Neuroscience 2010 - Presentations from Winnipeg scientists

Abstracts are below the schedule.

  Session Start Time
Presentation Time
Location 		Pres #
& Type		 Poster Board # Authors & Institutions Abstract Title
Session # & Title
Sat 11/13 1:00 PM
1:00 PM - 2:00 PM

Halls B-H 		42.1
Poster 		G55 *S. CURTI1, G. HOGE2, J. I. NAGY3, A. E. PEREDA2; 1Physiol., Sch. of Med., Montevideo, Uruguay; 2Neurosci., Albert Einstein Col. of Med., Bronx, NY; 3Physiol., Univ. of Manitoba, Winnipeg, MB, Canada Properties and molecular determinants of electrical coupling in the mesencephalic trigeminal nucleus (Mes-V)

42.Gap Junctions
Sat 11/13 1:00 PM
3:00 PM - 4:00 PM

Halls B-H 		42.3
Poster 		G57 *J. E. RASH1, N. KAMASAWA2, S. NANNAPANENI3, K. G. V. DAVIDSON1, C. FLORES3, A. DICARO1, T. YASUMURA1, J. O'BRIEN4, J. I. NAGY5, A. PEREDA3; 1Biomed. Sci., Colorado State Univ., Fort Collins, CO; 2Div. of Cerebral Structure, Natl. Inst. Physiol. Sci. (NIPS), Okazaki, Japan; 3Albert Einstein Coll. Med., New York, NY; 4Ophthalmology and Visual Sci., Univ. Texas Hlth. Sci. Ctr., Houston, TX; 5Physiol., Univ. Manitoba, Winnipeg, MB, Canada Neuronal gap junctions in goldfish hindbrain are primarily or exclusively at glutamatergic mixed synapses and are heterotypic, with Cx35 in axon terminals and Cx34.7 in somata and dendrites

42.Gap Junctions
Sat 11/13 1:00 PM
1:00 PM - 2:00 PM

Halls B-H 		42.5
Poster 		H1 W. BAUTISTA1, J. I. NAGY1, *D. A. MCCREA2; 1Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; 2Univ. Manitoba Fac Med., Winnipeg, MB, Canada Distribution of connexin36 in juvenile and adult rodent spinal cord: Co-localization with vglut-1 suggests primary afferent terminals form mixed chemical and electrical synapses

42.Gap Junctions
Sat 11/13 1:00 PM
3:00 PM - 4:00 PM

Halls B-H 		42.11
Poster 		H7 *X. LI1, J. I. NAGY2; 1Dow Neurobio., Legacy Clin. Res., Portland, OR; 2Physiol., Univ. of Manitoba, Winnipeg, MB, Canada Interaction of connexin36 with multi-PDZ-domain protein 1 (MUPP1)

42.Gap Junctions
Sat 11/13 1:00 PM
3:00 PM - 4:00 PM

Halls B-H 		64.3
Poster 		AA18 Y. LI, X. CHEN, S. LI, *G. J. KIROUAC; Dept of Oral Biol., Univ. of Manitoba, Winnipeg, MB, Canada Effect of blocking kappa opioid receptors on fear and anxiety-like behaviors in a rat model of post-traumatic stress disorder (PTSD)

64.Anxiety Disorders: Animal Models and Neuropeptide/Gaba/Glutamate Systems
Sun 11/14 8:00 AM
11:00 AM - 12:00 PM

Halls B-H 		148.8
Poster 		H66 R. ZACHARIAH, C. OLSON, *M. RASTEGAR; Biochem. and Med. Genet., Univ. of Manitoba, Winnipeg, MB, Canada Functional evaluation of MECP2 gene therapy vectors in differentiated neurons

148.Rett Syndrome
Sun 11/14 8:00 AM
8:00 AM - 9:00 AM

Halls B-H 		154.5
Poster 		M15 *E. EFTEKHARPOUR1, S. KARIMI-ABDOLREZAEE2, P. BOURTOS3, M. FEHLINGS4; 1Physiol., Spinal Cord Res. Center, Univ. of Manitoba, Winnipeg, MB, Canada; 2Physiol., Regenerative Med. Program and Spinal Cord Res. Center, Univ. of Manitoba, Winnipeg, MB, Canada; 3Ontario Inst. for Cancer Res., Toronto, ON, Canada; 4Surgery, Univ. of Toronto, Toronto Western Res. Institue, Krembil Neurosci. Res. Ctr., Toronto, ON, Canada Exploratory pathway analysis of temporal gene changes in rat spinal cord injury: Long lasting inflammatory response in chronic spinal cord injury

154.Spinal Cord Injury: Inflammation
Sun 11/14 8:00 AM
8:00 AM - 9:00 AM

Halls B-H 		156.21
Poster 		P12 *J. L. LEMAISTRE1,2, H. D. I. ANDERSON1,3, C. M. ANDERSON1,2; 1St Boniface Res. Ctr., Winnipeg, MB, Canada; 2Pharmacol. & Therapeut., 3Pharm., Univ. of Manitoba, Winnipeg, MB, Canada Astrocyte D-serine release influences NMDA receptor-mediated cerebral vasodilation

156.Cell Death Mechanisms: Excitotoxicty and Calcium
Sun 11/14 8:00 AM
9:00 AM - 10:00 AM

Halls B-H 		180.14
Poster 		SS1 *S. J. GARLAND1, S. KNORR3, C. L. POLLOCK2, C. W. MACDONELL4, T. D. IVANOVA1; 1Dept Physical Therapy, 2Rehabil. Sci., Univ. British Columbia, Vancouver, BC, Canada; 3Hlth. and Rehabil. Sci., Univ. of Western Ontario, London, ON, Canada; 4Spinal Cord Res. Ctr., Univ. of Manitoba, Winnipeg, MB, Canada Motoneuron afterhyperpolarization time course after chronic stroke

180.Motor Unit Recordings
Sun 11/14 1:00 PM
3:00 PM - 4:00 PM

Halls B-H 		247.27
Poster 		I3 *J. S. SCHAPANSKY1,2, G. ODERO4, T. ENNO3, M. DEL BIGIO3, G. GLAZNER4,2; 1St. Boniface Rese Ctr, Univ. Ma, Winnipeg, MB, Canada; 2Dept. of Pharmacol. and Therapeut., 3Dept. of Pharmacol., Univ. of Manitoba, Winnipeg, MB, Canada; 4St. Boniface Res. Ctr., Winnipeg, MB, Canada Reduced Alzheimer's disease pathology and enhanced insulin signaling in early type 1 diabetes in an overexpressing amyloid beta mouse model

247.Alzheimer's Disease: Abeta, Energy Metabolism, Cell Signaling, Autophagy
Sun 11/14 1:00 PM
2:00 PM - 3:00 PM

Halls B-H 		257.6
Poster 		T14 *W. ZHU, F. BEGUM, E. FROST, M. NAMAKA; Fac. of Pharm., Univ. of Manitoba, Winnipeg, MB, Canada Antigen induced expression of Fractalkine in an animal model of multiple sclerosis

257.Demyelination Disorders: Cellular Mechanisms of Pathology
Sun 11/14 1:00 PM
3:00 PM - 4:00 PM

Halls B-H 		258.11
Poster 		V6 L. ZHANG, Y. WU, J. KONG, I. SCHROEDTER, *M. VRONTAKIS-LAUTATZIS; Univ. of Manitoba, Winnipeg, MB, Canada Galanin protects cuprizone-induced demyelination via GalR1 and GalR2

258.Demyelinating Disorders: Cellular Mechanisms of Repair
Sun 11/14 1:00 PM
2:00 PM - 3:00 PM

Halls B-H 		259.14
Poster 		W7 *O. ALLUIN1, S. KARIMI-ABDOLREZAEE2, H. DELIVET-MONGRAIN1, M. MÉARD1, H. LEBLOND1, M. FEHLINGS3, S. ROSSIGNOL1; 1Dept. of Physiol., Univ. of Montreal, Montreal, QC, Canada; 2Dept. of Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; 3Toronto Western Res. Inst., Toronto Western Hosp., Toronto, ON, Canada Longitudinal study of locomotor recovery following chronic compressive spinal cord injury in rats

259.Spinal Cord Injury: Posture and Locomotion
Sun 11/14 1:00 PM
1:00 PM - 2:00 PM

Halls B-H 		259.17
Poster 		W10 U. SLAWINSKA1, *H. MAJCZYNSKI1, E. KISIELNICKA1, L. M. JORDAN2; 1Nencki Inst. Exp Biol, Warsaw, Poland; 2Physiol., Univ. of Manitoba, Winnipeg, MB, Canada Upright bipedal locomotion in rat: A suitable model for investigation of interventions after SCI?

259.Spinal Cord Injury: Posture and Locomotion
Sun 11/14 1:00 PM
4:00 PM - 5:00 PM

Halls B-H 		259.20
Poster 		W13 L. M. JORDAN1, H. FABCZAK2, E. KISIELNICKA2, A. LESZCZYNSKA2, H. MAJCZYNSKI2, J. I. NAGY1, *U. SLAWINSKA2; 1Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; 2Nencki Inst. of Exptl. Biol., Warsaw, Poland Segmental distribution of 5-HT2A and 5-HT2C receptor up-regulation one month after complete spinal cord injury

259.Spinal Cord Injury: Posture and Locomotion
Sun 11/14 1:00 PM
1:00 PM - 2:00 PM

Halls B-H 		290.9
Poster 		DDD10 *E. E. VASQUEZ-DOMINGUEZ, K. E. POWER, B. FEDIRCHUK; Physiol., Univ. of Manitoba, Winnipeg, MB, Canada Scratch-like fictive motor output can be elicited by drug application to cervical segments of the isolated neonatal rat spinal cord

290.Descending Control of Rhythmic Motor Pattern Generation
Sun 11/14 1:00 PM
3:00 PM - 4:00 PM

Halls B-H 		290.11
Poster 		EEE2 E. COUTO-ROLDAN1, Y. DAI1, J. LIU1,2, H. A. GRAVES1, E. S. DENERIS3, *L. M. JORDAN1; 1Dept Physiol., Univ. Manitoba, Winnipeg, MB, Canada; 2Brain Res. Ctr. and Dept. of Med., Univ. of British Columbia, Vancouver, BC, Canada; 3Dept. of Neurosci., Case Western Reserve Univ., Cleveland, OH Neurons of the parapyramidal region are active during a locomotor task and receive an excitatory cholinergic input

290.Descending Control of Rhythmic Motor Pattern Generation
Mon 11/15 8:00 AM
10:00 AM - 11:00 AM

Halls B-H 		332.15
Poster 		C22 A. SALEH1, S. BALAKRISHNAN1, E. MUTCH1, R. VAN DER PLOEG1, E. AKUDE1,2, D. R. SMITH1,2, *P. FERNYHOUGH1,2; 1St Boniface Res. Ctr., Winnipeg, MB, Canada; 2Pharmacol. & Therapeut., Univ. of Manitoba, Winnipeg, MB, Canada Abnormal NF-kB activity and associated impairments in axonal outgrowth in sensory neurons isolated from diabetic rats are corrected by cytokines

