Characterization of a zebrafish locus that regulates rod and ultraviolet cone photoreceptor cell fates during retinal development (oral presentation)

K. Alvarez-Delfin, A. C. Morris, and J. M. Fadool

Program in Neuroscience and Department of Biological Science, Florida State University, Tallahassee, FL 32306-4340

            Purpose:  The zebrafish (Dario rerio) has become a valuable organism for the genetic analysis of the development and function of the visual system. We have undertaken a large-scale genetic screen of chemically mutagenized free-swimming larvae to identify mutations affecting photoreceptor cell patterning. We identified the locus lot-of-rods (lor) that shows an increased number in rods photoreceptor compared with wildtype larvae and no other evident defects. This study describes the further characterization the lor phenotype.  Methods:  The initial screening was done in collaboration with collaboration with Drs. M. Mullins and M. Granato at the University of Pennsylvania.  lor mutants are identified by immunolabeling of whole mount larvae or frozen sections with specific-rod antibodies. The mutant phenotype was further characterized by labeling with cell specific markers for other photoreceptors. BrdU incorporation and Tunnel labeling were performed in frozen retinal sections. Confocal images of immunolabeled whole eyes were used for spatial pattern analysis. The initial mutagenesis strategy included a mapping panel that facilitates linkage analysis. Linkage mapping was performed by standard procedures in the Zebrafish Mapping Facility, University of Louisville.  Results:  The lor phenotype of an increased number of rods was associated with a decreased number of UV cones and indiscernible changes in other photoreceptors. Spatial pattern analysis suggests a change in fate from UV cone into rod photoreceptor. Tunnel and BrdU labeling showed no increase in apoptosis or cell proliferation, respectively, in the lor mutants. lor locus is linked to a region in chromosome 15 and candidate genes have been identifying.  Conclusions:  This study demonstrated the utility of the genetic screen to uncover recessive mutations affecting photoreceptor patterning in zebrafish. More specifically the analysis of the lor mutation revealed that a gene exists in zebrafish for controlling UV cone versus rod photoreceptor cell fate decision.

Dominant/subordinate relationships and brain-derived neurotrophic factor expression in the adult green anole (Anolis carolinensis) (poster)

M. P. Black,¹ K. J. Ressler,² R. L. Earley,³ and W. Wilczynski¹

¹Center for Behavioral Neuroscience, Department of Psychology, Georgia State University, Atlanta, GA 30303; ²Center for Behavioral Neuroscience, Department of Psychiatry and Behavioral Sciences, Emory University, Atlanta, GA 30329; ³Department of Biology, California State University, Fresno, CA 93740

            Brain-derived neurotrophic factor (BDNF) has been implicated in the modulation of aggression and plays an important role in learning and memory, activity-dependent plasticity, and social defeat stress (Lyons et al., 1999; Yamada et al., 2002; Lu, 2003; Berton et al., 2006). Because of these roles, we are interested in the function of BDNF in Anolis carolinensis, a neuroethological model for aggression and dominant/subordinate relationships. As a first approach to investigate the neural expression of BDNF, we mapped the distribution of BDNF expression in the anole forebrain and made comparisons between dominant and subordinate anoles. Nested primers based on a consensus sequence were used to isolate the Anolis BDNF gene. In situ using a riboprobe for A.carolinensis BDNF showed that mRNA expression was highest in the medial pallium, similar to the mammalian hippocampus, where BDNF expression is often highly expressed (Connor et al., 1997). Pharmacological manipulations of serotonergic activity in the medial pallium can reverse adult dominant-subordinate relationships in A.carolinensis (Summers et al., 2005), suggesting a key role of the medial pallium in the type of social behavior with which BDNF has been implicated. Other areas of high forebrain BDNF expression were the ventromedial and paraventricular hypothalamic nuclei.  Moderately high levels were present in the amygdala, habenula, other parts of the hypothalamus, and regions of the dorsal ventricular ridge. Lightest expression was in the basal ganglia and thalamic sensory nuclei. These results indicate that BDNF expression varies regionally within the anole forebrain, as in mammals, with the highest expression in limbic regions. In preliminary data, there was no difference between dominant and subordinate male anoles in the medial pallium, but dominant males had higher relative levels of BDNF mRNA in the amygdala. A power analysis indicates this would be significant at p = 0.05 with 6 subjects/group (95% power level). Research supported by the STC Program of the National Science Foundation under Agreement No. IBN-9876754, CBN postdoctoral fellowship to MPB, Georgia Research Alliance and GSU-RPE program, and NSF IBN-0090739 to WW.

Age differences in the threshold, but not in the magnitude of synaptic depression (poster)

Karthik Bodhinathan, Ashok Kumar, and Thomas Foster

Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610

            Intracellular calcium (Ca²⁺) levels play a pivotal role in regulating synaptic plasticity mechanisms and determining the degree and direction of synaptic strength in response to neuronal activity. Dysregulation of the Ca²⁺ homeostasis is thought to alter the mechanisms for induction of synaptic plasticity in the hippocampus of aged rats and as such may contribute to a decline in memory performance. However, the experimental paradigms for examining synaptic plasticity (Ca²⁺ levels, stimulation patterns) can vary across labs. Therefore, the current study examined the impact of Ca²⁺ levels and various stimulation paradigms on the synaptic depression over the course of aging. Extracellular excitatory postsynaptic field potentials were recorded from CA3-CA1 synapses in acute hippocampal slices obtained from young (55-65 days), young adult (5-8 months), and aged (22-24 months) male Fischer 344 rats. In recording medium containing 2 mM Ca²⁺ and 2 mM Mg²⁺, low frequency stimulation (LFS, 1 Hz 900 pulses) induced long-term depression (LTD) only in slices obtained from aged rats. Increasing the Ca²⁺:Mg²⁺ ratio in the recording medium (2.5mM:1.3mM) or employing paired-pulse LFS (PP-LFS, 1Hz 1800 pulses), also induced LTD in young adult animals and no significant age differences were observed under these conditions. Using multiple induction episodes under conditions which facilitate LTD-induction (PP-LFS and elevated Ca²⁺:Mg²⁺ ratio), no age-related difference was observed in the maximum level of LTD. These results indicate that the maximal magnitude of LTD is not difference across age groups. Rather, aged animals are more susceptible to LTD induction.

Effects of locomotor training on spinal cord injury (SCI) lesion (poster)

P. Bose,^1,2 R. Telford,² P. Nguyen,² R. Jain,² S. William,² R. Parmer,² D. K. Anderson,^1,2 P. J. Reier,² and F. J. Thompson²

¹North Florida/South Georgia VA Medical Center, Gainesville, Florida 32608; ²Department of Neuroscience,  McKnight Brain Institute, University of Florida and College of Medicine, Gainesville, FL 32610

            Our previous studies in animals following SCI revealed significant and equal improvements in locomotor ability and spasticity following treadmill and bicycle locomotor training. Our present studies were initiated to compare the lesion cavities and spared tissue following three months of locomotor training where treadmill and cycling locomotor training were used as therapy to manage SCI-induced spasticity and to improve locomotor function. Eighteen animals’ spinal cord segments contained lesions (midthoracic SCI, standard MASCIS protocol, 10 g 2.5 cm weight drop) were used for this purpose. They were divided equally into 3 groups: untrained control (UT-C), treadmill, and cycle trained. The sections were stained using conventional Luxol fast blue, cresyl violet and H&E staining techniques. To quantify injury lesion length, the numbers of sections containing the lesion were counted from rostral to caudal on serial sections. Further quantification involves volumetric measurement of the lesions. In order to obtain volume measurements, the lesion area was measured in every twentieth spinal cord section. After obtaining lesion area, a previously published mathematical formula was applied to calculate the volume. The amount of spared white matter, both dorsal and ventral quadrants of the cavity, was also measured on the serial sections. The proportion of the residual ventral white matter was expressed in relation to that observed in intact normal animal’s tissue (100% spared ventral white matter). Both locomotor trained groups revealed decreased lesion volume (rostro-caudal extension) and more spared tissue at the lesion site. Light microscopic qualitative studies of spared tissue revealed better preservation of myelin, axons and collagen morphology in locomotor trained animals. Importantly, these data indicate that the therapeutic efficacy of ergonomically practical cycle training was more effective in preserving spared tissue than more labor-intensive treadmill training. Locomotor training related improvements in spasticity and locomotor recovery were correlated with the decreased lesion volume and more spared white matter. We also found activity induced marked upregulation of BDNF, GABA, and monoamines (e.g., norepinephrine and serotonin) which might account for these decreased lesion volume and more spared tissue. Supported by The Christopher Reeve Paralysis Foundation (CRPF) # BA2-0202-2, BSCIRTF, and NIH R01 NS044293-01A1.

D-Aspartate as a neurotransmitter in Aplysia californica (oral presentation)

S. L. Carlson

Department of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami,Miami, FL 33149

            In recent decades D-aspartate (D-Asp) has been discovered in considerable quantities in the nervous tissue of numerous phyla. Although its role is unclear, D-Asp has been suggested to serve a purpose in the nervous system either mundane (a precursor for synthesis of the neurotransmitter NMDA) or exceptional (a neurotransmitter in its own right). Electrophysiological experiments were performed on cultured neurons of A. californica, with particular emphasis on specific cell clusters of the buccal and pleural ganglia. Cells were whole-cell current- and voltage-clamped, and potential changes or ionic currents in response to bath-application of D-Asp were recorded. D-Asp activated an inward-going conductance at resting membrane potentials that may be either a chloride current or a non-specific cation current. Current clamp experiments revealed that application of 1mM D-Asp induced action potentials. In a preliminary test of the hypothesis that D-Asp acts on glutamate receptors, a total of 88 cells were examined for responses to D-Asp and L-glutamate (L-Glu). D-Asp currents were observed in 32.9% of cells, while L-Glu currents were observed in 53.4% of cells. 52.3% of cells responded to only one agonist while 17% of cells responded to both agonists, and 30.7% lacked a response. In addition to the observation that more than half of cells responded to only one of the agonists, cells responded very differently to repeated applications of D-Asp or L-Glu. The current response usually desensitized upon repeated exposure of the cells to D-Asp. Repeated bath applications of L-Glu, in contrast, produced a potentiating current response. These data suggest that D-Asp does act as a neurotransmitter in Aplysia neurons, and it may have a role independent of glutamate receptors. In instances where both agonists might be acting at the same receptors, differences in the responses to D-Asp and L-Glu were noted.

