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Effects of Kappa-Opioid Agonism on Depressive-Like Symptoms in Mice

Thomas Adams ('10); Ioana Florea*; Nicole Weekes; Margaret Zellner**
*Girls Preparatory School; **Rockefeller University, New York, NY

The history of the neurobiology of depression is extensive and comparatively complex, with numerous neural substrates for its onset and symptoms having been proposed and studied. Yet despite the syndrome’s prevalence and its presence as a significant health risk, little conclusive, well-defined understanding exists concerning its pathophysiology. A recent, promising area of inquiry is that centered around the endogenous opioid peptide dynorphin and the primary receptor system it activates: the kappa opioid receptor (KOR) system. This study extends this area of inquiry by attempting to induce depressive- like symptomatology in mice by injecting a dynorphin-like drug into the brain. Preliminary results suggest that activation of KORs by the dynorphin-like drug induces a lack of motivation, a common symptom of depression, but perhaps not anhedonia (a lack of pleasure in previously pleasurable activities) as operationally defined in our study.
Funding provided by: The Aubrey H. and Eileen J. Seed Award

Ethanol Increases Motor Activity and Alters Post-Synaptic Response to Glutamate

Devika Bhatia ('11); Sean Chung ('12); Karen Parfitt

In previous years, Laura Johnson ’08 measured synaptic response to acute ethanol exposure; after analyzing recordings of glutamate-mediated spontaneous miniature excitatory junction potentials (mEJPs) in Drosophila melanogaster, she discovered that ethanol had a post-synaptic effect apparently mediated in part by the NMDA glutamate receptor. Drosophila is a very convenient model organism for ethanol studies because they display clear signs of intoxication resembling that seen in humans, including hyperactivity, lack of coordination, and eventual sedation (Heberlein, 2000); furthermore, the synapse is extremely well characterized. In our lab, we found that ethanol increases Drosophila larvaes’ motor activity in a dose-dependent fashion. We further observed that 60 mM ethanol significantly decreased the mEJP amplitude but did not significantly alter mEJP frequency, suggesting ethanol acts post-synaptically. We plan to extend Laura Johnson’s work further by determining the differences in post-synaptic responses of ethanol-sensitive and ethanol-resistant mutants of Drosophila melanogaster and comparing the electrophysiological.
Funding provided by: The Paul K. Richter and Evalyn E. Cook Richter Award (DB); The Hirsch Foundation (SC)

Jazzy Birds: The House Wren Likes to Improvise

Kristin Cettie ('11); Julia Gleichman ('10); Liz Cerny-Chipman ('09); Jade McGill ('09); Rachel Levin; Andre Cavalcanti

Most studies of bird song focus on species that sing repertoires of stereotyped songs. We are studying the structure of variable song using the house wren, Troglodytes aedon. We recorded two days of morning songs from birds at low and high elevation temperate zone sites. We then recorded their response to playback of songs from an unfamiliar house wren. We spectrographically analyzed 3,000 songs of six birds and compiled a dictionary of song phrases used. We developed a clustering program to examine note sequences and found little consistency within individuals across days. Although birds switched songs more frequently in response to simulated intruders, birds did not generally match their songs to those being broadcast. Since there seems to be no consistent song pattern over the course of a morning, we are developing a new clustering program to determine how notes appear within songs. We also plan to perform a broader analysis by generalizing note types.
Funding provided by: The Paul K. Richter and Evalyn E. Cook Richter Award (KC)

Novel Limited Access Two-Bottle-Choice Drinking Model Induces Clinically Significant Levels of Nicotine Consumption in C57BL/6 Mice

Rico Chenyek ('11); Nicole Weekes; Joan Holgate*; Tal Oppenheimer*; Nathan Santos*; Selena E. Bartlett*
*Ernesto Gallo Clinic and Research Center, University of California, San Francisco, 5858 Horton Street, Suite 200, Emeryville, CA 94608

Some advantages to studying nicotine in mice include availability of inbred strains with genetic alterations to various nicotinic acetylcholine receptor (nAChR) subunits and similarities to human nicotine metabolism. Current two-bottle-choice drinking models involve 24hr/day access to nicotine, gradual increases in nicotine concentrations and/or use saccharin to induce consumption. This results in an inconclusive cause of consumption, subsequent behavioral/receptor changes, and hinders studying nicotine consumption in combination with ethanol (80% of alcoholics also smoke). Our model, adapted from Rhodes, et al, “drinking in the dark,” is independent of saccharin and may eliminate incremental introduction. The model was validated by clinically significant plasma nicotine concentrations and decreased nicotine consumption following treatment with nAChR antagonist, CP-601,932 (analog of FDA approved smoking-cessation drug Varenicline/Chantix®). We demonstrate efficacy of this model in achieving concurrent consumption of ethanol and nicotine, which will be useful for understanding co- morbid alcohol and nicotine disorders and developing/improving smoking/drinking cessation therapies.
Funding provided by: The Paul K. Richter and Evalyn E. Cook Richter Award; State of CA for Medical Research through UCSF to S.E.B.; The α4-YFP mice in this project were generously provided by H.A. Lester and R. Nashmi.

