Neuroscience 2009 - 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 10/17 1:00 PM
4:00 PM - 5:00 PM

South Hall A 		76.8
Poster 		Y7 *B. L. SHAY1, H. WITTENBERG1, J. LARSSON2, M. HOFSTATTER1; 1Univ. Manitoba, Winnipeg, MB, Canada; 2Karolinska Inst., Stockholm, Sweden The effects of acupuncture on cutaneous blood flow, skin conductance and temperature in a model of acute mechanical nociception in healthy human participants

76.Musculoskeletal Pain
Sun 10/18 8:00 AM
10:00 AM - 11:00 AM

South Hall A 		164.11
Poster 		U7 *V. I. PINTO1,2, M. PIND2, D. D. EISENSTAT1,2; 1Univ. Manitoba, Winnipeg, MB, Canada; 2Manitoba Inst. of Cell Biol., Winnipeg, MB, Canada DLX homeobox transcriptional regulation of Cone-Rod homeobox (CRX) gene expression during vertebrate retinal development

164.Retina: Photoreceptors and Pigment Epithelium
Sun 10/18 8:00 AM
11:00 AM - 12:00 PM

South Hall A 		195.16
Poster 		FF46 Y. LI1,2, S. LI2, H. WANG1, N. SUI1, *G. J. KIROUAC2; 1Inst. of Psychology, Chinese Acad. of Sci., Beijing, China; 2Univ. of Manitoba, Winnipeg, MB, Canada Orexins act on paraventricular nucleus of thalamus to induce anxiety-like behaviors: Interaction with CRF and dynorphin systems

195.Emotion: Rat Models
Sun 10/18 8:00 AM
8:00 AM - 9:00 AM

South Hall A 		198.5
Poster 		FF114 *J. D. THIESSEN1, Y. ZHANG2, H. ZHANG2, L. WANG2, R. BUIST3, J. KONG4, X.-M. LI2, M. MARTIN1,5; 1Physics and Astronomy, 2Psychiatry, 3Radiology, 4Human Anat. and Cell Sci., Univ. of Manitoba, Winnipeg, MB, Canada; 5Physics, Univ. of Winnipeg, Winnipeg, MB, Canada Measuring myelin content in the corpus callosum of a cuprizone mouse model of demyelination with quantitative MRI methods

198.Imaging Techniques II
Sun 10/18 1:00 PM
3:00 PM - 4:00 PM

South Hall A 		233.3
Poster 		D35 *J. E. RASH1, K. G. V. DAVIDSON1, T. YASUMURA1, A. PEREDA2, J. I. NAGY3, J. O'BRIEN4; 1Dept Biomed Sci., Colorado State Univ., Fort Collins, CO; 2Neurosci., Albert Einstein Col. of Med. of Yeshiva Univ., Bronx, NY; 3Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; 4Ophthalmology and Visual Sci., Univ. of Texas, Hlth. Sci. Center, Houston, Houston, TX Neuronal gap junctions at goldfish club ending / Mauthner cell mixed synapses are heterotypic, with Cx35 in club endings and Cx34.7 in Mauthner cell hemiplaques

233.Gap Junctions
Sun 10/18 1:00 PM
4:00 PM - 5:00 PM

South Hall A 		233.4
Poster 		D36 *C. E. FLORES1, T. YASUMURA2, K. G. V. DAVIDSON2, S. ENE1, J. I. NAGY3, J. E. RASH2, A. E. PEREDA1; 1Dominick P. Purpura Dept. of Neurosci., Albert Einstein Coll of Med., Bronx, NY; 2Dept. of Biomed. Sci., Colorado State Univ., Fort Collins, CO; 3Dept. of Physiol., Univ. of Manitoba, Fac. of Medicine,, Winnipeg, MB, Canada Trafficking of gap junction channels at identifiable electrical synapses in vivo

233.Gap Junctions
Sun 10/18 1:00 PM
2:00 PM - 3:00 PM

South Hall A 		243.2
Poster 		J5 D. ZHANG1,2, R. BUIST1, J. PEELING1,3, B. C. ALBENSI1,2, *F. E. PARKINSON1; 1Univ. Manitoba, Winnipeg, MB, Canada; 2St Boniface Res. Ctr., Winnipeg, MB, Canada; 3Univ. of Winnipeg, Winnipeg, MB, Canada Increased endothelin-1 induced ischemic injury in hENT1 transgenic mice

243.Ischemia: Molecular Mechanisms
Sun 10/18 1:00 PM
3:00 PM - 4:00 PM

South Hall A 		275.15
Poster 		DD48 E. R. KIM, *T. M. MIZUNO; Univ. of Manitoba, Winnipeg, MB, Canada Activation of hypothalamic and brainstem cells by xenin

275.Regulation of Food Intake and Body Weight: Peripheral Regulators
Mon 10/19 8:00 AM
9:00 AM - 10:00 AM

South Hall A 		334.6
Poster 		J35 *W. ZHU1, F. BEGUM2, J. PATERSON1, E. FROST3, M. NAMAKA1; 1Fac. of Pharm., 2Human Anat. and Cell Sci., Univ. of Manitoba, Winnipeg, MB, Canada; 3Manitoba Inst. of Child Hlth., Winnipeg, MB, Canada Antigenic induction of BDNF within the DRG of an animal model of MS

334.Demyelinating Disorders: Cellular and Molecular Mechanisms
Mon 10/19 8:00 AM
8:00 AM - 9:00 AM

South Hall A 		334.29
Poster 		K20 W. ZHU1, F. BEGUM2, J. PATERSON2, E. FROST3, *M. P. NAMAKA4; 1Fac. of Pharm., 2Univ. of Manitoba, Winnipeg, MB, Canada; 3Manitoba Insititue of Child Hlth., Winnipeg, MB, Canada; 4Univ. Manitoba, Winnipeg, MB, Canada Antigenic induction of spinal cord BDNF responsive cells in animal model of MS

334.Demyelinating Disorders: Cellular and Molecular Mechanisms
Mon 10/19 1:00 PM
2:00 PM - 3:00 PM

South Hall A 		421.14
Poster 		D28 *D. A. MCCREA1, W. BAUTISTA GUZMAN2, J. I. NAGY2, B. FEDIRCHUK2; 2Physiol., 1Univ. Manitoba Fac Med., Winnipeg, MB, Canada Presynaptic inhibition in neonatal spinal cord is impaired in connexin36 knockout mice

421.Synaptic Transmission: Presynaptic Mechanisms
Tue 10/20 8:00 AM
9:00 AM - 10:00 AM

South Hall A 		565.14
Poster 		EE12 K. C. COWLEY, E. ZAPOROZHETS, *B. J. SCHMIDT; Physiol., Univ. of Manitoba, Winnipeg, MB, Canada Enhanced excitation of propriospinal neurons facilitates bulbospinal transmission of the locomotor command signal in the in vitro neonatal rat brain stem-spinal cord preparation

565.Rhythmic Motor Networks: Descending Control
Tue 10/20 1:00 PM
3:00 PM - 4:00 PM

South Hall A 		631.15
Poster 		L19 L. CLAYTON, X. CHEN, X. QIU, *J. KONG; Univ. of Manitoba, Winnepeg, MB, Canada SOD1 oxidation as a mechanism of motor neuron death in amyotrophic lateral sclerosis

631.Motor Neuron Disease Models and Mechanisms II
Tue 10/20 1:00 PM
2:00 PM - 3:00 PM