Characterization of the Basigin-MCT1 complex in vertebrate retina (poster)

Vilma Carson, NiCole Finch, Judith D. Ochrietor

Department of Biology, University of North Florida, Jacksonville, FL 32224

            Purpose:  Previous studies indicate that Basigin, a transmembrane protein, interacts with monocarboxylate transporter-1(MCT1), a lactate transporter.  This group hypothesizes that the two membrane proteins associate to form a lactate shuttle in the vertebrate retina.  Work from this laboratory and others indicates that the single transmembrane domain of Basigin interacts with MCT1, although it is not known what region of MCT1 interacts with Basigin. We have therefore begun a systematic process to determine which amino acids within MCT1 bind to the Basigin protein.  Methods:  The cDNA for MCT1 was divided in half via PCR using pTopo-MCT1, which contains the entire MCT1 cDNA sequence, as the template.  cDNA for MCT1-N (amino half) and MCT1-C (carboxy half) was cloned into the pET102 vector (Invitrogen) for recombinant protein expression.  This vector adds a 6X-histidine tag (6XHis) to the carboxy-terminus of recombinant proteins.  The cloned vectors were sequenced for accuracy and recombinant proteins were made via in vitro transcription/translation (PROTEINscript II, Ambion). A sandwich enzyme-linked immunosorbent assay (ELISA) was performed to test for MCT1 binding to Basigin.  Endogenous mouse retina Basigin was captured and probed with MCT1-N-6XHis or MCT1-C-6XHis.  Results:  Preliminary results of the ELISA analysis indicate that both the N-terminal region and the C-terminal region of MCT1 interact with Basigin protein.  Conclusions:  Our results support previous data demonstrating an interaction between MCT1 and Basigin.  Additional tests must be performed to determine which amino acids within MCT1 bind to the Basigin transmembrane domain.

Sphingosine-1-phosphate signaling in retinal amacrine cells (oral presentation)

S. M. Crousillac, P. N. Ojiaku and E. L. Gleason

Louisiana State University, Baton Rouge, LA 70820

            Sphingosine-1-phosphate (S1P) is a lipid signaling molecule that has been shown to produce transient Ca²⁺ elevations in amacrine cell processes (Lindstrom et al. 2004). To begin to understand the function of S1P in the retina, cultured amacrine cells were voltage-clamped at -70 mV and exposed to 10µm S1P. After about 75 seconds, a small (20-50 pA), noisy inward current developed. Both La³⁺ (25µM) and Gd³⁺ (10 µm), were effective in blocking the S1P-dependent current.  Measurement of the S1P-dependent current during voltage ramps indicates that the current is carried by cations.

            S1P can act as both a diffusible Ca²⁺ influx factor (Mattie et al. 1994) and as an extracellular ligand for the g protein-coupled receptor edg-1 (Lee et al. 1998; Meyer zu Heringdorf et al. 1998). Through these two mechanisms,  S1P has been shown to activate Ca²⁺ flux across the plasma membrane and to mobilize sequestered intracellular Ca²⁺ (Törnquist et al. 1997; An et al. 1999; Formigli et al. 2002). To determine whether the S1P-induced current could be receptor-mediated, we asked whether edg-1 was expressed by amacrine cells. A polyclonal antibody against edg-1 revealed expression of the receptor by amacrine cells.  Furthermore, we found that ~60% of amacrine cells in the inner nuclear layer expressing edg-1 are calretinin-positive. To further test the involvement of edg-1, we employed SEW 2871, a synthetic agonist for the edg-1 receptor. Upon application of 10µm SEW 2871 we observed a noisy, inward current similar to the SIP-induced current.  Activated Edg-1 receptors stimulate PLC (Zhou et al. 2004). Consistent with this, U73122 (a PLC inhibitor) reduced the S1P–induced charge movement by ~90%. In Ca²⁺ imaging experiments, S1P (10 μm) consistently elicited cytosolic Ca²⁺ elevations. These Ca²⁺ elevations were sensitive to 25µm La³⁺ and depended upon the presence of external Ca²⁺, indicating that at least a portion of the S1P-induced current is due to Ca²⁺ entry across the plasma membrane. 

            Sphingolipids similar in structure to S1P have been shown to activate members of the TRP super-family in heterologous expression systems (Grimm et al. 2004).  We have both immunocytochemical and molecular evidence indicating that multiple TRPC subunits are expressed by retinal amacrine cells. Identifying the complement of TRPC subunits expressed by these S1P-sensitive amacrine cells will allow us to determine whether they play a role in S1P signaling.

Time- and taste-dependent enhancement of conditioned taste aversion learning by d-cycloserine (oral presentation)

R. A. Davenport and T. A. Houpt

Program in Neuroscience, Department of Biological Science, Florida State University, Tallahassee, FL 32306

            NMDA receptors (NMDARs) have been implicated in conditioned taste aversion (CTA) learning, but the role of the NMDAR NR1 subunit, the "glycine-binding site," has not been well characterized. NMDAR neurotransmission in the forebrain is unique in that the presynaptic release of glutamate causes glial cells to synthesize d-serine, the endogenous and obligatory co-agonist at the postsynaptic NR1 subunit. D-serine does not saturate NMDARs in vivo. We have found that the potent systemic agonist d-cycloserine (DCS) potentiates short-delay, but not long-delay, CTA learning. This effect is not due to a short half-life of DCS, a build-up of learned safety during the long-delay, or the long temporal distance between the taste and toxin stimuli. In fact, the potency of DCS decreases with time elapsed since taste stimulus onset. We speculate that basal levels of endogenous d-serine in gustatory relays are low, but slowly increase after presynaptic glutamate release induced by a novel taste stimulation. Thus there may be a brief time period after taste stimulation when endogenous d-serine levels limit, and therefore exogenous DCS can augment, CTA acquisition.

Augmentation at neuromuscular junctions in crustacean walking legs occurs over behaviorally constrained burst frequencies (poster)

Richard B. Dewell

Biological Sciences, Louisiana State University, Baton Rouge, LA 70803

            Nervous systems use rhythmic bursting of motor neurons to produce locomotion.  In arthropod muscle strong summation of postsynaptic potentials can occur within these bursts due to facilitation.  While facilitation occurs at times under a second, longer term enhancement also occurs at these neuromuscular junctions, a process termed augmentation.  Augmentation produces an increase in both the postsynaptic potential and the contraction force of crustacean leg muscles with interburst intervals as long as a minute.  Very little research has focused on the physiological role of this process.  We used in vitro preparations of the walking legs of the crabs Carcinus maenas and Libinia emarginata and applied repetitive stimulation to individual motor neurons to measure this phenomenon.  Using burst frequencies, durations, and cycle periods within the ranges recorded from freely walking animals, identical bursts of neural activity produced significantly larger responses from the muscle on successive applications.  Area under the rectified EMG (used to estimate total postsynaptic current) and force of contraction both could double in magnitude within the behaviorally constrained range.  Both the EMG and contraction showed a greater increase at the start of the burst. The electrical response of the muscle from the first action potential in a train increased up to 300%, and the 50% rise time decreased by approximately 25%.  The exact amount of augmentation was dependent upon the firing pattern within bursts and the timing between bursts.  These data imply that the movement produced during a step from a given train of stimuli can depend on the pattern of activity preceding the burst.

Aging-related neuronal and urothelial changes in the rat urinary bladder (poster)

Natalia Dmitrieva and Guohua Zhang

Program in Neuroscience, Florida State University, Tallahassee, FL 32306

            Overactive bladder is a common urinary bladder problem in the elderly population. It is possible that adrenergic innervation, which plays a regulatory role in the urinary bladder is affected by aging. We have shown that α1D adrenergic activity increases in the bladder of old rats (Dmitrieva et al., 2007). We also found that expression of α1D receptor increases with aging especially in the urothelium (ephithelial layer of the bladder). We hypothesized that adrenergic innervation undergoes structural changes with aging, and different factors including vascular endothelial growth factor (VEGF), high affinity receptor (trkA) for nerve growth factor, and chemokines such as neurite outgrowth inhibitory protein A (NOGOA) and SLIT2 influence this process.

Expressions of these chemoactive molecules, as well as adrenergic (VMAT, vesicular monoamine transporter) and sensory (SGRP) neurotransmitters in the bladder of young (6-month old) and old (24-month old) rats were assessed by immunofluorescence. Images were visualized by epifluorescent microscopy. The results revealed that there were fewer neurites immunolabeled for VMAT2 in smooth muscles of bladders of old rats, but more VMAT2-labeled nerves in the suburothelial region in these rats. Unlike adrenergic innervation, CGRP contained nerves were found having numerous projections close to urothelim as well as in smooth muscles in both groups. There were no visible changes in CGRP innervation between old and young rats. Neurites double labeled for both CGRP and trkA, as well as for VMAT and trkA were observed in both groups. The bladder urothelium from old rats was strongly labeled with antibodies for trkA and VEGF. Opposite to this growth factor and NGF receptor, immunreactivity for NOGOA and SLIT2 proteins was stronger in the urothelium of young rats. Furthermore, the suburothelial region in young rats had considerably higher level of immunostaining for SLIT2.

            These results indicate that adrenergic innervation undergoes significant structural re-organization within the bladder wall with aging, which involves partial degeneration of adrenergic nerves in the smooth muscles and ingrowth of these fibers into the suburothelial region of the bladder. It is possible that these changes are produced by an aging-dependent decrease in expression of chemo-repulsive proteins NOGOA and SLIT2 in the urothelial and especially suburothelial layer, and an increase in VEGF production in the urothelium.

Expression of exogenous AChR subunits in receptor-null fish clarifies the role of AChR phosphorylation (poster)

K. E. Epley, R. Price, J. M. Urban, and F. Ono

The Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32080

            The zebrafish, Danio rerio, offers a unique opportunity to study the role of acetylcholine receptor (AChR) modification in vivo.  The zebrafish mutant Sofa potato (sop -/-) is completely paralyzed due to a lack of surface expression of endogenous AChRs in the neuromuscular synapse.  This absence of receptors is caused by a point mutation in the δ-subunit of the receptor. By expressing δ-subunits with mutations in receptor-less fish, we are able to examine the role of receptor modification in synapse development and synaptic transmission.  A tyrosine residue is phosphorylated in the intracellular loop of the AChR δ-subunit as the neuromuscular synapse is formed. The timing of this phosphorylation suggests that it plays a role in synapse formation, but the role has never been determined in a synapse in vivo.

