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ABSTRACTS OF POSTERS
PRESENTED BY 2006 FUN TRAVEL AWARDEES - ATLANTA, GA
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Lentiviral vectors expressing an inducible receptor are capable of regulating trkA signal transduction.
(522.3)
R. W. ALFA
1, M. H. TUSZYNSKI1,2, *A. BLESCH1
1. Dept Neurosci 0626, Univ California San Diego, La Jolla, CA, 2. Neurology, VAMC, San Diego, CA.
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Over the past decade, numerous studies have demonstrated that NGF gene delivery attenuates the degeneration of basal forebrain cholinergic neurons. The ability to regulate neurotrophin expression or signals downstream from trk receptors remains a desirable goal for in vivo gene transfer. To achieve precise pharmacological modulation of NGF activity, we have generated an inducible trkA receptor (itrkA) by fusing the entire intracellular domain of the trkA high-affinity NGF receptor to 2 binding domains for the synthetic dimerization agent, AP20187 (Ariad Pharm., Inc.). Rat PC12 cells were stably transduced with lentiviral vectors containing itrkA and GFP (via an internal ribosome entry site). Treatment of itrkA-expressing PC12 cells with AP20187 induced neurite outgrowth and differentiation in a time- and dose-dependent fashion, with an ID50 at a concentration of ~1 nM AP20187 with 70% of cells responding by day 3. In addition, western blots demonstrated that AP20187 treatment resulted in phosphorylation of Erk1/2 and Akt in itrkA-transduced PC12 cells, but not in untransduced, wild-type cells. Phosphorylation levels were comparable to levels obtained with 50 ng/ml NGF. These results demonstrate that small ligand-induced dimerization of the intracellular domain of trkA can efficiently simulate the biological activity of NGF in PC12 cells. Future studies will investigate whether this system can also ameliorate lesion-induced cholinergic neuronal degeneration in vivo.
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Calcium-release coupled to depolarization in the rat hippocampal CA1 neurons. (230.12)
J. BERROUT
, *M. ISOKAWA
Biological Sci, Univ Texas-Brownsville, Brownsville, TX.
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Calcium ions (Ca2+) play many important roles in various biological responses such as retrograde signaling. In the hippocampal neurons, voltage-gated calcium channels (VGCC) are a major source to increase the concentration of cytosolic calcium ([Ca2+]c). In addition, the endoplasmic reticulum (ER), an intracellular organelle, contains a high concentration of Ca2+ and acts as a Ca2+ source for the cytosol by releasing Ca2+. One of the Ca2+ release channels in the ER, the ryanodine receptor (RyR) was reported to be activated by VGCC-induced depolarization and the subsequent influx of Ca2+ in the hippocampal CA1 neurons leading to mobilization of endogenous cannabinoids (eCBs) (Isokawa and Alger, 2006). However, the type(s) of VGCCs that are linked to RyRs remains undetermined. We optically recorded RyR-mediated Ca2+ signals in hippocampal neuron slice cultures using acetoxymethyl (AM) ester derivatives of Ca2+ sensitive fluorescent indicator, fluo-3AM. The [Ca2+]c signals were acquired with an upright microscope, coupled to a cooled CCD camera, and analyzed using an imaging software, IPLab. A brief local application of caffeine, a RyR agonist, increased [Ca2+]c in a defined population of CA1 neurons. In order to activate VGCCs, in the presence of kynurenic acid (non-specific glutamate receptor blocker) in the bath, depolarization was induced by local puff application of artificial cerebrospinal fluid (ACSF) containing 200-500 mM KCl. Bath-application of ryanodine, which blocks RyR Ca2+-release channels, isolated VGCC-dependent [Ca2+]c-component from RyR-dependent [Ca2+]c-component in the KCl-induced [Ca2+]c-increase. We are currently investigating the VGCC subtype(s) that has a functional link to activate RyRs with the use of various combinations of VGCC blockers: nifedipine (L-type channel blocker), ?-conotoxin GVIA (N-type channel blocker), ?-agatoxin IVA (P/Q type channel blocker), nickel (T-type channel blocker), and cadmium (broad spectrum blocker). Conclusions from this project will determine the source(s) of Ca2+ that leads to a sequential activation of the RyRs in the hippocampal neurons in slice culture, and provide fuller understanding for the roles of RyRs in eCB-mediated retrograde regulation of neuronal network excitability.
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The effects of cognitive intervention in adolescence on behavioral abnormalities in a neurodevelopmental rat model of schizophrenia. (687.4)
*G. G. CALHOON
, A. M. BRADY
Department of Psychology and Neuroscience Program, St. Mary's College of Maryland, St. Mary's City, MD.
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Clinical data suggest that schizophrenic patients who achieve high levels of education prior to onset of psychotic symptoms have better prognoses than patients who accomplished lower levels of education. This raises the possibility that cognitive stimulation in adolescence may protect against the development of schizophrenic symptoms in adulthood. The present study assessed the effects of cognitive intervention in adolescence on disrupted adult behaviors in the neonatal ventral hippocampal lesion (NVHL) model, a neurodevelopmental model of schizophrenia in rats. At postnatal day (PD) 7, neonatal rats received bilateral infusions of the excitotoxin ibotenic acid (NVHL, n=12) or artificial CSF (sham-lesioned, n=8) into the ventral hippocampus. In adolescence (beginning at PD 28), NVHL rats and sham-lesioned rats were trained in an attentional set-shifting task in a T-maze, which served as the cognitive intervention. Rats in the intervention control condition were given equivalent time to explore the T-maze, but were not trained in the set-shifting portion of the task. Adolescent NVHL and sham-lesioned rats did not differ in their performance on the set-shifting task (p=.83). In adulthood (beginning at PD 56), rats were assessed on a number of behaviors known to be disrupted in the NVHL model. Compared to sham-lesioned rats, NVHL rats displayed impaired prepulse inhibition of the acoustic startle response (p=.003), decreased social interaction behaviors (p=.001), and hyperlocomotion in response to amphetamine (p=.015). These adult abnormalities were not alleviated by the adolescent cognitive intervention. As adults, NVHL rats also performed worse than sham-lesioned rats in a working memory task in an 8-arm radial arm maze, as indicated by total errors over 16 daily trials. However, the performance of NVHL rats that had received cognitive intervention in adolescence was markedly improved compared to NVHL rats that did not receive the intervention (p=.032). The results of the present study are consistent with previous findings of abnormalities in the NVHL paradigm, and suggest that premorbid cognitive intervention may protect against the cognitive symptoms of schizophrenia.
