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Calcium-related signal transduction systems in developing visual cortex Jia, Wei-Guo 1991

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& i CALCIUM- RELATED SIGNAL TRANSDUCTION SYSTEMS IN DEVELOPING VISUAL CORTEX By Wei-GuoJia B.Sc, Fudan University, 1982 M.Sc. Dalhousie University, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES NEUROSCIENCE PROGRAMME We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 1991 © Wei Guo Jia, 1991 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. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) C a 2 + - RELATED SIGNAL TRANSDUCTION SYSTEMS IN DEVELOPING VISUAL CORTEX (Abstract) Neuronal connections in cat visual cortex are highly susceptible to visual experience at early postnatal age and thus serve as a useful model of neural plasticity. The biochemical mechanisms underlying this cort ical plasticity remain unclear. In this thesis, the development of several elements in calcium-related signal transduction systems, including the type-1 muscarinic and alpha-1 adrenoceptor systems as examples of cell surface receptors and protein kinase C , calcium/calmodulin dependent kinase II and inositol 1,4,5 phosphotate receptors as second messenger targets, were investigated using the methods of immunocytochemistry and autoradiography. The results show that each receptor develops with its own time-table and laminar distribution; the various elements all culminate and display the maximal colocalization during the critical period; and, only at this age, the cortical levels of the receptors and kinases are dependent on subcortical afferents. The results suggest that cell surface receptors and their second messenger targets develop in specific temporal and spatial patterns, which may be both genetical ly and environmentally determined, and this specif ic sequence of development of the molecules for signal transduction results in a series of modifications in the morphology and physiology of the developing cortex leading to its maturation. ii Table of Contents Abstract 11 List of tables i v List of figures v Chapter one P i GENERAL INTRODUCTION Chapter two p7 DEVELOPMENT OF ALPHA ADRENOCEPTORS AND A COMPARISON WITH Ml CHOLINERGIC RECEPTOR Chapter three p24 POSTNATAL ONTOGENY OF PROTEIN KINASE C IN THE VISUAL CORTEX Chapter four p41 CALCIUM/CALMODULIN DEPENDENT KINASE II IN CAT VISUAL CORTEX AND ITS DEVELOPMENT Chapter five p5 3 POSTNATAL DEVELOPMENT OF INOSITOL 1,4,5-TRIPHOSPHATE RECEPTORS: A DISPARITY WITH PROTEIN KINASE C Chapter six p62 GENERAL CONCLUSIONS AND DISCUSSION Figures p80 Acknowledgement p i 27 Biblliography p i 28 List of Tables Table 1. Operated animals used in study of chapter one p l l Table 2. Bindings of [3H]prazosin and [3H]rauwolscine in the visual cortex of operated animals pi5.1 Table 3. Sequential development of cell surface receptors p6 9 Table 4. Morphological and physiological development of cat visual cortex p72 iv List of Figures Figure 1 Autoradiograms of [3H]prazosin (ccl)and [3H]rauwolscine (a2)binding in developing visual cortex p80 Figure 2 Autoradiograms of [3H]pirenzepine binding for M l cholinergic receptors in developing visual cortex. p8 0 Figure 3 Development of alpha adrenoceptors and M l cholinoceptors at different depth of visual cortex. p8 2 Figure 4 Autoradiograms of [3H]prazosin (al) and [3H]rauwolscine (oc2) bindings in operated animals. p8 3 Figure 5 Densitometric analysis of the ligand binding data of figure 4. p85 Figure 6 Comparison of development of alpha-1, alpha-2 adrenoceptors and Ml cholinoceptors in various cortical laminae. p87 Figure 7 Comparison in laminar distributions of the three receptors in developing visual cortex. p8 9 Figure. 8 Light micrographs of immunostaining for protein kinase C in area 17 (a) and area 18 (b) of kitten visual cortex p 9 1 Figure 9 Area 17 (top) and area 18 (bottom) of the visual cortex stained with the polyclonal antibodies to PKC at day 10. p93 Figure 10 High magnification of PKC immunostaining in area 17 of the visual cortex at day 40. p93 Figure 11 Bundles of fibers with PKC immunoreactivity in upper layer VI of area 17 at day 40. p9 3 Figure 12 Cytoplasm of a PKC immunoreactive cell from a postnatal day 10 kitten visual cortex. p95 Figure 13 Electron micrographs of immunoreactive profiles taken from the visual cortex of a postnatal day 30 kitten. p97 Figure 14 Distribution of PKC immunoreactive structures in a v postnatal day 4 kitten visual cortex. p9 9 Figure 15 PKC immunoreactivity in the visual cortex surgically isolated at day 14. p l O l Figure 16 Immunostaining of CAM-K II antibody in tissues perfused with two different protocols pi03 Figure 17 CAM-K II in cat visual cortex. p i 05 Figure 18 CAM-K II immunoreactivity in kitten visual cortex (area 17) at various ages. pi07 Figure 19 High magnification microphotograph taken from an area in upper layer IV of the animal at 24 days of age. p i 09 Figure 20 CAM-K II immunoreactivity in an animal with an early LGN-lesioned. p i 09 Figure 21 Cytoplasm of CAM-KII immunoreactive neurons from an adult cat (left) and a postnatal day 14 kitten (right) visual cortex. p i l l Figure 22 Electron micrographs of post-synaptic CAM-K II immunoreactive profiles taken from an adult cat (left) and a postnatal day 4 kitten (right) visual cortex. p 1 1 3 Figure 23 Electron micrographs of presynaptic CAM-KII immunoreactive profiles taken from an adult cat (left) and a postnatal day 4 kitten (right) visual cortex. pi 15 Figure 24 Charectelization of [ 3H]IP3 binding in cat visual cortex. p 117 Figure 25 Color coded optical density of autoradiography of [ 3 H ] IP3 and [3H]PDBu in adjacent sections of the visual cortex and hippocampus in developing kitten brain (left panel). pi 19 Figure 26 Development of [ 3 H]IP3 and [3H]PDBu binding sites in visual cortex. p 1 21 Figure 27 Autoradiography of [3H]IP3 and [3H]PDBu binding in undercut visual cortex (left) and the results of densitometry (right). pi23 vi Figure 28 Colocalization of receptors for PI turnover in developing visual cortex. p i 25 Figure 29 Comparison of development of receptors for PI turnover in the visual cortex. p i 25 vii Chapter One GENERAL INTRODUCTION During early age of postnatal life, the visual cortex displays a remarkable plasticity 198,199,200. Abnormal visual exposure at this early age can cause profound and permanent abnormalities in synaptic connections in the cortex4",41,182. Young children with peripheral visual disorders such as cataract, hyperopia or myopia will develop reduced visual capabilities (e.g. amblyopia) in the affected eye in adulthood, unless the optical defect is corrected early in life. The permanent defective consequences of abnormal vision have been widely studied in animal models 4 0.41,182,198,199,200. it has been well established that kittens reared with one eyelid sutured during the first three months of postnatal life sustain permanent blindness in the sutured eye when it is reopened, although the eye and the retina are normal. Further, anatomic and physiologic studies indicate that there are a series of changes in the central parts of the visual system. In the cerebral visual cortex, most cells become monocularly driven by stimulation through the normal eye and fewer than 10% of the cells respond to stimulation through the sutured eye. In addition, injection of [3H]proline in one eye, to label the areas on the visual cortex that receive the inputs from that eye, reveals a remarkable shrinkage in those areas of cortex representing the deprived eye and a corresponding expansion in the areas receiving input from the non-deprived eye85,i °9. These results indicate that monocular deprivation at an early age causes profound modification in synaptic connections in the visual pathways. In cat, this age is around postnatal week 3 to week 13 and this period of time characterized with the highest susceptibility to visual experience in organization of the visual cortex is called "the critical period" 1"' 2 0 0. 1 Based on the above discoveries, the visual system has provided a good model for the study of formation of neuronal synaptic connections which has become widely accepted as the basis of neuronal plasticity73,177. This plasticity enables organisms to adapt themselves to various environments and to adjust their responses to new situations. The formation of synaptic connections is a highly dynamic procedure depending on the activities of both presynaptic and postsynaptic ce l l s 2 9 . i ?? . In the visual cortex, one can prevent ocular dominance changes in monocularly deprived kittens either by overac t iva t ing or by cont inua l ly i n h i b i t i n g the c o r t i c a l n e u r o n s 1 5 1 . 1 5 2 - 1 6 8 . These results indicate that disruption of postsynaptic activity may interrupt formation of synapses. On the other hand, a blockade of presynaptic activity driven by the normal eye can also prevent the formation of ocular dominance c o l u m n s 1 8 1 . In addit ion, an established synaptic connection can be strengthened depending on activity. This phenomenon is best known as long term potentiation ( L T P ) 2 0 . 1 8 6 . i n cerebral cortex or hippocampus, tetanic stimulation of certain presynaptic pathways facilitates synaptic connections, and results in an elevated postsynaptic response which lasts hours or even days. The significance of L T P cannot be overemphasized?' L T P directly reflects -modification of synaptic transmission; therefore, studies on L T P provide important clues to the mechanisms of synaptic formation and strengthening. Evidence from the study of L T P suggests that the formation of a new synapse or its strengthening is based on the interaction between pre- and post-synaptic e lements 2 1 . This interaction is established mainly by chemical signals released from neuronal. terminals. The released substances (e.g. neurotransmitters) bind to their specific receptors, which transfer the signal into the cell either by directly opening certain ion channels to cause cell membrane depolarization/ 2 h y p e r p o l a r i z a t i o n or by act i v a t i n g GTP-dependent proteins (G-p r o t e i n s ) w h i c h are o f t e n c o u p l e d to s e c o n d m e s s e n g e r s y s t e m s 1 ^ , 1 9 7 or some ion channels33. The second messengers further activate specific protein kinases to phosphorylate a group of proteins i n c l u d i n g some DNA-binding proteins8,18,44,49,132,157,163. This signal pathway v i a second messenger systems usually causes relatively slow but long term changes in the c e l l , such as protein p h o s p h o r y l a t i o n , n e w p r o t e i n s y n t h e s i s o r g e n e transcription32,44,49,64,66,69,94,96,99,120,136,196. Since the formation of new synapses results f r o m a series of c e l l u l a r responses, the intracellular signal transduction from receptors to second messengers and corresponding protein kinases may play an important role during the development of the b r a i n 2 9 . In view of the importance of cellular signal transduction for the d e v e l o p m e n t of s y n a p t i c c o n n e c t i o n s i n v i s u a l cortex, the development of neurotransmitter receptors has been systematically investigated. Three conclusions have been drawn from these studies: 1. Many receptors in the visual cortex are not homogeneously di s t r i b u t e d throughout the layers of the c o r t e x i 69,170,201. For instance, using [3H] muscimol to label G A B A A receptors in adult cat, it has been shown that the highest density of binding sites appears to be in layer IV, while layers V and VI appear the least dense 133. i n the case of muscarinic acetylcholinergic receptors (mAChRs), the densest binding of a specific ligand [ 3 H]quinuclidinylbenzinlate (QNB), is found i n layer I through III but and the least dense b i n d i n g appears in layer IV171 . On the other hand, nicotinic acetylcholinergic receptors (nAChRs) labeled with [ 3 H]nicotine, are located s p e c i f i c a l l y in layer IV150. 2. The numbers o f neurotransmitter r e c e p t o r s and t h e i r distributions in visual cortex undergo a series of dynamic changes d u r i n g development 169,172. i n contrast to the laminar distribution of 3 mAChRs in adult cats, these receptors are concentrated in the middle layers in newborn kittens. During development, mAChR binding sites are expressed in superficial and deeper layers and gradually disappear in layer IV170,17 1,192. The redistribution of receptor binding sites during postnatal development is common in visual cortex. Almost two thirds of receptors studied in our laboratory, such as muscarinic, (3-adrenergic, opioid, C C K and glutamate receptors, show a similar form of redistributions. In all cases, the redistribution occurs during the critical period. Furthermore, the overall number of each type of receptors reaches the peak within that critical period. After that time, the number of receptors is either maintained or reduced to adult levels. 