The effects of sensitization on habituation using the olfactory jump reflex in Drosophila

Abstract only availableMemories can arise from simpler habituation and sensitization training as well as associative classical conditioning. However, in a complex environment, animals receive sensory cues in a fashion that can be more accurately described as having some habituation, sensitization and associative components. The relation between these types of memories at the molecular, systems and behavioral level remain largely unexplored. We can alter the timing of odor and electric shock presentation to induce all three types of memory in a defensive olfactory jump reflex. Habituation is a short-term change in behavior as a response to a repetitive stimulus. Using seven odors, we showed flies habituate their jump reflex to background levels of jump probabilities with ten odor presentations. Interestingly, the seven odors tested can be categorized into three groups based on their habituation rates: a high jump probability, a low jump probability, or a no-jump probability. Also, odors show some specificity as habituation of one odor does not lead to a total loss of jump response (complete cross-habituation) although it is reduced (partial cross-habituation). We chose to use six odors for further analysis. Sensitization is defined as an interference with habituation because of a dishabituating stimulus. Using electric shock as a potential sensitization cue, we presented shock and immediately tested jump probability. Interestingly, we found unpaired electric shock increased the jump probability with all odors tested, even those that do not induce a naïve jump. Classical (Pavlovian) conditioning arises when an animal associates a neutral stimulus with one that induces a reflex. Preliminary tests suggest that the paired presentation of electric shock and odor does not increase the jump probability of subsequent odor presentation. With the establishment of these three behavioral paradigms, the stage is set to investigate the interaction of habituation and sensitization on associate classical conditioning. Future experimentation should determine the relationship of the molecular and neural systems underlying these different forms of memory.Life Sciences Undergraduate Research Opportunity Progra

A century later another surprise: A non-visual behavioral function of the white gene

Abstract only availableDiscovery of the white mutation in Drosophila melanogaster has broadly influenced our understanding of the mechanisms of inheritance. We recently discovered a role of the white gene in memory formation. Thus, the white gene continues to provide insight into basic biological functions. We use two conditioning methods to routinely measure learning and memory in D. melanogaster, the heat-box, and classical olfactory conditioning. In the heat box experiments, white mutant flies' learning performance was notably impaired. However, in olfactory conditioning studies the mutant flies performed the same or better than wild-type flies. This differentiates the molecular mechanisms that support these conditioned behaviors. To better understand the regulatory elements that control white expression, we have initiated a molecular characterization of the white genomic locus. We identified the necessary regulatory elements by defining the deletion in the w1118 null allele. Using PCR methods we found that the deletion is about 7 kb long, and includes 5' regions, exon 1, and part of the first intron. Experiments to determine the sufficient set of regulatory elements for conditioned behavior were initiated. Two results argue that existing genomic transgenes do not contain all regulatory elements. First, mutations that affect eye color have molecular lesions outside a 14 kb genomic transgene. Second, attempted behavioral rescue experiments with this transgene fail. We interpret the failure of the 14 kb transgene to rescue as a consequence of incorrect white expression. Thus, we are creating a genomic construct that is 18 kb long that includes genomic DNA up to the next known gene. These approaches should define the regulatory regions necessary and sufficient for behaviorally important white expression.NSF-REU Program in Biological Sciences & Biochemistr

Using simple nervous systems to investigate the neural basis of behavior

Neuroscience - Vision and Functional Brain Imaging Poster SessionThe human brain is remarkable, both in the sense that it helps us with a lifetime of decisions and memories, but also that it allows us to contemplate how the brain itself works. One concludes, however, pretty quickly that the human brain is quite complicated. The brain has an estimated 1011 neurons, and more than a thousand times more connections. How these neurons and connections work together in systems is a grand challenge in the neurosciences. Fortunately, we can use organisms with simpler nervous systems to understand basic principles of nervous system function. For example, the first insights into the generation of nerve cell action potentials were determined in a squid giant neuron. Principles derived from these neurons are incorporated into most or all computational models used today. Thus, one expects lessons learned in simpler nervous systems to have application in more complex organisms, including humans. A successful approach in understanding nervous system function is to examine the role that different neural systems play in regulating behavior. Broadly speaking these include processes that support sensory encoding, motor activity, and multisensory and sensory-motor integration. Animals have developed sensory systems to sense the world around them. Audition and temperature perception are two of a few of the sensory modalities that are critical for communication and detecting ideal environments. Katydid hearing systems solve perceptual problems that are common to all hearing systems, such as the recognition of complex temporal patterns, or the detection of important signals in noisy backgrounds. Remarkably, katydids solve these problems with a sensory system encompassing only few neurons. In other studies, the ability to sense temperatures has been addressed in the fruit fly Drosophila. One can differentiate between neural systems important for sensing relatively cool and warm temperatures. Motor systems are critical for several animal behaviors, from regulating gut activity to locomotion. Studies of nervous system ganglia in the crab and lobster have identified principles of nerve cell interactions and modulation. Furthermore, mechanisms regulating brain circuits that initiate swimming behavior in the lamprey tell us that they are similar in a wide variety of vertebrates. Finally, one can begin to understand cellular mechanisms of nervous system regeneration after spinal cord injury in simpler vertebrates that are able to behaviorally recover following such injuries. Multisensory and sensory-motor integration provide essential elements for plasticity in the nervous system. The trigeminal system in the lamprey provide a starting point to determine how sensory inputs feed into brain locomotor command systems to initiate behavior. Also, plasticity underlying longer-lived changes in behavior with learning can be addressed in studies of memory formation. In Drosophila, several molecular and neural systems underlying multiple forms of memory have been identified, including implications of cAMP / PKA activity and serotonin reinforcement. Thus, one can use relatively simple organisms, from insects and crustaceans to lamprey, in determining principles of nervous system function. Results at the sensory, motor, and integrative levels are expected to influence our understanding of more complex systems

