Neuroscience 2008 Presentations from Winnipeg

Neuroscience 2008 - Presentations from Winnipeg scientists

Abstracts are below the schedule.

Saturday, November 15, 2008				Washington Convention Center: Hall A-C
	1:00 pm - 5:00 pm		Poster 29. Development: Motor, Sensory, and Limbic
1.		1:00 pm - 2:00 pm 29.17/C60 Transient expression of the neuronal gap junction protein connexin36 in the spinal cord of developing opossums, Monodelphis domestica *M. LEMIEUX¹, J. I. NAGY², T. CABANA¹, J.-F. PFLIEGER¹; ¹Univ. Montreal, Montreal, QC, Canada; ²Physiol., Univ. of Manitoba, Winnipeg, MB, Canada
	1:00 pm - 5:00 pm		Poster 62. Addiction: Neurobiology I
2.		2:00 pm - 3:00 pm 62.14/GG1 Anatomical and functional relationship between the paraventricular nucleus of the thalamus and dynorphin neurons in the nucleus accumbens *S. LI, G. KIROUAC; Univ. of Manitoba, Winnipeg, MB, Canada
	1:00 pm - 5:00 pm		Poster 63. Addiction: Neurobiology II
3.		3:00 pm - 4:00 pm 63.19/GG28 Orexin-A acts on the paraventricular nucleus of the thalamus to inhibit locomotor activity in rats Y. LI¹, S. LI², N. SUI¹, *G. J. KIROUAC²; ¹Key Lab. of Mental Hlth., Inst. of psychology, Chinese Acad. of Sci., Beijing, China; ²Dept of Oral Biol., Univ. of Manitoba, Winnipeg, MB, Canada
Sunday, November 16, 2008				Washington Convention Center: Hall A-C
	8:00 am - 12:00 pm		Poster 142. Huntington Disease and Prions: Mechanisms
4.		8:00 am - 9:00 am 142.17/T6 Molecular mechanism of prion protein-mediated myopathy J. LIANG¹, S. BOOTH³, *Q. KONG²; ¹Dept. of Pathology, ²Case Western Reserve Univ., Cleveland, OH; ³Div. ofHost Genet. and Prion Dis. Natl. Microbiology Lab., Winnipeg, MB, Canada
	8:00 am - 12:00 pm		Poster 174. Pain: Musculoskeletal, Incisional, and Visceral
5.		8:00 am - 9:00 am 174.9/NN9 Alternate pathways of induction of hyperalgesia in the rodent acidic saline model of chronic muscle pain L. L. JASPER, *B. J. MACNEIL; Physical Therapy, Univ. Manitoba, Winnipeg, MB, Canada
	1:00 pm - 5:00 pm		Poster 230. Cell Migration: Molecules Mediating Migration
6.		3:00 pm - 4:00 pm 230.7/A33 Extracellular regulated kinase regulation of oligodendrocyte progenitor migration *P. F. VORA^1,4, J. MUSTAPHA⁴, Z. ZHOU^2,4, E. E. FROST^1,2,3,4; ¹Human Anat. and Cell Sci., ²Pathology, ³Biochem. and Med. Genet., Univ. of Manitoba, Winnipeg, MB, Canada; ⁴Manitoba Inst. of Child Hlth., Winnipeg, MB, Canada
Monday, November 17, 2008				Washington Convention Center: Hall A-C
	8:00 am - 12:00 pm		Poster 334. LTP: Kinases and Intracellular Signalling II
7.		10:00 am - 11:00 am 334.19/E14 The induction of early growth response 2 (egr 2) is triggered by neuronal activity-dependent nf-kb activation S. NAFEZ^1,3, G. ODERO¹, K. OIKAWA¹, M. SPROULE^1,5, N. GE¹, D. ZHANG^1,3, B. ABRENICA², M. CZUBRYT^2,4, *B. C. ALBENSI^3,1; ¹Div'n. of Neurodegenerative Disorders, ²Inst. of Cardiovasc. Sci., St. Boniface Res. Ctr., Winnipeg, MB, Canada; ³Pharmacol & Therapeut., ⁴Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; ⁵Biol., Univ. of Winnipeg, Winnipeg, MB, Canada
	8:00 am - 12:00 pm		Poster 373. Rhythmic Motor: Cellular Properties
8.		11:00 am - 12:00 pm 373.8/NN3 Tetrodotoxin and dihydropyridine insensitive persistent inward currents are mediated by multiple components and activated at different membrane potentials in rodent spinal neurons *Y. DAI, L. M. JORDAN; Dept Physiol, Univ. Manitoba, Winnipeg, MB, Canada
	8:00 am - 12:00 pm		Poster 375. Rhythmic Motor: Sensory Regulation
9.		8:00 am - 9:00 am 375.13/OO12 Comparison of hindlimb motoneuron activities during fictive and normal locomotion in the cat *S. N. MARKIN^1,2, M. A. LEMAY¹, K. OLLIVIER-LANVIN¹, B. I. PRILUTSKY³, D. A. MCCREA⁴, I. A. RYBAK¹; ¹Dept. of Neurobiol. and Anat., Drexel Univ. Col. of Med., Philadelphia, PA; ²A.B. Kogan Res. Inst. for Neurocybernetics, South Federal Univ., Rostov-on-Don, Russian Federation; ³Sch. Applied Physiol., Georgia Tech, Atlanta, GA; ⁴Spinal Cord Res. Ctr., Univ. of Manitoba, Winnipeg, MB, Canada
	8:00 am - 12:00 pm		Poster 376. Rhythmic Motor: Network and Cellular Models
10.		9:00 am - 10:00 am 376.18/PP11 How a bipartite CPG can control the activity of bifunctional motoneurons: a modeling study with insights from deletions during fictive locomotion *K. HAMADE^1,2, N. A. SHEVTSOVA^1,3, S. N. MARKIN^1,3, S. CHAKRABARTY^4,5, D. A. MCCREA⁴, I. A. RYBAK¹; ¹Dept. of Neurobiol. and Anat., Drexel Univ. Col. of Med., Philadelphia, PA; ²Sch. of Biomed. Eng., Drexel Univ., Philadelphia, PA; ³A.B. Kogan Res. Inst. for Neurocybernetics, South Federal Univ., Rostov-on-Don, Russian Federation; ⁴Spinal Cord Res. Ctr., Univ. of Manitoba, Winnipeg, MB, Canada; ⁵Ctr. of Neurobiol. and Behavior, Columbia Univ., New York, NY
	1:00 pm - 5:00 pm		Poster 454. Sensory Disorders: Visual and Auditory
11.		4:00 pm - 5:00 pm 454.4/CC12 Improvements in binocular vision following a new binocularly-based treatment for amblyopia *B. MANSOURI^1,2, B. THOMPSON^1,3, R. HESS¹; ¹Dept Ophthalmol, McGill Vision Res. Unit, Montreal, QC, Canada; ²Neurol., Univ. of Manitoba, Winnipeg, MB, Canada; ³Optometry and Vision Sci., Univ. of Auckland, Auckland, New Zealand
Tuesday, November 18, 2008				Washington Convention Center: Hall A-C
	1:00 pm - 5:00 pm		Poster 646. Molecular Brain Ischemia
12.		2:00 pm - 3:00 pm 646.14/W27 Necrostatin-1 protects hypoxia-induced neuronal death by inhibiting BNIP3 J. WENG, *J. KONG; Human Anat. and Cell Sci., Univ. of Manitoba, Winnepeg, MB, Canada
Wednesday, November 19, 2008				Washington Convention Center: Hall A-C
	8:00 am - 12:00 pm		Poster 728. Cytokines: Expression, Regulation, and Function
13.		8:00 am - 9:00 am 728.5/D40 Multiple sclerosis (MS)-induced neuropathic pain via the dorsal root ganglia (DRG) M. MELANSON¹, Y. GONG², K. MADEC², X. GU², E. FROST³, F. BEGUM³, B. SHAY⁴, B. MCNEIL⁴, *M. P. NAMAKA²; ¹Neurol., Hlth. Sci. Ctr., Winnipeg, MB, Canada; ²Fac Pharm, Univ. Manitoba, Winnipeg, MB, Canada; ³Manitoba Inst. of Child Hlth., Winnipeg, MB, Canada; ⁴Sch. of Med. Rehabilitation, Fac. of Med., Winnipeg, MB, Canada
	8:00 am - 12:00 pm		Poster 734. Synaptic Transmission and Network Interactions
14.		10:00 am - 11:00 am 734.3/F11 Double-replica labeling reveals that connexin-45 is accompanied by connexin-36 adult rodent retina, forming bi-homotypic gap junctions *N. KAMASAWA¹, X. LI², K. G. V. DAVIDSON³, T. YASUMURA³, J. I. NAGY², R. SHIGEMOTO¹, J. E. RASH³; ¹DCS, NIPS, Okazaki, Japan; ²Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; ³Biomed. Sci., Colorado State Univ., Fort Collins, CO
	8:00 am - 12:00 pm		Poster 758. Neuroinflammation and Neurodegeneration II
15.		11:00 am - 12:00 pm 758.20/EE4 Multiple sclerosis (ms)-induced expression of brain derived neurotrophic factor (bdnf) in dorsal root ganglia (drg) *W. ZHU, K. MADEC, Y. GONG, X. GU, M. NAMAKA, W. ZHU; Fac. of Pharm., Univ. of Manitoba, Winnipeg, MB, Canada
	1:00 pm - 5:00 pm		Poster 825. Serotonin and Purine Receptors
16.		4:00 pm - 5:00 pm 825.24/D29 Hippocampal slices from human ENT1 transgenic mice show reduced temperature-induced and hypoxia-induced EPSP suppression B. C. ALBENSI^1,2, D. ZHANG^1,2, W. XIONG¹, *F. E. PARKINSON¹; ¹Dept Pharmacol, Univ. Manitoba, Winnipeg, MB, Canada; ²Div'n. of Neurodegenerative Disorders, St. Boniface Res. Ctr., Winnipeg, MB, Canada