            A δ-subunit sequence with yellow fluorescent protein (δ-YFP) was expressed in sop-/- mutant fish. YFP was conjugated to the intracellular loop of the δ-subunit to visualize its expression and distribution. δ-YFP was injected into fertilized eggs, and the expression was analyzed in sop -/- fish. Sop -/- embryos that express δ-YFP do not exhibit complete paralysis. Confocal microscopy and electrophysiological recordings show that receptors with δ-YFP subunits can form functional receptors at the synapse and synaptic transmission can occur. We then mutated the tyrosine residue of δ-YFP (δYtyr) to block phosphorylation. δYtyr was introduced into sop-/- fish in a fashion similar to δ-YFP. In the absence of tyrosine phosphorylation, transgenic subunits still form functional receptors and cluster at the synapse. Thus, in spite of the timing coincidence of synapse formation and tyrosine phosphorylation, functional synapses can form in the absence of tyrosine phosphorylation in δ-subunits.

            upport contributed by:  NINDS Grant 1R01NS050388-01A1 to FO and Muscular Dystrophy Association Grant MDA3818 to FO.

The C-terminal region of the Basigin transmembrane domain interacts with MCT1 (oral presentation)

NiCole A. Finch,¹ Paul J. Linser,² and Judith D. Ochrietor¹

¹Department of Biology, University of North Florida, Jacksonville, FL 32224; ²The Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32080

            Purpose:  It has been demonstrated that monocarboxylate transporter-1 (MCT1) interacts with Basigin using immunoprecipitation and fluorescence resonance energy transfer (FRET) techniques.  It has been hypothesized that these two membrane proteins interact via the transmembrane domain of Basigin.  We therefore sought to determine which of the amino acids are necessary for this interaction.  Methods:  A full-length 6X-histidine-tagged transmembrane protein (24 amino acids), as well as 6X-histidine-tagged deletion mutants were generated using the pET102/D vector (Invitrogen).  The domain was truncated from both the N- and C-termini.  ELISA analyses, in which endogenous MCT1 was captured and probed with the full length Basigin transmembrane domain or a deletion mutant, were performed.  Results:  We found that the probe containing the entire transmembrane domain of Basigin (amino acids 1 to 24) did interact with MCT1.  An interaction was also observed when a probe containing amino acids 13 to 24 of the Basigin transmembrane domain was used.  A comparable interaction was observed when amino acids 19 to 24 were used as a probe, but no signal was observed when amino acids 13 to 18 were used. Conclusions:  The results indicate that the last six C-terminal amino acids of the Basigin transmembrane domain bind to MCT1.  Future studies, including site directed mutagenesis will be aimed at determining the contribution of each of the six amino acids to this binding interaction.

The Drosophila giant fiber system as a model for synaptic competition (oral presentation)

Jason Hill^1,2 and Rodney K. Murphey²

¹Molecular & Cellular Biology Program, University of Massachusetts, Amherst, MA 01002; ²Department of Biological Sciences, Florida Atlantic University, Boca Raton, FL 33431

            While the various forms and basic mechanisms of synaptic competition have been described, there remains a lack of understanding at the molecular level.  By adapting a simple system, like the giant fiber circuit used in Drosophila melanogaster, we have generated a new model in which to screen for genes that play a role in synaptic competition.  The circuit is comprised of a pair of axons from homologous neurons known as the giant fibers which descend from the brain to the thorax and form monosynaptic connections exclusively with the motorneuron on the ipsilateral side.  The giant fibers are inhibited from crossing the midline during development by Slit/Robo repulsive signaling [Godenschwege et.al. 2002].  However, when Commissureless is over expressed in these cells, Robo is removed from the cell surface and the growth cones become insensitive to Slit signaling.  We have found that, in this genetic background, the giant fibers are able to cross the midline and form functional connections to either target, but most often do so in a one-axon-per-motorneuron fashion.  The fact that exclusivity is seen in the absence of a midline barrier suggests that the axons themselves are capable of competing for synaptic targets.  In other words, a giant fiber can access both sides, but can be prevented from synapsing with a motorneuron that is receiving input from a second giant fiber. The creation of this competitive interaction provides a platform on which to screen for proteins with a role in competition.  Preliminary candidate genes are being selected based on known functions such as cell-to-cell adhesion, synaptogenesis, and synaptic plasticity. These studies are being complemented with single cell laser ablations.  Selective removal of a giant fiber will illustrate the synaptic capability of a single axon in the absence of a competitor.  Unilateral and bilateral ablation of the postsynaptic target cells will allow us to examine the competition between two axons in situations where the preferred “one to one” is no longer possible.  Taken together, candidate screens and single cell ablations will allow us to study the molecular mechanisms behind the widely studied issue of synaptic competition in the simple circuit of Drosophila’s giant fiber system.

Identification of hereditary and congenital lens defects in zebrafish (poster)

Billy Hiller,* Alex Csizinszky,* Anna deCarvalho, Ann C. Morris, and James M. Fadool

Program in Neuroscience, Department of Biological Science, Florida State University, Tallahassee, FL 32306

            The purpose of the present study was to generate genetic models of hereditary and congenital defects of the lens. F2 generation mutiginized zebrafish were crossed and at 3, 4 and 5 days post-fertilization (dpf) F3 generation embryos and larvae screened for lens defects. After identification of the phenotypes, 1000 mutant and wild-type embryos are collected to map the genomic location of the mutant gene. Histological analysis is performed on mutant and wild-type larvae fixed in paraformaldhyde for frozen sections and gluderaldehyde and osmium tetroxide for plastic sections. Three different recessive mutations were isolated and characterized.  The first displayed ectopic lens (ufo), characterized by rupture of the lens capsule and drifting of the lens fiber cells. A second, pebbles, was associated with decreased lens size and later developed cataracts.  The third, barney, had the appearance of bubbles or vesicles formed below the lens epithelium.  For all three, homozygous mutant larvae could be grown to adults.  Mapping is currently being conducted to identify mutant loci. These should serve as useful models.

This project is currently being funded by a grant from the National Institutes of Health (NIH) R01 EY017753.

*Contributed equally to this project.

IEG expression in the juvenile songbird brain shows little relationship to singing (poster)

Tiffanie Holloway and Frank Johnson

Program in Neuroscience, Department of of Psychology, Florida State University, Tallahassee, FL  32306

            Immediate early genes (IEGs), zenk and fos, are often used to determine levels of singing-driven neuronal activation within the song control system of songbirds.  This specialized forebrain neural network contains two distinct telencephalic circuits responsible for vocal learning and production: a monosynaptic pathway termed the VMP (vocal motor pathway) and a polysynaptic pathway known as the anterior forebrain pathway.  The VMP in particular, is comprised of two cortical-like regions, the premotor region HVC (proper name) which sends projections to the motor output nucleus RA (robust nucleus of the acropallium).  Previously we have shown that singing by adult male zebra finches induces IEG expression within the VMP that is correlated with song production across the day.  Here, we tracked IEG expression of zenk and fos across an entire day of singing in juvenile males to ask whether the pattern of neuronal activation differs during song learning, when the juvenile brain is undergoing many neuronal and synaptic changes. 

            In the adult, singing-driven zenk expression peaks during the first 2h of morning singing within the VMP and is still present (although at significantly lower levels) after 10h of singing. However, our observations are consistent with Whitney et al. (2000) in that zenk protein expression within the VMP of the juvenile is nearly absent, despite hours of song production.  This suggests that zenk expression may track the development of song, since it is only expressed at high levels in the VMP of adults singing stereotyped song.  Interestingly, we found high levels of fos expression in the VMP of the juvenile, however it showed no correlation to song production across the day.  Recent work has revealed that electrophysiological activity within HVC of the juvenile is not correlated to song behavior as it is in the adult (Crandall et al., 2007).  The combination of electrophysiological recordings and our fos expression results may suggest that high levels of spontaneous baseline neural activity in the developing song control system may be important for processes of vocal learning.

Acute therapeutic intervention blocks development of SCI-spasticity (poster)

J. Hou, R. Jain, R. Parmer, Y. Cheng, , R. Telford, P. Bose, and F. J. Thompson

Possible functional role of rhythmically bursting olfactory receptor neurons (oral presentation)

S. H. Jezzini,¹ Y. V. Bobkov,¹ M. A. Reidenbach,² and B. W. Ache¹

¹Whitney Laboratory for Marine Bioscience and Center for Smell and Taste, University of Florida, Gainesville FL 32610;  ²Department of Integrative Biology, University of California Berkeley CA 94720

            Rhythmically oscillating or "bursting" neurons are fundamental to neuronal network function in both vertebrates and invertebrates, including neural networks associated with sensory processing.  Typically, however, they are not considered in the context of primary sensory signaling. We now report a novel subpopulation of lobster primary olfactory receptor neurons (ORNs) that exhibit spontaneous, rhythmic bursts of action potentials between 0.02 to 0.9 bursts/sec. The bursting is intrinsic: the structure of the bursts and the inherent frequency of bursting are consistent for any given cell and bursting is sensitive to membrane potential and Ca²⁺. The ORNs can be entrained by odorants. Odorants transiently applied to the cells evoke bursts similar to the spontaneous bursts in a phase-dependent manner. The efficacy of entrainment is concentration dependent, with more intense stimuli causing the cell to discharge earlier in the cycle. Data obtained by computer simulation suggest that subpopulations of bursting ORNs that fall within a close range of inherent bursting frequency could be selectively synchronized by matching frequencies of odor presentation. This, together with futher synchronization of the bursting ORN ensemble by periodic stimulus acquisition such as sniffing, could effectively enhance the detection and amplification of weak signals, generally assumed to be one of the hallmarks of olfaction. We argue that odor input from bursting ORNs could potentially provide a novel means for encoding odor information.

            Supported by the NIDCD through DC001655.

Physiological acclimation in crayfish among environment alterations and social interactions (poster)

J. Kolasa, S. Bierbower, M. Adami, and R. L. Cooper

Department of Biology, University of Kentucky, Lexington, KY 40506-0225

            Most animals assess the environment in which they live and consequently alter their behavior according to various stimuli.  Ecological behavioral studies usually  do not account for physiological measures of an animal's heart rate (HR) and ventilatory rate (VR).  Addition of physiological observations allow for an expanded analysis of the crayfish's behavioral as well as autonomic responses.  Measurements of its physiological responses were assessed and utilized to create an index of provoked response.  When no significant behavioral changes were observed the animal was previously characterized as unresponsive to a particular stimuli.  Introduction of environmental cues altered physiological responses that were not behaviorally observed.  Experiments conducted allowed for the crayfish's preference to be observed in conjunction with HR and VR recordings.  Results of this paradigm allowed for comparison of forced versus free-choice environment.  Preliminary evidence indicates crayfish experienced high levels of stress when prevented from moving freely between environments at their own discretion.  Further experiments focused on ventilatory regulation, in particular oxygen debt, involved removal of water from their environment.  Heightened ventilation rate was assessed upon reintroduction of water into the tank and used as a index of hypoxia.  [Support: G. Ribble Fellowship in Biology at the University of Kentucky (JK)].