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Synchrotron IR analysis of amyloid conformation in zinc- and iron-treated TgAPP2576 mice. (272.11)
*K. E. CANO
1, J. A. NEMEC1, N. S. MARINKOVIC2, M. V. SHERIDAN1, D. H. LINKOUS1, B. F. JONES3, J. M. FLINN1
1. Psychology, George Mason University, Fairfax, VA, 2. Center for Synchrotron Biosciences, Brookhaven National Laboratory, Upton, NY, 3. Water Resources, United States Geological Survey, Reston, VA.
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The progressive neurodegenerative disorder Alzheimer’s disease (AD) is characterised by the pathological accumulation of the beta-amyloid (AB) protein. AB is present in either alpha-helix or beta-pleated sheet conformation, and metals have been shown to alter the conformation of AB in vitro. The present study examined AB protein conformation in the hippocampus of transgenic APP2576 (Tg) mice expressing AD pathology, and in wild type (Wt; non-transgenic) mice. Males and females of both types were raised pre- and post-natally on either lab water or lab water enhanced with zinc carbonate (ZnCO3) or iron(II) nitrate (Fe(NO3)2). Animals were sacrificed at 12 months of age and their brains fresh frozen. Coronal sections were collected on silver-coated slides and processed using Fourier transform infrared microscopy (FTIRM), utilizing a high-powered ultraviolet synchrotron lightsource to detect protein conformation in the hippocampus. We report here that there were differences in amyloid conformation between the granule cell layer of the dentate gyrus (GrDG) and the adjacent molecular layer and hilar region (F(1,25)=30.4, p<.05). Post-hoc tests showed that the zinc-water mice had significantly less alpha-helical AB in the GrDG than in adjacent regions (p<.05), and the lab-water mice had less alpha-helical AB in the GrDG than in the adjacent hilar region. In contrast, no differences in alpha conformation in these regions were seen in mice raised on iron-enhanced water.
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Spontaneous recovery (SR) of a conditioned taste aversion (CTA) differentially alters extinction-induced changes in c-fos protein expression in rat amygdala (AMY) and neocortex. (752.7)
*G. A. MICKLEY, Z. HOXHA, S. BACIK, C. L. KENMUIR, J. A. WELLMAN, J. M. BIADA,
A. DISORBO
Neuroscience Program and Psychology Dept., Baldwin-Wallace College, Berea, OH.
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Conditioned taste aversions (CTAs) may be acquired when an animal consumes a novel taste (conditioned stimulus; CS) and then experiences the symptoms of poisoning (unconditioned stimulus; US). Animals will later avoid the taste that was previously associated with malaise. Extinction of a CTA is observed following repeated, non-reinforced exposures to the CS and represents itself as a resumption of eating/drinking the once-avoided tastant. SR of a CTA (a revival of the taste avoidance) occurs when the CS is offered after a latency period in which the CS was not presented. This study investigated changes in the Amygdala (AMY), Gustatory neocortex (GNC), and Medial Prefrontal Cortex (mPFC) functioning during acquisition, extinction and SR of a CTA. Brain c-Fos protein expression was analyzed in fluid-deprived rats that acquired a CTA [3 pairings of 0.3% oral saccharin (SAC) and 81mg/kg i.p. Lithium Chloride (LiCl)] followed by extinction training resulting in 90% reacceptance of SAC. Other animals were extinguished but were tested for SR of the CTA upon exposure to SAC following a 30-day latency period of water drinking. Rats were sacrificed on the final day of SAC exposure and brain c-Fos protein expression was evaluated via immunohistochemistry. Animals exhibited a significant SR of the CTA. The number of c-Fos-labeled neurons in GNC and mPFC was low following CTA acquisition, increased dramatically as rats fully extinguished the aversion, and then declined significantly following SR. Low levels of c-Fos expression in the central nucleus of AMY were observed throughout EXT with little change in expression detectable following SR. C-Fos expression in basolateral AMY decreased significantly from EXT to SR. These measurements suggest the dynamic nature of brain activity during acquisition, extinction and SR of a CTA and further reinforce an important role for cortical and amygdalar neurons in the reorganization of learned information.
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Mutation in clathrin-assembly Picalm gene alters striatal dopamine synaptic regulatory mechanisms (173.14)
E. DUBOUE
1
, G. MITTLEMAN2, E. CHESLER3, M. KLEBIG3, *C. D. BLAHA2
1. Biology, Tulane Univ., New Orleans, TN, 2. Department of Psychology, University of Memphis, Memphis, TN, 3. Bioinformatics, Oak Ridge Nat. Lab., Oak Ridge, TN.
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Elevated brain levels of iron in Parkinson’s disease (PD) patients are thought to increase oxidative stress-induced death of nigrostriatal dopamine (DA) neurons. Using www.genenetwork.org, we have identified a mouse gene (clathrin-assembly Picalm) with expression levels in mouse forebrain and striatum that inversely correlate with brain iron content. Mice with a truncated Picalm allele show abnormal iron disposition in multiple body tissues. We determined whether mutations in this gene are also associated with a dysfunction in striatal DAergic transmission in vivo. Alterations in DA terminal autoreceptor (DAR) sensitivity (max. autoinhibition of stimulation-evoked DA following pre-pulse stimulation), DA transporter (DAT) capacity (max. DAT inhibition with 10mg/kg nomifensine), and uptake efficiency (DA clearance following 15 pulse stimulation at 100, 75, and 50Hz) were determined in striatum of urethane (1.5gm/kg) anesthetized Picalm3452SB mutant (homozygous: -/-, heterozygous: -/+) and control (wildtype: +/+) mice using fixed potential amperometry. DAR sensitivity was 2.9 and 1.8 fold higher respectively in -/- and -/+ mice, compared to +/+ mice. DAT capacity was also significantly enhanced in mutant mice (-/-: 2.6 and -/+: 1.6 fold higher vs. +/+ mice). In contrast, uptake efficiency (50% decay time from max. DA evoked release) was significantly lower in mutant mice compared to control for all stimulation frequencies tested (e.g., -/-: 0.32s and -/+: 0.22s compared to +/+: 0.16s for 100Hz stimulation). As clathrin-mediated endocytosis normally mediates internalization and recycling of DAR and DAT, these data suggest a dysfunction in this mechanism resulting in a significant elevation in membrane bound DAR and DAT. These changes in transporter availability coupled to increased autoreceptor inhibition would be expected to decrease basal synaptic concentrations of DA. However, these changes were also accompanied by a decrease in DAT efficiency, which is known to increase synaptic DA concentrations. This reduced DA clearance rate may represent a compensatory mechanism working to maintain normal synaptic DA concentrations. These data show that a mutation in the clathrin-assembly Picalm gene results in important alterations in synaptic regulatory mechanisms leading to a dysfunction in dopaminergic neurotransmission.