ABSTRACTS

1.
29.17/C60 - Transient expression of the neuronal gap junction protein connexin36 in the spinal cord of developing opossums, Monodelphis domestica
*M. LEMIEUX¹, J. I. NAGY², T. CABANA¹, J.-F. PFLIEGER¹;
¹Univ. Montreal, Montreal, QC, Canada; ²Physiol., Univ. of Manitoba, Winnipeg, MB, Canada
Opossums Monodelphis domestica are born very immature after a 14-15 day gestation. The hindlimbs of a newborn are little more than immobile buds, but the rhythmic and alternate movements of its forelimbs allow it to climb on the mother’s belly and attach to a nipple to continue its development. The hindlimbs start moving during the second week. The neonatal spinal cord enlargements are very immature, especially at lumbar levels. Except for some bulbar reticular and lateral vestibular projections, the projections from the brain grow into the cord postnatally. Few chemical synapses are observed in the spinal enlargements in neonates, synaptogenesis unfolding largely postnatally. These findings led us to hypothesize that motor activity in neonatal opossums is initially mediated by electrical synapses (gap junctions), instead of by chemical transmission. Thus we have investigated connexin36 (Cx36), a gap junction protein widely expressed in neurons of mammalian CNS, in spinal enlargements by immunohistochemistry. Neurons were identified with the neuron specific protein NeuN and in some experiments motoneurons were retrogradely labeled with dextran-Texas Red injected in nerves innervating forelimbs muscles. At birth (P0), moderate Cx36 punctate labeling was detected in the presumptive grey matter and white matter in both enlargements, decreasing along an outside-in gradient. Puncta were seen ventrally at the location of presumptive motoneurons, which are small, undifferentiated and form one homogeneous ribbon along the border of the marginal zone. At P5 Cx36 expression decreased in the cervical enlargement, except dorsolaterally where it increased, and similar changes occurred later at lumbar levels. At P10 Cx36 labeling was still more promment in the dorsal horn and the dorsolateral funiculi, but decreased elsewhere, especially in the white matter. By P15 Cx36 decreased further in white matter, and was mainly located in substantia gelatinosa and to a lesser extent in nucleus proprius and lamina VII in grey matter. Labeled puncta were also seen close to motoneuronal somas and proximal dendrites. The high density of Cx36 labeling in the dorsolateral cord was observed until around P30, but had disappeared, as elsewhere, by P50. In adult opossums, Cx36 was below detection level in the spinal enlargements. The expression of Cx36 in postnatal opossums follows a reverse temporal pattern to that of the chemical synapses. This suggests that electrical synapses could mediate initial activity before chemical synapses are sufficiently in place.

