Molluscan Neuroscience in the Genomic Era: From Gastropods to Cephalopods

Scripps Research Institute, Jupiter, Florida, USA May 15 - May 19, 2012

Molluscan Neuroscience 2012 Organizing Committee:

Meeting Co-Chairs:

Tom Abrams, David Glanzman, Sathya Puthanveettil Meeting supported by NSF Grant to David Glanzman, UCLA Meeting Logo by Basia Goszczynska, MBL, Woods Hole

Tom Abrams Paul Benjamin Jack Byrne David Glanzman Roger Hanlon Len Kaczmarek Kelsey Martin Sathya Puthanveettil Wayne Sossin Klaude Weiss

- Sponsors –

The National Science Foundation Scripps Research Institute, Florida Leica Microsystems Olympus Hunt Optics & Imaging Promega Applied Biosystems - Life Technologies VWR International

    Molluscan Neuroscience Program Wednesday, May 16th, 2012  

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Molluscan Neuroscience in the Genomic Era: From Gastropods to Cephalopods

Wednesday, May 16th 2012

8:00 – 9:00 AM Continental Breakfast and Registration

9:00 – 9:15 AM Welcome

9:15 – 10:55 AM Cognition Chair: Paul Benjamin * Cognitive behavior in cuttlefish: conditional discrimination in maze learning, discrimination using a touch screen analog, learned control of body patterning, and sleep-like behavior Jean Boal * Pre- and post-natal development of visual cognition in cuttlefish Ludovic Dickel * The neurophysiological basis of motor function and learning and memory in the octopus - an animal with a complex 'embodiment' Binyamin Hochner * The cognitive mollusc: from gastropods to cephalopods, and beyond Rhanor Gillette

10:55 – 11:20 AM Break

11:20 AM – 1:00 PM Plasticity Chair: Kelsey Martin * Regulation of long-term excitability Aplysia neurons by Slack channels and the Fragile X Mental Retardation Protein FMRP Leonard Kaczmarek

* Possible roles of spontaneous transmitter release in homeostasis and growth related plasticity at Aplysia sensory-motor neuron synapses Robert Hawkins

* Synaptic efficiency: role of the anti-cell death protein Bcl-xL Elizabeth Jonas * Isoform specificity of PKCs and PKMs in synaptic plasticity in Aplysia californica. Wayne Sossin

    Molluscan Neuroscience Program Wednesday, May 16th, 2012  

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1:10 – 2:30 PM Lunch

2:30 – 4:35 PM Learning (Cellular and Molecular) Chair: Elizabeth Jonas

* The Synaptic Transcriptome: isolation and characterization of RNAs actively transported by the kinesin complex in sensory and motor neurons of the Aplysia gill-withdrawal reflex Sathya Puthanveettil

* Neurexin-neuroligin trans-synaptic interaction mediates learning-related synaptic remodeling and long-term facilitation in Aplysia Yun-Beom Choi

* Nitric oxide is necessary for reconsolidation of memory Pavel Balaban

* Reconsolidation of long-term sensitization memory in Aplysia: behavioral, synaptic and morphological consequences David Glanzman

*Characterization of ApEgrh, an activity-dependent transcript Robert J Calin-Jageman which is rapidly and persistently up-regulated by long-term sensitization training

4:35 – 5:15 PM Break

5:15 – 6:15 PM Special Lecture:

* Dynamic camouflage and communication in cephalopods: visual input, neural skin control and diverse behaviors Roger Hanlon

    Molluscan Neuroscience Program Thursday, May 17th, 2012  

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Thursday, May 17th 2012

8:00 – 9:00 AM Continental Breakfast

9:00 – 10:40 AM Sensory Signaling and Nociception Chair: Roger Hanlon

* Lessons for mammals from snails and squid: universal patterns of nociceptive plasticity and conserved signaling pathways in long-term sensory alterations induced by bodily injury Edgar Walters

* Peripheral injury produces long-term behavioral and neural sensitization in squid, Loligo pealei Robyn Crook

* Behavioral effects of clinical doses of the general anesthetic isoflurane on Octopus vulgaris Anna Di Cosmo

* Studies of interactions between molecular elements of a presynaptic switch that mediates attention-like sensory gating Thomas Abrams

10:40 – 11:00 AM Break

11:00 AM – 12:15 PM Genome Chair: Wayne Sossin

* A Proposed link between RNA editing and temperature adaptation in cephalopods Joshua Rosenthal

* Neurobiology of the Aplysia and cephalopod genomes: Insights into memory mechanisms and the multiple origins of complex brains Leonid Moroz

* Neuronal DNA amplification is polyploidization brought about by target innervation Ryota Matsuo

12:30 -2:00 PM Lunch

2:00 – 3:30 PM Workshop: Challenges of Cephalopod Research in a Laboratory Setting Robyn Crook, Ludivic Dickel, Roger Hanlon, Binyamin Hochner

3:30 – 5:30 PM Posters

5:30 – 6:30 PM Round Table: Genome, genome, wherefore art thou? Chair: John Byrne Kelsey Martin, Leonid Moroz, Sathya Puthanveettil, Wayne Sossin

    Molluscan Neuroscience Program Friday, May 18th, 2012  

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Friday, May 18th 2012

8:00 – 9:00 AM Continental Breakfast and Registration

9:00 – 10:40 AM Transmitters & Signaling Chair: Tom Abrams

* Long-term regulation of excitability and secretion by mitochondrial calcium Neil Magoski

* Localization of biogenic amines in the central nervous system and periphery of Biomphalariaglabrata, an intermediate host for schistosomiasis Mark Miller

* Nitric oxide as a regulator of Aplysia feeding Abraham Susswein * The involvement of D-aspartate receptors in aging in Aplysia californica Lynne Fieber

10:40 – 11:00 AM Break

11:00 AM – 12:15 PM Learning (Systems) Chair: Paul Katz

* Distinct roles for tonic inhibition and presynaptic facilitation in associative reward conditioning in Lymnaea Paul Benjamin

* Neurophysiological effects of aversive stimuli in Aplysia: plasticity beyond defensive neural circuits Riccardo Mozzachiodi * Role of electrical coupling and neuronal excitability in learning-induced, compulsive-like motor-pattern genesis in Aplysia Romuald Nargeot

12:30 PM – 2:00 PM Lunch and Focus Groups

2:00 – 5:30 PM Excursion

5:30 – 6:30 PM Special Lecture:

* The brain's logic for memory formation and forgetting Ronald Davis

    Molluscan Neuroscience Program Saturday, May 19th, 2012  

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Saturday, May 19th 2012

8:00 – 9:00 AM Continental Breakfast and Registration

9:10 – 10:40 AM Learning (Cellular and Molecular) Chair: David Glanzman

* Mechanisms underlying intermediate-term operant memory in Aplysia and its regulation by the circadian clock Lisa Lyons

* Decoding neuronal plasticity in Aplysia: eEF2 as an integrator coupling activity patterns to translational control Patrick McCamphill

* Computational design of enhanced learning protocols John Byrne * A differentially spliced neurotrophin and cognate Trk receptor mediate synaptic plasticity in Aplysia Stefan Kassabov

10:40 – 11:00 AM Break

11:00 AM – 12:40 PM Circuits Chair: Rhanor Gillette

* Maintaining functional articulation of network activity Andrew Dacks * The connectome is not enough: evidence from model invertebrate nervous systems Vladimir Brezina * Use of fast voltage sensitive dye imaging to study molluscan networks. William Frost * There's more than one way to swim a slug Paul Katz

12:50 PM – 2:20 PM Lunch

    Molluscan Neuroscience Program Saturday, May 19th, 2012  

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2:20 – 4:10 PM Development Chair: Sathya Puthanveettil

* A novel non‐chordate retinoic acid receptor (RAR) and its role in development and axon guidance. Gaynor Spencer

* Cell-specific expression and activation of bZIP transcription factors regulate the development and maintenance of synapse baseline Samuel Schacher

* Snail genes enhance axonal outgrowth of rodent central neurons Zhong-ping Feng * Synapse-specific translational regulation Kelsey Martin

6:30 PM Dinner

Special Lecture ROGER HANLON

Roger Hanlon, Ph.D. - Biography Roger Hanlon is Senior Scientist at the Marine Biological Laboratory in Woods Hole, MA and Professor (MBL) of Ecology and Evolutionary Biology at Brown University. He is a diving biologist who uses digital imagery (stills, video, hyperspectral) to analyze rapid adaptive camouflage and communication in cephalopods (squid, octopus, cuttlefish) and fishes. He was trained in marine sciences at Florida State University and University of Miami, and studied sensory ecology as a postdoctoral fellow at Cambridge University. Recently his laboratory has focused on a highly multidisciplinary effort to quantify animal camouflage, touching subjects as varied as visual perception, psychophysics, neuroscience, behavioral ecology, image analyses, computer vision, and art. Laboratory research involves live-animal, hypothesis-driven experimentation on several key aspects of the adaptive camouflage and signaling systems. Overall, 180 peer-reviewed scientific papers have been published on these and related subjects. Collaborations with materials scientists and engineers aim to develop new classes of materials that change appearance based on the pigments and reflectors in cephalopod skin. Active public outreach featuring these charismatic marine animals has been conducted recently with NOVA, BBC, Discovery, NatGeo, TEDx, and New York Times.

Special Lecture ROGER HANLON

Dynamic camouflage and communication in cephalopods: visual input, neural skin control and diverse behaviors ROGER HANLON, Trevor Wardill, Robin Crook, Paloma Gonzalez-Bellido Program in Sensory Physiology & Behavior Marine Biological Laboratory, Woods Hole, MA 02543

Cephalopods evolved differently from other mollusks, having lost the external shell protection and developed keen senses, a large complex brain, fast locomotion and diverse behaviors. Much of this behavior is manifest through their elaborate skin, which is richly innervated to provide fast, fine control of millions of chromatophores, iridophores and skin papillae. We will describe details of the biophotonic structures of the skin that produce such remarkable visual diversity, with emphasis on ultrastructure and neurophysiology of pigments and reflectors that interact to create rapid, diverse adaptive coloration. Many of the nearshore, shallow-water squids, octopuses and cuttlefish possess a unique visual ability to quickly analyze complex visual backgrounds and produce an effective camouflage pattern. We will present some new discoveries and simplifying principles of how this refined biological system operates. Using a “visual sensorimotor live animal bioassay” we have teased apart this process in some detail. We are currently addressing subjects such as color-blind camouflage, light sensors in the skin, and control of skin papillae and arm postures. The key points will be illustrated with field video and still images to convey the rapidity and sophistication of this visual sensorimotor system.

Special Lecture RONALD L. DAVIS

Ronald L. Davis, Ph.D. – Biography Ronald Davis completed his doctorate in genetics at the University of California, Davis, in 1980. He co-discovered that the prototypic learning mutant of Drosophila, named dunce, is deficient in the enzyme cAMP phosphodiesterase. This was one of the first indications of the importance of the cAMP signaling system in CNS processes underlying behavior. As a postdoctoral fellow with Norman Davidson at the California Institute of Technology, he began to use molecular biological tools to approach the problem of learning and memory. This led to the cloning of the Drosophila dunce gene and the clear demonstration through sequence analysis and expression that the gene encoded the enzyme. Two additional important observations emerged from the study of dunce. First, he demonstrated that the enzyme was preferentially expressed in the mushroom bodies of the adult brain. This was one of the first indications that mushroom bodies are the important neurons mediating olfactory learning in insects, which has led to the ability to conceptualize insect learning at the cellular level. Second, cloning of the mammalian homologs of dunce from the mouse, rat, and human led to the demonstration that the mammalian dunce homologs are the target of one type of antidepressant, the first indication that studies of Drosophila learning mutants might lead to insights into mammalian behavior. His basic research has broad implications for many psychiatric and neurological diseases, including schizophrenia, autism, Alzheimer’s Disease, attention deficit hyperactivity disorder, and mood disorders. He has supervised a laboratory for the last 30 years studying learning and memory at Michigan State University, Cold Spring Harbor Laboratory and the Baylor College of Medicine. His laboratory has also developed optical imaging procedures to visualize how memory is represented in the brain of the intact fly. He was the Vice Chair of Research in the Department of Psychiatry at Baylor College of Medicine and subsequently the Director for the Center for Memory and Learning at that institution. He is currently the Chair of Neuroscience at the newly founded campus of the Scripps Research Institute in Jupiter, Florida.

Special Lecture RONALD L. DAVIS

The brain's logic for memory formation and forgetting RONALD L. DAVIS The Scripps Research Institute, Jupiter, Florida, USA

We have recently addressed two important issues regarding memory formation and retention: What is the logic by which the brain stores memories associated with pleasing or positive cues vs negative or aversive cues? Is there an active biological process for removing or erasing memories after they form?

We have been able to peer into the brain of the living fruit fly, Drosophila melanogaster, before and after the fly learns about odor cues to visualize, using functional imaging techniques, the changes that occur in the brain due to learning. These changes, or memory traces, often present themselves as increases in calcium influx into specific populations of neurons in response to the learned odor. Our studies using a negative cue associated with an odor reveal that the brain represents the learned odor as multiple memory traces that form in different nodes of the olfactory nervous system. The aversive memory traces form at different times after learning and persist across different time windows, with some traces apparently representing short-term memory, one representing intermediate-term memory, and two representing long-term memory. More recent studies have focused on odor learning coupled with an appetitive cue, to address the underlying question of how the brain represents aversive vs positive events? We have found that the memory traces for positive events are longer lasting than aversive memory traces and that they exhibit broader expression in the neurons in which they form.

Memories can become stabilized after learning, or they can be forgotten. We have recently discovered that there exists an active forgetting mechanism to erase unwanted and unimportant memories. Dopamine signaling is required for learning about odors in the fly at the time that the odor is learned. Surprisingly, dopamine neurons remain active after learning and mediate a forgetting signal. Thus, the brain is designed to learn information, but also to actively forget using dopamine signaling.

Cognition JEAN BOAL

Cognitive behavior in cuttlefish: conditional discrimination in maze learning, discrimination using a touch screen analog, learned control of body patterning, and sleep-like behavior JEAN BOAL1, John Case2, Marcos Frank3

1Dept. of Biology, Millersville University, Millersville, PA 2Dept. of Computer Science, University of Delaware, Newark, DE 3Univ. of Pennsylvania School of Medicine, Philadelphia, PA

Recent experiments confirm and extend what we know of cognitively-related behavior in the cuttlefish Sepia officinalis. In Experiment 1, cuttlefish were trained to escape from two different maze configurations. Subjects learned to select the correct exit based on visual cues, demonstrating conditional discrimination. In Experiment 2, cuttlefish were trained with a simple 2-choice simultaneous discrimination; objects were presented inside the aquarium and subjects were rewarded with a live prey item delivered in a nearby food hopper if they grabbed the correct stimulus. The cuttlefish readily transferred this learning to stimuli presented outside the glass aquarium, and then to video presentations of the objects on a standard flat screen monitor. While it is not clear what cephalopods see when an image is projected on a monitor, this experiment demonstrates that video presentations can be used successfully. In Experiment 3, cuttlefish were trained to show body patterns that contrasted with their surroundings using simple shaping and food rewards. Cuttlefish normally use body patterns to camouflage; in this experiment, subjects trained in a black environment learned to show a contrasting white square on their mantle while those trained in a white environment learned to show a contrasting darkened mantle. Their success in this task demonstrates that cuttlefish can learn to control their body patterning for a food reward. In Experiment 4, sleep-like behavior was demonstrated, including regular activity cycles, quiescent states, rebound from sleep deprivation, and REM-like body movements and skin patterning during quiescent periods. Further investigations are underway to establish whether cuttlefish also show higher sensory thresholds during quiescent periods, as would be expected in sleep. Taken together, these four experiments expand what is known about cephalopod cognition, and the methods used are readily portable to other cuttlefish experimental objectives.

Cognition LUDOVIC DICKEL

Pre- and post-natal development of visual cognition in cuttlefish

LUDOVIC DICKEL University of Caen basse Normandy, France

Perinatal learning is of considerable ecological significance. Young or even embryonic animals may detect and learn features of their environment to shape their early behaviors. Cuttlefish are of major interest to investigate early development of brain and cognition since eggs and hatchlings do not benefit from any parental care, then they have to cope on their own to find food. Seven day-old cuttlefish prefer shrimp over crabs for their first meal but these preferences changed in individuals that have been familiarized with crabs during in ovo or just after hatching (sensitive period). This suggests very long-term retention capabilities in hatchlings despite their immature brain. We hypothesized the existence of food imprinting in cuttlefish.

