Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Thermal Disruption of Mushroom Body Development and Odor Learning in Drosophila

  • Xia Wang,

    Affiliation School of Life Sciences, University of Nevada, Las Vegas, Nevada, United States of America

  • David S. Green,

    Affiliations School of Life Sciences, University of Nevada, Las Vegas, Nevada, United States of America, Division of Biological Sciences, University of California at San Diego, La Jolla, California, United States of America

  • Stephen P. Roberts,

    Affiliation School of Life Sciences, University of Nevada, Las Vegas, Nevada, United States of America

  • J. Steven de Belle

    To whom correspondence should be addressed. E-mail: steven.debelle@unlv.edu

    Affiliation School of Life Sciences, University of Nevada, Las Vegas, Nevada, United States of America

Thermal Disruption of Mushroom Body Development and Odor Learning in Drosophila

  • Xia Wang, 
  • David S. Green, 
  • Stephen P. Roberts, 
  • J. Steven de Belle
PLOS
x

Abstract

Environmental stress (nutritive, chemical, electromagnetic and thermal) has been shown to disrupt central nervous system (CNS) development in every model system studied to date. However, empirical linkages between stress, specific targets in the brain, and consequences for behavior have rarely been established. The present study experimentally demonstrates one such linkage by examining the effects of ecologically-relevant thermal stress on development of the Drosophila melanogaster mushroom body (MB), a conserved sensory integration and associative center in the insect brain. We show that a daily hyperthermic episode throughout larval and pupal development (1) severely disrupts MB anatomy by reducing intrinsic Kenyon cell (KC) neuron numbers but has little effect on other brain structures or general anatomy, and (2) greatly impairs associative odor learning in adults, despite having little effect on memory or sensory acuity. Hence, heat stress of ecologically relevant duration and intensity can impair brain development and learning potential.

Introduction

Whereas the effects of environmental stress on developing nervous systems are well documented [1][3], few studies demonstrate causative influences on specific targets in the brain and their consequences for behavior. One familiar exception is the volumetric reduction of basal ganglia, cerebellum and corpus callosum due to in utero ethanol exposure in mammals [4]. These effects on the developing brain are associated with symptoms of fetal alcohol syndrome in humans, such as impaired verbal and visual-spatial learning, attention, reaction time, and executive functions [5]. Thermal stress is a more common and potentially hazardous feature of the natural environment for developing animals. Indeed, hyperthermia is also an especially powerful CNS teratogen in the laboratory [6], [7]. Adult male rats exposed to in utero hyperthermia display aberrant sexual behavior associated with disruptions of the sexually dimorphic nucleus of the preoptic area and the anteroventral periventricular nucleus [8]. However, the consequences of natural or ecologically-relevant heat stress for CNS development and function in organisms that normally experience extreme thermal heterogeneity are unknown. Drosophila melanogaster developing in necrotic fruit are subject to daily episodes of intense hyperthermia capable of causing significant mortality and disruption of external morphology [9], [10]. Here we show that the anatomy and function of Drosophila MBs, structures associated with sensory integration and higher processing in insects [11][13], are acutely sensitive to ecologically-relevant heat stress experienced during sub-adult stages.

Surprisingly little is known about invertebrate CNS and behavioral responses to thermal stress. In recent studies with honeybees, workers exposed to low temperatures within the range of normal experience showed reduced behavioral performance relative to their siblings raised at higher temperatures [14]. Deviations of only one degree from optimum induced striking developmental reductions in sensory mode-specific zones of the calyx, the dendritic input of the MBs [15], [16]. These findings imply that temperature-mediated MB plasticity may be important for regulating complex behavioral tasks. MBs are also remarkably responsive to sensory experience, with exposure to either enriched or deprived artificial environments inducing dramatic structural plasticity [17][20]. The current study expands our understanding of the acute sensitivity of the MB to stress and to thermal variation in particular. The implications of environment and experience for brain development and adult behavior are discussed.

Results

Heat Stress Influence on Development

D. melanogaster from a large orchard population reared at 23°C were exposed daily to a brief heat stress (39.5°C for 35 min) throughout larval and pupal development. This laboratory treatment mimics documented profiles of thermal oscillation experienced by developing flies in nature [9], [10], and like such intense natural hyperthermic episodes, yielded approximately 60% increases for both mortality and developmental time (data not shown). Eclosing heat-stressed (HS) adults nonetheless appeared entirely normal, with wild-type walking, flight, activity levels and reproductive capacity. However, the brains of these flies showed striking reductions in MB neuropil when viewed in paraffin sections under a fluorescence microscope (figure 1A). Using planimetric measurements to quantify this observation, we found that MB calyx volume (dendritic elements; figure 1B) and pedunculus cross section area (axonal elements; figure 1C) were both reduced by approximately 30% in HS flies relative to controls (CT) reared at a constant 23°C. In considering more peripheral brain structures associated with sensory input, antennal lobe (AL) volume was reduced by about 15% (figure 1D), while the much larger optic lobes appeared to be unaffected by heat stress treatment (figure 1E). The central complex, controlling aspects of motor output in flies and other insects [21], was 9% smaller in heat stressed males only (figure 1F). Except for a 6% wing area reduction in females, differences in external anatomical features, such as leg length, were indistinguishable between HS and CT flies (figure 1G and 1H).

thumbnail
Figure 1. Thermal Stress Disrupts Brain Development.