Group I metabotropic glutamate receptor agonist, DHPG, induced protein synthesis dependent longer lasting synaptic depression during senescence (oral presentation)

Ashok Kumar and Thomas C. Foster

Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610

            Group I metabotropic glutamate receptors (mGluRs) play an important role in learning/memory and processes which regulate the induction of synaptic plasticity. Synaptic plasticity mechanisms can change over development and maturation. Induction of long-term synaptic depression (synaptic-LTD) induced by pattern synaptic activation is increased in aged animals. Furthermore, changes in LTD induced by the group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG), have been observed during postnatal development. However, little is known concerning DHPG-LTD during senescence. The current study examined the influence of group I mGluRs on synaptic transmission during aging. Extracellular excitatory postsynaptic field potentials were recorded from CA3-CA1 synaptic contacts in hippocampal slices obtained from young (5-8 mo) and aged (22-26 mo) male Fischer 344 rats. Bath application of DHPG induced a long-term (60 min) synaptic depression in young and aged rats. The magnitude of DHPG-LTD was enhanced in aged compared to young adult rats. In addition, age-related differences in DHPG-LTD induction were observed such that the mGluR1 antagonist, LY367385 blocked DHPG-LTD only in aged animals. In contrast to young adults, induction involved a significant contribution of NMDA receptors and L-type Ca²⁺ channels indicating a role for Ca²⁺, however; induction of DHPG-LTD was not due to activation of phospholipase C and release of Ca²⁺ from intracellular Ca²⁺ stores. Prior induction of synaptic-LTD did not occlude DHPG-LTD and group I mGluR antagonist, AIDA, did not block synaptic-LTD indicating that the increased magnitude of DHPG-LTD in aged animals was not due to synaptic-LTD. The expression of DHPG-LTD in aged animals was dependent on protein synthesis and the increased expression was associated with a decrease in presynaptic function. The results provide evidence that DHPG-LTD is one of the few forms of synaptic plasticity that increases with advanced age.  In addition, age-related differences in induction and expression mechanisms were observed.

Neuropeptide expression patterns in the gastropod mollusc Tritonia diomedea (poster)

J. L. Lillvis,¹ A. B. Kohn,² L. L. Moroz,² and P. S. Katz¹

¹Georgia State University, Atlanta, GA 30303; ²Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32080

            Gastropod molluscs are excellent model organisms for studying neuronal signaling, synaptic plasticity, and neural circuit evolution.  A persistent limitation in the use of these animals, until recently, has been that no genetic information was available.  Fortunately, the Aplysia genome project is now well underway and a transcriptome of the anaspid, Aplysia californica, has been sequenced.  Building on the advances made with Aplysia, we initiated a CNS transcriptome project for the nudibranch Tritonia diomedea.  Tritonia uses a central pattern generator (CPG) to produce a rhythmic escape swim motor pattern that has been the subject of much investigation. cDNA libraries from the CNS Tritonia were constructed and 10,000 expressed sequence tag (EST) were collected.  These ESTs were assembled, Blasted, and a database was constructed from these data.  Ten full length cDNAs were cloned and identified as putative neurotransmitters, neuropeptides and components of neuronal signaling.  Using in situ hybridization, we identified expression patterns of FMRFamide, APGWamide, small cardiac peptide (SCP), an Aplysia R3-14 peptide homologue, pleurin, clionin, and Tritonia pedal peptide (Tpep).  The expression patterns revealed distinct subsets of neurons and narrowed the list of potential neuropeptide transmitters for one of the CPG components, cerebral neuron 2 (C2).  We will test the specificity of neuropeptide antibodies against the in situ expression patterns, which may provide us with new markers to aid in finding homologous neurons in various molluscan species.  Although this project is still in its early stages, it appears to have potential for future neurophysiological and comparative studies.

            Supported by a grant from NSF to PSK and by a grant from NIH to LLM.

Characterization of the Basigin-CyPA complex in retina (poster)

Stephen J. Lunz,¹ NiCole Finch,² and Judith D. Ochrietor²

¹Department of Chemistry and Physics and ²Department of Biology, University of North Florida, Jacksonville, FL 32224

            Cyclophillin A (CyPA) on HIV interacts with CD147 (Basigin) on T cells as the first step in the infection process. We sought to determine whether this interaction takes place in the retina as well. In retina, the basigin gene expresses two products. One product, Basigin, is expressed on Müller glial cells and the second product, Basigin-2, is expressed on photoreceptor cells. It was not known however, whether CyPA is expressed in retina. Mouse CyPA was cloned from a mouse retina cDNA library using primers designed against the CyPA known to interact with Basigin in blood cells. PCR was used to search for CyPA in cDNA from isolated populations of Müller cells and photoreceptor cells from mouse retina. Sandwich ELISAs were preformed to test for CyPA binding to Basigin. Endogenous Basigin was captured and probed with histadine-tagged recombinant CyPA. The OCR analysis confirmed the expression of CyPA in both Müller cell and photoreceptor cells. Our data indicates that recombinant CyPA binds to Basigin. This is a specific interaction that is not inhibited by the addition of Basigin-specific antibody or the lectin jacalin. We hypothesize that one of the helical domains in CyPA is responsible for the interaction with Basigin. Our continued research will test this hypothesis and provide a more detailed characterization of this interaction.

CRF pathways in the extended amygdala regulate brain reward function during nicotine withdrawal (oral presentation)

C. A. Marcinkiewcz,^1,2 M. M. Prado,¹ S. L. Constance,¹ S. K. Isaac,¹ M. S. Gold,^1,2 and A. W. Bruijnzeel^1,2

¹Department of Psychiatry and ²Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610

            Dysregulation of brain stress pathways has been implicated in the pathophysiology of nicotine dependence, a chronic relapsing disorder characterized by compulsive tobacco smoking and a withdrawal syndrome upon smoking cessation. Exposure to stress stimulates the release of corticotropin-releasing factor (CRF), the putative neuronal substrate for anxiogenic and depressive responses to acute nicotine withdrawal. These systems are predominantly active in subcomponents of the extended amygdala, including the central amygdala (CeA) and the bed nucleus of stria terminalis (BNST), which also comprise an integral part of the brain reward circuitry that mediates the negative reinforcing properties of drugs of abuse. The aim of our studies was to investigate the role of CRF in the extended amygdala in promoting deficits in brain reward function associated with nicotine withdrawal. Male Wistar rats were prepared with stainless steel electrodes in the medial forebrain bundle and bilateral guide cannulae above the CeA or a unilateral cannula above the BNST. Subsequently the rats were trained on an intracranial self-stimulation procedure (ICSS), which provides a quantitative measure of the affective aspects of drug withdrawal. An increase in brain reward thresholds is interpreted as a decrease in brain reward function. Animals were implanted with subcutaneous osmotic minipumps containing nicotine (9 mg/kg/day, 3.16 mg/kg/day, free base) or saline to induce nicotine dependence. D-Phe CRF_12-41, a nonspecific CRF antagonist, was infused into the CeA or BNST prior to precipitating withdrawal with the nicotinic receptor antagonist mecamylamine (3 mg/kg, sc) and brain reward thresholds were assessed by ICSS. Our results show that D-Phe CRF_12-41 infused into the CeA, but not the BNST, prevents elevations in brain reward thresholds associated with precipitated nicotine withdrawal. This indicates that increased CRF transmission in the CeA at least partly mediates the negative affective state associated with nicotine withdrawal.

Post synaptic density neuromodulation of Shaker channel current (oral presentation)

D. R. Marks and D. A. Fadool

Program in Neuroscience and Institute of Molecular Biophysics, Department of Biological Science, Florida State University, Tallahassee, FL  32306

            Previous work by our laboratory has demonstrated a pivotal role for the voltage-gated Shaker potassium channel (Kv1.3), highly expressed in the olfactory bulb (OB), in acuity, threshold, and odorant discrimination (Fadool et al., 2004).  The insulin receptor (IR) kinase is expressed at high levels in the mouse OB where it is found to suppress 36 ± 4% of Kv1.3 current via tyrosine phosphorylation of critical N and C terminal residues (Fadool et al. 1998).  We now show using patch-clamp electrophysiology that the adaptor protein post-synaptic density 95 (PSD-95) disrupts insulin-evoked Kv1.3 current suppression and restores peak current amplitude to 98 ± 5% of initial peak current.  PSD-95 also exhibits strong modulatory effects alone on mouse Kv1.3 channel inactivation kinetics by speeding the channel inactivation by 52 ± 4%.  We used immunocytochemistry and confocal microscopy to show that all three proteins are co-localized in the mouse OB, with PSD-95 showing heavy labeling across all neurolamina including the glomeruli.  Using SDS-PAGE and Western blot we found that PSD-95 co-immunoprecipitates with Kv1.3, as well as the IR kinase, demonstrating a multiple protein-protein interaction in the heterologous expression system of human embryonic kidney (HEK293) cells.  Confocal imaging revealed PSD-95 clusters Kv1.3 in transfected HEK293 cell membranes, as well as clusters the IR kinase, but only in the presence of Kv1.3.  A PSD-95 mutant lacking the SH₃ and guanylate kinase (GK) domain (PSD-95 ΔSH3) was constructed by cDNA truncation of PSD-95, as well as use of previous mutants created by A. El-Husseini (2000) to dissect the interaction of these three proteins through co-immunoprecipitation and confocal microscopy.  The PSD-95 ΔSH3 mutant fails to cluster the IR kinase with Kv1.3 in HEK293 cell membranes, suggesting the IR kinase-PSD-95 protein-protein interaction is mediated by the PSD-95 SH₃ domain.  PSD-95 ΔPM (minus palmitoylation mutant) was also used to demonstrate that PSD-95 may be involved in the trafficking and distribution of Kv1.3 by visualizing channel membrane and cytosol distribution in transfected HEK293 cells using confocal microscopy.  We propose a model of interaction of Kv1.3, IR kinase, and PSD-95 where Kv1.3 channels are bound by PDZ domains 1 & 2 of PSD-95 and the IR kinase is bound by the SH₃ domain.  These data suggest that PSD-95 may influence the excitability of synaptic connections in the mouse OB via K channel interaction and subsequent modulation.  Supported by National Institutes of Health (NIH DC03387; NIDCD).

