1.
*K. OIKAWA^1,2, M. SPROULE^1,5, B. ABRENICA⁶, M. CZUBRYT^6,3, B. C. ALBENSI^1,2,4..... 48.10/K27
Exploration of NF-kB dependent gene expression patterns in theta-burst stimulated hippocampal slices
F-kB mediated signaling is complex and plays a critical role in many biological processes including autoimmune diseases, cancer, inflammatory responses, and in cellular neuroprotection. Investigators have also reported that NF-kB is activated during the induction of Long term potentiation (LTP) and may be a requirement for synaptic plasticity and memory, which has up to now not been fully realized. To investigate a role of NF-kB in neuronal activity dependent gene expression, we analyzed transcriptomes of LTP induced hippocampal slices from 2-month-old NF-kB p50 knockout mice (p50-/-) and its littermate (p50+/+) using Affymetrix GeneChip^® Mouse Genome 430 2.0 DNA microarrays. LTP was evoked by applying theta-burst stimulation to Schaffer collateral axon in the CA1 region. There were no significant differences of both basal synaptic transmission and LTP magnitude in each group. At 3hr after conditioning, total mRNA samples were extracted from LTP-induced slices and non-stimulated control slices. These mRNA samples were subjected to the DNA microarray analysis. We identified NF-kB dependent gene expression among genes related to synaptic plasticity, such as Brain-derived neurotrophic factor (BDNF), early growth response 2 (Egr2), Homer-1a, and Calbindin.

2.
D. BOSCH¹, J. S. YEOMANS², *S. SCHMID^3,2,1....67.25/AA23
Nicotine inhibits startle mediating giant neurons in the PnC by activation of glycinergic interneurons
Behavioural and electrophysiological studies have shown that startle mediating giant neurons in the nucleus reticularis pontis caudalis (PnC) receive inhibitory cholinergic input from the pedunculopontine tegmental nucleus (PPTg), mediated via G-protein coupled receptors. This inhibitory projection was supposed to mediate prepulse inhibition (PPI) of the startle response. However, electrophysiological studies also showed an inhibitory effect of carbachol on PnC giant neurons that remained after blocking muscarinic receptors by scopolamine, implying that nicotinergic receptors might be involved (Bosch and Schmid, EJN, 2006). We therefore examined here whether PnC giant neurons in rat brain slices are inhibited by the activation of nicotinic receptors. Trigeminal and auditory afferent fibres were stimulated while we performed whole cell patch clamp recordings of PnC giant neurons.
Nicotine (10µm) was added to the bath solution to examine the effect on the presynaptically evoked EPSCs. Nicotine significantly reduced the evoked EPSC-amplitudes (for trigeminal stimulation to 79.4%±3.9 SEM, n=9 and for auditory stimulation to 70.5%±5.5 SEM, n=9 of control amplitude). Since nicotine is an excitatory transmitter receptor, we tested the hypothesis that it activates inhibiting glycinergic interneurons by additionally applying the glycinergic antagonist strychnine (10µm). The additional application of strychnine significantly reduced the nicotine induced inhibition of PnC giant neurons (for trigeminal stimulation to 100.6%±8.4 SEM, n=5, p=0.011 and for auditory stimulation to 88.5%±7.6 SEM, n=9 of control amplitude).
We conclude that startle mediating PnC giant neurons are inhibited by nicotine via activation of glycinergic interneurons. Nicotine may therefore play an important role in prepulse inhibition at certain ISIs. Further experiments will have to show if the inhibition observed here in the slice is indeed part of the startle inhibiting PPI pathway.

3.
C. A. ADDURI, *J. J. MAROTTA; ....69.5/BB28
Prosopagnosia in early stages of Alzheimer’s disease
Objectives: Typically, when people with Alzheimer’s disease (AD) are unable to recognize their friends and family members, it is attributed to a memory impairment. What is often overlooked is that AD can affect areas of the brain that are important in face perception. So, while memory breakdown in the later stages of AD will certainly affect the ability to recognize individuals, it is also possible that perceptual deficits are present in early AD that impair face recognition.
Methods: Three groups of participants, a young adult group (7M, 8F, mean 23 years), an older non-neurological control group (5M, 7F, mean 74.92 years), and a group diagnosed with AD (7M, 2F, mean 85.67 years), completed a computerized face-matching task with upright and inverted faces. The faces were presented in either a frontal, three-quarter or profile view. The task involved choosing which of two choice faces matched the target face presented above, regardless of orientation. Prior to testing, the older control and AD groups completed a famous face task.
Results: There was a significant difference in performance F_(2,33) = 34.459, p < 0.001, between all three groups with the young being the most accurate (92%) followed by the old (79.6%), and finally the AD group (61.5%). For both the young and older groups, accuracy decreased in the order of 0º > 45º > 90º, for both upright and inverted conditions. This consistent orientation effect was not shown with our AD group. Furthermore, even when the faces were all presented at the same angle, and no mental rotation was necessary, the AD group was significantly impaired relative to their control groups. On the famous faces task, the AD group scored significantly worse than our elderly controls, with no one scoring above 5/15 correct. In contrast, there was no significant difference between the AD patients and a set of elderly controls on a Boston Shape Naming task run after testing.
Conclusion: Not only did our AD group have difficulty with this face matching task, they were also impaired on the famous faces task. In contrast, they performed well on a shape naming task. These results suggest that with the onset of AD, perceptual deficits develop that lead to a face specific impairment.

4.
*S. N. MARKIN^1,2, B. GRIFFEL^1,3, M. A. LEMAY¹, B. I. PRILUTSKY⁴, D. A. MCCREA⁵, I. A. RYBAK¹;.... 188.11/SS19
Analysis of hindlimb motoneuron activity during fictive locomotion in cat and identification of possible motor synergies controlled by locomotor CPG
The fictive locomotor rhythm evoked in the absence of sensory input by midbrain stimulation in decerebrate cats results from activation of the spinal locomotor central pattern generator (CPG). Recent studies (Rybak et. al., J. Physiol. 577: 617-658, 2006) have proposed that this CPG has a two-level organization in which the “top-level” rhythm generator (RG) controls motoneuron activation through pattern formation (PF) networks. We suggest that interneuron populations within the PF networks directly project to motoneurons and activate groups of motoneuron pools to form motor synergies. In order to identify potential synergies defined by the CPG, we processed data on the activity of various motor pools recorded from peripheral nerves during fictive locomotion in adult cats. From this data we developed a unified table of the activity of hindlimb motoneuron pools during fictive locomotion. Cluster analysis was used to identify groups of motoneuron pools with similar activity profiles during fictive locomotion. Seven such synergist groups have been identified, including two separate groups for hip flexors and ankle flexors, and a single group for all hindlimb motoneurons with extensor related activity. Four separate groups of motoneurons acting on muscles spanning more than one joint (biarticular) were identified: one for posterior biceps-semitendinosis (PBSt, hip extensor and knee flexor), one for rectus femoris (RF, hip flexor and knee extensor), and two separate groups for extensor digitorum longus and flexor digitorum longus. As presented in another poster at this meeting, PBSt activity can be of four distinct patterns during fictive locomotion. Within these patterns, some relationships between the activities of PBSt and RF were identified. Analysis of EMG patterns recorded from uniarticular flexors and extensors during normal locomotion in intact cats showed striking similarities with the synergies identified during fictive locomotion with some differences in selected EMG timing that presumably reflected the effects of afferent input to spinal reflex circuitry below the level of the CPG. Two notable differences from activity recorded during normal locomotion were that PBSt never exhibited a short second burst at the onset of extension during fictive locomotion and the RF pattern in fictive locomotion differed from that recorded in intact cats. Our data are consistent with the idea that the firing patterns of biarticular muscles (especially RF) are defined by interactions between the CPG and afferent feedback. The results of this study are discussed in the context of possible organization of the spinal PF networks and their control by afferent feedback.