Unequal reinforcement values from equally warm and cool temperatures in Drosophila

Abstract only availableInsects inhabit extreme temperature environments and have evolved mechanisms to survive there. When temperatures either rise or fall, small insects alter their behavior within seconds to avoid, in extreme situations, freezing or high temperature induced death. Preference tests on gradients indicate a strong preferred temperature for Drosophila around 24°C. Warm temperatures can be used as effective negative reinforcers for place memory. The preference of 24°C over 18°C on a temperature gradient suggests low temperature might also have reinforcing qualities. The so-called heat-box was used to test the effectiveness of warm and cool temperatures as reinforcers. In this paradigm, single flies are allowed to run freely in a small chamber that is heated or cooled depending on that flies' behavior. If a fly goes to one-half of the chamber it warms and if the fly goes to the other half it cools. Using temperatures either nine degrees above or below the preferred 24°C, we found that both are effective reinforcers. Surprisingly, however, the 24/15°C pair induced a place preference for the cool associated half of the chamber. Systematic tests varying training duration and reinforcement temperature indicates the 15°C reinforcer reaches asymptotic memory levels of 0.2 while 33°C reinforcement plateaus at 0.4 on a scale from 0 to 1. To test for the conservation of molecular mechanisms underlying warm and cool induced memories, wild-type CS, rut-AC and white mutant flies were tested. As has been shown for warm temperature reinforcement, both rut-AC and white mutant flies are defective in place memory formation. Finally, to determine whether the warm and cool reinforcers could serve equally well in reversal learning, flies were trained to avoid one half of a chamber for 2-6 minutes, then trained to avoid the other chamber half. This procedure was repeated for a total of four training sessions with place memories tested after each session. Using a 33°C reinforcer, place memories increase with each training session even though flies are trained to avoid a different chamber-half each session. Interestingly, the effectiveness of the 15°C reinforcer was lost after the first reversal training session. This perhaps indicates that the additional experience in the chamber with only limited temperature extremes allows flies to conclude the temperature changes in the small, dark chambers are sub-lethal, and can ignore their rise and fall. Future experiments will explore this possibility. Together, the evidence indicates flies can use cool temperatures as reinforcement in forming place memories. Their effectiveness as reinforcers, however, are not equal as paired prolonged training and reversal training indicates. These cool induced memories depend on the rut-AC and white mutation, as warm induced memories do.NSF-REU Biology & Biochemistr

Characterizing serotonin in the fruit fly brain [abstract]

Abstract only availableThe biogenic amines serotonin (5-HT) and dopamine (DA) have been implicated in the formation of place memories in Drosophila melanogaster. However, stronger evidence exists in support of 5-HT as the more essential factor in mediating this process. My project focused on characterization of the serotonergic system in fly brains using immunocytochemistry. I probed for 5-HT using a monoclonal antibody and studied its co-localization with neurons expressing GFP driven by either Ddc-Gal4 or DdcThGal80. In an ongoing process, I have so far been able to count 31(+/- 3) 5-HT immunoreactive neurons per hemisphere, 10-15 of which also express GFP. This research works toward a well characterized 5-HT system which can corroborate the various genetic and pharmacological manipulations that are currently used to examine the effects of serotonin on memory formation. I investigated the effects of one of these pharmacological agents, alpha-methyl tryptophan, in addition to the immuno-labeling experiments. After a three-day treatment with the drug, flies showed a lower but not significant decrease in place learning performance as measured using the heat box

Debra, a protein mediating lysosomal degradation, is required for long-term memory in Drosophila

A central goal of neuroscience is to understand how neural circuits encode memory and guide behavior changes. Many of the molecular mechanisms underlying memory are conserved from flies to mammals, and Drosophila has been used extensively to study memory processes. To identify new genes involved in long-term memory, we screened Drosophila enhancer-trap P(Gal4) lines showing Gal4 expression in the mushroom bodies, a specialized brain structure involved in olfactory memory. This screening led to the isolation of a memory mutant that carries a P-element insertion in the debra locus. debra encodes a protein involved in the Hedgehog signaling pathway as a mediator of protein degradation by the lysosome. To study debra's role in memory, we achieved debra overexpression, as well as debra silencing mediated by RNA interference. Experiments conducted with a conditional driver that allowed us to specifically restrict transgene expression in the adult mushroom bodies led to a long-term memory defect. Several conclusions can be drawn from these results: i) debra levels must be precisely regulated to support normal long-term memory, ii) the role of debra in this process is physiological rather than developmental, and iii) debra is specifically required for long-term memory, as it is dispensable for earlier memory phases. Drosophila long-term memory is the only long-lasting memory phase whose formation requires de novo protein synthesis, a process underlying synaptic plasticity. It has been shown in several organisms that regulation of proteins at synapses occurs not only at translation level of but also via protein degradation, acting in remodeling synapses. Our work gives further support to a role of protein degradation in long-term memory, and suggests that the lysosome plays a role in this process