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Birefringence follows voltage-sensitive dye signals but not 90 degree scattering in isolated nerves. (513.3)
*A. J. FOUST
1, M. D. MCCLUSKEY2, D. M. RECTOR3
1. Dept Vet and Comparative Anat, Pharmacol and Physiol, Washington State University, Pullman, WA, 2. Physics, Washington State University, Pullman, WA, 3. Vet and Comparative Anat, Pharmacol and Physiol, Washington State University, Pullman, WA.
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We compared birefringence, 90 degree scattered light, and voltage sensitive dye (VSD) changes in isolated lobster nerves to elucidate mechanisms underlying fast optical signals. We applied different toxins during recordings to knock out specific mechanical, chemical, and electrical correlates of neuronal activation that could contribute to birefringence and scattering signals. Tetrodotoxin blocked voltage-gated sodium channels and abolished both electrical and optical signals, confirming the relationship between sodium conductance, neuronal activation, and optical changes. Tetraethylammonium (TEA) occluded voltage gated potassium channels and possibly aquaporin channels, increased the dI/I amplitude, and briefly delayed the falling phase of birefringence and VSD signals, linking birefringence to membrane potential altering mechanisms. In contrast, TEA elicited a long lasting reversal in the scattering signal, suggesting a different cellular mechanism. Dimethyl sulfoxide (DMSO) dissociated the membrane from the cytoskeleton, evoking an amplitude increase and late component in the birefringence signal similar to TEA trials. DMSO also delayed the 90 degree scattering signal recovery, indicating that DMSO affected both ion conductance and structural interactions. Protease, a glial sheath disruptor, caused no change in either optical signal, perhaps due to limited diffusion into the fascicles. Mercuric chloride, an aquaporin blocker, accelerated optical signal deterioration at high concentration, suggesting that the neurotoxin killed the nerve before an effective aquaporin blocking concentration was reached. Birefringence changes were tightly correlated with membrane potential as measured with VSDs in all cases tested, while 90 degree scattered light changes arose from other processes, perhaps cellular swelling. We conclude that multiple cellular mechanisms underlie rapid optical signals, which could be exploited to improve imaging of neural activity in-vivo with high temporal resolution.
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Regulation of hippocampal KCC2 protein levels in the first postnatal week in vivo and in culture. (729.18)
J. G. BRAY,
C. M. FREITAG,
*M. MYNLIEFF
Dept. of Biological Sciences, Marquette Univ, Milwaukee, WI.
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The first two postnatal weeks in rat hippocampus demonstrate a shift in the reversal potential of chloride and thus, a shift from an excitatory effect of GABA-A receptor activation to an inhibitory effect. This shift is caused by an upregulation of the potassium coupled chloride transporter KCC2 expression and a downregulation of the sodium potassium coupled chloride transporter NKCC1 expression leading to a decrease in intracellular chloride. KCC2 mediates chloride efflux while NKCC1 mediates chloride influx. Ganguly et al. (Cell, vol. 105, 521-532, 2001) demonstrated that treatment of embryonic rat cultures with L-type calcium channel antagonists prohibited the shift in chloride reversal potential suggesting that calcium influx through L-type channels is involved in the changes in chloride transporter expression. Experiments in our laboratory have demonstrated that activation of GABA-B receptors in acutely cultured neurons isolated from rat pups in the early postnatal period cause facilitation of L-type calcium currents in hippocampal neurons. Thus, facilitation of L-type current may enhance the upregulation of KCC2 expression. In the present study we used Western blot analysis of KCC2 on proteins extracted from postnatal day 1 and 8 rat hippocampus, as well as proteins extracted 24 hours and 8 days following culture of postnatal day 0 rat hippocampus. As previously shown by others, the level of KCC2 protein increased in vivo in the first postnatal week. Our data demonstrate that this increase in KCC2 expression is mimicked in culture when hippocampal neurons are isolated on postnatal day 0 and kept in culture for 8 days. The hippocampal cultures will be treated with GABA-B antagonists/agonists and L-type calcium current antagonists to determine whether calcium influx through L-type channels and GABA-B receptor activation affect the expression of KCC2 during this early neonatal period. Electrophysiological experiments will be performed to determine if the L-type calcium current facilitation by GABA-B receptors occurs at the same developmental time point as the upregulation of KCC2.
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Diffusion tensor imaging tractography of the human language pathways. (779.10)
M. F. GLASSER,
*J. RILLING
Dept Anthropol, Emory Univ, Atlanta, GA.
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Objective: Considerable evidence suggests that language processing is lateralized in the left hemisphere of the human brain. In this study, we explore asymmetries in the connections between the language areas as a possible neural substrate for the functional lateralization of semantic language processing found in neuroimaging studies and aphasic patients. We focus on the arcuate fasciculus, the main pathway thought to connect Broca’s and Wernicke’s language areas.
Methods: Twenty right-handed male subjects, aged 18-50, were scanned with a Siemens Trio 3 Tesla MRI scanner using an EPI diffusion weighted protocol (b=1000, 6 averages, 12 directions, 1.7 X 1.7 X 2.0 mm voxels). Images were analyzed with the deterministic tractography algorithm in the Siemens DTI Task Card software. In a coronal section of the DTI color map, an ROI was defined in the voxels containing the arcuate fasciculus and the pathway was reconstructed as streamlines. Results revealed that the arcuate had two distinct terminations in the temporal lobe and we divided the pathway into a smaller segment linking Broca’s area and classically defined Wernicke’s Area (BA 22), and a larger segment linking Broca’s area with an area posterior and inferior to Wernicke’s area (BA 21, 37 and 39) thought to be involved in semantic processing. The volume and cross-sectional area of the reconstructed pathways were quantified and compared across hemispheres.
