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Immunocytochemical studies of plasticity candidate proteins in the cat primary visual cortex during postnatal… Li, Pʻing 2002

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Immunocytochemical Studies of Plasticity Candidate Proteins in the Cat Primary Visual Cortex During Postnatal Development By Ping L i B.Med., Beijing Medical University, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES Graduate Program in Neuroscience We accept this thesis as conforming to the requir@d~5tandard The University of British Columbia © Ping L i , 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. De x^artment^ of 2_ The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT The developing visual cortex has served as a model system for understanding activity-dependent neural development, learning and memory, and the treatment and prognosis of some diseases. The anatomy, physiology, development, and plasticity of the visual cortex have been well characterized at the systems level. However, current knowledge of molecular mechanisms underlying visual cortical development remains limited. The purpose of this thesis is to investigate the cellular and laminar distributions of two proteins (OBCAM and IL-11) in the visual cortex during postnatal development, as there is evidence suggesting that these proteins may play important roles in visual cortical development and plasticity. The expressions of OBCAM and IL-11 were characterized in the primary visual cortices of normal cats at various postnatal ages as well as in 4-month-old dark-reared cats. The relative immunopositive cell density was determined across visual cortical layers. OBCAM and IL-11 expressions are regulated by age and rearing condition. OBCAM immunoreactivity is highest between 2 and 4 weeks of age and then rapidly reduced afterwards until 6 weeks of age, when it is then similar to adult levels. IL-11 immunoreactivity is highest between 1 and 2 weeks of age and then rapidly reduced after 2 weeks of age until 4 weeks of age, when it is similar to adult levels. Dark rearing slowed the decrease of both proteins. Our data indicated the expression levels of these two proteins were well correlated with the level of ocular dominance plasticity, and suggested that OBCAM and IL-11 might be involved in visual cortical plasticity. Unfortunately, the results from western blot and immunocytochemical analysis of AC-7 and Rap IB demonstrated that the antibodies were not specific to AC-7 and Rap IB in cat tissue. Therefore, the developmental studies of AC-7 and Rap IB were not performed. i i i T A B L E OF C O N T E N T S Abstract ii Table of Contents iv List of Figures.... vi Abbreviations viii Acknowledgements x Chapter I INTRODUCTION 1 1.1 General Introduction 1 1.2 Neuroanatomy and Physiology of the Developing Cat Visual System 3 1.2.1 Anatomical Organization and Development 3 1.2.2 Development of Ocular Dominance Columns 6 1.3 Cellular and Molecular Mechanisms Involved in Cortical Plasticity in the Developing Visual Cortex 9 1.3.1 Hebbian Mechanisms and the B C M Model 9 1.3.2 The Role of N M D A Receptors 11 1.3.3 The Role of Metabotropic Glutamate Receptors 13 1.3.4 The Role of Inhibitory Circuitry 14 1.3.5 The Role of Neuromodulatory Neurotransmitters 15 1.3.6 The Role of Neurotrophins 17 1.3.7 The Role of Immediate Early Genes 19 1.3.8 Summary 20 1.4 Properties of Plasticity Candidate Proteins 20 1.4.1 Opioid-Binding Cell Adhesion Molecule (OBCAM) 20 1.4.2 Interleukin 11 (IL-11) 22 1.4.3 Adenylate Cyclase 7 (AC-7) 24 1.4.4 Ras-Associated Protein IB (RapIB) 26 Chapter II MATERIALS AND METHODS 29 2.1 The Choice of the Cat as the Animal Model 29 2.2 Materials 30 2.2.1 Animals .j 30 2.2.2 Chemicals and Reagents 32 2.2.3 Equipments 33 2.3 Methods 33 2.3.1 Solution Preparation 33 2.3.2 Animal Preparation 35 2.3.3 Immunocytochemical Staining 35 iv 2.3.4 Western Blot 38 Preparation of Protein Samples 38 SDS-Polyacrylamide Gel Electrophoresis 38 Immunoblotting 40 2.3.5 Data Analysis 41 Chapter III RESULTS 43 3.1 Opioid-Binding Cell Adhesion Molecule (OBCAM) 43 3.1.1 Western Blot Analysis of O B C A M 43 3.1.2 Localization of O B C A M in Cat Primary Visual Cortex 43 3.1.3 Developmental Profile of O B C A M 48 3.1.4 Dark Rearing Effects on O B C A M Protein Expression 56 3.2 Interleukin 11 (IL-11) 62 3.2.1 Selection of the IL-11 Antibodies 62 3.2.2 Localization of IL-11 in Cat Primary Visual Cortex 63 3.2.3 Developmental Profile of IL-11 73 3.2.4 Dark Rearing Effects on IL-11 Protein Expression 78 3.3 Adenylate Cyclase 7 (AC-7) 83 3.3.1 Western Blot Analysis of AC-7 83 3.3.2 Immunocytochemical Staining Analysis of AC-7 83 3.4 Ras-Associated Protein IB (RapIB) 83 3.4.1 Western Blot Analysis of Rap 1B 83 3.4.2 Immunocytochemical Staining Analysis of Rap IB 86 Chapter IV DISCUSSION 89 4.1 The Involvement of O B C A M in Visual Cortical Plasticity in the Developing Visual Cortex 89 4.1.1 Developmental Changes in O B C A M Protein Expression During the Critical Period 89 4.1.2 Dark Rearing Effects on O B C A M Protein Expression 91 4.2 The Involvement of IL-11 in Visual Cortical Plasticity in the Developing Visual Cortex 91 4.2.2 IL-11 Protein Expression in the Developing Visual Cortex 91 4.2.3 Dark Rearing Effects on IL-11 Protein Expression 93 4.3 Cautions from AC-7 and Rap IB 94 4.4 Conclusion 95 4.5 Future Direction 95 REFERENCES 97 LIST OF FIGURES Figure 1. Anatomical Organization of the Visual System 5 Figure 2. The Rap Proteins, Members of the Ras Superfamily 26 Figure 3. Western Blot Analysis of the O B C A M Protein Expression in 4-week-old Cat Visual Cortex 44 Figure 4. Laminar Distribution of O B C A M Protein in Neonatal Cat Primary Visual Cortex 46 Figure 5. Immunoreactivity Localization of O B C A M in Neonatal Cat Primary Visual Cortex 49 Figure 6. Laminar Distribution of O B C A M Protein in 4-week-old Cat Primary Visual Cortex 50 Figure 7. Immunoreactivity Localization of O B C A M Protein in 4-week-old Cat Primary Visual Cortex 52 Figure 8. Laminar Distribution of O B C A M Protein in Adult Cat Primary Visual Cortex 53 Figure 9. Immunoreactivity Localization of O B C A M Protein in Adult Cat Primary Visual Cortex 55 Figure 10. Expression of O B C A M Protein in Postnatal Developing Cat Visual Cortex — A . Immunocytochemical Staining 57 Figure 11. Expression of O B C A M Protein in Postnatal Developing Cat Visual Cortex — B. Semi-quantitative Study 58 Figure 12. Developmental Changes in O B C A M Positive Neuron Densities Across Cat Visual Cortical Layers 59 Figure 13. Dark Rearing Effects on O B C A M Expression in the Cat Primary Visual Cortex at 4 Months of Age 60 Figure 14. Selection of the IL-11 Antibodies 64 Figure 15. Laminar Distribution of IL-11 Protein in Neonatal Cat Primary Visual Cortex 67 Figure 16. Immunoreactivity Localization of IL-11 Protein in 4-week-old Cat Primary Visual Cortex 69 Figure 17. Laminar Distribution of IL-11 Protein in 4-week-old Cat Primary Visual Cortex 70 Figure 18. Immunoreactivity Localization of IL-11 Protein in 4-week-old Cat Primary Visual Cortex 72 Figure 19. Laminar Distribution of IL-11 Protein in Adult Cat Primary Visual Cortex 74 Figure 20. Immunoreactivity Localization of IL-11 Protein in Adult Cat Primary Visual Cortex 76 Figure 21. Expression of IL-11 Protein in Postnatal Developing Cat Visual Cortex — A . Immunocytochemical Staining 77 Figure 22. Expression of IL-11 Protein in Postnatal Developing Cat Visual Cortex — B. Semi-quantitative Study ..78 Figure 23. Developmental Changes in IL-11 Positive Cell Densities Across Cat Visual Cortical Layers 79 Figure 24. Dark Rearing Effects on IL-11 Protein Expression in Cat Primary Visual Cortex at 4 Months of Age 81 Figure 25. Western Blot Analysis of AC-7 Protein in Cat Primary Visual Cortex 84 Figure 26. Immunocytochemical Study of AC-7 85 Figure 27. Western Blot Analysis of Rap IB Protein in Cat Primary Visual Cortex 87 Figure 28. Immunocytochemical Study of Rap IB 88 vii A B B R E V I A T I O N S A B C avidin-biotin-complex AC-7 adenylate cyclase 7 A N O V A analysis of variance APS ammonium persulfate ATP adenosine triphosphate B C M Bienenstock-Cooper-Munro BDNF brain-derived neurotrophic factor BSA bovine serum album cAMP cyclic adenosine monophosphate CNS central nervous system CP cortical plate cz compact zone D A B 3,3 '-diaminobenzidine tetrahydrochloride G A B A y-aminobutyric acid G A D glutamic acid decarboxylase GPI glycosylphosphatidylinositol H 2 0 2 hydrogen peroxide HRP horseradish peroxidase IEG immediate early gene immunoglobulin IgCAM Ig superfamily cell adhesion molecule IL-11 interleukin 11 IP phosphoinositides L A M P limbic system-associated membrane protein L G N lateral geniculate nucleus LTD long-term depression LTP long-term potentiation mGluR metabotropic glutamate receptor mRNA messenger R N A N C A M neural cell adhesion molecule NF nuclear factor NGF nerve growth factor N M D A N-methyl-D-aspartate N R N M D A receptor NP-40 nonidet P-40 NT neurotrophin O B C A M opioid-binding cell adhesion molecule PBS phosphate buffered saline PBS-T phosphate buffered saline (pH 7.5) with 0.1% Tween-20 PFA paraformaldehyde P K A cAMP-dependent protein kinase P K C protein kinase C PMSF phenylmethylsulfonyl fluoride PVDF hydrophobic polyvinylidene difluoride Rap IB ras-associtated protein IB SC superior colliculus SDS sodium dodecyl sulfate S E M standard error of the mean SP subplate T E M E D N, N, N', A^-tetramethylene-ethylenediaine TGF transforming growth factor trk tyrosine kinase receptor T T X tetrotoxin W M white matter A C K N O W L E D G E M E N T S There are several people whose assistance I would like to acknowledge. Firstly, I would like to express special gratitude to my supervisor Dr. Qiang Gu for his inspiration, encouragement, and enthusiasm. I will always appreciate the generous gifts of his time, advice, patience, and continuous support. Secondly, I would like to express my thankfulness to my co-supervisor Dr. Joanne A Matsubara for her time, very helpful suggestions, and continuous support. I also like to express my sincerest appreciation for working with such an excellent committee. I thank Drs. Joanne A . Matsubara, Alison M.J . Buchan, and William Jia for their time, very helpful comments and suggestions, and continuous support. I thank Dr. John O'Kusky for being on my final exam committee. I appreciate his knowledge and help. Many thanks go out to Dr. Shiv Prasad, Dr. Xuefeng Wang, Dr. Jing Cui, Bing Zhu, Lillian Luo, Tara Stewart, and Tim Blanche for their help. I thank Liz Wong for her warm-hearted secretary assistance throughout. Finally, I would like to express special appreciation and gratitude to my parents and my husband Yingru, who have provided me with encouragement and support over the years. Thank you. x Chapter I Introduction Chapter I INTRODUCTION 1.1 G E N E R A L I N T R O D U C T I O N The visual system has long been a model for the study of the functional organization of the brain. It has been extensively studied in many species from goldfish to primates (Gaze et al., 1972; Gordon and Stryker, 1996; Hubel and Wiesel, 1969; Levine and Schechter, 1993; Maffei et al., 1992; Meister et al., 1991; Shatz and Stryker, 1978; Van Sluyters and Stewart, 1974). In studying this system, we have the opportunity to explore the brain at many different levels: from activity-dependent synaptic rearrangements to parallel pathways, the columnar organization of cortex, and cortical plasticity. At each of these levels, the visual system has evolved to solve a number of difficult problems. For example, processes similar to synaptic rearrangements occurring during the development of the visual system are postulated to be the biological basis of learning and memory (Bear et al., 1987; Bienenstock et al., 1982). Additionally, the examination of mechanisms responsible for ocular representation has provided significant insight into the fundamental processes involved in activity-dependent plasticity. Visual cortical plasticity is a pivot area of research in the neurosciences in several respects. It comprises studies from developmental neuroscience concerning questions for growth and self-organization (Shook and Chalupa, 1986; Trachtenberg et al., 2000), from behavioral neuroscience with respect to sensitive periods and imprinting (Daw et al., 1992; Mower et al., 1985; Mower, 1991), and from computational neuroscience 1 Chapter I Introduction regarding information storage in distributed systems (Artola and Singer, 1987; Mittmann and Eysel, 2001). In addition, like few other areas of neuroscience, the visual system can be studied at many levels of investigation, from the molecular to the cognitive, and therefore represents a perfect area for an integrative approach. Finally, there is also an obvious clinical interest in research on visual plasticity, which concerns the prevention and treatment of visual abnormalities such as amblyopia, the restoration of sensory functions in the blind, or the understanding of memory deficits (Domenici et al., 1991; E l Mallah et al., 2000; Kapadia et al., 1994). Recent studies have reported many plasticity candidate genes, like opioid-binding cell adhesion molecule, interleukin-11, adenylate cyclase 7, and ras-associated protein IB (Corriveau et al., 1998; Nedivi et al., 1996; Prasad and Cynader, 1994; Prasad et al., 2002; Yang et al., 2002). Despite the wealth of knowledge available about visual cortical plasticity, it is still unknown whether these genes are involved in this type of plasticity. Since all the brain systems function in a similar way, we hypothesize that these four genes are involved in visual cortical plasticity. However, protein expression may more likely reflect the level of plasticity because it is the 'functional' effector of messenger R N A (mRNA). Nevertheless, the mRNA and proteins levels are not always consistent in every brain area. For example, Pollock et al. (2001) found a discrepancy between mRNA and protein levels of brain-derived neurotrophic factor (BDNF) in the visual cortex of dark-reared animals. Thus, we decided to study these plasticity candidate genes at the protein level. Since it is becoming increasingly clear that changes in visual cortical plasticity result from changes in the expression of molecules, and an older dark-reared animal has a more plastic visual cortex than an age-matched normally-reared one, our 2 Chapter I Introduction working hypothesis: is candidate plasticity proteins are expressed in higher abundance in the primary visual cortex of both younger animals which are at the age of peak visual cortical plasticity and dark-reared older animals relative to the adult. In order to prove our hypothesis, the expressions of these four proteins — opioid-binding cell adhesion molecule, interleukin-11, adenylate cyclase 7, and ras-associated protein IB, which have been demonstrated to have a possible involvement in activity-dependent plasticity, were examined by immunocytochemical studies. 1.2 NEUROANATOMY AND PHYSIOLOGY OF THE DEVELOPING CAT VISUAL SYSTEM 1.2.1 Anatomical Organization and Development The eyes of the cat are located in front of the head and face forward resulting in the overlap of the two visual fields required for binocular vision. The retinal axons are routed so that visual information from the same points in space coming from the two eyes can be combined. The axons of all of the ganglion cells in the retina come together to form the optic nerve, which leaves the eye through the optic disc and projects to the optic chiasm. At this point, fibers from each eye destined for opposite sides of the brain are sorted and rebundled in the bilateral optic tracts (axons from the inner parts of the retina cross over). The optic tracts project to three major subcortical targets: the superior colliculus, which controls saccadic eye movements, the pretectum of the midbrain, which controls papillary reflexes, and the lateral geniculate nucleus (LGN), which is the main terminus for input to the visual cortex. Although each LGN receives input from both eyes, the connections from each eye are kept segregated in eye specific layers. In the cat, the LGN has three 3 Chapter I Introduction layers: layers A and A l , which receive input from the contralateral and ipsilateral eyes, respectively, and layer C, which receives connections from both eyes (Guillery, 1970). As determined by anterograde transsynaptic tracing experiments, the cat L G N neurons mainly project to layer IV of the ipsilateral primary visual cortex with collaterals to layer VI (Komatsu et al., 1985; Shatz et al., 1977). Layer IV projects to layers II and III, which send signals to other areas of cortex. Layers II and III project to layer V , which sends signals back to the superior colliculus (SC). Layers II, III, and V project to layer VI , which sends signals back to the L G N (Daw, 1995). The complete story is far more complicated than this, but these are the predominant projections (Figure 1). The segregation of inputs to the L G N is retained in the cortex where the thalamocortical connections from the L G N form an alternating interdigitated series of eye specific columns referred to as "ocular dominance columns" (Hubel and Wiesel, 1969, 1972; LeVay et al., 1978). Ocular dominance columns are roughly 400 - 700 microns, respond mainly to stimuli from only one of the eyes, and run perpendicularly to the cortical surface (Hubel et al., 1977; Levay et al., 1985; Lowel and Singer, 1987; Swindale, 1988). A greater fraction of neurons in layer IV have distinct preferences for one eye or the other than in other cortical layers (Hubel and Wiesel, 1962). There is also evidence indicating the presence of functional columns in layer II, III, V , and VI of the visual cortex (Daw et al., 1992; Wiesel and Hubel, 1963). Physiological experiments have determined that cells located in the centers of ocular dominance columns are largely monocular and respond only to stimuli from one eye whereas cells bordering two adjacent ocular dominance columns are mainly binocular and respond equally well to stimuli from either eye (Shatz andStryker, 1978). 