332.Axon Guidance: Cellular and Organismal Mechanisms
Mon 11/15 8:00 AM
10:00 AM - 11:00 AM

Halls B-H 		339.19
Poster 		F14 *Y. DAI, L. M. JORDAN; Univ. Manitoba, Winnipeg, MB, Canada Tetrodotoxin, dihydropyridine, and riluzole resistant persistent inward current: A novel sodium channel in rodent spinal neurons

339.Sodium Channel Physiology I
Mon 11/15 8:00 AM
9:00 AM - 10:00 AM

Halls B-H 		342.10
Poster 		G16 D. ZHANG1, B. C. ALBENSI1,2, *F. E. PARKINSON1; 1Univ. Manitoba, Winnipeg, MB, Canada; 2St Boniface Res. Ctr., Winnipeg, MB, Canada Equilibrative nucleoside transporter 1 (ENT1) regulates hypoxic/ischemic adenosine levels in hippocampal slices

342.Synaptic Transmission: Modulation by Ach, Amino Acids, and GABA
Mon 11/15 8:00 AM
9:00 AM - 10:00 AM

Halls B-H 		364.10
Poster 		DD1 X. CHEN, Y. LI, *S. LI, G. J. KIROUAC; Univ. of Manitoba, Winnipeg, MB, Canada Individual differences in peritraumatic generalized fear as a predictor of post-traumatic stress disorder (PTSD) -like behaviors in rats

364.Anxiety Disorders: Animal Models
Mon 11/15 8:00 AM
10:00 AM - 11:00 AM

Halls B-H 		368.11
Poster 		HH18 *Y. LI1,2, H. WANG1, C. WEI1, K. QI1, N. SUI1, G. KIROUAC2; 1Inst. Psychol, CAS, Beijing, China; 2Dept of Oral Biol., Univ. of Manitoba, Winnipeg, MB, Canada Orexins in the paraventricular nucleus of the thalamus (PVT) as a mediator of dysphoria in morphine withdrawal

368.Neural Mechanisms of Drug Addiction
Mon 11/15 8:00 AM
9:00 AM - 10:00 AM

Halls B-H 		378.2
Poster 		TT15 *N. R. PADDOCK, P. SHEPPARD, P. F. GARDINER; Spinal Cord Res. Centre, Univ. of Manitoba, Winnipeg, MB, Canada The effect of 7 days of daily treadmill exercise on gene expression in large and small lumbar dorsal root ganglion neurons

378.Motor Neurons: Exercise, Injury, and Disease
Mon 11/15 8:00 AM
11:00 AM - 12:00 PM

Halls B-H 		403.8
Poster 		LLL64 M. G. WALLACE1, *D. W. CAMPBELL2, M. MODIRROUSTA2, J. SAREEN2, J. O. POLIMENI2, N. A. MCKEEN3, J. P. REISS4; 1Psychology, 2Psychiatry, Univ. of Manitoba, Winnipeg, MB, Canada; 3Cancer Care Manitoba, Winnipeg, MB, Canada; 4Psychiatry, Univ. of Western Ontario, Winnipeg, MB, Canada So funny I forgot to laugh: The neural correlates of cognitive humour processing

403.Social Cognition: Judgment and Attribution
Tue 11/16 1:00 PM
4:00 PM - 5:00 PM

Halls B-H 		661.12
Poster 		U15 *S. KARIMI-ABDOLREZAEE1, D. SCHUT2, J. WANG2, M. FEHLINGS2,3; 1Regenerative Med. Program, Univ. of Manitoba, Winnipeg, MB, Canada; 2Genet. and Develop., Toronto Western Res. Inst., Toronto, ON, Canada; 3Surgery, Univ. of Toronto, Toronto, ON, Canada Therapeutic strategies to stimulate proliferation and differentiation of the spinal cord precursor cells after spinal cord injury

661.Spinal Cord Injury: Therapeutic Strategies II
Wed 11/17 8:00 AM
8:00 AM - 9:00 AM

Halls B-H 		767.17
Poster 		Y9 *X. BI1, Y. ZHANG2, J. KONG3, X.-M. LI2; 1Shanghai, China; 2Dept. of Psychiatry, 3Dept. of Human Anat. and Cell Sci., Univ. of Manitoba, Winnipeg, MB, Canada Quetiapine regulates the proliferation and differentiation of oligodendrocytes in the hippocampus of global cerebral ischemia mice

767.Preclinical Experimental Therapeutics: Glutamate and Other Targets
Wed 11/17 1:00 PM
4:00 PM - 5:00 PM

Halls B-H 		865.8
Poster 		V12 *M. STOBART1,2, S. L. R. SIMON2, L. LAMOUREUX2, S. BRUNET1, J. D. KNOX1,2; 1The Univ. of Manitoba, Winnipeg, MB, Canada; 2Publ. Hlth. Agency of Canada, Winnipeg, MB, Canada Large scale screening of a shRNAmir library to identify genes essential to prion neurotoxicity

865.Prion Disease
Wed 11/17 1:00 PM
3:00 PM - 4:00 PM

Halls B-H 		866.15
Poster 		W11 J. WENG, R. SHI, L. ZHAO, *J. KONG; Univ. of Manitoba, Winnepeg, MB, Canada Knockdown of BNIP3 death gene family protects neurons from oxygen and glucose deprivation (OGD)-induced neuronal death

866.Other Neurodegenerative Disorders IV
Wed 11/17 1:00 PM
1:00 PM - 2:00 PM

Halls B-H 		873.13
Poster 		FF8 *K. GONCHARENKO1,3, E. EFTEKHARPOUR4, A. A. VELUMIAN1,2,3, P. L. CARLEN1,3, M. G. FEHLINGS1,2; 2Divisions of Neurosurgery, Cell and Mol. Biol., 1Toronto Western Hosp., Toronto, ON, Canada; 3Physiol., Univ. of Toronto, Toronto, ON, Canada; 4Dept. of Physiol., Univ. of Manitoba, Winnipeg, MB, Canada Changes in gap junction expression and function following ischemic injury of spinal cord white matter

873.Ischemia: Excitotoxicity


ABSTRACTS

42.1/G55. Properties and molecular determinants of electrical coupling in the mesencephalic trigeminal nucleus (Mes-V)
*S. CURTI1, G. HOGE2, J. I. NAGY3, A. E. PEREDA2;
1Physiol., Sch. of Med., Montevideo, Uruguay; 2Neurosci., Albert Einstein Col. of Med., Bronx, NY; 3Physiol., Univ. of Manitoba, Winnipeg, MB, Canada
One of the first examples supporting the presence of electrical synapses in the mammalian CNS arise from what are now considered classic experiments in the mesencephalic trigeminal (Mes-V) nucleus of the rat. The large size of Mes-V neurons provided the opportunity to obtain stable intracellular recordings in vivo, and to correlate them with ultrastructural analysis. Yet, the demonstration of the existence of functional coupling between these neurons relied on indirect electrophysiological evidence (Baker and Llinas, 1971), precluding detailed analysis of the properties of electrical transmission between these neurons. Taking advantage of current electrophysiological approaches and our knowledge of the molecular determinants of mammalian electrical transmission, we examined the properties of electrical coupling between Mes-V neurons. Using paired recordings in slices of rat brainstem, we provide the first direct evidence for the existence of electrical coupling between Mes-V neurons. Furthermore, immunochemical analysis demonstrated that large areas of contacts between Mes-V neuronal somata contain the gap junction protein connexin36 (Cx36), indicating that this widespread neuronal connexin also mediates electrical transmission between these neurons. No evidence for the presence of other connexins was obtained. While electrical recordings showed that coupling is usually strong and synergically interacts with the rich active properties of these neurons, tracer-coupling analysis and the distribution of Cx36 immunolabeling showed that they appear to be coupled in clusters of 2-3 cells. Interestingly, despite the presence of large areas of apposition containing Cx36, some cells were not coupled, suggesting that electrical coupling is under regulatory control. Taken together, our data indicate that electrical coupling between Mes-V neurons is mediated by Cx36 and that this coupling provides a mechanism to synchronize the activity of small sets of sensory afferents.



42.3/G57. Neuronal gap junctions in goldfish hindbrain are primarily or exclusively at glutamatergic mixed synapses and are heterotypic, with Cx35 in axon terminals and Cx34.7 in somata and dendrites
*J. E. RASH1, N. KAMASAWA2, S. NANNAPANENI3, K. G. V. DAVIDSON1, C. FLORES3, A. DICARO1, T. YASUMURA1, J. O'BRIEN4, J. I. NAGY5, A. PEREDA3;
1Biomed. Sci., Colorado State Univ., Fort Collins, CO; 2Div. of Cerebral Structure, Natl. Inst. Physiol. Sci. (NIPS), Okazaki, Japan; 3Albert Einstein Coll. Med., New York, NY; 4Ophthalmology and Visual Sci., Univ. Texas Hlth. Sci. Ctr., Houston, TX; 5Physiol., Univ. Manitoba, Winnipeg, MB, Canada
Goldfish giant club ending / Mauthner cell synapses (GCE / MC) provided the first system for studying the physiology and connexin composition at individual "mixed" (chemical plus electrical) synapses. Here, we used newly-developed connexin-specific antibodies against Cx35 and Cx34.7, combined with confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling (FRIL), to investigate the distribution of two evolutionarily divergent homologues of human connexin36 (Cx36) -- Cx34.7 and Cx35 -- at gap junctions (GJ) in more than a dozen identified goldfish hindbrain neurons. Both connexins showed a high degree of co-localization, but it was not possible by confocal microscopy to determine whether the GJs were composed of heteromeric or homomeric connexons, or formed heterotypic vs. homotypic GJs. However, by FRIL, anti-Cx35 exclusively labeled axon terminal GJ hemiplaques (>163), including at >20 giant GCE/MC synapses and in >15 reticulospinal (RS) and vestibulospinal (VS) synapses. Cx35 did not label any of >487 postsynaptic hemiplaques in MC, RS, or VS neurons. In the same replicas, anti-Cx34.7 exclusively labeled >425 hemiplaques in MC, and >40 hemiplaques in RS and VS. In matched double replicas, >60 GJs revealed both complementary hemiplaques, all labeled exclusively for Cx35 in the presynaptic hemiplaque and for Cx34.7 in the postsynaptic hemiplaque. This confirmed that all of these GJs were both heterotypic and homomeric. Equally important, all of these GJs were at mixed glutamatergic synapses, identified by their distinctive E-face clusters of glutamate receptor 10-nm particles and complementary P-face pits. No GJs were found at GABAergic synapses, which were identified by their distinctive clusters of 10-nm P-face particles. Moreover, despite extensive searches, including of the surrounding neuropil, no GJs were found forming exclusively electrical synapses. Currently unresolved is whether: a) Cx35 is the only connexin synthesized by sensory neurons and Cx34.7 is the only connexin synthesized by motor neurons (i.e., neuron sub-type specific expression of different connexins); or b) if motor neurons (MC, RS, and VS) synthesize both connexins, but sort Cx34.7 to somata and dendrites and Cx35 to their axon terminals in spinal cord (i.e., apical vs. basal sorting of connexins). Heterotypic gap junctions may provide the molecular basis for weak electrical rectification that we recently detected at these mixed glutamatergic GCE/MC synapses. Finally, we note that only by combining double replicas with multiple immunogold labeling could the question of heterotypic vs. bi-homotypic gap junctions be answered definitively.