2.
62.14/GG1 - Anatomical and functional relationship between the paraventricular nucleus of the thalamus and dynorphin neurons in the nucleus accumbens
*S. LI, G. KIROUAC;
Univ. of Manitoba, Winnipeg, MB, Canada
The paraventricular nucleus of the thalamus (PVT) is part of a group of midline and intralaminar thalamic nuclei implicated in brain arousal mechanisms. The PVT innervates a number of forebrain regions but provides an especially impressive projection to the nucleus accumbens (NAc). However, the types of neurons or neural mechanisms in the NAc influenced by the PVT remain unknown. A large number (95%) of neurons in the striatum are GABAergic medium spiny neurons (MSN) which contain either the enkephalin or dynorphin opiate peptides as co-transmitters. Compared to other areas of the striatum, staining for dynorphin is especially dense in the NAc. Using tract-tracing with the anterograde tracer biotin dextran amine (BDA) and immunohistochemical staining for prodynorphin (peptide precursor for dynorphin), we found that clusters of intense prodynorphin staining in the NAc received a dense fiber projection from the PVT. These overlapping clusters of PVT fibers and prodynorphin staining were found in different regions of both the core and shell of the NAc. We also found that neurons in the NAc could be activated following electrical stimulation of the PVT. Juxtacellular labeling with neurobiotin combined with immunofluoresence was used to show that neurons in the NAc that were strongly activated by stimulation of the PVT were MSNs which contain prodynorphin. In addition, the observation that prodynorphin-positive neurons were excited with short onset latencies (<15 ms) to PVT stimulation is consistent with the PVT providing monosynaptic inputs to these neurons in the NAc. Dynorphin neurons in the NAc are associated with aversive behavioral states and may play a role in psychiatric conditions such as depression and addiction. Since neurons in the PVT have been shown to be more active during arousal and stress, the results of the present study suggest that the PVT may relay arousal related information to dynorphin MSN of the NAc. This places the PVT in a key anatomical position to influence adaptive behaviors associated with stress and aversive states.

3.
63.19/GG28 - Orexin-A acts on the paraventricular nucleus of the thalamus to inhibit locomotor activity in rats
Y. LI¹, S. LI², N. SUI¹, *G. J. KIROUAC²;
¹Key Lab. of Mental Hlth., Inst. of psychology, Chinese Acad. of Sci., Beijing, China; ²Dept of Oral Biol., Univ. of Manitoba, Winnipeg, MB, Canada
Orexins (hypocretins) are novel peptides with arousal effects that have been shown to play a role in goal directed behaviors related to natural and drug reward. In addition, orexins have been implicated in the control of locomotion which forms an important component of exploratory and goal seeking behaviors. Our laboratory has previously shown that the paraventricular nucleus of the thalamus (PVT) receives a very dense orexin innervation, the function of which remains unknown. We have also demonstrated that neurons in the PVT that project to the nucleus accumbens receive appositional contacts from orexin fibers. Since the nucleus accumbens is involved in the control of locomotion, we tested the hypothesis that stimulation or blocking of orexin receptors in the PVT would influence locomotor activity (LA) in a variety of paradigms. Male Sprague-Dawley rats were implanted with a guide cannula directed at the posterior portion of the PVT (pPVT). All the rats then were injected with morphine (5mg/kg, i.p.) and immediately put into the box for 2 hours once a day for 5 days to develop behavioral sensitization. Conditioned LA (the day after last morphine injection) and expression of behavioral sensitization (priming with 5 mg/kg morphine 7 days after conditioned LA test) were observed respectively, after orexin-A (OXA, 3 mg/500 nl), orexin-1 receptor antagonist SB334867 (6 mg/ 500 nl), or vehicle (500 nl) injections in the pPVT. Microinjection of OXA in pPVT attenuated the LA produced by placing the rats in an environment previously paired with morphine and during a morphine challenge. In addition, OXA injections into the pPVT significantly attenuated the LA produced by exposing animals to a familiar environment and produced a similar but non-signifcant trend in rats exposed to a novel environment. In all cases, the attenuation was apparent only at times when peak LA is observed (first 10 min). The results of the present experiments indicate that stimulation of orexin receptors in the pPVT produces an inhibitory influence on LA in conditions where locomotion is enhanced. Since dopamine release in the nucleus accumbens plays a key role in stimulating LA in drug sensitized animals and during exposure to a novel environment, we speculate that orexins may act on the pPVT to inhibit dopamine release. The observation that orexin neurons are activated by emotional arousal and stress also suggests that this may represent a mechanism by which high levels of arousal can stop or inhibit ongoing behavior.