5.
*J. LIU¹, L. M. JORDAN¹, P. B. HEDLUND²; ....289.6/NN2
Locomotor-like activity evoked by neurotransmitters in 5-HT₇ receptor knockout mice
Several lines of evidence have shown that 5-HT₇ receptors play a critical role in modulation of 5-HT-inudced locomotor-like activity in postnatal rodent preparations in vitro. Using 5-HT₇ receptor knockout (5-HT₇ ^-/-) mice, we found that 5-HT failed to induce locomotor-like activity. Instead, uncoordinated rhythm was observed in most cases. It is well known that central pattern generators (CPGs) for locomotion could be activated by a variety of neurotransmitters. Here, we used the same strain of mice to examine whether the locomotor-like activity can be evoked by other agents in the neonatal mouse spinal cord in vitro. Experiments were performed on post-natal day 1-5 5-HT₇ ^-/- mice. The animals were anesthetized, eviscerated and the spinal cord (below T1) was isolated, pinned ventral side up in a recording chamber containing oxygenated aCSF. Electrophysiological recordings were made from the L2 (flexor) and L5 (extensor) ventral roots bilaterally with glass suction electrodes. All the preparations used in experiments were those where bath application of 5-HT (20-50µM) failed to evoke locomotor-like activity. Increasing excitability of spinal neurons with elevated extracellular concentrations of K+ (up to 8-9mM) in 2/5 5-HT₇ ^-/- mice produced locomotor-like activity, as did combined application (in 4/6 mice) of dopamine (60-100µM) and NMDA (4-10µM). Further application of 5-HT refined the dopamine/NMDA evoked locomotor rhythm, enhanced rhythmic stability and slowed the frequency of locomotion. Alpha-methyl-5-HT, a 5-HT₂ receptor agonist (4-8 µM), was effective for evoking locomotor-like activity, but the frequency was much slower than in wild-type mice. This locomotor rhythm induced by alpha-methyl-5-HT was blocked by either the 5-HT_2A antagonist ketanserin or the 5-HT_2B/C receptor antagonist SB 206553. A 5-HT_1A/7 receptor agonist, 8-hydroxy-DPAT hydroxybromide (8-OH-DPAT) (1-10 µM) had no effects on ventral root activity in both wild-type and 5-HT₇ ^-/- mice. Our results are consistent with the notion that 5-HT₇ receptors play an important role in 5-HT-induced locomotor-like activity, but they also show that other 5-HT receptors (e.g. 5-HT₂) and other transmitters can elicit locomotor-like activity in the absence of 5-HT₇ receptors.

6.
*N. J. PORITSANOS, D. G. WONG, T. M. MIZUNO; .... 301.13/WW9
Central administration of a melanocortin antagonist stimulates hepatic lipogenic gene expression independent of changes in body weight
Mutations in the melanocortin-4 receptor (MC4-R) are associated with obesity and the development of hepatic steatosis (fatty liver). However, it is currently unknown whether or not reduction in central melanocortin signaling causes abnormalities in hepatic lipid metabolism secondary to the development of obesity. It has been demonstrated that some of the metabolic effects mediated by central melanocortins are independent of body weight. Therefore, we hypothesized that reduced central melanocortin signaling would increase hepatic lipogenic activity independent of changes in body weight. To address this hypothesis, we investigated the effect of central administration of the MC4-/3-R antagonist, SHU9119, on hepatic lipid contents and the expression of genes involved in hepatic lipogenesis. Male C57BL/6J mice were given intracerebroventricular (icv) injections of artificial cerebrospinal fluid (aCSF) or SHU9119 every 24 h for 3 days. SHU9119 treatment significantly increased epididymal fat pad weight, compared with the control group, without having a significant change in body weight. Mice treated with SHU9119 had significantly elevated hepatic and serum triglyceride levels, and a concomitant increase in liver size. Real time PCR (RT-PCR) analysis indicated that SHU9119 treatment significantly increased hepatic mRNA levels of lipogenic enzymes acetyl-CoA carboxylase (ACC1), fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (SCD-1), which are responsible for the de novo lipogenesis. The expression of these genes is regulated by transcription factors, sterol regulatory element binding protein 1c (SREBP-1c) and carbohydrate responsive element binding protein (ChREBP). Hepatic expression of SREBP-1c was significantly increased in SHU9119-treated group compared to the control group. In contrast, SHU9119 treatment did not induce any changes in hepatic mRNA and protein levels of ChREBP. Additionally, SHU9119 treatment had no effect on mRNA expression of carnitine palmitoyl transferase-1 (CPT-1), suggesting that the accumulation of hepatic triglycerides is not due to impaired CPT-1-mediated fatty acid oxidation. These findings are consistent with the hypothesis that reduced central melanocortin signaling increases hepatic lipogenic activities by stimulating lipogenic gene expression independent of changes in body weight. Furthermore, the stimulatory effect on hepatic lipogenic gene expression is most likely regulated in a SREBP1c-dependent, but ChREBP-independent manner.

7.
*W. T. HUNT, C. M. ANDERSON;.... 380.23/AA9
Conjugated Linoleic Acid protects cultured cortical neurons from glutamate toxicity
Linoleic acid is an 18-carbon polyunsaturated fatty acid with cis double bonds at carbons 9 & 12. In contrast, conjugated linoleic acid (CLA) refers to an isomeric mixture of linoleic acid with conjugated double bonds in cis or trans configurations. Humans acquire CLA through dietary sources (mostly dairy and meat products from ruminant animals). Significant interest in CLA as a food constituent and nutraceutical arises from its reported benefits in cancer, diabetes, atherosclerosis, and weight-partitioning. The potential therapeutic benefits of CLA in CNS injury and neurodegeneration are poorly understood. Glutamate is a vital neurotransmitter in the CNS that is released in toxic excess in cerebral ischemia and brain injury. To model cerebral ischemia in vitro, we treated cortical neurons cultured from day-16 mouse embryos with increasing concentrations of glutamate for 1 hour in the presence and absence of CLA. Cell survival was identified 18-24 hours later as the proportion of cells able to take up and convert calcein acetoxymethyl ester to fluorescent calcein. Intracellular calcein fluorescence was assessed qualitatively by confocal imaging and quantitatively by a high-throughput fluorimeter. Glutamate dose-dependently reduced neuronal survival, having little effect at 0.3 mM and producing maximal cell death at 100 mM. CLA significantly attenuated glutamate toxicity of neurons in a concentration-dependent fashion, reducing neuron death from (73.6 ± 6.5%) for neurons treated with 3 µM glutamate alone to (32.1 ± 5.6%) for neurons co-treated with 10 µM CLA (p<0.01). The level of glutamate-induced neuron death observed with 10 µM CLA is not significantly different (p>0.05) from that observed in controls receiveing no glutamate, suggesting virtually complete protection at this level. Linoleic acid had no effect on glutamate-induced neuron death, inducating that neuroprotection requires the conjugated form of linoleic acid. Neither of the component positional isomers of CLA (cis 9-trans 11 nor trans 10-cis 12) protected neurons against glutamate toxicity individually up to 100 mM, suggesting that both isomers of CLA are required for protection against glutamate exitotoxicity. These studies show that CLA can protect neurons in vitro from glutamate toxicity. Since glutamate toxicity is a mechanism of neuron death common to both neuron injury and long-term degeneration of neurons, CLA is a potential target of therapeutic investigation as a nutraceutical and potentially as a dietary supplement in a wide range of neurological disorders, including stroke, Alzheimer's Disease and Amyotrophic Lateral Sclerosis (ALS).