South Hall A 		659.2
Poster 		BB32 *J. W. CHOPEK1,2, C. W. MACDONELL1, K. E. POWER1, K. GARDINER1, P. F. GARDINER1,2; 1Physiol., 2Kinesiology, Univ. of Manitoba, Winnipeg, MB, Canada Quipazine enhances the monosynaptic reflex to a greater extent in flexor versus extensor nerves in rat

659.Motor Neurons: Activity; Sensory, and Central Control - Exercise, Injury and Disease
Tue 10/20 1:30 PM
1:55 PM - 2:15 PM

Room S406B 		593.3
Minisymposium 		  S. L. Prime; Psychology, University of Manitoba, Winnipeg, MB, CANADA. The posterior parietal cortex plays a role in trans-saccadic memory of multiple objects

593.Nonspatial Functions of the Parietal Cortex in Monkeys and Humans
Wed 10/21 8:00 AM
8:00 AM - 9:00 AM

South Hall A 		728.5
Poster 		H34 G. L. ODERO1, S. NAFEZ2, K. OIKAWA1, D. GROSSMAN1, *B. C. ALBENSI2,1,3; 1Div'n. Neurogenerative Disorders, St. Boniface Res. Ctr., Winnipeg, MB, Canada; 2Pharmacol & Therapeut., 3Ctr. on Aging, Univ. of Manitoba, Winnipeg, MB, Canada Gene expression profiling in PS1 (M146V) mice and other transgenic Alzheimer's disease models

728.Alzheimer's Disease: Molecules and Mechanisms
Wed 10/21 8:00 AM
9:00 AM - 10:00 AM

South Hall A 		766.6
Poster 		CC75 U. SLAWINSKA1, H. MAJCZYNSKI1, K. MALESZAK1, *L. M. JORDAN2; 1Neurophysiol., Nencki Inst. of Exptl. Biol., Warsaw, Poland; 2Dept Physiol., Univ. Manitoba, Winnipeg, MB, Canada Improvement in coordination during hind limb locomotion produced by transplantation of 5-HT neurons into the spinal cord of paraplegic rats is mediated by 5-HT2 and 5-HT7 receptors

766.Spinal Cord Injury: Posture and Locomotion
Wed 10/21 1:00 PM
1:00 PM - 2:00 PM

South Hall A 		812.2
Poster 		B34 *T. L. IVANCO1, K. D. HARTLE2, M. S. JEFFERS2, M. PELOQUIN2, G. GERVAIS2; 2Psychology, 1Univ. Manitoba, Winnipeg, MB, Canada Differential effects of prenatal hypoxic insult on cortex and striatum

812.Development of Motor Systems: Mammalian Models and Human Studies
Wed 10/21 1:00 PM
3:00 PM - 4:00 PM

South Hall A 		860.15
Poster 		Z30 *K. E. POWER, B. FEDIRCHUK, W. BAUTISTA, D. MCCREA; Spinal Cord Res. Ctr., Univ. of Manitoba, Winnipeg, MB, Canada Intrapsinal regulation of motoneuron excitability during fictive scratch in the decerebrate cat

860.Motor Neurons: Development, Identification, Intrinsic Properties, and Mechanisms of Modulation
Wed 10/21 1:00 PM
4:00 PM - 5:00 PM

South Hall A 		860.16
Poster 		Z31 *C. MACDONELL1, K. E. POWER1, K. GARDINER1, P. F. GARDINER1,2; 1Spinal Cord Res. Centre, Dept. of Physiology, Fac. of Med., Univ. of Manitoba, Winnipeg, MB, Canada; 2Fac. of Kinesiology and Recreation Mgmt., Univ. of Maniotba, Winnipeg, MB, Canada Frequency current relationships in extensor and flexor motoneurons during fictive locomotion in the decerebrate rat

860.Motor Neurons: Development, Identification, Intrinsic Properties, and Mechanisms of Modulation


ABSTRACTS

76.8/Y7. The effects of acupuncture on cutaneous blood flow, skin conductance and temperature in a model of acute mechanical nociception in healthy human participants The effects of acupuncture on cutaneous blood flow, skin conductance and temperature in a model of acute mechanical nociception in healthy human participants
*B. L. SHAY1, H. WITTENBERG1, J. LARSSON2, M. HOFSTATTER1;
1Univ. Manitoba, Winnipeg, MB, Canada; 2Karolinska Inst., Stockholm, Sweden
Introduction: Acupuncture has been used for thousands of years in the treatment of many disorders. In the last decade acupuncture has been an increasingly more common treatment accepted in Western medicine, particularly in the treatment of pain. The mechanism and effects remain poorly understood. Purpose: To examine the effects of acupuncture on cutaneous blood flow (BF)and the sympathetic nervous system (SNS). Design and Methods: The study was a randomized repeated measures design. Ten healthy participants underwent unilateral acupuncture or placebo acupuncture at the acupuncture point LI10 (extensor carpi radialis brevis muscle of the forearm)with and without an acute mechanical nociceptive stimulus (clip). Laser Doppler blood flow (Perimed, Inc. Sweden), skin conductance (SC) and skin temperature (Thought Technology Ltd., Montreal Canada) were measured continuously throughout the experiment. Results: Acupuncture had a small transient effect on BF locally. The mechanical nociceptive stimulus increased BF 5 fold which appeared to be regulated by real acupuncture. Acupuncture increases skin conductance bilaterally as does the mechanical nociceptive stimulus. After application of the clip, both acupuncture and placebo acupuncture increase local skin temperature but not peripheral skin temperature i.e. local effect not a systemic or SNS effect. There appears to be minimal effect on temperature with the application of acupuncture in the absence of the nociceptive perturbation. Conclusions: In addition to a local effect there appears to be an activation of the SNS in the action of acupuncture. Monitoring cutaneous BF and the SNS are promising methods in determining the local and systemic effects of acupuncture. The use of Placebo acupuncture in these experiments may also provide insight into the higher brain regions involved in acupuncture analgesia.



164.11/U7. DLX homeobox transcriptional regulation of Cone-Rod homeobox (CRX) gene expression during vertebrate retinal development DLX homeobox transcriptional regulation of Cone-Rod homeobox (CRX) gene expression during vertebrate retinal development
*V. I. PINTO1,2, M. PIND2, D. D. EISENSTAT1,2;
1Univ. Manitoba, Winnipeg, MB, Canada; 2Manitoba Inst. of Cell Biol., Winnipeg, MB, Canada
Objectives: We are interested in identifying and characterizing DLX transcriptional targets during retinal development. The CRX (Cone-Rod homeobox) gene is required to differentiate and maintain retina cells into cone and rod photoreceptors. These photoreceptors are localized to the outermost layer of the retina where they are responsible for interpreting the image we see. In the Dlx1/Dlx2 mutant mouse, CRX is aberrantly expressed in the neuroblastic layer. We hypothesize that the Dlx homeobox genes directly regulate CRX expression during retinal development.
Methods: Gene and protein expression methods were performed on knockout tissue to visualize CRX expression. Chromatin immunoprecipitation (ChIP) of embryonic retina was utilized to identify DLX protein-genomic DNA complexes in situ. Quantification of expression was assessed by qRT-PCR and cell counting. In vitro assays such as electrophoretic mobility shift assays (EMSA) and luciferase reporter assays were used to observe the direct binding and activity of DLX2 on the Crx promoter in vitro.
Results: CRX is ectopically expressed in the Dlx1/Dlx2 knockout retina suggesting that these DLX transcription factors may be restricting CRX expression to the outer nuclear layer (ONL). ChIP assays demonstrated that DLX proteins are bound to regions of the CRX promoter in situ, supporting a direct role for Dlx genes in regulating CRX during development. These results are consistent with in vitro results obtained from EMSA and luciferase assays.
Conclusion: The Dlx1/Dlx2 knockout has aberrant and ectopic expression of CRX in the retina. Our data suggests that CRX is a transcriptional target repressed by the DLX transcription factors. DLX proteins may function to limit CRX expression to the ONL of the developing retina both spatially and temporally.