(A) Frontal 7 µm paraffin sections of MB calyces at their broadest point, viewed with a fluorescence photo microscope. MBs are smaller in HS flies than in the CT group. (B) Heat stress induced a significant 31% reduction in MB calyx volume (F[1,97] = 188.39, P<0.0001), estimated from planimetric measurements of serial sections of HS and CT flies shown in (A). (C) MB pedunculus cross-section area (the means of measurements from three serial caudal sections) was reduced by 29% in HS flies (F[1,97] = 123.43, P<0.0001). (D) AL volume [derived as in (B)] was reduced by 15% in HS flies (F[1,51] = 26.04, P<0.0001). (E) Optic lobe volume [medulla+lobula, derived as in (B)] was not significantly influenced by heat stress (F[1,40] = 1.59, P = 0.22). (F) Central complex volume [fan shaped body+ellipsoid body, derived as in (B)] was reduced by 9% in HS male flies only (F[1,51[ = 10.78, P = 0.002). (G) Wing area was reduced by 6% in HS female flies only (F[1,60] = 7.04, P = 0.01). (H) Forelimb length was not significantly affected in HS flies (F[1,60] = 1.21, P = 0.28). (B–H) Bars are mean±standard error (SE); n indicated on each bar. Different letters designate significant differences (SNK, P≤0.05).

https://doi.org/10.1371/journal.pone.0001125.g001

In D. melanogaster adults, MBs are paired neuropil structures each consisting of about 2500 intrinsic KC neurons [13], [22]. Four equivalent neuroblasts in each hemisphere of the developing brain generate three morphologically and spatially distinct classes of KCs in a specific temporal order [23][25]. Gamma neurons appear until the mid-3rd instar larval stage, followed by α′β′ neurons until puparium formation, with αβ neurons proliferating until adult eclosion. To address whether MB hypersensitivity to heat stress might be limited to any of these classes of neurons, we examined the brains of flies that were heat stressed according to the sequential pattern of KC generation (figure 2A). Adult MBs were reduced following heat treatment during all stages of larval and pupal development, and corresponding temporal windows of KC proliferation (figure 2B). MB calyx reductions induced during γ, α′β′, and αβ neuron proliferation periods were not significantly different, suggesting that all KC classes have equivalent heat stress sensitivity.

thumbnail
Figure 2. All Classes of Intrinsic MB Neurons Are Sensitive to Thermal Stress.

(A) Schematic illustration of heat stress treatment administered 35 min/day throughout larval and pupal development, or restricted to specific developmental stages that correspond with the birth of MB neurons projecting to γ, α′β′, or αβ-lobes. (B) MB calyx volume measurements (derived as in figure 1B). All three classes of MB neurons are sensitive to heat stress (F[4,138] = 17.92, P<0.0001). Calyx volume in flies receiving daily episodes of heat stress treatment throughout development reflected additive reductions of each of the three neuron classes exposed to heat stress as shown in (A). Bars are mean±SE; n indicated on each bar. Different letters designate significant differences (SNK, P≤0.05).

https://doi.org/10.1371/journal.pone.0001125.g002

To determine whether MB reduction in HS flies was due to either smaller or fewer KCs, we used the GAL4/UAS reporter gene system [26], [27] to visualize MB architecture [27][29] and count KC perikarya [30], [31]. In these experiments, cytoplasm-targeted green fluorescent protein (GFP) expressed by the T10 element [32] was used to label KC projection patterns, and nuclear-localized GFP expressed by the nls14 element [33] was used to label nuclei in KC perikarya. MBs in HS flies bearing T10 driven by one of three different P[GAL4] drivers (247 [34], 201Y [27], or C739 [27]) appeared slightly smaller, but otherwise normal in all respects. We observed paired neuropiles with wild-type structural features, including KC clusters, calyces, pedunculi, and lobes (figure 3A). In contrast, there were fewer labeled KCs counted in HS P[GAL4]/nls14 flies than in CT groups (figure 3B). Cell numbers differed by 29% in 247/nls14, 36% in 201Y/nls14, and 57% in c739/nls14 (figure 3C). Initially, heat stress appeared to influence numbers of GFP-expressing cells in some genetic backgrounds more than others, suggesting a possible distinction between KC classes. However, the analysis of variance (ANOVA) genotype×treatment interaction component was not significant (F[1,104] = 2.69, P = 0.07), indicating that intrinsic MB neurons have similar heat stress responses. Thus, heat stress disrupts MB development by either blocking KC proliferation or triggering abnormal KC death.

thumbnail
Figure 3. Thermal Stress Disrupts MB Development by Reducing KC Numbers.

