Identification of α-chimaerin as a candidate gene for critical period neuronal plasticity in cat and mouse visual cortex.

BACKGROUND: In cat visual cortex, critical period neuronal plasticity is minimal until approximately 3 postnatal weeks, peaks at 5 weeks, gradually declines to low levels at 20 weeks, and disappears by 1 year of age. Dark rearing slows the entire time course of this critical period, such that at 5 weeks of age, normal cats are more plastic than dark reared cats, whereas at 20 weeks, dark reared cats are more plastic. Thus, a stringent criterion for identifying genes that are important for plasticity in visual cortex is that they show differences in expression between normal and dark reared that are of opposite direction in young versus older animals. RESULTS: The present study reports the identification by differential display PCR of a novel gene, α-chimaerin, as a candidate visual cortex critical period plasticity gene that showed bidirectional regulation of expression due to age and dark rearing. Northern blotting confirmed the bidirectional expression and 5'RACE sequencing identified the gene. There are two alternatively-spliced α-chimaerin isoforms: α1 and α2. Western blotting extended the evidence for bidirectional regulation of visual cortex α-chimaerin isoform expression to protein in cats and mice. α1- and α2-Chimaerin were elevated in dark reared compared to normal visual cortex at the peak of the normal critical period and in normal compared to dark reared visual cortex at the nadir of the normal critical period. Analysis of variance showed a significant interaction in both cats and mice for both α-chimaerin isoforms, indicating that the effect of dark rearing depended on age. This differential expression was not found in frontal cortex. CONCLUSIONS: Chimaerins are RhoGTPase-activating proteins that are EphA4 effectors and have been implicated in a number of processes including growth cone collapse, axon guidance, dendritic spine development and the formation of corticospinal motor circuits. The present results identify α-chimaerin as a candidate molecule for a role in the postnatal critical period of visual cortical plasticity.

Timeline of Changes in Biomarkers Associated with Spinal Cord Injury-Induced Polyuria.

Deficits in upper and lower urinary tract function, which include detrusor overactivity, urinary incontinence, detrusor-sphincter dyssynergia, and polyuria, are among the leading issues that arise after spinal cord injury (SCI) affecting quality of life. Given that overproduction of urine (polyuria) has been shown to be associated with an imbalance in key regulators of body fluid homeostasis, the current study examined the timing of changes in levels of various relevant hormones, peptides, receptors, and channels post-contusion injury in adult male Wistar rats. The results show significant up- or downregulation at various time points, beginning at 7 days post-injury, in levels of urinary atrial natriuretic peptide, serum arginine vasopressin (AVP), kidney natriuretic peptide receptor-A, kidney vasopressin-2 receptor, kidney aquaporin-2 channels, and kidney epithelial sodium channels (ß- and γ-, but not α-, subunits). The number of AVP-labeled neurons in the hypothalamus (supraoptic and -chiasmatic, but not paraventricular, nuclei) was also significantly altered at one or more time points. These data show significant fluctuations in key biomarkers involved in body fluid homeostasis during the post-SCI secondary injury phase, suggesting that therapeutic interventions (e.g., desmopressin, a synthetic analogue of AVP) should be considered early post-SCI.

Activity-Based Training Reverses Spinal Cord Injury-Induced Changes in Kidney Receptor Densities and Membrane Proteins.

Complications in upper and lower urinary function arise after spinal cord injury (SCI), which creates a significant impact on quality of life for those affected. One upper urinary complication is SCI-induced polyuria, or the overproduction of urine, of which the underlying mechanisms have yet to be elucidated. Activity-based training (ABT) has been utilized in both animal and clinical settings as a rehabilitative therapy to improve many issues that arise after SCI, including more recently urogenital function. The goal of the current study was to identify potential mechanisms contributing to previously identified improvements in polyuria with ABT, using a male rat moderate-severe spinal contusion model. Although ABT had no significant effect on reversing injury-induced alterations of serum arginine vasopressin and urinary atrial natriuretic peptide levels, there was a dramatic effect upon the receptors of these fluid balance hormones (vasopressin receptor 2 and natriuretic peptide A receptor), as well as kidney aquaporin 2 and sodium channels. ABT changes in densities of key receptors and kidney membrane proteins involved in fluid balance after chronic SCI support the likelihood of multiple mechanisms through which exercise can positively influence urinary tract function after SCI. By understanding the mechanisms, amount, and timing regarding how ABT improves different aspects of urinary function, more targeted training strategies can be developed to optimize the functional gains within the SCI population.