4.
142.17/T6 - Molecular mechanism of prion protein-mediated myopathy
J. LIANG¹, S. BOOTH³, *Q. KONG²;
¹Dept. of Pathology, ²Case Western Reserve Univ., Cleveland, OH; ³Div. ofHost Genet. and Prion Dis. Natl. Microbiology Lab., Winnipeg, MB, Canada
The prion protein (PrP) level in muscle has been reported to be elevated in patients with inclusion-body myositis, polymyositis, dermatomyositis and neurogenic muscle atrophy. We have previously reported that over-expression of wild type PrP strictly limited to skeletal muscles in an doxycycline-inducible transgenic mouse model leads to a primary myopathy that appears to be correlated with muscular accumulation of N-terminally truncated PrP C1 fragment (Huang et al., PNAS 104: 6800-6805, 2007), but the underlying mechanism is unknown. Sunyach et al. (JBC 282:1956-1963, 2007) reported that the C1 fragment positively controls p53 transcription and increases p53-like immunoreactivity and activity in HEK293 cells. In addition, knockout of transcription factor myocyte enhancer factor 2c (MEF2c) has been reported to cause a myopathy characterized by central location of nuclei in muscle cells. To understand the molecular mechanism of PrP-mediated myopathy, we performed extensive Western blot analysis to examine the correlation of protein levels of the C1 fragment, p53 and MEF2c in skeletal muscles of the inducible transgenic mouse model, and found that increased C1 levels lead to dramatic increase of p53 and reduction of MEF2c. Our results suggest that, in our inducible transgenic mouse model, the PrP-mediated myopathy may be p53-dependent, and the down-regulation of muscular MEF2c expression may underlie the central nuclei location observed in the muscle cells of doxycycline-induced mice.

5.
174.9/NN9 - Alternate pathways of induction of hyperalgesia in the rodent acidic saline model of chronic muscle pain
L. L. JASPER, *B. J. MACNEIL;
Physical Therapy, Univ. Manitoba, Winnipeg, MB, Canada
Research into mechanisms of hyperalgesia is ongoing with the goal of improving clinical management of chronic pain. Although several animal models of chronic pain have been reported in the literature, few address painful muscle conditions such as fibromyalgia. One such model in Sprague-Dawley rats creates a chronic bilateral hyperalgesia following intramuscular injection of acidic saline. Two injections into a gastrocnemius muscle spaced five days apart induces a long-lasting bilateral reduction in paw withdrawal thresholds. The first objective of this study was to determine if the establishment of hyperalgesia required the two injections to occur in the same muscle. Paw withdrawal thresholds were measured by applying von Frey filaments to the plantar surface of the hindpaw; the development of hyperalgesia was indicated by a decrease in paw withdrawal threshold. Saline injections (pH 7.4 or 4.0, 100ul) were administered in either the right lateral (RL), right medial (RM) or left lateral (LL) gastrocnemius muscle followed 5 days later by an injection in the right lateral gastrocnemius muscle. Paw withdrawal thresholds decreased bilaterally in all animal groups receiving acidic saline (pH 4.0) when tested 24 hours after the second injection (RL F3,15=11.291, p<0.001; RM F3,21=29.239, p<0.001; LL F3,15=55.157, p<0.001). A similar effect was observed when the first muscle injection was substituted with a non-specific treatment (intraperitoneal injection of lipopolysaccharide). These results demonstrate that the mechanism of chronic pain in this model is neither dependent on sensitization of the peripheral injection site or primary sensory afferents nor specific to the acidic saline stimulus. Given the role of central glia cells in other pain models it was next assessed whether the development of hyperalgesia could be blocked by pretreatment with minocycline, an inhibitor of glia cell activation. Hyperalgesia did not develop following acidic saline injections in animals pretreated with minocycline before the first muscle injection (F3,15=2.498, p=0.099). In contrast, animals pretreated with minocycline prior to the second muscle injection displayed a clear decrease in paw withdrawal thresholds (F3,6=72.585, p<0.001). These data indicate that the hyperalgesia that develops in this model is not dependent on sensitization of the peripheral injection site or the associated primary sensory afferents. Rather, central processes including glia cell activation play important roles in the development of hyperalgesia in this model of chronic muscle pain.

6.
230.7/A33 - Extracellular regulated kinase regulation of oligodendrocyte progenitor migration
*P. F. VORA¹^,4, J. MUSTAPHA⁴, Z. ZHOU²^,4, E. E. FROST¹^,2^,3^,4;
¹Human Anat. and Cell Sci., ²Pathology, ³Biochem. and Med. Genet., Univ. of Manitoba, Winnipeg, MB, Canada; ⁴Manitoba Inst. of Child Hlth., Winnipeg, MB, Canada
Oligodendrocytes (OL) are the myelinating cells of the CNS. Migration of immature OL is a prerequisite for normal myelination, as the OL progenitors (OP) originate in discrete areas of the developing CNS and migrate significant distances to their final site of myelination. OL originate in the embryonic telencephalon and migrate across the subpallial layer to the sub-ventricular zone (SVZ). The OPs then migrate away from the SVZ to populate white matter tracts of the CNS in response to numerous local cues including soluble signaling proteins. One such factor, Platelet-derived growth factor (PDGF), is essential for normal development of the myelin system. PDGF modulates several different OP behaviours, including proliferation, migration, and survival. OP response to PDGF differs according to the signaling pathway activated by receptor phosphorylation, i.e. migration is regulated via activation of the ERK signaling pathway, whereas proliferation is regulated via the PI₃K signaling pathway. Short periods of PDGF exposure (10ng/ml), 10 minutes, are sufficient to initiate and maintain OP migration for up to 72 hours. Further, PDGF-A induced OP migration is concentration dependent, as it is initiated by transient exposure to 10ng/ml PDGF, but not 1ng/ml PDGF. However, ERK1/2 phosphorylation occurs after transient exposure to both 10 and 1ng/ml PDGF, suggesting that ERK1/2 activation alone is not sufficient to activate OP migration.Our hypothesis is that ERK activation requires a threshold level of receptor activation that is not achieved by exposing OPC to low concentrations of PDGF for short periods of time. This supports previous studies that show that duration of receptor occupancy determines the downstream effects of ligands. This work was funded by the Manitoba Children’s Hospital Foundation and the Manitoba Health Research Council.