8.
*L. A. BAUGH, J. J. MAROTTA; .... 397.16/MM17
Differential effects of aging on a visuomotor paradigm
Objectives. The present study describes the use of a viewing window paradigm to examine the effects of aging in a complex visuomotor adaptation scenario. The natural process of aging has been shown to have detrimental effects on the ability synthesize cross-modal information (a requirement in visuomotor transformations), but an in-depth examination using complex, realistic, goal driven tasks has yet to be performed.
Methods. Two groups of participants, a young adult control group (Age: 18 - 25) and an older adult group (Age: 60 - 80) completed a viewing window task that was controlled by the user via a touchscreen. Four visuomotor “flip” conditions were created by varying the relationship between the participant’s movement, and the resultant on-screen movement of the viewing window: 1) No flip 2) X-Axis and Y-axis body movements resulted in the opposite direction of movement of the viewing window. In each of the 3) Flip-X and 4) Flip-Y conditions, the solitary X- or Y-axes were reversed. Response times (ms) and movement of the window (represented by a series of x-axis and y-axis coordinates) were recorded.
Results. A significant main effect of aging was found (p<.05), with the older participants requiring more scanning of the image than the younger control subjects to correctly identify it. Additionally, a Flip by Age interaction effect was observed, with the older participants demonstrating much higher scanning times during all of the visuomotor flip conditions. Examining the complexity of scan-paths revealed the aged participants had considerable difficulty in adapting to all of the flip conditions, and a tendency to revisit regions of the image previously explored.
Conclusions. The present study demonstrates that task performance in a viewing window paradigm decreases as part of the natural aging process. Not only did our aged participants require significantly more time scanning the presented object to identify it, but they also were differentially affected by the requirement of a visuomotor flip. Qualitative examination of scan-path revealed the aged participants demonstrated a repetition not seen in the younger controls. The results provide additional evidence that the mechanisms involved in visuomotor transformation are negatively affected by age.

9.
*Y. DAI, L. M. JORDAN; .... 406.4/TT30
A study of tetrodotoxin and dihydropyridine insensitive persistent inward currents in locomotor activity-related neurons of Cfos-EGFP mice
Persistent inward currents (PICs) have been shown to play an important role in generation of rhythmic activities such as respiration and locomotion. PICs are widely expressed in spinal interneurons activated by locomotion, and these PICs are commonly composed of sodium and/or calcium components. The sodium component is tetrodotoxin (TTX) sensitive and the calcium component is dihydropyridine (DHP) sensitive. PICs mediated by either component or both have been studied intensively in spinal neurons. Here we report for the first time TTX & DHP insensitive PICs that are expressed in spinal interneurons related to locomotion. EGFP expression was induced by a locomotor task in cFos-EGFP transgenic mice (P6-P12), then whole-cell patch clamp recordings were obtained in spinal cord slices (200-250 um, T12-L4) from EGFP+ neurons distributed in lamina VII, VIII and X. The PICs were evoked by a family of 8-10 second voltage bi-ramps held at -70 mV to peak 10-40 mV with bath application of 0.5-3 µM TTX and 15-40 µM nifedipine (L-type calcium channel blocker). TTX & DHP insensitive PICs are observed in 23% of the EGFP neurons (12/51) recorded with TTX and/or nifedipine. They were high voltage activated (>-30 mV) currents that could be enhanced by blockade of calcium dependent potassium currents with apamin (200 µM) or non-specific calcium currents with cadmium (200 µM). These currents were expressed in neurons distributed in laminae VII (n=5), VIII (n=3), and X (n=4) from P6 to P11 animals. Some cells were recorded with Lucifer Yellow in the recording pipette for a study of morphological features of the cells. Intracellular labeling showed that neurons displaying the TTX & DHP insensitive PICs are usually multipolar with either round or pyramidal soma. We conclude that these novel TTX & DHP insensitive PICs occur in specific spinal locomotor interneurons and that they could provide a previously unknown means of controlling interneuron output during locomotion and other movements.

10.
*K. E. POWER, L. MARTINEZ, B. FEDIRCHUK, D. A. MCCREA; .... 408.15/WW2
Motoneuron firing is facilitated by reduction of the afterhyperpolarization during fictive scratch in the decerebrate cat
Hindlimb motoneuron firing is facilitated at the onset of fictive locomotion in the decerebrate cat by a lowering of the voltage threshold for action potential initiation (Vth, Krawitz et al., 2001) and a reduction of the post-spike afterhyperpolarization (AHP, Brownstone et al., 1992). A similar reduction of the AHP also occurs during pharmacologically induced locomotor-like activity in the isolated neonatal rat spinal cord (Schmidt, 1994). Previously we reported a reduction in Vth during fictive scratch (SFN, 2006). The present study extends the study of state-dependent changes in motoneuron excitability to changes in motoneuron AHP during fictive scratch in the decerebrate cat. Fictive scratch was induced by topical application of curare to the left C1 dorsal root entry region and light stroking of the left side of the face in adult decerebrate cats following neuromuscular blockade. Intracellular recordings were obtained from antidromically identified lumbar motoneurons using discontinuous current clamp. The AHP was assessed following spikes evoked by intracellular current injection and from spikes spontaneously occurring during fictive scratch. During both the approach (tonic flexion) and rhythmic phases of fictive scratch, there was a robust reduction of the AHP. This was repeatable with successive trials and recovered soon after the cessation of an episode of fictive scratch. AHP reduction was also observed during fictive scratch in a high spinal preparation. We are currently conducting experiments to determine the extent to which AHP reduction results from increased motoneuron conductance or an intraspinal modulatory mechanism.

11.
Q. ZHANG^1,2, G. DU^3,1, M. EKKER⁴, D. D. EISENSTAT^2,3; .... 461.16/F32
Transcriptional control by Dlx homeobox genes of evolutionarily conserved regulatory elements during vertebrate retina development.
Dlx homeobox genes expressed in the developing CNS are arranged in bigenic clusters, with Dlx1 and Dlx2 and Dlx5 and Dlx6 located on mouse chromosomes 2 and 6, respectively. Regulatory elements, including the Dlx5/Dlx6 intergenic enhancer (56ie) localized between Dlx5 and Dlx6 and the upstream regulatory element (URE1) localized upstream of Dlx1 are highly conserved across diverse vertebrate species, such as the zebrafish, mouse and human. Chromatin immunoprecipitation (ChIP) assays demonstrate that both DLX1 and DLX2 proteins bind to the 56ie in situ in the developing forebrain and retina (Zhou QP et al., 2004). Using transgenic reporter mice expressing lacZ and cell-specific markers, we show that the 56ie is co-expressed with Dlx1, Dlx2 and Dlx5 in selected populations of retinal ganglion cells (RGC), amacrine and horizontal cells. Crossing 56ie/lacZ reporter mice with Dlx1/Dlx2 mutants significantly reduces lacZ expression, providing genetic confirmation in vivo of our ChIP results in situ. Expression of the URE1 precedes the onset of both Dlx1 and Dlx2 in the developing retina. The earliest URE1/lacZ cells are expressed in the proximal optic nerve, a region that does not express Dlx genes. However, in the postnatal retina, URE1 expression is confined to the ganglion cell layer. Using ChIP assays, DLX1 but not DLX2, binds to the URE1 in situ. Binding specificity is confirmed by gel shift assays and the functional significance is shown by transactivation of a URE1/luciferase reporter gene in vitro. Crossing URE1/lacZ reporter mice with the Dlx1/Dlx2 mutant also results in significant reduction of lacZ expression. We provide direct evidence that DLX1 and DLX2 transcriptionally regulate conserved cis and trans elements in vivo during retinal development, underlying the significance of cross-regulatory interactions throughout vertebrate evolution.