Kv1.3 current suppression by tyrosine kinases may result from channel subunit multimerization (poster)

D. R. Marks, K. Tucker, and D. A. Fadool

Program in Neuroscience and Institute of Molecular Biophysics, Department of Biological Science, Florida State University, Tallahassee, FL  32306

            Modulation of outward potassium currents and ion channel biophysics is a method to alter membrane excitability, which can play a crucial role in the transmission, modulation, and processing of olfactory information from the olfactory sensory neurons (OSNs) to higher brain regions.  Cellular (CTK) and receptor (RTK) tyrosine kinases play essential roles in cell signaling and synaptic transmission, and show high expression in the olfactory bulb (OB).  The voltage-gated Shaker potassium channel, Kv1.3, is a major outwardly-rectifying channel within the OB, carrying 60-80% of the outward current in mitral cells and has been studied for its modulatory role in olfaction.  Previous work has documented the modulation of Kv1.3 by cellular and receptor tyrosine kinases, providing further insight into the mechanism of neuronal excitability and regulation in the OB.  Phosphorylation of Kv1.3 at key tyrosine residues by CTK and RTK suppresses Kv1.3 current, although the mechanism by which tyrosine phosphorylation evokes current suppression is unclear.  RTK-and CTK-induced current suppression of Kv1.3 is of significant interest to understand how voltage-gated activity may modulate the coding of olfactory information.  We now provide evidence that tyrosine phosphorylation of Kv1.3 evokes multimerization of Kv1.3 subunits in conditions in which current suppression is observed via patch-clamp analysis.  We show that tyrosine phosphorylation of key residues promotes tight association of channel subunits, which cannot be dissociated via intensive denaturing conditions, suggesting that the association is covalent in nature.  We employed an immunoprecipitation/Western analysis strategy to explore subunit multimerization of Kv1.3 by three kinases, as heterologously expressed in HEK293 cells.  Data indicate that the insulin receptor kinase, the neurotrophin receptor, TrkB, and the cellular TK v-Src all induce multimerization of Kv1.3 subunits; immunoreactive bands are observed at 54/ 62, 97/120, and 220 kDa, which represent the calculated weight of one, two, and four channel subunits, respectively.  These bands persist under 9M Urea, 4X KFL, and 10 min 95EC heating conditions, and are only recognized by antisera directed against Kv1.3 or antiphosphotyrosine.  We hypothesize that tyrosine phosphorylation of Kv1.3 induces subunit multimerization that is functionally tied to kinase-induced current suppression and that multimerization of the Kv1.3 is the event that induces channel current suppression.

            Supported by NIH grant DC003387 and a Robinson Foundation Grant from Tallahassee Memorial Hospital.

Directing expression of TRPC2 to the plasma membrane in cell culture (poster)

T. G. Mast and D. A. Fadool

Program in Neuroscience and Institute of Molecular Biophysics, Department of Biological Science, Florida State University, Tallahassee, FL  32306

            Transient receptor potential channel type C 2 (TRPC2) is a ligand-gated non-specific cation channel.  TRPC2 is expressed in the vomeronasal organ (VNO) of vertebrates and is thought to be essential for VNO function.  Mice with a gene-targeted deletion in TRPC2 channel have a 60-75% reduction in VNO transduction current elicited by either isolated or compound pheromones.  These TRPC2-deficient mice are also impaired in mating behavior.  Rodent vomeronasal sensory neurons (VSNs) also express the inositol-trisphosphate receptor type 3 (IP3R3).  IP3R3 and TRPC2 co-localize to VSN sensory cilia and have been shown to directly interact in immunoprecipitation experiments.  To investigate the protein-protein interactions between TRPC2 and other proteins, including IP3R3, we first investigated expression of TRPC2 in a heterologous expression system.  Utilizing a standard transient transfection protocol, TRPC2 was expressed in human embryonic kidney 293 cells (HEK293).   Under these conditions TRPC2 does not reliably insert into the plasma membrane and has a low transfection efficiency of 30% compared with 53% efficiency observed for a control channel (the Shaker-family potassium channel Kv1.3).  To increase TRPC2 channel insertion into the plasma membrane, HEK293 were co-transfected with either receptor expression enhancing protein 1 (REEP1) or receptor transporting protein 1 (TRP1).  REEP1 and RTP1 are chaperones expressed in the VNO and have been shown to facilitate the proper cell-trafficking and plasma membrane insertion of olfactory receptor proteins and bitter-taste receptors.  HEK293 cells co-transfected with TRPC2 and REEP1 or RTP1 have low transfection efficiencies (25 and 24%, respectively) and do not exhibit increased surface expression by either biotinylation or confocal imaging. Data suggest that neither chaperone is able to facilitate increased membrane insertion of TRPC2.

            Supported by: Florida State Neuroscience Fellowship (TGM) and NIH grant DC003381 (DAF).

Examining the role of Notch in retinal progenitor cell proliferation (poster)

A. Millet and J. M. Fadool

Program in Neuroscience, Department of Biological Science, Florida State University, Tallahassee, FL  32306

            Notch signaling is important for directing aspects of cell specification during development.  In the vertebrate retina, upon binding the ligand Delta, Notch signaling suppresses neuronal differentiation, and is also implicated in gliogenesis. In zebrafish (Danio rerio) mind bomb (mib) is an E3 ubiquitin ligase that interacts with Delta to promote its endocytosis when bound to the Notch extracellular domain and is essential for Notch signaling. The purpose of the present study was to determine the role of Notch signaling in retinal cell proliferation.  Mib mutants and their wild-type siblings were injected with bromodeoxyuridine (BrdU) to mark mitosis at 2-hour intervals between stages of 48 and 56 hours post fertilization (hpf). Immunolabeling of BrdU positive cells and various retinal cell types was performed.  Mib^-/- embryos display abnormal somite development, increased neurogenesis in the brain and spinal cord, and altered pigment formation. The mib^-/- retina contains only early neuronal cell types, namely red cones and ganglion cells.  Analysis of sections by fluorescence microscopy revealed an unexpected BrdU pattern. Injection at 48 hpf showed an absence of labeling in the ganglion cell layer, a few labeled cells in the inner nuclear layer, and robust labeling of the outer nuclear layer and retinal margins.  This is consistent with the inside to outside pattern of development seen in wild type embryos, but is in contrast to studies that suggest loss of Notch leads to premature exit from the cell cycle and an increase of neurogenic phenotypes. Our data suggest an alternative role for Notch in retinal neurogenesis.

            A. Millet supported by a Program in Neuroscience Fellowship.

Functional characterization of microglial amyloid degradation capacity in aged and Alzheimer's disease models (poster)

eMalick G. Njie, David R. Borchelt, and Wolfgang J. Streit

The Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32080

            Objectives:  (1) Develop an assay for measuring amyloid degradation by cultured microglia.  (2) Test whether there are differences in the capacity of microglia from rats of different ages to phagocytose and degrade amyloid.  (3) Study microglia from mice that are tTA/APP; a model of Alzheimer’s disease.  (4) Correlate observed changes in microglial capacity to degrade amyloid to occurrences of morphologically dystrophic microglia found in aged rats and Alzheimer’s disease mice.

            Methods: To study how aging may affect the ability of microglial cells to degrade amyloid, we will measure differences in the kinetics of amyloid degradation in microglial cells extracted from Fisher 344 rats at different ages and use mice that are transgenic models of Alzheimer’s disease.  The three experimental groups are; 1) neonatal rats, 2) aged rats (> 24 months of age), 3) symptomatic APPswe transgenic mice and 4) non transgenic littermates. 

            Microglial cells from the three experimental groups will be tested for amyloid phagocytosis (1 hour incubation) and degradative capacity using two novel assays.  This capacity will be plotted as a time course kinetic that shows the rate of phagocytosis and degradation quantity over 24hrs for each experimental group. We expect to see increased frustrated phagocytosis coupled with decreased degradative capacity in the aged rats and the transgenic APPswe mice.

            The assays what will be employed for above experiments are being refined and validated using easily obtainable N9 and RAW cell lines and also primary cultures of microglia isolated from neonatal mice.

            Results: The phagocytosis assay detects a dose dependant increase in signal when RAW cells are exposed to Aß₄₂FITC.  Using a 500nM concentration of Aß₄₂FITC, this assay indicates that N9 cells and RAW cells show a similar degree Aß₄₂FITC internalization in comparison to primary neonate microglia cultures, however the neonate microglia expel less of the amyloid back into the extracellular fluid.  This suggests that the immortalized N9 and RAW cells which are microglia-like and macrophage-like cell lines, respectively, have more frustrated phagocytosis than neonate microglial cells.  In addition, the degradation assay reports that neonate microglia are much more adept at degrading the amyloid.  Over the course of 24hrs, RAW cells expel internalized Aß₄₂ into the supernatant while neonate microglial cultures show a significant decrease of fluorescent Aß₄₂ and untagged Aß₄₂ lysate signal without a concomitant increase in extracellular fluid amyloid concentration.  This preliminary data indicates that the cell lines are not as proficient at degrading amyloid or have a greater propensity to expel undegraded amyloid in contrast to neonate microglia. 

            Past studies have shown that amyloid is degraded via the endocytic-lysosomal pathway (Hammad et al., JBC 1997).  Validation of the degradation assay comes from the fact that blockade of the lysosomal pathway with 100uM chloroquine treatment results in a significant depression of amyloid degradation and an increase in exocytosis (compared to non drug treated).  Expulsion of amyloid is an expected coping mechanism in response to intolerable buildup of intracellular undegraded amyloid.

            Conclusions: Using cells from immortal N9 cells, RAW cells and primary mouse neonate microglial cells, both the fluorescence based phagocytosis assay and the ELISA based degradation assay have yielded data that indicate neonatal microglial cells are more proficient at degrading amyloid following phagocytosis.  These newly developed assays will prove useful in quantifying microglial phagocytosis and degradation capacity in aged rats and mice with a disease model of Alzheimer’s disease.