7.
334.19/E14 - The induction of early growth response 2 (egr 2) is triggered by neuronal activity-dependent nf-kb activation
S. NAFEZ¹^,3, G. ODERO¹, K. OIKAWA¹, M. SPROULE¹^,5, N. GE¹, D. ZHANG¹^,3, B. ABRENICA², M. CZUBRYT²^,4, *B. C. ALBENSI³^,1;
¹Div'n. of Neurodegenerative Disorders, ²Inst. of Cardiovasc. Sci., St. Boniface Res. Ctr., Winnipeg, MB, Canada; ³Pharmacol & Therapeut., ⁴Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; ⁵Biol., Univ. of Winnipeg, Winnipeg, MB, Canada
NF-kB mediated signaling is complex where it plays an important role in many biological processes. Investigators have reported that NF-kB is activated during the induction of long-term potentiation (LTP), an experimental paradigm of synaptic plasticity, and may be a requirement for synaptic plasticity and memory encoding. In this study we explored target genes of NF-kB in LTP, and identified early growth response 2 (Egr 2) as one target of NF-kB activation. To this end, we analyzed transcriptomes of LTP-induced hippocampal slices (CA1 region) from 2-month-old NF-kB p50 knockout mice (p50^-/-) littermate (p50^+/+) to identify target genes of NF-kB. LTP was attempted using standard theta-burst stimulation protocols in the Schaffer collateral pathway. At 3hr after conditioning, total mRNA samples were extracted from LTP-induced slices and non-stimulated control slices. mRNA samples were then subjected to the DNA microarray analysis (Affymetrix GeneChip^® Mouse Genome 430 2.0) and real-time RT-qPCR. We also examined the mRNA and protein expression level of Egr 2 in HeLa cells using real-time RT-qPCR and Western blotting. In addition, TNFa was used for activating the NF-kB signaling pathway in HeLa cells. The P-Match software was used for sequence analysis of distal promoter region of Egr 2. There were no significant differences for either basal synaptic transmission or LTP magnitude in p50^-/-vs. p50^+/+following theta burst stimulation. We identified that Egr 2 was inducted by NF-kB activation following LTP induction. From the gene-structure analysis, we also found several NF-kB consensus binding sites around promoter region of Egr2. In addition, the upregulation of Egr 2 mRNA in HeLa cells treated with TNFa, an activator of the NF-kB signaling pathway, was observed. These data suggest that Egr2 expression level is controlled by direct transcriptional activity of NF-kB.

8.
373.8/NN3 - Tetrodotoxin and dihydropyridine insensitive persistent inward currents are mediated by multiple components and activated at different membrane potentials in rodent spinal neurons
*Y. DAI, L. M. JORDAN;
Dept Physiol, Univ. Manitoba, Winnipeg, MB, Canada
In recent study we have shown that persistent inward currents (PICs) are expressed in spinal interneurons activated by locomotion. These PICs are commonly composed of a sodium component, which is tetrodotoxin (TTX) sensitive, and a calcium component, which is dihydropyridine (DHP) sensitive. Furthermore, we have demonstrated a novel PIC which is insensitive to TTX and DHP (TDI-PIC). In this study we further explore the mechanism underlying the TDI-PIC in spinal neurons, using neonatal rat and cFos-EGFP transgenic mice. The whole-cell patch clamp recordings were obtained in spinal cord slices of 200-250 µm from T12-L4. The TDI-PIC was recorded by a family of 10 second voltage bi-ramps in the presence of 2-5 µM TTX and 15-30 µM DHP (nifedipine, nimodipine or isradipine). The following results were obtained in this study: (1) The TDI-PIC was widely expressed in spinal neurons from lamina I - X in both EGFP positive and negative neurons of cFos-EGFP transgenic mice (P1-12) and in spinal neurons of neonatal rats (P1-5). (2) There were two types of TDI-PIC: Ca⁺⁺ dependent and Ca⁺⁺ independent. The Ca⁺⁺ dependent TDI-PIC could be largely reduced or completely removed in the recording solution where the calcium was omitted or replaced by cobalt. A low sodium (27 mM) or replacement of sodium by choline in the recording solution could also reduce or remove this TDI-PIC, suggesting that the Ca⁺⁺ dependent TDI-PIC could be mediated by sodium current and might be a candidate of calcium activated non-specific cation current. The onset of Ca⁺⁺ dependent TDI-PIC was about -31.1±12 mV with an amplitude of 75.4±59 pA (n=34). (3) The Ca⁺⁺ independent TDI-PIC was observed in calcium free recording solution. This TDI-PIC is a high voltage activated current with onset voltage over -10 mV. This study demonstrates that the TDI-PIC is meditated by multiple components in rodent spinal neurons. The unique pharmacological and electrophysiological properties of the TDI-PIC would allow this current to play an important role in generation of rhythmic activities such as locomotion.

9.
375.13/OO12 - Comparison of hindlimb motoneuron activities during fictive and normal locomotion in the cat
*S. N. MARKIN¹^,2, M. A. LEMAY¹, K. OLLIVIER-LANVIN¹, B. I. PRILUTSKY³, D. A. MCCREA⁴, I. A. RYBAK¹;
¹Dept. of Neurobiol. and Anat., Drexel Univ. Col. of Med., Philadelphia, PA; ²A.B. Kogan Res. Inst. for Neurocybernetics, South Federal Univ., Rostov-on-Don, Russian Federation; ³Sch. Applied Physiol., Georgia Tech, Atlanta, GA; ⁴Spinal Cord Res. Ctr., Univ. of Manitoba, Winnipeg, MB, Canada
Recent studies (Rybak et. al., 2006; McCrea and Rybak 2008) have proposed that the spinal locomotor central pattern generator (CPG) has a two-level organization in which a half-center rhythm generator (RG) governs the activity of multiple pattern formation (PF) networks controlling groups of synergist motoneuron pools. Within this architecture motoneuron pools controlled by a common input from the PF level would display highly synchronized activity during locomotion and form a synergy. These pre-defined motor synergies could then be shaped and reorganized by descending control and peripheral afferent feedback.
The fictive locomotion preparation in decerebrate immobilized cats offers an opportunity to examine synergies emerging from CPG-generated locomotor activity in the absence of afferent feedback and cortical influences. The present report compares the profiles of motor pool activity derived from ENG recordings during fictive locomotion with the corresponding EMG activity observed during unrestrained locomotion. Minimum Spanning Tree (MST) based clustering methods were used to identify potential synergist groups operating during fictive and normal locomotion. The results of cluster analysis were further refined by analysis of delays in the onsets of ENG/EMG bursts of synergists and analysis of motoneuron behavior during deletions (in fictive locomotion). We have found striking similarities between motor pool activities during fictive and normal locomotion. For example, hip and ankle extensors belong to one synergy in both fictive and normal locomotion whereas knee extensors (Vasti) were identified as a separate cluster by absolute delay analysis in fictive and by MST clustering in normal locomotion. At the same time, the activities of the bifunctional motor pools of PBSt (posterior biceps, semitendinosis) and of RF (rectus femoris) were notably different during fictive and normal locomotion. For example, PBSt never exhibited a short burst at the onset of extension during fictive locomotion, and the flexor burst of RF, seen in fictive locomotion, was never expressed during normal locomotion.
The results of this study are discussed in the context of the PF network organization. They also provide insights for understanding of how afferent and descending inputs modulate spinal locomotor CPG synergies during locomotion.