12.
*Y. TAN¹, N. KUZENKO², D. D. EISENSTAT³; .... 561.12/B21
Boundary dispute in the developing vertebrate forebrain: Pax6 versus Dlx2
Introduction: The forebrain is required for complex functions in vertebrates, yet its development is incompletely characterized. Defects in human forebrain development can lead to severe neurodevelopmental disorders, such as autism and epilepsy.
Hypothesis: The extracellular matrix protein Tenascin-C (Ten-C) gene promoter contains candidate binding sites for proteins containing homeodomains, paired domains and paired class homeodomains. Dlx2 and Pax6 genes may control the expression of Ten-C at the striatal (sub-pallial)/neocortical (pallial) boundary, thereby affecting tangential interneuronal migration during forebrain development.
Objective: Our objective is to understand the gene regulatory network between the transcription factors, DLX1/DLX2 and PAX6, and the candidate target gene, Tenascin-C (Ten-C).
Methods: To determine Ten-C expression in mutant mice, we performed immunohistochemistry (IHC). To study the protein-DNA interactions of DLX1/DLX2 and PAX6 and the Ten-C promoter, we carried out chromatin immunoprecipition (ChIP) and electrophoretic mobility shift (EMSA) assays. To study the functional consequences of DLX1/DLX2 and PAX6 Ten-C promoter interactions, we performed luciferase gene reporter assays.
Results: TEN-C protein expression is unaffected in the Dlx1/Dlx2 double knockout mouse, whereas it is absent from the pallial-subpallial boundary in the forebrains of the Pax6 homozygous null mouse. Both DLX2 and PAX6 bind to regions of the Ten-C promoter in vitro and in vivo and transactivate expression of a Ten-C reporter gene construct in vitro. Using ChIP-reChIP assays, we have determined that both DLX2 and PAX6 bind to a common region of the Ten-C promoter.
Conclusions: Although DLX2 transactivates Ten-C expression in vitro, loss of Dlx1/Dlx2 function does not affect Ten-C expression at the pallial/sub-pallial boundary in vivo. It is possible that PAX6 and DLX2 compete for binding to the Ten-C promoter. Characterizing the interactions between these transcription factors and their target genes will improve our understanding of forebrain development and help elucidate the mechanisms underlying human neurodevelopmental disorders.

13.
*P. FERNYHOUGH^1,2, E. ZHEREBITSKAYA¹; .... 567.9/F34
Regenerating axons of adult sensory neurons from diabetic rats exhibit dystrophic axonal swellings that are labeled for adducts of 4-hydroxy-2-nonenal and consist of accumulated neurofilament and mitochondria
In diabetes hyperglycemia and lack of insulin signaling induce abnormalities in polyol pathway flux, protein glycation and neurotrophic support in the peripheral nervous system. These factors combine to enhance oxidative stress and trigger distal nerve damage in diabetic sensory polyneuropathy. A common feature of axonal pathology in diabetic neuropathies is the appearance of dystrophic axonal swellings in ganglia and nerve endings. These structures are comprised, in part, of accumulated neurofilament H and mitochondria, however, the pathobiology underlying the formation of these structures remains unknown. Therefore, we tested the hypothesis that diabetes-induced oxidative stress in sensory neurons would trigger abnormalities in axon structure and capacity for regenerative growth. Cultures of adult rat sensory neurons of lumbar dorsal root ganglia (DRG) from age matched normal or streptozotocin (STZ)-diabetic rats were grown in defined F-12 media supplemented with N2 additives under either normal (10mM) or high (25-50mM) glucose concentrations and levels of cell survival and axon outgrowth assessed. In addition, immunofluorescent staining was used to detect expression of phosphorylated neurofilament H (antibody SMI31), neuron-specific beta-tubulin III and amino acid adducts of 4-hydroxy-2-nonenal (4-HNE). The formalin-fixed cultures were also stained with MitoFluor green dye to colocalize the immunostaining with mitochondria in the axons. Cultured adult lumbar DRG neurons from normal rats did not exhibit cell death, oxidative stress or significant numbers of abnormal axonal structures when exposed to 25-50mM glucose for up to 1 month. Normal cultures did exhibit limited numbers of axonal varicosities/swellings but these structures did not stain positively for adducts of 4-HNE. The levels of axon outgrowth from diabetic neurons grown in 25mM glucose was impaired 2.3-fold (P<0.001) compared with control neurons. Analysis of axonal structure of diabetic neurons in 25mM glucose revealed abnormal growth cones, degenerating processes and formation of dystrophic swellings. The axonal swellings stained positively for 4-HNE and were filled with phosphorylated neurofilament H, beta-tubulin III and mitochondria. Antioxidant treatment (1mM N-acetyl-cysteine) of diabetic neurons exposed to 25 mM glucose prevented formation of aberrant axonal structures and enhanced axonal outgrowth by 2-fold (P<0.05). The results show that oxidative stress, as illustrated by elevated 4-HNE staining, in axons of diabetic neurons is associated with impaired axonal outgrowth that may be the result of reduced access of mitochondria to distal axonal sites.

14.
*E. K. AKUDE^1,2, E. ZHEREBITSKAYA¹, P. FERNYHOUGH^1,2; .... 567.14/F39
4-hydroxy-2-nonenal modifies neurofilament protein, causes aberrant axonal morphology, impairs axon outgrowth and mimics the effect of diabetes in cultured adult sensory neurons
Protein adducts of the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) have been associated with disease states involving oxidative stress. Modification of proteins by 4-HNE in peripheral nerve tissue occurs in diabetic sensory neuropathy, however, the target proteins and impact on nerve function are unknown. We, therefore, tested the ability of 4-HNE to induce amino acid adduct formation on axonal cytoskeletal proteins and determined if such modifications were associated with aberrant axon morphology and suboptimal axon outgrowth in cultured adult sensory neurons. The impact of type 1 diabetes on these processes was also investigated. Adult rat dorsal root ganglion (DRG) sensory neurons were cultured in defined F12-media supplemented with neurotrophic factors (NTFs;1nM insulin, 1ng/ml NGF, 10ng/ml GDNF and 10ng/ml NT-3) and treated with 4-HNE concentrations ranging from 1.0µM to 10 µM. Cell survival, axonal morphology and level of axonal regeneration were assessed at 24 hours in culture. Western blot and immunofluorescent staining were utilized to detect protein adduct formation by 4-HNE (anti-4-HNE) and phosphorylation levels of neurofilament H protein (antibody SMI31). 4-HNE induced formation of amino acid adducts on neurofilament H protein and impaired axon regeneration by approximately 50% (ED₅₀ 3mM) whilst having no effect on neuronal survival. 4-HNE initiated formation of aberrant axonal structures that mimicked those seen in axons of neurons under diabetic conditions (in animal models and humans). The formation of protein adducts also led to diminished levels of phosphorylation of neurofilament H protein. Sensory neurons from 3 month streptozotocin-diabetic rats showed abnormal axonal swellings which were filled with 4-HNE protein adducts and impaired levels of axon outgrowth; control neurons exhibited negative staining for 4-HNE in axons. This study demonstrates that 4-HNE induces amino acid adduct formation on neurofilament H protein and this modification is associated with impairment of axonal regeneration. The results show that 4-HNE might be an important link between oxidative stress triggered lipid peroxidation and subsequent modification of key neuronal cytoskeletal proteins in diabetic sensory neuropathy. It is proposed that 4-HNE mediated abnormalities in neurofilament function, and possibly other cytoskeletal proteins, may cause distal axon degeneration through suboptimal mitochondrial motility and localization.

