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.

Control of precerebellar neuron development by Olig3 bHLH transcription factor.

The rhombic lip (RL) is the neuroepithelium immediately adjacent to the roof plate of the fourth ventricle, and it gives rise to various brainstem and cerebellar cell types. Our study shows that the bHLH (basic helix-loop-helix) transcription factor Olig3 is expressed in the progenitors of RL, and ablation of Olig3 significantly affects the development of RL. In Olig3-/- caudal RL, the expression level of Math1 in the dorsal interneuron 1 (dI1) domain is reduced, and the formation of four mossy-fiber nuclei is compromised; dI2-dI3 neurons are misspecified to dI4 interneurons, and the climbing-fiber neurons (inferior olive nucleus) are completely lost. In addition, the formation of brainstem (nor)adrenergic centers and first-order relay visceral sensory neurons is also dependent on Olig3. Therefore, Olig3 plays an important role in the fate specification and differentiation of caudal RL-derived neurons.

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.

The relationship between relative eye usage and ocular dominance shifts in cat visual cortex.

A novel modification of the alternate monocular deprivation paradigm was used to quantitatively define the relationship between relative eye usage and the shift in visual cortical ocular dominance toward the advantaged eye. Both eyes of cats were alternately occluded by contact lenses during daily visual exposure sessions with varying ratios of relative eye usage: 1:1, 1.7:1, 3:1, 7:1, 50:1, 100:0. Only 100:0 and 50:1 ratios produced an ocular dominance shift in favor of the more experienced eye. The ocular dominance shift in 100:0 cats occurred in all cortical layers but only in extragranular layers of 50:1 cats. A steep power function described the data, indicating that an extreme imbalance in relative eye usage (>90%) is required for an ocular dominance shift.

Immediate early gene expression in the visual cortex of normal and dark reared cats: differences between fos and egr-1.

Northern blotting indicated that the level of induced fos was higher in normal than dark reared cat visual cortex (VC) at 5 weeks of age, comparable at 10 weeks, and higher in dark reared than normal cat visual cortex at 20 weeks. Fos egr-1, induction was higher in dark reared visual cortex at all ages. Thus, dark rearing has similar effects on fos, but not egr-1, expression as it has on physiological neuronal plasticity during the VC critical period.

Laminar distribution of NMDA receptor subunit (NR1, NR2A, NR2B) expression during the critical period in cat visual cortex.

Changes in NMDA subunit composition may be part of the molecular basis for critical period plasticity. The present study used immunohistochemistry to determine developmental changes in the laminar distribution of the three major cortical NMDA subunits (NR2A, NR2B, NR1) during the critical period in cat visual cortex. For all three subunits, at 1 week staining was concentrated in two bands: an upper band consisting of layer I, the compact zone and the upper half of the cortical plate; a lower band consisting of layers V and VI. In the lower part of the cortical plate (immature layer IV) staining was very low. For NR2A and NR2B, immunoreactivity in layer IV remained low until 10 weeks of age. At 20 weeks and adult, layer IV filled in and NR2A and NR2B label was rather uniform across all layers. NR1 showed a developmental pattern of expression different from NR2A and NR2B after 1 week. At 5 and 10 weeks, label was prominent in layer IV and superficial layers, but low in layers V and VI. The main change after 10 weeks was a progressive decrease in staining, such that in older animals label was markedly densest in superficial layers. Thus, during the rise of the critical period, NR1 is the dominant subtype in layer IV and could play a role in anatomical ocular dominance column formation and plasticity. At the same time, NR2A and NR2B subunits are concentrated outside layer IV, and could be related to physiological plasticity in extragranular layers, which precedes and outlasts plasticity in layer IV. For all three NMDA receptor subunits, the laminar distribution was similar in normal and dark reared visual cortex at 20 weeks, indicating that the developmental changes in laminar pattern of expression are independent of visual input.

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.

Cellular and laminar expression of Dab-1 during the postnatal critical period in cat visual cortex and the effects of dark rearing.

This study describes postnatal critical period changes in cellular and laminar expression of Dab-1, a gene shown to play a role in controlling neuronal positioning during embryonic brain development, in cat visual cortex and the effects of dark rearing (DR). At 1week, there is dense cellular staining which is uniform across cortical layers and very light neuropil staining. At the peak of the critical period (5weeks), dense cell staining is largely restricted to large pyramidal cells of deep layer III and layer V, there is faint cell body staining throughout all cortical layers, neuropil staining is markedly increased and uniform in layers III to VI. This dramatic change in laminar and cellular labeling is independent of visual input, since immunostaining is similar in 5-week DR cats. By 10weeks, the mature laminar and cellular staining pattern is established and the major subsequent change is a further reduction in the density of cellular staining in all cortical layers. Neuropil staining is pronounced and uniform across cortical layers. These developmental changes are altered by DR. Quantification by cell counts indicated that age and DR interact such that differences in cellular expression are opposite in direction between 5- and 20-week-old cats. This bidirectional regulation of cellular expression is the same in all cortical laminae. The bidirectional regulation of cellular expression matches the effects of age and DR on physiological plasticity during the critical period as assessed by ocular dominance shifts in response to monocular deprivation.

Effects of surgical induction of endometriosis on response properties of preoptic area neurons in rats.

Subfertility and severe pelvic pains are symptoms associated with endometriosis (ENDO), a common condition among women that is characterized by the growth of the uterine endometrium on the surface of organs within the pelvic region and abdominal cavity. The contribution of the CNS to symptoms associated with ENDO is not known. In the present study, the preoptic area (POA) of the hypothalamus was investigated, as this region of the forebrain is known to play an important role in the neuroendocrine control of the reproductive cycle, mating behavior, and antinociception. Female rats were either induced for ENDO by autotransplantation of uterine tissue (n=20) or uterine fat for surgical sham controls (n=11). Terminal extracellular electrophysiological recordings (urethane anesthesia) were conducted in the POA six weeks post-ENDO induction when the rats were in either the proestrus or metestrus stages of their estrous cycle. Significant differences were found between the ENDO versus SHAM groups of animals for the proportion of inhibitory responses as well as the percentage of neurons responding to stimulation of the abdominal branches of the vagus, which innervates portions of the female reproductive tract, including the ovaries. The endometriotic cysts were found to be significantly larger in proestrus rats (stage when hormones are elevated). These data demonstrate that the responses of POA neurons are influenced by the presence of endometriotic cysts in the abdominal cavity. Since the POA is known to be part of the neural circuitries that mediate nociception and fertility, any deviation from its normal activity under ENDO conditions could contribute to the constellation of symptoms that ensue.

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.