Results: No significant asymmetries were found in the smaller pathway linking Broca’s and Wernickes’ areas. However, a pronounced and highly consistent leftward asymmetry was found in the size of the larger pathway linking Broca’s area with the posterior temporal semantic regions.
Conclusions: When combined with existing evidence from fMRI studies and aphasic patients, our results support an extension to the classic model of the brain’s language network in which the arcuate tract linking Wernicke’s and Broca’s areas mediates speech repetition. Although we find a small pathway corresponding to the classic arcuate fasciculus, we also show that the bulk of the arcuate fasciculus projects to cortex inferior and posterior to classic Wernicke’ area. This portion shows strong leftward asymmetry and likely conducts semantic content between the posterior temporal lobe areas (BA 21, 37 and 39) and Broca’s area and surrounding cortex during complex semantic analysis and spontaneous speech.
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Three-dimensional video analysis of tail withdrawal responses evoked by heat stimuli in the rat. (251.4)
*J. B. HARROLD
, M. A. PARISE, C. L. CLELAND
Biology, James Madison Univ, Harrisonburg, VA.
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Previous results from our laboratory revealed that pinpoint heat stimuli applied to the rat tail evoked withdrawal movements whose direction depended on the location of the stimulus. These results were obtained either by recording isometric force (Weiss et al. 2006, this meeting) or the initial component of the movement (Cleland and Bauer, J. Neurosci.- Rapid Comm. 22:5265-5270, 2002). Both of these approaches, however, fail to capture the complex 3-dimensional nature of the complete withdrawal response. The goal of these experiments was to quantify the withdrawal response of the rat tail using high-speed, 3-dimensional video analysis. Rats were restrained vertically and the tail was marked 1/3 of the distance from the body for stimulation and movement tracking. Stimuli (980 nm laser; 2 mm and 1mm diameter) were delivered to the right side of the tail at 5 minute intervals (total 25 trails). Movement was recorded with two high-speed video cameras (250 fps; Redlake) and automatically tracked using ProAnalyst. The direction, speed and magnitude of movement were quantified over 300 ms following the stimulus. Results showed that the initial movement was in the dorsolateral direction, consistent with our previous studies. In the current experiments the initial response was observed to be followed by a strong ventral or left-ventral flexion of tail. These two responses also differed in the way they were produced. The early response arose from local bending just above the point of stimulation, with the lower tail remaining largely passive. The second, ventral response, however, arose from pronounced bending of the entire tail. The stability of the response was assessed by repeated stimulation over 25 trials. When using 2 mm diameter stimuli the direction, velocity and magnitude of response were stable. However, in experiments with 1 mm diameter stimuli there was significant habituation in movement velocity and magnitude, and direction shifted from dorsolateral to lateral. These results suggest that the tail withdrawal reflex in the rat utilizes a complex, multiphasic strategy to avoid injury.
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Phenytoin selectively reduces fear potentiated startle in rats with low serotonin. (67.10)
*C. R. HUGHES
1, N. B. KEELE2
1. Psychology and Neurosci, Baylor Univ, Waco, TX, 2. Psychology and Neuroscience, Baylor University, Waco, TX.
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The amygdala has well-defined roles in both epilepsy and emotional behavior, especially fear learning. We and others have suggested that abnormal emotional behaviors symptomatic of mental illness may result from epilepsy-like hyperexcitability in the amygdala. Anticonvulsants have been used in both humans and animals to normalize inappropriate emotional behavior. Also, many psychopathologies may involve serotonin (5-HT), a critical regulator of cell excitability in the amygdala. The present study investigates the effect of the anticonvulsant phenytoin on fear potentiated startle in animals with normal and low 5-HT.
Rats were conditioned to fear a light stimulus (CS) by standard fear conditioning procedures. Fear potentiated startle (FPS) was used to measure fear learning. Chronic depletion of 5-HT by administration of p-chlorophenylalanine (PCPA), a competitive inhibitor of tryptophan hydroxylase, selectively enhanced FPS in individually housed rats. In the group housed condition, PCPA-treated rats show 35 ± 7% FPS (n = 10) and saline treated controls show 33 ± 7% FPS (n = 10). However, in the individually housed condition, PCPA-treated rats show 60 ± 8% FPS (n = 13) compared to 38 ± 7% FPS (n = 12) in saline treated controls. Phenytoin (PHN), a widely used anticonvulsant, dose-dependently inhibited the PCPA-induced increase in FPS. FPS was measured 30 min. after injection of PHN (0, 10, or 30 mg/kg i.p.). In individually housed, PCPA-treated rats, FPS was 47 ± 7% in control, and reduced to 26 ± 10% and 6 ± 7% by 10 and 30 mg/kg PHN, respectively. In individually housed, SAL-treated rats FPS was 26 ± 6% in control, and 28 ± 13% and 19 ± 9% after 10 and 30 mg/kg PHN, respectively.
These data suggest that PCPA-induced low 5-HT results in a sub-seizure state of neuronal excitability that produces a hyperactive startle response in the presence of the light CS. This epilepsy-like (PHN-sensitive) state of neuronal hyperexcitability may contribute to abnormal emotional behaviors involving the amygdala, such as posttraumatic stress disorder. Understanding the neurobiological mechanisms of cellular excitability in the amygdala may improve treatment options for patients suffering from fear, anxiety or other disorders involving amygdala dysfunction.
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Endothelin-1-induced occlusion of the middle cerebral artery produces persistent forelimb deficits and bilateral dendritic structural plasticity in layers II/III and V of sensorimotor cortex in rats. (582.4)
L. C. HUSBANDS
1, T. J. PARIKH2, J. E. HSU3, *T. A. JONES1
1. Dept Psychol, Inst Neurosci, Univ Texas Austin, Austin, TX, 2. Biology Dept, Univ. of Texas, Austin, TX, 3. Inst Neurosci, Univ. of Texas, Austin, TX.