42.5/H1. Distribution of connexin36 in juvenile and adult rodent spinal cord: Co-localization with vglut-1 suggests primary afferent terminals form mixed chemical and electrical synapses
W. BAUTISTA1, J. I. NAGY1, *D. A. MCCREA2;
1Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; 2Univ. Manitoba Fac Med., Winnipeg, MB, Canada
Gap junctions made by channel-forming connexin proteins link the cytoplasmic compartments of coupled cells and allow cell-cell passage of ions, metabolites and tracer dyes. Neuronal gap junctions in mammalian CNS are formed primarily by connexin36 (Cx36) and represent the ultrastructural correlate of electrical synapses. Although the presence of electrical coupling between rodent spinal cord neurons has been described, a comprehensive analysis of the distribution of Cx36 in developing or mature cord has not yet been reported. We used immuno¬histochemical approaches to examine Cx36 localization in the cervical, thoracic and lumbar spinal cord at various developmental stages. The anti-Cx36 antibodies used have been validated for specificity by showing absence of Cx36 detection in Cx36 knockout mice. As elsewhere in the CNS, immunolabelling for Cx36 in spinal cord was exclusively punctate in appearance, which presumably reflects localization to sites of gap junctions. Cx36 was densely distributed throughout spinal cord gray matter during the second postnatal week. Although Cx36 expression was reduced in adult cord, it persisted in deep laminae of the dorsal horn and some regions of the ventral horn including areas containing motoneurons. At thoracic levels, immunolabelling for Cx36 was highly concentrated in the intermediolateral cell column, among the cell bodies and dendrites of sympathetic neurons which have been reported to be electrically coupled. Most striking at all spinal levels were densely distributed Cx36-positive puncta on cell bodies and initial dendrites of neurons located near the central canal at the transition between the dorsal and ventral horn. Similar labelling was seen on sparsely distributed neurons located in lamina VII. Preliminary observations suggest that the majority of Cx36-positive puncta in adult spinal cord is co-localized with the nerve terminal marker vglut-1. Since in the spinal cord vglut-1 is considered to be localized mainly in the terminals of myelinated primary afferents, our results suggest that Cx36 is contained in gap junctions formed by these terminals, raising the possibility that neurotransmission from a subpopulation of primary afferents occurs via mixed chemical/electrical synapses on rodent spinal neurons. Such mixed synapses between unidentified neuronal elements in spinal cord have been previously described by ultrastructural freeze-fracture approaches.



42.11/H7. Interaction of connexin36 with multi-PDZ-domain protein 1 (MUPP1)
*X. LI1, J. I. NAGY2;
1Dow Neurobio., Legacy Clin. Res., Portland, OR; 2Physiol., Univ. of Manitoba, Winnipeg, MB, Canada
The gap junction protein connexin36 (Cx36) is the first cloned neuronal gap junction protein and is widely expressed in neurons of the rodent and human central nervous system (CNS). It forms electrical synapses, mediates direct electrical communication between neurons of the CNS and has been implicated in juvenile myoclonic epilepsy, global ischemia and other neurological diseases. It was previously shown to interact with the first PDZ domain of zonula occludens-1 (ZO-1), zonula occludens-2 (ZO-2) and zonula occludens-3 (ZO-3). We investigated whether Cx36 is also able to interact with other PDZ domain containing proteins, including multi-PDZ-domain protein 1 (MUPP1), which contains thirteen PDZ domains and is a quantitative trait gene for drug withdrawal seizures. In lysates of mouse brain, MUPP1 was shown to co-immunoprecipitate with Cx36, Cx43 and Cx47, but not with Cx30 or Cx32. Double immunofluroscence labeling of Cx36 and MUPP1 showed co-localization of these two proteins in regions of mouse brain where Cx36-containing gap junctions between neurons are known to exist. However, Cx36-MUPP1 co-localization did not occur at all sites of Cx36 expression. In vitro pull-down assays revealed that Cx36 interacts with the PDZ10 domain of MUPP1. Truncated Cx36 lacking its c-terminus PDZ binding motif SAYV failed to bind PDZ10 of MUPP1. A fourteen amino acid peptide corresponding to the c-terminus of Cx36 inhibited the association of Cx36 with the PDZ10 of MUPP1, but a ten amino acid peptide that lacks the SAYV PDZ domain binding motif of Cx36 did not have this inhibitory capacity. These results indicate that Cx36 associates with the PDZ10 of MUPP1 and that this association requires the c-terminus SAYV sequence in Cx36. These findings, together with the known association of MUPP1 with more than 20 interacting proteins, which include various neurotransmitter receptors and signaling molecules in the CNS, suggest that MUPP1 may serve to anchor regulatory proteins at gap junctions composed of Cx36, including CamKII, which has been reported to interact with MUPP1 and to be localized to Cx36-containing gap junctions. Further, MUPP1 may play a linker role for regulatory relationships between chemical synapses and electrical synapses within gap junctionally coupled neurons. Research funded by Canadian Institutes for Health Research to JN and XL.



64.3/AA18. Effect of blocking kappa opioid receptors on fear and anxiety-like behaviors in a rat model of post-traumatic stress disorder (PTSD)
Y. LI, X. CHEN, S. LI, *G. J. KIROUAC;
Dept of Oral Biol., Univ. of Manitoba, Winnipeg, MB, Canada
Post-traumatic stress disorder (PTSD) is considered an anxiety disorder that can develop in individuals exposed to severe trauma. Therapeutic options are usually limited and targeted at alleviating the symptoms once they are established and not at the prevention of the development of the disorder. The kappa opioid receptor is a potential target to develop drugs for PTSD because of the link between this receptor, stress, and anxiety. In this study, we examined if chronic blockade of kappa receptors with the selective and long-acting antagonist nor-binaltorphimine (nBNI) prevented the development of fear, anxiety and avoidance behaviors in a rat model of PTSD. Male Sprague-Dawley rats (240-250 g) were exposed to acute foot shock (5 × 2 second episodes of 1.5 mA over 5 minutes with random interval 10-50 seconds between shocks) while control rats were placed in the chamber but did not receive footshocks. Half of the rats received saline and the other half received nBNI (15.0 mg/kg, s.c.) on day 2, 10 and 18 after footshock. The antagonist nBNI has slow onset (24 hours after injection) but long lasting (at least 7-10 days after systemic injection) pharmacological effect. A number of behavioral tests (measure fear and anxiety in the shock chamber, open field, novel chambers, social interaction situation, defensive withdrawal test, and exposure to novel palatable food) were done at different time points after the shock day (up to 30 days postshock). As previously reported, we found that exposure of rats to acute footshock induced more anxiety and fear-like behaviors in shock rats. Our results clearly show that nBNI had opposite effects on fear/anxiety in the footshock and non-shock rats. First, nBNI attenuated anxiety and fear-like behaviors in non-shock rats during all time points in which behaviors were assessed. In contrast, nBNI had no significant effect on anxiety and fear-like behaviors in shock rats during the earlier test period (8-10 days postshock). Furthermore, our results showed that nBNI caused more anxiety and fear-like behaviors during the tests done at later time points (25-31 days postshock). The results of our experiments are consistent with other studies showing an anxiolytic effect of nBNI in rodents that have no pre-exposure to stress. However, the observation that nBNI had anxiogenic effects in shock rats, suggests that the function of the dynorphin/kappa receptor system changes following exposure to an acute and intense stressor. The neural mechanism mediating the anxiogenic effect for nBNI in shock rats is unknown and requires further research. Based on our results, nBNI does not appear to be a suitable approach for the treatment of PTSD.



148.8/H66. Functional evaluation of MECP2 gene therapy vectors in differentiated neurons
R. ZACHARIAH, C. OLSON, *M. RASTEGAR;
Biochem. and Med. Genet., Univ. of Manitoba, Winnipeg, MB, Canada
One in 150 individuals suffers from Autism, a neurological disorder affecting the social interaction and communication skills of affected patients. Autism Spectrum Disorders (ASD) refer to different forms of autism usually diagnosed by 2 years of age, caused by harmful changes taking place in the brain as it grows and develops. Rett Syndrome (RTT) is the best-studied example of ASD and the primary cause of mental retardation in females. RTT is caused by mutations in the methyl-CpG binding protein-2 (MECP2) gene and does not have any effective treatment. However, reactivation of the Mecp2 gene after the onset of disease in mouse models rescues RTT phenotype [1,2]. This raises hope towards RTT therapy prospects either by delivering MECP2 into the affected neurons, or through drug treatments targeted towards proteins, both of which can compensate for MeCP2 loss in neurons. Gene therapy is a potent and cutting edge technology for human disease, where a single gene mutation has been identified. Therefore, RTT presents an excellent candidate disease for such therapeutic strategies.
Two MECP2 isoform variants exist; MECP2E1 and E2, which are both widely expressed. However, RTT symptoms are exclusively neuronal. Despite the exclusive RTT neuronal phenotype, MeCP2 isoform-specific expression and function in neurons is not known. We have developed advanced retroviral and lentiviral vectors for RTT gene therapy and performed functional studies in neural stem cells (NSC) of a RTT mice, where we showed a significant improvement in neuronal dendrite branching through retroviral delivery of the MECP2E1 isofrom [3]. However, we do not know whether or not MECP2E2 has similar or unique role in neurogenesis (the process by which new neurons are generated). We are now investigating MeCP2 isoform-specific expression in differentiated neurons and comparing the rescue effect of each isoform through gene therapy delivery of the MECP2E1/ E2 isoforms into Mecp2 deficient neurons. Our scientific approach is a combination of advanced neurobiology and gene therapy technologies with an ultimate goal of developing novel therapeutic strategies for Rett Syndrome.
1. Giacometti E, Luikenhuis S, Beard C, Jaenisch R (2007) Partial rescue of MeCP2 deficiency by postnatal activation of MeCP2. Proc Natl Acad Sci U S A 104: 1931-1936.
2. Guy J, Gan J, Selfridge J, Cobb S, Bird A (2007) Reversal of neurological defects in a mouse model of Rett syndrome. Science 315: 1143-1147.
3. Rastegar M, Hotta A, Pasceri P, Makarem M, Cheung AY, et al. (2009) MECP2 isoform-specific vectors with regulated expression for Rett syndrome gene therapy. PLoS One 4: e6810.