10.
376.18/PP11 - How a bipartite CPG can control the activity of bifunctional motoneurons: a modeling study with insights from deletions during fictive locomotion
*K. HAMADE¹^,2, N. A. SHEVTSOVA¹^,3, S. N. MARKIN¹^,3, S. CHAKRABARTY⁴^,5, D. A. MCCREA⁴, I. A. RYBAK¹;
¹Dept. of Neurobiol. and Anat., Drexel Univ. Col. of Med., Philadelphia, PA; ²Sch. of Biomed. Eng., Drexel Univ., Philadelphia, PA; ³A.B. Kogan Res. Inst. for Neurocybernetics, South Federal Univ., Rostov-on-Don, Russian Federation; ⁴Spinal Cord Res. Ctr., Univ. of Manitoba, Winnipeg, MB, Canada; ⁵Ctr. of Neurobiol. and Behavior, Columbia Univ., New York, NY
Motoneurons innervating muscles spanning two joints (i.e. bifunctionals) display a variety of activity patterns during fictive locomotion in decerebrate cats without afferent proprioceptive feedback. Bifunctional activity in these preparations can extend across flexion and extension phase boundaries or occupy only a portion of one phase. The present study used a computational model to investigate possible organizations of the circuitry controlling bifunctional motoneuron pools innervating posterior biceps and semitendinosis (PBSt; hip-extensor and knee-flexor) and rectus femoris (RF; hip-flexor and knee-extensor) muscles. The model is based on a two-level architecture of the locomotor CPG (Rybak et al. 2006; McCrea and Rybak 2007) in which a half-center rhythm generator (RG) controls pattern formation (PF) networks responsible for the alternating excitation and inhibition of flexor and extensor motoneurons. The hypothesis to be explored was whether a CPG with a bipartite RG could also create the more complex activities of bifunctional motoneuron pools. Our goal was twofold. First we wished to replicate the patterns of PBSt and RF motoneuron activity that occur during fictive locomotion evoked by midbrain stimulation in decerebrate adult cats (Markin et al. 2007). Second, the model needed to produce the changes in activity of PBSt and RF motor pools occurring during deletions. Deletions are spontaneously occurring periods during fictive locomotion in which motoneuron activity goes missing for a step cycles before returning to its regular activity pattern. Understanding the behavior of bifunctional motoneuron pools during deletions could provide important clues to the organization of the circuits sculpting their firing patterns. The circuits controlling PBSt and RF pools presented here incorporate additional interneuron circuits at the PF level that receive input from both the RG and PF networks. Using this architecture the full repertoire of PBSt and RF patterns observed during fictive locomotion as well as their activities during deletions can be reproduced by manipulating the weights of synaptic excitation and inhibition impinging on the specialized PF networks. Thus complex activity patterns of bifunctional motoneurons can be reproduced and explained within a framework of the two-level CPG with a bipartite organization of RG by incorporating circuitry that is only marginally more complex than that creating the strictly alternating activity of the pure flexor and extensor motor pools.

11.
454.4/CC12 - Improvements in binocular vision following a new binocularly-based treatment for amblyopia
*B. MANSOURI¹^,2, B. THOMPSON¹^,3, R. HESS¹;
¹Dept Ophthalmol, McGill Vision Res. Unit, Montreal, QC, Canada; ²Neurol., Univ. of Manitoba, Winnipeg, MB, Canada; ³Optometry and Vision Sci., Univ. of Auckland, Auckland, New Zealand
Purpose: Suppression of the amblyopic eye by the fellow fixing eye depends on the relative information content of the images that are presented to the two eyes dichoptically. Previously we have shown that when using a dichoptic RDK paradigm where signal and noise are presented to the AME and FFE separately, presenting higher contrast images to the AME can overcome interocular suppression in amblyopia and allow binocular combination to take place. We also showed that repeated measurements of the dicoptic coherence motion threshold under binocularly balanced conditions (i.e. high contrast to the AME and low contrast to the FFE) can improve both monocular and binocular vision in adult amblyopes. Here we tested whether the balanced performance condition is effective in improving the binocular combination of the information from the two eyes in amblyopic subjects.
Methods: Five adult amblyopes participated in the experiment. We used dichoptically and binocularly presented stimuli combined with signal/noise manipulations to test dichoptic and binocular combination. We used a motion task to assess dorsal visual pathway function. Firstly, signal and noise were presented dichoptically, signal to one eye and noise to the other. Secondly, signal and noise were presented binocularly (i.e. identical images were presented to the two eyes). Dichoptic presentation allowed for direct assessment of the contribution of each eye to perception and allowed the strength of the suppression of the AME to be quantified. Presenting stimuli binocularly allowed the extent to which information was combined between the two eyes to be assessed. The amount of information presented to each eye could be manipulated independently, allowing the stimuli to be biased in favour of one eye or the other.
Results: Our previous data (Mansouri et al., ARVO 2008) showed that repeated dichoptic measurement of the coherence motion threshold under balanced contrast conditions improves monocular and binocular vision in amblyopes. Here we found that when the contrast between the two eyes was balanced, binocular combination of stimuli was also improved.
Conclusions: Our data indicates that under conditions where the contrast of images presented to the AME and FFE is balanced to allow for normal binocular interaction, not only is suppression reduced but also the binocular integration of the images from the two eyes improves.