3. Redistribution of receptors is dependent on normal input to the c o r t e x i 7 3 . If part of the white matter underneath the visual cortical grey matter is surgically undercut, the cortical neurons that are innervated by those fibers will be isolated from the input of lateral geniculate nucleus (LGN) and other regions. In kittens with undercut visual cortex, some receptors do not show the redistribution that normally occurs during the critical period. One such example is mAChR: kittens were undercut at postnatal day 23, and mAChRs were labeled with [ 3H]QNB three weeks later. Instead of showing the adult pattern of binding (all layers, except layer IV), the receptors retained their immature pattern, i.e. layer IV is densely labeledi73. The heterogeneous distribution, the highly dynamic change, its presynaptic input dependence and especially the highest activity during the critical period,all of these characteristics of development of receptors in visual cortex suggest that the receptors play important roles in the organization of neuronal connections during development of the cortex. In order to induce long term cellular responses, receptors must couple to second messenger systems. Normally, this results in activation of various kinases, which should be considered as 4 receptors for second messengers. The present work is mainly focused on calcium-related second messenger systems, i.e., calcium-Ca + + /calmodulin dependent protein kinase II (Ca + + / C A M - K II system) and phosphoinositide (PI system). The C a + + / C A M - K II second messenger systemU.34.6i.i62.i88 is mobilized by elevations in intracellular free calcium level. Many neuronal activities can result in increases in the concentration of intracellular C a + + ([Ca+ +]i) by either activating C a + + influx from extracellular sources or mobilizing intracellular C a + + stores. Increased [Ca + +]i activates CAM-K II through an interaction between the kinase and the Ca++/calmodulin complex. Another second messenger system that is closely related with calcium is inositol trisphosphate/diacylglycerol ( I P 3 / D G ) 14,15,57,143,175. The I P 3 / D G second messenger system is triggered by occupation of several types of receptors, such as M l muscarinic cholinergic receptors, a, adrenergic receptors, 5-HTlc receptors, etc. Stimulation of these receptors results in the activation of phospholipase C (PLC) via G-protein(s)57,79. PLC hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to release two second messengers, DG and I P 3 . The former activates a calcium/lipid-dependent protein kinase, protein kinase C (PKC) and the latter reacts with its specific intracellular receptors to cause C a + + release from intracellular storesi6. Both the C a + + / C A M - K II and IP3/DG second messenger systems have been widely studied in the brain. It is accepted that the two are deeply involved in many neuronal functions, especially in neuronal plasticity and development (for C a + + / C A M - K II: 17,23,34,61,64,74,101,102,104,120,125,141,162,196; for I P 3 / D G : 2,32,49,64,66,94,96,115.-118,120,121,123,126.132,136,140,157.163,208). Both CAM-K II and PKC exist in high concentrations in the brain and are responsible for the phosphorylation of various neuronal specific 5 proteins that are important during development and synaptic formation or modulation. Some targets include synapsin I, cytoskeleton proteins and the growth associated protein (GAP 43). Both kinases are reported play a role in LTP1-110,120,123. An increase in I P 3 during LTP was also been reported?,25,117. It is possible that the three second messengers, calcium, IP3 and DG can have synergistic interactions under certain conditions to perform various cellular responses. It is particularly likely for DG and IP3 since they can be produced simultaneously from the hydrolysis of PIP2. Several groups have reported that there are additive effects of calcium and activators of PKC to induce full secretory responses in blood platelets and other cells 1 3. It is also well known that a number of proteins, such as synapsin I and microtubule associated protein 2 (MAP 2), are common substrates of both CAM-K II and PKC. However, little evidence has been collected to support this speculation in the nervous system. To throw a light on the issue, I compared the postnatal ontogeny and localization of C a + + / C A M - K II and IP3/DG systems in kitten visual cortex. This is under the presumption that if the two systems indeed play roles in cortical development in a synergistic manner, CAM-K II, the IP3 receptor, and PKC should be labelled at similar locations and show similar ontogenic profiles in developing visual cortex. To this aim, immunocytochemistry and autoradiography were utilized to localize the related molecules in cat visual cortex. The investigation was particularly focused on changes with age in the distributions and on the comparison among these molecules. In order to reveal the relationship between the development of these second messenger systems and neuronal activity, various manipulations were performed on the visual cortex at different ages and the effects of these manipulations on expression and distribution of these molecules were investigated. 6 Chapter Two DEVELOPMENT OF ALPHA ADRENOCEPTORS AND A COMPARISON WITH Ml CHOLINERGIC RECEPTOR INTRODUCTION: As the gates of intracellular signal transduction pathways, c e l l surface receptors play a crucial role. Among the receptors that are coupled to PI turnover, a l adrenergic receptors and M l cholinergic receptors have been most widely studied. Stimulation of the receptors with their agonists causes a rapid increase in i n o s i t o l phosphates and C a + + release from i n t r a c e l l u l a r stores24,97. In addition, there is also evidence that many ce l l u l a r responses to stimulation of these receptors are mediated by protein kinase C activation49. Both noradrenergic (NA) and cholinergic (ACh) systems are considered as cortical modulatory systems. NA and ACh fibers arise f r o m the locus coeruleus ( L C ) 1 1 2 and cells in the basal t e l e n c e p h a l o n ^ , respectively. These fibers run through dorsal bundles and project to various areas of the cerebral cortex. In cat visual cortex, Bear et al reported that combined destruction of the c o r t i c a l c h o l i n e r g i c and noradrenergic i n n e r v a t i o n results i n a reduced p h y s i o l o g i c a l response to monocular deprivation, suggesting a loss of the ocular dominance plasticity 9. On the other hand, it was f o u n d that v i s u a l s t i m u l a t i o n s y n c h r o n i z e d w i t h c o m b i n e d application of NA and ACh results in a long-lasting modification in ocular dominance, orientation selectivity and direction preference of the cells in kitten visual cortex 65. These results imply that the NA and ACh systems play roles in cortical plasticity in a cooperative manner. The postnatal development of muscarinic receptors i n kitten v i s u a l cortex has been reported p r e v i o u s l y 170. i n addition, the i d e n t i f i c a t i o n and laminar distribution of alpha-adrenoceptors in 7 adult cat visual cortex were reported by Parkinson et a l . 1 4 5 . In order to examine the functional contribution of adrenergic and cholinergic systems in the development of the visual cortex, the postnatal ontogeny of a l and a2 adrenoceptors was investigated in adjacent sections with autoradiography by utilizing [ 3H]prazosin and [ 3H]rauwolscine28, respectively. As a comparison, M l cholinergic receptors were labelled by [3H]pirenzepine in the visual cortex of animals in the same age groups. 8 METHODS AND MATERIALS  Animals A total of twenty-one cats of various ages (from postnatal day 1 to adult) were utilized in the present study. Animals were deeply anesthetized and perfused through the aorta with cold (8°C ) phosphate buffer (0.1M, pH 7.4) for 1 min. The perfused brains were rapidly removed and stored at -20°C. Some of animals were subjected to surgical manipulations before the perfusion. Kittens were anesthetized with helothane to effect via facemask and placed in stereotaxic frame. Four types of surgery were performed (Table 1). In the first group {front cut), a 1 mm thick trench of cortical tissue was aspirated by suction to a depth of 1 cm from the cortical surface (extending down to the corpus callosum) and extending from the midline to about 7 mm lateral of the midline on one side of the brain. The section was located at about A.P. 12.0 anterior and had the effect of interrupting modulatory fibers arising from brainstem, hypothalamic and basal forebrain sources9. In the second group (LGN lesion), LGN was aspirated on one side. In animals of the third group, the unilateral front cuts were combined with ipsilateral LGN lesion. In one additional kitten, the optic tract on the left side was severed at postnatal day 10 and the animal was perfused at day 59. The operations were performed at various ages and all operated kittens were given an injection of penicillin G (10,000 IU/kg) and a topical broad spectrum antibiotic is applied to the incision for 5 days post operatively. Animals were allowed to survive at least two weeks before sacrifice. The lesion sites were examined during sectioning process. Removal of LGN and severing of the dorsal bundles were confirmed by greatly reduced [3H]nicotine binding in the ipsilateral visual cortexl50 and by a negative result of cholinesterase (AChE) staining in the ipsilateral cortex posterior to the lesion site9, 9 respectively. Autoradiography Thin (12|im) coronal sections of the visual cortex from animals at various ages were cut on a cryostat, and thaw-mounted onto gelatin-subbed slides. Sections were incubated for one hour at 4°C in phosphate buffer (PB, 0.1 M, pH 7.7) containing 0.75nM [3H]prazosin (24.4 Ci/mmol) or 0.6nM [3H]rauwolscine (75.0 Ci/mmol, both from NEN) for a l and a2 receptors, respectively. Nonspecific binding was less than 15% of the total binding at current concentrations of ligands determined by adding phentolamine (Sigma, 10|iM, final concentration) into the incubation media. The incubations were followed by 3x10 min wash for [3H]prazosin and 3x5 min wash for [3H]rauwolscine in PB at 4°C. For Ml sites, sections were incubated in 0.02M Tris buffer (pH 7.5) with 0.01M MgCl2 and 5 nM [3H]pirenzepine (82.0 Ci/mmol, Sigma) for 60 min at room temperature followed by a 2X3 min wash. Sections were rapidly dried in a stream of room air and apposed to LKB Ultrofilm for 10 weeks. Quantitative analysis The autoradiography images were captured with a video camera, and input into a computer for densitometry analysis. Averaged optical density (OD) of any given region of cortex was measured and expressed in pseudocolor after subtracting background. The OD values were calibrated into concentration of the bound radioactive ligand using [3H]standards (Amersham). Student t-test or ANOVA were applied for statistical analysis. 10 Table 1 Operated animals used in study of chapter one Animal Operation type Operation (Postnatal age day) Sacrifice age RB50 left FC adult 90 days survival RB69 right FC PI 1 P40 RB71 right FC Pl l P40 RB52 right LGN(-) adult 14 days survival RB61 right LGN (-) adult 60 days survival RB68 right LGN (-) P l l P40 RB79 right LGN (-) Pl l P90 RB46 right FC/LGN (-) PI 1 P40 RB47 right FC/LGN (-) Pll P40 RB78 Left OT(-) P10 P59 FC, front cut; LGN(-), LGN lesion; FC/LGN(-), front cut combined with LGN lesion; OT(-), optic tract lesion. 