195.16/FF46. Orexins act on paraventricular nucleus of thalamus to induce anxiety-like behaviors: Interaction with CRF and dynorphin systems Orexins act on paraventricular nucleus of thalamus to induce anxiety-like behaviors: Interaction with CRF and dynorphin systems
Y. LI1,2, S. LI2, H. WANG1, N. SUI1, *G. J. KIROUAC2;
1Inst. of Psychology, Chinese Acad. of Sci., Beijing, China; 2Univ. of Manitoba, Winnipeg, MB, Canada
Orexins (hypocretins) are novel peptides produced in neurons of the lateral hypothalamus that regulate brain and behavioral arousal by acting on orexin receptors in arousal centers of the brain. The paraventricular nucleus of the thalamus (PVT) of the dorsal midline thalamus proposed to regulate arousal and like other arousal centers, the PVT receives a very dense projection from orexin neurons. In turn, glutamatergic neurons of the PVT project densely to areas of the extended amygdala (shell of the nucleus accumbens, bed nucleus of the stria terminalis, and central nucleus of the amygdala) which contain neurons that produce dynorphin and corticotropin releasing factor (CRF). Dynorphin and CRF neurons of the extended amygdala have been shown to play an important role in the regulation of negative emotional states including anxiety. Consequently, this study investigated if microinjections of orexin-A (OXA) and orexin-B (OXB) in the dorsal midline thalamus produced anxiety-like behaviors in rats tested in the elevated plus maze. We report that microinjections of OXA (3.0 and 10.0 μg/500 nl) and OXB (3.0 μg/500 nl) in or adjacent to the PVT reduced the open arm time and entries indicating an anxiety state. In addition, OXA and OXB increase the number of stretch attend postures and grooming duration which are behaviors normally expressed during aversive conditions. Control microinjections of OXA (3.0 μg/500 nl) in the lateral thalamus did not produce anxiety-like responses indicating that the effects were specific to the dorsal midline thalamus. Co-administration of an orexin-1 receptor (OX1R) antagonist with OXA did not attenuate the anxiogenic response induced by OXA. Since the OXA binds with similar affinity to the OX1R as the orexin-2 receptor (OX2R) and that OXB binds preferentially to the OX2R, we conclude that the OX2R is responsible for mediating the behavioral effects of both OXA and OXB. In addition, intracerebroventricular administration of a CRF receptor or an opioid kappa receptor antagonist attenuated the anxiety-like responses produced by orexins in the PVT. The results of the present study show that microinjections of OXA or OXB in the PVT produce anxiogenic effects which are mediated by central kappa and CRF receptors. Our study suggests that orexins released in the PVT may produce arousal in brain regions associated with emotions. The strong connection between the PVT and the extended amygdala provides an impressive anatomical framework for orexins to produce emotional arousal.



198.5/FF114. Measuring myelin content in the corpus callosum of a cuprizone mouse model of demyelination with quantitative MRI methods Measuring myelin content in the corpus callosum of a cuprizone mouse model of demyelination with quantitative MRI methods
*J. D. THIESSEN1, Y. ZHANG2, H. ZHANG2, L. WANG2, R. BUIST3, J. KONG4, X.-M. LI2, M. MARTIN1,5;
1Physics and Astronomy, 2Psychiatry, 3Radiology, 4Human Anat. and Cell Sci., Univ. of Manitoba, Winnipeg, MB, Canada; 5Physics, Univ. of Winnipeg, Winnipeg, MB, Canada
Introduction Magnetic resonance imaging (MRI) methods capable of quantifying the presence of myelin can improve both the diagnosis and understanding of white matter diseases such as MS. T2-weighted imaging, magnetization transfer imaging (MTI), and diffusion tensor imaging (DTI) were applied to the analysis of tissue in healthy mice and a cuprizone-fed mouse model of demyelination. MR measurements were correlated to pathohistology to quantify the influence myelin content and axonal geometry have on these quantitative MRI methods.
Methods Mouse Model: C57BL/6 mice were fed 0.2% cuprizone (w/w) at 8-weeks of age. After 6-weeks of feeding, extensive demyelination was observed in the corpus callosum. MRI: Control and cuprizone-fed mice underwent in vivo T2-weighted imaging (RARE sequence, 98 x 98 x 750 μm3 resolution, acquisition time = 10 minutes) and MTI (FLASH sequence, magnetization transfer ratio (MTR) calculated from proton density and magnetization transfer contrast images, 98 x 98 x 750 μm3 resolution, acquisition time = 2 x 14 minutes) at 2-, 4- and 6-weeks. Each set had 3 control and 3 cuprizone-fed mice (18 mice total). Longer scans were performed on both control and cuprizone-fed ex vivo brains from the 6-week set to acquire DTI (PGSE sequence, tetraorthogonal gradient-encoding scheme, 2 b-values: 300 and 800 s/mm2, 10 slices, 156x156x500 μm3 resolution, acquisition time = 5h20m) and quantitative MTI datasets. Image analysis was performed using MATLAB. Histology: Histological slices were stained with Luxol fast blue (LFB) to visualize myelin content.
Results T2-weighted images show contrast inversion in the extremities of the cuprizone-fed corpus callosum, indicative of demyelination. The MTR decreased in the absence of myelin. Reduced fractional anisotropy (a DTI metric) in the corpus callosum of the cuprizone-fed mouse may be indicative of demyelination or axonal degeneration. Histological slices stained with LFB show significant demyelination in the corpus callosum of the cuprizone-fed mouse.
Conclusion The MRI methods presented above could indicate demyelination, but they might also represent other physiological changes such as inflammation and axonal degeneration related to the cuprizone mouse model. In order to discriminate intact and degenerating myelin, a hybrid approach is necessary. This study lays the groundwork for correlating myelin-sensitive T2-weighted and MTI methods with axonal geometry-sensitive DTI metrics. Ultimately, a hybrid MRI approach could be an important diagnostic method for measuring myelin content in the CNS.