4 Chapter I Introduction A. Optic Nerve Optic Tract Optic Radiation Left Hemisphere Optic Chiamsm Lateral Geniculate Nucelus (LGN) Xj Right Hemisphere Primary Visual Cortex Layer IV ~f A B Cortex t Cortex Layer II/III «" l > La ver Layer VI w V Superior Colliculus L G N Figurel. Anatomical organization of the visual system. A. B. Overall view of the visual system. The retina projects to the L G N . The L G N projects to the visual cortex. Axons from nasal retina cross at the optic chiasm, and axons from temporal retina do not. Consequently, the left cortex deals with the right field of view, and vice versa. (Adapted from A n Introduction to The Visual System, Edited by Martin J. Tovee, 1996). Schematic diagram of connections in cat primary visual cortex. Most the thalamic input is concentrated in layer IV and to a lesser degree in layer VI. There is a fairly strict hierarchical pathway from layers IV —• II + III —* V —* VI with feedback connections from V —• II + III and VI —»IV. There are also many lateral connections between neurons within the same layer. (Adapted from Fundamental Neuroscience, Edited by Michael J. Zigmond et al., 1999). 5 Chapter I Introduction A newborn cat's visual cortex is immature to the extent that migratory events associated with the formation of superficial cortical layers (layers II and III) are not completed (Shatz and Luskin, 1986). The ventricular zone is present at birth and cell migration completed in the cat's visual cortex sometime between 2 and 3 weeks after birth (Shatz and Luskin, 1986). During fetal development in the cat thalamic axons invade the subplate and wait 2 to 3 weeks, before invading the cortical plate and forming synaptic connections with layer IV neurons (Shatz and Luskin, 1986). At birth, the connections to layer IV are polysynaptic and originate from the L G N and the subplate. At around 2 to 3 weeks of age, the connection becomes monosynaptic and originates in the L G N (Friauf and Shatz, 1991; Shatz and Luskin, 1986). Selective ablation of the subplate during the first postnatal week in cats prevents the formation of ocular dominance columns (Ghosh and Shatz, 1992, 1994; Lein et al. 1999; McAllister, 1999). This suggests a role for the subplate in ocular dominance column development. 1.2.2 Development of Ocular Dominance Columns In the first week after birth, geniculate fibers relaying input from each eye show none of the signs of segregation characteristic of the mature cat brain (Crair et al., 2001; LeVay et al., 1978; Shatz and Luskin, 1986). Segregation of the inputs into ocular dominance columns was first reported to be evident anatomically at postnatal three weeks of age (LeVay et al., 1978). However, improvements in anatomical and physiological techniques suggest that ocular dominance column formation begins between postnatal week one and week two, although the contralateral eye provides much stronger drive to the visual cortical neurons until about three weeks of age (Crair et al., 1998, 2001). 6 Chapter I Introduction The most striking feature of ocular dominance column development is its dependence on normal visual activity. If visual activity to only one eye is occluded early in life (induced by monocular lid suture and referred to as monocular deprivation), most neurons in the visual cortex lose their responses to the deprived eye and thereby respond exclusively to the eye that has remained open (Wiesel and Hubel, 1963). In severe cases, the animal is essentially blind in the deprived eye since there is little input left from it to the visual cortex. Anatomically, the cortical territory of the ocular dominance columns serving the nondeprived eye is dramatically expanded, while the territory serving the deprived eye shows a compensatory shrinkage (LeVay et al., 1978; Shatz and Stryker, 1978). At the cellular level, axons conveying input from the deprived eye decrease in total length and branch less in layer IV compared to normal, whereas axons conveying input from the nondeprived eye branch even more profusely than normal (Antonini and Stryker, 1993, 1996). The severity of this effect is dependent on the length of the deprivation and the age of the animal (Hubel and Wiesel, 1970; Shatz and Stryker, 1978). The effects of monocular deprivation only occur when the treatments are imposed early in life, leading to the notion of the "critical period" for ocular representation, which is a stage when normal visual activity is essential for the development of ocular dominance columns. In cats, the critical period for visual deprivation is considered to begin roughly 3 weeks after birth and end around 3 months of age (Hubel and Wiesel, 1970; Olson and Freeman, 1980; Wiesel and Hubel, 1963). However, the 3 week to 3 month time period is only a rough estimate of overall plasticity in the visual cortex of cat, and the length of the critical period varies significantly between different cortical layers (Daw et al., 1992). In the cat visual cortex, thalamocortical connections found in layer IV lose their ability to 7 Chapter I Introduction reorganize at around 50-60 days of age (Mower et al., 1985; Shatz and Stryker, 1978) while the remaining layers (layers II, III, V , and VI) exhibit a gradual reduction in plasticity beginning at 6 weeks of age until the end of the critical period (which is approximately at 1 year of age) (Daw et al., 1992). At 4 weeks of age, the cortex is maximally sensitive to monocular deprivation (Hubel and Wiesel, 1970; Movshon and Dursteler, 1977; Olson and Freeman, 1975). Once a cat is about 1 year old, which is after the critical period, monocular deprivation for even months no longer significantly affects ocular dominance in the cortex (Daw et al., 1992). The effects of monocular deprivation are reversible, i f both eyes are open or the original-deprived eye is opened and the original-open eye is sutured shut (reverse suture) early in life (Blakemore and Van Sluyter, 1974, Guillery and Stelzner, 1970; Mitchell et al. 2001). The critical period, however, is not a simple age-dependent maturational process, but is rather a series of events itself controlled in an activity-dependent manner. The end of the critical period in cats can be delayed substantially by raising animals in complete darkness (Cynader and Mitchell, 1980; Cynader, 1983; Mower et al., 1985; Mower, 1991). The effect of dark rearing is to slow the entire time course, both the rise and the decline, of visual cortical plasticity (Mower, 1991). Although dark-reared adult cats show clear shifts in ocular dominance in response to monocular deprivation, it is less severe than that seen in normally developing kittens (Cynader, 1983; Mower et al., 1985). The visual system of a dark-reared cat also matures and loses its plasticity quite rapidly once it is exposed to light (Swindale, 1988). In addition, dark-reared cats maintain plasticity primarily in cells located outside of layer IV as measured by electrophysiological recordings, which is not 8 Chapter I Introduction accompanied by a prolonged plasticity measured by anatomical methods (Mower et al., 1985). Spontaneous neural activity is believed to facilitate the establishment of ocular dominance columns. There is neural activity in the retina in the form of rhythmic bursts of action potentials spontaneous generated by retinal ganglion cells before the onset of vision (Galli and Maffei, 1988). Such activity persists until just before the onset of visually driven activity (Wong et al., 1993). Patterned visual experience in the cat begins when the eyes open which is at about 1 week after birth. A newborn cat has its retinal connections segregated into eye specific layers in the L G N (Shatz, 1983) and injection of tetrotoxin (TTX) into eyes of prenatal kittens prevents the formation of eye specific layers in the L G N (Shatz and Stryker, 1988; Sretavan et al., 1988). Thus segregation of retino-geniculate inputs into eye specific layers in the L G N is believed to be facilitated by spontaneous activity. By the time a primate is born, its visual cortex contains ocular dominance columns (Hubel et al., 1977). Since a newborn primate does not receive visual activity prenatally, spontaneous activity is also believed to give rise to the formation of ocular dominance columns in primates (Reiter et al., 1986; Stryker and Harris, 1986). 1.3 CELLULAR AND MOLECULAR MECHANISMS INVOLVED IN VISUAL CORTICAL PLASTICITY IN THE DEVELOPING VISUAL CORTEX 1.3.1 Hebbian Mechanisms and The BCM Model In the developing visual system there are correlated patterns of activity (Maffei and Galli-Resta, 1990; Meister et al., 1991; Schwartz et a l , 1998; Yuste et al., 1992) and there are cortical synapses that are capable of detecting such activity and responding with 9 Chapter I Introduction functional changes (Kirkwood et al., 1993; Miller et al., 1989; Shulz and Fregnac, 1992). Based on these Hebbian characteristics of visual cortical synapses during the critical period, most theories devised to explain ocular dominance plasticity (which is the best known type of visual cortical plasticity), are based on a Hebbian (Fregnac et al., 1988; Hebb, 1949; Shulz and Fregnac, 1992) concept of learning: that is, synaptic potentiation will occur i f the activities of the presynaptic and postsynaptic neurons are temporally correlated, or more simply stated, 'cells that fire together wire together'. Hebb's theory of synaptic modification was greatly supported by the discoveries of long-term potentiation (LTP), the stable enhancement of synaptic potentials following stimulation paradigms in which the presynaptic and postsynaptic neurons are concurrently active beyond some threshold level (Andersen et al., 1977), and long-term depression (LTD), the stable decrease of synaptic efficacy for a prolonged period of time following a low frequency stimulus (Lynch et al., 1977; Stanton and Sejnowski, 1989). Both LTP and LTD can be elicited electrophysiologically in visual cortical slices (Artola and Singer, 1987; Kirkwood et al., 1993; Kirkwood and Bear, 1994a, 1994b). Susceptibility to both LTP and LTD in visual cortical slices correlates with the critical period for development of ocular dominance columns (Dudek and Friedlander, 1996; Kirkwood et al., 1995). Additionally, visual experience can enhance or diminish LTP and LTD in visual cortical slices (Kirkwood et al., 1996). Hence, the properties of synaptic LTP and LTD may account for many aspects of visual cortical plasticity in the developing visual cortex. The Bienenstock-Cooper-Munro (BCM) model (Bear et al., 1987; Bienenstock et al., 1982; Clothiaux et al., 1991), a specific correlation modification of a hebbian type, has recently received more attention. According to the B C M theory, the connection strength 10 Chapter I Introduction of excitatory geniculocortical synapses varies as the product of a measure of input activity and a function ((()) of the summed postsynaptic response. For all postsynaptic responses greater than spontaneous but less than a critical value called the "modification threshold", (j) has a negative value. For all postsynaptic responses greater than the threshold, <|> has a positive value. The modification threshold changes during development, depending on the average firing rate of the postsynaptic cell (Bienenstock et al., 1982; Clothiaux et al., 1991). By the rules derived from the BCM theory, the modification of excitatory geniculocortical synapses can account for both the kinetics and outcome of a wide variety of experimentally observed visual deprivation effects (Clothiaux et al., 1991; Law and Cooper, 1994). For example, the theory can interpret why LTD-induced changes underlie the initiation of the shift in ocular dominance (Antonini and Stryker, 1993; Rittenhouse et al., 1999), why the initial recovery of vision after early monocular deprivation in kittens is faster when both eyes are open instead of reverse suture (Mitchell et al., 2001), why there are striking differences in the effects of monocular versus binocular deprivation in the plasticity of ocular dominance (Wiesel and Hubel, 1963), and why there are 'normal' features of neurons following binocular deprivation (Clothiaux et al., 1991; Kirkwood et al., 1995). Therefore, the BCM theory may be one type of mechanism contributing to developmental plasticity. 1.3.2 The Role Of NMD A Receptors Despite the large amount of information available regarding the physiological and anatomical development of the cat visual system, the molecular mechanisms responsible for this event have remained elusive. The NMDA receptors have received much attention during visual development because glutamate is the excitatory neurotransmitter in the 11 Chapter I Introduction visual cortex (Bear et al., 1990; Gu et al., 1989; Kleinschmidt et al., 1987; Rauschecker et al., 1990). In addition, the N M D A receptor can serve as a detector of coincident input activity (glutamate release) and postsynaptic response because it has voltage-dependent properties due to blockade of the channel by M g + + (Mayer and Westbrook, 1985; Nowak et al., 1984), and it is required for LTP in the hippocampus (Collingridge et al., 1983). N M D A receptors are present throughout the visual system during the critical period. The total number of the N M D A receptors in the visual cortex varies with age, and the number peaks at the same time that the critical period for shifts in ocular dominance peaks (Gordon et al., 1991). As would be expected of a molecule associated with visual cortical plasticity, autoradiographic studies of the distribution of the N M D A receptor in kitten visual cortex show a rise and fall in binding intensity that parallels the time course of the critical period (Bode-Greuel and Singer, 1989; Gordon et al., 1991; Reynolds and Bear, 1991) . The percentage of the visual response of individual neurons that is N M D A receptor-mediated decreases in layers IV, V , and VI between 3 and 6 weeks of age and dark rearing postpones this decrease (Fox et al., 1989, 1991, 1992; Fox and Daw, 1993). The N M D A receptor is a heteromer that consists of an obligatory NR1 subunit and NR2 subunits that impart functional properties (McBain and Mayer, 1994; Monyer et al., 1992) . Early in postnatal life, the NR2B subunit is predominant in visual cortex and N M D A receptor-mediated currents decay slowly, which facilitates the induction of LTP (Carmignoto and Vicini, 1992; Quinlan et al., 1999a). There is an experience-dependent inclusion of NR2A-containing N M D A receptors that shortens N M D A receptor currents with development in a manner paralleling the critical period (Carmignoto and Vicini, 1992; Flint et al., 1997; Nase et al., 1999). Thus, visual experience decreases the 12 Chapter I Introduction proportion of NR2B- to NR2A-containing receptors and shortens N M D A receptor currents during high-frequency stimulation, while visual deprivation exerts an opposite effect (Philpot et al., 2001; Quinlan et al., 1999a, 1999b). Direct support for N M D A receptor involvement in ocular dominance column development comes from three-eyed frog studies in which the application of N M D A receptor antagonists cause the eye-specific stripes to desegregate (Cline et al., 1987; Cline and Constantine-Paton, 1990). Similarly, the physiological shift toward the open eye that occurs in monocularly deprived kittens can be blocked by cortical infusion of N M D A receptor antagonists (Bear and Colman, 1990; Gu et al., 1989; Kleinschmidt et al., 1987). The effect of N M D A receptor antagonists is not simply due to blockade of neural activity like infusing T T X since the afferent activity is somewhat reduced but not abolished (Kleinschmidt et al., 1987; Daw, 1994). Thus, the N M D A receptors may play important roles in mediating visual cortical synaptic plasticity in the developing visual cortex. 1.3.3 The Role Of Metabotropic Glutamate Receptors The metabotropic glutamate receptors (mGluRs) have also been characterized during the critical period. They are distinguished from the N M D A receptors in that they act through G-proteins. The involvement of mGluRs in activity-dependent visual cortical plasticity has been suggested by evidence that mGluR-activated turnover of intracellular messengers, phosphoinositides (IP) and cyclic adenosine monophosphate (cAMP) vary with the critical period for ocular dominance plasticity (Dudek and Bear, 1989; Reid et al., 1996). The alterations on the cAMP levels mediated by the activation of multiple mGluRs correlate well with the critical period not only under normal but also under dark-13 Chapter I Introduction reared conditions (Reid et al., 1996). Infusion of a cAMP antagonist abolishes the ocular dominance shift by monocular deprivation (Beaver et al., 2001). Additionally, the laminar distributions of mGluR2/3 and mGluR5 change with the critical period and are sensitive to dark rearing (Reid and Daw, 1997; Reid and Romano, 2001). A recent study also showed that mGluR2/3s play a fundamental role in LTD of synaptic transmission within the primary visual cortex (Renger et al., 2002). Taken together, mGluRs may play important roles in activity-dependent visual cortical plasticity. However, since agonists and antagonists specific for individual mGluRs are still unavailable, the effect of each subtype of mGluR on monocular deprivation shifts remains unclear. 