154.5/M15. Exploratory pathway analysis of temporal gene changes in rat spinal cord injury: Long lasting inflammatory response in chronic spinal cord injury
*E. EFTEKHARPOUR1, S. KARIMI-ABDOLREZAEE2, P. BOURTOS3, M. FEHLINGS4;
1Physiol., Spinal Cord Res. Center, Univ. of Manitoba, Winnipeg, MB, Canada; 2Physiol., Regenerative Med. Program and Spinal Cord Res. Center, Univ. of Manitoba, Winnipeg, MB, Canada; 3Ontario Inst. for Cancer Res., Toronto, ON, Canada; 4Surgery, Univ. of Toronto, Toronto Western Res. Institue, Krembil Neurosci. Res. Ctr., Toronto, ON, Canada
Background:
Spinal cord injury (SCI) remains a devastating form of neurotrauma with no immediate therapy available. Inflammation has been shown to play a major role during acute stage of SCI, however its contribution to the chronic SCI pathology remains unclear. Approaches to promote or attenuate inflammation following the trauma have been actively investigated in pre-clinical models and clinical trials. These approaches are mainly focused on acute stages after injury. We sought to analyse the temporal pattern of underlying molecular mechanisms regulating the response of spinal cord tissue to injury during the acute to chronic stages.
Methodology
We used Affymetrix GeneChip Rat Genome chips at 1, 3, 7, 14 and 56 days after injury to distinguish differential gene expression using the BioConductor open-source project. The significantly deregulated data were then further analyzed using the Ingenuity Pathways analysis (Ingenuity© Systems, www.ingenuity.com) and the underlying molecular networks, their associated functions and canonical pathways were identified. A subset of genes was evaluated using quantitative Polymerase Chain Reaction.
Principal Findings /Importance:This is the first extensive global pathway analysis of temporal gene expression of genes after SCI. In this study, we report that inflammatory response continues to be one of the most deregulated functional networks in all stages of injury. Using the unique canonical pathway analysis of the Ingenuity system we show a timeline for molecular scenario that shape the spinal cord pathobiology. This is of great importance for both pro and anti inflammatory approaches in treatment of spinal cord injury. Our results suggest that inflammatory response is a continual process even at chronic stages of rat SCI model.



156.21/P12. Astrocyte D-serine release influences NMDA receptor-mediated cerebral vasodilation
*J. L. LEMAISTRE1,2, H. D. I. ANDERSON1,3, C. M. ANDERSON1,2;
1St Boniface Res. Ctr., Winnipeg, MB, Canada; 2Pharmacol. & Therapeut., 3Pharm., Univ. of Manitoba, Winnipeg, MB, Canada
Neuronal energy demand during synaptic activity is met by increasing local blood flow, a process which involves signaling between neurons, astrocytes and blood vessels, comprising the neurovascular unit. Astrocytes extensively encircle arterioles and function by releasing numerous gliotransmitters via calcium-dependent pathways in response to neuronal stimulation. D-Serine is one such gliotransmitter and an activator of the glycine regulatory site at NMDA-type glutamate receptors. D-Serine may participate in cerebral blood flow regulation, since NMDA receptors have been shown to influence cerebrovascular tone in vivo. We hypothesized that astrocyte activation (Ca2+ elevations) lead to D-serine-dependent, NMDA receptor-mediated vasodilation. To investigate this, we studied both isolated, pressurized mouse middle cerebral arteries (MCA) and cortical arterioles in acutely-isolated mouse brain slices. In pressure myography experiments using pre-constricted MCA, neither NMDA receptor agonists, glutamate or D-serine, was vasoactive. In contrast, the combination of both agonists caused dose-dependent vasodilation in a manner blocked by antagonsists of both the NMDA receptor glutamate binding site (2-amino-5-phosphonopentanoic acid) and the glycine/D-serine binding site (5,7-dichloro-kynurenic acid). NMDA receptor-mediated vasodilation persisted after chemical denervation, eliminating the possibility that remnant neuronal NMDA receptors influenced the change in vessel diameter. We also directly studied the effect of astrocyte Ca2+ elevations on local arteriole diameter using uncaging of o-nitrophenyl EGTA/AM in individual cortical slice astrocytes and 2-photon imaging. Slices were also loaded with rhodamine-2/AM, to monitor astrocyte Ca2+ levels, and isolectin B4-Alexa Fluor 488, to label the brain slice vasculature. Astrocyte Ca2+ uncaging produced intracellular Ca2+ transients and resulted in dilation of local arterioles in 20% ambient O2. Vasodilation was blocked by D-amino acid oxidase, which selectively degrades D-serine, strongly suggesting a role for D-serine in this mechanism. These results provide the first evidence that D-serine modulates vascular diameter by an NMDA receptor-dependent mechanism. Supported by operating funds from the Canadian Institutes of Health Research (CIHR) and salary awards from the CIHR (JL, HA) and Heart and Stroke Foundation of Canada (CA).



180.14/SS1. Motoneuron afterhyperpolarization time course after chronic stroke
*S. J. GARLAND1, S. KNORR3, C. L. POLLOCK2, C. W. MACDONELL4, T. D. IVANOVA1;
1Dept Physical Therapy, 2Rehabil. Sci., Univ. British Columbia, Vancouver, BC, Canada; 3Hlth. and Rehabil. Sci., Univ. of Western Ontario, London, ON, Canada; 4Spinal Cord Res. Ctr., Univ. of Manitoba, Winnipeg, MB, Canada
The time-course of the afterhyperpolarization (AHP) influences the capacity of the motoneurone to discharge action potentials. Typically there is a “speed-match” between the intrinsic properties of motoneurones and the contractile properties of their muscle (Gardiner, 1993; Gossen et al. 2003). Matthews (1996) developed a transform, referred to as the “Interval Death Rate (IDR) analysis”, for estimating the AHP time-course in humans from the interspike intervals of single motor units. The IDR has been validated in cat spinal motoneurons (Powers and Binder 2000) and in human hand muscle (Gossen et al. 2003). Because contractile properties and motor unit firing rates have been shown to slow after stroke (Frontera et al. 1997; Gemperline et al. 1995), and the AHP is known to influence motoneuron firing rate, the time course of the AHP may be altered in stroke survivors. The purpose of this study was to determine the time-course of the motoneuron AHP after stroke. Twelve subjects, who were between 10 months and 9 years post-stroke, performed low force isometric ankle dorsiflexion contractions for 600s. Single motor units from tibialis anterior (TA) were recorded from both the paretic and non-paretic legs. Participants were required to vary the firing rate of a single motor unit between its lowest firing rate and approximately 10 Hz, according to a predetermined protocol (MacDonell et al. 2007). The estimated AHP time constant was significantly slower (~35%) in the paretic leg than in the non-paretic leg. These results suggest that the slowing of motor unit firing rate after stroke may, in part, be the result of a lengthened motoneuron AHP.



247.27/I3. Reduced Alzheimer's disease pathology and enhanced insulin signaling in early type 1 diabetes in an overexpressing amyloid beta mouse model
*J. S. SCHAPANSKY1,2, G. ODERO4, T. ENNO3, M. DEL BIGIO3, G. GLAZNER4,2;
1St. Boniface Rese Ctr, Univ. Ma, Winnipeg, MB, Canada; 2Dept. of Pharmacol. and Therapeut., 3Dept. of Pharmacol., Univ. of Manitoba, Winnipeg, MB, Canada; 4St. Boniface Res. Ctr., Winnipeg, MB, Canada
Clinical studies have shown that insulin-insensitive diabetics are at greater risk for developing Alzheimer’s, and, conversely, people who develop Alzheimer’s disease can be partially insulin-insensitive. At the molecular level there is a close association between amyloid beta (Aβ) and insulin pathways, as oligomers will disrupt insulin signalling, and insulin insensitivity and elevated insulin levels contributes to amyloid accumulation and deposition. However, little is known as to the effects of low insulin on Alzheimer’s disease and amyloid pathology. In this study, early-stage type 1 diabetic transgenic Alzheimer’s disease mice showed elevated insulin signalling in the CNS, and less pathology than controls. Double APP mutant mice (TgCRND8) were injected with streptozotocin to reduce insulin production at two months of age, prior to Aβ plaque formation and cognitive disorders. Injected transgenics performed better at cognitive tests (Morris water maze) then their counterparts at 4 weeks post-injection. After 6 weeks of diabetes, qPCR revealed elevated insulin receptor mRNA expression in the hippocampus of STZ injected animals (controls and transgenics), and significantly higher phosphorylated insulin receptor and AKT proteins in injected transgenic animals versus controls. ELISA analysis demonstrated lower soluble Aβ levels in STZ-injected transgenic hippocampal tissue versus controls, and significantly lower plaque load as well. This data indicates a possible biological role for amyloid peptides, where high levels may activate the insulin pathway during insulin depletion, leading to reduced amyloid beta pathology in our animals.



257.6/T14. Antigen induced expression of Fractalkine in an animal model of multiple sclerosis
*W. ZHU, F. BEGUM, E. FROST, M. NAMAKA;
Fac. of Pharm., Univ. of Manitoba, Winnipeg, MB, Canada
The general objective of this research was to determine the expression of Fractalkine (CX3CL1) in antigenic induced multiple sclerosis (MS).
Background: MS is a chronic, inflammatory autoimmune disease characterized by targeted destruction of the central nervous system (CNS) myelin. Studies indicate that demyelination and oligodendrocyte death are mediated by immune cells. It has been reported that pain is second worst disease induced symptom and about 75% MS patients suffer from neuropathic pain prior to diagnosis. Fractalkine belonging to the chemokine famlily has several functions including the newly recognized role of mediating neuropathic pain conditions.
Methods: Adult Lewis rats (N=66) were divided into 3 groups: naïve control (NC), active control (AC) and active experimental autoimmune encephalomyelitis (EAE). NC animals did not receive any injections. AC animals received 2 intraperitoneal injections of pertussis toxin along with Freund’s adjuvant and Mycobacterium Tuberculosis. EAE animals received the same regimen as AC’s plus inoculation with guinea pig myelin basic protein (MBP). The neurological disability of all animals was accessed using neurological disability clinical scoring system and behavioral studies were conducted to test pain symptom in each rat. Comparative, time dependant protein analysis (day 0, 3, 6, 9, 12 &15) of Factalkine in spinal cord (SC) was conducted using AEC staining, immunohistochemistry (IHC) and western blotting, according to previously established methods.
Results: Antigenic induction involving an EAE model of MS does induce significant up-regulated protein expression of Fractalkine within the SC which reached a peak at day 12 post-antigenic induction relative to the other experimental groups. The sensory thermal hypoalgesia correlates to the elevations of Fractalkine protein expression within the SC thereby confirming the importance of Fractalkine in sensory processing such as neuropathic pain. IHC analysis of the SC showed that Fractalkine is co-localized with neuron and supported the function of neurons involving in the pain transmission.
Conclusions: The up-regulated protein expression of Fractalkine within SC may represent a critical link to advanced understanding of neuropathic pain in autoimmune disease such as MS.