(A) Cytoplasm-targeted GFP expression patterns driven by different GAL4-expressing elements in whole mount brains of CT (top) and HS (bottom) flies viewed with a laser scanning confocal microscope. All MB structural elements represented in each of three CT P[GAL4]/T10 genotypes were present (labeled) but clearly diminished in HS flies. We noted that cytoplasm-targeted GFP revealed low-level enhancer activity (labeled in blue) that is often not observed when targeting GFP expression to membranes (see references 50 and 52 for examples). (B) Nuclear-targeted GFP expression patterns driven by different GAL4-expressing elements in whole mount brains of CT (top) and HS (bottom) flies viewed with a laser scanning confocal microscope. We observed fewer KCs in the three HS P[GAL4]/nls14 genotypes compared with CT flies. (C) KCs counted in the brains of flies represented in (B). A two-way ANOVA found highly significant effects of genotype (F[2,104] = 42.36, P<0.0001) and treatment (F[1,104] = 143.00, P<0.0001), while the interaction component was not significant (F[1,104] = 2.69, P = 0.07). KC numbers were reduced by 29% in 247/nls14, 36% in 201Y/nls14 and 57% in c739/nls14. Bars are mean±SE; n indicated on each bar. Different letters designate significant differences (SNK, P≤0.05).

https://doi.org/10.1371/journal.pone.0001125.g003

Heat Stress Influence on Behavior

Since MBs are a secondary olfactory neuropil essential for mediating associative odor learning and memory in Drosophila [11][13], we compared the behavior of HS and CT flies using a Pavlovian conditioning assay [35][37]. Learning of odors paired with electric shock was profoundly reduced (28%) in HS flies relative to CT flies (figure 4A). While memory appears to decay more rapidly in HS flies, this effect is minor since the ANOVA treatment×time interaction component was not significant (F[2,56] = 2.00, P = 0.15). Performance indices averaged over all retention intervals for HS flies were 53% of the CT group. Similar olfactory conditioning defects and rates of memory decay have been described for several Drosophila mutants [38], [39], including those with observed reductions in MB anatomy [11], [37], [40].

thumbnail
Figure 4. Associative Odor Learning is Impaired by Thermal Stress.

(A) Olfactory learning and memory. The mean performance index calculated for HS flies was lower than CT flies at all time intervals. A two-way ANOVA detected significant effects of treatment (F[1,56] = 101.25, P<0.0001) and time (F[2,56] = 41.93, P<0.0001), while the interaction component was not significant; F[2,56] = 2.00, P = 0.15). (B) Shock reactivity. HS flies showed normal avoidance of 80 V dc electric shock used in (A) and a slight reduction in avoidance at 120 V (F[1,36] = 6.23, P = 0.017). (C) MCH odor avoidance. HS flies demonstrated a normal avoidance of MCH at the 1×10−2 dilution used in (A) and a slight reduction in avoidance at the 5×10−3 dilution (F[1,37] = 14.72, P = 0.0005). (D) OCT odor avoidance. HS flies demonstrated normal avoidance responses to OCT at both dilutions. (A–D) Symbols or bars are mean±SE; n indicated above each symbol or on each bar. Different letters designate significant differences (SNK, P≤0.05).

https://doi.org/10.1371/journal.pone.0001125.g004

Ablation studies show that Drosophila MBs are not required for normal responses to electric shock or noxious odors [36]. Although heat stress does have a minor influence on the development of other structures (figure 1D, 1F and 1G), and lengthens developmental time (figure 2A), HS flies did not have sensory acuity defects in control tests relevant to our conditioning paradigm. They avoided 80 V dc shock pulses normally, and responded to 120 V dc shock with only a slight reduction compared to CT flies (figure 4B). Similarly, HS flies showed normal avoidance of both 4-methylcyclohexanol (MCH) and 3-octanol (OCT) odorants at the 10×10−3 dilutions used in classical conditioning (figure 4C and 4D). Responses to a 5×10−3 dilution of MCH were slightly reduced (figure 4C). Thus, low performance of HS flies in conditioning experiments was not a secondary result of impaired shock reactivity or olfactory capacity as a consequence of AL reduction, but due to weak association of these stimuli paired during training.

Discussion

This study demonstrates that adult Drosophila brain anatomy and behavior are especially sensitive to acute, ecologically relevant heat stress during development. The effect was most evident in the MBs, which were smaller due to fewer KCs, but otherwise appeared structurally normal. Calyx volume measurements in flies recently derived from a natural population and counts of GFP-labeled KCs in P[GAL4]/nls14 brains suggested equivalent heat stress responses for all three classes of intrinsic neurons and corresponding γ, α′/β′ and α/β lobe systems. HS flies were also strongly impaired in associative odor learning, while memory decay, sensory acuity and basic motor behavior remained largely unaffected. Since odor avoidance was essentially normal in HS flies, associative functions that might be attributed to the ALs [41] were probably not markedly affected by heat stress. We saw no evidence of necrosis in paraffin sections of HS fly brains (figure 1A), and consequently favor the view that impaired KC proliferation, rather than aberrant KC mortality, was the source of MB and olfactory conditioning reduction. KCs may be especially sensitive to heat stress because they are derived from only four progenitor cells (of more than 100 in each brain hemisphere [42]) that divide asymmetrically [43] and continuously from embryo until adult eclosion [25], [44]. AL local and projection interneurons follow a similar temporal course of development [44], [45] and for this reason might be expected to show a similar sensitivity to heat stress. On the other hand, enhanced structural plasticity may be a fundamental feature of MB neurons, reflecting cellular changes that are particularly responsive to convergent sensory input, and having a profound impact on the behavioral characteristics of adults. The latter explanation may be more likely, since the optic lobes (about half of the brain) were evidently not affected by heat stress occurring throughout their development. The source of these stress response differences in the brain is a focus of our ongoing investigation.