Cognition BINYAMIN HOCHNER

The neurophysiological basis of motor function and learning and memory in the octopus - an animal with a complex ‘embodiment’

BINYAMIN HOCHNER Department of Neurobiology, Silberman Institute of Life Sciences and the Interdisciplinary Center for Neuronal Computation. The Hebrew University of Jerusalem

Analyzing the neurobiology of the octopus requires considering its special morphology. I first describe octopus ‘embodiment’ using the embodied view that "… the actual behavior emerges from the interaction dynamics of agent and environment through a continuous and dynamic interplay of physical and information processes” (Pfeifer et al., Science 2007). The octopus with its soft, flexible body and its great variety of active behaviors driven by a huge amount of sensory information is a ideal test-case for assessing this view in a biological system. I review the motor control strategies evolved in the octopus to cope with this special morphology. These include a uniquely organized neuromuscular system and a special distribution of control and computational labor between the central and an elaborated peripheral nervous system. In addition, the higher motor center is not organized somatotopically. I further discuss a comparative physiological analysis of a learning and memory network in the octopus and cuttlefish, which revealed dichotomous differences in the site of synaptic plasticity in a simple ‘fan-out fan-in network’. However, due to the linear input/output relationship of both networks, computational considerations suggest that the two networks are computationally similar. This suggests the involvement of ‘self-organizational’ processes to tune the network’s computational properties to fit the animal’s embodiment.

This work was supported by Smith Family Laboratory, The Hebrew University, the Binational US-Israel Science Foundation and by European FP7 Programme Project OCTOPUS

Cognition RHANOR GILLETTE

The cognitive mollusc: From gastropods to cephalopods, and beyond RHANOR GILLETTE Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Among the phyla, arthropods and chordates have produced multiple species quite developed in cognitive and social complexity. In the broadly diversified molluscs, while some evolved with markedly interesting aspects of cognition and sociality, they still fall far behind many arthropods and vertebrates. Why is this? Basic cost-benefit decision circuitry has been elucidated that may underlie behavioral economics of both simpler and more complex animals. Logic and simulations suggest that the decision module can be reasonably enhanced for markedly greater cognitive and social attainments – these are, however, quite limited in molluscs in comparison to arthropods and vertebrates. Simple circuit enhancements are discussed for increasing cognitive and social development that were only partly exploited within the phylum. Constraints are considered that could have limited molluscan cognitive and social evolution, and perhaps as well limited more successful radiation into fresh waters and land.

Plasticity LEONARD K. KACZMAREK

Regulation of long-term excitability Aplysia neurons by Slack channels and the Fragile X Mental Retardation protein FMRP LEONARD K. KACZMAREK Departments of Pharmacology and Cellular and Molecular Physiology, Yale University, New Haven, CT 06510 USA

Loss of expression of the RNA-binding protein FMRP (Fragile X Mental Retardation Protein) represents the most common form of inherited intellectual disability and autism. Our recent studies indicate that FMRP bound to RNA interacts directly with Slack Na+-activated K+ channels, producing an enhancement of channel activity. This suggests that activation of Slack channels could influence activity-dependent protein translation. We found that both FMRP and Slack channels are expressed in the bag cell (BC) neurons of Aplysia, which regulate reproductive behaviors in this species. FMRP and Slack immunoreactivity were co-localized at the periphery of isolated BC neurons and the two proteins could be reciprocally co-immunoprecipitated. In voltage-clamp experiments, injection of FMRP into bag cell neurons in culture produced an increase in a component of K+ current that was Na+-dependent and that matched Slack currents in its biophysical properties. We further used an siRNA approach to isolate the Slack-dependent currents, and found that a Slack-like component of current could be induced by intracellular injection of FMRP. In response to brief stimulation, BC neurons generate a ~30 minute discharge, which is followed by a period of inhibition that endures for ~18 hours. We found that recovery from the prolonged inhibited period requires new protein synthesis and is blocked by the protein synthesis inhibitor anisomycin (3 mM). Preincubation with anisomycin for 24 hrs without stimulation, however, had not effect on excitability. We tested the role of Slack channels in activity-dependent protein synthesis by suppressing Slack expression for five days using anti-Slack siRNA. This treatment reduced Slack protein levels detected by Western blotting and immunocytochemistry, and reduced KNa currents in BC neurons. Treatment with anti-Slack siRNA for 5 or 6 days did not prevent stimulation-induced discharge, but, like anisomycin, prevented the recovery of BC neurons from the subsequent prolonged inhibited state. Our studies indicate that FMRP is an evolutionarily conserved regulator of membrane excitability and raise the hypothesis that channel-FMRP interactions may link changes in neuronal firing to changes in protein translation.

Plasticity ROBERT D. HAWKINS

Possible roles of spontaneous transmitter release in homeostasis and growth related plasticity at Aplysia sensory-motor neuron synapses ROBERT D. HAWKINS1,2, E.R. Kandel1,2, and I. Jin1 Department of Neuroscience, Columbia University1, and New York State Psychiatric Institute2, New York NY 10032. We have previously found that spontaneous transmitter release is critical for the induction of long-term facilitation by 5HT at Aplysia sensory-motor neuron synapses, and that this process begins during an intermediate-term stage that is the first to involve postsynaptic as well as presynaptic mechanisms. Increased spontaneous release from the presynaptic neuron during short-term facilitation stimulates postsynaptic mechanisms of intermediate-term facilitation including metabotropic glutamate receptors, IP3, Ca2+, protein synthesis, and membrane insertion and clustering of AMPA-like receptors, which may be first steps in a cascade that can lead to synaptic growth during long-term facilitation. These findings have raised two new questions about the roles of spontaneous release, which we have now begun to address. First, in other systems spontaneous release and many of the same downstream mechanisms are also involved in synaptic homeostasis, but with the opposite sign of action: decreased spontaneous release produces an increase in synaptic strength. That is true at Aplysia sensory-motor neuron synapses as well: reducing spontaneous release or blocking postsynaptic metabotropic glutamate receptors (which decrease intermediate-term facilitation) increase the baseline EPSP and membrane insertion of AMPA-like receptors. We are now in a position to investigate how spontaneous release can contribute to both homeostasis and plasticity at the same synapses. Second, our results suggest that spontaneous release may also be an early anterograde signal in a growth cascade. If so, what other messengers are involved, and how do they relate to spontaneous release? We have begun to investigate an endogenous neurotrophin (ApNT) and its receptor (ApTrk), both of which were recently identified and found to be important for long-term facilitation (Kassabov et al., this meeting). Bath application of ApNT produces rapid increases in the EPSP and spontaneous release, whereas injection of an ApTrk antisense oligonucleotide into the sensory neuron decreases the 5HT-induced increase in spontaneous release. Furthermore, over-expression of ApNT in the sensory neuron or the motor neuron can produce facilitation. These results suggest that ApNT might function as an autocrine or retrograde signal to enhance spontaneous release, thus possibly forming feedback loops that could contribute to homeostasis as well as facilitation at these synapses. Supported by NIH grants GM097502, NS045108, and MH045923.

Plasticity ELIZABETH JONAS

Synaptic efficiency: Role of the anti-cell death protein Bcl-xL ELIZABETH JONAS Depts. Internal Medicine and Neurobiology, Yale University, New Haven, CT Following periods of exocytosis, the rate of recovery of neurotransmitter release is determined both by the rate of endocytotic retrieval of vesicles from the plasma membrane and by recruitment of new vesicles from internal reserve pools. The degree to which these two mechanisms contribute may be modified by neuronal activity. The Bcl-2 family protein Bcl-xL, in addition to its role in cell death, regulates neurotransmitter release through mechanisms that have previously been thought to be indirect and related to its effects on ATP availability from mitochondria. Adult neurons are generally long-lived and express Bcl-xL. Bcl-xL forms channels in mitochondrial membranes of squid presynaptic terminals. Using internal patch pipettes we found i) that presynaptic stimulation opens Bcl-xL channels in the outer mitochondrial membrane, ii) that application of Bcl-xL mimics such channel activity, enhances synaptic transmission and speeds recovery of transmission after repetitive firing iii) that inhibition of Bcl-xL blocks both presynaptically stimulated mitochondrial channel activity and slows the recovery of transmitter release following repetitive firing and that iv) ATP injection into presynaptic terminals occludes the effect of injection of Bcl-xL. We now find, however, that in addition to production and release of ATP by mitochondria, Bcl-xL directly regulates endocytotic retrieval through protein/protein interaction with components of the clathrin complex. Our evidence suggests that, during synaptic stimulation, Bcl-xL translocates to clathrin-coated pits in a calmodulin-dependent manner and forms a complex of proteins with the GTPase Drp1 and clathrin. This complex is necessary for normal endocytosis because depletion of Drp1 produces misformed endocytotic vesicles. Finally, our mutagenesis studies suggest that the formation of the Bcl-xL-Drp1 complex results in an increased rate of endocytosis of vesicles from the plasma membrane, thus providing a novel mechanism for presynaptic plasticity.

Plasticity WAYNE S. SOSSIN

Isoform Specificity of PKCs and PKMs in synaptic plasticity in Aplysia californica WAYNE S. SOSSIN, Carol A. Farah, Joanna K. Bougie and Margaret Hastings. Montreal Neurological Institute, McGill University, 3801 University Avenue, Montreal Quebec CANADA H3A-2B4. There are three classes of PKCs expressed in the nervous system of Aplysia, the classical PKC Apl I, the novel PKC Apl II and the atypical PKC Apl III. We have examined isoform specificity of PKCs in Aplysia using a combination of three techniques: (i) live imaging to determine when and how the PKCs are activated, (ii) isoform-specific dominant negative PKCs to block PKC activity and (iii) Pharmacological inhibitors that may be isoform specific. We have found that each isoform is tuned to activation by distinct cues. PKC Apl I is activated by the conjunction of calcium and diacylglycerol; PKC Apl II is activated by the conjunction of phosphatidic acid and diacylglycerol and PKC Apl III is activated downstream of PI-3 kinase pathways. Each of the isoforms has the ability in-vitro to be cleaved by calpains into PKMs and we have begun to use a live-imaging FRET-based cleavage assay to examine the isoform specificity of cleavage. Dominant negatives have been able to distinguish the roles of PKC Apl II in reversal of depression from the role of PKC Apl I in activity-dependent facilitation. In contrast, with two exceptions pharmacological inhibitors have been ineffective at distinguishing isoforms. Chelerythrine is better at inhibition of PKMs than PKCs, and Bis-I is more effective on classical and novel PKCs and PKMs than it is on atypical forms.

Learning (Cellular and Molecular) SATHYANARAYANAN V. PUTHANVEETTIL

The Synaptic Transcriptome: Isolation and characterization of RNAs actively transported by the kinesin complex in sensory and motor neurons of the Aplysia gill-withdrawal reflex SATHYANARAYANAN V. PUTHANVEETTIL, 2, 8, *, Igor Antonov1, Sergey Kalachikov3, Priyamvada Rajasethupathy1, Yun-Beom Choi1, 5, Andrea B. Kohn6, 7, Mathew Citarella6, 7, Fahong Yu6, 7, Kevin A. Karl2, Maxime Kinet1, Irina Morozova3, James J. Russo3, Jingyue Ju3, Leonid L. Moroz6, 7 and Eric R. Kandel1, 2,4, 5, * 1Department of Neuroscience, 2Howard Hughes Medical Institute, 3Department of Chemical Engineering and Columbia Genome Center, 550 West 120th St, New York, NY 10027, 4Kavli Institute for Brain Science, 5Department of Neurology, College of Physicians and Surgeons of Columbia University, 1051 Riverside Drive, New York, NY 10032, USA; 6The Whitney Laboratory for Marine Biosciences, 7Department of Neuroscience and McKnight Brain Institute, 100 S. Newell Drive, Building 59, University of Florida, Gainesville, FL 32611, USA; 8Department of Neuroscience, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, Florida, FL 33458. In response to learning, specific mRNAs are transported from the cell body of neurons to their terminals where their translation can modify communication at pre-existing synapses and induce formation of new synaptic connections. However, little is known about the identity of the RNAs that are actively transported, or when and how these RNAs are utilized during learning. By focusing on RNAs that are associated with kinesin, a motor protein that transports gene products from the cell body to synapses, we have now identified actively transported RNAs in the central nervous system (CNS) of Aplysia. Using deep sequencing of a library prepared from the kinesin complex immunoprecipitated from CNS, we have identified 5,657 unique sequences. These include 1,184 annotated RNAs, many of which have key roles in synaptic function and growth. Serotonin, a modulatory neurotransmitter important for learning, differentially regulates some of these RNAs both transcriptionally and translationally. Using a custom designed cargo-enriched microarray we have identified differentially expressed transcripts in the cell body and processes of sensory and motor neurons of the gill withdrawal reflex circuitry. Furthermore, we find that myosin heavy chain, an RNA cargo of kinesin, is specifically critical for the establishment of long-term facilitation (LTF), but not for its persistence.

Learning (Cellular and Molecular) YUN-BEOM CHOI

Neurexin-Neuroligin Trans-Synaptic Interaction mediates learning-related synaptic remodeling and long-term facilitation in Aplysia YUN-BEOM CHOI, Hsiu-Ling Li, Stefan R. Kassabov, Iksung Jin, Sathyanarayanan V. Puthanveettil , Kevin A. Karl, Yang Lu, Joung-Hun Kim, Craig H. Bailey and Eric R. Kandel Columbia University, Leonia, NJ

Neurexin and neuroligin, which undergo a heterophilic interaction with each other, are cell adhesion molecules involved in the regulation of synaptic maturation, specification, and stabilization. They are found to be mutated in some patients with autism which involves a deficit in social and emotional learning. We have explored directly the role of the neurexin-neuroligin trans-synaptic interaction in learning and memory storage in a model system, the gill-withdrawal reflex of the sea mollusc, Aplysia which exhibits a simple form of learned fear - sensitization,. We cloned the Aplysia homologs of neurexin (ApNRX) and neuroligin (ApNLG), and found they exhibit strong similarities with their vertebrate counterparts both in domain structure and subcellular localization. The simultaneous overexpression of ApNRX in the presynaptic sensory neuron and ApNLG in the postsynaptic motor neuron induces a long-term increase in synaptic strength. Depleting neurexin in the presynaptic sensory neuron or neuroligin in the postsynaptic motor neuron abolishes both long-term facilitation (a cellular analog of sensitization reconstituted in Aplysia sensory-motor neuron co-cultures) as well as the associated learning-related growth of new presynaptic varicosities that are induced by repeated pulses of serotonin. Moreover, when the R451C mutant of neuroligin-3 linked to autism is introduced into the motor neuron, it blocks both intermediate-term facilitation and long-term facilitation, thus disrupting the normal progression of memory storage. Our results suggest that activity-dependent regulation of the neurexin-neuroligin interaction is one of the mechanisms that govern trans-synaptic signaling required for the storage of long-term emotional memory.

Learning (Cellular and Molecular) PAVEL M. BALABAN

Nitric oxide is necessary for reconsolidation of memory *PAVEL M. BALABAN, M. V. Roschin, Gainutdinov Kh.L., Gainutdinova T.Kh., T. Korshunova Inst. Higher Nervous Activity & Neurophysiol., Moscow, Russian Federation

Nitric oxide (NO) is synthesized as needed by NO synthase and diffuses into adjacent cells. NO forms covalent linkages to a multiplicity of targets which may be enzymes, such as guanylyl cyclase (GC) or other targets. Influence of NO via GC activates intracellular signaling cascades and triggers increased synthesis of proteins, influencing the memory.

In our experiments in terrestrial snail Helix we tested the idea that NO is involved in erasure of memory during reconsolidation of context memory. Prior to the training session, the behavioral responses to tactile stimulation in two contexts were similar. One day after a session of electric shocks in one context only, the context conditioning was observed as the significant difference of behavioral response amplitudes in two contexts. On the day following testing of context learning, a session of “reminding” was performed, before which the snails were injected with drugs or vehicle. Next day testing of long-term context memory has shown that single injections of anisomycin (AN) or L-NNA impaired the context conditioning, while under combined injection of L-NNA+AN no impairment of the long-term context memory was observed. We have received similar results using combined injection of the AN and NO scavenger PTIO, NO-synthase inhibitors aminoguanidine, nitroindazole, L-NAME. Vehicle injections did not influnce context memory, as well as AN injections without reminding. Obtained results evidence that the NO is necessary for reconsolidation process triggered by “reminder”. No new memory can be formed under the AN, what evidences that the memory about noxious context observed under combined action of AN+NO inhibitors is some kind of not deleted (due to NO inhibition) previously existing memory. It leads us to a simple suggestion that NO is necessary for erasure of “old” memory. We hypothesize that besides involvement of NO in new memory via the GC pathway (necessary for reconsolidation phenomenon), NO can locally abolish “old” memory via the S-nitrosylation pathway.