233.3/D35. Neuronal gap junctions at goldfish club ending / Mauthner cell mixed synapses are heterotypic, with Cx35 in club endings and Cx34.7 in Mauthner cell hemiplaques Neuronal gap junctions at goldfish club ending / Mauthner cell mixed synapses are heterotypic, with Cx35 in club endings and Cx34.7 in Mauthner cell hemiplaques
*J. E. RASH1, K. G. V. DAVIDSON1, T. YASUMURA1, A. PEREDA2, J. I. NAGY3, J. O'BRIEN4;
1Dept Biomed Sci., Colorado State Univ., Fort Collins, CO; 2Neurosci., Albert Einstein Col. of Med. of Yeshiva Univ., Bronx, NY; 3Physiol., Univ. of Manitoba, Winnipeg, MB, Canada; 4Ophthalmology and Visual Sci., Univ. of Texas, Hlth. Sci. Center, Houston, Houston, TX
Goldfish club ending / Mauthner cell synapses (CE / MC) provide an easily accessible model system for studying the connexin composition and physiology at "mixed" (chemical plus electrical) synapses. We used freeze-fracture replica immunogold labeling (FRIL) to investigate the distribution of two evolutionarily divergent homologues of human connexin36 (Cx36) -- Cx34.7 and Cx35 -- at these synapses. One sample (FRIL#811) was double labeled for Cx34.7 (JOB #2930/I) vs. Cx35 (Chemicon MAB3043), which have been demonstrated to be connexin-specific (O'Brien et al., J. Neurosci. 2004). Anti-Cx35 exclusively labeled >10 CE hemiplaques but did not label MC hemiplaques, whereas in the same replica, anti-Cx34.7 exclusively labeled >50 MC hemiplaques in several synapses. Another sample (FRIL #812) was double labeled the same day under identical conditions using antibodies to glutamate receptor NMDAR1, plus an antibody to Cx36 that, based on it's sequence, likely cross-reacts with both Cx34.7 and Cx35 (Ab298; Pereda et al., J. Neurosci. 2003) using the same secondary antibodies as in #811. Anti-Cx36 heavily and equally labeled both MC and CE hemiplaques, comparable to those labeled for Cx35 in #811, but more strongly than the hemiplaques labeled for Cx34.7. These data demonstrate the usefulness of simultaneous semi-quantitative comparisons of cross-reacting vs. non-cross reacting antibodies made against shared vs. unique sequences in both Cx34.7 and Cx35. Equal labeling on both sides using the cross reacting antibody revealed that each connexin is present in equal amounts on opposite sides, suggesting that MC Cx34.7 forms 1:1 heterotypic channels with CE Cx35. It should be noted, however, that Cx34.7 could not be demonstrated using immunofluorescence confocal microscopy using this particular antibody. Thus, definitive labeling of Cx34.7 by FRIL (albeit weaker than for Cx35) may be due to the SDS washing step used in FRIL, which may expose epitopes that remain inaccessible in non-SDS-treated immunofluorescence preparations. To test this conjecture, additional antibodies to other unique sequences in Cx34.7 are currently being evaluated, as are different epitope exposure regimens. These experiments will allow us to determine whether this asymmetric distribution of Cx35 vs. Cx34.7 (pre- vs. post-synaptic) applies to all CE / MC synapses, to a subset of these terminals, or to pre- vs. postsynaptic hemiplaques in general. Finally, FRIL demonstration of neuronal subtype-specific expression of two different connexins that form heterotypic gap junctions raises the possibility of electrical rectification at these mixed synapses (experiments in progress).



233.4/D36. Trafficking of gap junction channels at identifiable electrical synapses in vivo Trafficking of gap junction channels at identifiable electrical synapses in vivo
*C. E. FLORES1, T. YASUMURA2, K. G. V. DAVIDSON2, S. ENE1, J. I. NAGY3, J. E. RASH2, A. E. PEREDA1;
1Dominick P. Purpura Dept. of Neurosci., Albert Einstein Coll of Med., Bronx, NY; 2Dept. of Biomed. Sci., Colorado State Univ., Fort Collins, CO; 3Dept. of Physiol., Univ. of Manitoba, Fac. of Medicine,, Winnipeg, MB, Canada
Previous studies tracking fluorescently-tagged connexins revealed active turnover of gap junction (GJ) channels. New channels (connexons) are inserted in the periphery of GJ plaques, whereas old channels are removed from the center. Some GJ plaques were also shown to be internalized to one of the coupled cells (annular GJs). Despite the wealth of evidence for this phenomenon in expression systems, no studies have addressed connexin turnover in native junctions, in particular in electrical synapses. We now show evidence that is consistent with the existence of similar connexon turnover at identifiable mixed synapses at identifiable Mauthner cell / Club ending synapses in vivo, where it is possible to correlate the ultrastructural features with their physiological properties and their biochemical composition. Freeze-fracture immunogold labeling (FRIL) analysis revealed exocytosis/connexon incorporation at the periphery of large GJs, whereas endocytotic profiles with labeled connexins were found at the center of those plaques. Endocytosis appears to occur within central areas that display disorganized / non-hexagonal arrays of connexons. Images consistent with the internalization of the double-membrane to one of the coupled cells were also observed. Consistent with these findings, intradendritic application of a dynamin inhibitory peptide (D15, aa 828-842 of rat dynamin) resulted in a progressive enhancement of the electrical component of the mixed EPSP evoked by the stimulation of these mixed synapses. Furthermore, using immunolabeling and high-resolution confocal microscopy, we found that dynamin labeling was not restricted to the periphery of Club ending synapses, where chemical synaptic components are distributed, but was also identified in puncta located in the center of the contacts, where GJs are concentrated. In contrast, intradendritic application of a peptide that mimics the last 15 aa of the CT of connexin35 (the fish ortholog of connexin36 and present at these terminals) produced a progressive decrease of the electrical component of the EPSP, suggesting that it might interfere with its binding to proteins responsible for the trafficking/insertion of new channels. Thus, both physiological evidence and the widespread co-existence of exo / endocytotic images in the same replicas suggests the existence of an active trafficking of gap junctions channels at these identifiable electrical synapses. The highly dynamic nature of electrical transmission at these terminals suggests that this trafficking could constitute an important target for regulation of electrical synapses.



243.2/J5. Increased endothelin-1 induced ischemic injury in hENT1 transgenic mice Increased endothelin-1 induced ischemic injury in hENT1 transgenic mice
D. ZHANG1,2, R. BUIST1, J. PEELING1,3, B. C. ALBENSI1,2, *F. E. PARKINSON1;
1Univ. Manitoba, Winnipeg, MB, Canada; 2St Boniface Res. Ctr., Winnipeg, MB, Canada; 3Univ. of Winnipeg, Winnipeg, MB, Canada
Adenosine is an important neuromodulator within CNS. Under ischemic conditions, adenosine levels increase by two or more orders of magnitude. The extracellular concentration of adenosine in brain is regulated by cytosolic and extracellular enzymes and plasma membrane located nucleoside transporters. However, the precise pathway leading from intracellular ATP to extracellular adenosine has not been fully established.
Objective: To test whether expression of human equilibrative nucleoside transporter 1 (hENT1) in mouse neurons affects stroke injury, using cortical injections of endothelin 1 as a stroke model.
Methods: Under isofluorane anesthesia, endothelin-1 (400 pmol; 1 µl) was injected unilaterally into cerebral cortex (1 mm anterior to Bregma, 1 mm lateral to Bregma, 1.2 mm ventral) of hENT-1 transgenic (Tg; n=8) and wild type (WT; n=8) littermate CD1 mice. Mice were monitored by magnetic resonance (MR) imaging. Perfusion weighted images were obtained pre-injection and at 4 hr and 2 days post-injection to assess blood flow changes. T2 weighted images were obtained 2 days post-injection to assess cerebral infarcts. Perfusion weighted images were acquired with the arterial spin labelling method using 2 second labelling of the carotid arteries. A HASTE imaging was used for image readout with a RARE factor of 36, FOV 3x3 cm2, slice thickness 1mm. T2 weighted images were acquired with a RARE factor of 8, echo time 80 ms, FOV 2.56x2.56 cm2 and SLTH 0.5mm.
Results: Perfusion weighted MR imaging confirmed that injection of endothelin-1 produced a localized loss of cerebral blood flow that persisted for a minimum of 4 hr. Our results show no significant differences between Tg and WT mice in CBF, at the injection site, at 4h (Tg:9.5±2.7%; WT:11±2%) or 2 days (Tg:33.6±4.7%; WT:42.9±6.4%) post-injection. Taking 20% of normal cerebral blood flow as ischemic threshold, WT mice had a smaller (p<0.05) ischemic region (6.3±0.6mm3) compared to Tg (8.1±0.6mm3). At 2 days post-injection, T2 weighted images revealed infarcts in the cortical tissue; the infarct volume was significantly greater (p<0.01) in hENT-1 transgenic than in WT mice: 9.47±0.73 mm3 and 5.69±0.97 mm3, respectively.
Conclusions: Expression of hENT-1 in mouse neurons increases ischemic injury in cerebral cortex. Nucleoside transporters may provide a therapeutic target for the modulation of adenosine levels for neuroprotection.