11 Results Postnatal development of alpha-1 adrenoceptors The laminar distribution of the alpha-1 adrenoceptors in the visual cortex varies with age (Fig.l). At postnatal day 1, binding of [3H]prazosin in the visual cortex was at a very low level, although a mild density of silver grains could be seen in cortical layer I. In contrast, high binding was found in the subcortical plate. By postnatal day 10, cortical layers I and IV were densely labeled by the ligand and the binding in the rest of the laminae remained low. Unlike the binding in layer I, the densely labelled middle layer particularly demarcated the visual cortex (areas 17 and 18). After postnatal day 1, levels of [3H]prazosin in the subplate consistently increased until P10. During this period, the largest proportion of the binding was located in the subplate. By postnatal day 30 to 40, [3H]prazosin binding increased in all cortical laminae (Fig. 3), particularly in the superficial and middle layers while layer VI showed the least radioactivity among all cortical layers. There was still a strong binding in the subplate although the width was greatly reduced. The number of binding sites in this zone declined gradually and finally disappeared in the visual cortex by postnatal day 60 (not shown) but they were still present in some cortical areas, such as the entorhinal cortex, to a lesser extent until adulthood. Between P40 and P75, there was still a substantial increase in density of binding sites in the cortex. However, there was not much change in the laminar distribution pattern of the binding. The total binding in the visual cortex started to decline between P75 and P120. During this period of time, the greatest reduction occurred in the middle layer while binding in the superficial layers declined less (Fig.3). This heterogeneous reduction in the binding among different layers resulted in a new laminar pattern, showing a higher density of silver grains in layers II and III than in layers IV, V and VI. This new 12 pattern remained into adulthood. Postnatal development of alpha-2 adrenoceptor Development of [3H]rauwolscine binding sites representing a 2 receptors in the visual cortex somewhat resembles that of [3H]prazosin. The binding achieved its maximum around the same age as that of [3H]prazosin (P75) and the number of binding sites declined subsequently. Again, this reduction was most remarkable in the middle layer and the superficial layers presented the densest binding of the ligand in adulthood. However, binding of the two ligands differs in several aspects. First of all, binding of [3H]rauwolscine in the subplate disappeared by postnatal day 30, much earlier than that of [3H]prazosin; Secondly, silver grains representing [3H]rauwolscine binding sites appeared in layer IV by P20, about 10 days later than the appearance of [3H]prazosin binding sites in the same layer (Fig. 3); Thirdly, the maximal binding density of [3H]rauwolscine during the critical period was 12-fold higher than during the first month while it was less than 3-fold for [3H]prazosin (Fig. 3); Finally, in adult visual cortex, the binding of [3H]rauwolscine in layer II was 3-fold higher than in layers IV-VI while the binding of [3H]prazosin in the top half of the cortex was only about 50% higher than that in the bottom half (Fig. 3 and Fig. 6). Development of muscarinic type-1 receptors The postnatal development of [3H]pirenzepine binding sites in the cat visual cortex has been reported previously 1 4 9 . The present work agrees with those results (Fig.2 and 3). Briefly, the [3H]pirenzepine binding sites were first apparent in layer IV in the neonatal cortex. The number of binding sites in this layer reached its maximum by P30 and subsequently reduced. In the superficial layers (II and III) and deep layers (V and VI), the binding was low in first two weeks, and rapidly increased around P20. The peak in layers I-III was 13 reached around P40, a week later than that in layer IV. Binding in layers V-VI reached a maximum at about P40 and remained essentially unchanged thereafter and the density of binding in these layers then declined to adult level. As a consequence of a greater decrease of binding in the middle layers comparing to the other layers, the adult laminar distribution of M l receptors was quite different from that of young animals, showing heavily labelled layers II, III and VI with relatively lower density of binding in the middle layers, especially layer IV. Effects of inputs deprivation on development of alpha adrenoceptor alpha-1 receptors (Figures 4, 5 and Table 2a) LGN lesions performed at a young age (Pll) resulted in a significant decrease (P<0.05) in binding of [3H]prazosin in the ipsilateral visual cortex when the animals were perfused either at P40 or at P90 (Fig.4a,c and Fig.5a,c). Although the "front cut" itself showed little effect (Table 2a), the loss in the binding resulting from combination of the front cut and ipsilateral LGN lesion was significantly greater (p<0.01, ANOVA, in all cortical layers) than that of LGN lesion only. There was no alteration in alpha-1 receptors when the LGN lesion was performed at adulthood (Fig. 4g, 5g and Table 2a). Contrary to the decline of binding in the grey matter, a striking increase in radioactivity was found in the ipsilateral subcortical plate in animals following the early LGN lesion with or without front cut (Fig. 4a,c; Fig. 5a, c and Table 2a). No effect was seen with the optic tract lesion (Fig.4e; Fig. 5e and Table 2a). Alpha-2 receptors (Figures 4, 5 and Table 2b) The effects of an early LGN lesion or combination of a LGN lesion and front cut on alpha-2 receptors was more remarkable than that of alpha-1 receptors. Again, the most striking effects were seen in animals having the combination of LGN lesion and front cut. Unlike alpha-1 14 receptors, the reduction in density of silver grains was not homogeneous across the cortical laminae. The most affected cortical layer was layer IV, where the binding of [3H]rauwolscine following LGN lesion was about half of the control value. In contrast, there was no change in layer I when the animal was sacrificed at P40, 4 weeks following the surgery. However, this heterogeneous reduction in different layers was less obvious in a kitten that was perfused at P90, 11 weeks after the operation (Fig. 5 b and d). In this animal, although there was still a greater decrease of binding in layer IV, the extent of decline in layer I was similar to that in layers II and III. The same LGN lesion in adult animals did not significantly change the binding density in the cortex (Fig. 5h). As with alpha-1 receptors, neither the optic tract lesion (Fig. 5f) nor a pure front cut had significant effect on the number of alpha-2 receptors. 15 Table 2 Binding of [3H]Prazosin and [3H]Rauwolscine in the Visual Cortex of Operated Animals a) [3H]prazosin 0T(-) FC Young LGN (-) LGN(-)/FC Adult LGN(-) Layers I-III 101.9±2.1n(4) 97.1±2.3n(8) 86.3±2.0b(9) 76.2±2.7C(6) 99.9±3.6(8)n Layer IV 103±l.l n(4) 96.8+2.8n(8) 91.2±3.7a(9) 73.7±3.1c(6) 96.0±2.7(8)n Layers V-VI 102±4.3n(4) 97.6±2.4n(8) 93.0±1.6b(9) 77.9±3.7b(6) 102.4±2.7(8)n Layers I-VI 102±2.3n(4) 96.1±2.2n(8) 90.8±2.4a(9) 76.1±3:3b(6) 99.8±2.7(8)n SP 99.0±1.8n(4) 105.0±3.5n(8) 202.8±15.4C(9) 147.1±8.8b(6) b) [3H]rauwolscine OT(-) FC Young LGN (-) LGN(-)/FC Adult LGN(-) Layers I-III 107 .7±7 .7 n (4 ) 1 0 6 . 5 ± 4 . 2 n ( 8 ) 74.0+2.6c(9) 74.8+8.la(4) 9 9 . 4 ± 3 . 5 n ( 8 ) Layer IV 109.0+4.8n(4) 9 7 . 5 ± 3 . 2 n ( 8 ) 50 .6±2 .9 C (9 ) 32.0+3. l c(4) 100+_4.8n(8) Layers V-VI 110 .4±4 .6 n (4 ) 1 0 5 . 5 ± 5 . 2 n ( 8 ) 54.3 + 3.4c(9) 58.2+1.9c(4) 1 0 6 ± 1 3 n ( 8 ) Layers I-VI 108 .2±2 .7 n (4 ) 1 0 7 . 6 ± 3 . 8 n ( 8 ) 66 .9±1 .3 C (9 ) 52 .1±2 .2 C (4 ) 9 5 . 5 ± 6 . 2 n ( 8 ) OT(-), optic tract lesion; FC, front cut; LGN(-), LGN lesion; SP, subcortical plate, n, P>0.05; a, P<0.05; b, P<0.005; P<0.0005 DISCUSSION Five alpha adrenoceptors have been cloned, namely three alpha-l's (la, lb, lc)i30 and two alpha-2's (2a, 2b)i08. Most recently, it was reported that alpha-lb but not alpha-la receptor was involved in PI turnoveri27. Prazosin does not distinguish these alpha-1 subtypes, so that the results reported above reflect overall changes of the alpha-l's. On the other hand, although rauwolscine has equal affinity to both 2a and 2b subtypes2 7, the autoradiograms of [3H]rauwolscine shown above probably represent alpha-2a sites since the majority of alpha-2 receptors in the cerebral cortex are believed to be alpha-2a2 7. Both alpha-2 subtypes are considered to be negatively coupled with adenylate cyclase via Gi, i.e., activation of either of the two can reduce cellular cAMP levels27,28. NA and ACh fibers arise from the locus coeruleus (LC)U2 and cells in the basal telencephalon^, respectively. Fibers containing noradrenaline or dopamine-[3-hydroxylase (DBH) have been widely studied in the cortex. In rat, NA axons derived from LC are observed in the cerebral cortex at very early developmental stage (El7) and cover the entire hemisphere by birthH6. in kitten visual cortex, endogenous monoamines are already measurable at birth and level of NA increases gradually with age and reaches 50% of its adult level by postnatal week 11-1398. This slow increase in endogenous NA at early postnatal age bears resemblance to the development of alpha adrenoceptors observed here. Interestingly, while the NA level keeps increase till adulthood, the ligand binding sites for the alpha adrenoceptors increases at early age followed by a dramatical drop in adulthood. This suggests that both subtypes of alpha receptors are transiently "over-expressed" at early postnatal ages. Compared with alpha receptors, a relatively early and fast increase in binding sites for [3H]pirenzepine was seen in neonatal cortex. Immunocytochemical visual ization of 16 cholineacetyltransferase (ChAT) in both cat179 and rat visual cortex45 indicated low concentrations of ChAT activity in the first week, with a significant increase during subsequent weeks. It was reported that unique long fibers with ChAT immunoreactivity run preferentially within layers I and IV of kitten visual cortex while, in adult cat, the superficial layers receive the strongest cholinergic innervation179. This redistribution of ChAT positive fibers resembles the changes in M l receptor binding pattern during development. Despite reportedly low levels of ChAT, the M l receptor-related intracellular signal pathway seems already fully functional at a neonatal age since high levels of phosphoinositide turnover can be induced by muscarinic agonists in both rat6,72 and kitten cortex 4 8 . Unlike alpha adrenoceptors, the binding of which in adult animals is only slightly higher than that in newborn kittens, the number of Ml binding sites in adulthood is four-fold that in newborn kittens (Fig.3 and Fig.6). Interestingly, the carbachol-stimulated PI turnover in adult cortex is similar to 4 8 or even lower than6.72 that in neonatal animals. The paradoxical changes in number of receptor binding sites and level of PI turnover stimulated by the receptor may indicate an alteration in efficacy of the coupling between the receptor and the second messenger system during development. None of the three receptor binding sites developed uniformly across the six cortical layers. This is manifested by difference in slopes of development curves for each cortical layer (Fig.3). A common feature of development shared by the three receptors is that layer IV presented the most binding sites early in life and that the binding densities were reduced in this layer greater than in other layers at late ages. This developmental pattern resulted in the adult animals showing the highest density of binding sites in the superficial cortical layers, which is consistent with previous results 1 4 5 . These laminar distributions of the receptors are also consistent with that of NA-containing terminals, which are 17 reportedly concentrated in upper and lower layers of cat visual cortex93. In all cortical layers, the a l sites showed a higher level of expression at postnatal day 1 and a slower rate of increase than did the a 2 or M l sites (Fig. 6). In the middle and deep layers, the increase in a2 binding sites was delayed at least a month beyond the time when the other two receptors started to increase. Differences were also noted in the falling phases of the developmental profiles. While the number of the two alpha receptors dropped rapidly, the number of M l receptors declined less and more slowly. Figure 7 illustrates the typical relative laminations of the three receptors in the visual cortex at various postnatal ages. It is clear that, in newborn kittens, the distribution of M l receptors is almost completely separate from that of alpha adrenoceptors. At later ages, the distributions of the three receptors are more or less overlapped. In adulthood, laminations of M l and a l receptors are almost complete ly identical . These data are consistent with the physiological results which showed synergistic effects of exogenous NA and ACh on single cortical neurons65. As both M l and a 1 receptors are coupled to the same PI second messengers, the colocalization of the two further suggests that the synergistic effects of the two may result from a boost in activation of this common second messenger system. These data also predict that, in newborn cortex, there would be little cooperation between adrenergic and chol inergic systems via M l and alpha receptors since the concentration of the latter is very low. Changes in laminar patterns of the binding sites during development indicate that different cell populations express the receptors at different ages. To wit, cells in the middle layer transiently show high levels of the receptors at early ages and cells in the superficial layers in turn become the populations with the most receptors in the adult. However, it