Learning (Cellular and Molecular) DAVID L. GLANZMAN

Reconsolidation of long-term sensitization memory in Aplysia: behavioral, synaptic and morphological consequences DAVID L. GLANZMAN1,2,3, D. CAI1, S. CHEN1, K. PEARCE1, P. Y. SUN1, A.C.ROBERTS1 1Integrative Biol. Physiol. 2Neurobiol. and Brain Res. Inst., David Geffen Sch. of Med. at UCLA 3Integrative Center Learn. Mem., UCLA, Los Angeles, CA

Long-term memories, even well consolidated ones, can be surprisingly labile under certain circumstances. For example, if an animal is given a reminder of a distant learned experience, and then receives a protein synthesis inhibitor (PSI), the long-term memory (LTM) for the learned experience will be disrupted. By contrast, treatment with a PSI in the absence of the reminder typically does not disrupt a consolidated memory. This phenomenon has led to the idea that the reminder triggers reconsolidation of the LTM, and during reconsolidation the LTM becomes subject to disruption by inhibitors of protein synthesis.

Although this idea is widely accepted, the biological basis of memory reconsolidation is poorly understood. This can be attributed to the lack of a system that permits rigorous examination of the synaptic consequences of memory reconsolidation. Toward the development of such a system, we examined reconsolidation of the LTM for sensitization in the marine snail Aplysia californica. Animals were given sensitization training, consisting of spaced bouts of tail shocks. The training produced long-term sensitization (LTS) of the siphon-withdrawal reflex (SWR) that persisted for ≥ 120 h. Some animals received reminder training (one bout of tail shocks) at 96 h after training followed by an intrahemocoel injection of the PSI anisomycin (ANISO). Animals that received the reminder training plus ANISO at 96 h exhibited no sensitization of the SWR at 120 h after the original training. By contrast, animals that received the ANISO injection without the reminder training exhibited significant LTS at this time. We also investigated whether long-term facilitation (LTF) of the sensorimotor synapse, the form of synaptic plasticity that mediates LTS, undergoes reconsolidation. Here, sensorimotor cocultures received training with two bouts of five 5-min pulses of serotonin (5X5-HT training); the bouts were spaced 30 min apart. All cocultures that received 5X5-HT training were treated with ANISO (10 µM, 2 h) at 48 h after training. Some of the cocultures also received reminder training (a single 5-min 5-HT pulse) immediately prior to the ANISO treatment. Cocultures given the 5X5-HT training plus ANISO exhibited significant LTF at 72 h after the training. But cocultures given the reminder pulse of 5-HT prior to the ANISO treatment did not exhibit LTF. LTF of in vitro sensorimotor synapses due to 5X5-HT training is accompanied by an increase in the number of presynaptic varicosities, the sites of presynaptic transmitter release. This learning-related long-term structural change was reversed by ANISO treatment when the PSI treatment was immediately preceded by a reminder pulse of 5-HT. Our data indicate that the apparent absence of LTM following the disruption of reconsolidation by PSI treatment results from the disruption of the synaptic changes that underlie the memory rather than to interference with retrieval.

Learning (Cellular and Molecular) ROBERT J. CALIN-JAGEMAN

Characterization of ApEgrh, an activity-dependent transcript which is rapidly and persistently up-regulated by long-term sensitization training ROBERT J. CALIN-JAGEMAN, Ashly Cyriac, Dmitry Belchenko, & Irina E Calin-Jageman Neuroscience Program, Dominican University, River Forest, IL Corresponding Author: Irina Calin-Jageman, icalinjageman@dom.edu

Learning is thought to induce long-term memory in part through changes in transcription. These transcriptional changes are complex, involving both an immediate set of transcriptional targets (immediate-early genes) which are usually activated transiently, and later sets of transcriptional targets which are usually activated for the duration of the long-term memory. We have recently identified a transcript in Aplysia californica (ApEgrh) which may bridge both these stages of transcriptional response, showing both rapid and possibly persistent up-regulation following long-term sensitization training. ApEgrh-1 was identified by screening the draft A. californica genome for homologs of activity-dependent genes. The gene codes for a predicted 593-amino acid protein with a highly conserved trio of zinc-fingered domains in the C-terminus, a characteristic of the Egr family of transcription factors. Qualitative PCR indicates basal expression of ApEgrh-1 in the nervous system as well as the skin, gut, ink gland, and reproductive organs. qPCR revealed that neural activity rapidly and bi-directionally regulates ApEgrh-1 expression in isolated pedal/pleural ganglia. Consistent with a role as an immediate-early gene, pre-incubation with anisomycin for 1 hour does not fully block the effects of activity on ApEgrh-1 expression. To begin examining a functional role for this transcript, we measured the expression of ApEgrh-1 both 1 and 24-hours after unilateral long-term sensitization training. We found strong and consistent up-regulation of ApEgrh-1 in the pleural ganglia 1 hour after training (comparing trained to un-trained sides). Surprisingly, this increase in expression seems to be maintained 24 hours after training. We are now working to localize the expression of ApEgrh-1 (using FISH) and to characterize its physiological role (using RNAi). Although it is still unclear if ApEgrh-1 plays a functional role in long-term memory, it seems to have a relatively distinctive transcriptional signature suggestive of possible roles in both encoding and maintaining memory.

Sensory Signaling and Nociception EDGAR T. WALTERS

Lessons for mammals from snails and squid: universal patterns of nociceptive plasticity and conserved signaling pathways in long-term sensory alterations induced by bodily injury EDGAR T. WALTERS and Robyn J. Crook University of Texas Medical School at Houston. It is likely that adaptive, long-term responses to bodily injury appeared during the earliest stages of animal evolution. If so, primitive mechanisms of injury-induced plasticity might be conserved across divergent phyla and modified during evolution for other long-term functions such as memory. Nociceptive sensory neurons detect but also “remember” peripheral injury. The most extensively studied adaptive response to bodily injury in Aplysia (with injury simulated by electric shock or 5-HT treatment) is long-term facilitation of synaptic transmission (LTF) from nociceptive sensory neurons. Equally important for sensory compensation and enhanced vigilance after bodily injury is long-term hyperexcitability (LTH) of nociceptors – in peripheral branches and the cell soma. Our studies of Aplysia nociceptors show that injury, depolarization, or 5-HT application in the periphery causes long-term behavioral hypersensitivity correlated with LTH in nociceptor axons and somata. Depolarization or 5-HT treatment of peripheral axons is more effective in producing long-term alterations than is treatment of central ganglia. Axonal LTH induced by peripheral depolarization depends upon local protein synthesis and many of the same intracellular signals that induce synaptic LTF. Peripheral depolarization also induces delayed somal LTH, which depends upon early protein synthesis at the depolarization site and later protein synthesis in the CNS. These findings indicate that mechanisms important for conventional synaptic memory models are prominent in injury-related axonal and somal LTH. How general are long-term behavioral hypersensitivity and LTH of somatic sensory neurons after bodily injury? Our recent studies show that the squid, Loligo pealeii, like Aplysia and mammals, exhibits enhanced defensive responses for at least 2 days after peripheral injury, and that responses of its sensory neurons to peripheral stimulation are similarly enhanced. An important implication of findings in molluscs is that nociceptor LTH and long-term behavioral hypersensitivity depend upon induction of memory-like alterations intrinsic to the nociceptor. This led us to test the novel prediction that a particularly long-lasting (life-long) form of chronic pain in mammals depends upon intrinsic LTH in nociceptors. We found that behavioral hypersensitivity after spinal cord injury in rats correlated with somal LTH in nociceptors, and that selectively blocking the activity of hyperexcitable nociceptors reversed behavioral signs of chronic pain. Supported by grants from the NIH, NSF, and Craig H. Nielsen Foundation.

Sensory Signaling and Nociception ROBYN J. CROOK

Peripheral injury produces long-term behavioural and neural sensitisation in squid, Loligo pealei ROBYN J. CROOK1, Roger T Hanlon2 and Edgar T Walters1 1University of Texas Health Science Center, Houston, TX 77030 2Marine Biological Laboratory, Woods Hole, MA 02543 Survivable injuries are a common yet costly experience. The ability to sense and respond to noxious or injurious stimuli (nociception) is an almost universal trait, and injury-induced sensitisation occurs in most animal taxa. In animals with complex brains nociception is the sensory precursor to pain experience. Cephalopods are the most neurologically complex invertebrates and their learning abilities are well studied, but nonassociative behavioural changes driven by injury have received little attention. Our work focuses on characterising behavioral and physiological changes elicited by minor injury in the squid, Loligo pealei. We have shown previously that squid express profound alterations to defensive behaviours that persist for at least 2 days after injury. These changes parallel forms of nociceptive plasticity in other animals, including general and site-specific sensitization. Injured squid also employ crypsis and escape defenses in different spatial and temporal patterns compared with uninjured conspecifics, indicating a generalised change in threat assessment that may be adaptive. After injury, peripheral sensory terminals show short- and long-term hyperexcitability. Long-term changes are expressed close to the injury site and contralaterally, thus generalisation of enhanced neural responsiveness parallels emergence of heightened defensive behaviours. Current experiments are testing the hypothesis that nociceptive sensitisation drives changes to antipredator behaviour that enhance escape probability, offsetting the increased mortality risk of minor injury. Our studies show that the patterns of expression of long-term nociceptive sensitisation in molluscs and mammals are similar, probably because they are highly adaptive. Similar hyperresponsiveness in nociceptors of molluscs and mammals suggests, moreover, that nociceptive sensitisation mechanisms may be highly conserved. This would mean that fundamental nociceptive plasticity mechanisms revealed in molluscs can have significant biomedical implications, e.g., for the treatment of chronic pain in vertebrate species, including humans.

Sensory Signaling and Nociception ANNA Di COSMO

Behavioral effects of clinical doses of the general Anesthetic isoflurane on Octopus vulgaris ANNA Di COSMO1, Gianluca Polese1, Francesco Paolo Ulloa Severino1, and William Winlow1,2 1 – Department of Structural and Functional Biology, University of Naples “Federico II” Complesso Universitario Monte S. Angelo, viale Cinthia, 80126 Naples, Italy 2 – Honorary research fellow, University of Liverpool, Liverpool, L69 7JZ, UK. Address for correspondence: NPC Newton, 32 Hill Crescent, Newton, Preston, PR4 3TR, UK Anesthesia of cephalopods is more in discussion than ever before due to the work in progress for their welfare legislation. Different approaches to anesthesia in cephalopods have been tried by a number of scientists, but in most cases the animals were not truly anaesthetized. For example, several workers have simply used muscle relaxants or simple hypothermia under the name “anesthesia”. This approach will not be adequate in the future. Inhalational anesthetics such as isoflurane (CF2HOCClHCF3) reduce L-type calcium currents and potassium currents in a dose-dependent manner in the pulmonate mollusk Lymnaea stagnalis and there is evidence from cell culture that such anesthetics also block excitatory chemical synapses, more effectively than inhibitory synapses. Here we report, for the first time, on the effects of clinical doses of the inhalational anesthetic isoflurane on the behavioral responses of Octopus vulgaris. The volatile anesthetic isoflurane (0.5-2.5% v/v) was equilibrated into seawater (1600 ml) via an air stone to adult Octopus vulgaris (n=8) of about 400 g. Using a web camera we recorded the animals response to a touch stimuli eliciting withdrawal responses of the arms and siphon and observed changes in the respiratory rate and the chromatophore pattern over time during the anesthetic application. We found that different animals of the same size respond showing with similar behavioral changes as the isoflurane concentration was gradually increased. Sudden application of the highest concentration of isoflurane can be lethal to the animal (n=1). After application of 2.5% isoflurane (for a maximum of 5 minutes), when all the responses indicated deep anesthesia, the animals recovered rapidly (within 10-15 minutes) in fresh aerated sea water. Gray (1970) proposed that the vertical lobe of Octopus vulgaris has integrative functions and receives two input fibers one of which comes from lower subvertical centers and is thought to mediate “pain”. The suggestion that the subvertical lobe is involved in pain is supported by our finding of the expression of estradiol receptors in the subvertical lobe allied to recent studies that demonstrate that estradiol can modulate the anesthetic actions.

Sensory Signaling and Nociception THOMAS W. ABRAMS

Studies of Interactions between Molecular Elements of a Presynaptic Switch that Mediates Attention-Like Sensory Gating THOMAS W. ABRAMS1,3, Shao-Gang Lu1, Pragya Shrestha1 and Thomas A. Blanpied2,3 1Department of Pharmacology, 2Department of Physiology and 3Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD, 21201

Synapses between high threshold mechanosensory neurons (SNs) and motor neurons (MNs) in Aplysia are have long been known to display rapid homosynaptic depression (HSD) with minimal activity. Several lines of evidence indicate that HSD at the SN-MN synapse occurs independent of vesicle release and involves a switching of release sites to a silent state. In contrast, when SNs are activated to fire brief bursts of several action potentials, the bursts prevent the development of HSD via a Ca2+- and PKC-dependent process. This burst-dependent protection (BDP) against synaptic depression is distinct from other known forms of activity-dependent plasticity. The choice between silencing sensory synapses or maintaining them in the active state is based on the salience of the stimulus that produces SN activity. Thus, this synaptic switch mediates a simple attention-like process in these simple marine snails.

We recently found that a central component of the switch that silences synapses during HSD is the small G protein Arf. We are examining how the classical, Ca2+-activated form of PKC, Apl-I, which mediates BDP, interacts with the proteins in the Arf pathway. In one series of experiments, we asked whether PKC Apl-I regulates the Arf pathway upstream of Arf, at the level of the Arf GEF, or downstream of Arf. Consistent with the possibility that PKC Apl-I regulates an Arf GEF, bursts of spikes that normally activate PKC had no effect on synapses that were previously depressed by inhibiting GEFs with the toxin brefeldin A (BFA). Like bursting, application of 5-HT to depressed synapses enhances transmission, but 5-HT acts via a distinct PKC isoform, the Ca2+-insensitive PKC Apl-II (Manseau et al, 2001). In contrast to the inhibitory effects of BFA on burst-dependent facilitation of depressed synapses, facilitation by 5-HT was not affected by BFA, indicating that PKC Apl-II acts downstream of the GEF-Arf switch. These results suggest that PKC Apl-I and PKC Apl-II target distinct molecular sites in the HSD pathway, which may be explained by localization of the two kinases in distinct nano-domains. Our electrophysiological evidence suggests that PKC Apl-I, the scaffolding protein PICK1, Arf and the GEF that regulates Arf are colocalized within a nanodomain proximal to Ca2+ channels. We have begun to examine the relationship between sites of Ca2+ influx, PKC and elements of the Arf pathway using super resolution microscopic techniques, PALM and STORM. This research is supported by NIH R01 grant MH-55880 and an NSF EAGER Award IOS-1151244 to TWA.

Genome JOSHUA ROSENTHAL

A Proposed link between RNA Editing and Temperature Adaptation in Cephalopods JOSHUA ROSENTHAL

Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, Puerto Rico, 00901

Among true metazoans, RNA editing by adenosine (A) deamination is a ubiquitous mechanism for diversifying the proteome. By changing adenosines to inosines (I), which are read as guanosines (G) during translation, codons can be recoded. The extent to which this system is used varies dramatically between taxa, and work from my lab has shown that cephalopods use it extensively. Why? The ability to edit codons gives an organism options: depending on physiological demand, it can choose which isoform of a protein to express. About ten years ago, we proposed that RNA editing was well positioned as a mechanism for temperature adaptation because of the makeup of the genetic code. In almost all cases, by changing an A→G in codons, the size of the R-group is reduced. We theorized that this would increase entropy in proteins, counteracting the effects of the cold. The large number of real editing sites that have been uncovered over the past decade allows us now to look at this question in greater detail. Indeed, the genetic code is set up so that editing should create lots of glycines, and organisms do in fact make lots of glycines. There are taxon specific biases, however, as cephalopods make an isoleucine to valine change far more frequently than expected. Almost all of the amino acid changes caused by editing agree with current theories of which amino acids are targeted for temperature adaptation by evolution at the DNA level. These ideas are supported by recent functional data from our lab on how RNA editing affects octopus K+ channels. A single edit in sequence encoding the channel’s pore domain, which changes an isoleucine to a valine, speeds up the channel’s closing rate, a parameter that is particularly sensitive to temperature. This edit appears in close to all of the mRNAs from polar species, at intermediate levels in temperate species, and scarcely at all in tropical species. This study is the first to link RNA editing with temperature adaptation.

Genome LEONID MOROZ

Neurobiology of the Aplysia and cephalopod genomes: Insights into memory mechanisms and the multiple origins of complex brains LEONID MOROZ The Whitney Laboratory, University of Florida, St. Augustine, FL, USA Department of Neuroscience, College of Medicine and Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Blvd, St. Augustine, FL 32080, USA In this presentation, I will summarize results of the completed sequencing of the Aplysia genome performed by the Aplysia genome consortium (more than 20 members from several institutions) as well as ongoing efforts to sequence 5 cephalopod genomes. Two key points will be outlined. First, we complemented the genome sequencing by deep transcriptome profiling of more than 40 molluscan species. These efforts allowed us (i) to validate more than 20,000 gene models in Aplysia, and (ii) reconstruct evolutionary relationships between key recognized molluscan lineages including all major models used in neurobiological research (e.g. Tritonia, Clione, Pleurobrachia, Lymnaea, Helisoma, Hermissenda, Loligo, Octopus, Sepia, Nautilus, bivalves, etc.). One of the most surprising results of this evolutionary reconstruction is a reconstruction of multiple events of the neural centralization suggesting that within the Molluscan phylum alone, the complex brains can be evolved at least 4-5 times independently. Second, I will outline recent advantages of the genomic dissection of one of the simplest known emory-forming circuits (the gill-withdrawal reflex) including analysis of combined RNA-seq/methylome profiling from individually identified neurons following 5-HT- and NO- applications. Here, we showed that the massive, rapid and active DNA demethylation of the neuronal genomes is a key component underlying long-term plasticity. This mechanism is responsible for integration of expression of more than 5,000 genes in both sensory and motor components of the circuit. Finally, I will outline the established molecular and genomic resources which we developed for the community to promote further exploration of molluscan genomes and advance molecular analysis of simpler neural circuits in representatives of this highly diverse phylum.