258.11/V6. Galanin protects cuprizone-induced demyelination via GalR1 and GalR2
L. ZHANG, Y. WU, J. KONG, I. SCHROEDTER, *M. VRONTAKIS-LAUTATZIS;
Univ. of Manitoba, Winnipeg, MB, Canada
Galanin, a 29 amino acid neuropeptide, is widely distributed in the central and peripheral nervous system. Studies over the last decade have demonstrated that galanin plays multiple regulatory roles in the nervous system. In this study, we investigated its myelin protective role in the cuprizone (CPZ) demyelination model by using galanin transgenic (Gal-Tg) mice. To induce demyelination, age-matched 8-week old wildtype and Gal-Tg mice were fed with 0.3% cuprizone (w/w) for 6 weeks. To allow remyelination, the groups were given cuprizone-free normal food for another 3 weeks after 6-week CPZ insult. The myelin sheath, mature oligodendrocytes, astrocytes and proliferating cells were immunostained by anti-MBP, anti-GST-pi, anti-GFAP and anti-BrdU antibodies respectively. De-myelination was measured by immunostaining and western-blot assay to MBP. Expression of galanin and its three receptors during demyelination and remyelination was quantitatively determined by comparative Cq Real-time PCR. Our results demonstrate that over-expression of galanin in Gal-Tg mice significantly inhibited cuprizone-induced demyelination compared to that in wildtype mice. Moreover, we have observed that the expression levels of Galanin Receptor 1 (GalR1) in Gal-Tg mice were highly activated after 6 weeks of demyelination, while GalR2 were up regulated later in the re-myelination period. In addition, over-expression of galanin also inhibited astrogenesis and proliferation of microglia. Together, these data suggest a putative role for Galanin in preventing myelin damage. The differential activation of galanin receptors in demyelination and remyelination processes suggests the potential pharmacological utility of their agonists against demyelination disease such as Multiple Sclerosis.
Key words: Galanin, Galanin receptors, cuprizone-induced demyelination, Galanin-Tg mice, Multiple Sclerosis



259.14/W7. Longitudinal study of locomotor recovery following chronic compressive spinal cord injury in rats
*O. ALLUIN1, S. KARIMI-ABDOLREZAEE2, H. DELIVET-MONGRAIN1, M. MÉARD1, H. LEBLOND1, M. FEHLINGS3, S. ROSSIGNOL1;
1Dept. of Physiol., Univ. of Montreal, Montreal, QC, Canada; 2Dept. of Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; 3Toronto Western Res. Inst., Toronto Western Hosp., Toronto, ON, Canada
The modification of the locomotor parameters following surgical spinal sections in adult animals has been extensively described before. However, there has been little such description after compressive spinal lesions which mimic more closely the complex spinal cord injuries (SCI) in Humans. Such lesions may be extensive but preserve various amounts of white matter.
In the present study, we investigate the evolution of the locomotion parameters in a chronic model of compressive incomplete SCI using treadmill training. This SCI model was induced by a modified aneurysm clip (23.8 gr) for 1 minute at T7-8, which is considered to cause a severely moderate SCI.
The experiment was performed on 22 adult female Wistar rats trained to walk on a treadmill at different speeds (14 m.min-1, 20 m.min-1 and 26 m.min-1) and eight of these were chronically implanted with EMG electrodes in the hindlimbs. Kinematics data were obtained using video recordings synchronized to EMGs when possible. Data were obtained before (baseline) and weekly for 6 weeks following SCI. Rats were randomized in 2 groups: 1) Trained group which received daily training for 15 minutes on a treadmill starting one day after SCI (n=11), and 2) Untrained group with no locomotor training (n=11).
At 6 weeks post SCI, our analysis revealed that a greater number of animals in the trained group (+10%) could walk on the treadmill at higher speeds (i.e., 20 and 26 m.min-1) compared to the untrained group. Antero-posterior coordination was lost while 1:1 alternation between both hindlimbs was recovered and cycle length and time were similar in both hindlimbs. Moreover, ankle angle excursion remained most affected at 6 weeks post-injury compared to the other hindlimb joints. At 6 weeks, intra-group variability of the kinematic parameters was similar to that of the baseline indicating a consistent recovery of the locomotor pattern. However, stereological analysis of the spinal lesion volume showed a relatively high variability across the animals in both groups (SD approx. 50% of the averaged volume for each group), suggesting that the spinal circuitry and peripheral afferent feedback could play a key role in the locomotor recovery despite the variability of the lesion.
In conclusion, after a compressive SCI in rat, some degree of consistent and robust spontaneous locomotor recovery of the hindlimb locomotion occurs and this should be taken into consideration when assessing the beneficial functional effects of any kind of added therapeutic interventions.



259.17/W10. Upright bipedal locomotion in rat: A suitable model for investigation of interventions after SCI?
U. SLAWINSKA1, *H. MAJCZYNSKI1, E. KISIELNICKA1, L. M. JORDAN2;
1Nencki Inst. Exp Biol, Warsaw, Poland; 2Physiol., Univ. of Manitoba, Winnipeg, MB, Canada
Upright bipedal locomotion in spinal rats has been used as a model for investigation of the effectiveness of various methods for restoration of function. We tested the hypothesis that locomotion of spinal rats in the upright posture differs substantially from locomotion in the normal horizontal position. We compared treadmill locomotion induced by tail pinching of spinal rats in the two positions, upright and horizontal. In addition, we investigated the effects of various serotonergic pharmacological agents on hind limb locomotor movements in both experimental conditions. The spinal cord was completely transected at the low thoracic level (Th9) in three month-old WAG rats under deep Equithesin anesthesia (3 ml/kg, i.p.), leaving a gap of 2-3 mm. Bipolar EMG electrodes were chronically implanted in the soleus and tibialis anterior muscles of both hind limbs. Video and EMG recordings were carried out during locomotor-like hind limb movements produced by tail stimulation. Polar plots showing the coordination among muscles of the hind limb during locomotion and plots of burst duration vs cycle duration were calculated from the EMG data. Spinal rats (3-4 months after spinal total transection) with upright posture can be easily induced to engage in bipedal plantar walking without any specific intervention other than tail pinching, while it was almost impossible to obtain any plantar walking using the same stimulus in the same animal when its body was kept in the normal horizontal posture. Quipazine (5-HT2 receptor agonist) application (i.p. 0.25 mg/kg b.w.) interfered with bipedal locomotoror-like hind limb movements of spinal rats in the upright posture, while it improved that of spinal rats in the horizontal posture. Cyproheptadine (5-HT2 receptor antagonist) application (i.p. 1 mg/kg b.w.) facilitated bipedal plantar walking of spinal rats with upright posture, while hind limb locomotor-like movements in a spinal rat in horizontal posture was additionally impaired by shortening of the extensor muscle burst duration. SB-269970 (5-HT7 receptor antagonist; i.p. 0.6 mg/kg b.w.) did not influence bipedal plantar walking with upright posture, while in the horizontal posture it disturbed coordination. We conclude that upright locomotion in the rat is much easier to elicit than locomotion in the horizontal posture, likely due to potentiation of afferent input from the plantar surface of the foot and muscle receptors activated during weight-bearing. Upright locomotion may, however, give misleading information about the effects of pharmacological interventions in relation to the normal locomotor task of rodents.



259.20/W13. Segmental distribution of 5-HT2A and 5-HT2C receptor up-regulation one month after complete spinal cord injury
L. M. JORDAN1, H. FABCZAK2, E. KISIELNICKA2, A. LESZCZYNSKA2, H. MAJCZYNSKI2, J. I. NAGY1, *U. SLAWINSKA2;
1Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; 2Nencki Inst. of Exptl. Biol., Warsaw, Poland
There is considerable evidence from research in neonatal and adult rat and mouse preparations to make the case that 5-HT2 receptor activation leads to increased motoneuron excitability and to activation of the central pattern generator (CPG) for locomotion. Both 5-HT2A and 5-HT2C receptors are implicated in these effects, but it is not clear how these receptors are involved in the responses to 5-HT observed after spinal cord injury. Here we examine the segmental distribution of both 5-HT2A and 5-HT2C receptors in intact spinal cord and their up-regulation in animals subjected to a complete spinal cord transaction. The spinal cord was transacted at Th9 in 3 month-old WAG rats under deep Equithesin anaesthesia. After 1 month, the animals were perfused with either PBS supplemented with protease and phosphatase inhibitors for Western blots or 4% paraformaldehyde for immnuhistochemistry. Proteins were transferred onto nitrocellulose membranes by wet electroblotting and labeled with primary polyclonal antibody raised against 5-HT2A or 5-HT2C receptors and horseradish peroxidase conjugated secondary antibodies. Immunoreactive bands were visualized using ECL system and their intensities were quantified using the InGenius system. Immunohistochemical labelling of 5-HT2A receptors was carried out in control and transected cords using a well-characterized antibody to 5-HT2A receptors.
Western blot analysis showed a differential distribution of the two receptor types in both control and spinal animals, with 5-HT2A receptors concentrated in the lumbar segments, while 5-HT2C receptors were most densely distributed in the sacral segments. One month after spinal cord injury, both receptor types were up-regulated, with no significant change in their rostro-caudal distribution. Up-regulation of 5-HT2A receptors could be readily demonstrated in the lumbar region using immunohistochemical techniques; maximal 5-HT2A receptor labelling was observed in small alpha and in gamma motoneurons of limb muscles, where both soma and dendritic labelling was prominent in cords from both intact and spinal animals. The up-regulation after spinal transaction appeared to be primarily in the dendrites of alpha motoneurons. Retrograde labelling with flurogold of motoneurons from ankle flexor and extensor muscles revealed 5-HT2A receptors in both types, while labelled tail motoneurons were rarely positive for 5-HT2A receptors. 5-HT2A receptors were also found on presumed interneurons, particularly in lamina VII. Lamina VIII interneurons did not appear to be labelled with the antibody to 5-HT2A receptors in cords from either intact or spinal animals.