275.15/DD48. Activation of hypothalamic and brainstem cells by xenin Activation of hypothalamic and brainstem cells by xenin
E. R. KIM, *T. M. MIZUNO;
Univ. of Manitoba, Winnipeg, MB, Canada
Xenin is a 25-amino acid peptide and is produced in gastric mucosa. We and others have shown that peripheral and central administration of xenin reduces food intake in rodents. However, the mechanism by which xenin reduces food intake is not well understood. A number of gut hormones reduce food intake by acting through the central nervous system (CNS). Therefore, we hypothesized that xenin suppresses food intake by acting through the CNS, in particular, the hypothalamus and the brainstem. However, the CNS sites that respond to xenin have not been examined comprehensively. In this study, we examined the distribution of xenin-activated cells throughout the mouse hypothalamus and the brainstem. Furthermore, we investigated the possible downstream mediator of xenin action in the hypothalamus. To determine if xenin activates hypothalamic cells, male C57BL/6 mice were injected intraperitoneally (i.p.) with xenin (50 μg/g b.w.) or saline and c-fos mRNA levels were measured. Xenin significantly increased c-fos mRNA levels in the hypothalamus, but not in the cortex, 30 min after injection. To further investigate the specific CNS sites that respond to xenin, we examined the effect of xenin on Fos expression in the mouse brain using immunohistochemistry. Mice were injected i.p. with xenin (50 μg/g b.w.) and perfused 2 hours after injection. Xenin significantly induced Fos expression in hypothalamic nuclei, including the arcuate nucleus (ARC), the paraventricular nucleus (PVH), the ventromedial nucleus (VMH), the dorsomedial nucleus (DMH), and the supraoptic nucleus (SON). In addition to these hypothalamic nuclei, xenin-induced Fos was found in the nucleus of the solitary tract (NTS) of the brainstem. The induction of Fos was not observed in the lateral hypothalamic area (LHA) and the area postrema (AP) of the brainstem. To further characterize xenin-activated cells, hypothalamic gene expression was compared between xenin-treated mice and saline-treated control mice using PCR array and real-time PCR. Hypothalamic interleukin 1β (IL-1β) mRNA levels were significantly increased in xenin-treated mice compared to saline-treated control mice. These results suggest that xenin stimulates specific CNS pathways that are involved in the regulation of food intake, and that hypothalamic IL-1β may mediate the effect of xenin on food intake.



334.6/J35. Antigenic induction of BDNF within the DRG of an animal model of MS Antigenic induction of BDNF within the DRG of an animal model of MS
*W. ZHU1, F. BEGUM2, J. PATERSON1, E. FROST3, M. NAMAKA1;
1Fac. of Pharm., 2Human Anat. and Cell Sci., Univ. of Manitoba, Winnipeg, MB, Canada; 3Manitoba Inst. of Child Hlth., Winnipeg, MB, Canada
The general objective of this research was to explore the role of brain derived neurotrophic factor (BDNF) in antigenic induced multiple sclerosis (MS).
Background: MS is a chronic, inflammatory neurological disease characterized by targeted destruction of the central nervous system (CNS) myelin. 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 (Copaxone) for this disease have recognized the importance of BDNF in re-myelination and protection of neurons.
Methods: A total of 66 adult female Lewis rats are divided into 3 experimental groups: naïve control, active control and active experimental autoimmune encephalomyelitis (EAE). Naïve control animals do not receive any injections. Active control animals receive 2 intraperitoneal injections of pertussis toxin (PT) and injections of Freund’s adjuvant (FA) and Mycobacterium Tuberculosis. Active EAE animals receive the same PT regimen administered to active controls plus full inoculation with FA and Guinea pig myelin basic protein. Comparative, time dependent analysis (day 0, 3, 6, 9, 12 &15) of BDNF gene and protein expression within dorsal root ganglia (DRG) and spinal cord is conducted using immunohistochemistry (IHC), western blot, In situ hybridization, RT-PCR and Real Time-PCR. The Cryostat techniques using 6% TCA solution was conducted for spinal column (10μm) tissue section including attached intact DRG and the specific BDNF antibody was tested with western blot.
Results: Antigenic induction involving an EAE model of MS does induce the gene and protein expression of BDNF within the DRG. BDNF expression reaches peak point at day 12 post-antigenic induction relative to the other experimental groups. The IHC and western blot analysis showed significantly up-regulated BDNF protein expression at day 12 post-antigenic induction group in spinal cord but no pronounced overall BDNF gene expression change in spinal cord using Real time PCR.
Conclusions: The up-regulated gene expression of BDNF within DRG and subsequent increased protein expression within spinal cord may represent a key element involved in myelin repair and neuroprotection in the CNS.



334.29/K20. Antigenic induction of spinal cord BDNF responsive cells in animal model of MS Antigenic induction of spinal cord BDNF responsive cells in animal model of MS
W. ZHU1, F. BEGUM2, J. PATERSON2, E. FROST3, *M. P. NAMAKA4;
1Fac. of Pharm., 2Univ. of Manitoba, Winnipeg, MB, Canada; 3Manitoba Insititue of Child Hlth., Winnipeg, MB, Canada; 4Univ. Manitoba, Winnipeg, MB, Canada
The general objective of this research was to explore the role of brain derived neurotrophic factor (BDNF) in the antigenic induction of multiple sclerosis (MS).
Background: MS is a chronic, inflammatory neurological disease characterized by targeted destruction of CNS myelin. 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 (Copaxone) for this disease have recognized the importance of BDNF in re-myelination. In addition, the active BDNF iso-form (14 Kda) binds the high affinity tyrosine kinase receptor B (TrkB) to cause most of the known biological pro-survival and neuroprotective effects.
Methods: A total of 66 adult female Lewis rats are divided into 3 experimental groups: naïve control, active control and active experimental autoimmune encephalomyelitis (EAE). Naïve control animals do not receive any injections. Active control animals receive 2 intraperitoneal injections of pertussis toxin (PT) and injections of Freund’s adjuvant (FA) and Mycobacterium Tuberculosis. Active EAE animals receive the same PT regimen administered to active controls plus full inoculation with FA and Guinea pig myelin basic protein. Comparative, time dependent analysis (day 0, 3, 6, 9, 12 &15) of BDNF gene and protein expression in spinal cord was conducted using immunohistochemistry (IHC), Western blot, in situ hybridization, RT-PCR and Real Time-PCR. The BDNF responsive cells in spinal cord are identified with IHC TrkB double labeling.
Results: IHC analysis of the spinal cord reveals elevated double labeling for TrkB receptor/neuron specific β-III tubulin expression that identifies a subpopulation of BDNF responsive spinal cord axons in EAE. Based on double labeling IHC analysis for TrkB & MBP, PDGFR-α or GFAP, the TrK B receptor was not co-localized with mature oligodendrocytes, oligodendrocyte precursors nor astrocytes. Our results have also identified another population of cells that express TrkB but are not co-labeled with any of the above CNS cellular markers.
Conclusions: The ability to identify a sub-population of BDNF responsive axons and an unknown cell type in the CNS may unveil key treatment strategies aimed at attenuating myelin damage along the axonal projections of these CNS spinal cord neurons.