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Intracerebral microinjection of the potent vasoconstrictor, endothelin-1 (ET-1), near the middle cerebral artery (MCA) has been shown to produce ischemic damage in neocortical and striatal brain areas that are vital for normal sensorimotor function (Biernaskie et al., 2005). The purpose of this study was (1) to further characterize the behavioral deficits incurred and (2) to assess cellular and structural changes in layers II/III and V of the forelimb representation region of the sensorimotor cortex (SMC) in both the peri-lesion and intact cortices following unilateral ET-1 induced MCA occlusion (MCAO). Lesion animals had significant impairments in the forelimb contralateral to the lesion (p’s<0.05 compared to sham-operates) in coordinated movement during locomotion, forepaw manipulation of food, and upright postural support, as shown by the footfault test, pasta-eating task, and Schallert cylinder test, respectively. These impairments persisted for the duration of the study (28 days after surgery). There was a significant increase (p’s<0.05) in the surface density of dendrites immunolabeled with microtubule-associated protein 2 (MAP2), a cytoskeletal structural protein associated with dendritic restructuring and growth, in SMC layers II/III and V of both hemispheres of lesion animals compared with sham-operates. These findings provide additional evidence that ET-1 induced MCAO results in persistent forelimb sensorimotor impairments and indicates that these effects are coupled with bilateral neocortical dendritic plasticity. Because of its low mortality rate and reliability, this approach may be particularly useful for exploration of neuronal reorganization in the chronic post-infarct period.
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Acute administration of the neurosteroid progesterone improves blood-brain barrier integrity leading to a reduction in secondary cell death after traumatic brain injury. (682.2)
S. JOHNSON
1, M. CEKIC1, *S. W. HOFFMAN2, E. WASHINGTON1, C. SCHLEGEL1, S. CUTLER1, S. WHITE1, D. STEIN1, J. VANLANDINGHAM1;
1. Department Emergency Medicine, Emory University School of Medicine, Atlanta, GA, 2. Dept Emerg Med, Emory Univ, Atlanta, GA.
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The primary impact of a traumatic brain injury (TBI) initiates a destructive wave secondary cell loss due to extravasation of neurotoxins through a compromised blood brain-barrier (BBB). Previous work has shown that the neurosteroid progesterone (PROG) maintains BBB integrity, decreases edema and improves cell survival (increases Bcl-2, decreases caspase-3). Interestingly, the cells that constitute the BBB express P-glycoprotein (PGP), an efflux pump that prevents the accumulation of cytotoxins in the neuronal milieu. PGP expression is dependent on the transcriptional actions of the pregnane-X receptor (PXR). NTFs further attenuate apoptosis and improve both BBB-associated (astrocytes and endothelial) and neuronal cell survival. These studies were designed to analyze the effects of systemic PROG administration on both NTF and PXR-induced PGP availability in the penumbral region of the contused brain. Male Sprague-Dawley rats were given a bilateral medial frontal cortex (MFC) contusion or a sham surgery. Injured groups (n=6/group) were injected at 1, 6, 24 and 48 h with either PROG (16 mg/kg) or vehicle (22.5% hydroxypropyl-ß-cyclodextrin). Brain tissue was freshly extracted separately from each hemisphere the injury site at 72h for both protein and gene expression analyses. While both injured groups had higher expression of PGP than the sham controls using real-time PCR (p<0.05), the highest expression was found in the injured rats given PROG as compared to the injured controls (p<0.05). This suggests that TBI activates protective mechanisms in BBB constituents, which is further enhanced by the administration of PROG. Western blot analyses further demonstrated that PXR was higher in the PROG-treated group compared to both sham and vehicle (p<0.05), paralleling the significant increase in PGP expression levels. Pro-survival NTFs (NGF and BDNF) were also significantly higher in the PROG-treated group compared to vehicle. Since NTFs not only promote survival of BBB-associated cells, but also indicate that active reparative processes are occurring. Therefore, these results suggest that PROG protects the BBB from TBI, which allows for earlier activation of brain repair mechanisms, which leads to improved recovery of function.
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The NMDA-receptor antagonist CPP, given systemically, impairs exploration but not the ability to complete a modified Hebb-Williams maze. (574.1)
*S. L. JUTILA
1, A. D. REDISH2;
1. Univ Minnesota, Minneapolis, MN, 2. Neuroscience, Univ Minnesota, Minneapolis, MN.
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Both exploration-reduction and goal-finding require learning processes. While exploration can improve performance, animals continue to explore beyond what is necessary to achieve a goal. Exploration-reduction and goal-finding may access different neural systems, and may be differentially dependent on manipulations. We tested the NMDA-receptor antagonist CPP on rats running a modified Hebb-Williams maze (HWM).
Mazes (75 x 75 cm) had moveable plexiglass walls used to form standard configurations (Rabinovitch and Rosvold 1951). The HWM was modified to include return paths on the outside, with food delivery sites at the start and end of the maze. Rats ran through a standard HWM configuration, then turned left (L) or right (R) through 11cm passages to return to the start of the maze. One of the two final returns was correct for each maze. If the rat chose the correct final choice, food was delivered during passage through the return rail.
Rats (n=6) were trained to navigate simple mazes and then were run through a 9-day protocol of 3 novel mazes, using differentiable mazes with unique solutions (2R, 5L, 6R, 7L, 10L, 12R). On the first day of each set, each rat received (IP) 3.5 mg/kg CPP, saline vehicle control, or no-injection control. Rats and mazes were counterbalanced. Rats received each injection type only once. Three types of paths were analyzed: correct final return choice to correct final return choice (laps), start of maze to start of maze (journeys), and times spent within maze but outside of optimum path (errors).
On CPP days, rats achieved the first lap earlier than saline controls. Average time between laps was less under CPP compared to saline, particularly early in the session. However, neither the number of laps nor the number of journeys increased significantly. The number of errors was also increased on the CPP day relative to the saline controls, in large part due to a persistence of errors beyond the first laps. Overall speed was increased slightly on CPP days, but not significantly. However, speed on error paths was greatly increased. When normal rats make errors, they slow down significantly. CPP rats, on the other hand, ran faster when off the optimum path.
CPP rats showed deficient exploration behavior, as well as persistence of error behavior. The ability of CPP rats to complete the maze at equivalent or faster rates than saline controls implies that saline controls are exploring more than necessary to complete the maze.
