Lmo genes regulate behavioral responses to ethanol in Drosophila melanogaster and the mouse

Background: Previous work from our laboratory demonstrated a role for the Drosophila Lim‐only (dLmo) gene in regulating behavioral responses to cocaine. Herein, we examined whether dLmo influences the flies’ sensitivity to ethanol’s sedating effects. We also investigated whether 1 of the mammalian homologs of dLmo, Lmo3, is involved in behavioral responses to ethanol in mice. Methods: To examine dLmo function in ethanol‐induced sedation, mutant flies with reduced or increased dLmo expression were tested using the loss of righting (LOR) assay. To determine whether mouse Lmo3 regulates behavioral responses to ethanol, we generated transgenic mice expressing a short‐hairpin RNA targeting Lmo3 for RNA interference‐mediated knockdown by lentiviral infection of single cell embryos. Adult founder mice, expressing varying amounts of Lmo3 in the brain, were tested using ethanol loss‐of‐righting‐reflex (LORR) and 2‐bottle choice ethanol consumption assays. Results: We found that in flies, reduced dLmo activity increased sensitivity to ethanol‐induced sedation, whereas increased expression of dLmo led to increased resistance to ethanol‐induced sedation. In mice, reduced levels of Lmo3 were correlated with increased sedation time in the LORR test and decreased ethanol consumption in the 2‐bottle choice protocol. Conclusions: These data describe a novel and conserved role for Lmo genes in flies and mice in behavioral responses to ethanol. These studies also demonstrate the feasibility of rapidly translating findings from invertebrate systems to mammalian models of alcohol abuse by combining RNA interference in transgenic mice and behavioral testing.     

A pair of dopamine neurons mediate chronic stress signals to induce learning deficit in Drosophila melanogaster

Chronic stress could induce severe cognitive impairments. Despite extensive investigations in mammalian models, the underlying mechanisms remain obscure. Here, we show that chronic stress could induce dramatic learning and memory deficits in Drosophila melanogaster. The chronic stress–induced learning deficit (CSLD) is long lasting and associated with other depression-like behaviors. We demonstrated that excessive dopaminergic activity provokes susceptibility to CSLD. Remarkably, a pair of PPL1-γ1pedc dopaminergic neurons that project to the mushroom body (MB) γ1pedc compartment play a key role in regulating susceptibility to CSLD so that stress-induced PPL1-γ1pedc hyperactivity facilitates the development of CSLD. Consistently, the mushroom body output neurons (MBON) of the γ1pedc compartment, MBON-γ1pedc>α/β neurons, are important for modulating susceptibility to CSLD. Imaging studies showed that dopaminergic activity is necessary to provoke the development of chronic stress–induced maladaptations in the MB network. Together, our data support that PPL1-γ1pedc mediates chronic stress signals to drive allostatic maladaptations in the MB network that lead to CSLD.

Stress has significant and complex effects on cognitive function. In general, these effects follow an inverted U–shaped dose–response relationship in intensity and duration. So that moderate acute stress could promote learning and memory, while chronic stress very often induces detrimental effects (1). Since chronic stress–induced learning and memory impairments are closely associated with many neural disorders, such as depression, schizophrenia, and Alzheimer''s disease, understanding the underlying neurobiology is of importance for developing effective drugs and treatments (2, 3). To this end, animal models, especially mammalian models, have been extensively investigated. Current findings suggest that the effects of chronic stress on learning and memory could be influenced by many internal and external factors that involve multiple brain regions, genes, and complex mechanisms that have not yet been fully elucidated (3–6).Stress could have consequential effects on aversive olfactory memory in Drosophila melanogaster. For example, moderate fast promotes long term memory (LTM) formation (7, 8), while sleep deprivation promotes forgetting and impairs memory capacity (9–11). Recent reports have shown that chronic stress can induce depression-like symptoms in Drosophila, as manifested by characteristic behaviors that indicate anhedonia, lack of motivation, prone to despair, and sleep disorder (12–14). However, investigation of the effect of chronic stress on Drosophila learning and memory is still lacking.In the present study, we report that a 4-d chronic stress treatment (CST) effectively induces strong learning and memory deficits in Drosophila. We focused on the learning deficit phenotype and found that the Drosophila dopaminergic (DAergic) system plays an important role in modulating susceptibility to chronic stress–induced learning deficit (CSLD), suggesting that DAergic modulation is an evolutionary conserved chronic stress–coping mechanism. We pinpointed the key CSLD regulating dopamine neurons (DANs) to a pair of PPL1-γ1pedc neurons that project to the mushroom body (MB) γ1pedc compartment and further showed that MBON-γ1pedc>α/β, the output neurons of γ1pedc compartment, modulates susceptibility to CSLD as well. Imaging studies identified chronic stress–induced abnormal neural activities in learning-related neurons, which require DAergic activity during CST. Overall, our studies delineate a model that chronic stress signals can be mediated by a pair of DANs, PPL1-γ1pedc, to drive maladaptations in the MB network that lead to CSLD.     