290.9/DDD10. Scratch-like fictive motor output can be elicited by drug application to cervical segments of the isolated neonatal rat spinal cord
*E. E. VASQUEZ-DOMINGUEZ, K. E. POWER, B. FEDIRCHUK;
Physiol., Univ. of Manitoba, Winnipeg, MB, Canada
In decerebrate cats, fictive scratch (FS) is induced after cervical application of curare and rubbing the face. A FS episode consists of ipsilateral rhythmic alternating flexion and extension at a frequency of 4-8 Hz. Recently, FS in acute spinal cats has been used to study state-dependent modulation of motoneurons. The aim of the present study is to test the ability of the in vitro neonatal rat spinal cord to generate FS-like motor output. Spinal cords isolated from neonatal rats (P1-5) were placed in a partitioned dish so the C1-C2 segments could be exposed to neurochemicals. Suction electrodes were used to record L2 and L5 ventral roots (VRs), and electrically stimulate the C1 dorsal columns (DC). Bicuculline (BIC), strychnine (STR) and/or curare (CUR) were applied individually or in combination. In some experiments, KCl was used to non-specifically increase spinal excitability. GRP(18-27), thought to be a spinal itch-specific mediator, was also tested. In absence of drugs, in some preparations DC stimulation could evoke brief periods of high frequency rhythmic discharge (>4 Hz) along with tonic activity. However, the rhythmic activity was not present in all VRs and faded quickly after stimulation. With BIC and/or STR, long-lasting bilateral episodes of fast frequency (4-12 Hz) bursts were usually evoked by stimulation. The response could take minutes to develop and the frequency usually decayed in seconds after stimulation. The fast frequency discharge could coexist with slower (<0.5 Hz) bursts. Fast frequency bursts could be out of phase, sometimes having clear ipsilateral L2/L5 alternation, but there were often periods of synchronous activity within the same preparation. Although CUR (30-50 µM) elicited similar responses following DC stimulation, it could also induce spontaneous high frequency bursts (~7 Hz), either ipsilaterally or in all VRs. Similarly, after brief exposure to GRP(18-27) (500 nM), spontaneous fast frequency (4-6 Hz) rhythmic bursts were observed. Unlike CUR however, GRP(18-27)-induced bursts usually occurred in either the left or right VRs, not bilaterally at the same time. Again, the activity could include periods of ipsilateral alternation. Using an approach similar to that used to elicit FS in the in vivo cat, we observed a variety of motor outputs in the in vitro neonatal rat spinal cord preparation. A fast frequency (~6 Hz) rhythmic output was evoked more specifically by CUR and GRP(18-27) and may be the equivalent of FS-like activity. The neonatal rat spinal cord preparation may serve as a useful tool for studying FS-like behavior and state-dependent modulation of neuronal properties using patch-clamp and pharmacological techniques.



290.11/EEE2. Neurons of the parapyramidal region are active during a locomotor task and receive an excitatory cholinergic input
E. COUTO-ROLDAN1, Y. DAI1, J. LIU1,2, H. A. GRAVES1, E. S. DENERIS3, *L. M. JORDAN1;
1Dept Physiol., Univ. Manitoba, Winnipeg, MB, Canada; 2Brain Res. Ctr. and Dept. of Med., Univ. of British Columbia, Vancouver, BC, Canada; 3Dept. of Neurosci., Case Western Reserve Univ., Cleveland, OH
We have shown that a descending serotonergic pathway that produces locomotor activity in the neonatal rat originates primarily, if not exclusively, in the serotonin (5-HT)-containing neurons located in the parapyramidal region (PPR) of the medulla. Here we test the hypothesis that the 5-HT neurons of the PPR are involved in the control of locomotion in adult animals using c-fos immunohistochemistry to reveal neurons that are activated during a locomotor task. The serotonergic neurons of the PPR are detected using ePet/EYFP mice in which EYFP is expressed in 5-HT neurons of the brainstem. In these mice, a Pet-1 enhancer region is used to direct reliable 5HT neuron-specific transgene expression in the CNS. We subjected the animals to a 60 minute locomotor trial, then the animals were anaesthetized, perfused transcardially with fixative (4% paraformaldehyde, 0.2% picric acid and 0.15M phosphate buffer, pH 6.9), and the brainstem was removed. The tissue was sectioned and then incubated with c-fos primary antibody (polyclonal rabbit anti-c-fos, Oncogene #PC38T, 1:10,000). The secondary antibody was donkey anti-Rabbit Cy3, 1:400, Jackson ImmunoResearch Laboratories. Primary antibody omission and antibody absorption were used as controls. The 5-HT neurons marked with EYFP were easily detectable in all parts of the brainstem, including the PPR. Numerous c-fos labelled EYFP positive neurons were found in the PPR region, but few double-labelled neurons were found in the raphe pallidus, raphe obscurus or magnus. These findings are consistent with our hypothesis that the major descending serotonergic pathway involved in the control of locomotion originates in the 5-HT neurons of the PPR. Using this animal model, we also performed whole cell patch-clamp recordings on EYFP+ cells in the PPR in neonatal. The EYFP+ cells in the PPR could be classified into 3 types based on their responses to depolarizing step currents: 1-2 spikes, phasic firing and tonic firing. Some spontaneous firing cells were also found. Bath application of 20µM Ach produced sustained depolarization of the membrane potential, evoking repetitive firing, and induced membrane oscillations (25 Hz) in EYFP+ cells in the PPR. Our results are consistent with the suggestion that neurons in this region, which can be stimulated to produce locomotion, receive excitatory cholinergic input from the Mesencephalic Locomotor Region.



332.15/C22. Abnormal NF-kB activity and associated impairments in axonal outgrowth in sensory neurons isolated from diabetic rats are corrected by cytokines
A. SALEH1, S. BALAKRISHNAN1, E. MUTCH1, R. VAN DER PLOEG1, E. AKUDE1,2, D. R. SMITH1,2, *P. FERNYHOUGH1,2;
1St Boniface Res. Ctr., Winnipeg, MB, Canada; 2Pharmacol. & Therapeut., Univ. of Manitoba, Winnipeg, MB, Canada
The transcription factor, NF-κB, is involved in the pathogenesis of a wide range of neurodegenerative disorders, including diabetic neuropathy. For example, lumbar dorsal root ganglia (DRG) of streptozotocin (STZ)-diabetic rats exhibit reduced activation of NF-κB. This defect may enhance neuropathy since in adult sensory neurons NF-κB is a regulator of survival and axonal regeneration, possibly mediated through anti-oxidant enzyme expression. We, therefore, tested the hypothesis that reduced NF-κB expression/activity and associated impairments in axonal outgrowth in sensory neurons in diabetes were instigated by sub-optimal neurotrophic support by the cytokines, IL-1α, IL-1β and CNTF.
To quantify levels of cytokines in DRG and nerve, the BioRad 9-plex rat cytokine kit combined with Luminex technology was used in age match control and up to 5 month STZ-diabetic rats. Neurons from normal or diabetic rats were cultured under defined conditions and transiently transfected with a wild-type NF-κB luciferase reporter construct to assess the ability of cytokines to modulate NF-κB activity. The effects of cytokines IL-1α, IL-1β and CNTF on axonal outgrowth were also assessed. Cytokine proteins showed altered expression levels in DRG and sciatic nerve isolated from diabetic rats. For example, IL-1β was 75.7±10.9 vs 32.1±5.2 pg/mg protein in control vs diabetic DRG (n=6; P<0.05). Sciatic nerve IL-1β was 64.15±8.1 pg/mg vs 25.68±5.3 in control vs diabetic (n=6; P<0.05). IL-1β, IL-1α and CNTF increased NF-κB reporter activity in adult sensory neurons cultured from normal and diabetic rats. This transcriptional activity was reduced in diabetic cultures (p<0.05). IL-1α, IL-1β and CNTF significantly increased axonal outgrowth in normal and, to a lesser extent, diabetic neurons. Finally, lentiviral-mediated shRNA knockdown of the NF-κB subunit p50 inhibited CNTF-induced axon outgrowth.
IL-1α and IL-1β levels were diminished in DRG/nerve and may underlie lower NF-κB activity in DRG in diabetes. In cultured adult sensory neurons, NF-κB activity was enhanced by different cytokines, such as IL-1α, IL-1β and CNTF, in association with enhanced axonal outgrowth. In neurons isolated from diabetic rats these effects of cytokines were sub-optimal.
Keywords: axon regeneration; CNTF; diabetes; lentivirus; neuropathy
Grant Number: Canadian Institutes for Health Research (grant#MOP-84214) and Juvenile Diabetes Research Foundation (grant#17-2009-722)



339.19/F14. Tetrodotoxin, dihydropyridine, and riluzole resistant persistent inward current: A novel sodium channel in rodent spinal neurons
*Y. DAI, L. M. JORDAN;
Univ. Manitoba, Winnipeg, MB, Canada
We have recently shown a novel persistent inward current (PIC) which is resistant to tetrodotoxin (TTX) up to 205 µM and dihydropyridine (DHP) up to 100 µM (TDR-PIC) in mouse spinal neurons. We also show that this current is calcium dependent and is mediated by sodium ions. In this study, we further explore the sensitivity of this current to riluzole, a specific antagonist of persistent sodium currents. The whole-cell patch clamp recordings were obtained in neonatal mouse spinal cord slices of 200-250 µm from T12-L4 in the presence of 2-5 µM TTX and 20-30 µM DHP (nifedipine, nimodipine or isradipine). TDR-PIC was recorded by a family of 10 second voltage bi-ramps and/or 2 second step voltages with 5 mV steps. Our results showed that the low concentrations of riluzole (<5 µM) did not induce any significant changes in onset voltage of TDR-PIC although a small reduction in amplitude was observed. But high concentrations of riluzole (10-30 µM) significantly reduced amplitude and depolarized onset of TDR-PIC. Statistical analysis indicated that the riluzole-induced reduction of TDR-PIC was dose-dependent and could be as high as 61% with 30 µM riluzole. On the other hand, the depolarization of TDR-PIC onset was small (≤ 4 mV) and did not appear to be concentration-related. Both low and high concentrations of riluzole reduced half-width of spikes induced by step voltages. These results suggested that the effect of riluzole on TDR-PIC was more observable in channel availability rather than the gating mechanism. On average (n=27), riluzole (3-30 µM) reduced the TDR-PIC by 31% (P<0.0005) and depolarized the onset by 1.8±2 mV (P<0.00005). The reduction of TDR-PIC by riluzole implicated that TTX and riluzole might not block the same type of persistent sodium currents because the TTX-sensitive component of PIC had been removed from TDR-PIC. This study demonstrated the persistence of TDR-PIC in high concentration of riluzole and was the first report to describe these unique pharmacological and electrophysiological properties of TDR-PIC in rodent spinal neurons. The presence of this novel PIC in spinal neurons suggests that this current could play an important role in generation of rhythmic activity.



342.10/G16. Equilibrative nucleoside transporter 1 (ENT1) regulates hypoxic/ischemic adenosine levels in hippocampal slices
D. ZHANG1, B. C. ALBENSI1,2, *F. E. PARKINSON1;
1Univ. Manitoba, Winnipeg, MB, Canada; 2St Boniface Res. Ctr., Winnipeg, MB, Canada
We have developed a transgenic mouse line that expresses human equilibrative nucleoside transporter type 1 (hENT1) in neurons. Relative to wild types, transgenic mice showed altered behavioural responses to ethanol and caffeine (Parkinson et al, J. Neurochem. 2009, 109:562-572), indicating that hENT1 affects endogenous adenosine levels and receptor signaling. The present study used in vitro methods to test whether hENT1 affects adenosine receptor signaling under ischemic conditions. Extracellular recording was used to measure field excitatory post-synaptic potentials (fEPSPs) in CA1 region of hippocampal slices from transgenic mice and their wild type littermates during transient oxygen deprivation (OD) or oxygen-glucose deprivation condition (OGD). Our results show that, compared to wild type mice, OD and OGD induced significantly (p<0.05) less synaptic inhibition in hippocampal slices from hENT1 transgenic mice. In the presence of NBTI, a selective inhibitor of ENT1, no differences between wild type and transgenic slices were apparent. These experiments indicate that while ischemic conditions increase adenosine levels, adenosine production and adenosine A1 receptor signaling were enhanced in wild type relative to transgenic mice. Thus, ENT1 in neurons may provide more cellular uptake than release of adenosine during ischemic conditions.