15.
N. YOUNG^1,2, E. ZHEREBITSKAYA², P. FERNYHOUGH^2,1, *G. W. GLAZNER^2,1; .... 567.16/F41
NF-kappaB activity in cultured normal and streptozotocin-diabetic dorsal root ganglia neurons regulates axonal growth and morphology
Diabetic peripheral polyneuropathy is characterized by distal neuropathic pain and numbness, and may lead to poor wound healing, infection, and eventually amputation of the affected area. Pathologically, this disease is characterized in peripheral nerve by reduced axonal diameter, dystrophic axonal swellings, increased oxidative stress, and loss of distal nerve endings. Consistent with these phenomena, dorsal root ganglia (DRG) neurons cultured from streptozotocin-diabetic adult rats demonstrate decreased neurite outgrowth, axonal dystrophy, and increased axonal reactive oxygen species (see associated poster this meeting), indicating that key pathological features of neuropathy continue in vitro. These events may be related to the observation that DRG neurons in experimental diabetic rats have approximately 50-60% reduced activation of nuclear factor kappa B levels, a transcription factor required for neuronal survival and stress resistance. Because experimental reduction of NF-kappaB in central neurons results in increased oxidative stress, loss of calcium homeostasis, and generalized reduction in stress resistance, we hypothesize that decreased NF-kappaB in diabetic DRG neurons may lead to increased oxidative stress which contributes to reduced and dystrophic neurite growth in vitro. DRG neurons were cultured in F-12 media supplemented with N2 additives from normal or streptozotocin (STZ)-diabetic rats. In our preliminary studies, normal (non-diabetic) neurons treated with the NF-kappaB inhibitor SN50 demonstrated reduce neurite outgrowth and increased appearance of neurite swellings and terminal beads compared to non-treated neurons. In contrast, diabetic DRG neurons treated with IkappaB antisense, which enhances NF-kappaB activity, reversed the neurite outgrowth deficit observed in untreated diabetic DRG neurons. These initial findings indicate that deficits in neurite outgrowth observed in cultured diabetic DRG neurons can be mimicked in normal neurons by reduction of NF-kappaB activity, or abolished in diabetic neurons by enhancing NF-kappaB activity. These early data support the hypothesis that reduced NF-kappaB DNA binding activity observed in diabetic DRGs plays a critical role in axonal growth and function, and may contribute to diabetic neuropathy.

16.
*K. S. TANG^1,2, S. W. SUH^3,4, R. A. SWANSON^3,4, C. M. ANDERSON^1,2; ....579.8/M17
Severe inhibition of glutamate uptake by PARP-1-induced ATP depletion in astrocytes
The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is activated in astrocytes and neurons in response to oxidative DNA damage, and leads to cell death in cerebral ischemia. Thus, reduction of PARP-1 activity could be a promising therapeutic target for stroke. The pathways by which activation of PARP-1 leads to neuronal death in mixed cell populations are unclear. Since activation of PARP-1 leads to nicotine adenine dinucleotide (NAD⁺) and ATP depletion, we hypothesized that active glutamate uptake in astrocytes is compromised in conditions of elevated PARP-1 activity. To examine this, we measured NAD⁺ and ATP levels, and glutamate uptake capacity in cultured neonatal mouse astrocytes following PARP-1 activation by the DNA alkylating agent, 1-methyl-3-nitro-1-nitroguanosine (MNNG). Astrocyte death occurred at 6 hours after 30 min of MNNG exposure (100 µM), therefore we limited our analysis to 4 hours after MNNG treatment, when astrocyte death was not significantly observed. ATP levels decreased with time, diminishing by ~30% by 3 hours and ~90% by 4 hours after MNNG treatment. NAD⁺ depletion preceded declining cellular ATP content, as NAD⁺ levels were reduced by ~30% when the half-an-hour MNNG exposure was complete, and by ~40% by 2 hours after MNNG removal. Na⁺-dependent glutamate uptake capacity in astrocyte cultures was decreased in a manner that correlated with the degree of ATP depletion, declining progressively to < 1% at 4 hours after MNNG treatment. Bioenergetic depletion and declines in glutamate uptake were not observed in PARP-1^-/- astrocyte cultures. These results suggest that activation of PARP-1 in astrocytes may contribute to elevated glutamate levels and increased risk of neuronal glutamate excitotoxicity.

17.
*Z. SHAO, C. M. GLIDDON, C. ANDERSON; .... 579.21/N2
Expression of D-serine transporters in mouse brain cultures and slices
D-Serine is an endogenous agonist of N-methyl-D-aspartate receptors that plays an important role in regulation of synaptic function and may be important in excitotoxicity. We are interested in how extracellular D-serine levels are regulated and therefore investigated D-serine uptake kinetics in cultured cortical neurons and astrocytes. Cells were exposed to ³H-D-serine with varying amounts of unlabeled D-serine and intracellular ³H content measured following uptake. Uptake was linear with time and predominantly Na⁺- dependent in both neurons and astrocytes in culture. Both cell types displayed low affinity D-serine uptake and this uptake was inhibited by amino acids known to be substrates for ASCT1 and ASCT2. In cultures, both neurons and astrocytes stained positive for low affinity Na⁺-dependent transporters ASCT1 and ASCT2. In support of this ASCT1 immunofluorescence was associated with two markers of astocytes (i.e., GFAP and S100b) and NeuN (a marker of neuronal cell bodies) in mouse cortical and hippocampus slices. In contrast, ASCT2 immunofluorescence was associated with MAP-2 (a predominant marker of neuronal dendrites) only, and not astrocyte GFAP or S100bin slices. Neurons also stained for the high affinity transporter, asc-1 in both cutlures and slices, but no evidence of functionally was found. These data contribute to our understanding of D-serine transport and therefore provide insight into how extracellular D-serine levels are regulated.

18.
*C. E. FLORES¹, X. LI², J. I. NAGY², A. E. PEREDA¹;....581.13/O6
SPR analysis of connexin35 (Cx35) and zonula occludens-1 (ZO-1) interaction
While protein-protein interactions are involved in regulating chemical synapses, little is known about such interactions in regulating gap junction-mediated electrical synapses. Auditory afferents terminating as "Club Endings" on the distal portion of the lateral dendrite of the goldfish Mauthner cells are identifiable "mixed" (electrical and chemical) synaptic terminals that constitute a valuable model for the study of vertebrate electrical synapses, as it is possible to correlate physiological properties with structural and biochemical composition of individual synapses. Electrical synapses at these terminals are formed by Cx35, the fish ortholog of the mammalian neuronal gap junction protein Cx36. We have previously shown that Cx35 co-localizes with ZO-1 in goldfish brain, a scaffold protein of the MAGUK family. Double-immunolabeling and co-immunoprecipitation showed that Cx35 and ZO-1 are associated at Club endings and other regions of goldfish brain. In addition, affinity precipitation and peptide competition experiments showed that this association is specific to PDZ 1 domain of ZO-1 and the carboxyl-terminal domain of Cx35. We further studied this interaction using Surface Plasmon Resonance (SPR), a technique in which interactions between the Cx35 carboxyl-terminal domain peptides and the PDZ domains of ZO-1 can be studied in real time. SPR analysis confirmed a direct interaction between the last 15 carboxy-terminus amino acids of Cx35 (Cx35-CT peptide) and PDZ 1 domain of ZO-1. This interaction was fast and of relative low affinity, and it was not observed when using a truncated version of Cx35-CT (lacking the PDZ binding motif) or a scrambled peptide. In addition, no significant interactions were observed with PDZ 2 or PDZ 3 domains of ZO-1. In contrast to this specific interaction via PDZ-1, as previously reported, Cx43 CT was found to interact strongly with PDZ 2 of ZO-1 with higher affinity and slower kinetics. The lower affinity of the ZO-1/Cx35 association suggests the existence of a dynamic interaction between these two proteins, consistent with a possible regulatory role of ZO-1. Although the function of Cx35/ZO-1 association is not yet known, its direct interaction through conserved regions of both Cx35 and Cx36 carboxy-terminus, suggests it might constitute a widespread property, relevant to all Cx35/Cx36-mediated electrical synapses.