12.
646.14/W27 - Necrostatin-1 protects hypoxia-induced neuronal death by inhibiting BNIP3
J. WENG, *J. KONG;
Human Anat. and Cell Sci., Univ. of Manitoba, Winnepeg, MB, Canada
Necrostatin-1 (Nec-1) has been shown to be highly neuroprotective in ischemic neuronal death in vivo and in glutamate-induced cell death in vitro. Previously we reported that the mitochondrial death-inducing protein BNIP3 was upregulated in hypoxia and stroke, and caused neuronal cell death in a caspase-independent manner. The BNIP3-induced cell death pathway involves the interaction of dimerized BNIP3 with mitochondria to cause mitochondrial dysfunction, and subsequent release and nuclear translocation of the mitochondrial protein endonuclease G (EndoG). Here we show that Nec-1 inhibits BNIP3 function by preventing BNIP3 from interacting with mitochondria. Recombinant BNIP3 protein, when incubated with freshly isolated mitochondria, was able to integrate with mitochondria, possibly through its interaction with the mitochondrial membrane protein VDAC, and induce release of mitochondrial proteins including EndoG. When Nec-1 was added to the incubation, it significantly disassociated the binding of BNIP3 to mitochondria and prevented the release of EndoG. In primary cortical neuronal cultures, hypoxia induced significantly accumulation of BNIP3 expression and cell death. Inhibition of BNIP3 alone by RNAi significantly reduced neuronal cell death by 25%. Treatment with Nec-1 at the concentrations of 25 mM and 50 mM showed similar reduction of cell death rates as by the knockdown of BNIP3 in the same hypoxic condition. Nec-1 treatment did not change the levels of BNIP3 expression, but decreased the amount of mitochondria-bound BNIP3 dose-dependently. Our data suggests that Nec-1 could be an inhibitor for the BNIP3 cell death pathway.

13.
728.5/D40 - Multiple sclerosis (MS)-induced neuropathic pain via the dorsal root ganglia (DRG)
M. MELANSON¹, Y. GONG², K. MADEC², X. GU², E. FROST³, F. BEGUM³, B. SHAY⁴, B. MCNEIL⁴, *M. P. NAMAKA²;
¹Neurol., Hlth. Sci. Ctr., Winnipeg, MB, Canada; ²Fac Pharm, Univ. Manitoba, Winnipeg, MB, Canada; ³Manitoba Inst. of Child Hlth., Winnipeg, MB, Canada; ⁴Sch. of Med. Rehabilitation, Fac. of Med., Winnipeg, MB, Canada
Background: MS is a chronic, neurological disease characterized by targeted destruction of central nervous system (CNS) myelin. Circulating Th1-cells in the blood become activated upon exposure to specific CNS antigens such as myelin basic protein (MBP). Once activated they secrete inflammatory cytokines such as tumor necrosis factor alpha (TNF), which plays a pivotal role in the immune mediated destruction of myelin. The resultant disruption of electrical nerve impulse transmission produces a variety of clinical symptoms such as: weakness, fatigue and neuropathic pain. Hypothesis: Elevated production of inflammatory cytokines such as TNF in the early stage of MS is responsible for the initial activation of DRG and/or spinal cord that facilitate the downstream cellular events involved in the underlying pathogenesis of neuropathic pain. Objectives: 1) To determine if gene expression levels of TNF are up-regulated within the DRG and/or spinal cord of rats induced to a state of MS. 2) To correlate the temporal changes of gene expression to behavioral assessments of pain. Methods: 66 adolescent female Lewis rats were divided into 3 groups: naïve control (NC), active control (AC) and experimental autoimmune encephalomyelitis (EAE). NC animals (n=6) did not receive any injections and were sacrificed at day 15. AC animals (n=30) received intraperitoneal injections of pertussis toxin (0.3 mg in 200 ul of phosphate buffered saline at the identified time point’s day 0 & 2. AC animals also received 2 x 50 ml s.c injections of Freund’s adjunvant + 500 mg of Mycobacterium Tuberculosis H37Ra + saline into the area above the base of the tail at day 0. EAE animals (n=30) received the same treatment as that of the AC animals with the addition of 100 mg guinea pig MBP. A total of 6 AC and EAE animals were sacrificed respectively at each of the 5 time points (days 3, 6, 9, 12 and 15). DRG and spinal cord tissue were harvested for histological and real time reverse transcription polymerase chain reaction analysis. Results: Antigenic induction of MS resulted in significant elevations of mRNA for TNF within the DRG but not spinal cord. TNF mRNA expression reached peak significance at day 12 only in the EAE group relative to all other experimental groups and time points. A strong correlation between temporal TNF mRNA expression and specific behavioral assessments were established. Significance: Antigen-induced production of TNF within the DRG and/or spinal cord identifies a novel mechanism for MS-induced pain. This research represents a pivotal step towards designing new strategies that attenuate MS-induced neuropathic pain.

