. A successful reach is defined as the rat obtaining the piece of Fruit Loop. Each time the Fruit Loop is retrieved, another piece is immediately placed into the tube. Upon completion of the testing session, rats are taken out of the reaching apparatus and placed back in their cages, where they have free access to food for three hours and are then food-deprived until the next session. Paw preference scores are estimated as the number of right paw entries (RPE). If the animal quits before 50 entries, the score will be adjusted appropriately. This score combines both direction (number of right paw entries) and degree of asymmetry (calculated by subtracting 25 from the RPE and dropping the algebraic sign). Thus, subjects with RPE scores of 0 or 50 would have the same asymmetry score of 25 but different direction scores. Statistics. In these types of experiments it is important to control for litter effects. This is accomplished by using only one sex pair per treatment per litter following alcohol treatment and by using only one subject from each litter in any cell of the experimental design. Data will be analyzed with SPSS statistical programs for the PC. The level of significance will be set at p<.05 and Newman-Keuls post hoc tests (p<.05) will be done where appropriate. All of these experiments lend themselves to analysis of variance. In all studies, ethanol treatment (EtOH, SHAM), IGF treatment (IGF-I, saline vehicle) and sex (male, female) act as independent measures. Depending on the behavioral measure, one or more repeated measures will be included in the analysis. For example, in the open-field analysis, day and 5-min blocks will be used as within-subject variables. In the parallel bar studies, the maximum distance will be analyzed with day as a repeated, within-subject measure. Correlational analyses will be conducted between behavioral performance and DTI FA and diffusivity data in select regions of interest. Sample sizes were determined using preliminary data subjected to power analyses (InStat) and were conducted with alpha = .05 and 1- beta (power) = 0.80. B. ACCELERATE THE TEMPO OF SCIENTIFIC RESEARCH AND ALLOW FOR JOB CREATION AND RETENTION. Per the descriptions above, hopefully, the reviewers will see that this is a very labor intensive project, and as such will provide for two technical support people. Given the downturn in the economy, there is an ample supply of unemployed or underemployed individuals in the biotechnology industry in San Diego who could be employed in these positions. In discussing this application with the SDSU Foundation, the number of employment applications for laboratory work has increased tremendously over the past six-months. There are typically 20 applicants for each technical position, up from fewer than 6 a year ago. Previously, well qualified individuals had no difficulty in obtaining employment with one of the many biotechnology companies in San Diego, however, these positions have dried up and in fact, many well qualified individuals are now unemployed. Unemployment in California has gone from 6.4% to 10.9 % in the last year, and San Diego has not faired much better, going from 5.3% to 8.8%. We will be able to hire two highly qualified indiviudals for for this project. In addition, we will have to purchase supplies and rats and buy some additional equipment. Also, the interest in impact of IGF as a potential treatment for various disorders has accelerated in the past few years.


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