is also possible that the 18 observed shift in laminar pattern of the binding sites may reflect a translocation of the receptors at subcellular level, i.e. terminals versus cell bodies that are located in different cortical layers. In addition, a shift in cell types expressing the receptors can also occur during development within the same cortical layers. This was observed in the postnatal ontogeny of beta receptors, which are expressed mainly by pyramidal cells in early life and by astrocytes and nonpyramidal cells in adulthood113. The autoradiographic technique utilized in the present work does not have sufficient resolution to detect such changes for alpha adrenoceptors. The most striking transient expression of the receptors was seen in the subcortical plate. Both ligands for the alpha subtypes heavily bound to the subplate region at early postnatal ages. The band of binding in this region gradually shrunk and disappeared by postnatal day 20 for oc2 receptors and by day 60 for a l . Based on results of [3H]thymidine birthdating experiments, Shatz and her colleagues reported that, in cat visual cortex, the subplate was formed between embryonic day 40 to 60 and cells in this region finally disappeared by 2 months after birth166. Neurons in the subplate contain various neurotransmitters and peptides and receive synaptic contacts at early developmental ages165. Although the role of these transiently existing neurons in the subplate remains to be elucidated, it is suggested that these neurons may be transient targets of geniculate afferents and of callosal projections during early developmental stages while the axons are waiting to invade the cortical platens. The time course of disappearance of the alpha binding sites in the subplate coincides almost exactly with that of subplate neurons, suggesting that the receptors probably are located on these neurons or on terminals making transient contacts on them, and that these receptors may be involved in the novel functions of the subplate cells. As alpha-2 receptors disappeared in this region much earlier than alpha-1 type, the two types of receptors might play distinct 19 roles in the subplate. The difference in time courses also indicates that either the two are located on separate cell populations on subplate cells with different life-times or expression of the two types of receptors are regulated by distinct mechanisms. Data from operated animals provided further information about the regulation of the receptors. Radioactivity of bound [3H]prazosin doubled in the subplate from 29 to 79 days following an early LGN lesion. Although it is difficult to estimate the exact extent of the lesion besides LGN, it should be safe to say that alpha-1 adrenoceptors in the subplate were up-regulated by abolition of subcortical inputs, particularly the geniculocortical input. This up-regulatory effect is not likely to be due to blockade of neuronal activity stimulated by vision since optic tract lesions, which abolished retinogeniculate input, did not affect the binding. Therefore, expression of alpha-1 receptors in the subplate may be regulated either by direct LGN afferents including its basal non-visual activity, or by other factors dependent on LGN activity. Alternatively, since the density of binding in the subplate on the lesion side resembled that of animals at younger ages, it is possible that these "extra" al binding sites are associated with a population of subplate cells that would have died under normal condition and that the increased binding was a consequence of delayed cell death following abolition of thalamic afferents on the lesion side. This latter possibility perhaps is less likely since Nissl staining did not show any notable difference between the two hemispheres in total number of subplate cells, a comparison of the number of subplate cells in the two hemispheres shall be necessary to distinguish between these two possibilities. The fact that binding for a l receptor sites increased after a LGN lesion strongly indicates that the majority of a l receptors in the subplate are not located on LGN terminals. The majority of a l receptors in the grey matter may also not be 20 associated with LGN terminals since a LGN lesion only resulted in a minor reduction (about 10%, p<0.01) of the binding density. Even this minor reduction was not seen in adulthood, suggesting that either this small portion of a l receptors only transiently exists on LGN terminals or the subcortical regulatory capability for the cortical a l receptors is age-dependent. Alpha 2 binding sites were more sensitive to LGN lesions than were alpha l's. In the same operated animals, much larger portions of [3H]rauwolscine binding sites were lost, particularly in the middle and deep cortical layers, after LGN lesions. It should be noted that the rapid increase of cortical a2 receptors does not start until the end of the first month, about two weeks after the operation was performed (Pll). Abolishing subcortical inputs prior to the development of receptors might be more effective in blocking the expression. Again, attenuated visual activity may be not responsible for the loss of a2 receptors, since there was no reduction following an unilateral optic tract lesion. The loss of a2 receptors was also age-dependent, as adult LGN lesions had no significant effect. As stimulation of a2 receptors results in reduced release of NA in the cortex, it is believed that some of these receptors are located on adrenergic terminals and cause an inhibition of NA release, i.e. serve as autoreceptorsi76. However, although the front cut should seven adrenergic fibers of the dorsal bundle, no marked loss in a2 sites was observed in the operated animals at any age. This could be that the lesion did not entirely abolish adrenergic fibers despite the substantial loss in AChE staining, an index of the total ACh fibers in the same bundle9. Another possible explanation is the number of autoreceptors is such a small proportion of the receptor number that any change in this subpopulation could not be detected by the technique used here. As there was often a slight shrinkage (about 10-20%) in the hemispheres of operated side, decrease in the binding might be 21 partially due to possible cell death. However, this may only contribute a small portion to the total loss of binding sites since number of cortical cells in operated side did not seem much less than that in the control side as assessed by Nissl staining. The effect of interruption of cortical inputs on development of M l receptors has previously been reported by our group173. It has been shown that isolation of a portion of the visual cortex from all afferents (undercut, see next chapter for detail) prevents the redistribution of the receptor, resulting in a high density of M l sites remaining in layer IV as seen in younger animals. This effect was not observed after adult lesions. Undercut reduced the bindings of [3H]prazosin and [3H]rauwolscine to a similar extent (data not shown) as observed in young LGN lesion animals. Differences in the effects of cortical input blockade on development of Ml receptor and alpha adrenoceptors in cat visual cortex suggests that expression of these sites is regulated in different ways. The present study showed both similarities and dissimilarities in the development of cortical M l cholinoceptor and alpha adrenoceptors. In summary, both alpha and M l receptors are maximally expressed at young ages. These receptors show similar changes in their laminar distribution patterns during development. Effects of blockade of subcortical input on these receptors are all age-dependent. On the other hand, timetables of development of the three receptors are quite different; they are therefore not colocalized at certain ages. In some regions, e.g. the subplate, M l and a 1 receptors never co-exist, indicating different roles played by the two. The effects of abolition of subcortical afferents on these receptors are quite different, suggesting that either they have different subcellular locations (e.g. terminals versus cell bodies) or that their expressions are regulated by distinct mechanisms. As both M l and a l receptors are gates to the same PI second 22 messenger pathway, the differences in development leads to a question of how these unique patterns match the development of the second messenger system to which they are coupled. This issue is to be addressed in the following chapters. 23 Chapter Three POSTNATAL ONTOGENY OF PROTEIN KINASE C IN THE VISUAL CORTEX INTRODUCTION Binding of ACh or NA to M l or a l receptors initiates the intracellular signal cascade that results in activation of protein kinase C (PKC) via PI second messenger system. The PKC family is one of the kinases that are especially abundant in the brain, particularly in the cerebral cortex and hippocampus81.138. This kinase was first reported in 1977 as a proteolytically activated kinase by Inoue ej a l . 9 1 . Two years later, it was further revealed that this kinase was calcium-activated and its activation was also dependent on phospholipid! 8 4. The function of the enzyme in intracellular signal transduction became clearer when it was found that activity of the kinase was greatly increased by diacylglycerol, an early product of inositol phospholipid hydrolysis induced by stimulation of cell surface receptors'85. Since then, six isozymes of the kinase (a, (31, [32, y, e and Q have been isolated68,82,105,160 and two additional mRNAs (8 and e')i 4 2 have been found in rat brain. By phosphorylating various proteins, PKC plays important roles in many cellular processes including cell differentiation, neurite outgrowth, synapse formation and receptor expression and regulationi4-52,53,69,90,96,99,1 18,1 19,137. The development of PKC has been studied in the brain, and evidence shows that its activity varies with age?0,83,134,180,207. In the present work, the postnatal ontogeny of PKC in the visual cortex was investigated with polyclonal antibodies against P K C 8 4 . The results show that PKC level in kitten visual cortex is not only regulated by developmental age, but is also use-dependent. 24 MATERIALS AND METHOD  Animals Twenty-one cats of various ages (from postnatal day 1 to adult) were deeply anesthetized and perfused through the left ventricle or aorta with a 4% paraformaldehyde and 0.1% glutaraldehyde solution. The perfused brains were post-fixed in 4% paraformaldehyde overnight at 4°C. Three cats were surgically manipulated before the perfusion. In one 14-day-old kitten and a second adult cat, a portion of visual cortex in one hemisphere was completely isolated from the rest of the brain by three 1 cm-deep scalpel cuts (undercut): two cuts extended from the midline to the lateral edge of the marginal gyrus, while the third cut ran parallel to the midline but was directed at a 45° angle in such a way that the white matter underlying the portion of visual cortex was completely cut. In another 14-day-old kitten, the front cut was performed (see chapter one for detailed surgical description). Both kittens were sacrificed at day 90. The adult undercut animal was allowed to survive for two weeks before it was perfused for immunocytochemical examination. Immunocytochemistry The tissue was cut into 50 |im sections with a vibratome and incubated with goat polyclonal antibodies against rat protein kinase C provided by F. Huang84 for periods ranging from overnight to 72 hours at 4°C. The dilution of the antibodies varied from 1:2,000 to 1:12,000 depending on the age of kittens. In general, the minimum antibody concentration required for optimal immunoreaction increased consistently after day 40. In animals that had been operated at an early age, a 1:8000 dilution of the primary antibodies was used in order to increase the contrast in immunoreactivity between the operated region and the control areas. Following 25 incubation with biotinylated rabbit-anti-goat antiserum (Biocan, 1:400) for two hours at room temperature, the sections were processed with the avidin-biotin (ABC) system (Vector) for one hour at room temperature, and the immunoreaction was visualized with 0.01% 3\3'-diaminobenzidine (DAB) and 0.003% H 2 0 2 . Adjacent sections were stained with cresyl violet to identify cortical layers. In control experiments, normal goat serum was substituted for the antisera in adjacent sections. No immunoreactivity was found in those sections, indicating that the immunostaining was not due to a cross reaction of secondery antibody. Sections used for light microscopic observations were then dehydrated with ethanol and mounted with DPX. Sections for electron microscopic observations were washed in phosphate buffer (PB, pH7.4), and postfixed for 30-60 min in 1% osmium tetroxide dissolved in 0.1M PB (pH 