19.
*J. D. TOUPIN^1,3,6, K. OIKAWA^2,4, G. ODERO², D. R. OLIVER³, B. C. ALBENSI^2,4,5; .... 595.2/X15
The effect of electrical stimulation frequency on hippocampal epileptiform activity
About one third of patients with epilepsy display refractory behavior towards pharmacological treatments. In order to treat this pharmacologically non-responsive subset of patients alternative treatments such as deep brain stimulation have been explored. This study aims to clarify which stimulation frequency works best for the relief of epileptic seizure-like activity in acute hippocampal slices. All treatment protocols used in this study employed a constant number of electrical pulses delivered to the slice ensuring that the total electrical work done on the slice during seizure activity suppression was unchanged over the broad range of stimulation frequencies (0.5 Hz, 0.75 Hz, 10 Hz, 25 Hz, 50 Hz) considered. The frequency-dependent effect on transient and long-term suppression of seizure-like events (SLE) was observed along with the effects on interictal-like events (ILE). Slice electrical activity was observed in in vitro hippocampus slices, a critical brain structure for epileptic foci, in a chemically-induced (low Mg²⁺) model of epilepsy. The results showed that frequency does have a differential effect on the number and intensity of SLE. In general, slices exposed to high stimulation frequencies (> 25 Hz) exhibited fewer SLE and ILE than was observed in slices stimulated with lower frequencies. Despite these reductions in the incidence of SLE and ILE, there was no change in the magnitude of the events that occurred. Very low frequency stimulation (0.75 Hz) resulted in a reduction in the amplitude of both invoked field excitatory postsynaptic potentials (EPSP) and SLE. The effectiveness of frequency was not found to vary monotonically; rather, the extrema were found to be more effective than the mid-range frequencies (2-25 Hz). Finally, the level of seizure suppression observed was not as large as reported in other studies, suggesting that the total energy delivered to the slice by the stimulation may be as important as the frequency at which the stimulation is delivered. Other comparisons of high and low frequency stimulation protocols have yielded greater contrast in suppression. This work suggests that the effectiveness of a stimulation protocol may depend on the total energy (number of pulses) delivered to the tissue sample. Further clarification of this is a subject for ongoing investigation.

20.
*G. J. KIROUAC, S. LI; .... 628.23/XX26
The paraventricular nucleus of the thalamus innervates corticotropin releasing factor (CRF) and dynorphin rich regions of the extended amygdala and nucleus accumbens
Dynorphin and corticotropin releasing factor (CRF) are two neuropeptides associated with aversive behavioral states which have been linked to a variety of psychiatric conditions including depression, anxiety disorders and addiction. The paraventricular nucleus of the thalamus (PVT) is part of a group of midline and intralaminar thalamic nuclei implicated in arousal and attention. In addition, neurons in the PVT have been shown to be activated following the exposure of rats to a variety of physiological and psychological stress protocols. This study examined the connections between the PVT and regions of the nucleus accumbens and extended amygdala with a focus on neurons containing the dynorphin and CRF. The anterograde tracer biotin dextran amine (BDA) was injected in the anterior and posterior regions of the PVT in the rat. Brain sections were subsequently processed and stained for BDA, dynorphin and CRF. The PVT was found to provide a strong projection to the dorsolateral bed nucleus of the stria terminalis (BST) and central nucleus of the amygdala (CeA), two regions associated with the extended amygdala which contains both dynorphin and CRF neurons. The PVT was also found to innervate regions of the shell of the nucleus accumbens containing dense staining for both dynorphin neurons and fibers. We observed that BDA fibers made numerous apparent contacts on the soma and dendrites of CRF and dynorphin neurons in BST and CeA. The results of the present study suggest that the PVT may relay arousal and stress related information to dynorphin and CRF neurons in the nucleus accumbens, BST and CeA. The projection from the PVT to dynorphin and CRF neurons in the basal forebrain places the PVT in a key anatomical position to influence adaptive behaviors associated with stress and aversive states.

21.
*J. M. KALMAR, D. C. BUTTON, K. GARDINER, F. CAHILL, P. F. GARDINER; .... 726.3/NN2
Effects of aging and caloric-restriction on the biophysical properties of rat lumbar motoneurons
A loss of alpha-motor neurons (MN) and/or changes in their biophysical properties may contribute to age-associated changes in neuromuscular function. It has been proposed that cellular damage imposed by reactive oxygen species may contribute to age-related deficits in CNS function. Thus, we hypothesized that aging would alter the functional properties of MNs and that caloric restriction would offset these changes. In situ intracellular recordings were made from lumbar MNs of anaesthetized (ketamine/xylazine) female Fisher Brown Norway (FBN) fed ad libitum (oldAL, n=11 MNs, 31.5±1.3mo) as well as aged FBN rats maintained on a fortified calorie-restricted diet from 16-weeks of age (oldCR, n=11 MNs, 32.0±1.8mo). The passive and rhythmic firing properties recorded from MNs of aged rats were compared to MN properties of young FBN (conFBN, n=4 MNs, 9mo,) and young Sprague-Dawley (conSD, n=20 MNs, 4mo) controls. Although oldCR (255.1±17.8g) bodyweight (326.9±71.1g) was lower (p<0.XX) than oldAL, relative lower limb muscle mass (gmuscle/kgBW) was 30.6% higher in the CR group (p<0.05), indicating that caloric restriction was successful. ANOVA revealed no differences between oldAL and oldCR or between conFBN and conSD electrophysiological properties; therefore, data were pooled and independent t-tests were used to test for differences between old and young animals. Compared to young MNs, old MNs also had a 123% greater (p<0.0001) afterhyperpolarization (AHP) amplitude, a 40% longer AHP half-decay time (p<0.0001), 29.5% greater (p<0.05) input resistance, 41.9% (p<0.0001) less spike frequency adaptation during a sustained 30-s intracellular current injection, and significant reductions in minimal ( 41.5%, p<0.0001) and maximal (42.8%, p<0.0001) steady-state firing frequencies recorded during an incremental series of 500-ms current pulses. These data are consistent with previous reports of an age-induced selective loss of high threshold MNs. However, the slope of the frequency-current relationship, which is normally similar for high and low threshold MNs, was 65% lower in the old MNs. Furthermore, the old and young MNs demonstrated the same range for most MN properties and aging had no effect on rheobase, suggesting that factors other than the loss of high threshold MNs contribute to age-induced changes in MN function. We conclude that caloric restriction has no specific effect on the biophysical properties of old MNs and that the lumbar MN population maintains heterogeneity that is similar to young MNs.

22.
*B. J. MACNEIL; .... 733.22/SS23
Neurological level of spinal cord injury alters experimental arthritis in Lewis rats
The nervous system can alter inflammatory joint conditions such as rheumatoid arthritis in humans. Animals models of immune-mediated arthritis have revealed a specific role for the sympathetic nervous system (SNS); reducing systemic SNS activity decreases joint inflammation and bone erosion while enhanced SNS activity increases arthritic changes. It is unknown whether these effects are due to centrally arising SNS activity or agents present within sympathetic postganglionic terminals that are released through a local process. The current study determined whether centrally-mediated SNS activity influenced the development of joint edema in a rat model of arthritis. Female Lewis rats (125-150g, Charles River, Quebec) were used in all studies. Streptococcal cell wall extract (SCW) (5-25 ug PG-PS 10S/g body weight, Lee Laboratories, New Jersey) was injected ip and animals were monitored for a maximum of 21 days. Animals received a complete spinal cord injury (SCI) at either the 1st thoracic (T1) or 1st lumbar (L1) neurological level of the spinal cord to assess the effects of interrupting supraspinal input to preganglionic sympathetic neurons on the arthritic process. One week after surgery animals underwent arthritis induction. The medial-lateral widths of both ankle joints were measured bi-weekly using a digital caliper and analyzed by repeated measures ANOVA. In naïve animals ip injection of SCW extract induced a dose-dependent, bilateral ankle arthritis (F3,16=11.28, p<0.001). Animals undergoing sham spinal cord injury (laminectomy only) and subsequently injected with SCW extract (25 ug/g body wt) developed an arthritis that was identical to that of controls receiving the same dose of SCW extract. A robust bilateral arthritis was also induced in L1 SCI animals that was indistinguishable from control animals receiving the same dose of SCW extract (25 ug/g body wt). Both control and L1 SCI groups displayed significant bilateral ankle edema at every time point following SCW extract injection (p<0.006 for all). In contrast, T1 SCI animals had no significant increase in ankle dimensions at any time point following injection of SCW extract (25 ug/g body wt)(p>0.229 for all). Thus, interrupting supraspinal input at the T1 spinal level, but not the L1 spinal level, prevented the development of ankle edema in this model. These data indicate that supraspinal input to sympathetic preganglionic neurons may play a major role in regulating peripheral inflammatory processes such as arthritis.