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Chronic stress-induced suppression of neurogenesis and impairments in spatial memory are prevented by prenatal choline supplementation in rats. (467.4)
*E. D. KIRBY
, M. J. GLENN, C. L. WILLIAMS
Psychological and Brain Sciences, Duke University, Durham, NC.
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Long-term exposure to glucocorticoids associated with chronic stress has a particularly devastating impact on the hippocampus, leading to suppressed adult neurogenesis and deficits in spatial memory. Interestingly, prenatal supplementation with the nutrient choline in rats prevents declines in spatial memory caused by aging and seizures, suggesting that prenatal choline supplementation may protect the hippocampus from neural assault. In the present study, we examined whether prenatal choline supplementation could protect the hippocampus from chronic stress-induced exposure to glucocorticoids. On embryonic days 11-17, Sprague-Dawley rat dams received a control or supplemented diet (1.1 or 5 g/kg choline chloride, respectively). Mixed treatment, cross-fostered litters, raised by control mothers were weaned on postnatal day 25 and housed in same-sex pairs. In adulthood, hippocampal response to chronic stress in female prenatally supplemented and control-fed rats was evaluated using both spatial memory retention and new cell survival. Before other manipulations, all rats received five days of bromodeoxyuridine (BrdU) injections. They were then pre-exposed to the water maze and fixed-platform task for 5 days. The next day, half of the rats began 2 weeks of chronic, variable restraint stress, after which all animals learned a novel platform location. Animals were assessed for retention of that novel location 24 hours later and then sacrificed. The efficacy of our chronic stress regime was confirmed by both a suppression of new cell survival and a marked retention deficit in stressed control rats when compared to unstressed controls. In contrast, supplemented animals showed protection from the effects of stress on both of these measures of hippocampal function: the prenatally choline supplemented stressed animals had similar cell survival levels and memory retention as the supplemented unstressed animals. Based on these findings, it is possible that prenatal choline supplementation may prevent age-induced declines in cognition by protecting the hippocampus from the negative effects of increasing levels of glucocorticoids associated with aging. Preliminary data suggest that this protection may be mediated by alterations in brain derived neurotrophic factor (BDNF) in the hippocampus.
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Repeated exposure to PCP alters stress-induced behavior and striatal c-Fos. (768.11)
T. LIN
, M. SUBRAMANIAN, *S. M. TURGEON
Dept Psych, Amherst Coll, Amherst, MA.
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Prior exposure to PCP has been reported to produce a number of schizophrenia-like behaviors in rats, including alterations in the behavioral responses to stress-inducing stimuli (Noda, et. al., 1995, Br J Pharmacol: 116, 2531-2537 and Jentsch, et al., 1998, Neuropsychopharmacol: 19, 105-113). The present experiments examined the effects of prior exposure to a single or repeated injections of PCP on stress-induced behavior and striatal c-Fos. Twenty-four hours after a single injection (15 mg/kg) or the end of a series of injections (10 mg/kg for 14 days) of PCP, male rats were exposed to either mild stress (novel environment) or a more moderate stress (forced swim test). Animals displayed predicted behavioral changes in both environments with decreases in activity over time in the novel environment and decreases in active behavior accompanied by increases in immobility over time in the forced swim test. In addition, exposure to the stressors increased striatal c-Fos as compared to home cage controls with larger increases seen in the FST than in the novel environment. Single injections of PCP 24 hours prior to stress did not alter either the behavioral or the c-Fos response to the stressors. However, repeated injections of PCP decreased the number of cage-crossings and rears during exposure to the novel environment and increased the number of immobilities in the forced swim test. In addition, repeated PCP increased the number of c-Fos positive cells in the striatum following the forced swim test. These results, although not in complete concordance with prior findings, suggest that prior exposure to PCP alters the behavioral and neurochemical response to stress and are thus consistent with the hypothesis that repeated exposure to PCP produces schizophrenia-like behavior in rats.
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Differential contribution of electrically-evoked dorsal root reflex to peripheral vasodilatation and plasma extravasation. (51.11)
*O. V. LOBANOV
1, Y. B. PENG2
1. Department of Biology, Univ Texas Arlington, Arlington, TX, 2. Department of Psychology, Univ Texas Arlington, Arlington, TX.
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Dorsal root reflexes (DRRs) are believed to be involved in the generation of neurogenic inflammation. DRR can be generated by peripheral stimulation and supraspinal stimulation. DRRs are produced by activation of GABAA receptors in the presynaptic terminals of both myelinated and unmyelinated primary afferent fibers. Plasma extravasation and vasodilatation are two main signs of inflammation. The hypothesis of this study is that electrical stimulation of the proximal stump of a cut dorsal root will lead to generation of DRRs in the neighboring dorsal roots, resulting in plasma extravasation and vasodilatation in the hind paw. We used still pictures to capture color changes related plasma extravasation, and laser Doppler imager to detect vasodilatation. Following intravenous injection of Evans Blue in male Sprague-Dawley rats, stimulation of the distal stump of a cut dorsal root produced plasma extravasation in the ipsilateral paw. Electrical stimulation of the proximal stump of a cut dorsal root generated significant increase of DRRs (from 0.09±0.03 Hz for spontaneous activity to 2.28±0.76 Hz) in the recorded fiber of the neighboring root, but failed to induce plasma extravasation. By using the color intensity before stimulation to normalize the subsequent data, the percentage change of color intensity of the left paw in response to proximal L4 stump stimulation ranged from 2.09±1.09 to 8.50±4.65%; the percent change of color intensity of the right paw ranged form -0.57±1.34 to 3.68±2.42%. The percentage change of color intensity of the left paw in response to distal L5 stump stimulation ranged from -21.28±4.73 to -9.38±1.56%. The negative values represent a darker color and, consequently, plasma extravasation. However, electrical stimulation of the proximal stump induced significant and long lasting increase in blood perfusion. It is concluded that DRRs may be involved in vasodilatation but not plasma extravasation in neurogenic inflammation.
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TrkB immunoreactive cells in the intermediolateral cell column of the spinal cord: effects of exogenous nerve growth factor. (126.20)
*A. M. McCARTNEY
, V. L. ABEJUELA, L. G. ISAACSON
Center for Neuroscience / Department of Zoology, Miami Univ, Oxford, OH.