7.4). They were washed again in PB, then dehydrated in ethanol at concentrations of 50% for 5 min, 70% (containing 1% uranyl acetate) for 20 min, 90% and 95% for 5 min each, and 100% (2 x 10 min). They were then immersed in propylene oxide (2 x 10 min) and finally embedded on glass slides in Araldite (Durcupan ACM; Fluka) resin. Portions of interest were cut from the slides and reembedded. Serial ultrathin sections were cut on an ultramicrotome, mounted on Formvar-coated single-slot grids, and viewed under a Phillips 400 at 40 kv. C 26 RESULTS Light microscopic observations PKC-immunoreactive cells were mainly neurons rather than glia or other supportive cells based on the morphological characteristics of the cells. During postnatal development of the visual cortex, most of the PKC-immunopositive neurons in the grey matter were pyramidal cells of different sizes, although stellate cells were sometimes found. Stained cells in the white matter were concentrated in the subplate, especially in a region underneath the crown of the lateral gyrus. Although the staining intensity varied in individual cells, this may not be due solely to variations in antibody penetration of cells at different depths of tissue, since cells in the same focal plane also showed variable density of staining when observed by light microscopy. Hence the variation in staining density was at least partly determined by the level of kinase in each cell. The pattern of PKC immunoreaction in the visual cortex of kittens varied with age as follows: Postnatal day 1-4 (n=5). In kittens of this age group, both area 17 and area 18 were heavily stained (Fig.8ia,b)). In area 17 (Fig.8ia)), the most densely stained cells were concentrated in layers II, III, V and VI. In addition, the densely packed cells and fibers at the border region between layers I and II were strongly immunoreactive. Cells in this region had small somata with unclear profiles. No immunoreactive processes could be visualized originating from these somata. This densely-stained zone more or less remained until day 20. Moving from this region toward layers II and III, the density of stained cells decreased and the cells in these layers showed clearly-outlined somata with short apical dendrites. Layer IV showed less reaction product. This layer contained lightly-stained neurons and a matrix. The latter was also seen in layers II and VI but not in layers III and V. In area 18 27 (Fig.8 l b ) ) , layers II and III were most strongly stained. At the border of layer I/II, particularly, numerous cells with immunoreaction product were densely packed and, among these cells, many neuronal fibers were stained. Many densely-stained pyramidal cells were found in layers II, III and V. Compared to the neurons that were PKC-positive in area 17, immunostained cells in area 18 were larger, and the number of immunoreactive cells in layers II and III was much greater than in the corresponding layers of area 17. In both areas, the majority of stained cells were pyramidal, while in layer IV of area 17, cells with round cell bodies showed weaker immunoreactivity. At this age, particularly on day 4, areas 17 and 18 were also characterized by many large and small puncta that were densely stained and distributed mostly in layers V and VI (Fig.14). Layer I in both areas was pale but in some sections, a few small stained cells were sparsely scattered in this layer. Postnatal Day 10-20 (n=2). In area 17 (Fig.82a)), layers V and VI were most densely stained. Layers II and III were lightly stained. Unlike the staining pattern of day 1-4, layer IV appeared rather clear. Cells in this layer presented the least immunoreactivity if any within the cortex, and the matrix was not stained. As in area 17, cells in layer IV of area 18 (Fig. 8 2 b ) ) appeared the least immunoreactive, while layer I showed more intense staining than at earlier ages. There were more neuronal fibers, that were probably dendrites, exhibiting PKC immunoreactivity in both areas at this age than in one-day-old animals. Another characteristic of the staining pattern in the visual cortex at this age was the strong difference in intensity of immunoreactivity between areas 17 and 18 (Fig. 9). The reaction in the latter area was much stronger than in the former, both in the number of PKC-immunopositive neurons (especially in layer IV) and in the intensity of staining of individual cells. This difference remained to a smaller extent until at least postnatal day 28 90. As shown in Figure 82a,b), the immunoreaction product was restricted to the periphery of the nucleus and to dendrites (particularly in proximal portion of the dendrites), in both area 17 and area 18 of 10-day-old animal. Both light and electron microscopic observations (Fig. 12) suggested that the reaction product was concentrated in the perinuclear cytoplasm and dendrites but not in the nuclei. Postnatal day 30-60 {n-6) (Fig. 83a,b)). As seen at earlier ages, cells in layer IV of both area 17 and area 18 in this age group presented the weakest immunoreactivity relative to other laminae. This difference became greater after postnatal day 40, as evidenced by a continual reduction in immunoreactivity in the middle layer which extended to the lower part of layer III and the upper part of layer V at later ages. By counting number of immunoreactive cells in randomly chose regions, it was estimated that about 40% to 50% of the neurons, mainly pyramidal cells of various sizes, in superficial (II and III) and deeper ( V and VI) layers, exhibited strong immunoreactivity, while a further 20% of the cells showed mild staining. Compared to the extent of immunoreaction at day 10, a higher proportion of the most densely stained neurons were concentrated in layers II and III in area 17 at these ages. In area 18, by contrast, the relative intensity of staining in layers II and III decreased and most of cells with the strongest immunoreactivity were found in layers V and VI. Layer I in both areas exhibited only mild staining. Observations at higher magnification indicated that, unlike the situation at day 10, the entire cell body and the dendrites were labelled in each PKC-immunopositive cell. Staining in a kitten at day 40 gave rise to the most striking observation among animals in this age group. Long apical dendrites were clearly stained in area 17. The dendrites originated from the 29 cells in layer V and bifurcated in layers II,III,and IV of the cortex (Fig. 84a) and Fig. 10 for high magnification). Another interesting finding at day 40 is shown in Figure 11 at higher magnification. Many PKC-immunopositive axon-like fibers were found in both area 17 and area 18. These fibers, with swelling varicosities that were densely stained by the antibodies, run vertically in bundles between layer III and layer V. The long apical dendrites and axon-like fibers were occasionally seen in animals of P30 but not at any other ages. Postnatal Day 90 (n=3)and Adult (n=5). The staining profile of the polyclonal antibodies against PKC at day 90 resembled that of adult cats95(Fig. 85a,b,)). No marked difference in immunoreactivity between area 17 and area 18 was found in animals of these later ages. In general, the extent of the PKC reaction was much less than in the younger kittens, and a higher concentration of the primary antibodies was necessary to visualize the stained cells. The PKC-positive cells were distributed mainly in layer II, upper layer III, lower layer V and layer VI. Cells in the middle layers showed little immunoreactivity at normal dilution (1:4000) of the primary antibody. Some cells in these laminae, however, were weakly immunoreactive at a dilution of 1:2000, while background staining remained the same. Most of the stained cells at these ages were pyramidal, although some nonpyramidal cells were also seen. Examination by light microscopy indicated that the immunoreaction product in the majority of cells was located on the entire cell body, and probably on the membrane and in the cytoplasm, but not in the nucleus (although a few exceptions were noted). Numerous fibers were found in a variety of laminae. The diameters of these fibers suggested that they were more likely dendrites than axons. This was supported by electron microscopic examinations (see below). Electron microscopic observations 30 Cytoplasm. Fig. 12 shows an electron micrograph of an immunopositive cell body in layer VI of the visual cortex of a 10-day-old kitten labelled with the PKC antibodies. It should be noted that, in a young kitten, the brain tissue tended to be much more fragile than in an adult animal. Consequently, it was very difficult to achieve excellent preservation of the tissue (especially with the incubating time required for the immunoreaction). Nevertheless, some very useful observations were made. At the level of the cell body, end-product could be found throughout the cytoplasm (Fig. 12). The membranes of cytoplasm, mitochondria, Golgi apparatus, and endoplasmic reticulum were immunoreactive (arrowheads, Fig. 12). This staining pattern of the cell body was present in every age group studied, and no striking differences could be found during development. Dendrites. Positive staining was also found in dendrites (Figs. 13C and 14D). The end-product was seen mainly on the microtubules and on the membranes of dendritic mitochondria. No obvious changes in the distribution of the immunoreactivity in dendrites could be found among the different ages studied. Occasional synapses which were PKC-negative were found contacting positive dendrites (Fig. 13C). This type of synapse was more frequently encountered in the 30-60 day age group and in adult animals than in the group of newborn kittens (day 1-4). This is not surprising, since the overall number of synaptic contacts is very low in newborn kittens38. When a positive dendrite received a synaptic contact (Fig. 13C) the post-synaptic opacity was usually similar to or, in some cases, greater than that for a typical asymmetrical contact. Vesicle-containing profiles. During development, occasional vesicle-containing profiles were found to be immunoreactive for PKC. Fig. 13A,B illustrates two positive terminals making synaptic contacts with unlabelled postsynaptic elements. In such profiles, the 31 immunoreaction product was present in the presynaptic terminal, especially concentrated on the membranes of the synaptic vesicles. It is interesting to note that many synapses with a positive terminal had adult features: Terminals were usually large and contained many synaptic vesicles; they sometimes showed perforated synaptic contacts (Fig 13A) and had a well-defined postsynaptic opacity (Fig 13B). All synapses with immunoreactive presynaptic terminals found at this age (P30) were judged to belong to the asymmetrical category. Immunopositive vesicle-containing profiles making synaptic contacts were also found (but much more rarely) in newborn animals. In these animals, however, it was difficult to classify these synapses. Despite extensive searches in older animals (postnatal day 90 to adult), no PKC-positive terminals were found. It thus seems that there is a transient expression of PKC in synaptic terminals which peaks around postnatal day 30-60 in cat visual cortex. Undetermined profiles. Light microscopic observations of newborn kittens revealed that some large puncta (Fig. 14E) were heavily labelled. Electron microscopic observations showed that these large puncta were profiles that were different from the PKC-positive dendrites because they were much more immunoreactive for PKC, contained more mitochondria, and emitted branches (Fig. 14 A,B,C). Compare Figure 14A to D for example. Although these two profiles were taken from the very same section, the larger profile (Fig 14A) is much more immunoreactive and larger in size, and sends prolongations. Although we could not exclude the possibility that these large profiles were some type of mature dendrites, they were more likely immature ones since these profiles were not found in older animals. The prolongations could also be growing heads of dendritic growth cones, or be possibly dendritic spines, although no synapses or vesicle-containing profiles were seen in apposition to the ends of these branches. Alternatively, these profiles could be growth 32 cones from axons. Further investigations will be needed to identify these profiles clearly. The presence of these elements was observed only in young animals (newborn until postnatal group day 20) under both light microscopy and electron microscopy. In older animals, neither large immunoreactive puncta nor profiles with appendages could be seen. Operated animals Figure 15 compares immunoreactivity in control regions with that in the part of the visual cortex that was surgically isolated from other areas of the brain on postnatal day 14 in the animal that was perfused on day 90. The extent of reaction in the isolated area was strikingly stronger than in the corresponding area of the control hemisphere (not shown) and in the areas of the cortex surrounding the isolated zone. The isolated zone had both a greater number of PKC-positive cells and a higher density of staining. Interestingly, this increase in immunoreactive cells did not occur in layer IV. Here the low immunoreactivity level was comparable to that in the control tissue. Therefore, in spite of the increase in immunoreactivity in the isolated area, the laminar pattern of the staining was basically unchanged. In contrast to these findings, no alteration in PKC immunoreactivity was found in visual cortex that had been similarly isolated in adulthood95. Immunostaining in the animal in which modulatory inputs were interrupted by a front cut on day 14 also showed no difference between the operated hemisphere and the control side (data not shown) when the animal was sacrificed on day 90. 