Genome RYOTA MATSUO

Neuronal DNA amplification is polyploidization brought about by target innervation RYOUTA MATSUO, Miki Yamagishi, Kyoko Wakiya, Yoko Tanaka, Etsuro Ito Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University The brain of gastropod mollusks contains giant neurons whose nuclei are enlarged with a large amount of genomic DNA. Such DNA is produced by repeated endoreplication, and its frequency is correlative to the body growth of the adult land slug and to the increase in the amount of transcripts within the neuron (Yamagishi et al. (2011) J Neurosci 311, 5596). However, it is not known whether the neuronal DNA endoreplication entails whole genome amplification (polyploidy) or only the necessary genomic loci are amplified (polyteny, polysomy, or cis-DNA amplification by repeated unequal recombinations etc.). Nor is it known what induces DNA endoreplication in neurons during the body growth – (A) some soluble molecules contained in the hemolymph that reflect the nutritive condition of the animal (such as glucose or insulin), or (B) some neurotrophic factors retrogradely transported from the target organs. To address the first question, we adopted quantitative genomic PCR to clarify the mode of DNA endoreplication (polyploidy or polyteny), and demonstrated that multiple genomic loci were amplified to the same extent irrespective of the transcriptional activities at these loci. The BrdU incorporation experiment also indicated that DNA synthesis occurred all throughout the whole genome. Moreover, the visceral giant cell, the biggest neuron in the slug’s brain, was estimated to contain approximately 10000-times as much genomic DNA as the haploid amount. Next, to clarify the mechanism that induces DNA endoreplication in neurons, we transplanted a whole brain from another slug into the body cavity. Thereby we could see the pure effect of the hemolymph on the DNA endoreplication by analyzing the frequency of the DNA endoreplication in the transplanted brain because it is devoid of the target innervation to the host organs. Our results showed that the frequency of the DNA endoreplication is irrelevant to the nutritive status of the host slug, implying that the target innervation plays an important role in the body growth-dependent DNA endoreplication in neurons.

Transmitters and Signaling NEIL MAGOSKI

Long-term regulation of excitability and secretion by mitochondrial calcium NEIL MAGOSKI Queen’s University, Kingston, Ontario, Canada

A remarkable property of many neurons is that they respond to transient input with long-term changes in excitability, such as plateau potentials, prolonged depolarizations, or afterdischarges. These events are critical to learning, sensory processing, and motor control, as well as drinking, eating, ovulation, lactation, and parturition. The bag cell neurons of Aplysia californica control reproduction through an afterdischarge, where brief synaptic stimulation causes a lengthy burst, hormone secretion, and the initiation of egg-laying behaviour. The response is provoked by activation of ligand-gated channels; specifically, acetylcholine opened an ionotropic receptor to depolarize cultured bag cell neurons. This was dose-dependent and sensitive to the cholinergic blockers, mecamlyamine and alpha-conotoxin ImI. Application of acetylcholine to the intact bag cell neuron cluster resulted in genuine afterdischarges, while clusters stimulated to fire an afterdischarge failed to respond to acetylcholine and vice versa. Moreover, cholinergic antagonists prevented synaptically-induced afterdischarges. Once underway, the afterdischarge results in calcium influx and release, which triggers cation channels to extend bursting as well as the secretion of reproductive peptides. The mitochondria play a key role in regulating calcium availability during activity. Following a train of depolarizing stimulation, cultured bag cell neurons presented a protracted calcium-induced calcium-release. Liberating mitochondrial calcium with the protonophore, FCCP, or blocking mitochondrial calcium exchange with the lipophilic cation, TPP, eliminated stimulation-induced calcium-release. Prior stimulation also increased the amount of calcium liberated by FCCP. These data suggest that calcium entering through voltage-gated channels is taken up by mitochondria and released back into the cytosol. Furthermore, mitochondrial calcium arising from FCCP activated a non-selective cation current capable of sustaining depolarization and caused apparent peptide secretion, as monitored by tracking membrane capacitance. In summary, transient acetylcholine stimulation elicits a long-term change to bag cell neuron excitability, and this is potentially influenced by mitochondrial calcium handling. The latter may represent a general means to control firing and secretory output in the absence of ongoing synaptic input. SUPPORT: Canadian Institutes of Health Research and Natural Sciences & Engineering Research Council of Canada

Transmitters and Signaling MARK W. MILLER

Localization of biogenic amines in the central nervous system and periphery of Biomphalaria glabrata, an intermediate host for schistosomiasis MARK W. MILLER Institute of Neurobiology, University of Puerto Rico, Medical Sciences Campus Schistosomiasis is one of most devastating parasitic diseases affecting humankind. By most estimates, up to 10% of the people on Earth are infected by schistosome blood flukes (World Health Organization, Media Centre Fact Sheet 2011). The disease is most widespread in tropical Africa, Asia, and South America where it imposes severe limitations on the productivity and quality of countless lives. All schistosomes have an obligatory molluscan intermediate host, i.e. a freshwater snail in which asexual reproduction produces the cercariae that are capable of infecting humans. Schistosoma mansoni, which causes the form of schistosomiasis that occurs in the Western Hemisphere, requires the planorbid snail Biomphalaria glabrata as its intermediate host. As human infections would cease if parasite infections in snails were prevented, it is crucial to further our understanding of the interactions between S. mansoni and B. glabrata. It has been proposed that the transition from the free-living S. mansoni miracidium to parasitic mother sporocyst depends upon uptake of biogenic amines from the snail host. However, little is known about potential sources of serotonin and dopamine in the B. glabrata integument where this transition occurs. This investigation examined the localization of serotonin-like immunoreactivity (5HTli) and tyrosine hydroxylse-like immunoreactivity (THli) in the central nervous system (CNS) and peripheral tissues of B. glabrata. Emphasis was placed upon the cephalic and anterior pedal regions that are common sites of S. mansoni miracidium penetration. The anterior foot and body wall were densely innervated by 5HTli fibers but no peripheral immunoreactive neuronal somata were detected. In contrast, large numbers of peripheral THli neurons were present in the cephalic sensory organs. Double-labeling experiments of the tentacular nerve and the three major pedal nerves disclosed central serotonergic neurons that project to the cephalopedal periphery. The projections to the foot and body wall indicate that, as in other gastropods, serotonin may participate in defensive, nociceptive, or inflammation responses. Dopamine, in contrast, appears to serve an afferent, possibly chemosensory function. These observations identify potential sources of host-derived serotonin in this parasite-host system. Supported by the National Institutes of Health: RCMI RR-03051, NIGMS MBRS: GM-08224 & GM-061838; National Science Foundation DBI-0115825.

Transmitters and Signaling ABRAHAM J. SUSSWEIN

Nitric oxide as a regulator of Aplysia feeding

ABRAHAM J. SUSSWEIN Goodman Faculty of Life Sciences and Gonda Multidisciplinary Brain Research Center, Bar Ilan University, Ramat Gan 52 900, Israel Nitric oxide (NO) regulates Aplysia feeding, via NO production at rest, as well as via NO production in response to neural activity. Blocking NO production elicits feeding even when food is not present, indicating that background NO production tonically inhibits feeding. On a cellular level, NO is produced in the absence of spiking by neurons C-PR and B31/B32, which respectively control appetitive and consummatory feeding behaviors. NO production contributes to these neurons’ resting potentials. Feeding causes an increase in hemolymph concentration of the NO precursor L-arginine, and this increase is a post-feeding inhibitory signal, presumably acting via increased background NO production. Neural activity producing NO occurs in response to attempts to swallow food. NO then acts to maintain arousal, as well as biasing the motor system to reject and reposition food that resists swallowing. If mechanically resistant food is not successfully swallowed, NO mediates the formation and expression of memory that food is inedible. NO also is released in response to other stimuli that inhibit feeding, such as tissue damage and nociception. NO also signals aspects of egg-laying, which is associated with feeding inhibition. The different functions of NO may reflect the evolution of NO signaling from a response to tissue damage that was then elaborated and used for additional functions that produce effects similar to those caused by damage. NO signaling in Aplysia feeding has 6 novel features: 1) NO is produced by non-active neurons; 2) background NO production contributes to the resting potential of neurons; 3) elicited and background NO release have parallel effects on behavior; 4) A circulating factor presumed to affect background NO production regulates spatially separated neurons controlling different aspects of a behavior; 5) L-arginine is a regulator of neural activity; 6) L-arginine is a post-ingestion inhibitor of feeding. Since initially novel features of neural regulation in Aplysia are often subsequently found in other animals, it is likely that these novel features of NO signaling in Aplysia will be recognized as general features controlling animal behavior.

Transmitters and Signaling LYNNE A. FIEBER

The involvement of D-aspartate receptors in aging in Aplysia californica LYNNE A. FIEBER, Stephen L. Carlson and Andrew T. Kempsell University of Miami - RSMAS – Marine Biology and Fisheries

D-Aspartate (D-Asp) activates a non-specific cation current in sensory neurons of Aplysia californica. Results with L-glutamate (L-Glu) receptor blockers indicate that D-Asp and L-Glu have some overlap in the receptors they activate. Half of sensory neurons exposed to both agonists one at a time, however, had only D-Asp- or L-Glu-induced currents, not both. This observation suggests that these 2 agonists can act at different sites. Ion selectivity and pharmacological results advocate for a D-Asp channel that is not uniformly characteristic of any known agonist associated channel type, but that has certain characteristics in common with Aplysia NMDA-like and AMPA-like receptors. D-Asp electrophysiological recordings were made from primary cultures of the pleural ganglion (PVC) and buccal ganglion S cluster (BSC) neurons in 3 egg cohorts at sexual maturity and senescence as a potential correlate of aging. The frequency of D-Asp-induced currents and their density were significantly decreased in senescent PVC cells but not in senescent BSC cells. These changes in sensory neurons of the skin and tail predict functional deficits that may contribute to an overall decline in reflexive movement in aged Aplysia.

Learning (Systems) PAUL R. BENJAMIN

Distinct roles for tonic inhibition and presynaptic facilitation in associative reward conditioning in Lymnaea PAUL R. BENJAMIN School of Life Sciences, University of Sussex, Brighton, Sussex, BN1 9QG, UK.

Although the study of modifications of excitatory synaptic connections has been a major emphasis in studies of molluscan learning, it is also clear that inhibitory processes have an important role as well. The feeding system of Lymnaea is modulated by tonic inhibitory synaptic inputs that suppress feeding during quiescence. This inhibitory input arises from a CPG interneuron, N3t, that suppresses the CPG during quiescence but fires phasically during feeding as part of the CPG [1]. We have shown recently that this tonic inhibition is reduced by one-trial chemical conditioning leading to a reduced threshold for feeding activation by the CS [2]. We were interested in relating the temporal pattern of this threshold-controlling mechanism to phases of memory. We show that the reduced inhibition is restricted to Intermediate-term through to Long-term memory (1-4 hours) but is absent during the earlier Short-term memory time point at 10 minutes after conditioning. Presynaptic facilitation of the CBI command neurons by the modulatory CGCs (equivalent the metacerbral giant cells of Aplysia) for feeding was observed only from 16 to 24 hours after conditioning [3] and is thought to play a role in the maintenance of Long-term memory.

[1] Staras, K., Benjamin, P.R. and Kemenes, G. (2003) Curr. Biol. 13,116-124. [2] Marra, V., Kemenes, I, Vavoulis, D., Feng, J-F, O’Shea, M. and Benjamin, P.R. (2010) Fron. Behav. Neurosci. 4: doi: 10.3389/fnbeh.2010.00161. [3] Kemenes, I., Straub, V.A., Nikitin, E.S., Staras, K., O’Shea, M., Kemenes, G. and Benjamin, P.R. (2006) Curr. Biol. 16, 1269-1279.

Learning (Systems) RICCARDO MOZZACHIODI

Neurophysiological effects of aversive stimuli in Aplysia: plasticity beyond defensive neural circuits RICCARDO MOZZACHIODI Department of Life Sciences, Texas A&M University - Corpus Christi, TX Although the mechanisms by which aversive stimuli produce adaptive changes in defensive neural circuits have been explored, their effects on non-defensive behaviors remain largely unknown. We examined the consequences of noxious stimuli, which induce enhancement of defensive responses in Aplysia (i.e., sensitization), on a non-defensive behavior (feeding) and its underlying neural circuit. Behavioral training, consisting of one or multiple trials of noxious electrical stimuli, produced concurrent sensitization and suppression of feeding. To characterize the cellular mechanisms responsible for the changes in feeding observed in vivo, we used a training protocol, which induces both long-term (24 h) sensitization (LTS) and long-term suppression of feeding. We examined the role of B51, a decision-making neuron in the feeding neural circuit, which is critical for the expression of the feeding response. B51 exhibits a sustained burst (i.e., plateau potential), which correlates with ingestive motor activity. B51 threshold to elicit a plateau potential was greater in trained animals, as compared to untrained (control) animals, and was not accompanied by any change in resting properties and synaptic input to B51. These findings suggest that LTS training reduced B51 excitability in a manner that is consistent with the suppression of feeding. When feeding and B51 excitability were examined at a time point in with LTS is no longer observed (i.e., 72 h post treatment), no differences were measured between trained and untrained animals, strengthening the role of B51 as a locus of plasticity underlying the suppression of feeding. We also began to explore possible biochemical pathways responsible for the long-term suppression of feeding induced by LTS training. Because serotonin (5-HT) mediates sensitization in Aplysia, we tested the hypothesis that it could also play a role in the suppression of feeding. Bath application of 500 µM 5-HT for 1.5 h induced LTS, but did not alter feeding or B51 excitability 24 h after treatment, thus suggesting that distinct biochemical pathways mediate LTS and the long-term suppression of feeding. In summary, the above findings indicate that aversive stimuli have widespread neurophysiological effects in Aplysia, altering not only defensive circuits, but also those controlling non-defensive response. Support: NIH-EARDA 5G11HD046353-05; NSF-IOS 1120304; Texas Research Development Funds.

Learning (Systems) ROMUALD NARGEOT

Role of electrical coupling and neuronal excitability in learning-induced, compulsive-like motor-pattern genesis in Aplysia ROMUALD NARGEOT1, Fred H Sieling2, Alexis Bédécarrats1 & John Simmers1

1 Univ. Bordeaux, INCIA, CNRS UMR 5287, Bordeaux F-33000, France 2 Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA

Appetitive operant conditioning in Aplysia’s feeding behavior switches the underlying buccal motor patterns from erratic and variable expression to accelerated and stereotyped recurrences. This motor plasticity was previously found to be correlated with an increase in the excitability of, and electrical coupling between, the three anticipatory interneurons B63, B65, B30 of the buccal central pattern generator. In the present study, we implemented a dynamic clamp procedure to artificially change the excitability and electrical coupling among these identified interneurons and explore the active role that these cellular changes might play in the learning process.

In isolated buccal ganglia from naïve animals that spontaneously generated

irregular and infrequent motor patterns, an artificial decrease in the leak conductance of the three interneurons immediately accelerated motor pattern genesis without changing their irregular expression. In contrast, an artificial increase in the coupling conductance between the three cells immediately switched network activity from irregular to regular burst patterns, but without changing the frequency of pattern genesis. However, a coincident decrease in leak conductance and an increase in coupling conductance switched erratic motor pattern genesis to an accelerated and regularized expression as observed after operant learning. Conversely, in isolated preparations from previously trained animals, accelerated and stereotyped motor pattern production can be converted into the erratic motor output observed in naïve preparations by increasing the leak conductance in the anticipatory interneurons and decreasing their coupling conductance. These data therefore indicate a causal relationship between the strength of electrical coupling and the temporal regularity of buccal motor pattern genesis, while a separate mechanism implicating a change in membrane leak conductance underlies the learning-induced changes in pattern cycle frequency.