A prevailing neural circuit model for olfactory discrimination and learning proposes that KCs serve as temporal coincidence detectors for odors paired with inherently meaningful or conditioned reinforcement [13], [46]. KCs might learn and represent odors as memories in their signaling to downstream neurons. In consideration of this model, we expect that training flies to avoid one simple odor will recruit relatively few neurons, whereas the vastly more complex natural olfactory environment should engage large overlapping KC arrays. In HS flies, fewer KCs had a diminished capacity for odor learning, but these remaining neurons had superficially normal projections and sustained relatively normal representations of odor memory. Correlated reductions of MB structure (figure 1B and 1C, figure 2B, figure 3C) and learning (figure 4A) by about 30% may reflect a simple relationship between the numbers of KCs capable of representing specific conditioned odors and learning performance, at least for the pure odorants used in our experiments. Moreover, since both MB structure and memory decay were apparently spared in HS flies, we argue that normal KC projection and connectivity are critical for memory storage and retrieval. Several observations support these simple arguments. In MB ablation studies, Drosophila larvae fed the cytostatic agent hydroxyurea developed into adults having only a small fraction of the normal KC complement and correlated reductions in odor learning [36]. A number of these flies had partially ablated MBs that were reduced in size but otherwise appeared anatomically normal. Similarly, mutations that reduce MB neuropil but have no obvious additional structural phenotypes also impair olfactory conditioning but not memory [37], [40]. More recent transgenic studies showed that synaptic transmission from KC terminals in the lobes is required for memory retrieval but not acquisition or storage [47], [48]. In view of these observations, we propose that lower memory scores in HS flies reflects a reduced sum of conditioned KC signals received by extrinsic neurons downstream of the MBs.

Heat stress appears to phenocopy defects described for several Drosophila MB anatomy mutants [11], [37], [49], providing a practical non-invasive tool for dissecting brain structure-function relationships. The significance of different KC classes, with their discrete temporal and spatial patterns of proliferation and projection to the three lobe systems of the Drosophila MB, is largely unknown. Mutant and transgenic studies suggest a possible distinction between them as neural substrates for representations of memories consolidated at different stages of development [18], discrete phases of memory, [28], [31], [50][52; see discussion in ref. 38], or conduits to extrinsic sites downstream of the MBs for memory storage and retrieval [47], [48]. Since temporal windows of heat stress can reliably induce significant and equivalent reductions of each KC class (figure 2, figure 3), this method should distinguish behavioral functions of these neurons and MB structures formed by their projections.

Although the mechanism(s) by which heat stress disrupts neural development and behavior are unknown, the apparent phenocopy of MB mutant defects may provide important clues for understanding how the brain responds to normal environmental variation. Our results suggest that KC proliferation during development is especially sensitive, while KC plasticity in adults may respond with more subtle changes [17][20]. Whole genome analyses (e.g., DNA microarrays) should identify potential links between both types of neuronal plasticity and environmental triggers of gene activity that may either drive or accompany them.

In the wild, flies encounter stress from many sources, but also receive a broad spectrum of complementary enrichment. Stimulating environments augment MB development in a learning mechanism-dependent manner [18], while stressful environments disrupt MB anatomy and impair function. Hence, genetic influences and a combination of beneficial and deleterious environmental exposures during development likely have significant roles in determining the neural and behavioral characteristics of adults. Since all nervous systems demonstrate acute sensitivity to environmental stress, our findings have broad implications for brain development and cognitive ability in all animals, including humans.

Materials and Methods

Flies

Wild-type D. melanogaster adults were collected from a large orchard population in southern Nevada. The lineage of these flies was used for all paraffin histology and behavior. We generated heterozygous GFP-expressing flies for confocal laser scanning microscopy by crossing either P[UAS-GFP.S65T]T10 (T10; Bloomington Stock Center) [32] or P[UAS-GFP.nls]14 (nls14; Bloomington Stock Center) [33] with three different enhancer trap strains in which GAL4 expression was reported in distinct subsets of MB neurons: P[Mef2-GAL4.247] (247; γ, α′/β′ and α/β lobe neurons; Robert Schulz) [34], P[GAL4]201Y (201Y; γ and αβ lobe neurons; Douglas Armstrong) [27], or P[GAL4]C739 (C739; αβ lobe neurons; Douglas Armstrong) [27]. Cytoplasm-targeted GFP expression was examined in HS and CT 247/T10, 201Y/T10 and C739/T10 heterozygotes. Nuclear-localized GFP expression in HS and CT 247/nls14, 201Y/nls14, and C739/nls14 heterozygotes was used to count KC nuclei. We cultured flies at equal density in plastic vials with cotton plugs on 8 ml of standard Drosophila cornmeal and molasses medium at 23°C (except for heat stress treatment, below).

Heat stress

HS treatment consisted of a single daily 39.5°C pulse for 35 min throughout larval and pupal development. We administered HS by immersing culture vials of flies in a circulating water bath. In staged HS experiments, daily heat pulses were limited to (1) early 1st instar to early 3rd instar, stressing γ-lobe neuron development, (2) late 3rd instar to puparium formation, stressing α′β′-lobe neuron development, and (3) pupal development, stressing αβ-lobe neuron development, respectively.