33 DISCUSSION The polyclonal antibodies used in the present study were raised in a goat and against rat brain PKC by Dr. F. Huang at NIH 8 4 . The antibodies bind to both purified or crude rat brain PKC and incubation of these PKC preparations with the antibodies showed 100% inhibition of the enzyme activity. Furthermore, these antibodies preferentially inhibit type I and type II PKC isozymes with a lower titer against type III. It has been shown that the activity of PKC in rat brain is low at birth. Thereafter, it gradually increases and reaches its maximum in the first few weeks of postnatal Hfe70.83,190,207. In cat visual cortex, Stichel 1 ? 8 reported that the activity of protein kinase C peaked at about 5 weeks of age and maintained this level into adulthood. Although the present immunoreaction data are not well suited for quantification of the activity of the kinase, it was noticed that the best immunostaining was obtained in tissues at ages from day 10 to day 40 and the immunoreactivity decreased at later ages. The decrease in the immunoreactivity was shown by: (1) the decline in the number of stained neurons and in the intensity of the staining after postnatal day 40 in the primary visual cortex, while the immunoreactive level in the hippocampus and other associated cortical areas showed little change; (2) the higher concentrations of the antibodies needed to obtain results in tissue older than 50 days postnatal (1:4000 for adult animals vs. 1:10,000 for younger kittens). Neither treatment with Triton-100-X (0.3 to 1%) nor longer incubation times (up to 72 hr.) noticeably improved the staining in adult tissue; (3) with the 1:8000 dilution of the antibodies used for the undercut animal on day 90, the isolated area of the visual cortex displayed good immunostaining while the immunoreaction in control areas was rather poor (again consistent with reduced immunoreactivity in the normal visual cortex at later ages). In 34 addition, the reduction in immunoreactivity at later ages was heterogeneous in different laminae, showing a greater decrease in the middle layers (especially layer IV) than in the superficial and deeper layers. Therefore, the reduction in immunoreactivity appears to indeed reflect a decreased amount of PKC isozymes detectable by our antibodies in adult visual cortex, although the effect of poorer penetration of the antibodies in adult tissue cannot be completely ruled out. Similar results were observed by Stichel and S i n g e r i 8 0 , and in this laboratory (data not shown) with a monoclonal antibody purchased from Amersham that appears to be more specific for PKC isozyme III 1 9 5. Immunoreactivity of this latter monoclonal antibody was found in certain populations of neurons only at younger ages and decreased later in postnatal life. It is not clear whether the decrease in PKC immunoreactivity results from the disappearance of a particular population of cells or from the reduced expression of the kinase in these cells. However, since cell death occurs mainly at earlier developmental stages, it is more likely that intracellular levels of PKC (at least the isozyme(s) recognized by our antibodies) vary, or the kinase is only transiently expressed in certain populations of neurons in the postnatal visual cortex. One of the regions that transiently displayed a PKC immunoreaction product was the border of layers I/I I, presumable cortical plate, where there was a group of cells that appeared morphologically immature in newborn kittens. This region was heavily stained in neonatal kittens; immunoreactivity was then gradually lost, finally disappearing by day 20. It is believed that cells in this region are the latest-generated ones, and are predominantly destined for layer IIH6. Disappearance of this highly immunoreactive region may result from the migration of the cells in the region. PKC has been shown to be important in stimulation of neuronal sprouting80,90 and in regulation of cytoskeletal processes26. In accordance with these functions, the high level of expression of 35 PKC in this population of cells in newborn kittens suggests that the kinase may participate in maturation of the neurons in this region. In another example of transient appearance of PKC immunoreactivity, the reaction product was seen in the long apical dendrites of some pyramidal cells in layer V only in animals of postnatal ages four to seven weeks, particularly around day 40. Interestingly, the same type of cells were observed by Tsujino et all89 using a monoclonal antibody directed against PKC g-isozyme in rat neocortex. It is therefore possible that the PKC immunoreactivity seen in these dendrites represents the g-subtype, which has been shown to be expressed slowly and does not reach its maximum level until 3-4 weeks postnatal in rat brain70,83. Furthermore, the disappearance of the stained long apical dendrites at later ages suggests that this subtype of PKC is transiently expressed in this particular population of pyramidal cells, or alternatively, that the isozyme changes its subcellular distribution during development of kitten visual cortex. More evidence for the developmentally-regulated expression of PKC at the subcellular level was provided by the electron microscopic observations. A PKC immunoreaction product could be localized on presynaptic terminals in the cortex in neonatal animals and was most frequently encountered around day 30-40. It was not found, however, in presynaptic terminals of adult tissue^. On the postsynaptic side, PKC immunoreactivity was consistently localized i n dendrites, in perikarya, and on the postsynaptic membranes of both young and adult animals. The finding of the large unclassified puncta with strong immunoreactivity is interesting. Morphologically, they resembled dendrites rather than axonal terminals, but were characterized by prolongations and microtubules. In particular, they appeared only in young kittens, and showed denser immunostaining than normal dendrites. These features imply 36 that they were possibly growth cones of neurites containing high concentrations of PKC, whose roles in elongation process have been studied elsewhere 80,90,135. It is unclear why the kinase is only transiently seen in the presynaptic membrane, but persistently found in postsynaptic sites and perikarya. The peak of expression of PKC at the presynaptic sites (P30-40) occurred at the same time as the maximum development of synapses in the visual cortex. It has been shown that the number of synapses increases during the first few weeks, and peaks during the critical period38. Meanwhile, use-dependent adjustment of synapses to form ocular dominance columns also takes place at an early developmental stage. It is most active at around 3-4 weeks of age43.60,87 and then decreases again. The correlation between appearance of presynaptic PKC and development or stabilization of synapses strongly argues that PKC in the presynaptic location may participate in synapse formation or modification in developing visual cortex, as has been suggested for long term potentiation (LTP) in the hippocampus! 10.1 15,1 22,123. In LTP, the neurotransmitter that is involved in synaptic modification is mainly glutamate3 7. Interestingly, all synapses with presynaptically located PKC-immunoreactivity found in the visual cortex of young animals were classified as asymmetric. On the assumption that terminals containing round vesicles and making asymmetrical contacts are e x c i t a t o r y 3 6 , it can be suggested that PKC-positive synapses use an excitatory neurotransmitter within the cortex. Furthermore, the fact that the end-product is concentrated on the membranes of synaptic vesicles agrees to the note that synapsin I, a synaptic vesicle associated protein, is one of the substrates of PKC and implies that PKC may participate in neurotransmitter release during synaptic formation in developing visual cortex. A similar function for PKC has been reported in LTP studies of the hippocampusi J 9 . However, unlike the kinase in hippocampus and cerebellum, where a high 37 plasticity remains and PKC has been reported to be present on both pre- and post-synaptic membranes in adulthood1 0 6 , 2 0 3 , the presynaptically located PKC in the primary visual cortex appears to be diminished once the use-dependent synaptic organization is established. It is interesting that the transient presynaptic location of PKC in the developing visual cortex is temporally correlated with transient expression of GAP-43-like immunoreactivity in this structure12. Since GAP-43 is known to be a substrate for PKC^. i se , it would be interesting and important to determine if they are localized within the same specific terminal sites. Double-label electron microscopic studies would be required to answer this question. The argument that PKC is involved in use-dependent synaptic organization is more directly supported by the results of the undercut experiments. These results indicate that the number of immunopositive cells in layers II, III, V and VI of the isolated area was much greater than in the control area for the animal that had been undercut at postnatal day 14, but not for the animal operated in adulthood. It was clear that the level of the immunoreactivity in the isolated area at day 90 closely resembled that in younger animals. Therefore, in this portion of the visual cortex, the decrease in immunoreactivity of PKC that normally occurs during postnatal development was interrupted by the isolation at an early age. This suggests that neuronal activity from subcortical and/or other cortical areas is responsible for the decline in the level of PKC in the normal cat visual cortex. This effect of afferent activity seems to be absent in adulthood, at least as measured two weeks after the undercut95. The level of the kinase in the cortex may well be regulated by the input at an early postnatal developmental stage, i.e., the critical period, when synaptic organization in the visual cortex is most plastic and most susceptible to visual experience. I hypothesize that PKC 38 may be involved in synaptic formation in developing visual cortex and, as a consequence of establishment of the synaptic connections, continuing neuronal activity then diminishes the level of the kinase. It is interesting to note that regulation of muscarinic receptors by input activity also appears to occur. As mentioned earlier, [ 3 H]QNB binding in the isolated kitten visual cortex showed a laminar pattern that resembles that of immature animals. This will be fully discussed later in Chapter 6. What component(s) in the external input to the visual cortex are responsible for regulation of the PKC level? The undercut procedure isolated the cortical area from two types of input, one from callosal afferents and from other cortical areas including neuromodulatory inputs of the forebrain, and the other from the thalamic nuclei, primarily the lateral geniculate nucleus. Since no effect was found in the contralateral visual cortex (in which the callosal projection from the operated hemisphere should also be abolished in the corresponding area of the unoperated cortex), the callosal fibers are unlikely to be involved in the regulatory effects of the undercut. The input from the forebrain also does not appears to be crucial, since the "front cut" severed the modulatory neuronal pathways from the forebrain early in life yet no alteration in PKC immunoreactivity was seen. Hence, the component most likely to be responsible is the geniculate input. If the geniculate activity directly regulated the level of PKC detected by the current antibodies in the developing visual cortex, the layer most affected by the undercut would have been layer IV, the major termination lamina of the geniculate input. As shown in Fig. 15, there was no obvious difference in this layer between the isolated zone and the control areas, although the immunoreactivity in other laminae was strikingly higher and resembled the level found in younger animals. Thus, it is more likely that the geniculate input indirectly regulates the level of PKC in the visual cortex. The lack of 39 geniculate input may result in reduced neuronal activity in layer IV of the isolated area, which alters the intrinsic neuronal activities of those superficial and deeper layers which are innervated by the cells in layer IV. As a consequence of this change in activity, the decrease of PKC in these laminae (which occurs in normal tissue) is blocked, and a high level of the kinase is maintained. PKC level could be regulated at various stages from the gene expression to the enzyme degradation. An experimental study using in situ hybridization with cDNAs for PKC mRNAs to detect the level of expression of the kinase in the developing visual cortex would be valuable. The results of immunocytochemistry from the animals operated as both young and adults also suggests that PKC labelled by our antibodies is localized in neurons of the visual cortex but not on the terminals of subcortical inputs or association fibers. This is indicated by the increase or lack of change in PKC immunoreactivity after undercutting of the visual cortex. Although further investigations are necessary, the increase in the level of PKC immunoreactivity in the area isolated from neuronal activities of other CNS structures at early ages further suggests that the change in PKC level in the developing visual cortex is strongly influenced by the postnatal experience of the animal, and is not determined solely by genetic factors. 