Learning (Cellular and Molecular) LISA C. LYONS

Mechanisms underlying intermediate-term operant memory in Aplysia and its regulation by the circadian clock LISA C. LYONS, Maximilian Michel, Charity L. Green, Jacob S. Gardner and Chelsea L. Organ Department of Biological Science, Program in Neuroscience, Florida State University

Aplysia californica represents an ideal system for investigating the mechanisms underlying memory formation and the factors that modulate those mechanisms. Using an operant learning paradigm that takes advantage of the extensive neural plasticity associated with feeding behaviors, we investigated the mechanisms critical to intermediate-term memory (ITM) and the modulation of ITM by the circadian clock. In the learning that food is inedible paradigm (LFI), the animal associates a specific seaweed with the failure to swallow, generating short (30 min), intermediate (4-6 h) and long-term (24 h) memory. Through in vivo experiments, we determined that the formation of ITM required multiple signaling cascades including protein kinase A, protein kinase C and MAPK activity. Moreover, the maintenance of ITM required PKA, the persistently active form of PKC (PKM Apl III) and MAPK. Intermediate-term LFI memory is strongly phase restricted by the circadian clock. ITM is evident only when animals are trained in the early hours of the (subjective) day with training during the late day or night resulting in no memory. As multiple kinase pathways are necessary for the induction and maintenance of ITM, we investigated whether inhibition of protein phosphatase activity affected circadian regulation of memory. We found that inhibition of protein phosphatase 1 or 2A with okadaic acid or calyculin blocked ITM during the early subjective day at a time when memory is normally formed. However, inhibition of protein phosphatase 1 or 2A induced phase specific memory rescue when training occurred late in the subjective day or early evening. In contrast, inhibition of protein phosphatase 2B with FK506 did not affect ITM during the early day. Interestingly, administration of FK506 prior to training permitted ITM at all time points including the late subjective day and time points throughout the night. These results demonstrate that levels of protein phosphatase activity are critical regulators of memory formation and identify kinase and phosphatase activity as mechanisms through which the circadian clock regulates ITM. This research was supported by NIMH Grant MH081012 to L.C.L.

Learning (Cellular and Molecular) PATRICK McCAMPHILL

Decoding neuronal plasticity in Aplysia: eEF2 as an integrator coupling activity patterns to translational control PATRICK McCAMPHILL, Sanjida Hoque, and Wayne Sossin McGill University, Montreal, Quebec, Canada

One proposed mechanism for modulating synaptic strength during memory formation is by rapidly increasing the levels of specific proteins through the control of their translation. At the sensory-motor neuron synapse of Aplysia a serotonin (5-HT) induced intermediate term facilitation (ITF) exists that requires new protein synthesis but not gene transcription. Two alternative intracellular pathways can be activated during ITF. Synapses activated by spaced 5-HT training generate ITF dependent on persistent protein kinase A (PKA), whereas massed training generates persistent protein kinase C (PKC). Recent work from our lab has shown that a spaced pattern of 5-HT training leads to a greater desensitization of PKC activation than does massed 5-HT and the difference depends on the differential production of proteins by the various stimuli (Farah et al., 2009). We hypothesize that the divergent upstream signaling seen in spaced vs. massed stimulation may activate different translational mechanisms. In this study we show that spaced and massed 5-HT training induces distinct translational activation in the cytoplasm of cultured mechano-sensory neurons from the pleural ganglia. We demonstrate that phosphorylation of eukaryotic elongation factor 2 (eEF2), a modification known to inhibit translational elongation, is increased by massed training and conversely decreased by spaced training. Consistent with regulation of eEF2 as a downstream effector for differentiating spaced and massed applications of 5HT, the increase in eEF2 phosphorylation observed after massed 5-HT was blocked with inhibitors of PKC and the decrease in eEF2 phosphorylation observed after spaced 5-HT was blocked by inhibitors of PKA. Surprisingly, rapamycin blocked both the increase and decrease in eEF2 phosphorylation, although previous results showed that 90 min of massed 5-HT induced a rapamycin-insensitive ITF (Yanow et al., 1998). Intracellular recordings of ITF induced by a massed 25 min 5-HT protocol at cultured sensory-motor neuron synapses reveal that this temporal domain of facilitation is partially rapamycin sensitive. We propose that specific translational regulators are differentially induced by spaced and massed stimulation and that eEF2 acts as a control point integrating diverse activity patterns.

Learning (Cellular and Molecular) JOHN BYRNE

Computational Design of Enhanced Learning Protocols JOHN BYRNE , Yili Zhang, Rong-Yu Liu, George Heberton, Paul Smolen, Douglas Baxter, Leonard Cleary

Department of Neurobiology and Anatomy, The University of Texas Medical School at Houston

Learning and memory are influenced by the temporal pattern of training stimuli. The mechanisms that determine the effectiveness of a particular training protocol are not well understood, however. The hypothesis that the efficacy of a protocol is determined, in part, by interactions among biochemical cascades that underlie learning and memory was examined. Previous studies suggest that the PKA and ERK cascades are necessary to induce long-term synaptic facilitation (LTF) in Aplysia, a neuronal correlate of long-term memory (LTM). A computational model of the PKA and ERK cascades was developed, and used to identify a training protocol that maximized PKA/ERK interactions. The predicted protocol had non-uniform interstimulus intervals (ISIs), which is in marked contrast to the fixed intervals that are generally used in experimental psychology and in previous studies of LTF and LTM in Aplysia. Four lines of empirical evidence support the model predictions. First, the novel training protocol, referred to as the Enhanced protocol (5, 5-min duration pulses of 5-HT with ISIs of 10, 10, 5 and 30 min), induced LTF in sensorimotor cultures, which was significantly greater and longer lasting than that produced by a previously well-established protocol, referred to as the Standard protocol (5, 5-min duration pulses of 5-HT with an ISI of 20 min). Second, in isolated sensory neurons, the Enhanced protocol significantly increased long-term excitability (LTE), a second correlate of LTM. Third, in isolated sensory neurons, the Enhanced protocol significantly increased the levels of phosphorylation of CREB1, a transcription factor essential for LTF, as compared to the Standard protocol. Finally, the Enhanced training protocol significantly improved LTM following behavioral training.

In the fields of neuroscience and experimental psychology, multiple learning trials spaced over time generally produce LTM more effectively than a single trial or multiple trials massed together. However, virtually all of the learning protocols and their neuronal analogues used in animal and human studies have been developed on an ad hoc basis. The optimal procedure or spacing of trials is not predicted by any learning theory. The present study demonstrates the feasibility of using computational models of biochemical signaling to design enhanced training protocols that increase LTF, LTE, transcriptional activation, and LTM.

Learning (Cellular and Molecular) STEFAN R. KASSABOV

A Differentially Spliced Neurotrophin and Cognate Trk Receptor Mediate Synaptic Plasticity in Aplysia STEFAN R. KASSABOV1, Yun-Beom Choi 1, Kevin Karl 1, Harshad D Vishwasrao1,

Craig H. Bailey1,3 and Eric R. Kandel1,2,3,#. 1 Department of Neuroscience, 2 Howard Hughes Medical Institute, 3Kavli Institute for Brain Science, College of Physicians and Surgeons of Columbia University, New York State Psychiatric Institute, 1051 Riverside Drive, New York, NY 10032

Neurotrophins and their tropomyosin-related (Trk) receptors play fundamental roles in the development and activity-dependent synaptic modulation of the vertebrate nervous system but whether neurotrophin signaling exists and functions similarly in invertebrates has remained unanswered. Here we report the discovery of a Trk receptor (ApTrk) and its cognate neurotrophin ligand (ApNT), expressed in the central nervous system of Aplysia, which share a high degree of sequence, structural and functional homology with their vertebrate counterparts. ApNT –ApTrk signaling mediates serotonin dependent long term facilitation in sensory-motor synapses, and is sufficient for induction of structural synaptic growth and long lasting synaptic enhancement. Unlike vertebrate neurotrophins, which are encoded by a single exon, the single ApNT gene has alternatively spliced coding exons giving rise to splice variants that are differentially processed and secreted as mature and pro-forms, which exert distinct enhancing effects on synaptic function. Our findings demonstrate that neurotrophic signaling has ancient evolutionary origins, predating the split between protostomes and deuterostomes with control of synaptic growth and plasticity being one of its core, ancestral functions. Our finding of a mechanism aimed specifically at the generation of mature and pro-neurotrophins in Aplysia, underscores the physiological significance and independent biological roles of both neurotrophin forms.

Circuits ANDREW DACKS

Maintaining functional articulation of network activity ANDREW DACKS Mount Sinai School of Medicine, New York, NY, USA Behavioral output is a product of the integration of sensory information and the current internal state, which is biased by factors such as previous experience with a given stimulus. At the level of the nervous system, this internal state causes the alteration of the response properties and interactions between neurons in specific neural circuits resulting in the establishment of a “network state”. Despite the dramatic changes imposed by the internal state, the nervous system still produces functionally articulated, multi-phasic output. The maintenance of functional articulation can be implemented by regulating the excitability of neurons active only during discreet phases of network activity, however modulating excitability cannot maintain phase specification of neurons that are active across multiple phases. Therefore a different mechanism must be implemented by the network. Using the feeding CPG of Aplysia we examined the cellular mechanisms that implement the functional articulation of neural output during the dynamical process underlying the establishment of the network state. Over the course of repeated biting motor programs the activity of the multi-phasic motor neurons B8 progressively increases in only one phase, a form of network dynamics called repetition priming. We found that while priming increases B8 excitability, resulting in the increase in B8 activity, a decrease in inhibitory drive from the inhibitory neurons B4/5 also occurs, allowing the expression of the increased B8 activity in only one phase. Depolarizing B4/5 during motor programs post-priming decreases B8 firing rate to near pre-priming levels and hyperpolarizing B4/5 causes B8 activity to reach post-priming levels at a faster rate. By regulating B8 activity in only one phase, the feeding CPG can modulate neuronal excitability as a means to alter motor output, yet retain control over the expression of the changes in B8 intrinsic biophysical properties via B4/5.

Circuits VLADIMIR BREZINA

The connectome is not enough: evidence from model invertebrate nervous systems VLADIMIR BREZINA Department of Neuroscience and the Friedman Brain Institute, Mount Sinai School of Medicine, New York, NY

Great efforts are currently being exerted to map the wiring diagram, or “connectome,” of the vertebrate nervous system. The ultimate aim is functional. It is assumed that the structural connectome is the major determinant of the functional output, so that instantiating the connectome in a computer model will recreate the brain with all of its sensory processing, behavior generating, and cognitive capabilities. How realistic is this hope?

The experience in invertebrates is instructive. Many invertebrate nervous systems,

with their experimental tractability and small numbers of neurons, have been studied for decades. Their connectomes have been mapped, with a completeness as yet unmatched in vertebrates, down to the level of the individual neurons and their synaptic connections. The complete connectome is available for one entire invertebrate nervous system, that of Caenorhabditis elegans.

Yet these invertebrate nervous systems, whose connectomes are the closest to

being “solved”, also offer the best understanding of processes that make the connectome a very incomplete determinant of nervous system function. In this talk I will highlight some of these processes.

Even if functional, rather than structural, connections are mapped, connectomes

are essentially static snapshots. But the nervous system is highly dynamic even in its connectivity. Synaptic plasticity can dramatically alter the sign, strength, even the very existence of functional connections, thus reconfiguring the connectivity entirely. This can happen quickly, so that the network may be reassembled with a different connectivity for each repetition of a motor act. Another complication for the concept of a well-defined wiring diagram is the ubiquitous presence of neuromodulators, released in various combinations by the network’s own activity, whose spatiotemporal profiles then establish channels of communication, store information, and even perform computations that are at least partially independent of the neuronal wiring. Finally, even supposedly stereotyped networks can operate with widely different connectivity patterns: thus, a unique wiring diagram may neither exist nor be necessary for function.

Altogether, these processes create a substantial explanatory gap between the

connectome and function. The nervous system is likely to prove far more variable, self-reconfiguring, and dynamic than can be captured by a static wiring diagram.

Circuits WILLIAM FROST

WILLIAM FROST Department of Cell Biology and Anatomy, Rosalind Franklin University, The Chicago Medical School, North Chicago, IL, USA Use of fast voltage sensitive dye imaging to study molluscan networks

Progress in understanding the neural basis of behavior requires a detailed knowledge of the underlying neural networks. A powerful and rapidly improving tool for deciphering neural networks is large-scale imaging with fast voltage sensitive dyes. While the hardware for imaging action potentials from dozens of neurons simultaneously was developed twenty years ago, laboratories have been slow to adopt this technology, in part due to a lack of suitable tools for processing the acquired data.

This talk will demonstrate our ability to process the information in raw imaging

files into single neuron traces, and then to organize these into statistically significant neuronal ensembles, all during the time course of a live experiment. Our approach uses a three step procedure, which we are currently applying to rhythmic motor networks in the marine mollusks Aplysia and Tritonia. In the first step, the isolated brain preparation is stained with the fast voltage sensitive absorbance dye RH155, and nerve-evoked rhythmic motor programs are imaged using a 464-element photodiode array. Because many diodes record multiple neurons, and many neurons are recorded by multiple diodes, it is difficult to decode the underlying networks by inspecting the raw data alone. In the second step, we solve this problem by using independent component analysis to process the raw data into single neuron action potential traces, a fully automated method that takes just a few minutes per data file. This procedure also returns maps of the ganglion locations of all resolved neurons. By concatenating files collected at different times during a single preparation, the firing behavior of ~ 100 individual neurons can be followed across separate acquisition files spanning different experimental treatments. In the third step, we apply statistical correlation methods to reveal the significantly related ensembles of neurons present in the recorded population.

This combined three step approach represents a powerful new tool for studies of

the neural basis of behavior. The talk will demonstrate its use to: 1) locate new neurons of interest; 2) characterize and map the different neuronal ensembles comprising a network; 3) identify and study neurons with multifunctional roles in behavior; 4) track neuronal and network changes over time with modulation or learning.

Circuits PAUL S. KATZ

There’s more than one way to swim a slug PAUL S. KATZ, Joshua L. Lillvis, Charuni A. Gunaratne, Arianna Tamvacakis, and Akira Sakurai Neuroscience Institute, Georgia State University

In Gastropods, homologous neurons can be identified across species. Yet animals exhibit a diversity of behaviors. Here we explore the roles of homologous neurons in swimming behaviors of the Nudipleura (Opisthobranchia). Of more than 3000 species, only about 60 have been reported to swim. Of those, about 40 species swim with rhythmic left-right (LR) body flexions, including Melibe leonina and Dendronotus iris. Some of the remaining species swim with dorsal-ventral (DV) body flexions, including Tritonia diomedea and Pleurobranchaea californica.

Homologues of interneurons in the Tritonia DV swim central pattern generator

(CPG) have been identified in a diversity of species regardless of the behavior. In Pleurobranchaea, homologues of the Tritonia DSI and C2 neurons are also part of the DV swim CPG (Jing & Gillette, J. Neurophysiol. 1999). In contrast, these neurons are not members of the LR swim CPG in Melibe or Dendronotus.

In Tritonia, serotonergic neuromodulation is necessary for DV swimming. We

found that it is also present in Pleurobranchaea and most prevalent in individual Pleurobranchaea that swim longer bouts. The serotonin receptor antagonist methysergide reduced the number of flexion cycles. In contrast, the neuromodulatory action is absent from the LR swimmer, Hermissenda crassicornis.

It was previously shown that in Melibe the LR swim CPG contains two bilaterally

symmetric neurons, Si1 and Si2. We have identified a third swim CPG member, Si3. We have also identified homologous neurons in Dendronotus, but found that their synaptic connections are fundamentally different. Furthermore, in Dendronotus, Si1 does not function as a member of the LR swim CPG but initiates and accelerates rhythmic activity. The functional interactions of Si3 also differ between the species. Thus, despite producing similar two-phase motor outputs and having homologous neurons, the swim CPGs in Melibe and Dendronotus differed substantially in network architecture and internal dynamics.

In conclusion, DV swimming and LR swimming are produced by completely

different sets of neurons. Analogous DV swimming behaviors in Tritonia and Pleurobranchaea involve not only the same neurons, but also the same neuromodulatory actions. In contrast, LR swimming in Melibe and Dendronotus involves homologous neurons organized with different circuit architectures. Supported by NSF- IOS-1120950, 0814411

Development GAYNOR E. SPENCER

A novel non-chordate retinoic acid receptor (RAR) and its role in development and axon guidance GAYNOR E. SPENCER, Christopher J Carter, Christopher D Rand, Robert L Carlone

Retinoic acid (RA), the Vitamin A metabolite, is an important molecule during neural development and regeneration of the nervous system in chordates. There is limited evidence for a similar role of RA in some non-chordate species, in which some components of the retinoid signalling pathway have been identified. RA exerts many of its functional effects by binding to receptors that usually act as nuclear transcription factors to influence gene transcription. Retinoid X receptors (RXRs) have previously been shown to exist in non-chordates, but the retinoic acid receptors (RARs) were, until recently, considered a chordate innovation, shown only to exist in vertebrates. We have, however, now cloned the first full-length, non-chordate RAR (termed LymRAR) from the adult CNS of the protostome Lymnaea stagnalis. The LymRAR protein shares high amino acid similarity with known vertebrate RARs and is expressed during Lymnaea embryogenesis, as well as in the adult CNS.