Histology and anatomy

We used paraffin mass histology to process flies for neuroanatomical analyses as described previously [36], [37], [53]. Three-4-day-old Drosophila adults were cold-anaesthetised and placed in collars. They were then fixed in Carnoy's solution, dehydrated in ethanol, embedded in paraffin, cut in 7 µm serial frontal sections, and photographed under a fluorescence microscope with an AxioCam digital camera (Zeiss). Brain structure volumes were derived from planimetric measurements of serially-sectioned brains [36], [37] using AxioVision software (Zeiss). Pedunculus cross section area was derived from the means of measurements taken from three serial sections anterior to the calyx. The means of all paired structures were used for each fly. To examine GFP expression in whole mounted fly brains, heads were dissected in PBS and maintained in Focus-Clear (Pacgen) for 15 min. They were then mounted and viewed under a fluorescence microscope with a far-blue (FITC) filter. Z-series confocal images were collected (Zeiss LSM510) to cover the whole MB for viewing structure (1.5 µm virtual sections), or perikarya clusters (0.75 µm virtual sections) for counting cells. GFP-labeled KC nuclei in HS and CT brains were counted manually in every 10th section with the assistance of Image-J software [54], ensuring that all perikarya (diameters<6 µm) in each of these sections would each be counted only once.

We measured right wing area and right fore limb length to assess the effects of heat stress on external anatomy. Appendages were removed using micro scissors from cold-anaesthetised flies being processed for paraffin mass histology (above). These were mounted on glass microscope slides with cover slips sealed with nail polish. Images were photographed under a light microscope with an AxioCam digital camera and measured using AxioVision software (Zeiss).

Behavior

Associative odor learning, memory and sensory acuity controls were assayed using a Pavlovian conditioning T-maze paradigm as described previously [35][37]. Groups of approximately 100 3-4-day-old flies were aspirated into a training tube embedded with an internal double-wound electrifiable copper grid. To assay odor learning and memory, flies were exposed to an air current (750 ml/min) bubbled through one odor [1×10−2 dilutions of either MCH (Sigma) or OCT (Sigma) in heavy mineral oil (Sigma)] paired temporally with 1.25 sec pulses of 80V dc electric shock delivered every 5 sec for 1 min. They were then exposed to an air current bubbled through a second odor without electric shock for an additional 1 min. We assessed learning and memory by presenting trained flies with both odors in converging air currents for 2 min. Performance was measured as a function of shock-paired odor avoidance at a variety of time points ranging from 1 min (giving an approximation of learning at the earliest testable time in the T-maze) to 3 hr after training. A second group of flies was trained in a reciprocal manner and tested. Scores from both tests were averaged to account for odor preferences among different populations of flies. In electric shock-avoidance controls, one arm of the T-maze was electrified with 80 or 120 V dc for 2 min. In odor-avoidance controls, flies were exposed to 5×10−3 or 1×10−2 dilutions of MCH or OCT versus air for 2 min. A performance index represents the average normalized percent avoidance of the shock-paired odor (learning, memory) or individual stimulus (sensory acuity).

Statistical analyses

The Shapiro-Wilk test [55] showed that all 57 data samples in this report are distributed normally. Comparisons were made using ANOVA followed by the Student-Numan-Keuls (SNK) multiple range test [55] (SAS Institute software).

Acknowledgments

We are grateful for flies from Douglas Armstrong, Robert Shultz and the Bloomington Drosophila Stock Center. T-mazes and components were meticulously constructed by Hans Kaderschabek. We thank Andrew Andres and Michael Stebbins for reading preliminary versions of the manuscript, and JSdB and SPR lab members for helpful discussion. Part of this work was conducted by JSdB while serving as a Visiting Scientist at the National Science Foundation.

Author Contributions

Conceived and designed the experiments: Jd XW SR. Performed the experiments: XW DG. Analyzed the data: Jd XW SR. Contributed reagents/materials/analysis tools: Jd SR. Wrote the paper: Jd XW SR.