Physiological,anatomical, and behavioral changes after acoustic trauma in Drosophila melanogaster

Noise-induced hearing loss (NIHL) is a growing health issue, with costly treatment and lost quality of life. Here we establish Drosophila melanogaster as an inexpensive, flexible, and powerful genetic model system for NIHL. We exposed flies to acoustic trauma and quantified physiological and anatomical effects. Trauma significantly reduced sound-evoked potential (SEP) amplitudes and increased SEP latencies in control genotypes. SEP amplitude but not latency effects recovered after 7 d. Although trauma produced no gross morphological changes in the auditory organ (Johnston’s organ), mitochondrial cross-sectional area was reduced 7 d after exposure. In nervana 3 heterozygous flies, which slightly compromise ion homeostasis, trauma had exaggerated effects on SEP amplitude and mitochondrial morphology, suggesting a key role for ion homeostasis in resistance to acoustic trauma. Thus, Drosophila exhibit acoustic trauma effects resembling those found in vertebrates, including inducing metabolic stress in sensory cells. This report of noise trauma in Drosophila is a foundation for studying molecular and genetic sequelae of NIHL.Noise-induced hearing loss (NIHL) is a pervasive and growing health issue arising from occupational and recreational hazards, with significant costs in health care and personal quality of life. Despite this, the molecular and physiological mechanisms involved in the etiology or recovery from injury are not yet fully understood. Importantly, intense acoustic trauma can induce permanent damage—unlike other vertebrates, mammals cannot regenerate auditory hair cells (1, 2). NIHL associated with permanent changes in auditory sensitivity causes multiple consistent effects: stereocilia bundle disruption, inner (IHC) and outer hair cell (OHC) death or damage, supporting cell tissue disruption, and eventual spiral ganglion cell damage or loss (3–7). Most studies to date used mammalian model organisms such as mice (8, 9), rats (10), and guinea pigs (11–14). These animals have difficult access to the inner ear inside the temporal bone and high maintenance costs coupled with relatively long generation times.Drosophila is a compelling alternative model system with strong genetic tools, inexpensive production of large numbers of animals, and an accessible auditory system that is becoming better understood genetically and physiologically. During courtship, Drosophila males vibrate their wings to produce a courtship song composed of pulse and sinusoidal components (15, 16). This song facilitates species identification and mate selection (16, 17). Drosophila males and females detect airborne vibrations via Johnston’s organ (JO) in the second antennal segment (18). The JO is an array of chordotonal mechanoreceptors (or scolopidia; Fig. 1 A–C). Via the aristae, acoustic energy is transformed to rotational movement of the third antennal segment, activating mechanosensitive channels on JO neuron dendrites. Like vertebrate hair cells, JO neurons are ciliated and respond to mechanical stimulation. Although JO has morphologically diverged from hair cells in the human inner ear, the genetic program for its development shares a strong homology (19, 20). For example, the Atoh1 gene required for vertebrate auditory hair cell specification was found by direct homology to the fly atonal gene required for JO specification and atonal/Atoh1 genes can be functionally exchanged between mice and flies (21, 22). The advantages of studying hearing in Drosophila are that the genome is fully sequenced, genetic tools for extensively manipulating the genome are at hand, genetic background effects can be effectively eliminated, and large numbers of individuals can be tested.Open in a separate windowFig. 1.Organization of Drosophila JO and physiological response to sound. (A) Deconvolution micrograph of labeled scolopidia in JO. The actin scolopale rods are labeled with phalloidin (magenta), mitochondria in some JO neurons are labeled with mito-GFP (green), and nuclei are labeled with TOPRO-3 (blue). (B) Schematic diagram of an individual scolopidium, oriented and colored similarly to scolopidia in A. (C) Approximate longitudinal section of JO in an untraumatized control 40AG13 fly. bb, basal bodies; cap, dendritic cap; cd, ciliary dilation; m, membranous structure; mt, mitochondria; N, nuclei of JO neurons; ScN, nuclei of scolopale cells; t, trachiole. (Scale bar: 1 μm.) (D) Example of SEPs recorded in response to acoustic stimulation (stim). The top trace is the synthetic courtship song pulse stimulus; immediately below is the resulting SEP, with the analyzed amplitude and latency parameters indicated. The bottom trace shows multiple SEPs from a wild-type fly in response to a pulse train.In this study, we establish Drosophila as an inexpensive and flexible model system for genetic and physiological study of NIHL. We exposed two control strains [Canton-S (CS) and 40AG13] to acute acoustic trauma and examined physiological, behavioral, and anatomical effects. Our findings show immediate effects on auditory function, with reduced and delayed evoked activity. Although evoked potential amplitudes were restored after 7 d, the latency of these potentials did not fully recover and we found significant changes in JO neural mitochondrial morphology. We also tested mutant flies with a reduced copy number of nervana 3 (nrv3) encoding a Na⁺/K⁺ ATPase β subunit expressed in JO neurons (23). We hypothesized that compromised JO ionic homeostasis would confer susceptibility to noise trauma. Indeed, nrv3 heterozygotes showed increased sensitivity to trauma and a significantly reduced auditory functional recovery.     