In order to investigate the role of LymRAR in development, we treated Lymnaea

embryos with various RAR antagonists (or DMSO as a control) for 6 to 7 days. The RAR antagonist treatment resulted in shell malformations, eye defects and in some cases, even halted development. Immunoreactivity for LymRAR was detected in the late veliger/metamorphic embryo within the shell region and the lining of the pharynx/esophagus.

The LymRAR protein was also expressed in the adult CNS, as well as in

regenerating cultured motorneurons, where, interestingly, its expression pattern showed a non-nuclear distribution. LymRAR protein expression was also found in regenerating growth cones and we provide evidence that it plays a role in RA-mediated chemoattraction. We have previously shown that RA can induce neurite outgrowth and growth cone turning of cultured Lymnaea neurons. We report here that in the presence of a RAR pan-antagonist, LE540, there is a significant reduction in the RA-induced growth cone turning. Taken together, these findings show that RARs must have originated as early as bilaterians and suggest a role for LymRAR in molluscan development and growth cone guidance. Acknowledgements: NSERC (Canada) Discovery Grant to G.E. Spencer; NSERC CGS-D to C.J. Carter and Ontario Graduate Scholarship to C.D. Rand.

Development SAMUEL SCHACHER

Cell-Specific Expression and Activation of bZIP Transcription Factors Regulate the Development and Maintenance of Synapse Baseline SAMUEL SCHACHER, Jiang-Yuan Hu, Amir Levine, and Yang Chen Departments of Neuroscience and Psychiatry, Columbia University College of P & S and New York State Psychiatric Institute, New York, NY 10032 Activity-dependent long-term synaptic plasticity and its contributions to learning and memory are dependent on the initial baseline strength of the circuit’s synapses. Moreover, when plasticity wanes, synapse strength generally returns to the initial baseline. What is the molecular basis for the development and ‘memory’ of synapse baseline? To address this we used the Aplysia cell culture preparation consisting of a sensory neuron plated with motor neuron L7. Sensorimotor synapses with stable baselines develop by day 4 and can then undergo long-term facilitation or depression lasting days mediated by stimulus-induced changes in the expression and activation of bZIP transcriptions factors CREB1 and CREB2. We therefore examined how cell-specific alterations in the expression and activity of bZIP factors at different time points might impact synapse baseline. We found both temporal- and cell-specific effects of these factors. Overexpressing wild type CREB1 or cJun in sensory neurons or L7 on day 1 produced an increase in baseline, while overexpressing a dominant negative form of CREB1 reduced baseline. In contrast, overexpressing CREB1 or the dominant-negative form on day 4 failed to alter synapse strength. Overexpressing CREB2 in sensory neuron or L7 on day 1 produced a transient decline in synapse strength that was correlated with protein degradation of the GFP-tagged construct. Overexpression of a mutant form of CREB2 that cannot be ubiquitinated blocked both CREB2 protein degradation and its capacity to reduce baseline. Thus reducing synapse baseline by CREB2 may require its modification first by phosphorylation and then mono-ubiquitination. On day 4, overexpression of CREB2 failed to materialize in L7 because of rapid protein degradation, while CREB2 overexpression in sensory neurons no longer altered baseline. When CREB2 and cJun were overexpressed simultaneously, cell specific changes were detected at each time point. Simultaneous overexpression of both factors in L7 significantly increased synapse baseline despite persistent CREB2 expression. In contrast, simultaneous overexpression of both factors in sensory neurons significantly reduced synapse baseline. Thus tight control of expression and activation of bZIP factors during both synapse formation and maintenance in presynaptic and postsynaptic neurons regulates the baseline strength of sensorimotor synapses.

Development ZHONG-PING FENG

Snail genes enhance axonal outgrowth of rodent central neurons ZHONG-PING FENG Department of Physiology, University of Toronto, Toronto, Ontario, Canada

Adult central neurons in most invertebrates can regenerate and form synaptic connects following axonal injury. Thus, identifying molecules that enable this regeneration may suggest new directions to stimulate axonal growth in the CNS in vertebrates, which typically do not regenerate. Freshwater pond snail, Lymnaea stagnalis, has served as a successful model for morphological and functional studies in axonal regeneration. However, the lack of adequate transcriptome information of the snail limits its use in functional and comparative molecular studies. To overcome this limitation, we have conducted a partial neuronal transcriptome sequencing analysis and deduced 7,712 distinct EST sequences. We further created the first gene chips covering ~15,000 of L. stagnalis EST sequences and our microarray analysis showed 67 sequences with more than 2-fold changes following in vivo nerve injury. In this talk, I will discuss the newly identified novel pro-regenerative molecules in snail and our first evidence that the snail molecule promotes the outgrowth capacity of rodent axons. Our findings provide a potential alternative approach to identify new molecular mechanisms promoting intrinsic regenerative properties of mammalian neurons.

Development KELSEY C. MARTIN

Synapse-specific translational regulation KELSEY C. MARTIN, Sang Mok Kim, Elliott Meer Department of Biological Chemistry, University of California, Los Angeles mRNA localization and regulated translation provides a means of spatially restricting gene expression to individual synapses within a given neuron. We have developed methods to dynamically visualize RNA trafficking and local translation in cultured Aplysia sensory-motor neurons and have monitored these processes during synapse formation and synaptic plasticity. Using culture preparations in which sensory neurons are paired with target and nontarget motor neurons, such that they form glutamatergic synapses with the target and fasciculate with, but do not form chemical synapses with, the nontarget motor neuron, we show that localized RNAs are trafficked to both target and nontarget sensory-motor connections, but that the RNA is only translated at synapses. These results indicate that the activity-dependent spatial regulation of gene expression in neurons is mediated at the level of translation, and not via stimulus-induced mRNA trafficking. We show that stimulus-induced translation requires a calcium-dependent trans-synaptic signals from the motor neuron to the sensory neuron. Our recent work indicates that gene expression is spatially regulated primarily at the level of translational regulation rather than mRNA localization, and implicate a role for netrin-DCC interactions in translational regulation at synapses.

Posters ZHONG-PING FENG

A sodium leak channel regulating rhythmic activity of respiratory pacemaker neuron in Lymnaea stagnalis

ZHONG-PING FENG, T.Z. Lu

Department of Physiology, Faculty of Medicine, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada, M5S 1A8.

NALCN (Na+ leak current, nonselective) is a newly described nonselective cation channel. It conducts Na+ current at rest, and potentially contributes toward neuronal excitability and synaptic transmission. Targeted deletion of NALCN gene in mice resulted in a fatal postnatal phenotype characterized by an abnormal respiratory rhythmic activity (Lu B et al., Cell, 2007, 129:371). However, whether the NALCN channels is involved in pacemaker neuron activity has not been addressed. An NALCN orthologue, U-type channel (Spafford JD et al., J Biol Chem, 2003, 278: 4258) was reported from L. stagnalis, a freshwater pond snail. L. stagnalis is a bimodal breather: its respiratory activity is controlled by a well-described CPG network, including a pacemaker neurons, RPeD1. In this study, we determined the role of U-type channels in the rhythmic activity of RPeD1 neuron in adult L. stagnalis using RNAi gene silencing approach in combination of electrophysiology. Aerial respiratory behavior was compared between the control and the U-type channel suppressed snails. Our whole-cell voltage-clamp recordings showed U-type channel RNAi treated RPeD1 cells exhibited a reduced inward hyperpolarizing cation current component. As compared with the control RPeD1, a Na+ conductance recorded in the cells with U-type dsRNA/siRNA treatments was abolished, accompanied with a reduction in rhythmic firing activity, and a more hyperpolarized resting membrane potential. Respiratory behavioral study showed a reduction of basal aerial respiratory activity following partial U-type gene knockdown, which was confirmed by real-time PCR analysis. Taken together, our results provided the first evidence of the function expression of the NALCN-like channels in the respiratory CPG neurons of L. stagnalis and the involvement of the channel in the respiratory rhythmic activity via regulation of intrinsic membrane properties.

Posters CHARUNI A. GUNARATNE

Comparative mapping of GABA-immunoreactive neurons and identification of homologous neurons in Nudipleura Molluscs CHARUNI A. GUNARATNE* and P. S. Katz Neuroscience Institute, Georgia State University, Atlanta, GA

Comparative mapping of neurotransmitter systems in related species can provide important insights into the evolution of brain organization. In this study, we compared GABA-immunoreactive neurons in the central ganglia of four nudibranch mollusc species (Tritonia diomedea, Dendronotus iris, Melibe leonine and Hermissenda crassicornis). We also mapped the GABA-immunoreactivity in the buccal ganglia of the above species, an additional nudibranch (Tochuina tetraquerta) and a distantly related pleurobranchomorpha species (Pleurobranchaea californica). We found consistent patterns of GABA-immunoreactivity in the pedal and cerebral-pleural ganglia across species. In particular, there are bilateral clusters in the anterior and medial regions of the dorsal surface of the cerebral ganglia. However, we found inter-species differences in the number of neurons in each cluster. We also report the presence of a distinctive GABA-ir cell in the ventral pleural ganglia across species. The GABA-ir in the buccal ganglia of the Nudibranch molluscs in this study are similar across species except for Melibe, where we find considerably fewer GABA-ir neurons. This is consistent with the reduced nature of the Melibe buccal ganglia that corresponds to Melibe’s lack of a radula and buccal mass. These feeding structures are controlled by the buccal ganglia in other nudibranchs. The out-group Pleurobranchaea has significantly more buccal GABA-ir neurons and a different distribution pattern from the nudibranchs.

Comparative mapping of neurotransmitter phenotypes combined with anatomical characteristics can also be used to identify homologous neurons across related species. Identification of homologous neurons in different species is a first step towards understanding the neural basis for species-specific behaviors. We found that none of the identified neurons controlling rhythmic left-right (LR) and dorsal-ventral (DV) escape swimming behavior in the Nudipleura molluscs in this study are GABAergic. As such, GABA-ir cannot be used as a characteristic to identify homologues of these central pattern generator (CPG) neurons in other Nudipleura molluscs. However, we were able to use immunoreactivity for the peptide neurotransmitter FMRF-amide combined with anatomical features to identify homologues of the LR swim CPG neurons in Tritonia and Pleurobranchaea, species that swim with DV flexions. Support: NSF IOS-1120950

Posters STAVROS P. HADJISOLOMOU

Dynamics of chromatophore response to visual stimulation

STAVROS P. HADJISOLOMOU, Frank W. Grasso

BioMimetic and Cognitive Robotics Laboratory Psychology: Brain, Cognition and Behavior Sub-Program Brooklyn College of the City University of New York The Graduate Center of the City University of New York shadjisolomou@gc.cuny.edu

Coleoid cephalopods (octopus, squid, and cuttlefish) are able to change their external appearance in milliseconds for visual crypsis and communication. Color changing is the result of the combined action of intradermal chromatophores, iridophores, and leucophores. This behavior is driven by a sensorimotor system consisting of visual input from the eyes and large, sophisticated central nervous system for information processing and a muscular skin for display. Coleoids have well-developed eyes and acute vision, which provide the central nervous system (CNS) with input on spatial patterning, contrast, and luminance of the environment. Networks of the optic lobes, basal lobe, and chromatophore lobes “select” and modulate the most efficient body pattern for camouflage. While the anatomical arrangement of the neuro-muscular components and the sensory contributions of the visual system have been documented, the underlying computation of this sensorimotor control system is still unknown. We captured the spatial and temporal variations of chromatophore activity on a patch of cuttlefish skin in response to a transient input (brief, intense white light flash). This stimulus was adequate to trigger chromatophore responses. We observed consistent, variable spatial gradients of contrast across the mantle which were not present in pre-flash chromatophore activity. Some responses included a brief expansion of chromatophores with a short latency (130 milliseconds) after the flash, followed by immediate retraction lasting up to 4 seconds. The long duration of these responses may reflect the contributions of several central processes. This is consistent with a system that adapts to ambient light level and the interactions of the central state of the cuttlefish (e.g., fight or flight) with the chromatophore system. System identification techniques will allow us to explore the (first order) dynamics of brain function that control the chromatophore system response to light input.

Posters MARGARET H. HASTINGS

Investigating the mechanism of formation and downstream substrates of protein kinase Ms during memory in Aplysia MARGARET H. HASTINGS, Cherry Gao, Xiaotang Fan, Joanna K. Bougie, Danay Baker-Andresen and Wayne S. Sossin McGill University, St. Lambert, Quebec, Canada

Persistently active truncated forms of protein kinase C (PKC) known as PKMs play crucial roles in memory maintenance in both vertebrates and invertebrates. The mechanism by which PKMs maintain memory may be conserved, as in both systems PKMs play roles in the regulation of post-synaptic glutamate receptors. However, while rodent PKMzeta is transcribed from a promotor within the PKCzeta gene, the Aplysia homolog, PKM Apl III, is thought to be produced by calpain cleavage of the full-length PKC. This study further investigates events upstream and downstream of PKM formation in Aplysia. We have identified four calpains expressed in the Aplysia nervous system and have begun to test their ability to cleave Aplysia PKCs. We found that PKC Apl III was cleaved by mammalian calpain-1 in vitro, confirming previous reports, yet we did not observe PKC cleavage by the Aplysia classical calpain, despite the calpain’s apparent activation and autolysis. Future experiments will continue to test the abilities of this and other Aplysia calpains to cleave PKC in vitro and in neurons. Downstream of PKM, we investigated the possible role of Numb, a conserved adaptor protein involved in endocytosis and regulated by atypical PKCs, as an Aplysia PKM substrate. Using a phospho-specific antibody we have obtained the following evidence supporting Numb phosphorylation by PKM Apl III: (1) PKM Apl III phosphorylated Aplysia Numb in vitro, and this phosphorylation was blocked by the pseudosubstrate inhibitor ZIP. (2) Cultured Aplysia sensory neurons co-expressing Numb with PKM Apl III exhibited increased phospho-Numb immunoreactivity compared to neurons expressing Numb alone. This increase was not observed when a non-phosphorylatable Numb was co-expressed with PKM Apl III. This work will contribute to the understanding of PKM formation and its downstream targets in Aplysia.

Posters JIANG-YUAN HU

Development of Synapse Baseline: Cell-Specific Expression and Activation of CREB1 and CREB2 Regulates Synapse Strength JIANG-YUAN HU, Amir Levine, Yang Chen and Samuel Schacher Departments of Neuroscience and Psychiatry, Columbia University College of P & S and New York State Psychiatric Institute, New York, NY 10032

During development, a number of factors including activity-dependent and Hebbian-like mechanisms regulate the final strength of synapses (stable synapse baseline). The timely activity is likely to affect the expression or activation of bZIP transcription factors and regulate synapse baseline. In Aplysia, sensorimotor synapses undergo long-term facilitation or depression lasting days that are accompanied by the growth or elimination of synaptic connections that are mediated by stimulus-induced changes in the expression and activation of bZIP transcriptions factors CREB1 and CREB2. We used the Aplysia cell culture preparation consisting of a sensory neuron and a motor neuron L7 to examine whether these bZIP factors also regulate the development of sensorimotor synapses. Since stable synapse baselines are established by 4 days, we examined how synapse baseline was affected by early cell-specific manipulations of bZIP factor expression or activation (injections of oligonucleotides or GFP-tagged constructs). Injection of CRE oligonucleotides (dominant negative regulator of bZIP factor binding at DNA sites) produced opposite consequences. Injection into sensory neurons resulted in a decline in synapse baseline, while injection into L7 produced an increase in synapse baseline. In sensory neurons, the actions of CRE appear to be mediated primarily by blocking CREB1 binding. Overexpressing wild type CREB1 produced an increase in baseline, while overexpressing a dominant negative form of CREB1 reduced the baseline. Overexpressing wild type CREB2 produced a transient decline in synapse baseline that was correlated with protein degradation of the GFP-tagged construct. The actions of the CRE blockade of bZIP factor binding in L7 appear to be more complex. CREB1 overexpression and activation in L7 produced the same increase in synapse baseline, while CREB2 overexpression produced the same transient decline in synapse baseline. The simultaneous overexpression of bZIP factors can mimic the consequences of CRE injections. Synapse baseline decreased when CREB2 and cJun were overexpressed simultaneously in sensory neurons, but increased when they were overexpressed in L7. Thus tight control of expression and activation of CREB1 and CREB2 in both neurons and their potential interactions with other bZIP factors regulate the strength of the developing sensorimotor synapse.