421.14/D28. Presynaptic inhibition in neonatal spinal cord is impaired in connexin36 knockout mice Presynaptic inhibition in neonatal spinal cord is impaired in connexin36 knockout mice
*D. A. MCCREA1, W. BAUTISTA GUZMAN2, J. I. NAGY2, B. FEDIRCHUK2;
2Physiol., 1Univ. Manitoba Fac Med., Winnipeg, MB, Canada
In the mammalian spinal cord presynaptic inhibition of transmission from sensory afferents has long been considered to involve GABAergic dorsal horn interneurons that make axo-axonic synapses with afferent fiber terminals. Activation of these dorsal horn interneurons causes a prolonged depolarization of sensory afferents that can be recorded extracellulary as the dorsal root potential (DRP). Recently, attention has been drawn to the presence of gap junctions forming electrical synapses between neurons in the spinal cord. Elsewhere in the CNS, the major connexin protein forming such synapses is connexin36 (Cx36), which we find to be widely distributed in spinal cord laminae IV and V in both young and adult mice. To explore the possibility that Cx36 may play a role in presynaptic inhibition, we compared physiological measures of presynaptic inhibition in wild-type and Cx36 knockout (KO) mice.
Isolated spinal cord in vitro preparations from mice at postnatal age 9 to 11 days were used to asses cord dorsum potentials (CDP), the DRP and the ability of conditioning stimuli to inhibit the monosynaptic ventral root reflex. In the Cx36 KO mice there was a 79% decrease in DRP duration, a 66% decrease in DRP amplitude (n=19) and a 77% (n=4) reduction in the amplitude of the CDP measured at a latency of 10 ms. The ability of conditioning stimulation of an adjacent dorsal root at appropriate intervals to presynaptically inhibit the monosynaptic ventral root reflex was compared in wild-type and KO mice. In wild-type mice, conditioning stimulation (single shock at 2.5 times the threshold for the monosynaptic reflex) delivered 50ms before the test shock reduced the ventral root reflex by 30% (n = 8). This presumably presynaptic inhibition of the monosynaptic reflex was blocked by bath application of 20 mM bicuculline. In all eight Cx36 KO mice tested, such conditioning stimulation failed to reduce the ventral root reflex, i.e. failed to evoke a presynaptic inhibition of the monosynaptic activation of motoneurons by muscle spindle afferents.
We suggest that Cx36 is involved in sensory-evoked presynaptic inhibition in the spinal cord where the presence of gap junctions may serve to facilitate and synchronize activity between GABAergic interneurons that mediate presynaptic inhibition.



565.14/EE12. Enhanced excitation of propriospinal neurons facilitates bulbospinal transmission of the locomotor command signal in the in vitro neonatal rat brain stem-spinal cord preparation Enhanced excitation of propriospinal neurons facilitates bulbospinal transmission of the locomotor command signal in the in vitro neonatal rat brain stem-spinal cord preparation
K. C. COWLEY, E. ZAPOROZHETS, *B. J. SCHMIDT;
Physiol., Univ. of Manitoba, Winnipeg, MB, Canada
It is widely accepted that long direct bulbospinal projections contribute to spinal locomotor network activation. Using an in vitro neonatal rat brainstem-spinal cord preparation, we showed that a propriospinal relay system of neurons also contributes to transmission of the descending locomotor signal, and can be sufficient in this role (Cowley et al., J. Physiol. 586: 2008). Thus, electrical stimulation of the brainstem evoked hindlimb locomotor-like activity in 27% of preparations in which all long direct descending bulbospinal projections were disrupted by staggered contralateral hemisections. Given that 73% of the staggered hemisection preparations did not allow sufficient propagation of the bulbospinal signal to elicit locomotor activity, we are now focusing on this preparation as a model of partial spinal cord injury. We hypothesize that artificial excitation of propriospinal neurons may facilitate propagation of the locomotor command signal and produce hindlimb locomotor-like activity in preparations that otherwise fail to respond to brainstem stimulation alone. In the present work, hemisections were made in the rostral (right T1) and contralateral caudal (left T11) thoracic regions. Bath barriers were placed such that thoracic segments (T2 through T10 inclusive) were selectively exposed to neurochemicals. These were applied at concentrations subthreshold for evoking locomotor-like activity in the absence of brainstem stimulation and included: 5-HT (10 µM) with NMDA (2 µM), acetylcholine (10-15 µM) with the acetylcholinesterase inhibitor edrophonium (50-60 µM), and the alpha adrenergic receptor agonist clonidine (5-60 µM). The effect of increasing neuronal excitability through elevation of bath potassium concentration (up to 7 mM) or lowering magnesium ion concentration was also examined. Brainstem electrical stimulation alone failed to evoke hindlimb locomotor-like activity in 26/39 preparations. In two of three such preparations, application of 5-HT and NMDA to the thoracic region promoted locomotor-like activity in response to brainstem stimulation. This facilitatory effect was reversed by drug wash-out. On the other hand, acetylcholine / edrophonium (n=0/7), clonidine (n=0/5), increased bath potassium (n=0/5), and reduced magnesium (n=0/9) failed to facilitate the effect of brainstem stimulation. The results thus far suggest that at least some of the thoracic propriospinal neurons involved in propagation of the descending locomotor command signal are 5-HT and glutamate sensitive. These neurons may be suitable targets for therapeutic interventions to restore locomotor function after injury.



631.15/L19. SOD1 oxidation as a mechanism of motor neuron death in amyotrophic lateral sclerosis SOD1 oxidation as a mechanism of motor neuron death in amyotrophic lateral sclerosis
L. CLAYTON, X. CHEN, X. QIU, *J. KONG;
Univ. of Manitoba, Winnepeg, MB, Canada
ALS is an adult-onset, progressive and fatal neurodegenerative disorder. Pathologically it is characterized by loss of motor neurons leading to progressive muscle weakness, atrophy, and death . ALS presents as both sporadic (SALS) and familial (FALS) illness. To date, more than 100 mutations of the SOD1 gene have been reported in ALS families.
SOD1 is a 17KD protein that contains one copper and one zinc atom. The known function of this enzyme is to convert superoxide to water and hydrogen peroxide. It was first thought that the toxicity of different SOD1 mutants linking to ALS could result from decreased free radicals scavenging activity. However, studies show that mutant SOD1 enzymes cause motorneuron degeneration by a gain of toxic property. The nature of the gain-of-function toxicity in mutant SOD1 is not clear.
Recent studies suggest that SOD1 itself is a target of oxidative damage. Human SOD1 has four cysteine residues, Cys6, Cys57, Cys111, Cys146. An internal disulfide bond exists between Cys57 and Cys146. This disulfide bond is highly conserved in SOD1, while the remaining two cysteine residues are free and prone to posttranslational modifications.
Here we show that free cysteine residues in SOD1 are available to be modified by malPEG and AMS, and that this modification decreases with disease progression. We also show that the mutation of cysteine 111 residue resulted in reduced cross-linking activity. Additionally we report that SOD1-related aggregation are more stable in G37R transgenic mice than in wild type mice and this stable aggregation increases as the disease progresses in the neuronal tissues of G37R transgenic mice. Our data suggests that cysteine residues in SOD1 are post translationally modified, and Cys111 plays a role in SOD1 aggregation.