Short and long-lasting behavioral consequences of agonistic encounters between male Drosophila melanogaster

In many animal species, learning and memory have been found to play important roles in regulating intra- and interspecific behavioral interactions in varying environments. In such contexts, aggression is commonly used to obtain desired resources. Previous defeats or victories during aggressive interactions have been shown to influence the outcome of later contests, revealing loser and winner effects. In this study, we asked whether short- and/or long-term behavioral consequences accompany victories and defeats in dyadic pairings between male Drosophila melanogaster and how long those effects remain. The results demonstrated that single fights induced important behavioral changes in both combatants and resulted in the formation of short-term loser and winner effects. These decayed over several hours, with the duration depending on the level of familiarity of the opponents. Repeated defeats induced a long-lasting loser effect that was dependent on de novo protein synthesis, whereas repeated victories had no long-term behavioral consequences. This suggests that separate mechanisms govern the formation of loser and winner effects. These studies aim to lay a foundation for future investigations exploring the molecular mechanisms and circuitry underlying the nervous system changes induced by winning and losing bouts during agonistic encounters.Across the animal kingdom, aggression between conspecifics often accompanies the competition for food, mates, and territory. Although an innate behavior, aggression is a highly adaptive trait as well, with animals learning from previous experience and changing their behavior in response to new challenges. In competition for rank, for example, previous fighting experience influences the outcome of subsequent contests: prior defeat decreases whereas prior victory increases the probability of winning later contests. These have been called “loser” and “winner” effects (1). Such effects have been observed in many species, including fish (2), birds (3), and mammals (4). In general, loser effects persist longer than winner effects (5). The durational asymmetry observed between loser and winner effects has been hypothesized to participate in stabilizing social hierarchies among conspecifics (6).Fruit flies (Drosophila melanogaster) exhibit a variety of simple and complex social behaviors, including aggregation (7), courtship (8), and aggression (9) in which learning and memory have been demonstrated or postulated to serve important roles (10–12). Thus, characterizing the molecular basis of memory formation, retention, and retrieval is crucial to ultimately understanding the adaptability of these social behaviors. In Drosophila, a variety of operant and classical training paradigms have been used to subdivide memory into distinct categories. Short-term memory (STM) lasting minutes to hours is induced by a single training session, whereas long-term memory (LTM) lasting days usually requires repeated training sessions and involves de novo protein synthesis (13). A large number of studies have been carried out using olfactory, visual, social, and place memory paradigms. These have allowed the functional and molecular characterization of neuronal circuits and the identification of numerous genes underlying learning and memory (14–16). Included are mutations in rutabaga (rut, type 1 adenylyl cyclase) that interfere with learning and STM formation (17); amnesiac (amn, peptide regulator of adenylyl cyclase) that affect STM retention (18); and crammer (cer, inhibitor of a cathepsin subfamily) that prevent LTM formation (19). Whether rut, amn, and cer serve roles in the learning and memory that accompanies aggression remains unknown.Male–male aggression in fruit flies was first described almost 100 y ago (20). Since then, considerable progress has been made in understanding its expression and regulation (21–26). In competition for food resources and territory, male–male agonistic encounters, composed of stereotyped behavioral patterns, usually result in the formation of dominance relationships (9). During the progression of fights, both flies modify their fighting strategies: The ultimate winners chase and lunge at their opponents to gain sole access to the resources, whereas the losers retreat from the resources after receiving such attacks (9, 10).In second fights (30 min after first fights), losing flies display greater submissive behavior and never win against naïve or experienced opponents, revealing short-term loser effects (10). Evidence for winner effects, however, was not found (11). Recently, in olive fruit flies (Bactrocera olea) it was found that previous losing and winning experiences both increased the aggressiveness of the flies. This suggests that the consequences of losing or winning may vary across species (27).We previously suggested that fights between male flies function as operant learning situations in which males learn to use their most advantageous fighting strategy during fights and then continue to do so in subsequent contests (28). In an attempt to optimize the learning and memory associated with aggression, we designed new “handling-free” behavioral chambers (29). These proved to be more desirable for studying the formation of loser effects (12). By using these experimental arenas and pairing familiar opponents in second fights we previously showed that changes in fighting strategies could be developed by both winning and losing flies. This allowed us to suggest the existence of short-term winner effects along with the previously demonstrated loser effects (12). A more detailed examination of these short-term effects is presented here along with experiments attempting to measure the intrinsic changes in fighting abilities of losing and winning flies.In the present study, we ask (i) whether a single fight can lead to the formation of loser and winner effects and how long these effects persist, (ii) whether flies adopt different fighting strategies in second fights depending on their opponents, (iii) whether longer-lasting behavioral effects result from sequential repeated defeats or victories, (iv) whether protein synthesis is required for the short- or long-term effects observed, and (v) whether mutations in genes involved in learning and memory affect aggressive behavior.     