Posters JIAN JING

Functional differentiation of a population of electrically-coupled heterogeneous elements in a microcircuit JIAN JING, Kosai Sasaki, Klaudiusz R Weiss Department of Neuroscience, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029

Electrical coupling is present in many microcircuits, including mammalian cortical circuits. Although there is a general consensus that electrical coupling promotes synchronous firing of coupled neurons, it is not well understood how the strength of electrical coupling, particularly, within a population of heterogeneous elements, relates to specific network functions of these neurons. In the Aplysia californica feeding motor network, five circuit elements, B64, B4/5, B70, B51 and a newly identified interneuron B71 are electrically coupled. Here we study how the strength of electrical coupling between these five neurons relates to their different activity patterns and network functions. These 5 neurons exhibit distinct activity patterns during the radula retraction phase of feeding motor programs. In a subset of motor programs, retraction can be flexibly extended by adding a phase of network activity, named hyper-retraction. This is manifested most prominently as a burst of firing of the radula closure motoneuron B8. Two excitatory neurons B51 and B71, and one inhibitory neuron B70 of B8 are active during hyper-retraction, and may potentially control B8 activity. Consistent with their near synchronous firing, B51 and B71 showed one of the strongest coupling ratios in this group of neurons. However, by manipulating their activity, we show that, during hyper-retraction, B51 preferentially acted as a driver of activity in B64 and B71, whereas B71 played a larger role in driving B8 activity. In contrast, B70 was weakly coupled to other neurons and its inhibitory action onto B8 counter-acted the excitatory drive that B8 receives from B71. Finally, the distinct firing patterns of the electrically coupled neurons were fine-tuned by the largely chemical cross-inhibition between them. Thus, network activity is controlled by a group of heterogeneous neurons, whose activity patterns are in turn determined by both electrical and chemical connections. More generally, the small microcircuit of Aplysia feeding network enables us to understand how a population of electrically-coupled heterogeneous neurons may fulfill specific network functions.

Posters ANDREW T. KEMPSELL

Behavioral deficits accompanying aging in Aplysia californica ANDREW T. KEMPSELL and Lynne A. Fieber University of Miami - RSMAS – Marine Biology and Fisheries

The California sea hare, Aplysia californica, provides an excellent model for studies of nervous system aging with a relatively short and easily manipulated lifespan, well-mapped neural networks, a set of previously studied behaviors, and a recently completed genome sequence. Tail withdrawal following mechanical stimulation is a well-documented behavior in Aplysia with a known neural circuit, including primary mechanosensory neurons of the pleural ganglia, making the tail withdrawal reflex (TWR) optimal for correlating behavioral responses with individual neuronal output as a function of age. Habituation in the TWR, a form of non-associative learning, is marked by a decrease in the amplitude of tail withdrawal upon repeated tactile stimulation to the tail. The TWR and habituation in the TWR were monitored in freely behaving animals from sexual maturity to senescence. The tail remained withdrawn for significantly longer periods of time in senescent animals. Furthermore, whereas animals at sexual maturity were capable of habituating to tactile stimulation of the tail by 10 trials, measured as a significant decrease in time to recover from tail touch, senescent animals did not significantly habituate before 15 trials. The biting response was also measured in freely behaving Aplysia as a function of age. Biting amplitude decreased significantly in senescent animals while biting latency significantly increased. Such changes in reflexive movement of the tail and buccal mass may correspond to age-related deficits in nervous system functioning at the physiological level. Also, changes in habituation of the TWR in senescent animals may indicate alterations in learning and memory that are evident at the cellular level, for example changes in synaptic facilitation of sensory neurons innervating the tail.

Posters KOMOLITDIN AKHMEDOVETAL

Electrophysiological analysis of aging of Aplysia neuron R15 KOMOLITDIN AKHMEDOVETAL, Rizzo V., Kadakkuzha B., *Puthanveettil SV Department of Neuroscience, The Scripps Research Institute, Jupiter, FL33458 *Correspondence: SPuthanv@scripps.edu Understanding age-dependent physiological and molecular changes in neuronal connectivity in the brain is a major challenge in basic neuroscience and neurological medicine. Despite several elegant studies to understand aging, the physiological and genomic correlates of aging are poorly understood at single neuron or circuit level. The long-term goal of our research is to determine the molecular basis of age-dependent changes in individual neurons and neural circuits in addressing two specific questions concerning aging in the nervous system: 1) How does aging modify the function of neural circuits? And 2) what molecular pathways are substrates of age-related changes? To address these questions, we explored the identified neuron R15 of marine mollusk Aplysia Californica. R15 is a bursting neuron (Adam et al., 1985) and is involved in regulating locomotion (Romanova, et al., 2007), water balance (Skinner and Peretz, 1989) and egg laying (Alevizos et al., 1991). Using a modified ganglia preparation that preserves intact neuronal circuits, we find that fundamental properties of action potentials and expression of specific genes change during aging. Specifically, aging decreased response of R15 neuron to acetylcholine (Ach). R15 neuron from old and mature animals showed significant differences in depolarization, amplitude and hyperpolarization of Ach induced action potentials. Furthermore we find that aging affects the expression of neuropeptides R15 alpha and beta, produced by R15 neuron whereas expression of Ef1alpha, kinesin and Ach receptor are not affected.

Posters BJOERN C. LUDWAR

Imaging of widespread intracellular calcium during homosynaptic facilitation

BJOERN C. LUDWAR Mt. Sinai School of Medicine, Department of Neuroscience, New York, NY, USA

Sensorimotor transmission in the Aplysia feeding network is a valuable model system to study synaptic plasticity. Previously we demonstrated potentiating effects of holding potential on transmission between the mechanoafferent B21 and the follower neuron B8. However, even with maximal physiologically relevant subthreshold depolarizations, effects were small when compared to effects of repetitive spiking (i.e., induction of homosynaptic facilitation). Interestingly, however, holding potential modified the dynamics of facilitation, by increasing its rate of induction. Previously, we demonstrated that effects of subthreshold depolarization are mediated via an influx of calcium through DHP sensitive channels. This increase in background calcium then potentiates spike mediated transmission.

Here we asked if we could image an increase in intracellular calcium that could be

responsible for potentiation during repeated B21 spiking. We imaged intracellular free calcium in B21 while simultaneously recording PSPs in follower B8. Individual B21 spikes produced a measurable calcium signal which declined with a slow time constant. With repeated stimulation at 2 and 6 Hz we observed summation of the calcium signal. The time course of the decay of the calcium signal and PSP amplitude showed a close correlation. Furthermore, increasing the holding potential of B21 increased the summated calcium signal, i.e., the summated signal was larger when the holding potential was 20 mV above resting potential as opposed to 10 mV. If the generation of the summated calcium is a necessary first step in the induction of facilitation, reducing it should also reduce B8 PSP amplitude. We injected buffered 250 mM EGTA, a calcium chelator that reduces the available free calcium. As predicted, we observed a progressive reduction of the calcium signal over the time course of ~10 minutes, accompanied by a reduction in PSP size.

To summarize, under stimulus conditions that lead to facilitation we found a large

change in presynaptic intracellular calcium due to the summation of signals induced by individual spikes. The amplitude of the summated calcium signal is membrane potential dependent. Experimental reduction reduces facilitation. It therefore seems likely that summation of intracellular calcium plays a role in the induction of homosynaptic facilitation at the B21-B8 synapse.

Posters GIANLUCA POLESE

Topology, morphology and function of olfactory organ in Octopus vulgaris GIANLUCA POLESE, Francesco Paolo Ulloa Severino, Luca Troncone, Carla Bertapelle and Anna Di Cosmo Department of Structural and Functional Biology, University of Naples “Federico II” Complesso Universitario Monte S. Angelo, viale Cinthia, 80126 Naples, Italy The cephalopod olfactory organ was described for the first time in 1844 by Kollinker who was attracted from the pair of pits, one on each side of the head, in both octopuses and squids. In 1974 Woodhams and Messenger wrote “A note on the ultrastructure of the Octopus olfactory organ” in which they used the electron microscopy techniques to describe the structure of the Olfactory Epithelium (OE). More information about the OE has been gained in Octopus joubini in which Emery (1976) characterized six types of Olfactory Receptor Neurons (ORNs), but to date not much is known about its functions. Recently, in squid two pathways of signal transduction have been found mediate the odor responsiveness. Just like O. jubini the O. vulgaris olfactory organ is localized on each side of the head anteriorly to the dorsal junction of the mantel with the body, it appear to be a tiny white hole hard to be recognized. At light microscope the OE result to be organized in bagged bulge whose surface layer is made of ORNs and sustentacular cells, underneath a group of ring shape cells give turgor to the entire structure. The Olfactory Marker Protein (OMP) is an abundant, phylogenetically conserved, cytoplasmic protein of unknown function expressed in vertebrate mature olfactory sensory neurons, we found a strong OMP immunoreactivity in almost all the octopus ORNs. Neuropeptide Y (NPY) is widely distributed in vertebrate brains, where it appears to be involved in the control of appetite and feeding behavior, its presence in the olfactory system has been linked to a modulatory activity of the olfactory perception and to a proliferative capability of olfactory epithelia, we observed scattered NPY immunoreactive ORNs within the octopus OE. Moreover, by means Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry, we observed a diffuse proliferative activity within the superficial layers of ORNs with a slightly more intense immunoreactivity along the marginal side of the bag. Overall our data suggest that these peptides have a conserved role also in cephalopods.

Posters CAILIN M. ROTHWELL

A role for retinoic acid in long-term memory formation and synaptogenesis in Lymnaea stagnalis CAILIN M. ROTHWELL and Gaynor E Spencer Brock University, St. Catharines, Ontario, Canada

The vitamin A metabolite, retinoic acid (RA), regulates gene transcription by binding with retinoic acid receptors (RARs) and retinoid X receptors (RXRs). The conservation of retinoid signalling between vertebrates and invertebrates is supported by recent evidence identifying specific metabolic enzymes and retinoid receptors required for retinoid signalling in a number of invertebrate non-chordate species. For example, in the pond snail Lymnaea stagnalis, endogenous RA is present in the CNS and haemolymph, and we have also identified the RA-synthesizing enzyme RALDH, and fully cloned a RXR, and the first non-chordate RAR from the Lymnaea CNS.

In vertebrates, RA has been implicated in hippocampal plasticity and long-term

potentiation, but a role for RA in invertebrate learning and memory has not previously been described. In these studies, we inhibited the synthesis of RA by incubating Lymnaea in two different RALDH inhibitors, citral and DEAB (and EtOH as control), for 72 hours. Following the incubations, we then operantly conditioned the aerial respiratory behaviour to examine the effects of RALDH inhibition on both learning and memory formation. Inhibition of RA synthesis did not affect learning, but prevented the animals from forming long-term memory (LTM). Interestingly, the formation of intermediate-term memory (ITM) was not disrupted. Incubation of animals in either pan-RAR or pan-RXR antagonists also impaired LTM formation, suggesting that RA might act to regulate gene transcription during LTM formation of this operantly conditioned behaviour.

RA exerts trophic effects to stimulate neurite outgrowth in this mollusc (as in

vertebrates), but no studies have previously investigated whether RA acts as a trophic factor to support either synaptic plasticity or synaptogenesis in invertebrate species. Previous studies with Lymnaea indicate that excitatory synapse formation in vitro requires the presence of extrinsic trophic factors (Hamakawa et al., 1999). Using the same soma-soma configuration as these previous studies, our preliminary results demonstrate that RA alone can indeed support synaptogenesis between excitatory partner cells in vitro, and, thus, may have the potential to exert trophic influences on synapse formation during memory formation.

Hamakawa et al. (1999) J Neurosci 19: 9306-9312

Acknowledgements: NSERC Discovery Grant to G.E.S. and NSERC Scholarship to C.M.R.

Posters BRYAN K. ROURKE

The search for the 5-HT receptor important for translocation of PKC Apl II BRYAN K. ROURKE, Carole A Farah, Tyler W Dunn and Wayne S Sossin Sossin Lab – McGill University PKCs are a large family of serine-threonine kinases important for the molecular memory trace. In the marine mollusk, Aplysia californica, the calcium-independent novel PKC Apl II is activated in sensory neurons by serotonin (5HT), where it is required for the reversal of synaptic depression. However, while five 5HT receptors have been identified in Aplysia, none of these receptors appear to play a role in activation of PKC Apl II. In Aplysia, there is a family of 7 G-protein coupled receptors, originally called B receptors. From bioinformatic studies, these receptors appear to have arisen from a duplication of the Aplysia D1 receptor, but then diverged quickly. We have cloned two B receptors present in the Aplysia nervous system, and are examining their role in PKC activation. Unlike 5HT2Apl and 5HT7Apl, the B receptors do not translocate PKC Apl II in heterologous Sf9 cells in response to 5HT or Dopamine. While the concentration of 5HT required to induce PKA responses has been well characterized, there have been few studies examining the concentration of 5HT required for translocation of PKC Apl II. We examined the translocation of endogenous PKC Apl II using an antibody to PKC Apl II and confocal microscopy. In isolated sensory neurons, maximal translocation occurred at 10 uM and there was a significantly smaller response to 1 uM 5HT. In contrast, translocation in the processes in response to 1 uM 5HT when the sensory neuron was cultured with a motor neuron was similar to that seen in response to 10 uM. Possible explanations include soma-specific inhibitors of translocation or distinct 5HT receptors used in isolated sensory neurons and after pairing.

Posters AKIRA SAKURAI

Distinct neural circuit architectures produce analogous rhythmic behaviors in related nudibranch species AKIRA SAKURAI and Paul S. Katz Neuroscience Institute, Georgia State University Nervous systems are relatively conserved with homologous neurons being recognized across species. Therefore, it might be expected that circuit architectures underlying similar behaviors produced by homologous neurons would also be similar. However, recent modeling studies have shown that similar patterns of activity can be generated by distinct neural circuit architectures. We have found that the nudibranchs, Melibe leonina and Dendronotus iris, which exhibit similar swimming behaviors, have homologous neurons but use different circuit architectures to produce the behaviors. Melibe and Dendronotus each exhibit swimming behaviors consisting of rhythmic alternation of left and right body flexions. It was previously shown in Melibe that the central pattern generator (CPG) for swimming contains 2 bilaterally symmetric neurons, Swim Interneuron 1 (Si1Mel) and Swim Interneuron 2 (Si2Mel). Si1Mel and Si2Mel are electrically coupled ipsilaterally and mutually inhibitory with both contralateral counterparts. We have identified their homologues in Dendronotus (Si1Den and Si2Den) but found that their synaptic connections are fundamentally different. The Si1Den neurons do not form mutual inhibitory synapses with the contralateral neurons, but are electrically coupled to all other swim interneurons. Furthermore, Si1Den does not function as a member of the swim CPG; rather its tonic firing initiates and accelerates the swim motor program. In addition to Si1 and Si2, we have identified another bilateral pair of CPG neurons, Si3, in both species, and Si4 in Melibe. The Si3 pair forms another half-center oscillator element that is interconnected with the Si1/2 complex. The Si3 neurons in Melibe and Dendronotus differ in aspects of synaptic connectivity, phase relationships, and functional interactions. In Melibe, the Si1/2Mel and Si3Mel half centers can burst independently, but in Dendronotus, Si3Den synaptic input was essential for Si2Den bursting. The Si4 pair in Melibe provides further complexity in the circuit; they are not mutually inhibitory but electrically-connected to the contralateral Si2Mel while making inhibitory synapses onto Si3Mel. Thus, despite producing similar two-phase motor outputs and having homologous neurons, the swim CPGs in Melibe and Dendronotus differed substantially in network architecture and internal dynamics. Supported by NSF- IOS-1120950, 0814411

Posters MICHAEL C. SCHMALE

The National Resource for Aplysia MICHAEL C. SCHMALE, Thomas Capo, and Lynne Fieber Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149 USA.

The National Resource for Aplysia is an NIH sponsored resource which provides high-quality cultured sea hares, Aplysia californica, at all stages of development throughout the year for research and teaching programs. The resource typically ships 20,000 – 30,000 animals per year to researchers throughout the USA, to Europe, Israel and other locations. The resource also produced inbred animals (5 generations) for use in the recently completed Aplysia genome sequencing project. The mission of the resource is to provide the research community with the materials needed for studies of Aplysia by continually improving culture conditions to maximize efficiency and reliability of production and to facilitate new uses for this model system. Research conducted at the resource includes studies of how diet and other factors affect growth of juvenile and adult animals and evaluation of biotic and abiotic factors affecting growth, survival and metamorphosis of larval Aplysia. Recent studies at the resource have focused on developing Aplysia as a neurophysiological and genetic model of aging. The annual life cycle of these animals ends with a period of rapid senescence characterized by reduced body size and weight, reduction or cessation in egg laying and other behavioral changes. These end of life stages can be reliably produced at the resource and provide an ideal substrate for comparative studies of neurological function and gene expression in aging. Supported by PHS grant P40 RR10294.