1.3.4 The Role Of Inhibitory Circuitry Activity-dependent plasticity in the developing visual cortex is not limited to excitatory circuitry. y-Aminobutyric acid ( G A B A ) is well established as the major inhibitory neurotransmitter in the mammalian visual cortex (Barnstable et al., 1992; Daniels and Pettigrew, 1975; Naegele et al., 1988; Sato and Tsumoto, 1984). As expected, expressions of subunits of the G A B A A receptor are developmentally regulated. The switch in relative dominance of expression from the 'immature' subunits 012 and 013 to the 'mature' CM subunit occurs near the peak of the critical period for developmental plasticity in cat visual cortex and these changes are dependent on visual input (Paysan and Fritschy, 1998; Chen et al., 2001). The direct evidence that inhibitory interactions are necessary for the manifestation of experience-dependent plasticity comes from transgenic mice lacking the 65-kDa isoform of the G A B A - s y n t h e s i z i n g enzyme glutamic acid decarboxylase (GAD) which is specialized to respond to a short-term increase in demand for G A . B A in axon terminals (Erlander and Tobin, 1991; Esclapez et al., 1994; Gold and 14 Chapter I Introduction Roth, 1979; Kaufman et al., 1991; Martin and Rimvall, 1993). In these animals the ocular dominance shifts in response to monocular deprivation are deficient and normal plasticity can be rescued if GABAergic transmission is enhanced in the visual cortex by means of benzodiazepines (Fagiolini and Hensch, 2000; Hensch et al., 1998). A shifting balance between excitation and inhibition has been suggested as a mechanism for changes in neuronal plasticity during the critical period in the visual cortex (Artola and Singer, 1987; Chen et al., 2001; Fagiolini and Hensch, 2000; Guo et al., 1997; Hensch et al., 1998; Huang et al., 1999; Kirkwood and Bear, 1994a). The development of inhibition lags behind that of excitation and provides a time window — a critical period — during which the organization of cortical circuitry can be particularly influenced by sensory experience. Additionally, a recent study using pharmacological and genetic tools showed that increased intracortical inhibition not only promotes plasticity in primary visual cortex, but is also a major factor responsible for triggering the initial onset of the critical period (Fagiolini and Hensch, 2000). However, autoradiographic studies showed that in the visual cortex G A B A A receptor binding sites and binding affinity fluctuate widely during development, which is not correlated with the time course of the critical period (Shaw et al., 1984, 1986). Taken together, inhibitory circuitry is likely to be involved in activity-dependent synaptic plasticity in the developing visual cortex although the exact mechanism is still unclear. 1.3.5 The Role Of Neuromodulatory Neurotransmitters In addition to excitatory and inhibitory circuitries, neuromodulatory neurotransmitters are also implicated in visual cortical plasticity (Gu, 2002). Neuromodulatory neurotransmitters can have either excitatory or inhibitory effects depending on the 15 Chapter I Introduction postsynaptic cell type and receptor composition (Hasselmo, 1995; Kimura and Baughman, 1997). There are four main neuromodulatory pathways to the visual cortex using acetylcholine, noradrenaline, serotonin, and dopamine. Acetylcholine signals come from the basal forebrain (Irle and Markowitsch, 1984; Mizuno et al., 1969; Troiano and Siegel, 1978), noradrenaline signals from the locus coeruleus (Watabe et al., 1982), serotonin signals from the raphe nuclei (Chazal and Ralston, 1987; Mulligan and Tork, 1988), and dopamine signals from the brain stem (Higo et al., 1996; Parkinson, 1989; Tork and Turner, 1981). The neuromodulatory neurotransmitter receptors have age-specific laminar distribution in the visual cortex, and the expressions and the distributions of these receptors correlate with the critical period (Aoki et al., 1986; Dyck and Cynader, 1993; Gu et al., 1990; Jia et al., 1994; Liu and Cynader, 1994; Prusky et al., 1988; Prusky and Cynader, 1990; Stichel and Singer, 1987). Moreover, the selective blockade of these receptors by pharmacological agents affects ocular dominance plasticity. The infusion of these neurotransmitters into aplastic visual cortex can restore the plasticity. These provide evidence for the involvement of neurotransmitters such as acetylcholine (Bear and Singer, 1986; Gu and Singer, 1993), noradrenaline (Bear and Singer, 1986; Kasamatsu et al., 1979, 1981; Kasamatsu and Shirokawa, 1985; Shirokawa and Kasamatsu, 1987), and serotonin (Gu and Singer, 1995; Wang et al., 1997) in visual cortical synaptic plasticity. Additionally, these neurotransmitters can facilitate the induction of LTP or LTD in visual cortex under different stimulations (Kirkwood et al., 1999; Kojic et al., 1997, 2001), and they can increase N M D A induced elevation of intracellular calcium levels (Nedergaard et al., 1987; Yang et al., 1996). The contribution of neuromodulatory neurotransmitters to 16 Chapter I Introduction visual plasticity is likely to underlie a control system gating the activity-dependent changes of synaptic organization in the developing visual cortex. 1.3.6 The Role Of Neurotrophins Recently, a role for neurotrophins in regulating cortical developmental plasticity has clearly emerged (Cellerino and Maffei, 1996; Gu, 1995; Pizzorusso and Maffei, 1996; Thoenen, 1995). The neurotrophins are a family of structurally related proteins that are essential for survival, growth, and differentiation of distinct populations of neurons in the central and peripheral nervous system. Each neurotrophin binds to a specific tyrosine kinase receptor (trk) through which it exerts its biological functions. These specific receptors are trkA for nerve growth factor (NGF) (Cordon-Cardo et al., 1991), trkB for BDNF and neurotrophin (NT) 4-5 (Berkemeier et al., 1991; Ip et al., 1992; Klein et al., 1991), and trkC for NT3 (Lamballe et al., 1991). The afferents originating from the LGN have been suggested to compete for these neurotrophic factors produced by and released from cortical cells in an activity-dependent manner (Elliott and Shadbolt, 1998; Harris et al., 1997, 2000; Maffei et al., 1992). According to this model, afferents which successfully receive neurotrophins are able to form synapses whereas afferents which do not take up enough neurotrophins are eliminated. The finding that cortical infusion of NT4 in the ferret is capable of preventing LGN cell shrinkage associated with monocular deprivation supports this hypothesis (Riddle et al., 1995). Evidence for NGF involvement in visual cortical plasticity originally came from observations that an exogenous supply of NGF prevents the effects of monocular deprivation (Maffei et al., 1992), whereas blockade of endogenous NGF action severely interferes with development of the visual system and prolongs the critical period (Berardi 17 Chapter I Introduction et al., 1994; Domenici et al., 1994). Activation of NGF receptor trkA in the visual cortex is sufficient to prevent the effects of monocular deprivation (Pizzorusso et al., 1999). In addition, NGF infusion is capable of inducing plasticity in the adult visual cortex (Gu et al., 1994). However, since the level of trkA receptor expression in the visual cortex is very low compared to trkB (Allendoerfer et al., 1994; Cabelli et al., 1996), the importance of NGF in the developing visual system is questionable. The fact that relatively high levels of NGF infusion are required to have an effect on visual cortical plasticity is also of concern because NGF can interact with trkB at high concentrations as well as with its 'own' receptor trkA (Rodriguez-Tebar et al., 1990), The production and release of BDNF in the visual cortex depends, at least in part, on afferent activity (Bozzi et al., 1995; Castren et al., 1992; Schoups et al., 1995), and the ontogeny of trkB expression is consistent with the critical period (Allendoerfer et al., 1994; Cabelli et al., 1996). Infusion of BDNF or blockade of trkB in normal kittens during the critical period interferes with ocular dominance column development and 'normal' synaptic rearrangements induced by monocular deprivation (Cabelli et al., 1995, 1997; Galuske et al., 1996). Recent work on transgenic mice models overexpressing BDNF has found that an excess of BDNF results in a shorter critical period for ocular dominance plasticity and the visual cortex appears to mature faster (Hanover et al., 1999; Huang et al., 1999). Since in these mice the maturation of GABAergic innervation and inhibition was accelerated, it is proposed that BDNF promotes the maturation of cortical inhibition during early postnatal life, thereby regulating the critical period for visual cortical plasticity. 18 Chapter I Introduction Despite evidence showing that neurotrophins are involved in the activity-dependent plasticity in the developing visual cortex, the mechanisms of action are still unclear. Since the neurotrophins have a diverse number of effects and are involved in various stages of development, different neurotrophins may affect visual cortical plasticity through different mechanisms. 1.3.7 The Role Of Immediate Early Genes Immediate early gene (IEG) activity may also contribute to visual cortical plasticity. Since several characteristics of IEG expression indicate a role in translating extracellular input into lasting changes in cellular function (Johnson and McKnight, 1989; Mitchell and Tjian, 1989), it has been proposed that IEGs initiate the genomic response underlying long-term modification of neuronal physiology (Goelet et al., 1986; Morgan and Curran, 1991; Sheng and Greenberg, 1990). The postnatal expression of IEGs in the kitten visual cortex is developmentally regulated during the critical period (Beaver et al., 1993; Kaplan et al., 1995, 1996; McCormack et al., 1992), and high levels of IEG expression can be maintained in older animals by dark rearing (Beaver et al., 1993; Mower and Kaplan, 1999). In addition, the expressions of IEGs are regulated by light-driven neural activity (Kaczmarek et al., 1999; Mitchell et al., 1995; Rosen et al., 1992). The magnitude of IEG induction after dark-reared cats are exposure to light is higher in the visual cortex of dark-reared kitten than dark-reared adult cats (Kaplan et al., 1996; Mower, 1994; Rosen et al., 1992). Unfortunately, the precise physiological roles of the proteins encoded by them remain largely unknown although much progress has been made toward understanding the intracellular processes that guide the expression of these genes. While intervention approaches — such as gene knockout and anti-sense knockdown — are 19 Chapter I Introduction potentially powerful in revealing their function, they have not been performed due to the difficulties associated with modifying gene expression in the cat. 1.3.8 Summary A large amount of work has been performed identifying the cellular and molecular mechanisms involved in visual cortical plasticity in the developing visual cortex. Although there is some evidence showing that there are several molecular factors involved in the plasticity in the developing visual cortex, the mechanisms by which these molecules contribute to visual cortical plasticity are still unclear. Therefore, our understanding of the cellular and molecular basis of this plasticity remains dim. Clearly, a substantial amount of work has to be performed in order to unravel the cellular and molecular mechanisms responsible for the activity-dependent plasticity in the developing visual cortex of cat. 1.4 PROPERTIES OF PLASTICITY CANDIDATE PROTEINS — OPIOID-BINDING C E L L ADHESION MOLECULE, INTERLEUKIN-11, ADENYLATE CYCLASE 7, AND RAS-ASSOCIATED PROTEIN IB. 1.4.1 Opioid-Binding Cell Adhesion Molecule Opioid-binding cell adhesion molecule (OBCAM), a neuron-specific protein, contains three immunoglobulin (Ig)-like domains anchored to the membrane through a glycosylphosphatidylinositol (GPI)-tail (Hachisuka et al., 1996; Schofield et al., 1989). Based on the cDNA sequence O B C A M is predicted to be a 345-amino acid polypeptide (Schofield et al. 1989). Using a monoclonal antibody against O B C A M , it has been shown to have a molecular weight of 51- and 58-kDa and to be localized throughout almost the 20 Chapter I Introduction entire gray matter, including the hippocampus, cerebral cortex, striatum, cerebellar cortex, and spinal cord (Hachisuka et al., 1996). O B C A M was initially thought to be a type of opioid receptor in the brain since indirect evidence implied its role in opioid actions (Ann et al., 1992; Cho et al., 1986; Govitrapong et al., 1993; Roy et al., 1988; Schofield et al., 1989). However, its primary structure deduced from the cDNA nucleotide sequence is entirely different from other opioid receptors that belong to the superfamily of seven-transmembrane domain receptors functionally coupled to G-protein (Savarese and Fraser, 1992; Schofield et al., 1989; Uhl et al., 1994; Wang et al., 1994). The high homology to the Ig-like domains of neural cell adhesion molecule (NCAM) makes O B C A M a member of Ig superfamily C A M s (IgCAMs). Like cadherins and integrins which are the other two superfamilies of C A M s , IgCAMs have been found to be involved in many aspects of nervous system development. For instance, IgCAMs play roles in the migration of neuronal precursors, axon guidance, contact-dependent induction/inhibition of neurite outgrowth, and neurite fasciculation (Faivre-Sarrailh and Rougon, 1997; Tessier-Lavigne and Goodman, 1996; Walsh and Doherty, 1997). Recently, several proteins that possess only three Ig domains and a GPI-anchor, like O B C A M , have been discovered in the central nervous system (CNS) including L A M P (limbic system-associated membrane protein), CEPU-1/neurotrimin (chick and rat orthologues, respectively) and neurotractin (chick)/kilon (rat) (Funatsu et al., 1999; Marg et al., 1999; Pimenta et al., 1995; Spaltmann and Brummendorf, 1996). They are named IgLON family (Struyk et al., 1995). Members of this family on opposing cell membranes can interact homophilically and heterophilically with each other (Lodge et al., 2000). Although the role of most IgLON proteins is 21 Chapter I Introduction currently unclear, they have been presumed to play a role in the guidance of growing axons by providing molecular cues which enable them to accurately form synapses with their appropriate targets (Lodge et al., 2000). A recent study examined the expression pattern of O B C A M in rat brain during development and in the adult (Hachisuka et al., 2000). It suggested that O B C A M is related to synaptogenesis. Based on the reported functions of IgCAMs and the IgLON family and the available studies of O B C A M , we propose that O B C A M is likely to be a plasticity candidate protein involved in the visual cortical plasticity during early postnatal development. Interestingly, O B C A M mRNA level in the cat visual cortex is developmentally regulated (Prasad et al., 2002). However, previous experiments have indicated that GPI-linked glycoproteins have a long half life compared to most transmembrane and cytoplasmic proteins, suggesting they have low rates of degradation (Lemansky et al., 1990). Therefore, O B C A M protein expression might not parallel its mRNA level in the visual cortex. Thus, in order to prove our conjecture, we examined O B C A M protein level to determine whether it is also developmentally regulated and affected by dark rearing. 