23.
K. D. HARTLE, *T. L. IVANCO; .... 789.13/J1
Anatomical consequences of early damage across the lifespan in a rat model of stroke
Models of human brain damage often provide insight into how plastic the brain is. We have been interested in how early damage influences neuronal reorganization through the lifetime. Understanding prenatal injury such as stroke, for example, involves evaluation of how reorganization in the brain evolves as the child ages and moves into the adult and later adult years. Currently, there is little evidence on the long-term reorganization following early developmental injury. To address this issue, male Long Evans rat pups (n = 30) underwent a unilateral photochemically-induced stroke at 10 days of age, a time at which the brain is developmentally similar to that of a full term human infant. Animals were given an intraperitoneal injection of light sensitive Rose Bengal dye, the skull was exposed and a laser beam (wavelength 532nm) was positioned over the right motor cortex. The laser was left on for 20 minutes. Control animals underwent the same surgical procedure except the laser was not turned on. Pups were sacrificed at either 2 months of age (young adult) or 6 months of age (mature adult) 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 microscope. Using NeuroLucida software, five layer II pyramidal cells from each of the right and left motor cortex were drawn from each animal for a total of 10 cells per animal. Analysis of dendritic length, complexity and volume was conducted on the apical and basilar dendrites of each cell using NeuroExplorer software. Results revealed that at 2 months of age experimental animals exhibited significant reorganization of basilar and apical dendrites relative to control animals. At 6 months of age the experimental animals did not differ from controls. This study indicates that extensive reorganization after early developmental damage is still evident in young adult animals and then neuronal processes resemble those in control animals later in later adulthood.

24.
*J. L. LEMAISTRE^1,3, H. D. ANDERSON^2,4, C. M. ANDERSON^1,3; .... 875.24/F37
NMDA receptor-mediated dilation of isolated mouse middle cerebral arteries by glutamate and D-serine
Functional hyperemia is the increase in cerebral blood flow observed during elevated neuronal activity, coupling energy supply and demand. Growing evidence suggests that astrocyte gliotransmitters mediate functional hyperemia. D-Serine is a gliotransmitter and activator of glycine regulatory site at NMDA-type glutamate receptors that is released by astrocytes in response to increased intracellular Ca²⁺ produced by neuronal activity. We hypothesized that D-serine dilates cerebral resistance arteries by activating endothelial NMDA receptors and stimulating nitric oxide production. Middle cerebral arteries (MCAs) of 15 week old male CD1 mice were isolated, mounted between glass cannulae, pressurized to 30 mmHg and preconstricted with norepinephrine (1 µM). Dilatory responses to NMDA receptor ligands and other tests compounds were measured by video microscopy. Neither glutamate nor D-serine alone produced significant vasodilation up to concentrations of 10 mM. However, glutamate and NMDA produced dilation of MCAs in a concentration-dependent fashion (31 ± 5.1% at 100 µM glutamate) in the presence of D-serine (100 µM). Similarly, D-serine dose-dependently increased vessel diameter in the presence of a fixed concentration of glutamate. Vasodilatory effects of glutamate and D-serine together (both 100 µM) were significantly reduced by both the competitive NMDA receptor antagonists, 2-amino-5-phosphonopentanoic acid (AP5, 100 nM, 10 µM) and the selective glycine site antagonist, 5,7-dichloro-kynurenic acid (DCKA, 300 nM, 30 µM), while blockers of AMPA/kainate receptors (6-cyano-7-nitroquinoxaline-2,3-dione, CNQX; 10 µM) and metabotropic glutamate receptors (LY341495, 10 µM) did not affect glutamate/D-serine induced vasodilation. Denuding the endothelium abolished vasodilation induced by glutamate/D-serine, as did pre-treatment of MCAs with the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). These results provide functional evidence that NMDA receptors are expressed by cerebral arterial endothelium, and that the gliotransmitter D-serine is capable of producing NMDA receptor-mediated cerebral vasodilation in combination with glutamate. Furthermore, our results indicate that mouse cerebrovascular NMDA receptors mediate vasodilation by signaling production of endothelial nitric oxide.

25.
*E. ZHEREBITSKAYA¹, Z. WANG¹, P. FERNYHOUGH^1,2; .... 897.1/U5
Adult sensory neurons from diabetic rats exhibit alterations in reactive oxygen species generation that is dependent on the cellular compartment
Diabetes in humans and in experimental animal models induces sensory neuropathy that presents as sensory loss, foot ulceration and infection and can precipitate lower limb amputation. The etiology may involve oxidative stress and mitochondrial dysfunction leading to a dying-back process of degeneration of distal axons. Increased reactive oxygen species (ROS) generation has been proposed as a critical factor in nerve degeneration in diabetes. To date no studies have demonstrated alterations in ROS in adult sensory neurons in diabetes. We tested the hypothesis that sensory neurons exposed to long term type 1 diabetes in vivo would exhibit enhanced ROS and oxidative stress in perikarya and axons and tested if this stress was associated with impaired regenerative axon outgrowth in cell culture. Lumbar (L1-L6) dorsal root ganglia (DRG) sensory neurons from age matched normal and 3 month streptozotocin (STZ)-diabetic rats were cultured in defined F-12 media -/+ neurotrophic factors (NTFs: 1nM insulin, 1ng/ml NGF, 10ng/ml GDNF and 10ng/ml NT-3). The following parameters were measured at 1 and 4 days in culture: (1) total axon outgrowth, (2) level of ROS in perikarya and axons using dihydrorhodamine (detects primarily superoxide generation by mitochondria) with real time video confocal microscopy, and (3) mitochondrial preparations from whole DRG were analyzed for rates of electron transport by measuring rates of oxygen consumption (Clarke electrode). DRG sensory neurons isolated from diabetic rats exhibited a 2-fold (P<0.001) elevation of mitochondrially-associated ROS levels in axons after 24 hrs of culture compared with control. However, the perikarya exhibited no change in ROS levels. At 4 days levels of NTF-induced axonal growth were significantly reduced by 2.3-fold (P<0.001) in diabetic cultures compared with control. Acute and longer term (24 hr) treatment with 1mM N-acetyl-cysteine significantly lowered axonal ROS levels and prevented the deficit in axonal outgrowth in diabetic neurons. Mitochondrial preparations from DRG of diabetic rats demonstrated enhanced rates (P<0.05) of coupled and uncoupled electron transfer. We show for the first time that DRG sensory neurons with a history of diabetes express high levels of ROS in their axons but not in the perikarya. We also discovered that axonal morphology of diabetic neurons was abnormal with appearance of dystrophic swellings that were filled with neurofilament, mitochondria and amino acid adducts of 4-hydroxy-2-nonenal. Oxidative stress is limited to the axonal compartment in diabetic neurons, is associated with elevated mitochondrial electron transport and results in impaired axon outgrowth.

26.
*W. ZHU, K. MADEC, Y. GONG, X. GU, M. NAMAKA; .... 901.2/Y1
Multiple sclerosis (ms)-induced expression of brain derived neurotrophic factor (bdnf) in dorsal root ganglia (drg)
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.