Posters NAAMA STERN

Morphological and functional characterization of the fast neurotransmission in the learning and memory network of the Octopus vulgaris NAAMA STERN¹, T. SHOMRAT¹, N. NESHER¹, N. FEINSTEIN², S. KIMURA³, M. BELENKY², B. HOCHNER¹ 1 Department of Neurobiology, Life Science Institute and the Interdisciplinary Center for Neural Computation, Edmond J Safra Campus, Givat Ram Hebrew University, Jerusalem, Israel 2 Deptment of Cell & Developmental Biology, Life Science Institute, Hebrew University, Jerusalem, Israel 3 Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu 520-2192, Japan The understanding of the evolution of neural circuits mediating complex behaviors may be broadened through comparative analysis of brain function in invertebrates with sophisticated behavior, such as the Octopus Vulgaris. The superior frontal lobe -vertical lobe (SFL-VL) system of this species plays a predominant role in learning and memory associated with their complex behaviors. Our previous studies revealed activity dependent LTP in the input synapses to the VL (Hochner et al. 2003) and that the VL and its LTP are important for acquisition of long term memory (Shomrat et al 2008). Gray and Young's anatomical results suggested the VL system is organized as a network where the inputs from the SFL innervate en passant millions of small amacrine interneurons (AM) which in turn converge onto a relatively small number of large neurons (LN); the only VL output. Here we investigate the organization of the VL circuitry by using immunohistochemistry to identify possible neurotransmitters. Our previous physiological experiments suggested that the synaptic input AMs is mainly glutamatergic and indeed positive immunoreactivity supports glutamatergic input to the VL. Whole-cell recordings in the large LN or measuring their axonal activity suggest that the excitatory input to the LN is cholinergic as it is blocked by hexamethonium. In this work, cholinergic cells were targeted by immuno labeling of choline acetyltransferase (cChAT) and their possible postsynaptic targets with fluorescently tagged ACh receptor blocker, α-bungarotoxin. Confirming the physiological results, ChAT and α-bungarotoxin positive staining are observed within the VL neuropil. We could not detect ChAT reactivity in the AM cell bodies, raising the possibility that cChAT molecules are localized at the AMs’ terminals. Whole cell recordings reveal that some of the LNs are also innervated by inhibitory inputs, however none or only few cells in the AM layer showed GABA reactivity. GABA reactivity is revealed within the population of the LN efferent and thus suits the idea that the VL output inhibits the attack behavior. In summary, these neuroanatomical results support our physiological results (Shomrat et al 2011), suggesting the architecture of the VL as a simple fan-out fan-in feed-forward learning and memory network in the configuration of: glutamatergic synaptic input à cholinergic interneurons à GABAergic output. This work was supported by Smith Family laboratory, the Binational US-Israel Science Foundation (2007-407), by Project OCTOPUS, a European Commission 7th Framework Programme (FP7) 231608

Posters ARIANNA N. TAMVACAKIS

Serotonergic neuromodulation provides phylogenetic plasticity for behavior ARIANNA N. TAMVACAKIS, J.L. Lillvis, and P.S. Katz Neuroscience Institute, Georgia State University, Atlanta GA

The presence or absence of neuromodulatory signalling may underlie the expression of particular behaviors. The sea slugs Tritonia diomedea and Pleurobranchaea californica, which belong to the monophyletic clade Neudipleura, perform analogous, though independently evolved, dorsal-ventral (DV) flexion swimming behaviors. Most other members of Neudipleura do not perform DV swimming. In Tritonia, the central pattern generator (CPG) for swimming is composed of three identified neuron types (C2, DSI, and VSI). DSI is serotonergic and modulates the strength of C2 synapses; this modulation is necessary for swimming in Tritonia. Homologues of DSI and C2 have been identified in several species of Neudipleura including Pleurobranchaea, which uses these neurons in its DV swim CPG. Hermissenda crassicornis is more closely related to Tritonia than to Pleurobranchaea, yet does not swim like either even though C2 and serotonergic DSI homologues are present. We hypothesize that serotonergic neuromodulation of C2 homologues may underlie the ability to produce DV swimming.

We found that in Pleurobranchaea, as in Tritonia, the amplitudes of C2-evoked

synaptic potentials increased in response to DSI stimulation or bath-applied 5-HT. Furthermore, DV swimming was inhibited by the 5-HT receptor antagonist methysergide. In contrast, in the non-DV swimmer Hermissenda, neither DSI stimulation nor bath-application of 5-HT affected the size of C2-evoked synaptic potentials. Thus, serotonergic neuromodulation correlates with DV swimming behavior, suggesting that the presence of modulation may be necessary for homologous neurons to function as a CPG circuit for swimming.

The presence of neuromodulation and DV swimming might be caused by the

expression of particular serotonin receptors in C2 homologues. To test this, we have identified four partial receptor sequences from 5-HT receptor families 1 and 2 in each of the three species. We are in the process of determining their localization and relative expression levels in identified neurons. Altogether, the results of this project suggest that differential neuromodulation is a mechanism for phylogenetic plasticity in behavior. Supported by NSF- IOS-112095, IOS-0814411, IOS-10114176, and a GSU Brains and Behavior Seed Grant.

Posters AYAKO TONOKI

Age-dependent memory impairment analyzed in Drosophila AYAKO TONOKI, Ronald L. Davis Department of Neuroscience, Scripps Research Institute Florida, Jupiter, FL How the functional activity of the brain is altered during aging to cause age-related memory impairments is unknown. We used functional cellular imaging to monitor two different calcium-based memory traces that underlie olfactory classical conditioning in young and aged Drosophila. Functional imaging of neural activity in the processes of the dorsal paired medial (DPM) and mushroom body (MB) neurons revealed that the capacity to form an intermediate-term memory (ITM) trace in the DPM neurons after learning is lost with age, while the capacity to form a short-term memory trace in the a′/b′ MB neurons remains unaffected by age. Stimulation of the DPM neurons by activation of a temperature-sensitive cation channel between acquisition and retrieval enhanced ITM in aged but not young flies. These data indicate that the functional state of the DPM neurons is selectively altered with age to cause an age-related impairment of ITM, and demonstrate that altering the excitability of DPM neurons can restore age-related memory impairments.

Posters LEI RAY ZHONG

Role of Acetylcholine in regulating growth cone filopodial dynamics LEI RAY ZHONG, Stephen Estes, Liana Artinian, Vincent Rehder Biology Department, Georgia State University, Atlanta, Georgia 30302 During early developmental stages, neurons extend neurites to connect to appropriate target cells. Growth cones at the tip of growing neurites are important for pathfinding and its filopodia serve as sensors to probe the environment for guidance cues. In addition to acting as a classical neurotransmitter in synaptic transmission, Acetylcholine (Ach) has also been shown to play a role in axonal growth and growth cone guidance. What is not well understood is how Ach acts at the level of the growth cone to affect growth cone filopodial dynamics. Here we address this question using an identified neuron (B5) from the buccal ganglion of the pond snail Helisoma trivolvis grown in cell culture. We found that addition of Ach (1 µM) to the culture dish caused pronounced filopodial elongation within minutes, and the blockade of nicotinic Ach receptors (nAchRs) by tubucurarine eliminated the effect of Ach on filopodia. The Ach-induced filopodial elongation was likely mediated through the second messenger calcium, since calcium imaging studies showed that Ach caused an elevation in intracellular calcium levels ([Ca]i) in growth cones that preceded the filopodial responses to Ach. Neuronal electrical activity is known to be important in mediating some behavioral responses of growth cones to guidance cues. Using whole-cell patch clamp recording, we demonstrate that Ach caused a depolarization of the membrane potential, an increase in spiking frequency, and a reduction in input resistance in B5 neurons. Whereas the nAchR agonist DMPP mimicked the effect of Ach, the nAchR antagonist tubucurarine attenuated the Ach-induced changes in electrical activity, further supporting the hypothesis that Ach acted via the nAchR. Lastly, Ach acted locally at the growth cone, because growth cones that were physically isolated from their parent neuron responded to Ach by filopodial elongation with a similar time course as growth cones that were part of an intact control neuron. Our data reveal a critical role for Ach as a modulator of growth cone filopodial dynamics. Ach signalling is mediated via nAchR and results in an increase in [Ca]i, which, in turn, causes filopodial elongation.

Conference Participants Dr. Thomas Abrams Department of Pharmacology University of Maryland School of Medicine Baltimore, MD, USA tabrams@umaryland.edu Dr. Komolitdin Akhmedov The Scripps Research Institute Jupiter, FL, USA kakhmedo@scripps.edu Dr. Pavel Balaban Inst. Higher Nervous Activity Moscow, Russia pmbalaban@gmail.com Dr. Paul Benjamin Sussex Centre for Neuroscience Brighton, University of Sussex East Sussex, UK p.r.benjamin@sussex.ac.uk Dr. Jean Boal Millersville University Millersville, PA, USA jean.boal@millersville.edu Dr. Jana Boerner Florida Atlantic University Boca Raton, FL, USA jboerner@fau.edu Ms. Melissa Borgen Florida Atlantic University Boca Raton, FL, USA mborgen1@fau.edu Dr. Vladimir Brezina Department of Neuroscience Mt. Sinai School of Medicine New York, NY, USA vladimir.brezina@gmail.com

Dr. John Byrne University of Texas Medical School at Houston Houston, TX, USA John.H.Byrne@uth.tmc.edu Dr. Robert Calin-Jageman Dominican University River Forest, IL, USA rcalinjageman@dom.edu Dr. Irina Calin-Jageman Dominican University River Forest, IL, USA icalinjageman@dom.edu Mr. Tom Capo Rosenstiel School of Marine and Atmospheric Science University of Miami Miami, FL, USA tcapo@rsmas.miami.edu Dr. Christopher Carter Queen's University Kingston, ON, Canada christopher.carter@queensu.ca Dr. John Case CIS Department University of Delaware Newark, DE, USA case@udel.edu Dr. Isaac Cervantes Sandoval The Scripps Research Institute Jupiter, FL, USA isandova@scripps.edu Dr. Yun-Beom Choi Columbia University Leonia, NJ, USA yc2029@columbia.edu Dr. Robyn Crook University of Texas Health Science Center Houston, TX, USA robyn.crook@uth.tmc.edu

Dr. Andrew Dacks Mount Sinai School of Medicine New York, NY, USA adacks@gmail.com Dr. Ronald Davis The Scripps Research Institute Jupiter, FL, USA rdavis@scripps.edu Dr. Anna Di Cosmo University of Naples "Federico II" NA, Italy dicosmo@unina.it Dr. Ludovic Dickel University of Caen Caen, Basse Normandie, France ludovic.dickel@unicaen.fr Dr. Zhong-Ping Feng University of Toronto Toronto, ON, Canada zp.feng@utoronto.ca Dr. Lynne Fieber Rosenstiel School of Marine and Atmospheric Science University of Miami Miami, FL, USA LFieber@rsmas.miami.edu Dr. William Frost Department of Cell Biology and Anatomy Rosalind Franklin University, The Chicago Medical School North Chicago, IL, USA william.frost@rosalindfranklin.edu Dr. Rhanor Gillette Department of Molecular & Integrative Physiology University of Illinois at Urbana-Champaign Urbana, IL, USA rhanor@illinois.edu Dr. David Glanzman UCLA Los Angeles, CA, USA dglanzman@physci.ucla.edu

Dr. Tanja Godenschwege Florida Atlantic University Boca Raton, FL, USA godensch@fau.edu Ms. Charuni Gunaratne Georgia State University Atlanta, GA, USA cgunaratne@gmail.com Mr. Stavros Hadjisolomou Psychology Department City University Of New York - Brooklyn College Brooklyn, NY, USA shadjisolomou@gc.cuny.edu Dr. Roger Hanlon Marine Biological Laboratory Woods Hole, MA, USA rhanlon@mbl.edu Ms. Margaret Hastings McGill University St. Lambert, QC, Canada margaret.hastings@mail.mcgill.ca Dr. Robert Hawkins Columbia University New York, NY, USA rdh1@columbia.edu Dr. Binyamin Hochner Department of Neurobiology Silberman Institute of Life Science Hebrew University Jerusalem, Israel bennyh@lobster.ls.huji.ac.il Dr. Jiangyuan Hu Department of Neuroscience Columbia University New York, NY, USA jh2004@columbia.edu

Dr. Jian Jing Department of Neuroscience Mount Sinai School of Medicine New York, NY, USA jingj01@gmail.com Dr. Elizabeth Jonas Yale University New Haven, CT, USA elizabeth.jonas@yale.edu Dr. Leonard Kaczmarek Yale University School of Medicine New Haven, CT, USA leonard.kaczmarek@yale.edu Dr. Beena Kadakkuzha The Scripps Research Institute Jupiter, FL, USA bkadakku@scripps.edu Dr. Stefan Kassabov Columbia University New York, NY, USA srk2110@columbia.edu Dr. Paul Katz Georgia State University Atlanta, GA, USA pkatz@gsu.edu Mr. Andrew Kempsell University of Miami Key Biscayne, FL, USA akempsell@rsmas.miami.edu Dr. Xin-an Liu The Scripps Research Institute Jupiter, FL, USA xinanliu@scripps.edu Mr. Brandon Lloyd Florida Atlantic University Boca Raton, FL, USA blloyd6@fau.edu

Dr. Bjoern Ludwar Mt. Sinai School of Medicine Department of Neuroscience New York, NY, USA bjoern@ludwar.net Dr. Lisa Lyons Department of Biological Sciences Florida State University Tallahassee, FL, USA lyons@bio.fsu.edu Dr. Neil Magoski Queen's University Kingston, ON, Canada magoski@queensu.ca Dr. Ryota Matsuo Tokushima Bunri University Sanuki, Kagawa, Japan matsuor@kph.bunri-u.ac.jp Mr. Patrick Mc Camphill McGill University Montreal, QC, Canada pkmccamphill@gmail.com Ms. Monica Mejia Florida Atlantic University Coral Springs, FL, USA mmejia8@fau.edu Dr. Mark Miller Institute of Neurobiology University of Puerto Rico San Juan, Puerto Rico mark.miller@upr.edu Dr. Leonid Moroz The Whitney Laboratory University of Florida St. Augustine, FL, USA moroz@whitney.ufl.edu

Dr. Riccardo Mozzachiodi Texas A&M University - Corpus Christi Corpus Christi, TX, USA riccardo.mozzachiodi@tamucc.edu Dr. Rod Murphey Florida Atlantic University Boca Raton, FL, USA rmurphey@fau.edu Ms. Janet Nagorski The Scripps Research Institute Jupiter, FL, USA jnagorsk@scripps.edu Dr. Romuald Nargeot University of Bordeaux Segalen, Bordeaux, France romuald.nargeot@u-bordeaux2.fr Mr. Brian Orr Florida Atlantic University West Palm Beach, FL, USA borr5@fau.edu Dr. Gianluca Polese University of Naples "Federico II" Naples, Italy gianluca.polese@unina.it Dr. Sathya Puthanveettil The Scripps Research Institute Jupiter, FL, USA sputhanv@scripps.edu Dr. Bindu Raveendra The Scripps Research Institute Jupiter, FL, USA rbindu@scripps.edu Dr. ValerioRizzo The Scripps research Institute Jupiter, FL, USA vrizzo@scripps.edu Dr. Joshua Rosenthal Institute of neurobiology San Juan, Puerto Rico joshua.rosenthal@upr.edu

Ms. Cailin Rothwell Brock University St. Catharines, ON, Canada cr04kc@brocku.ca Mr. Bryan Rourke Montreal Neurological Institute McGill University QC, Canada bryan.rourke@mail.mcgill.ca Dr. Akira Sakurai Georgia State University Neuroscience Institute Atlanta, GA, USA akira@gsu.edu Dr. Samuel Schacher Department of Neuroscience Columbia University College of P & S New York, NY, USA sms2@columbia.edu Dr. Michael Schmale Rosenstiel School of Marine and Atmospheric Science University of Miami Miami, FL, USA mschmale@rsmas.miami.edu Ms. Olesya Slipchuk Florida Atlantic University Ft. Lauderdale, FL, USA oslipchu@fau.edu Dr. Wayne Sossin McGill University Montreal Neurological Institute Montreal, QC, Canada wayne.sossin@mcgill.ca Dr. Gaynor Spencer Department of Biological Sciences Brock University St Catharines, ON, Canada gspencer@brocku.ca

Ms. Naama Stern Hebrew University of Jerusalem Jerusalem, Israel naamas05@gmail.com Dr. Avy Susswein Goodman Faculty of Life Sciences Bar Ilan University Ramat Gan, Israel abrham.susswein@biu.ac.il Ms. Arianna Tamvacakis Georgia State University Atlanta, GA, USA tamvacakis@gmail.com Dr. Seth Tomchik The Scripps Research Institute Jupiter, FL, USA stomchik@scripps.edu Dr. AyakoTonoki The Scripps Research Institute Florida Jupiter, FL, USA ayamaguc@scripps.edu Dr. Edgar Walters University of Texas Medical School at Houston Houston, TX, USA edgar.t.walters@uth.tmc.edu Dr. Klaudiusz Weiss Department of Neuroscience Mt. Sinai School of Medicine New York, NY, USA klaudiusz.weiss@gmail.com Dr. Keqiang Xie The Scripps Research Institute Jupiter, FL, USA kxie@scripps.edu Mr. Lei Zhong Georgia State University Atlanta, GA, USA lzhong1@gsu.edu