364.10/DD1. Individual differences in peritraumatic generalized fear as a predictor of post-traumatic stress disorder (PTSD) -like behaviors in rats
X. CHEN, Y. LI, *S. LI, G. J. KIROUAC;
Univ. of Manitoba, Winnipeg, MB, Canada
Post-traumatic stress disorder (PTSD) is a disabling psychiatric condition that can develop when individuals are exposed to a stressful and life-threatening event. The disorder is characterized by three clusters symptoms: re-experiencing (fear and anxiety produced by memories and cues associated the trauma); avoidance/numbing (avoidance of people and places) and hyperarousal (hypervigilance, irritability, insomnia). It is also well recognized that not all individuals exposed to trauma develop chronic PTSD. For this reason, a viable animal model of PTSD should account for individual variability in the expression of PTSD-like symptoms following exposure to a traumatizing event. Previous studies have shown that rodents exposed to acute footshocks can develop a behavioral profile that resembles some aspects of the clinical PTSD. To our knowledge, these studies have not shown individual differences in the response to trauma. In this study, we exposed male Sprague-Dawley rats (240-250 g) to acute footshock (5 x 2 second episodes of 1.5 mA presented randomly over 5 min) and divided the animals in low and high responders based on their freezing response when placed in a novel open field (40 x 65 cm) one day after shock exposure. Control animals were placed in the shock chamber however no shock was delivered. Results show that shock rats showed increased freezing when placed in the same context in which the shock was given (Days 10 and 27 post-shock) or when placed in a novel chamber (Days 9 and 25). High responders showed significantly more freezing at all test points when compared to low responders and non-shock rats. In addition, high responders exposed to the same context or novel chamber emitted more ultrasonic vocalizations in the range of 20-30 KHz, which are associated with fear, anxiety and dysphoria. We also show that shock rats displayed less contact with another rat in a social interaction test at Days 8 and 24 post-shock. Furthermore, shock rats were found to exhibit more avoidance behavior or anxiety in the defensive withdrawal and novelty suppressed feeding tests at Day 30. These effects were more pronounced in the high responders at all time points and the difference between low responders and non-shock rats were found to be non-significant at the end of the test period. The finding that high levels of fear in a novel environment (as a measure of hyperarousal) immediately after shock exposure is predictive of the development of more pronounced PTSD-like behaviors in the shock rats is consistent with recent clinical data which suggest that the presence of hyperarousal is the best predictor of the development of chronic PTSD.



368.11/HH18. Orexins in the paraventricular nucleus of the thalamus (PVT) as a mediator of dysphoria in morphine withdrawal
*Y. LI1,2, H. WANG1, C. WEI1, K. QI1, N. SUI1, G. KIROUAC2;
1Inst. Psychol, CAS, Beijing, China; 2Dept of Oral Biol., Univ. of Manitoba, Winnipeg, MB, Canada
Acute abstinence from morphine or other opioid drugs in dependent individuals produces negative physiological and emotional states. While the physiological effects produced by withdrawal quickly dissipate over days, the negative emotional state remains and represents a major factor contributing to relapse. Previous studies have implicated the bed nucleus of the stria terminalis and the central nucleus of the amygdala (collectively called the extended amygdala) as well as the shell of the nucleus accumbens as playing an important role in mediating the dysphoria associated with opioid withdrawal. The paraventricular nucleus of the thalamus (PVT) provides a very dense input to the extended amygdala and the shell of the nucleus accumbens and may represent a thalamic region involved in morphine withdrawal. For example, the PVT is activated following morphine withdrawal and receives a dense projection from orexin (hypocretin) neurons which are also activated by withdrawal from morphine. Work in our laboratory has provided evidence for a role for orexins in the PVT in the modulation of negative emotional behaviors. The present study was done to determine if blockade of orexin receptors in the PVT region attenuated the dysphoric effects of morphine withdrawal as measured using a conditioned place preference paradigm. Rats were treated with escalating doses of morphine given twice daily for 4 days. The orexin-1 receptor (OX1R) antagonist SB334867, the orexin-2 receptor (OX2R) antagonist TCSOX229, or vehicle were microinjected into the PVT region through an implanted cannula either 30 min before receiving naloxone during the conditioning trial to assess the acquisition of conditioned place avoidance (CPA) or one day later 30 min before placing the subject in the chamber to assess the expression of CPA. Administration of naloxone produced avoidance of the naloxone-paired compartment. Rats pretreated with the OX1R or OX2R antagonist (5.0 or 15.0 μg) had no effect on the acquisition of CPA. However, mircoinjections of TCSOX229 (15.0 μg) in the PVT region significantly attenuated the expression of naloxone-induced CPA while SB334867 had no effect. Results from these experiments indicate a role for OX2R in the PVT on the expression of the dysphoric state associated with morphine withdrawal. This is consistent with previous work in our laboratory showing that OX2R in the PVT regulates negative emotional behavior.



378.2/TT15. The effect of 7 days of daily treadmill exercise on gene expression in large and small lumbar dorsal root ganglion neurons
*N. R. PADDOCK, P. SHEPPARD, P. F. GARDINER;
Spinal Cord Res. Centre, Univ. of Manitoba, Winnipeg, MB, Canada
It has been previously demonstrated that the expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), synapsin I (SYNI), and growth associated protein (GAP-43), is upregulated in rat whole dorsal root ganglia (DRG) after 7 days of voluntary exercise. We wished to extend these observations to include forced treadmill exercise, to add expression of the genes syntaxin-IA (STX1A), nerve growth factor (NGF), and sodium ion channel alpha subunits (Nav 1.2 and 1.9), and to determine if small (<=30um) and large (>=40um) DRG neurons respond differently. Female Sprague-Dawley rats were exercised on a treadmill daily for 7 days, with the final treadmill challenge being at a speed of 22 meters/minute for 30 minutes, at a 10 degree grade. Rats were sacrificed 4 hours after the last treadmill bout, and L4 and L5 DRGs from both sides were quickly removed and flash-frozen. Sections of DRGs were cut at 12 micrometres, and RNA was isolated from combined samples of large (500 cells) and small (700 cells) DRG neurons using laser capture microdissection. Samples were then prepared for real-time polymerase chain reaction (qPCR) experiments. Large DRG cells differed from small in expression of STX1A (small>large), SYNI (small>large), GAP-43 (small>large) and Nav1.9 (large>small). Exercise resulted in a decreased expression STX1A in small cells only, as well as a decrease in Nav1.2 expression in large cells only. No significant small-large cell differences, or exercise effects, were noted for NGF or BDNF. Small and large DRG cells differ significantly in the expression of several genes investigated in this study, as well as in their responsiveness to a short (7 days) daily exercise challenge. Based on previous literature, it appears that short-term daily voluntary and forced treadmill exercise may have different effects on DRG gene expression.



403.8/LLL64. So funny I forgot to laugh: The neural correlates of cognitive humour processing
M. G. WALLACE1, *D. W. CAMPBELL2, M. MODIRROUSTA2, J. SAREEN2, J. O. POLIMENI2, N. A. MCKEEN3, J. P. REISS4;
1Psychology, 2Psychiatry, Univ. of Manitoba, Winnipeg, MB, Canada; 3Cancer Care Manitoba, Winnipeg, MB, Canada; 4Psychiatry, Univ. of Western Ontario, Winnipeg, MB, Canada
Background. The ability to understand and enjoy humour is a fundamental characteristic of the human experience. The construct of humour is commonly associated with enjoyment; however, individuals are frequently presented with jokes that are not considered to be funny. Although recent neuroimaging studies have attempted to investigate humour comprehension and appreciation, none have focused on the examination of brain regions involved in not funny jokes. Cognitive humour processing has not been assessed in the absence of affect-related humour appreciation. By segregating the brain’s response to jokes rated as funny and jokes rated as not funny, the present study evaluates a method to unambiguously examine the neural correlates of cognitive humour processing.
Hypothesis. Jokes that are understood, but judged as not funny, will activate brain regions commonly associated with cognitive humour processing, including the left inferior frontal gyrus (IFG) and the bilateral posterior-temporal junctions (TPJ).
Methods. Twenty-three adult volunteers were presented with 120 visual captioned comics. Thirty comics were modified so that their captions provided a literal comment of the visual content to reduce the comedic impact. Participants rated each comics in the scanner as a funny joke, a joke but not funny, or not a joke. We performed voxel- and cluster-based analyses on mean BOLD responses to contrast responses to comics judged as not funny jokes relative to non-jokes.
Results. Repeated-Measures ANOVAs revealed that comics judged as not funny jokes, in comparison with those judged as non-jokes, activated a set of regions previously identified to be involved in humour comprehension: left IFG (p < .005, Z-Score = 3.2), left TPJ (p < .005, Z-Score = 3.0, right TPJ (p < .005, Z-Score = 3.2). Furthermore, subcortical reward regions associated with funny jokes were not activated during not funny jokes.
Implications. This study supports our hypothesis and builds upon recent neuroimaging humour studies by establishing a method for isolating the neural correlates of cognitive humour processing independent of affective processing. This new design helps resolve some conflicting results obtained in prior fMRI humour studies on humour comprehension. This method can be applied in future studies to dissect the cognitive and affective neural aspects of humour processing. As humour processing deficits are associated with certain psychiatric conditions, future studies should assess whether these deficits can be attributed to humour comprehension, appreciation, or a combination of both processes.