659.2/BB32. Quipazine enhances the monosynaptic reflex to a greater extent in flexor versus extensor nerves in rat Quipazine enhances the monosynaptic reflex to a greater extent in flexor versus extensor nerves in rat
*J. W. CHOPEK1,2, C. W. MACDONELL1, K. E. POWER1, K. GARDINER1, P. F. GARDINER1,2;
1Physiol., 2Kinesiology, Univ. of Manitoba, Winnipeg, MB, Canada
5-HT has the ability to modulate motor output by activating both sodium and calcium persistent inward currents (PICs). Recently it has been shown that in neonatal rats, identified flexor and extensor motoneurons express different intrinsic firing properties. Specifically, a significant portion of extensor but not flexor motoneurons were able to generate self-sustained firing, consistent with the activation of PICs. Apart from this, little is known about the potential differences in sensitivity between flexor and extensor motoneurons to 5-HT in adult rats. Therefore, the purpose of this study is to determine if Quipazine, a 5-HT2R agonist, has a preferential effect on either the flexor or extensor monosynaptic reflex (MSR). A 5-HT2R agonist was used since it has been shown that 5-HT2R activation induces long term facilitation of the MSR in vitro and in vivo. Female Fisher Brown Norway rats were used. Surgical preparation consisted of a precollicular decerebration followed by spinal transection at C4 and sectioning of the L4/L5 dorsal roots. The MSR was elicited by stimulation of the L4/L5 dorsal roots and monitored by the tibial (extensor) and peroneal (flexor) nerves using bipolar silver electrodes. Baseline measurements of the MSR were recorded prior to drug administration. After baseline, an intraperitoneal injection of Quipazine (0.5mg/kg) was given and the MSR at Threshold (T), 1.25T, 2T and 3T were recorded every five minutes for two hours. The MSR recordings were rectified and the area under the responses for 1ms, 1.5ms and 2ms was determined. Immediately after the administration of Quipazine, a significant increase (p <0.05) in the MSR was observed and maintained for two hours. The tibial and peroneal nerves showed 3.76- and 5.97- fold increases respectively. The effect of Quipazine on the MSR response was significantly greater for the peroneal nerve when compared to the tibial nerve (p <0.05). The results indicate that the MSR pathway for flexor motoneurons is more sensitive to Quipazine than extensor motoneurons. Quipazine may increase MSR excitability either presynaptically by altering neurotransmitter release or post synaptically by activating motoneuron PICs, to a greater extent in hindlimb flexors than extensors.



593.3. The posterior parietal cortex plays a role in trans-saccadic memory of multiple objects The posterior parietal cortex plays a role in trans-saccadic memory of multiple objects
S. L. Prime;
Psychology, University of Manitoba, Winnipeg, MB, CANADA.
Prime SL, Vesia M, Crawford JD. (2008): Transcranial magnetic stimulation over posterior parietal cortex disrupts transsaccadic memory of multiple objects. J Neurosci. 28(27):6938-49.



728.5/H34. Gene expression profiling in PS1 (M146V) mice and other transgenic Alzheimer's disease models Gene expression profiling in PS1 (M146V) mice and other transgenic Alzheimer's disease models
G. L. ODERO1, S. NAFEZ2, K. OIKAWA1, D. GROSSMAN1, *B. C. ALBENSI2,1,3;
1Div'n. Neurogenerative Disorders, St. Boniface Res. Ctr., Winnipeg, MB, Canada; 2Pharmacol & Therapeut., 3Ctr. on Aging, Univ. of Manitoba, Winnipeg, MB, Canada
Introduction. An increasing number of studies now show that age-related memory deficits can be caused by early functional neurochemical changes, without the appearance of major structural alterations (such as neuronal cell death). This suggests that alterations, such as transcriptional level changes in the hippocampus, may precede structural decline in both normal aging and age-related diseases. We hypothesize that modulation of transcription factor activation occurs in age-related diseases that leads to alterations in the expression of specific molecules normally used for synaptic reconstruction, which ultimately results in impairments in synaptic plasticity and memory. Methods. To test this hypothesis we have been using gene chips (Affymetrix), RT-PCR, Western blots, inmmunohistochemistry, and standard staining and imaging techniques, etc. to profile possible changes in gene expression, cellular anatomy, and pathways of hippocampal networks in transgenic mouse models of Alzheimer’s disease (AD). Results. We find that homer1 expression is increased approximately three-fold in isolated hippocampus from PS1 (M146V) mice and is associated with other changes in the PS1 brain that have implications for synaptic plasticity and memory. These results in combination with our past studies showing enhanced LTP and increased calbindin D28k expression point towards dysregulation of calcium homeostasis as an early event that influences subsequent gene expression in this model. Significance. Understanding how memory-associated gene expression may change is critical for our understanding of normal and pathological processes of synaptic plasticity in age-related disorders.



766.6/CC75. Improvement in coordination during hind limb locomotion produced by transplantation of 5-HT neurons into the spinal cord of paraplegic rats is mediated by 5-HT2 and 5-HT7 receptors Improvement in coordination during hind limb locomotion produced by transplantation of 5-HT neurons into the spinal cord of paraplegic rats is mediated by 5-HT2 and 5-HT7 receptors
U. SLAWINSKA1, H. MAJCZYNSKI1, K. MALESZAK1, *L. M. JORDAN2;
1Neurophysiol., Nencki Inst. of Exptl. Biol., Warsaw, Poland; 2Dept Physiol., Univ. Manitoba, Winnipeg, MB, Canada
Previous experiments have shown that locomotor-like hind limb movements in spinal rats are improved by grafting fetal 5-HT neurons into the spinal cord below the level of the lesion. The involvement of 5-HT2 receptors in this recovery is indicated by the finding that Cyproheptadine, a 5-HT2 antagonist, alters the recovered locomotion in grafted rats, and by the fact that Quipazine, a 5-HT2 agonist, reverses the effects of Cyproheptadine. Here we examine in detail the effects of the grafts on coordination among flexor and extensor muscles and between the two hind limbs, and show that 5-HT2 and 5-HT7 receptors are involved in the improvement in coordination produced by the grafts. Wistar rats 3- 4 months old were spinalized at the T8 - T10 level. One month later, fetal neurons taken from the raphe region were grafted into the spinal cord below the lesion (at the T9 - T12 level). Control spinal animals were sham operated without tissue transplantation. EMG electrodes were implanted into the soleus and tibialis anterior muscles of both hind limbs. Locomotor trials were commenced one month after transplantation. Recovery of locomotion was evaluated with behavioural observations and EMG recordings during locomotor-like hind limb movements induced by tail pinching in rats suspended over the treadmill. Coordination was evaluated using polar plots of the relationships between the onsets of activity in pairs of muscles. Plantar walking occurred in the grafted animals, but not in the control animals. EMG analysis revealed markedly improved coordination among flexor and extensor muscles, and between the two hind limbs. IP injections of SB269970 (5-HT7 antagonist) or Cyproheptadine produced impaired coordination among flexor and extensor EMGs and between the two hind limbs, and abolished plantar walking. The disturbed coordination produced by Cyproheptadine was reversed by subsequent injections of Quipazine. We hypothesize that the changes in coordination produced by these drugs are due to actions at 5-HT receptors on coordinating interneurons of the central pattern generator (CPG) for locomotion. At least a portion of these neurons are likely to be inhibitory interneurons responsible for reciprocal inhibition among flexor and extensor components of the CPG, and for coordination between the left and right hind limbs. These results provide increased understanding of the actions on the CPG of grafted 5-HT neurons and new insights into the organization of the CPG for locomotion in mammals.