1.4.2 lnterleukin-11 Interleukin-11 (IL-11) is a multifunctional growth factor originally identified in conditioned medium from a primate bone marrow stromal cell line, and cloned from a cDNA library generated from the same cell line (Paul et al., 1990, 1991). IL-11 precursor protein consists of 199 amino acids including a 21-amino acid leader sequence (Ohsumi et al., 1991; Paul et al. 1990). The theoretical molecular weights of recombinant human and murine IL-11 are 19,144 daltons and 19,154 daltons (Morris et al., 1996; Ohsumi et al., 1991), respectively. Mature human and primate IL-11 proteins share 94% identity 22 Chapter I Introduction whereas human and murine proteins share 88% identity in the amino acid sequence (Kawashima et al., 1991; Morris et al., 1996; Ohsumi et al., 1991). Analysis of normal murine tissue indicates the presence of low levels of IL-11 mRNA in the hematopoietic organs (such as bone marrow, spleen, and thymus) as well as in the brain, heart, lung, small and large intestine, kidney, testis and ovary (Davidson et al., 1997; Du et al., 1996). In the CNS, IL-11 can be expressed by both neurons and glial cells (Du et al., 1996; Murphy etal., 1995). The IL-11 receptor complex consists of the ligand binding a chain and the signal transducing subunit, gpl30 (Hilton et al., 1994), which makes IL-11 a member of the gpl30 family. This family has been indicated to play roles in macrophage differentiation, expression of acute-phase proteins by hepatocytes, and neuronal survival and differentiation (Kishimoto et al., 1995). Previous studies have demonstrated IL-11 can affect growth and differentiation of several hematopoietic cell types, including early pluripotent stem cells, megakaryocyte progenitors and megakaryocytes, erythrocyte progenitors, and granulocyte progenitors (Du et al., 1995; Lemoli et al., 1993; Rodriguez et al., 1995; Taguchi et al., 2001; Weich et al., 2000). The biological effects of IL-11, however, are not limited to cells of hematopoietic origin. IL-11 has been reported to inhibit adipogenesis in preadipocytes (Kawashima et al., 1991), and to stimulate production of several acute phase plasma proteins in hepatocytes (Baumann and Schendel, 1991) and the tissue metalloproteinase inhibitor in connective tissue cells (Maier et al., 1993). IL-11 is also an anti-inflammatory agent, mediating many of its effects by inhibition of the transcriptional activator nuclear factor (NF)-kappa B (Trepicchio et al., 1997). IL-11 has also been shown to regulate the proliferation and 23 Chapter I Introduction differentiation of neural cells (Carvey et al., 2001; Du et al., 1996; Farm and Patterson, 1994; Mehler et al., 1993). For example, in the presence of IL-11 and some other hematopoietic cytokines, the mesencephalic progenitor cells can be clonally expanded in culture and differentiated to dopamine neurons (Carvey et al., 2001). IL-11 can also promote neuronal differentiation of an immortalized neural stem and progenitor cell line (MK31) from dissociated embryonic mouse hippocampus (Mehlet et al., 1993). IL-11 alone or in combination with transforming growth factor-a (TGF-a) enhances cellular polarity, neurite outgrowth, and nuclear enlargement and MK31 cell complexity. IL-11 alone can also induce inward currents and the propagation of immature action potentials in MK31 cells. These data indicate that IL-11 induces intermediate stages of neuronal maturation in the mammalian brain. Moreover, IL-11 is structurally and functionally related to ciliary neurotrophic factor which is a polypeptide that promotes the survival and differentiation of a number of neural cell types (Negro et al., 1994; Shimazaki et al., 2001). Since neuronal differentiation and maturation are very important in the development of the brain and cortical plasticity, we propose that IL-11 is likely to play a role in the developmental visual plasticity. 1.4.3 Adenylate Cyclase 7 At least nine membrane-bound isoforms and one soluble form of adenylate cyclases (ACs), the enzymes responsible for the synthesis of cAMP from adenosine triphosphate (ATP), have been cloned and characterized in mammals (Bakalyar and Reed, 1990; Cali et al., 1996; Feinstein et al., 1991; Gao and Gilman, 1991; Ishikawa et al., 1992; Katsushika et al., 1992; Krupinski et al., 1989, 1992; Paterson et al., 1995; Premont et al., 1992; Watson et al., 1994; Yoshimura and Cooper, 1992). The membrane-bound ACs 24 Chapter I Introduction share a primary structure consisting of two hydrophobic domains with six transmembrane spans and two cytoplasmic regions (Krupinski et al., 1989). AC-7 is a member of type II A C subfamily which exhibits the property of 'coordinate' stimulation by the active a subunit (Gsa) of the stimulatory GTP-binding protein Gs and the Py subunits of the heterotrimeric G proteins (Feistein et al., 1991; Tang and Gilman, 1991; Yoshimura and Cooper, 1992). The activity of AC-7 can also be modulated by protein kinase C (PKC) (Watson et al., 1994). AC-7 mRNA is present in a large number of tissues including lung, liver, kidney, heart and brain (Hellevuo et al., 1995; Nomura et al., 1994). The presence of AC-7 in the brain suggests that AC-7 might be coupled to specific signal transduction pathways and regulate certain functions in the brain. Supporting evidence for a possible role of A C in cortical plasticity has been obtained from somatosensory cortex where it has been shown that disruption of AC-1 in mutant mice prevented the developmental formation of the whisker barrels that characterize this structure (Abdel-Majid et al., 1998). Moreover, activation of A C has been reported to restore ocular dominance plasticity in adult visual cortex (Imamura et al., 1999), while inhibition of cAMP-dependent protein kinase (PKA) can block ocular dominance shifts that occur following monocular deprivation early in the critical period (Beaver et al., 2001). Taken together, this indicates that A C is involved in visual cortical plasticity although it remains unknown which isoform plays the role. A recent study showed that AC-7 mRNA level is developmentally regulated in the visual cortex (Prasad et al., 2002). However, the levels of mRNA for a particular protein do not always reflect the levels of the protein in the examined tissues. Thus, we decided to explore the presence of AC-7 protein in the cat 25 Chapter I Introduction primary visual cortex during postnatal development in order to support the idea that AC-7 plays an important role in activity-dependent plasticity in the developing visual cortex. 1.4.4 Ras-Associated Protein IB Ras-associated protein IB (RapIB) was first identified as a 184-amino acid protein by screening a Raji cell line cDNA library (Pizon et al., 1988). Subsequently, it was also purified from bovine brain (Kawata et al., 1988), human platelets (Ohmori et al., 1989), and ovine aortic smooth muscle (Kawata et al., 1989). Rap IB is a 22-kDa low molecular mass GTP-binding protein which belongs to the ras superfamily. Members of the ras superfamily are essential components of receptor-mediated signaling pathways controlling cell proliferation and differentiation (Hall, 1990). The homologies among the Rap proteins are very high (Figure 2). Rapl and Rap2 proteins are 70% identical at the amino acid level. Rapl A and IB differ by only nine out of 184 amino acids (95% identity), with the sole region of substantial nonidentity being between positions 171-189 of the C-terminus. Similarly, Rap2A and 2B differ by 18 out of 183 amino acids (90% identity) (Bokoch, 1993). Rapl proteins were detected in various mammalian tissues including cerebrum, cerebellum, adrenal gland, thymus, lung, heart, liver, small intestine, kidney, and testis (Kim et al., 1990). Rapl A Rap IB Rap2A Rap2B Figure 2. The Rap proteins, members of the ras superfamily Four rap subtypes have been identified, as described in the text. The percentage of sequence similarly at the amino acid level between the indicated proteins is shown. 26 Chapter I Introduction As a GTP-binding protein, Rap IB functions as a GTP/GDP-regulated switch that cycles between inactive GDP- and active GTP-bound states. RaplB is associated with the cytoskeleton during cell activation, linked to phospholipase C activation, and can regulate C a 2 + ATPase function (Corvazier et al., 1992; Lacabaratz-Porret et al., 1998; Lazarowski et al., 1990; White et al., 1993). RaplB has also been reported to be phosphorylated by P K A (Siess et al., 1990), cyclic GMP-dependent protein kinase (Miura et al., 1992), and a neuronal calcium/calmodulin-dependent protein kinase (Sahyoun et al., 1991). Since it is clear that P K A is involved in visual cortical plasticity, RaplB might be its downstream effector working as a third messenger to regulate cellular processes. Moreover, activation of Rapl is required for the enhanced neurite outgrowth observed in the Shb-overexpressing PC 12 cells, and transient blockade of the Rapl pathway reduces the NGF-dependent neurite outgrowth in these cells (Lu et al., 2000). This suggests that NGF-dependent Rapl signaling may be of significance for neurite outgrowth under certain conditions. While the involvement of NGF in visual cortical plasticity is well established, we hypothesize that RaplB is also involved in this plasticity. Additionally, since Rap proteins are associated with the regulation of vesicle trafficking and selective membrane fusion (Chavrier et al., 1990; Kelly and Grote, 1993), RaplB may be important for the transport and fusion of synaptic vesicles and for exocytosis and endocytosis which may lead to activity-dependent synaptic formation and /or elimination. The overall experimental objective of this thesis is to examine the cellular and laminar distributions of these four proteins in the cat primary visual cortex during postnatal development using immunocytochemical methods. The involvement of these 27 Chapter I Introduction plasticity candidate proteins in the visual cortical plasticity will be discussed. The remainder of this thesis will describe our findings in detail. Chapter II Materials and Methods Chapter II MATERIALS AND METHODS 2.1 THE CHOICE OF THE CAT AS THE ANIMAL MODEL There are both benefits and problems associated with using the cat as an experimental animal model in visual neuroscience. Historically, the cat has been a popular animal model in visual neuroscience research. It is estimated that more is known about the cat visual cortex than about any other cerebral area, or even any other structure in the brain of any other species (Payne and Peters, 2002). Accordingly, the cat has a well-described postnatal anatomical and physiological development, and a well-defined critical period for ocular dominance plasticity which is limited to the first several months of life and can be manipulated by rearing conditions (Cynader and Mitchell, 1980; Mower et al., 1981, 1985; Sherman and Spear, 1982). The ocular dominance plasticity in cat visual cortex is the best-known and most intensively studied type of visual cortical plasticity. Compared to mice and rats, the organization of the cat visual system closely resembles that of the human: unlike rodents who have two lateral eyes and have very small binocular zone in the visual cortex, the cat has two frontal eyes and well-developed stereoscopic depth vision (Fagiolini et al., 1994; Gordon and Stryker, 1996; LeVay et al., 1978; Shatz and Stryker, 1978). Therefore, the cat is a more relevant animal model for the human visual system than rodents. Additionally, compared to the primates, the cat visual system is fairly immature at birth and hence it is a more convenient system to study early events in visual development (LeVay et al., 1978; Shatz and Stryker, 1978). Also, cats are relatively inexpensive to purchase and to house compared to primates. Finally, since the 29 Chapter II Materials and Methods cat visual cortex develops more rapidly than that of the primate, time dependent studies are much easier to perform on the cat (Blakemore and Van Sluyter, 1974; Hubel and Wiesel, 1970; LeVay et a l , 1978; Shatz and Stryker, 1978). Unfortunately, the cat is not a suitable animal model for molecular biology. Gene replacement or gene knockout experiments are difficult to perform in cats because homologous recombination occurs very rarely in cells of highly complex animals (Alberts et al., 1994). In addition, the cost associated with establishing transgenic cat strains is unreasonably high. Therefore, very little information is available regarding the sequences of cat genes. Thus, there are few antibodies generated against peptides originated from cat. The above statements outline the current dilemma in visual neuroscience. The cat possesses a sophisticated visual system, resembling that of the human, which is well characterized both at the neuroanatomical and neurophysiological levels. However, this system is hard to be manipulated at the molecular level. We chose the cat as our animal model because the focus of our research is to identify the involvement of plasticity candidate proteins in the visual cortical plasticity of highly complex animals. The fact that cat gene sequence information is lacking is not a critical problem since many of the important neural molecules are highly conserved in mammals, and many antibodies produced for use in other animals cross-react with cat proteins due to the high levels of evolutionary conservation. Therefore, the cat is the most suitable animal model for our study. 2.2 M A T E R I A L S 2.2.1 Animals 30 Chapter II Materials and Methods A total of 14 postnatal cats aged from 1-week-old to adult (older than 1-year-old) were used in this study. The normally-reared 3-month-old, 4-month-old, adult cats, and the pregnant cats were obtained either from University of British Columbia Animal Services Center inbreed colonies, commercially purchased from Laka (Montreal, Quebec), University of California (Davis, California), or Harlan Sprague Dawley (Madison, Wisconsin). The pregnant cats were bred until the kittens were born and reached the desired ages (from 1-week to 6-weeks old). The dark-reared 4-month-old cat was obtained from Dr. Donald Mitchell's lab (Dalhousie University). For the list of the number and ages of the cats used in this study please see table 1. Table 1. The list of the cats used in this study. Age Of Cats Number Of Cats 1 -week-old 1 2-week-old 2 4-week-old 2 6-week-old 2 3-month-old 2 4-month-old Normal-Reared 2 Dark-Reared 1 Adult (> 1-year-old) 2 A l l cats except the one 4-month-old dark-reared cat were reared under normal conditions. We chose the cats' ages because: postnatal 1 week is before the eyes are open, postnatal 2 weeks is when eyes are open but the visual cortex has not reached the peak of plasticity, postnatal 4 weeks is when the visual cortex reaches the peak of plasticity, 31 Chapter II Materials and Methods postnatal 6 weeks is when the plasticity starts to decline, postnatal 3 months is when the "critical period" for ocular dominance plasticity is almost over, and adulthood is when the visual cortex is mature and no longer plastic. 2.2.2 Chemicals And Reagents The following items were purchased from Amersham Pharmacia Biotech (Baie d' Urde, Quebec): ECL™ Western blotting analysis system, horseradish peroxidase (HRP) conjugated anti-mouse IgG, HRP conjugated anti-rabbit IgG, and hydrophobic polyvinylidene difluoride (PVDF) membrane. The following items were purchased from Bio-Rad laboratories (Hercules, CA): protein assay, N, N, N1, A^-tetramethylene-ethylenediaine (TEMED), sodium dodecyl sulfate (SDS), ammonium persulfate (APS), and 2-mercaptoethanol. The following items were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): anti-mouse IL-11 polyclonal antibody from rabbit, anti-human IL-11 polyclonal antibody from goat and its blocking peptide, anti-mouse AC-7 polyclonal antibody from goat and its blocking peptide, and anti-human Rap IB polyclonal antibody from goat and its blocking peptide. The following items were purchased from Sigma (St. Louis, MO): biotinylated anti-mouse IgG from sheep, bovine serum album (BSA), 3,3'-diaminobenzidine tetrahydrochloride (DAB), hydrogen peroxide (H2O2), paraformaldehyde (PFA), HRP conjugated anti-goat IgG from rabbit, ethanolamine, Triton X-100, sodium azide, bromophenol blue, aprotinin, and phenylmethylsulfonyl fluoride (PMSF). The following items were obtained from Vector Labs (Burlingame, CA): biotinylated anti-goat IgG from rabbit, biotinylated anti-rabbit IgG from goat, vectastain avidin-32 Chapter II Materials and Methods biotin-complex (ABC) kit, normal goat serum, normal horse serum, and HRP conjugated anti-goat IgG from horse. Anti-mouse O B C A M monoclonal antibody was obtained from Dr. Jun-ichi Sawada's lab (Tokyo, Japan). Pentobarbital (Euthanyl) was purchased from M T C Pharmaceuticals (Cambridge, ON). Nonidet P-40 (NP-40) was purchased from Calibiochem-Novabiochem Corporation (La Jolle, CA). BenchMark™ Protein Ladder was purchased from GIBCO B R L (Grand Island, N Y ) . Kodak BioMax M R film was purchased from Eastman Kodak Company (Rochester, NY) . Developer and replenisher, fixer and replenisher were purchased from Kodak Canada Inc. (Toronto, ON). 2.2.3 Equipment The minigel apparatus for protein electrophoresis was Mini-Protean II system from Bio-Rad Laboratories (Mississauga, ON). The electroblotting apparatus for protein transferring was Trans-Blot SD semi-dry transfer cell also from Bio-Rad Laboratories (Mississauga, ON). The vibratome for slicing was the DSK microslicer from Dosaka E M CO., LTD. (Japan). The spectrophotometer U-2000 was manufactured by Hitachi, Japan. The sonicator and vortex were purchased from Fisher Scientific (Nepean, ON). The orbital rocker and centrifuge were obtained from V W R (Bridgeport, NJ). The slides were studied using a Nikon microscope, and photographed with a Nikon digital camera E-990. 2.3 M E T H O D S 2.3.1 Solution Preparation • Phosphate Buffered Saline (PBS) (0.1 M , pH 7.4): 2.89 g sodium dihydrogen orthophosphate (NaHiPO^^O) (21 mM), 11.5 g disodium hydrogen orthophosphate 33 Chapter II Materials and Methods anhydrous (NaiHPC^) (80 mM), and 9 g sodium chloride (0.9% (w/v)) were dissolved in dh^O to make 1 liter PBS. • Phosphate Buffered Saline (pH 7.5) with 0.1 % Tween-20 (PBS-T) contains: 2.76 g sodium dihydrogen orthophosphate (NaHiPO^HiO) (20 mM), 11.5 g disodium hydrogen orthophosphate anhydrous (Na2HP04) (80 mM), 5.85 g sodium chloride (100 mM), and 1ml Tween-20 detergent (0.1% (v/v)) in 1 liter PBS-T. • Triple Detergent contains: 50mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 0.02% sodium azide, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 100 ug/ml PMSF, and 1 pg/ml aprotinin. PMSF and aprotinin were added immediately before use. Before PMSF and aprotinin were added, the solution was stored at room temperature. • 30% (w/v) Acrylamide Mix: dissolve 29.2 g acrylamide (29.2% (w/v)) and 0.8 g N, N'-methylene-bisacrylamide (0.8% (w/v)) in dH 20, put at 37°C for 5-10 minutes to improve dissolution, filter and bring to 100 ml with dFbO. Store at 4°C in a dark container for at least 30 days. • 10% (w/v) SDS: dissolve 10 g SDS in dH 20 with gentle stirring and bring to 100ml with dH20, store at room temperature. • Stacking Gel: 2 ml stacking gel contains 1.4 ml dH.20, 0.33 ml 30% Acrylamide Mix, 0.25 ml 1.0 M Tris (pH 6.8), 0.02 ml 10% SDS, 0.02 ml 10% APS, and 0.002 ml TEMED. 