14.
734.3/F11 - Double-replica labeling reveals that connexin-45 is accompanied by connexin-36 adult rodent retina, forming bi-homotypic gap junctions
*N. KAMASAWA¹, X. LI², K. G. V. DAVIDSON³, T. YASUMURA³, J. I. NAGY², R. SHIGEMOTO¹, J. E. RASH³;
¹DCS, NIPS, Okazaki, Japan; ²Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; ³Biomed. Sci., Colorado State Univ., Fort Collins, CO
Gap junctions are essential in initial processing of visual information in the retina. In the inner plexiform layer (IPL), the two principal neuronal connexin proteins are Cx36 and Cx45. Confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling of both single- and double-replicas were used to identify Cx36- and Cx45-containing gap junctions in adult mouse and rat retina. In the IPL, 87% of Cx45 immunofluorescent puncta were co-localized with Cx36. In single-replicas, Cx45 was detected in 9% (64/671) of gap junction hemiplaques, 90-100% of which also contained Cx36. Cx45-containing gap junctions formed primarily plaques, with a few reticular configurations, compared to a wider variety of morphological types of Cx36-only gap junctions, as previously reported (i.e. plaque, reticular, ribbon and string configurations, Kamasawa et al., Neuroscience 142:1093-1117, 2006). To document the compositions of both sides of individual Cx45-containing gap junctions, we examined double-replicas showing complementary pairs of hemiplaques. In one set of matched replicas, we found >160 immunogold-labeled gap junctions, including 11 Cx45-containing gap junctions that had matching complements. Eight of these pairs were labeled for both Cx45 and Cx36 in both matching hemiplaques. In six of the eight mirrored doubly-expressing gap junctions, Cx45 and Cx36 appeared to be segregated into domains, with labels for both connexins beneath their respective complementary sectors in both mirror hemiplaques. These data demonstrate that Cx45-expressing neurons also express Cx36, and that rather than forming heterotypic gap junctions, Cx45 and Cx36 are present together on both sides, with Cx45 coupling to Cx45 and Cx36 coupling to Cx36, as bi-homotypic gap junctions.

15.
758.20/EE4 - Multiple sclerosis (ms)-induced expression of brain derived neurotrophic factor (bdnf) in dorsal root ganglia (drg)
*W. ZHU, K. MADEC, Y. GONG, X. GU, M. NAMAKA, W. ZHU;
Fac. of Pharm., Univ. of Manitoba, Winnipeg, MB, Canada
The general objective of this research was to explore the role of BDNF expression in MS. The specific objective of this research was to determine if antigenic induction of MS induces the expression of BDNF within the DRG.
Background: MS is a chronic, neurological disease characterized by targeted destruction of CNS myelin. The exact cause of MS has yet to be fully characterized; however, in all cases, demyelinated lesions are present throughout the CNS. As a result, the normal propagation of electrical impulses along nerve axons that are essential for physiological functioning are interrupted resulting in a variety of clinical deficits. While the cellular and molecular mechanisms responsible demyelination are not well understood, studies indicate that demyelination and oligodendrocyte death is mediated by immune cells and by activated parenchymal CNS cells. Current treatment strategies involving the use of glatiramer acetate for the relapsing remitting form of the disease have recognized the importance of BDNF in myelin repair.
Methods: A total of 66 adult female Lewis rats will be divided into 3 experimental groups: naïve control, active control and active experimental autoimmune encephalomyelitis (EAE). Naïve control animals (n=6) will not receive any injections. Active control animals (n=30) will receive 2 intraperitoneal injections (I.P.) of pertussis toxin (PT) (0.3 mg in 200 ul of phosphate buffered saline (PBS) at the identified time point’s day 0 & 2. In addition, these animals will also receive 2 x 50 ml s.c injections of Freund’s adjuvant (FA) + 500 mg of Mycobacterium Tuberculosis H37Ra (MT) + saline into the area above the base of the tail at day 0. Active EAE animals (n=30) will receive the same PT regimen administered to active controls plus full inoculation with FA + 500 mg MT + 100 mg Guinea pig myelin basic protein (MBP) given as 2 x 50 ml s.c injections into the area above base of the tail. Comparative, time dependent analysis (day 0, 3, 6, 9, 12 &15) of BDNF expression within DRG will be conducted using immunohistochemistry, semi-quantitative RT-PCR and Real Time-PCR techniques.
Results: Antigenic induction involving an EAE model of MS does induce the expression of BDNF within the DRG. BDNF expression reaches peak significance at day 12 post-antigenic induction relative to the other experimental groups.
Conclusion: The antigenic-induced expression of BDNF within the DRG may represent a key element involved in facilitating central myelin repair.

16.
825.24/D29 - Hippocampal slices from human ENT1 transgenic mice show reduced temperature-induced and hypoxia-induced EPSP suppression
B. C. ALBENSI¹^,2, D. ZHANG¹^,2, W. XIONG¹, *F. E. PARKINSON¹;
¹Dept Pharmacol, Univ. Manitoba, Winnipeg, MB, Canada; ²Div'n. of Neurodegenerative Disorders, St. Boniface Res. Ctr., Winnipeg, MB, Canada
Background Mice expressing human equilibrative nucleoside transporter subtype 1 (hENT1) under the control of a neuron specific enolase promoter were developed and characterized in biochemical and hippocampal slice experiments.
Methods Experiments were conducted to verify transgene expression and included tritiated adenosine uptake assays, tritiated nitrobenzylthioinosine binding assays, and reverse transcriptase polymerase chain reactions (RT-PCR) with hENT1 specific primers. For synaptic function assessment, the Schaffer collateral pathway was stimulated and EPSPs were recorded using standard brain slice electrophysiological techniques in CA1 hippocampal slices that were equilibrated, then either exposed to increasing temperature, from 32.5 C to 38.5 C over 15 min, or incubated in hypoxic aCSF (ie., bathed with 95% N₂- 5% CO₂) for 15 min.
Results Tritiated adenosine uptake into synaptosomes was increased 2-3 fold and tritiated nitrobenzylthioinosine binding to cortical membranes was increased up to 20-fold in samples from hENT1 transgenic mice, relative to wild type littermates. hENT1 expression was detected, by RT-PCR, in cortex, striatum, hippocampus and cerebellum but not in liver, heart, lung, kidney or skeletal muscle. Hippocampal slices from both wild type and transgenic mice showed suppression of EPSP amplitude with exposure to increasing temperatures or to hypoxia; in both conditions the suppression of EPSPs was significantly greater (p < 0.05) in slices from wild type mice relative to slices from transgenic mice.Conclusions hENT1 transgenic mice have increased ENT1 activity in neurons. This increase in ENT1 activity was associated with reduced inhibition of synaptic transmission in hippocampal slices exposed to progressive increases in temperature, or to hypoxic conditions. These data suggest that hENT1 expression reduces adenosine levels and adenosine signalling in electrically stimulated hippocampal slices.