Midline Axon Guidance and SSP4: A Potential Player?

Katherine DeJong ('10); Karl Johnson

The majority of neurons in bilaterally symmetric organisms send axons that make synaptic contacts on the opposite side of the body. Mutations in the chemorepellent, Slit, or its receptor, Robo, cause axon guidance defects at the midline. Because of previously reported genetic interactions, and a predicted role in microtubule severing, we hypothesized that SSP4 mutants would exhibit a Slit/Robo phenotype. We created lines of flies with excisions and P-element hops in an attempt to mutate SSP4. We prepared genomic DNA from each line of flies, performed PCR, and analyzed PCR products on a gel. Two lines of flies produced PCR products suggesting a possible mutation in SSP4. Following reproduction of our results, the DNA for the two lines of flies will be sequenced to see if the mutation is in the coding region of SSP4. Once mutated, we will be able to determine SSP4’s role in the Slit/Robo repulsion pathway.
Funding provided by: The Paul K. Richter and Evalyn E. Cook Richter Award

HSPGS at the Drosophila Neuromuscular Junction

Victoria Gillet ('10); Karl Johnson

Syndecan (Sdc) and Dallylike (Dlp) are membrane-anchored heparan sulfate proteoglycans (HSPGs) which are believed to regulate synapse formation through interactions with the transmembrane phosphatase LAR. However, little is known about the molecular mechanisms by which they function, particularly the role of the highly conserved cytoplasmic domain of Sdc. Previous students in the Johnson lab performed a yeast-two-hybrid screen and identified 60 potential interactors with cytoplasmic Sdc. We have identified the 11 most promising hits for interaction with Sdc from the Y2H screen that do not have a delineated role in nervous system development through a series of bioinformatics screens. Preliminary analysis of mutant phenotypes has begun. We also attempted to confirm the current model for the interactions of Dlp and Sdc with LAR through tissue-specific expression of RNAi. Preliminary results place Sdc function in the muscle during synaptic development, which is at odds with the preexisting model.
Funding provided by: Howard Hughes Medical Institute

Threat Appraisal Partially Mediates Relationship Between State Anxiety and Declarative Memory Functioning

Jaime Guillot ('10); Roxanna Salim (CGU); Nicole Weekes

Numerous studies have demonstrated that exposure to a stressor influences memory performance. Additionally, cognitive appraisal of that stressor as either threatening or challenging affects how stress is experienced. In order to investigate the impact of appraisal on the relationship between stress and memory, 55 undergraduate students were examined under either a low or high stress testing session. The Trier Social Stress Task (TSST) was used as the stressor. Measures included a number of psychological and physiological indicators of the stress response, an appraisal measure, and a declarative memory task. We found that memory performance was dependent both on the nature of the appraisal made and the level of state anxiety experienced. Specifically, appraising the stressor as a threat reduced declarative memory performance and was a slightly better predictor of memory functioning than state anxiety. Future analysis of the physiological data should help to inform the exact nature of these relationships.
Funding provided by: Rose Hills Foundation

PPT-1 Deficiency Caused Altered Synaptic Recycling in Drosphila Melanogaster

Kayleigh Kaneshiro ('10); Nick Kramer ('11); Jani Kim ('10); Karen Parfitt; Christopher Korey*
*College of Charleston, Charleston, SC

Infantile Neuronal Ceriod Lipofuscinoses (INCL), a hereditary neurodegenerative disorder, results from a deficiency in palmitoyl protein thioesterase 1 (PPT1). PPT1 cleaves thioester linkages of S-acylated proteins and removes palmitate residues, and therefore may be involved in synaptic vesicle recycling. Previous studies have shown that a PPT1 deficiency in mammalian cultured neurons results in an increase in the number of membrane-docked vesicles and a decrease in endocytosis, but its substrate and therefore its mechanism is still unknown. We performed electrophysiological recordings of miniature and evoked excitatory junction potentials (EJPs) at the neuromuscular junction of Drosophila larvae in order to elucidate the effects of PPT1 mutation on neuronal function. The frequencies of the PPT1-mutant mEJPs increased while the amplitudes did not change significantly, indicating a presynaptic change in PPT1- deficient synapses. Our experiments with evoked EJPs and high frequency repetitive stimulation showed that PPT1-mutant larvae had deficiencies in calcium-mediated neurotransmitter release and endocytosis.
Funding provided by: Rose Hills Foundation (KK, JK); The Elgin Fund for Summer Student Research (NK)

Ginsenosides Regulate Long-Term Potentiation in the Rat Hippocampus

Tingyu Liu ('10); Meghan Flanigan ’(11); James Yang (’10); Alex Moscicki (’10); Eric Chang (’09); Jonathan King