10% APS and TEMED were added just before use. • Separating Gel (8%): 5 ml 8% separating gel contains 2.3 ml dH/jO, 1.3 ml 30% Acrylamide mix, 1.3 ml 1.5M Tris (pH 8.8), 0.05 ml 10% SDS, 0.05 ml 10% APS, and 0.003 ml TEMED. 10% APS and TEMED were added just before use. 34 Chapter II Materials and Methods • Separating Gel (10%): 5 ml 10% separating gel contains 2.0 ml dH 2 0 , 1.7 ml 30% Acrylamide mix, 1.3 ml 1.5M Tris (pH 8.8), 0.05 ml 10% SDS, 0.05 ml 10% APS, and 0.002 ml TEMED. 10% APS and TEMED were added just before use. • Electrophoresis Buffer: 9 g Tris-base (15 g/1), 43.2 g glycine (72 g/1), and 3 g SDS (5 g/1) were dissolved in dHiO to make 600 ml 5x stock solution which was stored at 4°C. • Sample Buffer: mix 0.6 ml 1 M Tris-HCl (pH 6.8) (60 mM), 5 ml 50% glycerol (25%), 2 ml 10% SDS (2%), 0.5 ml 2-mercaptoethanol (14.4 mM), 1 ml 1% bromophenol blue (0.1%), and 0.9 ml dFbO to make 10 ml 5x sample buffer. It is stable for weeks in the 4°C refrigerator or for months at -20°C. • Transfer Buffer: dissolve 5.82 g Tris (48 mM), 2.93 g glycine (39 mM), and 0.0375 g SDS (0.0375%) in dH 2 0 , add 200 ml of methanol (20% (v/v)), adjust the volume to 1 liter with dF^O, and store at 4°C. Do not add acid or base to adjust pH. 2.3.2 Animal Preparation A l l cats were euthanized with an intraperitoneal injection of pentobarbital (Euthanyl, 150 mg/kg body weight). For immunocytochemical staining use, they were perfused with PBS, followed by a fixative solution containing 4% paraformaldehyde. Subsequently, the brains were removed and preserved in the fixative at 4°C before use. For Western blot analysis, one 4-week-old kitten and one adult cat were perfused briefly with ice-cold PBS, and their primary visual cortices were isolated on dry ice. The frozen brains were stored at -80°C until use. 2.3.3 Immunocytochemical Staining 35 Chapter II Materials and Methods The primary cortices were sectioned in the frontal plane with a vibratome. The section thickness of one-week-old kitten was 50 pm while all sections from other ages were 40 pm thick. During the procedure, the tissue was submerged in PBS and not allowed to dry. The procedures for O B C A M staining are as follows: 1) The sections were washed in PBS for 15 minutes four times at room temperature. 2) Endogenous peroxidase activity was blocked by incubating the sections in PBS with 3% (v/v) H2O2 and 10% (v/v) methanol for 20 minutes, followed by four washes with PBS for 15 minutes each. 3) To block non-specific binding the sections were incubated with normal goat serum for 1 hour at a concentration of 20% (v/v) in PBS with 3% (w/v) B S A and 1% sodium azide at room temperature. 4) The sections were then incubated with primary anti-OBCAM antibody (mouse monoclonal anti-OBCAM antibody) diluted 1:2500 in PBS with 3% B S A and 1% sodium azide at 4°C overnight. 5) After removing the primary antibody and rinsing with PBS for 15 minutes four times, the sections were incubated with biotinylated goat anti-mouse IgG (1:200) for 1 hour at room temperature. 6) Following four rinses 15 minutes each with PBS, the sections were finally incubated for 1 hour at room temperature in the solution which was 2 drops of solution A and 2 drops of solution B from A B C kit in 5 ml PBS and was made at least 30 minutes before use. 7) After removing the solution and four washes with PBS, the sections were placed in D A B solution (0.7 mg D A B per ml PBS, 5 ml per well) for 5 minutes, and incubated for another 10 minutes after 0.01% H2O2 (final concentration) was added. 8) After a final four rinses with PBS, the sections were mounted on the slides and dried overnight. 9) The slides were put in dF^O for 1 minute for rehydration the next day, and then dehydrated in 36 Chapter II Materials and Methods increasing graded ethanol solutions, transferred to xylene for 20 minutes, and coverslipped with permount. A l l the wash and incubation procedures were with shaking. For IL-11 immunostaining, after four washes with PBS the sections were incubated in PBS with 1M ethanolamine (pH 7.4) and 1% (v/v) Triton X-100 for 1 hour at room temperature to reduce the non-specific staining. The following procedures were the same as above except 0.5% Triton X-100 was put in primary antibody solution; and both primary anti-IL-11 antibodies were diluted to 1:100. For the rabbit polyclonal antibody, 10% normal goat serum was used instead of 20% normal goat serum to block non-specific bindings, and the secondary antibody was biotinylated anti-rabbit IgG. For the goat polyclonal antibody, 10% normal horse serum was used instead, and the secondary antibody was biotinylated anti-goat IgG. For AC-7 and Rap IB immunostaining, the primary antibody concentrations were 1:600. The secondary antibody (biotinlated anti-goat antibody) was anti-goat IgG. 0.5% Triton X-100 was added to the first wash step and the primary antibody solution. Other procedures were the same as for O B C A M staining. For IL-11, AC-7, and Rap IB, two types of negative controls were performed. One followed the same procedures and only replaced the primary antibody with PBS. The other one also followed the same procedures and put blocking peptide into the primary antibody solution. The concentration of blocking peptide was 5 times that of the concentration of the primary antibody. The primary antibodies and their corresponding blocking peptides were combined at 4°C overnight with shaking in order to fully neutralize the primary antibody. Since the primary anti-OBCAM was a monoclonal 37 Chapter II Materials and Methods antibody, only one type negative control was performed, which replaced the primary antibody with PBS. For each antibody, sections from different-aged cats were performed simultaneously. For each protein examined, all sections were treated under the same conditions (e.g. incubation times including primary antibody, secondary antibody, A B C , and D A B incubation times). 2.3.4 Western Blot A. Preparation Of Protein Samples Ten volumes of ice-cold triple detergent were added to one unit per weight of frozen brain tissue (10 ml triple detergent per gram tissue). Triple detergent was cooled on ice and the protease inhibitors PMSF and aprotinin were added immediately before use. The brain tissue was teased apart using a pair of forceps and the solution was incubated on ice for 30 minutes. Every ten minutes the solution was pipetted several times to improve solublization of the tissue. High molecular weight chromosomal D N A was sheared by sonication for 1 minute at maximum speed. The sonication was performed on ice. The solution was then centrifuged at 15,000 g for 5 minutes at 4°C. The supernatant was aliquoted in order to avoid repeated freeze-thaw cycles and placed into a -20°C freezer for short-term storage or -80°C freezer for long-term storage. B. SDS-Polyacrylamide Gel Electrophoresis The procedures for SDS-polyacrylamide gel electrophoresis include pouring a gel, preparing and loading samples, running the gel, and transferring to the PVDF membrane. The procedures for pouring a gel are as follows: 1) Thoroughly wash and dry both gel plates and spacers. Make sure their bottoms were against a flat surface before tightening 38 Chapter II Materials and Methods clamp assembly. 2) Make separating gel. Add 10% APS and T E M E D just before use as mentioned above. 3) Carefully introduce the gel into gel sandwich using a pipette. Make sure there were no air bubbles in the gel. 4) Gently add about l-5mm of water on top of the separating gel solution. 5) Wait for about 40 minutes at room temperature to allow the separating gel to polymerize. 6) Make stacking gel. Also add 10% APS and T E M E D just before use. 7) Pour off the water covering the separating gel and pipet the stacking gel solution onto the separating gel until solution reaches top of front plate. 8) Carefully insert comb into gel sandwich until the bottom of the teeth reach the top of the front plate. Make sure no bubbles were trapped on ends of the teeth. 9) Wait for about 30 minutes to allow the stacking gel to polymerize. 10) Remove comb carefully. Make sure not to tear the well ears. 11) Place the gel into electrophoresis chamber. 12) Add electrophoresis buffer to inner and outer reservoir. Make sure both the top and the bottom of the gel were immersed in the buffer. For O B C A M and AC-7, 8% separating gel was used. For IL-11 and RaplB, 10% separating gel was used. In order to load the same amount of protein in each well, the protein samples were added to 1:7 diluted protein assay (5 pi protein sample was added to 1 ml diluted protein assay), and then the relative protein concentrations were measured from absorbance at 595 nm by spectrophotometer. After combining the protein sample with sample buffer, they were boiled for 5 minutes to denature the protein. Following spin down protein solution for 30 seconds, each well was introduced about 30 pi of the protein sample according to the relative concentration. 10 pi protein molecular weight ladder was loaded into one well. 39 Chapter II Materials and Methods The gel was run under 200 V for about 40 minutes until the dye front migrated to about 5mm from the bottom of the gel. The gel then was washed with transfer buffer for 15 minutes. The dry PVDF membrane was soaked in 100% methanol for 5 seconds, washed in d H 2 0 for 5 minutes, and then incubated in transfer buffer for 10 minutes with shaking. At the same time, two thick filter papers were also soaked in transfer buffer. Then a blotting membrane/filter paper sandwich was assembled by putting one filter paper, PVDF membrane, gel, and the other filter paper in the order from bottom to top. Make sure there are no air bubbles inside which would affect the transfer. Proteins were electrophoretically transferred to the membrane under 20 V and 135 mA for about 30-45 minutes according to their molecular weights. C. Immunoblotting The PVDF membrane with protein was placed into a heat sealable plastic bag containing 5% B S A in PBS-T and incubated at 4°C overnight with shaking. For immunoblotting, PBS-T was used for all washes, rinses, and to dilute solutions. The next day, the membrane was quickly rinsed twice, washed once for 15 minutes and twice more for 5 minutes each at room temperature with shaking. After the washes, the membrane was incubated in a heat sealed bag with the diluted primary antibody (1:10,000 dilution for O B C A M , 1:400 dilution for IL-11, AC-7, and RaplB) for 1 hour at room temperature with shaking. Unbound primary antibody was washed off by two quick rinses, followed by a 15-minute wash and then two more washes for 5 minutes each at room temperature with shaking. The membrane then was incubated in diluted HRP conjugated secondary antibody (1:1000 for HRP conjugated anti-mouse and anti-rabbit antibody, 1:20,000 for HRP conjugated anti-goat antibody from Sigma, 1:12,000 for HRP conjugated anti-goat 40 Chapter II Materials and Methods antibody from Vector Labs) for 1 hour at room temperature with shaking. Unbound secondary antibody was washed off by two quick rinses, followed by a 15-minute wash and then two more washes for 5 minutes each at room temperature with shaking. During the washes, the detection solution (consisting of equal volumes of solution 1 and solution 2 from Amersham ECL™ Western blotting analysis system) was prepared. The excess liquid from the washed membrane was drained onto a paper towel and the membrane was placed protein-side up on a piece of Saran wrap. The remainders of the procedures were performed in the dark room. The membrane was then covered with the detection solution for exactly 1 minute. The membrane was removed from the detection solution and wrapped in a fresh piece of Saran wrap. The membrane was put in a cassette with the protein side up and a film (Kodak BioMax M R film) was placed on the membrane. The exposure times for the film varied from 30 seconds to 5 minutes. The E C L signal was detected by developing the film. 2.3.5 Data Analysis Sections adjacent to those used for immunocytochemical staining were stained by the Nissl method with cresyl violet. For classification of cortical areas and laminae, we relied on the criteria of Otsuka and Hassler (1962). Considering the laminar thickness is not the same within the cortex, and the thickness of the same layer changes during development, the immunopositive cell numbers in each layer cannot be compared (the difference could simply be the difference between the laminar thicknesses). Thus, the goal of our quantitative analysis was to determine relative changes in the laminar distribution of the plasticity candidate protein-positive cell densities across ages. Because antibodies do not penetrate evenly through the 40 urn 41 Chapter II Materials and Methods sections and preferentially stain structures close to the cut surfaces, absolute numbers of cells could not be determined. However, relative differences across the primary visual cortical laminae between ages could be determined. Two 100 pm wide strips from each animal's primary visual cortex were randomly selected. Photomicrographs were taken by a digital camera at a magnification of 40x. They were visualized using Photoshop Software at a final magnification of 80x. According to the scale bars taken under the same conditions, 100 pm wide strips were defined. And each visual cortical layer was distinguished according to the Nissl stained sections. The immunopositive cells were counted i f they were positioned within the defined layer or intersected by its inclusion edges (e.g., the top and right edges). Data were calculated to the relative cell numbers per 100 x 100 pm. In the 1-week old kitten, sampling was done in layer I, the compact zone, the cortical plate, layer V , and layer Vl/subplate. In older cats, where laminae were identifiable, sampling was done in layers I, II+III, IV, V , and VI. We used a 2 x 5 (repeat measures x laminae) analysis of variance (ANOVA) for the intra-animal comparison at each age, and a 2 x 5 (animal numbers x laminae) A N O V A for the inter-animal comparison at each age (except 1-week-old and dark-reared 4-month-old cats). The statistical significance of differences among the means for relative immunopositive cell densities between ages was determined with a 6 x 5 (ages x laminae) A N O V A . Following A N O V A , differences between pairs of group means were tested using Fisher's PLSD post hoc tests. 42 Chapter III Results CHAPTER III RESULTS 3.1 OPIOID-BINDING C E L L ADHESION M O L E C U L E (OBCAM) 3.1.1 Western Blot Analysis Of O B C A M The O B C A M primary antibody was obtained from Dr. Jun-ichi Sawada's lab (Tokyo, Japan). This antibody is a monoclonal antibody against a synthetic O B C A M peptide. It was reported that the antibody could specifically recognize both 58- and 51-kDa OBCAMs in the brains of the cow, bovine, rat, mouse, guinea pig, and rabbit O B C A M protein (Hachisuka et al., 1996). And it has been suggested that the difference in molecular weight between 58- and 51-kDa OBCAMs is due to the degree of glycosylation (Hachisuka et al., 1996). Figure 3 shows the western blot analysis performed in the visual cortex of 4-week-old cat with this anti-OBCAM monoclonal antibody. Two bands of roughly 51- and 58-kDa were detected confirming the fact that the antibody from Dr. Jun-ichi Sawada's lab reacts with the cat O B C A M protein. Also, since mainly 51- and 58-kDa bands are present in this Western blot analysis, which is in good agreement with the data from other species, this antibody appears to specifically recognize the O B C A M protein in the cat. Therefore, this antibody is suitable for performing immunocytochemistry in the cat visual cortex to localize the distribution of the O B C A M protein. 3.1.2 Localization Of O B C A M In Cat Primary Visual Cortex 43 Chapter III Results 58-kDa 51-kDa 4 W Figure 3. Western blot analysis of OBCAM protein expression in 4-week-old cat visual cortex. Western blot analysis performed on 4-week-old cat visual cortex using the O B C A M antibody obtained from Dr. Jun-ichi Sawada's lab (Tokyo, Japan). This antibody specifically recognizes mainly two bands of around 51- and 58-kDa in visual cortex protein preparation obtained from 4-week-old adult cat. Chapter III Results The monoclonal O B C A M antibody from Dr. Jun-ichi Sawada's lab, whose specificity in the visual cortex was established by western blot analysis, was used to localize the O B C A M protein expression in the developing cat primary visual cortex. To provide a semi-quantitative comparison of the laminar distribution of O B C A M immunopositive neurons during postnatal development, the relative cell density (cells/ 0.01 mm2) was determined as described in Chapter II. We compared the densities determined from each animal (intra-animal) and from the two animals (inter-animal) at each age except in the 1-week old cat. It showed no significant difference (p > 0.05). In neonatal cat primary visual cortex: To analyze the immunoreactivity in neonatal (1-week-old) kitten primary visual cortex, it is necessary to account for the immature lamination pattern at this age. As shown in the upper left panel of Figure 4, we followed the designations of Luskin and Shatz (1985a, b). In neonates, layers V and VI are identifiable and a significant number of cells in layer VI are remnants of the embryonic subplate (SP). The cortical plate (CP) consists mainly of cells destined to become the adult layer IV. Most of the cells that will form layers II and III are densely packed in the compact zone (CZ). Layer I, which corresponds to the embryonic marginal zone, is identifiable. The upper right panel of figure 4 shows O B C A M immunoreactivity in neonatal kitten primary visual cortex. Although there were O B C A M positive neurons in all layers of neonatal visual cortex, few stained O B C A M cells were distributed in layer I compared to other layers (Figure 4B). 