40 Chapter Four CALCIUM/CALMODULIN DEPENDENT KINASE II IN CAT VISUAL CORTEX AND ITS DEVELOPMENT INTRODUCTION PKC is not the only kinase which undergoes changes during cortical development. Ca2+/calmodulin-dependent kinase II (CAM-K II) represents another important calcium dependent kinase. This is an oligomeric enzyme consisting of subunits of various sizes6i.i04. it was first purified from rat brain in 198311. Since then, five subunits of C A M - K II, the a , f3/(3', y, and 5 subunits have been isolated34,i32,i62,i88. Among them, the a and pVp" subunits are expressed primarily in brain!88 in great abundance. It is estimated that the kinase represents 0.3% of total protein in brainii and is especially concentrated in postsynaptic densities, where it comprises up to 30-50% of the total protein 101.103. CAM-K II has a fairly broad spectrum of substrates including synapsin I, tyrosine hydroxylase, and m i c r o t u b u 1 e - a s s o c i a t e d prote in ( M A P -2)34,102,104,132,162,164,183,194,196. Accordingly, the kinase has been suggested to play important roles in many neuronal functions, such as regulation of catecholamine synthesis, facilitation of neurotransmitter release, and strengthening o f synaptic transmission74,l02,i20,i22. Considering the abundance of CAM-K II in the brain, its calcium-dependent activity, and the suggested important role the kinase plays in neuronal plasticity in the hippocampus3,i9,i20,i22,i35, one might expect that CAM-K II would be involved in the development of the visual cortex as an effector of calcium-related intracellular signal cascades. It is therefore interesting to understand the postnatal ontogeny of the kinase in kitten visual cortex and to compare its development with that of PKC and other elements in 41 calcium dependent signal pathways. Here, a monoclonal antibody against the oc-subunit of CAM-K II was utilized to localize the kinase and the postnatal development of immunoreactivity of the kinase was studied at both the light and electron microscopic levels. 42 METHODS AND MATERIALS  Animals Fifteen kittens of various ages (postnatal day 1, day 4, day 14, day 15, day 24, day 30, day 40, day 90 and adult) were used in the experiments. In one kitten, the lateral geniculate nucleus (LGN) was completely removed by an extensive lesion of the ipsilateral thalamus at day 14. The kitten was allowed to survive for another 11 weeks before it was sacrificed at postnatal day 90. In most cases, the brain tissue was prepared as described in chapter two. In addition, one 14-day-old and two adult animals were perfused with 500 ml of 2% l-ethyl-3-(3-dimethylamino-propyl)carbodimide (Sigma) and 2% paraformaldehyde in PB followed by another 500 ml of buffered (pH 7.4) 4% paraform-aldehyde (EDC-PFA). We chose this method since adding 0.1% glutaraldehyde into 4% paraformaldehyde resulted in reduced immunoreaction with the antibody. Compared with conventional PFA perfusion, the EDC-PFA perfusion did not change the laminar pattern of the immunostaining (Fig. 16), tended to allow more cellular staining, and gave better preservation of the ultrastructure. Therefore, tissue prepared by this procedure was used for electron microscopy. Immunocytochemistry The monoclonal antibody against the cc-subunit of CAM kinase II was generously provided by Dr. M. Kennedy51. The tissue was cut into 50 \im sections with a vibratome and incubated with the antibody (1/500) and 4% normal horse serum in PB at 4°C overnight. The sections were then processed with conventional procedures for both light and electron microscopic, observations (see chapter two). No immunoreactivity was found in these sections when the primary antibody was omitted during the process. 43 44 RESULTS C A M - K II in adult cat visual cortex(n=4): Fig. 17a is a representative section illustrating the distribution of CAM-K II positive neurons in cat visual cortex. Neurons with immunoreactivity for CAM-K II were found in all cortical laminae in adult cat. In particular, a group of nonpyramidal cells in lower layer IV were strikingly stained (Fig. 17b) while some lightly labelled cells could be seen in upper layer IV and layer V. Cells in layers II and VI also presented strong immunoreactivity. In layers III and V, there were a few large pyramidal cells with weak immunoreaction product. In general, the incidence of nonpyramidal cells appeared higher among the total population of labelled neurons than pyramidal cells. No immunoreactive glia-like cells were encountered. The immunoreaction product was mainly present in cell bodies, and dendrites. Axons were not seen under light microscopic observation although axonal terminals were found at the electron microscopic level (Fig. 22). CAM-K II in developing visual cortex Postnatal day 1-4 (n=3)(Fig. 18a-b): By postnatal day 4, strong C A M - K II-immunopositive cells were found in layers V and VI, although numerous lightly stained cells were also found in upper layers of the visual cortex. Pyramidal cells in layer V probably are the earliest cells expressing CAM-K II immunoreactivity since in one of the two animals studied at day 4, large pyramidal cells in layer V were more distinctly stained than other neurons (Fig. 18a) and this pattern somehow resembles that seen in a PI kitten (not shown). Neuropil and matrix in the middle layers also showed strong immunoreactivity, while the most superficial lamina (layer I) was rather pale at day 4. Many bipolar-like cells in the white matter were also found to be immunopositive. Most of these cells were 45 present in the region closely adjacent to layer IV. Postnatal day 14-24 (n=3) (Fig. 18c,d): Many neurons of various sizes and morphologies showed CAM-K II immunoreactivity in animals of this age group. These neurons were located through layers II to VI with particular concentration in layers II to IV. Many pyramidal cells in layer III and V presented immunoreactivity, while non-pyramidal cells in layer II and IV were also stained. Numerous densely labelled particles of various sizes were seen in the visual cortex of this age group, especially around day 24 (fig. 18d and Fig. 19). Most of these particles were found in the superficial and middle cortical layers but not in the deep layers. Under higher magnification (Fig. 19), these particles appeared to be terminals of fibers, possibly dendritic growth cones and/or axonal terminals. This conclusion was supported by electron microscopic observations (data not shown). Postnatal day 30-40 (n=3) (Fig. 18e): Unlike the staining pattern observed at earlier ages, cells in layers V and VI appeared much less strongly immunoreactive at this age. Along the border of layer IV/V, large pyramidal cells were occasionally positive for CAM-K II. However, the immunoreaction in these cells was much weaker than that in cells of other layers. Many large pyramidal cells with strong C A M - K II immunoreaction product were seen in layer III, while small non-pyramidal cells in lower layer IV were also densely stained. Postnatal day 90 (n=l) (Fig.l8f): The most surprising finding in this animal was a reappearance of many immunopositive neurons in the deeper layers that were barely stained in animals at postnatal days 30-40. These neurons were also seen in control hemisphere of the operated animal (perfused at P90) and adult animals. However, the intensity of the immunostaining was weak in lower layer Ill/upper layer IV and layer V. A group of cells in deeper layer IV was most densely labelled. This laminar pattern was almost identical 46 to that of adult animals. Operated animal (n=l) (Fig. 20): In the animal in which an ipsilateral LGN lesion had been performed at 14 days of age and which was allowed to survive until day 90, C A M - K II immunoreactivity in the visual cortex showed striking differences between the two hemispheres. On the control side, the staining pattern resembled the normal adult pattern, with most immunopositive neurons concentrated in layers II, deeper IV, and VI. On the operated side, the number of cells that presented CAM-K II immunoreaction product was approximately double that of the control cortex. However, upper layer IV and layer V still showed the least immunoreactivity, suggesting that the laminar pattern appropriate to the animal's age was maintained. Electron microscopic observations. Cytoplasm. Figure 21 shows electron micrographs of immunopositive cell bodies taken from layer VI of the visual cortex of an adult cat ( A ) and of a 4-day-oId kitten (B) labelled with the CAM-K II antibody. In the cell body, end-product could be found throughout the cytoplasm but not on plasmic membrane. While the membranes of mitochondria, Golgi apparatus, and endoplasmic reticulum were immunoreactive, no obvious immunoreactivity could be found inside these cell organelles. The nucleoplasm also presented some degree of immunoreactivity. This staining pattern of the cell body was present in every age group studied, and no striking differences could be found during development. Dendrites. Positive staining was also found in dendrites at the different ages studied. In adult tissue (Fig. 22), the end-product was seen mainly adjacent to the synaptic contact. It formed a dense and wide aggregate, which was many times larger than the non-labelled post-synaptic opacity. In addition, the kinase was localized along the 47 dendritic microtubules as seen on transverse (Fig. 22A) and longitudinal (Fig. 22B) sections of immunopositive dendrites. Dendrites from young tissue showed a different pattern. The postsynaptic density was not particularly labelled and the kinase did not seem to concentrate on the microtubules. In fact, the kinase was rather uniformly distributed in the dendritic trunk. Vesicle-containing profiles. In the adult cortex, many vesicle-containing profiles were found to be immunoreactive for CAM-K II. Figure 23a illustrates a positive terminal making synaptic contacts with unlabelled postsynaptic elements in layer III of the visual cortex. The end product was concentrated mainly on the membranes of the synaptic vesicles and mitochondria. Adult immunopositive terminals were usually large and contained many synaptic vesicles. When the synaptic contact was cut at an angle appropriate for visualising the synaptic cleft, we found primarily asymmetrical contacts, i.e., the postsynaptic membrane had a well-defined postsynaptic opacity (Fig 23a). Immunopositive vesicle-containing profiles making synaptic contacts were also found in younger animals. The kinase was always present on the synaptic vesicle membranes. It is difficult to evaluate quantitatively the number of immunopositive synapses during development but, since the number of positive varicosities is higher in the day 20-40 age group as indicated by both electron and light microscopic observations, it is probably that the number of CAM-K II vesicle-containing profiles is much higher at these ages. Growth cones. At very young ages (day 1 and day 4) , large varicosities were also found in the subplate region and layer VI. Electron microscopic observations showed that these large puncta were profiles which contained many vacuoles and mitochondria (Fig.23b). Ultrathin serial sections showed that many of them had a bulbous ending attached to a long tail. For these reasons, we believe that these profiles are neuronal growth cones. 