References

  1. 1. Rice D, Barone S Jr (2000) Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect 108: Suppl 3511–533.D. RiceS. Barone Jr2000Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models.Environ Health Perspect108Suppl 3511533
  2. 2. Weinstock M (2001) Alterations induced by gestational stress in brain morphology and behaviour of the offspring. Prog Neurobiol 65: 427–451.M. Weinstock2001Alterations induced by gestational stress in brain morphology and behaviour of the offspring.Prog Neurobiol65427451
  3. 3. Welberg LA, Seckl JR (2001) Prenatal stress, glucocorticoids and the programming of the brain. J Neuroendocrinol 13: 113–128.LA WelbergJR Seckl2001Prenatal stress, glucocorticoids and the programming of the brain.J Neuroendocrinol13113128
  4. 4. Mattson SN, Riley EP (1998) A review of the neurobehavioral deficits in children with fetal alcohol syndrome or prenatal exposure to alcohol. Alcohol Clin Exp Res 2: 279–294.SN MattsonEP Riley1998A review of the neurobehavioral deficits in children with fetal alcohol syndrome or prenatal exposure to alcohol.Alcohol Clin Exp Res2279294
  5. 5. Roebuck TM, Mattson SN, Riley EP (1998) A review of the neuroanatomical findings in children with fetal alcohol syndrome or prenatal exposure to alcohol. Alcohol Clin Exp Res 2: 339–244.TM RoebuckSN MattsonEP Riley1998A review of the neuroanatomical findings in children with fetal alcohol syndrome or prenatal exposure to alcohol.Alcohol Clin Exp Res2339244
  6. 6. Milunsky A, Ulcickas M, Rothman KJ, Willett W, Jick SS, et al. (1992) Maternal heat exposure and neural-tube defects. J Am Med Assoc 268: 882–885.A. MilunskyM. UlcickasKJ RothmanW. WillettSS Jick1992Maternal heat exposure and neural-tube defects.J Am Med Assoc268882885
  7. 7. Suarez L, Felkner M, Hendricks K (2004) The effect of fever, febrile illnesses, and heat exposures on the risk of neural tube defects in a Texas-Mexico border population. Birth Defects Res A Clin Mol Teratol 70: 815–819.L. SuarezM. FelknerK. Hendricks2004The effect of fever, febrile illnesses, and heat exposures on the risk of neural tube defects in a Texas-Mexico border population.Birth Defects Res A Clin Mol Teratol70815819
  8. 8. Rhees RW, Al-Saleh HN, Kinghorn EW, Fleming DE, Lephart ED (1999) Relationship between sexual behavior and sexually dimorphic structures in the anterior hypothalamus in control and prenatally stressed male rats. Brain Res Bull 50: 193–199.RW RheesHN Al-SalehEW KinghornDE FlemingED Lephart1999Relationship between sexual behavior and sexually dimorphic structures in the anterior hypothalamus in control and prenatally stressed male rats.Brain Res Bull50193199
  9. 9. Feder ME (1997) Necrotic fruit: A novel model system for thermal ecologists. J Therm Biol 22: 1–9.ME Feder1997Necrotic fruit: A novel model system for thermal ecologists.J Therm Biol2219
  10. 10. Roberts SP, Feder ME (1999) Natural hyperthermia and expression of the heat shock protein Hsp70 affect developmental abnormalities in Drosophila melanogaster. Oecologia 121: 323–329.SP RobertsME Feder1999Natural hyperthermia and expression of the heat shock protein Hsp70 affect developmental abnormalities in Drosophila melanogaster.Oecologia121323329
  11. 11. de Belle JS, Kanzaki R (1999) Protocerebral Olfactory Processing. In: Hansson BS, editor. Insect Olfaction. Stuttgart: Springer. pp. 243–281.JS de BelleR. Kanzaki1999Protocerebral Olfactory Processing.BS HanssonInsect OlfactionStuttgartSpringer243281
  12. 12. Zars T (2000) Behavioral functions of the insect mushroom bodies. Curr Opin Neurobiol 10: 790–795.T. Zars2000Behavioral functions of the insect mushroom bodies.Curr Opin Neurobiol10790795
  13. 13. Heisenberg M (2003) Mushroom body memoir: from maps to models. Nat Rev Neurosci 4: 266–275.M. Heisenberg2003Mushroom body memoir: from maps to models.Nat Rev Neurosci4266275
  14. 14. Tautz J, Maier S, Groh C, Rössler W, Brockmann A (2003) Behavioral performance in adult honey bees is influenced by the temperature experienced during their pupal development. Proc Natl Acad Sci U S A 100: 7343–7347.J. TautzS. MaierC. GrohW. RösslerA. Brockmann2003Behavioral performance in adult honey bees is influenced by the temperature experienced during their pupal development.Proc Natl Acad Sci U S A10073437347
  15. 15. Groh C, Tautz J, Rössler W (2004) Synaptic organization in the adult honey bee brain is influenced by brood-temperature control during pupal development. Proc Natl Acad Sci U S A 101: 4268–4273.C. GrohJ. TautzW. Rössler2004Synaptic organization in the adult honey bee brain is influenced by brood-temperature control during pupal development.Proc Natl Acad Sci U S A10142684273
  16. 16. Groh C, Ahrens D, Rössler W (2006) Environment- and age-dependent plasticity of synaptic complexes in the mushroom bodies of honeybee queens. Brain Behav Evol 68: 1–14.C. GrohD. AhrensW. Rössler2006Environment- and age-dependent plasticity of synaptic complexes in the mushroom bodies of honeybee queens.Brain Behav Evol68114
  17. 17. Technau GM (1984) Fiber number in the mushroom bodies of adult Drosophila melanogaster depends on age, sex and experience. J Neurogenet 1: 113–126.GM Technau1984Fiber number in the mushroom bodies of adult Drosophila melanogaster depends on age, sex and experience.J Neurogenet1113126