45 Chapter III Results Figure 4. Laminar distribution of OBCAM protein in neonatal cat primary visual cortex. A . Immunocytochemical localization of O B C A M in neonatal (1-week-old) cat primary visual cortex. Photographs of 1-week-old cat primary visual cortex showing a cresyl violet stained section and a section processed immunocytochemically for O B C A M . Cortical layers were designated according to Luskin and Shatz (1985a, b). Scale bar = 0.2 mm. B. Laminar distributions of O B C A M immunopositive neurons in 1-week-old cat primary visual cortex. The relative immunopositive neuron densities (cells/0.01 mm2) were determined as mentioned in chapter II. Each bar represents the mean density of each layer ± S E M of two sections from one animal. ) 46 Chapter III Results A. Immunocytochemical localization of OBCAM in neonatal cat primary visual cortex V VI/SP 1 '.»:<- \ C P VI /SP WM WM Nissl Staining Immunocytochemical Staining < £ u g pa v o o > p ••C £ — <U OS Z B. Laminar Distribution of OBCAM in Neonatal Cat Visual Cortex 20 15 10 0 layer I CZ CP layer V layer VI/SP 47 Chapter III Results Higher magnification photomicrographs (Figure 5) showed that more neurites were labeled compared to older animals. There were both pyramidal and non-pyramidal neurons which were labeled. In 4-week-old cat primary visual cortex: Figure 6A shows the lower magnification photomicrographs of immunostaining in 4-week-old cat primary visual cortex by the antibody to O B C A M . The O B C A M positive neurons were distributed uniformly throughout most visual cortical layers, and they were rarely located in layer I (Figure 6B). As shown in Figure 7, the higher magnification photomicrographs of O B C A M immunocytochemical staining in visual cortical layers II/III, IV, V , and VI, both pyramidal and non-pyramidal neurons were labeled, both cell bodies and neurites were labeled, and both neurons and neuropil were labeled. In adult cat primary visual cortex: Figure 8A shows photomicrographs of the O B C A M immunostaining in adult cat primary visual cortex. The O B C A M immunoreactivity showed a little labeling of the extracellular matrix. The O B C A M positive neurons were distributed throughout all visual cortical layers. The O B C A M immunopositive neuron density was low in layer I, while it was slightly higher in layers II/III, IV, and VI (Figure 8B). Higher magnification photomicrographs revealed details of the O B C A M immunoreactivity in adult cat primary visual cortex across cortical layers as shown in Figure 9. Only faint cell body staining and faint neuropil staining were seen. Almost no neurite profiles were labeled. 3.1.3 Developmental Profile Of OBCAM 48 Chapter III Results compact rone cortical plate layer V layer Vl/subplate Figure 5. Immunoreactivity localization of OBCAM protein in neonatal cat primary visual cortex. Higher magnification photomicrographs of immunostaining in neonatal cat primary visual cortex by the antibody to O B C A M in CZ, CP, layer V , and layer VI/SP. More neurites were labeled. There were both pyramidal (arrow) and non-pyramidal (arrowhead) neurons. Scale bar = 30 um. 49 Chapter III Results Figure 6. Laminar distribution of OBCAM protein in 4-week-old cat primary visual cortex. A . Immunocytochemical localization of O B C A M in 4-week-old cat primary visual cortex. The upper left panel is Nissl staining. The upper right panel is the immunocytochemical staining. Visual cortical layers were determined from the adjacent Nissl stained section. The O B C A M antibody rarely labeled cell bodies in layer I. Scale bar = 0.2 mm. B. Laminar distributions of O B C A M immunopositive neurons in 4-week-old cat primary visual cortex. The relative immunopositive neuron densities (cells/0.01 mm2) were determined as described in Chapter II. Each bar represents the mean density of each layer ± S E M of four sections from two animals. 50 Chapter III Results A . Immunocytochemical localization of O B C A M in 4-week-old cat primary visual cortex W M W M Nissl Staining Immunocytochemical Staining B. Laminar Distribution of O B C A M in 4-week-old Cat Visual Cortex • 5 20 n U 1 15 -09 « I 1 , T , ^ © « i o -> o i 9 5-— o oi z o I r ^ n , I L_^J L_^J L ^ J L layer I layer II/III layer IV layer V layer V I 51 Chapter III Results layer V layer VI Figure 7. Immunocytochemical localization of OBCAM protein in 4-week-old cat primary visual cortex. Higher magnification photomicrographs of immunostaining in 4-week-old cat visual cortex by the antibody to O B C A M in layer II/III, layer IV, layer V , and layer VI. Both pyramidal (arrow) and non-pyramidal (arrowhead) neurons were labeled. Both cell bodies and neurites were labeled. Both neurons and neuropil were labeled. Scale bar = 30 pm. 52 Chapter III Results Figure 8. Laminar distribution of OBCAM protein in adult cat primary visual cortex. A . Immunocytochemical localization of O B C A M in adult cat primary visual cortex. The left panel is Nissl staining. The right panel is the immunocytochemical staining. Visual cortical layers were determined from the adjacent Nissl stained section. The O B C A M antibody rarely labeled cell bodies in layer I. Scale bar = 0.2 mm. B. Laminar distributions of O B C A M immunopositive neurons in adult cat primary visual cortex. The relative immunopositive neuron densities (cells/0.01 mm2) were determined as described in chapter II. Each bar represents the mean density of each layer ± S E M of four sections from two animals. 53 Chapter III Results A . Immunocytochemical localization of O B C A M in adult cat primary visual cortex Nissl Staining Immunocytochemical Staining u g sa S o o OS c o 1-3 Z B. Laminar Distribution of O B C A M in Adult Cat Visual Cortex 10 -| 8 -6 -4 -2 0 I T—' 1 —i—' '—r layer I layer II/III layer IV layer V layer VI 54 Chapter III Results layer II/III layer IV layer V layer VI Negative Control Figure 9. Immunocytochemical localization of O B C A M protein in adult cat primary visual cortex. Higher magnification photomicrographs of immunostaining in adult visual cortex by the antibody to O B C A M in layer II/III, layer IV, layer V , and layer VI. The lower right photomicrograph is the negative control (without primary anti-OBCAM antibody). Only faintly labeled neurons and nerupil were seen. Almost no neurite was labeled. Scale bar = 30 pm. 55 Chapter III Results Figure 10 shows the O B C A M protein expression in the postnatal developing cat primary visual cortex. The O B C A M protein was highly expressed during early postnatal development. The strongest labeling of O B C A M was found between 2 and 4 weeks of age, while it was at a minimal level in adult primary visual cortex. The relative total O B C A M immunopositive neuron density was high in younger animals' visual cortices and low in those of older animals (Figure 11). O B C A M neuron density began to decline very quickly after 4 weeks of age until 6 weeks of age, when the density was similar to the adult. Although O B C A M positive neurons were present in all visual cortical layers at all ages, there were laminar changes in their relative densities across postnatal development. Figure 12 shows the laminar difference and the overall O B C A M immunopositive neuron density difference between the cat primary visual cortices of different ages. The relative O B C A M neuron densities in layers II/III, layer IV, layer V , and layer VI of 2-week-old and 4-week-old cats were significantly higher than in corresponding layers of adult cats (p < 0.001 for layers II/III, layer IV, and layer V;p< 0.05 for layer VI). The difference in layer I was not significant. However, since this layer, which is relatively cell sparse, shows a very low relative O B C A M immunopositive neuron density, it may obscure the changes. Taken together, the overall densities of O B C A M immunopositive neurons in 2-week-old and 4-week-old primary visual cortices were significantly higher than the density in adult primary visual cortex (p < 0.001). 3.1.4 Dark Rearing Effects On O B C A M Protein Expression Raising animals in the dark leads to a diminution, but not complete absence, of optic nerve activity and causes changes in most proteins involved in visual cortical plasticity. 56 Chapter III Results 6-week-old 3-month-old Normal Adult Figure 10. Expression of OBCAM protein in postnatal developing cat visual cortex — A. Immunocytochemical staining. Lower magnification of photomicrographs of O B C A M immunocytochemical staining in the postnatal developing cat visual cortex including 1-week-old, 2-week-old, 4-week-old, 6-week-old, 3-month-old, and adult visual cortex. The O B C A M protein was highly expressed during early postnatal development. The strongest labeling of O B C A M was found between 2 and 4 weeks of age, while it was at a minimal level in adult primary visual cortex. Scale bar = 0.2 mm. 57 Chapter III Results 20 Developmental Changes in O B C A M Immunopositive Neuron Densities in Cat Primary Visual Cortex 10 15 5 0 1W 2W 4W 6W 3 M NA Age Figure 11. Expression of OBCAM protein in postnatal developing cat visual cortex — B. Semi-quantitative study. The relative O B C A M neuron densities (cells/0.01 mm ) were determined as described in Chapter II. The overall O B C A M immunopositive neuron density was the highest in the visual cortex of cat between 2 and 4 weeks of age, and the lowest in that of adult cat. n = 2 for each age (except at 1-week old n = 1). For each age, data are shown as mean ± S E M . The X-axis is the animal ages (1-week-old (1W), 2-week-old (2W), 4-week-old (4W), 6-week-old (6W), 3-month-old (3M), and normal adult (NA)). Raising animals in the dark also extends the critical period during which abnormal visual experience can cause anatomical and physiological changes in cortical connectivity (Cynader and Mitchell, 1980; Mower et al., 1985, Stryker and Harris, 1988). To examine whether dark rearing regulates the O B C A M protein expression, immunocytochemical studies were performed on the primary visual cortices of normal and dark-reared cats at the same age (4-month-old). As shown in Figure 13, in both rearing conditions, O B C A M immunopositive neurons were distributed throughout all visual cortical layers. There were more OBCAM-labeled cells seen in the primary visual cortex of the dark-reared cat than in that of normal animals. The O B C A M immunopositive neuron densities 58 Chapter III Results Developmental Changes in O B C A M Positive Neuron Densities Across Visual Cortical Layers s o -3 Z u S m « o a > _« "33 layer I layer II/III layer IV layer V layer VI Total Figure 12. Developmental changes in OBCAM positive neuron densities across cat visual cortical layers. The relative OBCAM immunopositive neuron densities (cells/0.01 mm2) were determined as described in Chapter II. Each bar represents the mean ± SEM of four sections from two animals. The OBCAM neuron densities in layer II/III, layer IV, layer V, layer VI, and overall cortical layers of 2 and 4-week-old cats visual cortices were significantly greater compared to those of normal adult cats. (*p < 0.05, *** p < 0.001). 2 • • (cells/0.01 mm ) of each layer of the primary visual cortex of normal and the dark-reared cat were obtained as described in Chapter II in order to provide a semi-quantitative comparison of the laminar distribution of OBCAM positive neuron densities under two rearing conditions. Despite no statistically significant difference in the cell density of each layer (p < 0.001 for layers II/III, p < 0.005 for layer IV and layer VI, p > 0.05 for layer I and layer V), the total OBCAM-labeled neuron densities were significantly different between the primary visual cortices of dark-reared and normal cats (p < 0.001, Figure 13B). 59 Chapter III Results Figure 13. Dark rearing effects on OBCAM expression in cat primary visual cortex at 4 months of age. A. Photomicrographs showing the expression of O B C A M in the visual cortex of normal and dark-reared cat both at 4 months of age. In both rearing conditions, OBCAM-labeled cells were distributed throughout all layers of visual cortical layers, and rarely located in layer I. More O B C A M positive neurons were seen in the primary visual cortex of the dark-reared animal. Scale bar = 0.2 mm. B. The relative O B C A M immunopositive neuron densities (cells/0.01 mm ) between the two cats reared in different conditions in each layer and all cortical layers were compared. Although there was no statistically significant difference between the density in each layer, the relative overall densities were significantly different (**p< 0.005, ***p< 0.001). 60 Chapter III Results B. Dark Rearing Effect on O B C A M Expression Across Visual Cortical Layers il/lll Chapter III Results The question arises as to whether the higher O B C A M immunopositive neuron counts in dark-reared cats reflect a true increase in O B C A M cells or an increase in total cellular packing density, since cellular packing density is higher in dark-reared than normal cats at 4-5 months of age (Mower et al., 1988). Mower and his colleague determined the total cellular density using similar methods as described in Chapter II. In dark-reared cats, the average cellular density across all cortical layers (cells/0.01 mm ) is 29.5 ±2.1 and 20.7 + 1.4 in normals. In order to take the difference into account, the density of O B C A M neurons of both dark-reared and normal cats was corrected by the ratio of the total cellular density. The corrected average O B C A M neuron density in dark-reared cats was 34.57% ± 0.55% compared to 28.61% ± 0.71% in normals. The difference was still statistically significant {p < 0.001). This indicated that dark rearing slowed the decrease of O B C A M expression in the cat primary visual cortex. 3.2 INTERLEUKINE 11 (IL-11) 3.2.1 Selection Of The IL-11 Antibodies Two types of IL-11 antibody from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) were used for immunocytochemical and western blot analysis. They are both suitable for western blotting, immunoprecipitation, immunocytochemistry, and ELISA according to the manufacturer. The differences between each type are that: one is a polyclonal IgG from rabbit, which is against amino acids 31-199 mapping to an internal region of IL-11 of human origin, and the other one is a polyclonal IgG from goat, which is against amino acids near the carboxy terminus of IL-11 of human origin. The first one is specific for IL-11 of mouse, rat, and human origin, while the second one is specific for IL-11 of human 62 Chapter III Results origin only. Additionally, for the first one, there is no blocking peptide available because the peptide used to generate the antibody is too big while the second one has a blocking peptide. We performed western blot with both antibodies. The western blot result using the first one showed only one band which was about 20-kDa, while the result using the second one showed multibands (Figure 14A). However, the negative control of immunocytochemistry, which used the blocking peptide to neutralize the primary antibody (the second one), showed no labeling at all (Figure 14B). This suggests that the reason for the multibands could be non-specific binding of the secondary antibody, or the primary antibody could recognize more than one protein when the proteins are denatured. Importantly, the immunocytochemical staining using the two antibodies showed a similar pattern of IL-11 expression (Figure 15, 17, 19). The semi-quantitative study results using the two antibodies showed no statistically significant difference between the IL-11 positive cell densities across all visual cortical layers at each age, except a few layers showed a small difference (5 out of 42 comparisons, 0.03 <p< 0.05). Therefore, we concluded that both of these two antibodies are specific to cat IL-11 protein. 3.2.2 Localization Of IL-11 In Cat Primary Visual Cortex The IL-11 antibodies from Santa Cruz Biotechnology Inc., which were characterized in the last two sections, were used to localize IL-11 protein expression in the postnatal developing cat primary visual cortex. The relative IL-11 immunopositive cell numbers were counted from the sections using either antibody. Since the two groups of numbers were not significantly different, they were averaged to determine the IL-11 positive cell densities. 