812.2/B34. Differential effects of prenatal hypoxic insult on cortex and striatum Differential effects of prenatal hypoxic insult on cortex and striatum
*T. L. IVANCO1, K. D. HARTLE2, M. S. JEFFERS2, M. PELOQUIN2, G. GERVAIS2;
2Psychology, 1Univ. Manitoba, Winnipeg, MB, Canada
Models of damage often provide insight into how plastic the brain is. Of particular interest is how early damage influences neuronal reorganization. During prenatal development the fetus is highly vulnerable to hypoxia. Exposure to hypoxic insults result in cerebral palsy, motor difficulties and developmental delay, however, the underlying morphological changes the brain undergoes remain poorly understood. To address this issue, pregnant dams (n = 2) were placed in a hypoxic chamber where they were exposed to 10% O2 beginning at gestational day 20 and continuing until the pups (n = 14) were 2 days old. During this time neurons are still actively migrating to their final, mature positions. Control animals (n = 14) were gestated under normoxic (21% O2) conditions. Pups were sacrificed at 65 days of age and the brains processed using the Golgi-Cox method. This method allows the cell body, dendrites, and dendritic spines to be clearly visible under a light microscope. Using NeuroLucida software, ten layer II and layer V pyramidal cells from motor cortex and ten medium spiny neurons from the dorsolateral striatum were drawn from each animal. Analysis of dendritic length, complexity and volume was conducted on the apical and basilar dendrites of each cell using NeuroExplorer software. Results revealed layer II neurons of experimental animals had shorter apical and basilar dendrites with less cell volume than controls. Apical dendrites of experimental animals were also less complex than those of control animals; however basilar dendrites did not show this trend. No differences were observed on any measure between groups for layer V pyramidal neurons in the motor cortex. Striatal regions of the brains of experimental animals had longer dendritic processes as well as an increased density of dendritic spines than control animals. This study indicates that the global effects of hypoxia induce area-specific changes in cell morphology and function as opposed to a global trend of morphological change.



860.15/Z30. Intrapsinal regulation of motoneuron excitability during fictive scratch in the decerebrate cat Intrapsinal regulation of motoneuron excitability during fictive scratch in the decerebrate cat
*K. E. POWER, B. FEDIRCHUK, W. BAUTISTA, D. MCCREA;
Spinal Cord Res. Ctr., Univ. of Manitoba, Winnipeg, MB, Canada
Motoneuron excitability is greatly increased during fictive scratch through a lowering of the voltage threshold for action potential initiation (Vth), a reduction in the afterhyperpolarization and voltage dependent enhancement of scratch drive potentials (SDPs). These effects occur in both flexors and extensors immediately at the onset of scratch and in extensors even during the initial phase of prolonged inhibition. Motoneuron excitability returns to control levels soon after a fictive scratch episode. We now report on the excitability of lumbar motoneurons during contralateral scratch and during scratch in acute high spinal preparations. Following neuromuscular blockade and topical application of curare to the C1 dorsal root entry region, fictive scratch was induced by light stroking of the face and ear.
During scratch in the contralateral limb, ipsilateral extensor motoneurons discharge vigorously to provide ipsilateral body weight support (BWS). Unlike the hyperpolarization of Vth that occurs during ipsilateral fictive scratch, Vth is either unaffected or depolarized in extensors during contralateral rhythmic scratching. Thus 4/5 motoneurons examined during both ipsi and contra-lateral scratch showed a Vth hyperpolarization during ipsilateral scratch but none displayed a reduction if Vth during BWS with contralateral scratch. This difference occurred despite the intense firing during BWS. Following an acute spinalization at C1, the magnitude of Vth lowering was similar (mean hyperpolarization -7.1 mV; n=12) to that in spinal intact preparations (mean hyperpolarization -5.1 mV; n=36). We also examined whether ipsilateral fictive scratch was associated with voltage dependent amplification of SDPs in acute spinal preparations by comparing the effect of intracellular current injection on the amplitude of the SDPs. With fast sodium channels blocked by QX314 in the electrode and under discontinuous current clamp, scratch drive potential amplitude could be increased with membrane depolarization or reduced with hyperpolarization. We postulate that these increases in motoneuron excitability in acute spinal preparations reflect the operation of previously unknown intraspinal mechanisms regulating motoneuron excitability.



860.16/Z31. Frequency current relationships in extensor and flexor motoneurons during fictive locomotion in the decerebrate rat Frequency current relationships in extensor and flexor motoneurons during fictive locomotion in the decerebrate rat
*C. MACDONELL1, K. E. POWER1, K. GARDINER1, P. F. GARDINER1,2;
1Spinal Cord Res. Centre, Dept. of Physiology, Fac. of Med., Univ. of Manitoba, Winnipeg, MB, Canada; 2Fac. of Kinesiology and Recreation Mgmt., Univ. of Maniotba, Winnipeg, MB, Canada
In rat motoneurons, firing rate increases in a linear manner with increased excitation via current injection through the intracellular microelectrode. During fictive locomotion, however, the frequency/current (F/I) relationship may be modified. For instance, Brownstone et al. (1992) found that the F/I relationship, present in the quiescent (non-locomoting) state was abolished during fictive locomotion in the cat. A more recent preliminary report has shown that the slope of the F/I relationship is largely reduced during fictive locomotion (Fedirchuk, SfN 1998). As such, the association between current input and frequency during fictive locomotion remains to be fully investigated. The goal of the present research was to induce fictive locomotion for the purpose of examining the F/I relationship of flexor and extensor hind-limb motoneurons in the adult rat. Recordings from antidromically-identified flexor and extensor motoneurons in the L3-L4 region of paralyzed decerebrated Sprague-Dawley rats were collected with the use of glass microelectrodes. Motoneuron discharge during ramp current injection (5 s up, 5 s down) was recorded prior to, during, and following periods of fictive locomotion lasting 20-40 s (mesencephalic locomotor regioin stimulation 80 to 200 µA; 0.5 ms duration; 15 to 30 Hz). The use of bilateral flexor (peroneal) and extensor (tibial) silver-chloride hook electrodes allowed both verification of locomotor-like activity in peripheral nerves and a means to stimulate and identify spinal motoneurons antidromically. F/I relationships obtained prior to and following fictive locomotion were calculated by plotting the instantaneous firing rate as a function of current amplitude. During fictive locomotion, motoneuron discharge occurring on either the peak or trough of the locomotor drive potential (LDP) was averaged and plotted against the mean intracellular current injected during that epoch. Results show that the F-I relationship is not absent during fictive locomotion. For both flexor and extensor motoneurons, a relationship between frequency and current was evident during firing on the peak of the LDP, but the slope of this relationship was lower than that of the non-locomotor condition by 30-50 %. The slope of the F-I relationship when firing occurred in the trough of the LDP was similar to that of the non-locomotor condition. The F-I relationship in the decerebrate rat during fictive locomotion is modified but only during the peak of the LDP.