The signal transduction cascade for arrestin translocation (oral presentation)

W. Orisme and W. Clay Smith

Department of Ophthalmology, University of Florida, Box 100284, Gainesville, FL 32610

            Visual arrestin is a protein that shows a dynamic light-dependent localization between the rod outer segments (ROS) and the rod inner segments (RIS) of the rod photoreceptor. In the dark, arrestin is predominately localized in the RIS and migrates to the ROS upon exposure to light. We are interested in the molecules that are associated with the signaling cascade that promotes arrestin translocation between the ROS and RIS. Protein kinase C (PKC) and phospholipase C (PLC) have been identified in the rod photoreceptor. However, their roles in signal transduction in any cascade have not been determined. We treated arrestin-GFP transgenic tadpoles with both activators and inhibitors of PKC and PLC to determine if these proteins were associated with arrestin translocation. Arrestin translocation to the ROS was stimulated by both phorbol 12, 13-diacetate (diacylglycerol analog) and m-3M3FBS (activator of PLC) in the absence of light. Interestingly, treatment with chelerythrine (PKC inhibitor) and U73122 (PLC inhibitor) resulted in the localization of arrestin to the ROS instead of being fully distributed in the ROS in response to light. These molecules may play a role in regulating arrestin translocating to the ROS. We also conducted biochemical analysis to determine if there are any proteins affected by drug treatment. Retinal extracts treated with m-3M3FBS showed an increase in phosphorylation of a ~30-kDa protein. We are currently characterizing this protein.

Prolonged odorant environment sculpts neural circuits in the olfactory bulb (poster)

E. F. Padgett,¹ D. Singh,¹ J. H. Hoffman,¹ S. J. Godbey,¹ K. C. Biju,¹ and D. A. Fadool^1,2

¹Department of Biological Science, ²Programs in Neuroscience and Molecular Biophysics, The Florida State University, Tallahassee, FL 32306

            Mitral cell neurons, the largest cells in the olfactory bulb (OB), are second order neurons that are responsible for transmitting sensory information from primary olfactory sensory neurons of the olfactory epithelium (nose) to higher centers of the brain.  Mice with gene-targeted deletion of a predominant Shaker ion channel (Kv1.3), which is typically expressed in mitral cell neurons, have an increased olfactory ability in terms of both threshold and discrimination.  Kv1.3-null animals were crossed with M72TauLacZ mice to create a double mutant mouse that expresses a genetic tag (visualized by β-galactosidase staining) in tandem with the odor receptor (OR) called M72.  Mice at two different ages (adult and postnatal) were chronically stimulated with peppermint, citralva, or acetophenone for a one-month period.  Four different, one month trials have thus far been performed to compare the variables of odorant quality, age, and genotype on the ability to shape mitral cell density or olfactory sensory neuron (OSN) abundance.   Unstimulated postnatal Kv1.3-null mice have a significantly greater mitral cell density compared with that of WT mice but no notable change in mitral cell area or circularity.  Chronically stimulated adult mice demonstrate a reduction in mitral cell density independent of the presence or absence of the Kv1.3 channel.  The opposite is found in postnatal mice (P30-P60), in which chronically stimulated Kv1.3-null mice have no significant change in mitral cell density in comparison to that of WT controls that show significant reduction.  Thus, there is a time-dependent window in which odorant environment can activate normal pruning processes observed in WT controls, but the absence of Kv1.3 activity may prevent this pruning.  This altered anatomical response might be a component of their “supersmeller” phenotype.  Currently we are analyzing our fourth and final trial that takes advantage of a known ligand-OR pair (acetophenone/M72) and an advantageous genetic model for which we will be able to elucidate changes in neural sculpting at both the central (OB) target and the periphery (OSNs).

            This work was funded by NIH DC003381 and HHMI Undergraduate Computational Biology Program at FSU.

Rapid actions of BDNF activation of TRPC currents and capacitative Ca²⁺ entry (keynote address)

Lucas Pozzo-Miller

Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294-2182

            Brain-derived neurotrophic factor (BDNF) exerts prominent effects on hippocampal neurons, but the mechanisms that initiate its actions are poorly understood. We report here that BDNF evokes a slowly developing and sustained non-selective cationic current (IBDNF) in CA1 pyramidal neurons. These responses require PLC, IP3Rs, Ca²⁺ stores, and Ca2+ influx, suggesting the involvement of TRPC channels.  Indeed, IBDNF is absent after siRNA-mediated TRPC3 knockdown. The sustained kinetics of IBDNF appears to depend on PI3K-mediated TRPC3 membrane insertion, as shown by surface biotinylation assays. Slowly emerging membrane currents following theta burst stimulation are sensitive to the scavenger TrkB-IgG and TRPC inhibitors, suggesting IBDNF activation by evoked released of endogenous, native BDNF.  Lastly, TRPC3 channels are necessary for BDNF to increase dendritic spine density. Thus, TRPC channels emerge as novel mediators of BDNF- mediated dendritic remodeling through the activation of a slowly developing and sustained membrane depolarization.

Inhibitors of Hsp90 modulate the formation of peripheral myelin protein 22 aggregates (poster)

Sunitha Rangaraju, Irina Madorsky, Jocelyn Go, and Lucia Notterpek

Department of Neuroscience, College of Medicine, McKnight Brain Institute, University of Florida, Gainesville, FL 32610

            The correct expression of peripheral myelin protein 22 (PMP22) is required for normal Schwann cell function and myelination. Duplication (C22 mouse), deletion or point mutations (TrJ mouse) in PMP22 are associated with a range of demyelinating peripheral neuropathies. Neuropathic Schwann cells exhibit abnormal protein trafficking with decreased turnover rate of PMP22 resulting in intracellular accumulation and subsequent aggregate formation. To aid the folding and trafficking of the wild type PMP22 in neuropathic models, we modulated the levels of heat shock proteins (Hsps), Hsp90 and Hsp70, by activating the heat shock response. In this study, we screened 15 novel Hsp90 inhibitor compounds (analogs of geldanamycin, GA) provided by Conforma Therapeutics. These novel GA-analogs at 50-500 nM show no significant cellular toxicity, as determined by MTT assays. Sixteen hour exposure of cells to these concentrations induces Hsp70 levels. To explore the influence of elevated Hsps on the formation of PMP22 aggregates, Schwann cells were treated simultaneously with the proteasome inhibitor, lactacystin (10 µM) and Hsp90 inhibitor compounds (50 nM) for 16 h. Biochemical analyses of total cell lysates revealed a modest reduction in slow-migrating polyubiquitinated proteasome substrates in response to combined treatment, as compared to Lc alone. The aggregate reducing effect of these compounds (50 nM) upon spontaneous aggregate containing neuropathic cells was also investigated and was associated with a modest reduction in polyubiquitin.  Significantly, improved trafficking and processing of PMP22 in neuropathic mouse Schwann cells correlated with increased myelin production in dorsal root ganglion neuron explant cultures. These results demonstrate that Hsp90 inhibitors are potential therapeutic agents for PMP22 neuropathies.

Detection of complex chemosignals by the main olfactory system (keynote address)

Diego Restrepo¹, Weihong Lin,² Wilder Doucette,¹ Robert Margolskee,³ Gerald Donnert,⁴ and Stefan W. Hell⁴

¹Neuroscience Program, University of Colorado Health Sciences Center, Aurora, CO 80045; ²Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, MD 21250; ³Department of Neuroscience, Mount Sinai School of Medicine, New York, NY 10029; ⁴Department of Biophotonics, Max Planck Institute for Biophysical Chemistry, 37070, Göttingen, Germany

            A rodent’s survival depends upon its ability to perceive odor cues necessary to guide mate selection, sexual behavior, foraging, territorial formation, and predator avoidance. The need to obtain reliable information necessitates an olfactory system capable of discriminating among multiple odors within a complex olfactory environment. In this presentation I will discuss two features that enable the main olfactory system to respond in an efficient manner to the stimuli present in this highly dimensional odor space. First I describe how input to the olfactory bulb is mediated by multiple olfactory subsystems – presumably a divide and conquer approach. I will show that a subset of olfactory sensory neurons (OSNs) express the transient receptor potential channel M5 (TRPM5, an effector of the phospholipase C pathway) and that these OSNs respond to semiochemicals –odors involved in animal communication. In addition, I will show behavioral experiments that indicate that noradrenergic fibers activated by contextual cues modulate processing at an early stage - within the olfactory bulb, before the second synapse in olfactory cortex- resulting in tuning of the ability to discriminate between similar odors. Our data indicate that the olfactory system has evolved into an adaptive multifaceted system in order to cope with the stringent demand imposed by the multidimensional odorous environment.

            The work was funded by NIH grants DC00566, DC004657 and DC006070 (DR), DC03055 and DC03155 (RM), DC006828 (WL) and DC008066 (WD) and by an Exzellenzfond grant from the Max Planck Society (SH).

Characterization of behavioral response to cold and hot facial stimulation in an operant assay (oral presentation)

H. L. Rossi, C. J. Vierck, R. M. Caudle, and J. K. Neubert

Departments of Orthodontics and Neuroscience, University of Florida, Gainesville, FL 32610

            A hallmark of many orofacial pain disorders is cold sensitivity, but unlike heat-related pain, the mechanism of cold perception are not clearly understood.  Molecular mediators of cold sensation such as TRPM8 have been recently identified and characterized using in vitro studies.  Some are beginning to focus on characterizing behavioral responses to cold.  However, reflex-driven assays typically used for evaluating heat-induced pain have been difficult to translate to cold temperatures and do not necessarily evaluate higher cortical processing of nociceptive stimuli.  Operant assays have proven easier to translate to cold temperatures and overcome some of the problems associated with reflex tests.  We previously developed an operant behavior system to evaluate thermal sensitivity in the face (Neubert et al. 2005).  Here we characterized operant behavior with respect to a range of cold and hot stimuli, each presented in a separate test session.  We also characterized the behavior with respect to a choice between a cold or hot stimulus.  Rats were trained to drink sweetened milk while pressing their shaved faces against a thermode. This presents a conflict paradigm between milk reward and thermal stimulus. Here we demonstrate that the cold stimulus response is more modest compared to the heat response.  We also demonstrate that despite the fact that a single cold stimulus only modestly hinders task performance, rats strongly prefer the hot stimulus to the cold stimulus in a thermal preference task.  These results indicate a strong role for an affective component in processing of cold stimuli; perhaps more so than for heat.  Taken together these finding suggest that the single-stimulus task evaluates the sensory component of pain, whereas the thermal preference task can be used to evaluate the affective component of pain in rats.  This study provides the basis for future studies involving orofacial pain and analgesics.