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

Identification of Munc13-3 as a candidate gene for critical-period neuroplasticity in visual cortex.

The first several months of life are a critical period for neuronal plasticity in the visual cortex during which anatomic and physiological development depends on visual experience. In cats, electrophysiologically assessed neuronal plasticity is minimal until approximately 3 weeks, peaks at 5 weeks, gradually declines to low levels at 20 weeks, and disappears at approximately 1 year of age (Daw, 1994). Rearing in darkness slows the entire time course of this critical period, such that at 5 weeks of age, normal cats are more plastic than dark-reared cats, whereas at 20 weeks, dark-reared cats are more plastic (Mower, 1991; Beaver et al., 2001). Thus, a stringent criterion is that genes that are important for plasticity in visual cortex will show differences in expression between normal rearing and dark rearing that are of opposite direction in young versus older animals. The present study reports the identification by differential display PCR of Munc13-3, a mammalian homolog of the Caenorhabditis elegans "uncoordinated" gene (unc-13), as a candidate gene for critical-period neuronal plasticity, the expression of which is regulated according to this criterion specifically in visual cortex and not in frontal cortex. Other members of the Munc13 family (Munc13-1 and Munc13-2) do not meet this criterion in visual cortex, indicating that Munc13-3 is the only family member that is regulated by age and dark rearing in the same manner as physiological plasticity during the visual cortical critical period.

Age and dark rearing bidirectionally regulate the level and laminar pattern of expression of Abelson interacting protein 2 (Abi-2): a novel candidate visual cortical plasticity gene.

Electrophysiological studies indicate that cat visual cortical critical period neuronal plasticity peaks around 5 weeks and largely disappears by 20 weeks. Dark rearing slows this time course. Normal cats are more plastic than dark-reared cats at 5 weeks, but the opposite is true at 20 weeks. Thus, a stringent criterion for identifying genes controlling neuronal plasticity is that normal and dark rearing produce opposite direction differences in expression between young and older animals. Differential display polymerase chain reaction identified Abelson interacting protein 2 (Abi-2) as a candidate plasticity gene regulated according to this criterion. Western blotting showed bidirectional regulation of Abi-2 protein levels in cats and mice that was specific to visual cortex and did not occur in frontal cortex. Immunohistochemistry indicated developmental changes in Abi-2 laminar expression in cat visual cortex. Dark rearing altered laminar expression such that at 5 weeks, dark-reared cats were similar to 1-week normally reared cats, and at 20 weeks, dark-reared cats were similar to 5-10-week normally reared animals. The effect of dark rearing on both Abi-2 expression levels and laminar expression patterns was to slow the normal developmental process, the same effect seen on physiologically assessed plasticity in visual cortex.

Identification of disabled-1 as a candidate gene for critical period neuroplasticity in cat and mouse visual cortex.

Rearing in darkness slows the time course of the critical period in visual cortex, such that at 5 weeks of age normal cats are more plastic than dark-reared cats, whereas at 20 weeks dark-reared cats are more plastic [G. D. Mower (1991)Dev. Brain Res., 58, 151-158]. Thus, a stringent criterion is that genes that are important for plasticity in visual cortex will show differences in expression between normal and dark-reared visual cortex that are of opposite direction in young vs. older animals. The present study reports the identification by differential display PCR of Dab-1, the mammalian homolog of the drosophila disabled-1 gene, as a candidate gene for critical period neuronal plasticity, expression of which is regulated according to this criterion in cat visual cortex. Evidence for this bidirectional direction regulation is extended to Dab-1 protein in cat and mouse visual cortex and shown to be specific to visual cortex, not occurring in frontal cortex. The Reelin/Dab-1 pathway has well-documented functions in cell migration during prenatal life and increasing evidence indicates that in postnatal brain the pathway plays a role in synaptic plasticity. The present results extend this evidence by directly implicating Dab-1 in postnatal critical period plasticity of visual cortex.