A mild stress, hypergravity exposure, postpones behavioral aging in Drosophila melanogaster.

Flies were submitted to two weeks of hypergravity in a centrifuge (3 or 5 g), starting at the second day of imaginal life, and their behavior (spontaneous locomotor activity, patterns of movement, and climbing activity) was observed from removal of the centrifuge to an older age; the usual effects of age on these behaviors were generally observed. Hypergravity-kept flies had worse behavioral scores on removal of centrifuge than those always kept at 1 g. When they aged, they got either similar or better scores than 1 g flies, which indicates that their behavioral aging may be slower. These results show that a mild stress such as hypergravity, which has been previously shown to increase the longevity of males and resistance to heat shock in both sexes, is an environmental manipulation postponing aging in flies.     

Multicopper oxidase-1 is a ferroxidase essential for iron homeostasis in Drosophila melanogaster

Multicopper ferroxidases catalyze the oxidation of ferrous iron to ferric iron. In yeast and algae, they participate in cellular uptake of iron; in mammals, they facilitate cellular efflux. The mechanisms of iron metabolism in insects are still poorly understood, and insect multicopper ferroxidases have not been identified. In this paper, we present evidence that Drosophila melanogaster multicopper oxidase-1 (MCO1) is a functional ferroxidase. We identified candidate iron-binding residues in the MCO1 sequence and found that purified recombinant MCO1 oxidizes ferrous iron. An association between MCO1 function and iron homeostasis was confirmed by two observations: RNAi-mediated knockdown of MCO1 resulted in decreased iron accumulation in midguts and whole insects, and weak knockdown increased the longevity of flies fed a toxic concentration of iron. Strong knockdown of MCO1 resulted in pupal lethality, indicating that MCO1 is an essential gene. Immunohistochemistry experiments demonstrated that MCO1 is located on the basal surfaces of the digestive system and Malpighian tubules. We propose that MCO1 oxidizes ferrous iron in the hemolymph and that the resulting ferric iron is bound by transferrin or melanotransferrin, leading to iron storage, iron withholding from pathogens, regulation of oxidative stress, and/or epithelial maturation. These proposed functions are distinct from those of other known ferroxidases. Given that MCO1 orthologues are present in all insect genomes analyzed to date, this discovery is an important step toward understanding iron metabolism in insects.     