  18. 18. Balling A, Technau GM, Heisenberg M (1987) Are the structural changes in adult Drosophila mushroom bodies memory traces? Studies on biochemical learning mutants. J Neurogenet 4: 65–73.A. BallingGM TechnauM. Heisenberg1987Are the structural changes in adult Drosophila mushroom bodies memory traces? Studies on biochemical learning mutants.J Neurogenet46573
  19. 19. Heisenberg M, Heusipp M, Wanke C (1995) Structural plasticity in the Drosophila brain. J Neurosci 15: 1951–1960.M. HeisenbergM. HeusippC. Wanke1995Structural plasticity in the Drosophila brain.J Neurosci1519511960
  20. 20. Barth M, Heisenberg M (1997) Vision affects mushroom bodies and central complex in Drosophila melanogaster. Learn Mem 4: 219–229.M. BarthM. Heisenberg1997Vision affects mushroom bodies and central complex in Drosophila melanogaster.Learn Mem4219229
  21. 21. Strauss R (2002) The central complex and the genetic dissection of locomotor behaviour. Curr Opin Neurobiol. 12: 633–638.R. Strauss2002The central complex and the genetic dissection of locomotor behaviour.Curr Opin Neurobiol.12633638
  22. 22. Technau GM, Heisenberg M (1982) Neural reorganisation during metamorphosis of the corpora pedunculata in Drosophila melanogaster. Nature 295: 405–407.GM TechnauM. Heisenberg1982Neural reorganisation during metamorphosis of the corpora pedunculata in Drosophila melanogaster.Nature295405407
  23. 23. Ito K, Awano W, Suzuki K, Hiromi Y, Yamamoto D (1997) The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124: 761–771.K. ItoW. AwanoK. SuzukiY. HiromiD. Yamamoto1997The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells.Development124761771
  24. 24. Armstrong JD, de Belle JS, Wang Z, Kaiser K (1998) Metamorphosis of the mushroom bodies; large-scale rearrangements of the neural substrates for associative learning and memory in Drosophila. Learn Mem 5: 102–114.JD ArmstrongJS de BelleZ. WangK. Kaiser1998Metamorphosis of the mushroom bodies; large-scale rearrangements of the neural substrates for associative learning and memory in Drosophila.Learn Mem5102114
  25. 25. Lee T, Lee A, Luo L (1999) Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast. Development 126: 4065–4076.T. LeeA. LeeL. Luo1999Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.Development12640654076
  26. 26. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415.AH BrandN. Perrimon1993Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.Development118401415
  27. 27. Yang MY, Armstrong JD, Vilinsky I, Strausfeld NJ, Kaiser K (1995) Subdivision of the Drosophila mushroom bodies by enhancer-trap expression patterns. Neuron 15: 45–54.MY YangJD ArmstrongI. VilinskyNJ StrausfeldK. Kaiser1995Subdivision of the Drosophila mushroom bodies by enhancer-trap expression patterns.Neuron154554
  28. 28. Zars T, Fischer M, Schulz R, Heisenberg M (2000) Localization of a short-term memory in Drosophila. Science 288: 672–675.T. ZarsM. FischerR. SchulzM. Heisenberg2000Localization of a short-term memory in Drosophila.Science288672675
  29. 29. Ito K, Sass H, Urban J, Hofbauer A, Schneuwly S (1997) GAL4-responsive UAS-tau as a tool for studying the anatomy and development of the Drosophila central nervous system. Cell Tissue Res 290: 1–10.K. ItoH. SassJ. UrbanA. HofbauerS. Schneuwly1997GAL4-responsive UAS-tau as a tool for studying the anatomy and development of the Drosophila central nervous system.Cell Tissue Res290110
  30. 30. Mader MT (2004) Analyse von Expressionsmustern in den Pilskörpern von Drosophila melanogaster. Würzburg, Germany: Universität Würzburg. MT Mader2004Analyse von Expressionsmustern in den Pilskörpern von Drosophila melanogaster.Würzburg, GermanyUniversität WürzburgDiplom thesis,. Diplom thesis,.
  31. 31. Akalal D-BG, Wilson CF, Zong L, Tanaka NK, Ito K, et al. (2006) Roles for Drosophila mushroom body neurons in olfactory learning and memory. Learn Mem 13: 659–668.D-BG AkalalCF WilsonL. ZongNK TanakaK. Ito2006Roles for Drosophila mushroom body neurons in olfactory learning and memory.Learn Mem13659668
  32. 32. Ahmad K, Henikoff S (2001) Modulation of a transcription factor counteracts heterochromatic gene silencing in Drosophila. Cell 104: 839–847.K. AhmadS. Henikoff2001Modulation of a transcription factor counteracts heterochromatic gene silencing in Drosophila.Cell104839847
  33. 33. Robertson K, Mergliano J, Minden JS (2003) Dissecting Drosophila embryonic brain development using photoactivated gene expression. Dev Biol 260: 124–137.K. RobertsonJ. MerglianoJS Minden2003Dissecting Drosophila embryonic brain development using photoactivated gene expression.Dev Biol260124137
  34. 34. Schulz RA, Chromey C, Lu MF, Zhao B, Olson EN (1996) Expression of the D-MEF2 transcription in the Drosophila brain suggests a role in neuronal cell differentiation. Oncogene 12: 1827–1831.RA SchulzC. ChromeyMF LuB. ZhaoEN Olson1996Expression of the D-MEF2 transcription in the Drosophila brain suggests a role in neuronal cell differentiation.Oncogene1218271831
  35. 35. Tully T, Quinn WG (1985) Classical conditioning and retention in normal and mutant Drosophila melanogaster. J Comp Physiol [A] 157: 263–277.T. TullyWG Quinn1985Classical conditioning and retention in normal and mutant Drosophila melanogaster.J Comp Physiol [A]157263277
  36. 36. de Belle JS, Heisenberg M (1994) Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies. Science 263: 692–695.JS de BelleM. Heisenberg1994Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies.Science263692695