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The presence of trkB-immunoreactive(-ir) oligodendrocytes in the ventral horn of the lumbar spinal cord was recently reported (Skup 2002), yet there have been few studies detailing trkB expression in the intermediolateral cell column (IML). The objective of the present study was to examine trkB immunoreactivity in the IML at upper thoracic levels (C8-T4) and to determine any effects of exogenous nerve growth factor (NGF). Similar to the previous reports examining the ventral horn, trkB immunoreactivity was localized to small cells in the IML having characteristics of oligodendrocytes. These small cells maintained close apposition to dendritic profiles and cell bodies of ChAT-ir neurons, the marker for pre-ganglionic neurons, but no co-localization was observed. However, co-localization of trkB and ChAT was observed in the preganglionic neurons. Following a two week intracerebroventricular administration of the neurotrophin nerve growth factor (NGF), trkB immunoreactivity was significantly increased in the IML in the small cells (up to 50 fold) and trkB was enhanced in the double labeled trkB/ChAT-ir pre-ganglionic neurons. The present results suggest that brain-derived neurotrophic factor and/or neurotrophin-4 regulate the preganglionic neurons in the thoracic IML. Further, trkB oligodendrocytes and pre-ganglionic neurons in the IML apparently are responsive to exogenous NGF, suggesting that NGF from peripheral targets may influence neurotrophin expression in the spinal cord.
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The substantia nigra reticulata is over-inhibited in a murine model of Huntington's disease. (677.4)
*A. MURPHY-NAKHNIKIAN,
J. L. DORNER, G. V. REBEC
Program in Neuroscience and Dept. of Psychological & Brain Sciences, Indiana Univ, Bloomington, IN.
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Huntington’s disease (HD) is an autosomal dominant, neurodegenerative disorder primarily affecting the basal ganglia. As a primary basal ganglia output structure, the substantia nigra reticulata (SNr) provides inhibitory modulation of the motor thalamus. Apart from internal mechanisms, control of SNr activity depends on the flow of information from the striatum, which previous work suggests is dysfunctional in mice that model HD. To assess SNr activity in HD, we implanted micro-wire electrode bundles bilaterally in 140 CAG knock-in mice (KI) and their wild type littermate controls (WT). Single-unit activity was isolated by autocorrelation and principle component analysis along with qualitative analysis of waveform and signal-to-noise ratio. Data were gathered from awake, free-behaving mice ranging from 12 to 25 weeks of age with a mean age of 19 weeks for both groups. During the recording sessions, the animals displayed a variety of behaviors including locomotion, grooming, and digging. Ethological assessment of KI animals conducted in our lab suggests that KI mice in this age range are in the early stages of symptomatic HD, displaying subtle but significant motor abnormalities. In SNr, neuronal activity appears suppressed in KI mice as our data to date indicate a significantly lower number of spikes/s compared to WT mice (p <0.05). We did not find a significant effect of age, nor an age-by-group interaction, suggesting either that changes in the SNr are not age-related in this model, or that age effects are not discernable within the 12 to 25 week range. Our results are consistent with models of the basal ganglia in which HD is associated with an imbalance of striatal outflow via the direct/indirect pathways. Although the precise influence of these pathways on SNr activity during behavior is unclear, our results suggest that SNr over-inhibition plays a key role in the HD behavioral phenotype.
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Prospective removal of cardiac-induced signal contaminants in fMRI time series. (492.1)
*N. H. NEUFELD
1, S. MEHTA2, L. BOLINGER3, T. J. GRABOWSKI2,4, M. C. MCINTYRE1
1. Psychology, Univ Winnipeg, winnipeg, MB, CANADA, 2. Neurology, Univ Iowa, Iowa City, IA, 3. MR Research and Development, National Research Council Canada, Institute for Biodiagnostics, Winnipeg, MB, CANADA, 4. Radiology, Univ Iowa, Iowa City, IA.
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OBJECTIVE: Cardiac pulsation causes magnetic resonance signal changes that act as noise in functional magnetic resonance imaging (fMRI) time series, reducing the sensitivity of data analysis. Temporal autocorrelation of residual terms may also be introduced, threatening the validity of statistical inference. The shift from block design to event-related design, with its lower sensitivity, emphasizes the need to address cardiac-induced noise. This study compares three approaches to modeling cardiac-induced noise as an effect of no interest within the general linear model.
METHODS: Using the I/OWA system (Smyser et al., 2001), anatomical and functional images were obtained for three subjects on a 1.5 Tesla GE LX CV/i scanner. The image acquisition and cardiac channels were filtered to gather timing information. It was determined at what phase of the cardiac cycle each image was acquired. A voxel-wise deconvolution was then performed using three basis sets (Meizin et al., 2000): Fourier deconvolution using a truncated Fourier series with the relative position within the R-R interval of the cardiac cycle as the phase (Glover et al., 2000), bin deconvolution using the relative time within the cardiac phase indicated by the R-wave, and bin deconvolution using the absolute time from the last R-wave.
RESULTS: Adjusted R2 images showed the three approaches similarly modeled cardiac-related signal changes in brain blood vessels across three subjects. Cardiac-induced noise was modeled in grey matter and showed spatial heterogeneity within and between subjects. Grey matter close to blood vessels and pulsation-susceptible tissues were better modeled by all approaches than grey matter regions more distant from said regions.
CONCLUSIONS: The three approaches all accounted for signal variance to an equal or greater extent in all examined regions than a single cosine function. Spatial heterogeneity in the amount of cardiac-induced signal variance between and within subjects underscores the importance of individual differences and provides further rationale for prospective modeling. Any one of the approaches may be used in fMRI statistical packages or real-time analysis to prospectively remove cardiac-induced signal changes.
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Divalent zinc interacts with leptin pathways in pituitary and hypothalamic models. (355.4)
*K. M. PICKOFF
, A. BETTICA, S. TRUSSA
Dept Biol, Manhattanville Coll, Purchase, NY.