63 Chapter III Results Figure 14. Selection of the IL-11 antibodies. A . Western blot analyses performed in adult cat visual cortex. The upper left panel is the result using the rabbit polyclonal IL-11 antibody, which showed only one band around 20-kDa. The upper right panel is the result using the goat polyclonal IL-11 antibody, which showed multibands. B. Immunocytochemical staining with the goat polyclonal IL-11 antibody. A l l three sections were from the same 4-week-old cat primary visual cortex. The lower left panel (Bl) shows a section which followed the regular immunochemistry procedure, and showed IL-11 immunopositive cells. The lower middle panel (B2) is one negative control with the blocking peptide to the primary antibody. The lower right panel (B3) is the other negative control which was not incubated with the primary antibody but was subjected to all other steps in the immunocytochemistry procedure. The photomicrographs of the two negative controls showed no labeled cell at all. Scale bar = 0.2 mm. 64 Chapter III Results B. Bl B2 Chapter III Results To provide a semi-quantitative comparison of the laminar distribution of IL-11 immunopositive cell density during postnatal development, the cell densities (cells/0.01 mm2) were determined as described in Chapter II. Similar to O B C A M , the intra-animal and inter-animal comparisons at each age showed no significant difference (p > 0.05). In neonatal cat primary visual cortex: As mentioned before, the visual cortical layers in the neonatal (1-week-old) cat were distinguished as layer I, compact zone (CZ), cortical plate (CP), layer V , and layer Vl/subplate (VI/SP) according to Luskin and Shatz (1985a, b). The IL-11 immunopositive cells were mainly located in CZ, CP, layer V , and layer VI/SP (Figure 15). Most IL-11 protein was in the cell bodies (Figure 16). The nucleus-like labeling indicated that there might be IL-11 proteins located in the nuclei of cortical cells, or the nuclei were very small in the neonatal visual cortex. In 4-week-old cat primary visual cortex: As shown in Figure 17, the IL-11 immunopositive cells in the 4-week-old cats' primary visual cortices were also distributed throughout all visual cortical layers. And the IL-11 positive cell densities were similar in all visual cortical layers except layer I, in which the density was very low (Figure 17B). Figure 18 shows photomicrographs of a 4-week-old cat primary visual cortex tissue section, which has been subject to IL-11 immunocytochemical analysis, taken at higher magnification. The IL-11 immunoreacitivity was mainly associated with cell bodies. The different labeling pattern might be there was some nucleus labeling in the superficial layers, or the nuclei were still small in these layers. 66 Chapter III Results Figure 15. Laminar distribution of IL-11 protein in neonatal cat primary visual cortex. A . Immunocytochemical localization of IL-11 in neonatal (1-week-old) cat primary visual cortex. Photomicrographs of 1-week-old cat primary visual cortex showing a cresyl violet stained section (the upper left panel), a section processed immunocytochemically for IL-11 with the polyclonal goat anti-IL-11 antibody (the upper middle panel), and a section processed immunocytochemically for IL-11 with the polyclonal rabbit anti-IL-11 antibody (the upper right panel). The IL-11 immunoreactivity patterns labeled with both antibodies were similar. Cortical layers were designated according to Luskin and Shatz (1985a, b). Scale bar = 0.2 mm. B. Laminar distribution of IL-11 immunopositive cells in 1-week-old cat primary visual cortex. The relative immunopositive cell densities (cells/0.01 mm2) were determined as mentioned in chapter II. The results from the two antibodies were averaged and plotted. Each bar represents the mean density of each ± S E M of four sections from one animal. 67 Chapter III Results A . Immunocytochemical localization of IL-11 in neonatal cat primary visual cortex Nissl Staining Immunocytochemical Staining Immunocytochemical Staining (goat anti-IL -11 antibody) (rabbit anti-IL -11 antibody) —1 Q — _ & I © B. Laminar Distribution of IL-11 in Neonatal Cat Primary Visual Cortex 25 n 20 15 -10 -5 0 layer I CZ CP layer V layer VI/SP 6 8 Chapter III Results compact zone cortical plate layer V layer VI/SP Figure 16. Immunoreactivity localization of IL-11 protein in neonatal cat primary visual cortex. Higher magnification photomicrographs of immunostaining in neonatal cat primary visual cortex by the goat antibody to IL-11 in CZ, CP, layer V , and layer VI/SP. There were nucleus-like labeling. Scale bar = 30 fim. 69 Chapter III Results Figure 17. Laminar distribution of IL-11 protein in 4-week-old cat primary visual cortex. A . Immunocytochemical localization of IL-11 in 4-week-old cat primary visual cortex. The upper left panel is Nissl staining. The upper middle panel is the immunocytochemical staining using the goat polyclonal IL-11 antibody. The upper right panel is the immunocytochemical staining using the rabbit polyclonal IL-11 antibody. The IL-11 immunoreactivity patterns labeled with the two antibodies were very similar. Visual cortical layers were determined from the adjacent Nissl stained sections. Scale bar = 0.2 mm. B. Laminar distributions of IL-11 immunopositive cells in 4-week-old cat primary visual cortex. The relative immunopositive neuron densities (cells/0.01 mm2) were determined as described in chapter II. The results from the two antibodies were averaged and plotted. Each bar represents the mean density of each layer ± S E M of eight sections from two animals. 70 Chapter III Results A . Immunocytochemical localization of IL-11 in 4-week-old cat primary visual cortex Nissl Staining Immunocytochemical Staining Immunocytochemical Staining (goat anti-IL-11 antibody) (rabbit ant i - IL -11 antibody) B. Laminar Distribution of IL-11 in 4-week-old Cat Primary Visual Cortex > 09 o a. -NN ii > "•C OS en C ii Q a U 10 8 6 -4 -2 -0 1 T layer I layer II/III layer IV layer V - i — — i layer VI 71 Chapter III Results layer 11/111 layer IV layer V layer VI Figure 18. Immunoreactivity localization of IL-11 protein in 4-week-old cat primary visual cortex. Higher magnification photomicrographs of immunostaining in 4-week-old cat primary visual cortex by the goat antibody to IL-11 in layers II/III, layer IV, layer V , and layer VI. The IL-11 immunoreactivity was mainly associated with cell somas. Scale bar = 30 ( A m . 72 Chapter III Results In adult cat primary visual cortex: Figure 19A shows the immunocytochemical staining of the adult cat primary visual cortex using IL-11 antibody. IL-11 immunopositive cells were seen at all visual cortical layers, while they were mainly located in layers II/III, layer IV, layer V, and layer VI (Figure 19B). Few cells were labeled in layer I. Higher magnification photomicrographs in Figure 20 show labels of cell bodies and neuropils. No pyramidal neuron was labeled by the antibody. The IL-11 immunoreactivity was mainly associated with cell bodies. 3.2.3 Developmental Profile Of IL-11 As it is shown in Figure 21, IL-11 was highly expressed during early postnatal development. The strongest labeling of IL-11 was found between 1 and 2 weeks of age, while it was at a minimal level in adult primary visual cortex. The IL-11 immunopositive cell densities in layer I were low at all ages. There was possible labeling pattern change with age. While most IL-11 immunoreactivity was nucleus-like in the younger animals, IL-11 immunoreactivity was associated with cell somas in the older animals. The overall IL-11 immunopositive cell density was the highest in cat visual cortex between 1 and 2 weeks of age and the lowest in that of the adult cat (Figure 22). IL-11 positive cell density began to decline very quickly after 2 weeks of age till 4 weeks of age, when the number was just slightly higher than that of the adult. While IL-11 positive cells were present in all visual cortical layers at all ages, there were laminar changes in their relative densities across postnatal development. Figure 23 shows the laminar difference and the total IL-11 immunopositive cell density difference between the cat primary visual cortices of different ages. The differences between the visual cortices of 2-73 Chapter III Results Figure 19. Laminar distribution of IL-11 protein in adult cat primary visual cortex. A . Photomicrographs of adult cat visual cortex showing a cresyl violet stained section and sections processed immunocytochemically for IL-11 with two different primary antibodies. The upper left panel is Nissl staining, which is used to distinguish cortical layers. The upper middle panel is the immunocytochemical staining using the goat polyclonal IL-11 antibody. The upper right panel is the immunocytochemical staining using the rabbit polyclonal IL-11 antibody. The staining patterns of the IL-11 immunoreactivity using two different IL-11 antibodies were similar. Both antibodies rarely labeled cell bodies in layer I. Scale bar = 0.2 mm. B. Laminar distribution of IL-11 immunopositive cells in adult cat primary visual cortex. The relative immunopositive cell densities (cells/0.01 mm2) were determined as described in Chapter II. The X-axis is the layers of the visual cortex. The results from both antibodies were averaged and plotted. Each bar represents the mean density of each layer ± S E M of eight sections from two animals. 74 Chapter III Results A . Immunocytochemical localization of IL-11 in adult cat primary visual cortex Nissl Staining II/III IV V VI WM II/III IV V VI WM Immunocytochemical Staining Immunocytochemical Staining (goat anti-IL-11 antibody) (rabbit anti-IL-11 antibody) > 'mm *5 o > 'mm B. Laminar Distribution of IL-11 in Adult Cat Primary Visual Cortex 10 -| 8 -e 6 -Q 4 -u u 2 -0 -layer I layer II/III layer IV layer V layer VI 75 Chapter III Results layer V layer VI Figure 20. Immunoreactivity localization of IL-11 protein in adult cat primary visual cortex. Higher magnification photomicrographs of immunostaining in adult cat primary visual cortex by the antibody to IL-11 in layers II/III, layer IV, layer V , and layer VI. No pyramidal neurons were labeled by the antibody. The IL-11 immunoreactivity was mainly associated with cell bodies. Scale bar = 30 urn. 76 Chapter III Results 1-week-old 2-week-old 4-week-old 6-week-old 3-month-old Normal Adult Figure 21. Expression of IL-11 protein in postnatal developing cat visual cortex — A. Immunocytochemical staining. Lower magnification of photomicrographs of IL-11 immunocytochemical staining in the postnatal developing cat visual cortex including 1-week-old, 2-week-old, 4-week-old, 6-week-old, 3-month-old, and adult visual cortex. IL-11 was highly expressed during early postnatal development. The strongest labeling of IL-11 was found between 1 and 2 weeks of age, while it was at a minimal level in adult primary visual cortex. Scale bar = 30 pm. 77 Chapter III Results Developmental Changes in IL-11 Positive Cell Densities in Cat Primary Visual Cortex o w 0 4 , , , 1 , 1 , 1W 2W 4W 6W 3M NA Age Figure 22. Expression of IL-11 protein in postnatal developing cat visual cortex — B. Semi-quantitative study. The relative IL-11 immunopositive cell densities (cells/0.01 mm ) were determined as described in Chapter II. The relative total IL-11 immunopositive cell density was the highest in cat visual cortex between 1 and 2 weeks of age and the lowest in that of adult cat. n = 2 for each age (except at 1-week old n = 1). For each age, data were shown as mean ± SEM. The X-axis is the animal age. week-old and adult cats in all visual cortical layers were significant (p < 0.001). Thus, the overall density of IL-11 immunopositive cell in 2-week-old primary visual cortex was significantly higher than the overall density in adult primary visual cortex (p < 0.001). Although the IL-11 immunopositive cell densities between the primary visual cortices of 4-week-old and adult were significant different only in layer IV and layer V (p < 0.05), the overall density in 4-week-old visual cortex was still significantly higher than that in adult visual cortex (p < 0.05). 3.2.5 Dark Rearing Effects On IL-11 Protein Expression Immunocytochemical study was performed on the primary visual cortex of normal and dark-reared cats at the same age (4-month-old) to determine the effect of dark rearing 78 Chapter III Results Developmental Changes in IL-11 Positive Neuron Densities Across Visual Cortical Layers Qi u ii e OH • - J HH .> Pi 25 20 £ 15 a QJ Q 10 • 2W • 4 W ^ 6 W E33M B N A 5 0 *** X a *** i i layer I layer II/III layer IV layer V layer VI total Figure 23. Developmental changes in IL-11 positive cell densities across cat visual cortical layers. The relative IL-11 immunopositive cell densities (cells/0.01 mm2) were determined as described in Chapter II. Each bar represents the mean ± S E M of eight sections from two animals. The differences between 2-week-old and normal adult cats in all visual cortical layers were significant. Although the IL-11 immunopositive cell densities between the primary visual cortices of 4-week-old and adult were significantly different only in layer IV and layer V , the overall density in 4-week-old visual cortex was still significantly higher than that in adult visual cortex (*p < 0.05, ***p < 0.001). on IL-11 expression. For the dark-reared cat primary visual cortex tissue, the immunocytochemical staining was performed only with polyclonal anti-IL-11 rabbit antibody since there was no more tissue available for the staining with polyclonal anti-IL-11 goat antibody. We treated the results from the dark-reared cat the same as the normally-reared cats because the results from normal-reared animals from 1 week of age to adult showed similar immunostaining patterns and the relative IL-11 immunopositive cell numbers across visual cortical layers had no statistically significant difference. 79 Chapter III Results Figure 24 compares IL-11 protein expression in normal and dark-reared cat visual cortices at 4 months of age. As shown in Figure 24 A , in both rearing conditions IL-11 immunopositive cells were distributed throughout all visual cortical layers. There were more IL-11-labeled cells seen in the primary visual cortex of the dark-reared cat than in that of age-matched normal animals. The IL-11 immunopositive cell densities (cells/0.01 mm2) of each layer of the primary visual cortices of normal and dark-reared cats were determined as described in Chapter II in order to provide the semi-quantitative comparison of the laminar distribution of IL-11 positive cells under the two rearing conditions. There were significant differences between the IL-11 positive cell densities in the primary visual cortex of the dark-reared cat and normal cats across the visual cortical layers (p < 0.001 for layers II/III, layer IV, layer V , and layer VI; p < 0.05 for layer I; Figure 24B). The overall IL-11 immunopositive cell densities were also significantly different (p < 0.001). The level of IL-11 protein was significantly affected by dark rearing. Similar to O B C A M , the question also arises as to whether the higher IL-11 immunopositive cell density in dark-reared cats reflects a true increase in IL-11 labeled cells or an increase in total cellular packing density. As mentioned before, the average cellular density across all visual cortical layers in dark-reared cats is 29.5 ± 2 . 1 , and is 20.7 ± 1.4 in normals (Mower et al., 1988). Therefore, the corrected IL-11 cell density in dark-reared cats was 51.78% ± 7.15% compared to 18.51% ± 0.75% in normals. The difference was still statistically significant (p < 0.01). This indicated that dark rearing slowed the decrease course of IL-11 expression in the cat primary visual cortex. 80 Chapter III Results Figure 24. Dark rearing effects on IL-11 expression in cat primary visual cortex at 4 months of age. A . Photomicrographs showing the laminar pattern of IL-11 expression in the visual cortex of normal and dark-reared cat both at 4 months of age. In both rearing conditions, the IL-11 labeled cells were distributed throughout all visual cortical layers. More IL-11 positive cells were seen in the primary visual cortex of dark-reared animal. Scale bar = 0.2 mm. B. The relative IL-11 immunopositive cell densities (cells/0.01 mm ) between the two cats reared in different conditions in each layer and all visual cortical layers were compared. There were significant differences between the relative IL-11 positive cell numbers in the primary visual cortex of dark-reared and normal cat across all visual cortical layers (*p <0.05, ***p < 0.001). 81 Chapter III Results , WM WM w j n Normal Dark-Reared 4-monl h-olil 4-month-old B. Dark Rearing Effect on IL-11 Expression Across Visual Cortical Layers "3 25 -| • > ~ 1 t 15 I Ij 10 ft! S 5 • Dark-Reared *** • N o r m a l U I I I layer I layer II/III layer IV layer V layer VI total 82 Chapter III Results 3.3 ADENYLATE CYCLASE 7 (AC-7) 3.3.1 Western Blot Analysis of AC-7 The AC-7 antibody was an anti-mouse AC-7 polyclonal goat IgG from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). According to the manufacturer, it is against the carboxy terminus of AC-7 of mouse origin, reactive to mouse, rat and human tissue, and suitable for western blot and immunocytochemical studies. However, the results from western blot analysis showed multibands ranging from 20-kDa to 150-kDa (Figure 25) despite the fact that several HRP conjugated anti-goat secondary antibodies (from Sigma and Vector labs) were employed. This indicated the antibody was not specific to the cat AC-7 protein. 