Physiology declines prior to death in Drosophila melanogaster

For a period of 6–15?days prior to death, the fecundity and virility of Drosophila melanogaster fall significantly below those of same-aged flies that are not near death. It is likely that other aspects of physiology may decline during this period. This study attempts to document changes in two physiological characteristics prior to death: desiccation resistance and time-in-motion. Using individual fecundity estimates and previously described models, it is possible to accurately predict which flies in a population are near death at any given age; these flies are said to be in the “death spiral”. In this study of approximately 7,600 females, we used cohort mortality data and individual fecundity estimates to dichotomize each of five replicate populations of same-aged D. melanogaster into “death spiral” and “non-spiral” groups. We then compared these groups for two physiological characteristics that decline during aging. We describe the statistical properties of a new multivariate test statistic that allows us to compare the desiccation resistance and time-in-motion for two populations chosen on the basis of their fecundity. This multivariate representation of the desiccation resistance and time-in-motion of spiral and non-spiral females was shown to be significantly different with the spiral females characterized by lower desiccation resistance and time spent in motion. Our results suggest that D. melanogaster may be used as a model organism to study physiological changes that occur when death is imminent.     

Conditioned Behavior in Drosophila melanogaster

Populations of Drosophila were trained by alternately exposing them to two odorants, one coupled with electric shock. On testing, the flies avoided the shock-associated odor. Pseudoconditioning, excitatory states, odor preference, sensitization, habituation, and subjective bias have been eliminated as explanations. The selective avoidance can be extinguished by retraining. All flies in the population have equal probability of expressing this behavior. Memory persists for 24 hr. Another paradigm has been developed in which flies learn to discriminate between light sources of different color.     

Reproductive fitness and longevity in Drosophila melanogaster

It has been suggested that senescence could have evolved by selection of genes with beneficial effects early in life and detrimental ones later in life (pleiotropy theory of the evolution of senescence). To test that theory, the egg production of 322 females of the Oregon strain of Drosophila melanogaster was recorded daily throughout their life. At the individual level, no relation could be detected between early components of fitness and longevity. For the time being it appears that there are no unequivocal reasons to accept the pleiotropy theory of the evolution of senescence.     

Host-plant specialization in the Drosophila melanogaster species complex: a physiological, behavioral, and genetical analysis.

Drosophila sechellia, endemic to the Seychelles, breeds in a single resource, Morinda citrifolia, whereas its close sympatric relative, Drosophila simulans, is a cosmopolitan generalist breeding in a great variety of resources. The effects of morinda on various fitness traits of these two species, their F1 hybrids, and reciprocal backcrosses were analyzed. Morinda fruit is highly toxic to Drosophila species, except D. sechellia. The toxicity is expressed in adults, embryos, and larvae. In embryos, early mortality is a maternally inherited trait, depending only on mother's genotype. The tolerance of D. sechellia to morinda is fully dominant in F1 hybrids. Egg production is stimulated by morinda in D. sechellia but inhibited in D. simulans; in hybrids, the inhibition observed in D. simulans is dominant. Morinda is an oviposition attractant for D. sechellia but a repellent for D. simulans; F1 hybrids and backcross individuals exhibit intermediate, approximately additive, behavior. In the field, adult flies of the two species exhibit opposite behavior in that D. sechellia is attracted to morinda and D. simulans is attracted to banana; hybrids have an intermediate behavior. These differences between the species explain why they do not hybridize in nature although living in sympatry. The various traits have different genetic bases: three or four different genes, or groups of genes, differentiate the ecological niches of the two species.     

Spontaneous Recombination in Drosophila melanogaster Males

A second chromosome of Drosophila melanogaster (symbol T-007) isolated from a natural population in Harlingen, Texas, was found to undergo recombination in heterozygous males. Heterozygous males transmit this chromosome with a frequency, k, of about 0.4, considerably reduced from the expected value of 0.5. The frequency of male recombination and the k value are negatively correlated, indicating that the two phenomena are in some way related. The complementary recombinant products are recovered in equal frequency and the recombination is not restricted to the heterochromatic regions. The time of recombination is not certain, but the distribution of recombinants is more suggestive of meiotic than of premeiotic occurrence. In the natural population of these flies, the frequency of chromosomes with male recombination is 20% or more.     