661.12/U15. Therapeutic strategies to stimulate proliferation and differentiation of the spinal cord precursor cells after spinal cord injury
*S. KARIMI-ABDOLREZAEE1, D. SCHUT2, J. WANG2, M. FEHLINGS2,3;
1Regenerative Med. Program, Univ. of Manitoba, Winnipeg, MB, Canada; 2Genet. and Develop., Toronto Western Res. Inst., Toronto, ON, Canada; 3Surgery, Univ. of Toronto, Toronto, ON, Canada
The adult spinal cord harbours a population of multipotential stem/progenitor cells (NPCs) in ependymal/subependymal and paranchymal regions. Despite the intrinsic ability of NPCs for cell replacement, the extent of their activation in the microenvironment of spinal cord injury (SCI) is inadequate to result in substantial spontaneous recovery. Moreover, under the influence of the post-injury microenvironment, activated NPCs mainly differentiate into reactive astrocytes. These findings suggest the regulatory effects of the post-injury niche on the behaviour of NPCs after SCI. We recently reported that chondroitin sulphate proteoglycans (CSPGs) in the glial scar restrict the outcomes of NPC transplantation after SCI. Here, we hypothesized that targeting CSPGs by chondroitinase (ChABC) and growth factor (GF) treatments would optimize the activation of endogenous NPCs after SCI. Five days after a compressive SCI, we intrathecally delivered vehicle, ChABC and/or GFs (EGF, bFGF and PDGF-AA) for seven days. On the treatment day, we started daily injections of BrdU. Using slot blot analysis, we showed the efficacy of ChABC treatment in degradation of CSPGs. By multiple labeling immunohistochemistry against BrdU and several neural markers, we examined the number and the phenotype of BrdU-labeled cells in five regions of the spinal cord section including the ependymal/subependymal regions. We show a significant increase in the number of BrdU-labeled cells in ChABC+GFs group compared to the vehicle group. In the base-line injured rats, astrocytes and NG2 cells with the phenotype of macrophages/microglia were the dominant BrdU-labeled cells. Interestingly, after treatments there was a significant increase in the number of Nestin+/GFAP-/BrdU+ cells (indicative of NPCs) in ChABC and GFs+ChABC groups. Moreover, the number of BrdU labeled astrocytes was significantly reduced in all three treatment groups compared to the vehicle groups. GFs and GFs+ChABC treatments favoured the number of BrdU-labeled cells within the oligodendrocyte lineage (Olig2+/ GFAP- /BrdU+). While the total number of NG2+/BrdU+ cells was unaffected by the treatments, the number of NG2+/Oligo+/BrdU+ (suggestive of oligodendrocyte precursor cells) was significantly higher in the GFs+ ChABC group. ChABC and GFs+ChABC treatments also resulted in a higher percentage of newly generated endothelial cells. In conclusions, our data emphasizes a key role for CSPGs in regulating the behaviour of stem/progenitor cells in the microenvironment of the post-injury extracellular matrix. Therefore, targeting CSPGs could potentially harness the therapeutic benefits of NPCs for the repair of SCI.



767.17/Y9. Quetiapine regulates the proliferation and differentiation of oligodendrocytes in the hippocampus of global cerebral ischemia mice
*X. BI1, Y. ZHANG2, J. KONG3, X.-M. LI2;
1Shanghai, China; 2Dept. of Psychiatry, 3Dept. of Human Anat. and Cell Sci., Univ. of Manitoba, Winnipeg, MB, Canada
Our previous research indicated that quetiapine, an atypical antipsychotic drug, prevented neuronal loss induced by cerebral global ischemia (GCI) in mice. In current study, we studied the effects of quetiapine on oligodendrocyte survival and development in the brain of GCI mice. The mice were treated with quetiapine (10 mg/kg/day; i.p.) or vehicle for two weeks prior to GCI surgery. BrdU (100mg/kg/day; i.p.) was administrated to the mice on postoperative day 4, 5 and 6 to detect proliferating cells. Mice were sacrificed on postoperative day 7 or day 40 and the brains were collected for immunohistochemistry staining. Mature oligodendrocytes and oligodendrocyte progenitor were identified using anti-GST-pi, anti-NG2 antibodies respectively. Neurons and astrocytes were detected by anti-NeuN and anti-GFAP antibodies respectively. Cell proliferation and differentiation were analyzed by co-localization of NeuN, GFAP, GST-pi, NG2 and Bromodeoxyuridine (BrdU). GCI decreased the mature oligodendrocyte number in hippocampus and corpus callosum and impaired the maturation of oligodendrocyte linage. Quetiapine pretreatment significantly prevented the oligodendrocyte loss and enhanced the oligodendrocyte progenitor cells genesis and maturation. These results indicate a new mechanism of quetiapine in the therapy of vascular dementia and other psychiatric disorders and suggest wider perspectives for the clinical use of quetiapine.



865.8/V12. Large scale screening of a shRNAmir library to identify genes essential to prion neurotoxicity
*M. STOBART1,2, S. L. R. SIMON2, L. LAMOUREUX2, S. BRUNET1, J. D. KNOX1,2;
1The Univ. of Manitoba, Winnipeg, MB, Canada; 2Publ. Hlth. Agency of Canada, Winnipeg, MB, Canada
Prion diseases are invariably fatal amyloidogenic disorders of the central nervous system. The amyloid plaques result from fibrillization of the host-encoded prion protein and the molecular mechanisms underlying prion-induced neurodegeneration remain elusive. This is in part due to the absence of an appropriate cell culture model system where neuronal cell death occurs in a manner similar to that observed in vivo. The objective of this study was to develop a cell culture model of PrP106-126 mediated neurotoxicity, and to use this model to screen a shRNAmir library targeting all known open reading frames in the human genome in an attempt to identify non-essential host genes required for prion-mediated neurotoxicity. This resulted in the identification of 97 gene targets conferring resistance to PrP106-126 mediated neurotoxicity. The gene target associated pathways and interactions are discussed.
In the developed model the application of the PrP106-126 neurotoxic peptide results in the death of ~99% of the neuronal cells. An apoptosis PCR primer array was used to demonstrate that the mechanism of cell death was caspase 3-mediated, potentially as a result of mitochondrial dysregulation, as observed in vivo. The model was then used to screen a shRNAmir library to identify non-essential host genes required for PrP106-126 mediated neurotoxicity. Sequencing of the shRNAmir present in surviving clones has led to the identification of a total of 97 gene targets, 59 of which are listed at NCBI as confirmed genes. An association of the 59 gene targets with prion neurotoxicity has not previously been reported. The networks and associations of the novel targets were determined using the Ingenuity Pathway Analysis software. Direct interactions of multiple gene targets with the Huntingtin (Htt) and Amyloid precursor (APP) proteins were observed. In order to demonstrate their relevance in an animal model the expression levels of the identified gene targets in brains from control and prion-infected mice at 8 time points, with 6 biological replicates per time point were determined using quantitative real-time PCR.
Overall, these studies show that our model of prion neurotoxicity mimics the accepted in vivo killing mechanism, and enabled the identification of previously unreported gene targets proposed here to play a role in prion-mediated neurotoxicity.



866.15/W11. Knockdown of BNIP3 death gene family protects neurons from oxygen and glucose deprivation (OGD)-induced neuronal death
J. WENG, R. SHI, L. ZHAO, *J. KONG;
Univ. of Manitoba, Winnepeg, MB, Canada
BNIP3 family is a group of death-inducing mitochondrial proteins that includes BNIP3, NIX (also called BNIP3L) and BNIP3h. Previously we reported that BNIP3 was upregulated and played a role in delayed neuronal death in in vivo and in vitro models of hypoxia and experimental stroke. Here, we show the protective effects of inhibition of the BNIP3 death gene family on primary neuronal cultures exposed to oxygen and glucose deprivation (OGD) for 6 hours followed by reperfusion (RP) for 0, 12, 24, 36, 48, 60 and 72 hours respectively. Members of the BNIP3 family were highly upregulated after OGD/RP insults. To inhibit the BNIP3 family, we developed a lentiviral microRNA vector for BNIP3 and a lentiviral shRNA vector for NIX and BNIP3h (NIX and BNIP3h have identical mRNA). Knockdown of BNIP3 or NIX alone protected about 15% - 23% of neurons from OGD-induced cell death. Combined use of the two vectors to knockdown the BNIP3 family (BNIP3L and BNIP3h) reduced death of neurons by 40% in cells exposed to ODG for 6 h followed by reperfusion for 72 h. Mitochondrial membrane potential loss, permeability transition pore opening and ROS production were all significantly attenuated by BNIP3 inhibition. We further show that EndoG and AIF were released from mitochondria and translocated into nuclei in neurons treated with OGD/ RP. Over-expression of BNIP3 and BNIP3L caused AIF and EndoG translocation and subsequent neuronal death. Inhibition of BNIP3 by RNA silencing blocked EndoG and AIF tanslocation and prevented neuronal death. A time course of increase of autophagy was observed as determined by the ratio of LC3-II to LC3-I, an autophagy marker protein. Using Fluoro-Jade C and MDC double-staining, and electron microscopy we found that the increment in autophagy after OGD and RP injury was accompanied by increased autophagic cell death rates, while this increase could be attenuated by the specific autophagy inhibitor, 3-Methyladenine (3-MA). We also proved that the time course and expression levels of Beclin-1, an autophagy related protein, correlated with the expression of BNIP3. Knockdown of BNIP3 by miRNA reduced OGD-induced autophagic cell death in neurons. In conclusion, our results indicate that the BNIP3 death gene family are regulators of caspase-independent autophagic neuronal death in the OGD model of stroke.



873.13/FF8. Changes in gap junction expression and function following ischemic injury of spinal cord white matter
*K. GONCHARENKO1,3, E. EFTEKHARPOUR4, A. A. VELUMIAN1,2,3, P. L. CARLEN1,3, M. G. FEHLINGS1,2;
2Divisions of Neurosurgery, Cell and Mol. Biol., 1Toronto Western Hosp., Toronto, ON, Canada; 3Physiol., Univ. of Toronto, Toronto, ON, Canada; 4Dept. of Physiol., Univ. of Manitoba, Winnipeg, MB, Canada
Axonal dysfunction after spinal cord injury (SCI) is associated with ischemia, which can be modeled in vitro as oxygen-glucose deprivation (OGD). Under such conditions the Na+/Ca2+ exchanger reverses and leads to increased intracellular levels of calcium. Previous attempts at reducing such excitotoxic damage by blocking the exchanger provided only partial axonal protection, foreshadowing the involvement of other channels in propagation of axonal injury. Gap junctions are widely present in the spinal cord white matter; however, their role in post-injury axonal dysfunction remains largely unexplored. The purpose of the present study was to explore the role of gap junctions in exacerbating the deleterious effects of OGD on spinal cord white matter. We hypothesized that inhibition of gap junctions would improve axonal conduction in spinal cord white matter during OGD. The functional role of gap junctions was assessed by electrophysiological recordings of compound action potentials (CAP) in rat spinal cord slices using the sucrose-gap technique. To model secondary SCI, in vitro slices were subjected to 30 min oxygen-glucose deprivation. Gap junction connexin mRNA expression was determined using qPCR and normalized to β-actin. Localization of connexins in white matter spinal cord tissue was performed using immunohistochemistry. In response to 30 min OGD, there was a rapid and reversible decline in the amplitude of the CAP compared to pre-injury. Carbenoxolone (100µM), a gap junction blocker, significantly delayed both the rate and the extent of the CAP decline, suggesting that blocking gap junctions reduces conduction loss during OGD. Carbenoxolone alone did not change the amplitude of CAP in non-injured tissue and thus does not seem to interfere with intrinsic axonal conduction. The results show no difference in mRNA expression of connexins 32, 36 and 43 immediately following or one hour after OGD. However, upon application of carbenoxolone in the setting of OGD, connexin 43 expression was downregulated (p<0.005). Immunohistochemistry showed that both connexins 36 and 43 are present in longitudinal spinal cord slices and connexin 43 overlaps with the astrocytic marker GFAP, confirming its location in astrocytic gap junctions. In summary, blocking gap junctions enhances axonal conduction during OGD. Moreover, dynamic changes in mRNA expression occur during OGD perhaps due to alterations in their open states. In light of these data, blocking gap junctions may represent a promising therapeutic strategy for ischemic or traumatic SCI.