3.3.2 Immunocytochemical Staining Analysis of AC-7 To confirm the antibody was not specific to the cat AC-7 protein, we performed two negative controls: one replaced the primary antibody with PBS; the other one used the blocking peptide of the primary antibody. Although the negative control without the primary antibody showed no immuno-labeling (as expected), the negative control using the blocking peptide still showed immunopositive cells similar to those found in the sections following the regular procedures (Figure 26). Thus, this antibody was not specific to the cat AC-7 protein. Therefore, we didn't perform the developmental study. 3.4 RAS-ASSOCIATED PROTEIN IB (RAPIB) 3.4.1 Western Blot Analysis of RaplB The RaplB antibody was an anti-human RaplB goat polyclonal IgG from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). It is against the carboxy terminus of RaplB of 83 Chapter III Results N A Figure 25. Western blot analysis of AC-7 protein in cat primary visual cortex. The western blot analysis performed on the adult cat visual cortex using the AC-7 antibody from Santa Cruz Biotechnology, Inc. The antibody recognized more than one protein in visual cortex protein preparations obtained from adult cat, which indicated the antibody was not specific to the cat AC-7 protein. 84 Chapter III Results Figure 26. Immunocytochemical study of AC-7. Photomicrographs of immunocytochemical staining in adult visual cortex by the antibody to AC-7. The left panel (1) showed several cells which were labeled by the antibody. The middle panel (2) showed a section which was treated with the blocking peptide. Instead of not labeling any cells, it still showed some immunopositive cells. The right panel (3) is a section which was treated without the primary antibody. As expected, there was no labeled cell. Scale bar = 30 pm. 85 Chapter III Results human origin, reactive to mouse, rat, and human tissue, and suitable for western blot and immunocytochemical studies, according to the manufacturer. The results from the western blot study also showed multibands ranging from 22-kDa to 70-kDa (Figure 27) even though several different HRP conjugated anti-goat secondary antibodies were used. Therefore, we considered this antibody not specific to the cat RaplB protein. 3.4.2 Immunocytochemical Staining Analysis of RaplB To be cautious, we also performed two negative controls (the procedures similar to those used in the analysis of AC-7 above). Figure 28 showed that the blocking peptide couldn't neutralize the primary antibody. There were still immunopositive cells although the concentration of the blocking peptide was 5 times higher than that of the primary antibody. Therefore, the antibody was not specific to the cat RaplB protein, and we didn't perform the developmental study. 86 Chapter III Results 65-kDa 50-kDa 40-kDa 20-kDa Figure 27. Western blot analysis of RaplB protein in cat primary visual cortex. The western blot analysis performed on the adult cat visual cortex using the RaplB antibody from Santa Cruz Biotechnology, Inc. Instead of specifically recognizing RaplB protein, the antibody recognized several proteins in visual cortex protein preparations obtained from adult cat. This indicated the antibody was not specific to cat RaplB protein. 87 Chapter III Results • Figure 28. Immunocytochemical study of RaplB. Photomicrographs of immunocytochemical staining in adult visual cortex by the antibody to RaplB. The left panel (1) showed a section with several immunopositive cells. The middle panel (2) is a section which was treated with the blocking peptide. It also showed some immunopositive cells. The right panel (3) is a section which was treated without the primary antibody. There were no immunopositive cells. Scale bar = 30 pm. 88 Chapter IV Discussion CHAPTER IV DISCUSSION 4.1 T H E I N V O L V E M E N T O F O B C A M IN VISUAL C O R T I C A L PLASTICITY IN T H E D E V E L O P I N G VISUAL C O R T E X 4.1.1 Developmental Changes In O B C A M Protein Expression During The Critical Period O B C A M immunocytochemical analysis revealed that the expression of this protein in the visual cortex was developmentally regulated. O B C A M immunoreactivity was highest between 2 and 4 weeks of age, decreased quickly following 4 weeks of age, and remained low in adulthood. The semi-quantitative study agreed with these results. While the visual plasticity for ocular representation peaks at about 4 weeks of age in cat and is low in adulthood, the immunocytochemical results indicated that the O B C A M protein expression peaks near the peak of ocular dominance plasticity and decreases as the plasticity decreases. This supports the conjecture that O B C A M is involved in visual cortical plasticity. This finding is consistent with the work of Schoop et al. (1997), which claimed the adhesiveness of the visual cortex parallels the critical period. Conceivably, the cortex may lose its plastic capabilities due to the down-regulation of cell adhesion molecules. Although the volume of the primary visual cortex increases during early postnatal development, it is unlikely to be the reason for the reduction of the relative O B C A M immunopositive cell densities in the older animals. The semi-quantitative data showed not only the density reduction but also a reduction in the relative number of O B C A M 89 Chapter IV Discussion positive neurons. In addition, between 4 weeks of age and adulthood, Duffy and his colleagues (1989) determined that there was a 58% increase in the surface area of cat primary visual cortex; and our data showed that there was a 6% increase in the visual cortical thickness. Thus, there would be a 68% increase in the volume with age. Assuming there was no cell genesis in the visual cortex after 4 weeks of age, there would be a 40% decrease in the cell density due to the volume increase. Since the O B C A M immunopositive neuron density decreased 66% from 4 weeks of age (11.3 cells/0.01 mm ) to adult (3.9 cells/0.01 mm ), it further indicates the density reduction is not simply because of the volume increase, and is at least partly because of less cells expressing O B C A M during postnatal development. During early postnatal development, there is a substantial amount of cell death in the primary visual cortex (approximately 30%; Finlay and Pallas, 1989). It raises the question of whether the O B C A M immunopositive neuron density decrease is due to the cell death. Since the density of O B C A M immunopositive neuron decreases 68% from 1 week of age (11.9 cells/0.01 mm2) to adulthood (3.9 cells/0.01 mm2), cell death is unlikely to be the reason for the decrease of density. The O B C A M protein is expressed more on cell bodies and neurites in the visual cortex of younger cats than in that of adult cats. Since O B C A M is believed to be related to synaptogenesis (Hachisuka et al., 2000), this distribution suggests O B C A M may regulate the synapse formations between cortical neurons, and between neurons in visual cortical layer IV and axons originating from L G N neurons during the critical period. O B C A M protein expression was also high in the neonatal cat visual cortex, which is not correlated with the critical period of ocular dominance plasticity since the critical 90 Chapter I V Discussion period begins roughly 3 weeks after birth in cats. This suggests that O B C A M may play a role in visual cortical plasticity other than plasticity for ocular representation. O B C A M and other members in IgLON family can have bi-directional effects on neurite growth by homophilic and heterophilic interactions (Gil et al., 1998; Kamiguchi and Lemmon, 2000). Since the neonatal cortex is immature, O B C A M may play a role in brain maturation by regulating axonal growth and pathfinding. 4.1.2 Dark Rearing Effects On OBCAM Protein Expression The hypothesis of O B C A M involvement in visual cortical plasticity is bolstered by the fact that the O B C A M protein expression is not simply age-dependent, it is also sensitive to visual deprivation by dark rearing since the expression remains high in the visual cortex of the dark-reared 4-month-old cat. Not only the O B C A M immunoreactivity but also the relative O B C A M immunopositive neuron density in the visual cortex of the dark-reared cat are greater than those in normal age-matched cats. It is believed that the effect of darkness on physiologically assessed visual cortical plasticity is to slow the entire course of the critical period such that near the end of the critical period (4 months) dark-reared cats are more plastic than age-matched normal cats (Mower, 1991). Thus, higher O B C A M protein expression in the visual cortex of the dark-reared cat than in that of normal cats at 4 months of age correlates with our working hypothesis that plasticity candidate proteins are expressed in higher abundance in the more plastic visual cortex. 4.2 THE INVOLVEMENT OF IL-11 IN VISUAL CORTICAL PLASTICITY IN THE DEVELOPING VISUAL CORTEX 4.2.2 IL-11 Protein Expression In The Developing Visual Cortex 91 Chapter I V Discussion The western blot and immunocytochemical data presented in Chapter III indicated that the IL-11 protein was expressed in the primary visual cortex in a developmentally regulated manner. Interestingly, IL-11 protein expression in the visual cortex correlates with the critical period for ocular representation. Although the correlation is not perfect, the lower IL-11 protein expression levels in the visual cortex of older cats supports the conjecture that this protein is involved in visual cortical plasticity. The known functions of IL-11 such as promotion of neuronal differentiation and intermediate stages of neuronal maturation induction (Carvey et al., 2001; Mehler et al., 1993) further support a role for this protein in visual cortical development. Similar to the case of O B C A M , the reductions of the IL-11 immunopositive cell density in older cats' visual cortices are not due to the increase in the visual cortex volume with age during postnatal development. From 2-weeks old to adult, there was an 85% increase in the surface area of cat primary visual cortex (Duffy et al., 1989), and a 13% increase in the visual cortical thickness (our data). Thus, there would be a 109% increase in the visual cortex volume from 2 weeks of age to adulthood and a 48% decrease in the cell density due to the volume increase with age. However, the IL-11 immunopositive cell density decreased 69% (2 weeks of age, 14.3 cells/0.01 mm ; adult, 4.4 cells/0.01 mm ), which was not parallel with the volume increase. Cell death in the primary visual cortex can also not account for the density decrease, since the density decreased 73% from 1 week of age (16.1 cells/0.01 mm ) to adulthood (4.4 cells/0.01 mm ) while there was only approximately 30% cell death in this time frame (Finlay and Pallas, 1989). 92 Chapter I V Discussion The possible nucleus labeling in the visual cortex of younger animals suggests that there might be IL-11 protein located in the nuclei of the visual cortical cells. It indicates that IL-11 might function as or with a transcription factor to promote the expression of other genes that are involved in ocular dominance plasticity in younger animals. The change in expression pattern starts between 2 and 4 weeks of age. Often the regulatory changes caused by transcription factors do not immediately alter the phenotypic behavior of cells and this may explain why nuclear IL-11 protein was detected in the visual cortex until slightly before the peak of ocular dominance plasticity. For example, i f IL-11 activity leads to the activation of a transcription cascade, the proteins involved need to be synthesized and allowed to take effect. More importantly, genes up-regulated by IL-11 may exhibit phenotypic lag. Interestingly, the expression pattern change proceeds from the inner layers (layer IV to layer VI) to the superficial layers (layers II/III). This suggests a correlation in layers II/III between high levels of nuclear IL-11 immunoreactivity and known periods of visual cortical plasticity. Although in these superficial layers, the pattern of lateral axonal connections develops over the same time course as that for the geniculocortical axons (Callaway and Katz, 1990; Ruthazer and Stryker, 1996), the effects of monocular deprivation on the ocular dominance preference of neurons in the superficial layers last longer than in layer IV (Daw et al., 1992; Shatz and Stryker, 1978). The finding here that the time of the possible nuclear IL-11 immunoreactivity remaining in layers II/III is longer than in layer IV is consistent with an ongoing role for IL-11 protein in modulating visual cortical plasticity. 4.2.3 Dark Rearing Effects On IL-11 Protein Expression 93 Chapter IV Discussion Dark rearing has been shown to prolong the critical period for susceptibility to monocular deprivation (Cynader and Mitchell, 1980; Mower et al., 1981). The effect of dark rearing is to slow the entire time course of the critical period. If IL-11 were involved in the visual cortical plasticity for ocular representation, it would be predicted that the normal changes in IL-11 protein level would be slowed in dark-reared animals. Our findings support this hypothesis. Dark rearing slowed both the decrease of IL-11 immunoreactivities and the decrease of the IL-11 immunopositive cell density. It suggests IL-11 is involved in visual cortical plasticity. The IL-11 immunoreactivity pattern in the dark-reared visual cortex is more like the pattern in the younger animals with possible nucleus labeling, while the IL-11 immunoreactivity pattern in normal-reared visual cortex is like the pattern in the adult with most IL-11 protein associated with the cell somas. This suggests that dark rearing slowed the change of the IL-11 expression pattern. This further supports the hypothesis that IL-11 plays an important role in modulating visual cortical plasticity. 4.3 C A U T I O N S F R O M A C - 7 A N D R A P IB Our western blot data as well as the results from the negative controls of immunocytochemical studies for AC-7 and RaplB suggest that the two antibodies are not specific to cat AC-7 and RaplB. However, there are some issues we should address here. First, i f we suppose the antibodies could bind AC-7 or RaplB and other proteins (because the negative control with the blocking peptides did show lower immunoreactivity), the immunocytochemical analyses indicated the protein levels were very low in the visual cortex since the immunoreactivity was low even with nonspecific labeling. Therefore, in 94 Chapter IV Discussion the future, i f people want to study these two proteins in the visual cortex of other species, the precautions should be taken regarding low levels of protein expression. Second, we should have considered the fact that the homologies among the Rap proteins are very high (as mentioned in Chapter I). Although it is claimed that the RaplB antibody we used is specific for RaplB and not cross-reactive with Rapl A and Rap2 in mouse, rat, and human tissue, it is unknown whether the antibody is cross-reactive with Rapl A and Rap2 in other species, like cat. Thus, precautions should be taken i f one wants to study a protein like RaplB in an 'unpopular' species. Third, the multibands shown in western blot analyses could be caused by non-specific binding of the secondary antibody. However, since different secondary antibodies were tried, and they showed similar multibands, this is unlikely to be the case. The reason for the multibands could also be that the primary antibody cross-reacted with other proteins when they were denatured. However, i f the primary antibody can only bind proteins when they are denatured, the negative control for immunocytochemistry using blocking peptides should not show positive labeling. Since the negative controls did show some immunopositive cells despite a concentration of the blocking peptide that was 5 times that of the primary antibody, the primary antibody did not likely bind to other denatured proteins. 4.4 C O N C L U S I O N Our findings in this thesis provide several lines of evidence supporting the involvement of O B C A M and IL-11 in the visual cortical plasticity. First, both O B C A M and IL-11 protein expressions are developmentally regulated during early postnatal 95 Chapter IV Discussion development. Second, the changes of O B C A M and IL-11 protein expressions do not change simply in an age-dependent manner but rather are correlated with the level of cortical plasticity and regulated in an activity-dependent manner. Thus, we have established the expression of O B C A M and IL-11 proteins in the cat primary visual cortex during the critical period and provided evidence for their involvement in visual cortical plasticity. 4.5 FUTURE DIRECTION Although it is, known that members in IgLON family can have bi-directional effects on neurite growth by homophilic and heterophilic interactions (Gil et al., 1998; Kamiguchi and Lemmon, 2000), their role in neuronal plasticity is still unclear. Our data showed that there is abundant O B C A M protein expressed in the visual cortex of younger animals, it is also worth studying the temporal and spatial distributions of other members in IgLON family, such as L A M P , neurotrimin, and kilon. This will give some clue to their roles in visual cortical plasticity. Further, i f an O B C A M knockout mouse model can be created, or O B C A M anti-sense can be delivered to the visual cortex, the particular role of O B C A M in visual cortical plasticity can be tested. 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