  37. 37. de Belle JS, Heisenberg M (1996) Expression of Drosophila mushroom body mutations in alternative genetic backgrounds: a case study of the mushroom body miniature gene (mbm). Proc Natl Acad Sci U S A 93: 9875–9880.JS de BelleM. Heisenberg1996Expression of Drosophila mushroom body mutations in alternative genetic backgrounds: a case study of the mushroom body miniature gene (mbm).Proc Natl Acad Sci U S A9398759880
  38. 38. Margulies C, Tully T, Dubnau J (2005) Deconstructing memory in Drosophila. Curr Biol 17: R700–713.C. MarguliesT. TullyJ. Dubnau2005Deconstructing memory in Drosophila.Curr Biol17R700713
  39. 39. McGuire SE, Deshazer M, Davis RL (2005) Thirty years of olfactory learning and memory research in Drosophila melanogaster. Prog Neurobiol 5: 328–347.SE McGuireM. DeshazerRL Davis2005Thirty years of olfactory learning and memory research in Drosophila melanogaster.Prog Neurobiol5328347
  40. 40. Pinto S, Quintana DG, Smith P, Mihalek RM, Hou ZH, et al. (1999) latheo encodes a subunit of the origin recognition complex and disrupts neuronal proliferation and adult olfactory memory when mutant. Neuron 23: 45–54.S. PintoDG QuintanaP. SmithRM MihalekZH Hou1999latheo encodes a subunit of the origin recognition complex and disrupts neuronal proliferation and adult olfactory memory when mutant.Neuron234554
  41. 41. Yu D, Ponomarev A, Davis RL (2004) Altered representation of the spatial code for odors after olfactory classical conditioning; memory trace formation by synaptic recruitment. Neuron 42: 437–449.D. YuA. PonomarevRL Davis2004Altered representation of the spatial code for odors after olfactory classical conditioning; memory trace formation by synaptic recruitment.Neuron42437449
  42. 42. Urbach R, Schnabel R, Technau GM (2003) The pattern of neuroblast formation, mitotic domains and proneural gene expression during early brain development in Drosophila. Development 130: 3589–3606.R. UrbachR. SchnabelGM Technau2003The pattern of neuroblast formation, mitotic domains and proneural gene expression during early brain development in Drosophila.Development13035893606
  43. 43. Campos-Ortega JA (1993) Early neurogenesis in Drosophila melanogaster. In: Bate M, Martinez-Arias A, editors. Development of Drosophila melanogaster. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. pp. 1091–1130.JA Campos-Ortega1993Early neurogenesis in Drosophila melanogaster.M. BateA. Martinez-AriasDevelopment of Drosophila melanogasterCold Spring HarborCold Spring Harbor Laboratory Press10911130
  44. 44. Ito K, Hotta Y (1992) Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster. Dev Biol 149: 134–148.K. ItoY. Hotta1992Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster.Dev Biol149134148
  45. 45. Stocker RF, Heimbeck G, Gendre N, de Belle JS (1997) Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of antennal target interneurons. J. Neurobiol. 32: 443–456.RF StockerG. HeimbeckN. GendreJS de Belle1997Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of antennal target interneurons.J. Neurobiol.32443456
  46. 46. Gerber B, Tanimoto H, Heisenberg M (2004) An engram found? Evaluating the evidence from fruit flies. Curr Opin Neurobiol 14: 737–744.B. GerberH. TanimotoM. Heisenberg2004An engram found? Evaluating the evidence from fruit flies.Curr Opin Neurobiol14737744
  47. 47. Dubnau J, Grady L, Kitamoto T, Tully T (2001) Disruption of neurotransmission in Drosophila mushroom body blocks retrieval but not acquisition of memory. Nature 411: 476–480.J. DubnauL. GradyT. KitamotoT. Tully2001Disruption of neurotransmission in Drosophila mushroom body blocks retrieval but not acquisition of memory.Nature411476480
  48. 48. McGuire SE, Le PT, Davis RL (2001) The role of Drosophila mushroom body signaling in olfactory memory. Science 293: 1330–1333.SE McGuirePT LeRL Davis2001The role of Drosophila mushroom body signaling in olfactory memory.Science29313301333
  49. 49. Heisenberg M, Borst A, Wagner S, Byers D (1985) Drosophila mushroom body mutants are deficient in olfactory learning. J Neurogenet 2: 1–30.M. HeisenbergA. BorstS. WagnerD. Byers1985Drosophila mushroom body mutants are deficient in olfactory learning.J Neurogenet2130
  50. 50. Pascual A, Préat T (2001) Localization of long-term memory within the Drosophila mushroom body. Science 294: 1115–1117.A. PascualT. Préat2001Localization of long-term memory within the Drosophila mushroom body.Science29411151117
  51. 51. Isabel G, Pascual A, Préat T (2004) Exclusive consolidated memory phases in Drosophila. Science 304: 1024–1027.G. IsabelA. PascualT. Préat2004Exclusive consolidated memory phases in Drosophila.Science30410241027
  52. 52. Krashes MJ, Keene AC, Leung B, Armstrong JD, Waddell S (2007) Sequential use of mushroom body neuron subsets during drosophila odor memory processing. Neuron 53: 103–115.MJ KrashesAC KeeneB. LeungJD ArmstrongS. Waddell2007Sequential use of mushroom body neuron subsets during drosophila odor memory processing.Neuron53103115
  53. 53. Heisenberg M, Böhl K (1979) Isolation of anatomical brain mutants of Drosophila by histological means. Z Naturforsch C 34: 143–147.M. HeisenbergK. Böhl1979Isolation of anatomical brain mutants of Drosophila by histological means.Z Naturforsch C34143147
  54. 54. Abramoff MD, Magelhaes PJ, Ram S (2004) Image processing with ImageJ. Biophotonics International 11: 36–42.MD AbramoffPJ MagelhaesS. Ram2004Image processing with ImageJ.Biophotonics International113642
  55. 55. Zar JH (1996) Biostatistical Analysis, 3rd edition. Englewood Cliffs: Prentice Hall. . JH Zar1996Biostatistical Analysis, 3rd edition.Englewood CliffsPrentice Hall. 662