Interactions between heterosynaptic and homosynaptic plasticity of the Tritonia swim CPG synapses (poster)

A. Sakurai and P. S. Katz

Department of Biology, Georgia State University, Atlanta, GA 30303

            Synaptic strength can be modified by both heterosynaptic neuromodulation and homosynaptic plasticity. In this study, we found a complex interplay between these two types of synaptic modification in the swim circuit of the mollusc, Tritonia diomedea. In this neural circuit, the serotonergic Dorsal Swim Interneurons (DSIs) produce heterosynaptic spike timing-dependent neuromodulation of Ventral Swim Interneuron-B (VSI). Specifically, DSI activation produces a transient potentiation phase (<15 sec) followed by a depression phase (up to 2 min) of VSI synaptic strength. In addition to this heterosynaptic neuromodulation, we found that the VSI synapse exhibited homosynaptic post-tetanic potentiation (PTP) that lasted for approximately 10 min after a VSI spike train. The magnitude of the PTP increased proportional to VSI spike frequency during the train. Heterosynaptic plasticity by DSI had diverse effects on the production and maintenance of this homosynaptic PTP, which depended upon the relative timing of the DSI and VSI spike trains. The magnitude of PTP increased when the DSI spike train preceded the VSI spike train. In contrast, the DSI spike train eliminated the PTP when DSI was stimulated after the induction of PTP. The neuromodulatory effect of DSI also depended upon the sate of the VSI synapse; before the induction of PTP, DSI caused only a transient potentiation with no depression phase. After PTP induction, DSI caused a transient potentiation followed by a pronounced depression that apparently eliminated the PTP. Finally we examined the effect of DSI after a series of alternating bursts of VSI and DSI in a fashion that mimicked their activity during the swim motor pattern. The swim-like pattern by itself produced PTP in the VSI-to-VFN synapse. This PTP was blocked by tonic firing of DSI after the swim-like pattern, suggesting the functional role of DSI to turn off the effect of the homosynaptic potentiation. Altogether these results showed that the effect of DSI modulation has either a positive or a negative effect on the homosynaptic plasticity at the VSI synapse, depending on the pattern and the timing of the activity of these two neurons. This work was supported by a grant from NIH.

Zebrafish rod progenitor cells are specified while still in the cell cycle (poster)

Tamera L. Scholz, Ann C. Morris, and James M. Fadool

Program in Neuroscience, Department of Biological Science, Florida State University, Tallahassee, FL 32306

            In mammals, central nervous system damage is typically irreversible.  Unlike mammals, teleost fishes posses the ability to repair retinal neurons following acute injury.  A further understanding of this process may be beneficial to humans with acute retinal damage, retinitis pigmentosa, or other retinal disorders.  We have characterized a transgenic line of zebrafish, the XOPS-mCFP line, whose rod cells degenerate early in development.  Throughout their lifetimes, they continuously and unsuccessfully try to replace the dying rods from a population of proliferative cells known as rod progenitors.  We are studying this line of fish to learn more about the properties of rod progenitor cells.  Retinal cryosections from 10, 15, and 30 days post-fertilization (dpf) wild type and transgenic zebrafish were immunolabeled to examine how early the XOPS-mCFP zebrafish began responding to rod cell death.  While there were virtually no BrdU-positive cells in the 10 dpf wild type retinas, significant numbers of BrdU-positive cells were found in the outer nuclear layer (ONL) of the XOPS-mCPF retinas at 10 dpf.  In contrast to previous studies of retinal injury in zebrafish, we did not observe an increase in BrdU-positive cells in the inner nuclear layer of XOPS-mCFP retinas in response to rod photoreceptor degeneration.  Rod progenitors in wild type and XOPS-mCFP adult retinas were molecularly characterized by immunolabeling and in situ hybridization.  We found that the proliferating rod progenitor cells expressed markers of the rod photoreceptor lineage, such as Nr2e3, neuroD, and crx.  This suggests that, unlike mammals, rod progenitors are specified as rod photoreceptors while they are still in the cell cycle.  Further research on the signals that regulate the rod progenitor cells in wild type and XOPS-mCFP zebrafish should yield important information that could lead to new avenues of treatments for human retinal disease.

            This work is supported by NIH grant R01 EY017753.

Systematic computational exploration of the parameter space of the multi-compartment model of the lobster pyloric pacemaker kernel suggests that the kernel can achieve functional activity under various parameters configurations (poster)

T. G. Smolinski,¹ C. Soto-Treviño,² P. Rabbah,³ F. Nadim,^2,3 and A. A. Prinz¹

¹Department of Biology, Emory University, Atlanta, GA 30322; ²Department of Mathematical Sciences, New Jersey Institute of Technology, Newark, NJ 07102; ³Department of Biological Sciences, Rutgers University, Newark, NJ 07102

The tumor suppressor p53 regulates downstream gene expression to control cellular proliferation and apoptosis in zinc deficient human neuronal precursor cells (oral presentation)

Rikki C. Somers, Nadine M. Tassabehji, Cathy W. Levenson

Program in Neuroscience and Department of Food, Nutrition and Exercise Science, Florida State University, Tallahassee, FL  32306

            In the adult human stem cell proliferation and neurogenesis take place in several regions of the brain, the most prominent of which are the subgranular zone of the dentate gyrus and the subventricular zone. Despite the tremendous therapeutic potential of these cells, little is known about dietary factors that may regulate them.  To study the role of essential trace metal zinc in neuronal proliferation, we employed a chelator (TPEN) to induce zinc deficiency in the human neuronal precursor cell line, Ntera-2 (NT-2). Treatment of 8 µM TPEN for 18 h, resulted in a significant reduction in cell number.  BrdU uptake, a marker of cellular proliferation, was reduced from approximately 50% in untreated controls to 3% in zinc deficient cells (p < 0.001). Nuclear morphology exhibited the hallmark signs of apoptosis including nuclear shrinkage, chromatin aggregation and nuclear blebbing.  Furthermore, dihydrorhodamine123 labeling showed increased production of mitochondrial reactive oxygen species (ROS) after TPEN treatment. Immunohistochemistal staining revealed increases in abundance and nuclear translocation of the tumor suppressor protein p53.  Because p53 has been shown to regulate the cell cycle and apoptotic pathways, we used oligonucleotide array designed to identify down-stream target genes of p53 at 0, 6, and 18 hrs following chelation of zinc. Consistent with our BrdU data, in the initial phases of zinc deprivation, p53 induces regulators of the cell cycle (e.g. 14-3-3sigma, lats2) to arrest cellular proliferation. With time, both anti-apoptotic and pro-apoptotic genes were induced, a balance of which appear to determine cell fate during zinc deficiency. A dominant negative construct was used to knock down expression of p53 and confirm that these molecular mechanisms are dependent on p53 in zinc deficient cells.

Effects of acute treatments with estradiol on anxiety in female rats: role of individual differences (oral presentation)

A. Stack, H. Wang, D. Dietz, C. Bauer, J. Sorenson, and M. Kabbaj

Program in Neuroscience and Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306

            In this study, we aimed to examine the effects of acute injections of different doses of 17-β estradiol (2 μg, 5 μg, and 10 μg E2) or vehicle on anxiety-like behaviors in ovariectomized rats categorized as High Responders (HR) or Low Responders (LR) based on their locomotor activity in a novel environment. In addition, the expression of immediate-early gene zif268 mRNA was used to examine the effects of anxiety on neuronal activation. Ovariectomized adult female Sprague-Dawley rats (n = 96) were divided into HR and LR groups, then injected with either sesame oil (OIL) or 2, 5, or 10 μg 17-β estradiol (E2) and run in the open field test and social interaction 48 hours later. Adult male Sprague-Dawley rats (n = 24) received sham surgery and were used as positive controls. In situ hybridization was performed using a mouse zif268 cDNA clone (sub cloned in our lab) that yields a 382-nt cRNA probe. In the open field, OIL-treated HR females and HR males displayed less anxiety-like behavior than control LR females and LR males. Generally, E2 treatment eliminated individual differences in anxiety-like behavior, demonstrated by the lack of HR/LR differences in measures of time spent in center and number of crosses into the center of the open field. However, when compared to OIL-treated females, 10 μg E2 treatment increased anxiety-like behavior in the open field in HR rats.  When tested for social interaction, females generally displayed more social anxiety than males, although this effect was not significant. Finally, in both the prefrontal cortex and the dorsal striatum, there was a treatment effect in which female E2 10ug rats had significantly higher expression of zif268 than OIL-treated females. Male rats also had significantly higher zif268 expression than OIL-treated females. In conclusion, our data suggest that E2 may be anxiogenic in the open field test.  Our data also suggest that E2 has no effect on social interaction.

The role of fast potassium currents in shaping the activity of pituitary cells (poster)

J. Tabak, N. Toporikova, A. E. Iglesias-Gonzales, M. E. Freeman, and R. Bertram

Program in Neuroscience and Department of Biological Science, Florida State University, Tallahassee, FL  32306

            Many pituitary lactotrophs generate spontaneous patterns of spiking or bursting. This electrical activity translates into calcium (Ca²⁺) entry and prolactin (PRL) release. It is modulated by hypothalamic factors such as dopamine (DA) which inhibits PRL secretion, in part by opening K⁺ channels. We show, using a Hodgkin-Huxley-type model of the lactotrophs, that fast K⁺ currents such as the current flowing through large conductance (BK) channels, or the inactivating A-type current, can surprisingly have a stimulatory effect on Ca²⁺ entry and PRL release. Both I_A and I_BK can transform the electrical activity of the pituitary cells from spiking to bursting, leading to larger Ca²⁺ transients. However, they operate through distinct mechanisms. This paradoxical effect of I_A or I_BK may explain the stimulatory effect of low doses of DA. We are now using perforated patch recordings in conjunction with the dynamic clamp technique to verify that I_A and I_BK can indeed have such dramatic effect on electrical activity and Ca²⁺ entry.

Recovery of song bout structure following HVC microlesion requires auditory feedback (oral presentation)

J. A. Thompson and F. Johnson

Program in Neuroscience, Florida State University, Tallahassee, FL 32306

            Bilateral microlesions to HVC of adult male zebra finches produce a brief (~1 week) destabilization of the vocal pattern characterized by increased variability in note morphology and sequence (Thompson & Johnson, 2006).  The underlying mechanisms that allow birds to recover their song pattern are unknown.  Auditory feedback as well as a basal ganglia circuit (the anterior forebrain pathway, AFP) are both known to be important for vocal plasticity, although each could play distinct roles in the onset and recovery from vocal destabilization.  For example, we have found that lesions within the AFP prior to HVC microlesions prevent vocal destabilization, suggesting that the AFP contributes to the onset of vocal variability following HVC microlesions. 