Ginseng is one of the most popular herbal remedies today. In particular, ginsenosides, the active components of ginseng, are implicated in learning and memory. We examined the effect of direct exposure of ginsenosides Rb1, Rb2, Rc, Rg1, and Rg2 on rat hippocampal slices on long-term potentiation (LTP), the main molecular mechanism of learning and memory. Short term exposure (10 minutes) of Rb1, Rb2, Rc, and Rg1 demonstrated a significant decrease in LTP while Rg2 exposure significantly increased LTP as compared to control. However, long term exposure (1 hour) of Rb2 revealed no effect on LTP while Rg1 exposure revealed a significant decrease compared to control and short term Rg1 exposure. This suggests additional mechanisms and/or substrates are triggered depending on the length of exposure. Given the significant effects of ginsenosides on the hippocampus, more studies are needed to assess its value as a herbal remedy.
Funding provided by: The Paul K. Richter and Evalyn E. Cook Richter Award (TL); Howard Hughes Medical Institute (AM)

Restraint Stress Shows Trend Toward Increased Anxiety As Indexed by the Novelty Induced Hypophagia Test, But Does Not Reduce LTP in the CA1 Region of the Rat Hippocampus

Alexander Moscicki ('10); James Yang (’10); Tanya Liu (’10); Meghan Flanigan ('11); Jonathan King

Recent evidence has suggested that clinical depression may occur when neuronal systems cannot adapt appropriately via normal mechanisms of neuroplasticity to external stimuli like stress. Much of this evidence has been provided by the fact that chronic stress reduces neuroplasticity in the hippocampus, but that antidepressants work to reverse these reductions. The novelty induced hypophagia (NIH) test is a behavioral assay used to assess the therapeutic effects of antidepressants and is thought to be most responsive to changes in neuroplasticity. The test evaluates anxiety by measuring the suppression of food intake (latency to feed and amount consumed) caused by an anxiety inducing exposure to a novel environment. The current study investigates whether chronic restraint stress suppresses one mechanism of neuroplasticity, long term potentiation (LTP), in the CA1 region of the rat hippocampus. Additionally, in order to examine whether stress-induced changes in LTP corresponded to the behavioral deficits characteristic of depression.
Funding provided by: Howard Hughes Medical Institute; The Paul K. Richter and Evalyn E. Cook Richter Award (TL)

Regulating Cleavage of Syndecan by MMP1 and MMP2

Margaret Nguyen ('10); Karl G. Johnson

Syndecan (sdc) is a heparan sulfate proteoglycan (HSPG) that controls processes ranging from wound healing to promoting neurite growth in vertebrates. At the Drosophila melanogaster midline, ipsilateral and commissural axons reach their targets by receiving guidance cues from their environments. Here, axons are repelled through slit and robo interactions. Syndecan mediates this interaction by shaping the gradient of slit and by serving as a molecular bridge between Slit and Robo. In vertebrates, sdc-1 is cleaved by matrix-metalloproteinase type 1 (MT1-MMP). However, in invertebrates it is unknown whether sdc is cleaved or intact in vivo. In this study, we look at gain and loss of function of MMP1 and MMP2, which cleave extracellular matrix (ECM) proteins. Overexpression of MMP1 causes midline defects but sdc staining is still visible, while MMP2 overexpression mutants are similar to WT. We are currently examining the phenotype of MMP1 and MMP2 mutants.
Funding provided by: Rose Hills Foundation

The Role of Dallylike During Axon Guidance in Drosophila

Ashley Smart ('10); Meredith Course ('12); Karl Johnson

To create a nervous system, axons must travel throughout the organism and make connections at the correct places. Proteins and other molecules are responsible for guiding these axons to their destination. The heparan sulfate proteoglycan (HSPG), syndecan (Sdc) is required for axon guidance at the central nervous system (CNS) midline. Sdc regulates the interactions between Slit and Roundabout (Robo). Since HSPGs contain very similar heparan sulfate side chains we set out to determine if they are functionally specific. Previous studies on another HSPG, dallylike (Dlp), suggest that Dlp may also be involved in Slit/Robo repulsion. In this study, we will determine if Slit interacts with Dlp by staining for Slit in Dlp mutants, if Dlp interacts with other midline guidance factors by analyzing dose-sensitive genetic interactions and we will determine if we can rescue Dlp mutants with Dlp and Sdc.
Funding provided by: The Paul K. Richter and Evalyn E. Cook Richter Award (AM)

Exploring the Roles of the Syndecan Binding Protein Synbindin in Nervous System Development

Joseph Wilson ('10); Karl Johnson

The heparan sulfate proteoglycan Syndecan regulates both axon guidance at the CNS midline and synapse growth at the neuromuscular junction in Drosophila. Previous research in vertebrates has implicated Synbindin as a binding partner for Syndecan. We aimed to create a mutation in Drosophila Synbindin, through an imprecise excision of a transposon in the first intron of Synbindin, in order to elucidate Synbindin’s role in neural development. After isolating DNA of each candidate mutant, we ran PCR and agarose gel electrophoresis to detect deletions. Although we did not find any mutations that generated amorphic alleles of Synbindin, larval and embryonic phenotypes of a Synbindin hypomorphic allele suggest a function for Synbindin at both the neuromuscular junction and at the midline. Continuing experimentation will include phenotype rescue, an analysis of other candidate Synbindin alleles, and an attempt to generate a Synbindin mutation using ethyl methanesulfonate (EMS).
Funding provided by: National Institue of Health (KJ)

Research at Pomona