Transplantation of Nuclei in Drosophila melanogaster

Nuclei surrounded by ooplasm of the syncytial stage of developing eggs of wild-type Drosophila melanogaster were implanted into freshly laid fertilized eggs of females of a y w stock. More than half of the recipient eggs produced larvae, but few of the larvae hatched or developed further. The best sets of experiments gave about twelve percent of imagos, mostly y w in appearance. Several larvae were mosaics with yellow Malpighian tubes, and two flies had part of the abdominal segments of the wild type. Half of the flies were fertile, but they produced only y w offspring, except for two males that had y w appearance, but wild-type gonads. When crossed with y w females, they gave wild-type females and y w males.     

Age-associated cardiac dysfunction in Drosophila melanogaster

The fruit fly, Drosophila melanogaster, has served as a valuable model/organism for the study of aging and was the first organism possessing a circulatory system to have its genome completely sequenced. However, little is known about the function of the heartlike organ of flies during the aging process. We have developed methods for studying cardiac function in vivo in adult flies. Using 2 different cardiovascular stress methods (elevated ambient temperature and external electrical pacing), we found that maximal heart rate is significantly and reproducibly reduced with aging in Drosophila, analogous to observations in elderly humans. We also describe for the first time several other aspects of the cardiac physiology of young adult and aging Drosophila, including an age-associated increase in rhythm disturbances. These observations suggest that the study of declining cardiac function in aging flies may serve as a genetically tractable model for genome-wide mutational screening for genes that participate in or protect against cardiac aging and disease.     

A gene adjacent to satellite DNA in Drosophila melanogaster.

Several copies of a sequence adjacent to 1.688 g/cm3 satellite DNA in the Drosophila melanogaster genome have been isolated by molecular cloning. This sequence, called the Dm142 gene, is homologous to a 1.6-kilobase RNA found in both D. melanogaster embryos and tissue culture cells. One cloned DNA segment includes two copies of the Dm142 gene and 1.688 g/cm3 satellite DNA sequences, which are located between and flanking both gene copies. The Dm142 gene is repeated many times in the D. melanogaster genome, and some copies are not flanked by 1,688 g/cm3 satellite DNA.     

Postponed aging and desiccation resistance in Drosophila melanogaster

Studies with the fruit fly, Drosophila melanogaster, have repeatedly shown that selection for postponed reproduction leads to increases in mean life span and increased stress resistance; including increased resistance to desiccation, starvation and ethanol vapors. We show that desiccation resistance declines with age in both short- and long-lived flies suggesting that desiccation resistance may serve as a useful biomarker for aging-related declines in physiological performance. We examined the physical basis of desiccation resistance in five replicate populations selected for postponed reproduction and five replicate control populations. The variables examined were water content, rates of water loss during desiccation, and water content at time of death due to desiccation. In the absence of desiccation stress, both the flies exhibiting postponed senescence and their controls maintained constant water content throughout their lifetimes. In the presence of desiccation stress, the short-lived flies showed significantly higher rates of water loss at all ages than did the long-lived flies. Flies from the two treatments did not differ in water content at death. Our results indicate that water loss rates are the major determinant of desiccation resistance. Water loss rates are under genetic control and covary with age in populations with genetically-determined postponed senescence.     

Patterns of movement and ageing in Drosophila melanogaster

The paths of young and old Drosophila melanogaster have been photographed from the center of an arena, the release point. The results indicate that old flies do not move as far away from the release point and have more sinuous paths than young ones. A high proportion of old flies trace loops during their course, while this is the case for only a few young files. It can be argued that young flies show patterns of movement which maximize the dispersion, while old flies explore the environment in such a way as to withdraw not too far from the release point.     

Propyl gallate delays senescence in Drosophila melanogaster

The effect of propyl gallate (PGL) on life span in Drosophila was investigated. Four groups of flies were supplemented as follows: group 1, no PGL; group 2, no PGL supplement until 28 days followed by 0.3% PGL for remaining life span; group 3, 0.3% PGL from 7 days to 28 days, then none for remaining life span; and group 4, 0.3% PGL from 7 days until death. In all cases, PGL significantly increased mean life span. The largest increase in mean life span (34.2%) was in the group receiving PGL for the entire life span (group 4). Increases of 14.6% and 14.7% were measured in groups 2 and 3, respectively.