27.
*A. MACKENZIE-GRAHAM¹, S. TIWARI-WOODRUFF¹, C. AGUILAR¹, K. T. VO¹, L. V. STRICKLAND¹, L. MORALES¹, B. FUBARA², M. MARTIN³, R. E. JACOBS⁴, G. A. JOHNSON², R. R. VOSKUHL¹, A. W. TOGA¹; .... 901.8/Y7
Purkinje cell death underlies cerebellar atrophy in experimental autoimmune encephalomyelitis
Grey matter atrophy is an important correlate to clinical disability and disease duration in multiple sclerosis. The objective of this study was to link brain atrophy to its underlying pathology using the MS model, experimental autoimmune encephalomyelitis (EAE). To quantify volumetric changes in the brains of EAE mice compared to normal controls, post-mortem high-resolution T2-weighted magnetic resonance microscopy images were collected, a minimum deformation atlas was constructed and a deformable atlas approach was used. A significant decrease in cerebellar and cerebellar cortex volumes during EAE was observed. Actively-stained magnetic resonance histology images localized the volume loss to the molecular layer of the cerebellar cortex. Immunohistochemistry demonstrated Caspase-3 staining of Purkinje cells and a significant decrease in Purkinje cell numbers. Cross modality correlations revealed a significant association between Purkinje cell loss and cerebellar cortical atrophy. This approach may be useful in a variety of neurodegenerative disease models.

28.
*D. A. MCCREA¹, S. CHAKRABARTY²; .... 925.3/QQ15
Activity patterns in bifunctional PBSt motoneuron pools during fictive locomotion in decerebrate cats: clues to CPG organization
Locomotion is characterized by strongly coupled alternating bursts of activity in hindlimb flexors and extensors. It is well known, however, that hindlimb motoneurons innervating bifunctional muscles such as posterior biceps and semitendinosis (PBSt, hip extensor and knee flexor) display more complex activity patterns than motoneurons innervating uniarticular muscles. For example during real locomotion, the timing of PBST bursts within the locomotor cycle varies with gait and locomotor conditions. A variety of PBSt activities have also been reported during fictive locomotion in decerebrate preparations in the absence of proprioceptive afferent feedback. This indicates that the central locomotor circuitry (i.e. the CPG) can activate PBSt motoneurons in a number of ways. Here we report on the patterns of PBSt activity recorded in decerebrate cat preparations during fictive locomotion evoked by continuous electrical stimulation of the midbrain locomotor region. Our analysis shows that PBSt activity falls into four patterns: activity during only the initial portion of the flexor phase, activity at the onset of flexion continuing throughout most of flexion, activity throughout the extensor phase, or activity during both the flexion and extension phases. These different patterns were encountered during experiments carried out under more or less identical conditions. It was rare for a pattern to change during an experiment. Intracellular motoneuron recordings confirmed that these different patterns result from differences in the pattern of CPG input to PBSt motoneurons and not simply from variations in the level of sub and supra-threshold membrane depolarizations. Some PBSt activity patterns are difficult to reconcile with previous suggestions of how CPG input to bifunctional motoneurons is organized. For example, the large depolarization of PBSt motoneurons at the onset of flexion that rapidly decays into a hyperpolarization while uniarticular flexors are still vigorously active is not consistent with a simple summation of the excitation and inhibition that controls flexor and extensor motoneuron activities. We hypothesize that the complex activity patterns of PBSt during locomotion result from a special organization of pattern formation networks within the CPG. A computational model of the locomotor CPG incorporating such a network has been developed that can reproduce all of the patterns of PBSt activity observed during fictive locomotion (see poster by Shevtsova et al. this meeting).

29.
*N. A. SHEVTSOVA^1,2, S. CHAKRABARTY^3,4, K. HAMADE^1,5, S. N. MARKIN¹, D. A. MCCREA³, I. A. RYBAK¹; .... 925.4/QQ16
Computational model of mammalian locomotor CPG reproducing firing patterns of flexor, extensor and bifunctional motoneurons during fictive locomotion
Recent studies have proposed a two-level organization of the locomotor CPG in which a single half-centre rhythm generator (RG) controls the operation of pattern formation (PF) networks that in turn produce the coordinated rhythmic excitation and inhibition of multiple motoneuron pools during locomotion (Rybak et. al., J. Physiol. 577: 617-658, 2006). This organization was implemented in a computational model that generated a realistic locomotor rhythm and produced the alternating activity of a pair of flexor and extensor motoneuron pools (Ibid.). The model could reproduce many characteristics of the fictive locomotor pattern evoked in the immobilized, decerebrate cat by midbrain stimulation. These characteristics included spontaneous deletions of motoneuron activity (missing bursts), occurring with and without rhythm resetting (changing the phase of locomotor oscillations), and various effects of afferent stimulation on the locomotor phase durations and phase transitions. We now show that, while maintaining a bipartite organization of RG, this model can be extended to generate the more complex activity patterns of motoneurons activating muscles spanning more than one joint (biarticular). Specifically, incorporating additional neural circuits within the PF network allowed our extended model to reproduce the locomotor activity patterns of posterior biceps and semitendinosis (PBSt, hip extensor and knee flexor).
During real locomotion, the activity of PBSt varies according to gait and locomotor conditions. While afferent feedback may contribute to some of these variations, PBSt also shows a variety of activity patterns during fictive locomotion in decerebrate cats. Thus in the absence of sensory input, the CPG is able to produce PBSt activity in the beginning of the flexor phase, throughout the extensor phase, or during both phases. Our extended model demonstrates the full spectrum of PBSt activity with particular firing patterns selected by adjusting the weights of synaptic connections within the extended PF network. The model also reproduces the changes in PBSt activity occurring during spontaneous deletions of flexor or extensor activity during fictive locomotion and the effects of afferent stimulation on PBSt activity. We hypothesize that sensory inputs to the selected PF interneuron populations provide a mechanism for the proprioceptive control of PBSt activity. The proposed organization of PBSt circuits predicts the behavior of PBSt motoneurons under different conditions that may be tested experimentally in fictive locomotion preparations.

30.
*K. A. COLLISTER^1,3, G. ODERO³, K. OIKAWA^3,1, G. GLAZNER^3,1, B. C. ALBENSI^3,1,2; .... 932.13/WW8
Spatial memory formation and hippocampal function are influenced by background strain but not gender in the TgCRND8 mouse model of Alzheimer’s Disease
Alzheimer’s disease (AD), the most common form of age-related dementia, is characterized by loss of memory formation and impaired hippocampal function. Though no natural rodent model exists, recent advances in molecular biology have allowed the creation of multiple genetic models of AD. The pathological and physiological effects of AD-related mutations may be influenced by background strain and/or gender. In order to determine the effect of these potentially important confounding variables, we investigated memory encoding and synaptic plasticity using a transgenic mouse model of AD, the TgCRND8 strain. We evaluated spatial hippocampal-dependent memory using the Morris Water Maze (MWM) in vivo and brain slice electrophysiological recordings in vitro. For this study, we tested both males (M) and females (F) of two different age-matched background strains, (129Svevtac & C3HBl6).
In both background strains, transgenic animals demonstrated reduced spatial memory performance compared to their respective controls. However, the 129Svev transgenic took nearly twice as long as their C3H transgenic counterparts to find the hidden platform. During retention phase we observed no significant difference between Tg and controls of either strain as measured by the time spent in target quadrant. However, both control and transgenic C3H demonstrated greater retention than both control and transgenic 129Svev, again demonstrating greater learning ability in the C3H strain. We calculated the Annulus Crossing Index (ACI), another measure of memory retention, and found that there was a larger difference between control and transgenic on the C3H background than on the 129Svev. Electrophysiology was performed on both background strains. The 129 Svev transgenic mice showed impaired basal function in the hippocampus when stimulated in the CA1 region. This was not observed in the C3H transgenic mice. When we stimulated with 3 trains of 100Hz (LTP inducing protocol), we found that the C3H transgenic mice exhibited attenuated LTP, while the 129 Svev transgenic mice generated a weak LTP response.
These preliminary results suggest that impairments in spatial memory are significantly influenced by background strain. There was no effect attributed to gender.