            Here we have tested the role of auditory feedback in the recovery from destabilized vocal production in adult male zebra finches.  Vocal patterns were recorded preoperatively and birds then received bilateral HVC microlesions.  Following a day of destabilized singing to confirm the impact of lesion damage birds were deafened by removal of cochleae.  Immediately after deafening, post-operative vocalizations were monitored continuously for three weeks; birds with HVC microlesions alone normally recover their vocal pattern within this time period. 

            We found that following deafening, HVC-damage induced destabilization persisted and, more interestingly, did not recover.  This demonstrates that vocal recovery from HVC damage is not simply the product of neuronal repair.  More importantly, our results show that an adult bird can be induced to recapitulate the critical features of juvenile vocal learning:  1) a transition from highly variable to highly consistent vocal production that is 2) dependent on auditory feedback.  We conclude that the memory of the song pattern persists in the event of induced vocal change, but must be referenced to recover. 

            Supported by NIH DC02035 to FJ and by NIH DC008028 to JT

Nitric oxide released from a physiological source affects the motility of neuronal growth cones in vitro (oral presentation)

K. Tornieri and V. Rehder

Department of Biology, Georgia State University, Atlanta, GA 30303

            The gaseous messenger, nitric oxide (NO), has been suggested to play an important role during neuronal development. We have previously shown that global and local application of NO donors on growth cones of identified B5 neurons of the fresh water snail Helisoma trivolvis in vitro induce an increase in filopodial length, a decrease in filopodial number and a concomitant decrease in neurite outgrowth  through the soluble guanylyl cyclase (sGC) pathway (Van Wagenen and Rehder, 1999; Trimm and Rehder, 2004; Welshhans and Rehder, 2005). However, it is presently unclear whether NO released from a physiological source would be able to affect growth cone morphology as well. To address that question cell bodies of identified neurons expressing the enzyme nitric oxide synthase were maneuvered with a poly-L-Lysine coated electrode to the vicinity (20-30 μm) of growth cones, which were tested for their response to NO. B5 cell bodies induced a rapid and reversible increase in filopodial length, with no effect on filopodial number. B19 somata, which do not express NOS, had no measurable effect on B5 growth cones. Inhibition of NOS with 10 µM 7-Ni blocked the filopodial elongation induced by the B5 cell body, suggesting further that the increase in filopodial length was due to NO. Inhibition of sGC with 20 µM ODQ blocked the filopodial elongation in B5 growth cones induced by B5 somata, confirming that NO acted via the sGC pathway. These results suggest that NO released from a single cell can affect growth cone dynamics, confirming further a potential role of NO as a signaling molecule regulating neuronal pathfinding.

            Supported by NSF grant 0343096 to V.R.

Kv1.3, a possible pharmaceutical target for obesity? (poster)

K. Tucker,^1,3 J. M. Overton,^2,3 and D. A. Fadool^1,2,3,4

¹Department of Biological Science, ²Department of Biomedical Sciences, and ³Programs in Neuroscience and ⁴Molecular Biophysics,  Florida State University, Tallahassee, FL 32306

            Mice with gene-targeted deletion of Kv1.3, a voltage-gated potassium channel of the Shaker family, are thin and do not gain weight when challenged with a moderately high fat diet.  Kv1.3 has been shown to modulate glucose uptake in white adipose tissue and be expressed by mitochondria.  Kv1.3 is also expressed in brain regions important for feeding and metabolism such as the hypothalamus and olfactory bulb, where its activity is modulated by insulin.  To address hypothalamic Kv1.3 contribution, we generated doubly homozygous MC4R/Kv1.3-null mice (mmkk) to ascertain if deletion of Kv1.3 channel protein could abrogate weight gain in a model of hypothalamic obesity.  At 9 months, mmkk mice weighed 18% less than MC4R-null mice. This phenomenon began to emerge around postnatal day 60 (P60) at which point there is lower visceral and subcutaneous fat pad deposition in the mmkk mice with no change in nose to anus length.  To characterize the onset of the reduced adiposity phenotype, locomotor activity, mass-specific metabolic rate (VO₂), and ingestive behaviors were monitored in P60-P75 mice.   These experiments revealed no differences in ingestive behaviors but locomotor activity (66% in females, 50% in males) and VO₂ (21% in females, 12% in males) were increased in mmkk mice above that of MC4R-null mice.  These results indicate Kv1.3 deletion reduces fat deposition and total body weight in a model of hypothalamic-driven, late-onset obesity possibly through increased locomotor activity and VO₂.

            This work was supported by:  NIH DC 003387 and DC 00044, and FSU CRC Competitive Planning Grant.

Does reliable neurotoxin action require that neuronal network parameters be tightly regulated? (poster)

Jan Vargas and Astrid A. Prinz

Department of Biology, Emory University, Atlanta, GA 30322

            Previous experimental results and simulation studies show that similar spontaneous electrical activity can arise from different cellular and synaptic properties, both at the level of single neurons and at the level of neuronal circuits. Neuronal circuits thus appear to have large “solution spaces” at their disposal, rather than having to fine-tune their cellular and synaptic parameters to specific values in order to function properly. On the other hand, toxins and neuromodulators often have reliable effects on the same circuit in different animals. If different animals generate the same circuit output on the basis of different circuit properties, how can they react in the same way to application of a toxin or modulator? To address this question we simulated the effects of the I_A channel blocker 4-AP in 452,516 models of the crustacean pyloric pattern-generating network. These three-cell circuit models differed in their cellular membrane conductance composition and their synapse strengths, but all 452,516 circuit models had previously been shown to generate spontaneous network activity that closely mimics the biologically observed pyloric rhythm. We then identified those pyloric network models that responded to application of 4-AP in the same way as the biological circuit with respect to rhythm criteria such as period, burst frequencies, and duty cycles. We found that only a subset of the original 452,516 network models showed a response similar to that of the biological circuit. This implies that although similar spontaneous circuit activity can arise from different circuit properties, the requirement that a circuit model respond correctly to a channel blocker can impose additional constraints on circuit parameters and thus decrease the size of the solution space available to a neuronal circuit. However, the subset of network models that performed correctly during simulated application of 4-AP contained models that differed widely in some of their cellular and synaptic parameters. This suggests that even neuronal network models that need to be able to generate a variety of behaviors in the presence of different toxins or neuromodulators can do so without having to narrowly tune their circuit parameters. 

            We gratefully acknowledge support from the Burroughs Wellcome Fund, the Sloan Foundation, and NIH R01 NS054911-01A1.

Brief and prolonged dietary sodium deprivation reduce chorda tympani nerve responses to NaCl (oral presentation)

J. M. Vaughn, K. S. Curtis, and R. J. Contreras

Program in Neuroscience, Florida State University, Tallahassee, FL 32306

            Eight to ten days of dietary Na⁺ deprivation are necessary to increase 24 h intake of a concentrated NaCl solution. Na⁺ deprivation of similar duration also decreases the sensitivity of the chorda tympani nerve (CT) to NaCl, suggesting that changes in CT responses are necessary for increased NaCl intake. However, our studies indicate that behavioral taste responses change following as little as two days of dietary Na⁺ deprivation. Specifically, short-term lickometer tests and microstructural analysis showed that after two days of Na⁺ deprivation, rats increased licking to concentrated NaCl solutions. Accordingly, the goal of the current study was to determine whether brief dietary Na⁺ deprivation decreases CT responses to NaCl, and to assess CT amiloride-sensitivity after brief (2 days) or prolonged (10 days) dietary Na⁺ deprivation. We recorded whole nerve electrophysiological activity from the CT in response to lingual application of NaCl (75, 150, 300, 450, 600 mM) and to NaCl mixed with 100 μM amiloride, an epithelial Na⁺ transport blocker. CT responses to NaCl were reduced at all concentrations after both brief and prolonged Na⁺ deprivation compared to Na⁺-replete controls. Moreover, amiloride, which suppressed CT responses to NaCl by 35% in controls, had virtually no effect on CT responses in Na⁺-deprived rats. These results suggest that both brief and prolonged Na⁺ deprivation lead to changes in CT responses to NaCl that may selectively involve the amiloride-sensitive component of NaCl taste. Supported by NIH Grants DC 04785 (RJC), T32 NS07437 (JMV).

Adaptations for sideways and forwards walking in decapod crustaceans (oral presentation)

A. G. Vidal-Gadea and J. H. Belanger

Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803

            The production of adaptive behavior is dependent on the successful interaction between several organ systems in an animal. In order to fully understand locomotion, it is necessary to study the different components of behavior. We study the kinematics, physiology, neural, muscular, and skeletal anatomy of the walking machinery of spider crabs. Libinia emarginata is a brachyuran crab that, unlike most of its close relatives, preferentially walks forwards. We show here differences in anatomy and walking behavior between Libinia, a sideways-walking brachyuran (Carcinus maenas), and the forwards-walking crayfish (Procambarus clarkii). We found kinematic and skeletal differences for all three animals that reflected their walking preferences. The merus-carpus joint in Carcinus crabs makes the largest contribution to sideways locomotion. Forwards locomotion, in contrast, is produced by more equal contributions from forwards-articulating joints. We found that the joints that articulated the leg in the preferred direction of locomotion had greater ranges of motion in all three species. The muscles responsible for longer ranges of motion were housed in longer leg segments.

Nitric oxide regulates growth cone morphology via ryanodine receptor-mediated intracellular calcium release (poster)

K. Welshhans and V. Rehder

Department of Biology, Georgia State University, Atlanta, GA 30303

            Nitric oxide (NO) is a gaseous intercellular messenger that is involved in a number of processes during neuronal development, including regulation of growth cone pathfinding. We have previously shown that a brief, local application of an NO donor, NOC-7, onto individual growth cones from Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number, and an increase in the intracellular calcium concentration ([Ca²⁺]). In addition, our previous work demonstrated that NO causes these changes in growth cone morphology via an intracellular second messenger pathway involving soluble guanylyl cyclase, protein kinase G, and cyclic adenosine diphosphate ribose (cADPR). We have now further extended these results to demonstrate that injection of cADPR into B5 neurons mimics NO’s effects on growth cone morphology and [Ca²⁺]. Additionally, we have found that NO causes a release of intracellular calcium via the ryanodine receptor (RyR). Although the initial release of calcium seems to be triggered via the RyR-accessed store, it appears that influx of calcium across the plasma membrane also plays a role in the increase of the [Ca²⁺]. Therefore, this study demonstrates that NO can cause changes in growth cone morphology via an intracellular cascade that results in a calcium increase triggered by the RyR. This work provides a mechanism through which NO can directly and locally affect neuronal growth cone pathfinding during development. The work was supported by NSF grant 0343096 to VR and a stipend from the Brain and Behavior Area of Focus to KW.