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Leptin, a 16 kDA adipocyte-derived peptide, is part of the hypothalamic-pituitary-thyroid (HPT) axis, contributing in several ways to metabolic regulation, mainly through appetite suppression. Several studies indicate that zinc bioavailability plays an integral role in the regulation of peptide levels implicated in feeding circuits including leptin, although direct involvement of divalent zinc (Zn2+) in leptin pathways has not yet been established. It is suggested that Zn2+ activity involves stabilizing receptor conformation and modulating ligand-receptor binding. Chelation of Zn2+ has been shown to further enhance these mechanisms by protecting against ion-induced toxicity and in some cases, by increasing binding affinity. The concentration of leptin activity in the hypothalamus and anterior pituitary indicate these as potential sites of zinc-mediated enhancement of leptin. As a model for leptin activity in the pituitary, GH4C1 rat pituitary tumor cells were utilized, since they can be easily stimulated to secrete detectable levels of growth hormone (GH) in response to leptin. In this study, 10nM leptin significantly increased GH secretion after 48 hours. Exposure to 40 µM Zn2+, with or without EDTA-chelation, produced a similar increase, indicating that Zn2+ may enhance other mechanisms for GH secretion, and that chelation did not increase the effects of Zn2+. However, exposure to a combination of leptin and chelated Zn2+ produced GH levels of 400 pg/ml, compared to the 50 pg/ml GH stimulated by leptin, Zn2+, or EDTA/ Zn2+, suggesting direct involvement of Zn2+ in pituitary-based leptin activity. In the hypothalamus, leptin stimulates the production and secretion of proopiomelanocortin (POMC) through a JaK-STAT cascade, resulting in increased anorexigenic signaling. Simultaneously, leptin inhibits the synthesis and secretion of neuropeptide Y (NPY), responsible for the propagation of orexigenic signals. To determine Zn2+ involvement in hypothalamic leptin mechanisms, human neuroblastoma SH-SY5Y cells were exposed to 10nM leptin and 10µM Zn2+ alone and in combination for 1 and 5 hours. Leptin significantly decreases NPY levels after 4 hours in SH-SY5Y, confirming their expression of the long isoform leptin receptor (ObR) and catabolic NPY capabilities.
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Specific cytokines are synthesized and released by sympathetic neurons in cell culture. (128.5)
J. TREECE
, S. MISHRA, *M. SRIVATSAN
Department of Biological Sciences, Arkansas State University, Arkansas State Unive, AR.
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Increasing evidence indicates that the immune system and the nervous system interact extensively. Peripheral sympathetic neurons innervate lymphoid tissue. Neurons of the superior cervical sympathetic ganglion (SCG) express receptors for cytokines, interleukin-3 (IL-3), interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Kannan et al., 2000; Soliven and Wang, 1995). These findings prompted us to investigate if SCG neurons synthesize as well as release specific cytokines. SCG neurons from postnatal day 1 Sprague Dawley rat pups were isolated and maintained in cell culture in serum free Neurobasal medium with B-27 supplement and NGF-2.5S (25ng/ml). To obtain and maintain a neuron enriched culture, neurons were pre-plated three times on uncoated culture dishes before the final plating . Further the culture medium contained 20 µM fluorodeoxyuridine and 20 µM uridine to eliminate non neuronal cells. After 48 hours in culture, the culture medium was removed, neurons were washed with phosphate buffered saline and cell lysates were obtained using Pearce lysis buffer. The cell lysate and the culture supernatant samples were centrifuged at 5000 RPM at 4 º C and the supernatant was used for analysis of cytokine profile. Cytokines were detected and quantified by using the Bio-Rad Bio-Plex Suspension Array System and the Bio-Plex 100 array reader run by the Bio-Plex Manager 4.0 software. The Bio-Plex Rat Cytokine 9-Plex A Panel consisting of the cytokine specific beads and detection antibodies was used to bind and detect respective cytokine. Among the nine cytokines, SCG cell lysate contained IL-1ß > IL-4 > TNF-a > GM-CSF and the culture medium contained IL-1ß > TNF-a > IL-4 > GM-CSF. Three fourth of the IL-1ß, TNF-a and half of GM-CSF synthesized in the neurons appeared to have been released into the culture medium. These results show that in cell culture postnatal SCG neurons of rat actively synthesize selective cytokines and release significant amounts of it into the medium. We are currently extending these experiments to investigate if exposure to nicotine alters these cytokine expression and release profile.
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Seizures increase neurogenesis in the dentate gyrus by shortening progenitor cell cycle length. (418.15)
F. P. VARODAYAN
1, M. MARONSKI1, *B. E. PORTER1,2
1. Dept Neurol, Children's Hosp Philadelphia, Philadelphia, PA, 2. Dept Neurol, University of Pennsylvania, Philadelphia, PA.
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Neurogenesis in the dentate gyrus of the hippocampus occurs throughout life and is affected by drugs, stress, depression and seizures. A prolonged seizure, status epileptics (SE) is a potent stimulus for increasing neurogenesis and may contribute to the formation of an epileptic network. The molecular mechanisms that regulate cell birth in the dentate gyrus are unknown, though of great interest for their potential role in depression and epilepsy.
For all experiments, male post-natal day 20 CD rats underwent SE as previously reported (Porter et al 2004). Rats were injected with BrdU at (2 hours, 8 hours, 24 hours, 14 days and 40 days) for time course and sacrificed 4 hours later. Cell cycle length was calculated using a repetitive BrdU labeling protocol (Nowakowski et al 1989) for 12 hours following SE. p27/Kip1 antibody from Cell Signaling Technology was used for immunohistochemistry and cell counts.
SE causes a ~2.5 fold increase in cell birth in the dentate gyrus within 2 hours after SE and lasting for 2 weeks. A 30% but still significant increase in cell birth is present at 40 days following SE. To determine whether the increase in neurogenesis was due to a shortening of the cell cycle in dividing progenitor cells we measured cell-cycle length. We find that the cell cycle is shortened by 25% in the SE exposed animals. To determine if the G1 phase of the cell cycle is altered in the progenitor cells we studied expression of p27/ Kip1 an inhibitor of G1 to S transition. We found that density of p27/Kip1 labeled cells in the subgranular region of the dentate gyrus was significantly increased 30% at 2 hours following SE; but within 4 hours of SE and lasting for 2 weeks, there is a 30% reduction in p27/ Kip1 cell density.
Pilocarpine increases by ~2.5 fold the number of cells born in the dentate gyrus for up to 2 weeks after SE. Increased neurogenesis is due to a 25% reduction in total cell cycle length after SE. Alteration in 27/Kip1 expression in subgranular region of the dentate gyrus following SE suggests that transition from G1 to S is part of the regulatory mechanism for SE shortening of total cell cycle length.
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