4Q DISCUSSION The monoclonal antibody used in the study showed high affinity to the a subunit of rat brain CAM-K II (50 kD) and recognizes a single protein band of the same molecular weight in crude rat brain homogenates^^. Our results show that, in cat visual cortex, this antibody gives rise to a specific immunoreaction and that intensity of staining seems to depend on concentrations of the antibody in a range of 1/2000 to 1/50 dilution. The pattern of the immunoreactivity in the visual cortex varies with development. The kinase appears to be highly expressed at early postnatal developmental stages and to decline thereafter, at least in some subpopulations of cortical cells, especially the pyramidal neurons in layer V. Subcellularly, immunoreactivity of the kinase was found in locations that are closely related with the functions revealed / n vitro. It has been suggested that the kinase phosphorylates synapsin I, a synaptic vesicle-associated phosphoproteini i , to facilitate neurotransmitter release?4. This is consistent with its localization in presynaptic terminals and, especially, on membranes of synaptic vesicles. The kinase can also phosphorylate MAP 2 at a high rate n . i 6 i to suppress microtubule assembly and actin filament gelation. This results in regulation of the neuronal cytoskeleton, which can affect neurite outgrowth and modulate the shape of both dendrites and dendritic spines 5 1 . Our findings that the immunoreaction product of the kinase was concentrated on microtubules agree with this possible function. Strong immunoreactivity was also found in the post-synaptic opacity. This is consistent with the abundant evidence that CAM-K II is a major postsynaptic density protein 101,102. The pattern of immunoreactivity in adult cat visual cortex is similar to that found in monkey with the same antibody?5. in the 49 monkey visual cortex, small nonpyramidal cells with C A M - K II immunoreactivity were found in layers II,III, VI and low layer IV( IVCb) and few cells were found in layer V. It is interesting to note that, in cat visual cortex, cells in lower layer IV present strong immunoreactivity while this layer contains moderately-stained cells in normal monkey visual cortex. Particularly, after unilateral enucleation, cells of this sub-lamina that were driven by the deprived eye showed an increased immunoreactivity?5. With a polyclonal antibody62 or monoclonal antibodies against both a and P subunits144, a different distribution of immunoreaction product was found in rat visual cortex. In these cases, the most prominently stained neurons were pyramidal cells in layer V and the least prominently labelled were the cells in lower layer IV. The distinct distributions of immunoreaction product with the various antibodies may reflect differential expression of various subunits in different cell populations. As the a and P subunits are present in a 4:1 ratio in rat forebrain and a 1:4 ratio in cerebellum 1 2 5 . 1 2 9 , it is possible that the two subunits may also present in different cortical cell populations with different ratios. Thus, cells that contain the kinase, but with a high degree of p subunit composition, might show only weak staining or be undetectable with the antibody utilized in the present experiments. Thus, the low levels of C A M - K II-immunoactivity found in pyramidal cells of adult cat visual cortex could be due to a decline in the ratio of a to p subunits in this cell type or to a lower overall level of the kinase. The different patterns of immunostaining for C A M - K II at different ages strongly imply that the kinase is involved in the cortical development. As many pyramidal cells in deep layers were densely stained at early ages, and the staining of these neurons was reduced at a later stage of the critical period, the kinase may play a specific role in the development of this cell type. In addition, the kinase immunoreactivity was highly concentrated in growth cone-50 like terminals at early postnatal ages. The labelling in fact reached its height at the peak of the critical period for cortical plasticity, suggesting that C A M - K II is involved in neurite outgrowth, synaptogenesis or the activity dependent synapse elimination that occurs at this time. It is interesting that PKC, another Ca++-dependent protein kinase, has an age-dependent, but different subcellular and laminar distribution in the developing cat visual cortex95,i80. I have shown in the previous chapter that PKC is mainly located in superficial (II/III) and deep (V/VI) cortical layers but not in the middle layers (IV) of adult cat visual cortex. In adult animals, PKC was found at postsynaptic locations only, and it was particularly concentrated on cytoplasmic membranes of cell bodies, where C A M - K II immunoreactivity was rather weak. However, there are some similarities in the distribution of the two kinases. In young animals, when PKC is transiently found at presynaptic locations, it is also concentrated on synaptic vesicles involved with making asymmetric synapses, just as is CAM-K II. These similarities suggest that presynaptically-located PKC and CAM-K II may both be involved in excitatory neurotransmitter release. Since PKC was only found presynaptically in visual cortical neurons near the height of the critical period (postnatal day 20-40) while presynaptic CAM-K II was found in both young and adult animals, the presynaptic PKC may play some special role in synaptic formation during the peak of synaptogenesis and CAM-K II may be involved in a more general way with neuronal plasticity in the cortex. As early abolition of LGN input results in menifest increased levels of both PKC (chapter 2) and CAM-K II immunoreactivities in the developing cortex, it is unlikely that these kinases are associated directly with the thalamic afferent terminals, rather they are probably both localized in intracortical neurons. Furthermore, these 51 results indicate that the levels of immunoreactivity of the two kinases during development are down-regulated by the subcortical input. Interestingly, the increase in CAM-K II immunoreactivity is seen in all cortical layers except layer V, implying that the level of the kinase in different layers is regulated by different factors. As mentioned earlier, the dependence of CAM-K II down-regulation on subcortical input has also been observed in monkey visual cortex75. The elevation of CAM-K II immunoreactivity may reflect an increase in level of the kinase, implying that the expression of the kinase is regulated by the subcortical activity. Alternatively, since the antibody binds twice as well to the phosphorylated a subunit as to the unphosphorylated form5*, so the increase in immunoreactivity may reflect an increased ratio of phosphorylation/dephosphorylation of the kinase in the layers, suggesting that this ratio may be regulated by the LGN input. To distinguish these two regulatory mechanisms, measuring mRNA levels of the a subunit in control and lesioned hemispheres will be valuable. 52 Chapter Five POSTNATAL DEVELOPMENT OF INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS: A DISPARITY WITH PROTEIN KINASE C INTRODUCTION Calcium dependent protein kinases are activated by elevated intracellular calcium levels. Intracellular calcium concentrations can be raised by either Ca+ +-influx from extracellular space or C a + + -release from intracellular pools. One type of such intracellular stores is inositol 1,4,5-trisphosphate (IP3)-sensitive. IP3 is generated by breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) when a specific phospholipase C (PLC) is activated by receptor/ligand interaction. This receptor-stimulated phosphoinositide turnover was first reported by Hokin and Hokin in 195578. Based on accumulating evidence from a variety of tissues, Berridge and Irvine suggested IP3 as a second messenger that links between receptor stimulation and increased intracellular calcium^5. This hypothesis has been widely accepted. Intracellular application of IP3 induces a calcium activated potassium current in neuroblastoma cells77 and neurons of the dorsal raphe nucleus59. IP3-induced C a + + release was directly evidenced in Aplysia bag cells, w h i c h showed a long-lasting e l e v a t i o n of intracellular calcium following IP3 injection55. Recently, receptors for I P 3 have been purified and cloned from r a t 1 2 8 and mouse c e r e b e l l a 6 3 . The molecular weight of the receptor protein is about 260 kDa and it possibly contains transmembrane regions that may form the calcium channel intrinsic to the receptori28. Stimulation of the purified IP3 receptors (IP3R) can cause C a + + flux through reconstituted lipid vesicles54. The localization of IP3Rs is best known in Purkinje cells, which have the highest concentration of the receptor. Immunocytochemical studies using an antibody against 5 3 IP3R showed that the receptors are located on special portions of the e n d o p l a s m i c r e t i c u l u m membranes and p o s s i b l y other smooth surfaced structures, particularly near the n u c l e u s e s . Generated IP3 can be e i t h e r f u r t h e r p h o s p h o r y l a t e d i n t o inositol - 1 , 3 , 4 , 5 -tetrakisphosphate (IP4) by inositol - 1 , 4 , 5-trisphosphate - 3-kinase or d e g r a d a t e d i n t o i n o s i t o l - 1 , 4 - p h o s p h a t e by i n o s i t o l - 5 -p h o s p h a t a s e s 16,57. The IP4 has been recently reported to act as another second messenger in regulating extracellular C a + + influx in various types of cells3°,50,56,159. Since the breakdown of PIP2 generates diacylglycerol (DG) and IP3 s i m u l t a n e o u s l y 16, it leads to a bifurcating signal transduction pathway. Since IP3-stimulated calcium elevation may facilitate the activation of PKC by DG, it is believed that the two limbs of the PIP2 signal pathway cooperate with each other to regulate cellular activity by a c t i n g s y n e r g i s t i c a l l y i 3 6 . To demonstrate this contention, evidence of colocalization of the two second messenger receptors is necessary. In rat brain, Worley et al. reported that PKC was enriched in certain areas which lack IP3 r e c e p t o r s 2 0 6 , suggesting some independence between them. As has been described in previous chapters, PKC is transiently expressed in certain populations of neurons in the developing cat visual cortex, especially between 3-11 weeks postnatal during the c r i t i c a l periodi34,i80. it has also been reported that, in kitten visual cortex, the PI turnover stimulated by a subtype of glutamate receptor reaches its peak during the c r i t i c a l p e r i o d 4 8 . In an attempt to determine whether development of IP3R matches that of glutamate-sensitive PI turnover and whether PKC and IP3 may act synergistically in cortical development, [ 3 H]IP3 and [ 3 H]phorbol - 1 2 , 1 3-dibutyrate ([3H]PDBu) were used to localize IP3 receptors and PKC, respectively, in adjacent sections of the developing kitten visual cortex (areas 17 and 18) and hippocampus. 5 4 MATERIALS AND METHODS Fourteen cats of various ages (from postnatal day 1 to adult) were used in the present experiment. For characterization of [ 3 H ] I P 3 binding, the visual cortex (areas 17 and 18) was dissected and homogenized in 20 volumes of ice-cold buffer (50 mM Tris-HCl, pH 8.5, 1 mM EDTA). Tissue was pelleted by centrifugation (10,000 g for 5 min.), and resuspended in 50 volumes of the buffer. Saturation binding assays were run by incubation with [ 3 H]IP3 (NEN, specific activity: 17 Ci/mmol) at concentrations of 0.7 to 50 nM in 1 ml of the same buffer for 20 min. at 4°C, and were stopped by centrifugation (10,000 g for 1 min.). To evaluate non-specific binding, 10 mM of unlabelled IP3 (Sigma) was added to the incubation medium. The pellets were washed in 1 ml of the buffer and resuspended in 2 ml of Formula 963 (NEN) for counting of radioactivity in a Beckman LS 2800 liquid scintillation counter. Brain tissue for autoradiography was prepared as described in chapter one. For the IP3 receptor, sections were incubated with 20 nM of [ 3H]IP3 in Tris buffer (50 mM Tris-HCl, pH 8.5, 1 mM EDTA) for 20 min. at 4°C, followed by 2x30 s wash in the same buffer. For [3H]PDBu binding, adjacent sections were incubated with 20 nM [3H]PDBu (NEN, specific activity: 13.2 Ci/mmol) in Tris-HCl, pH 7.4 for 6 hours, .a.t 4 ° C 1 3 4 followed by 3x10 min. washes in the same buffer. Sections were rapidly dried in a stream of room air and apposed to LKB Ultrofilm for 7 weeks for [ 3H]IP3and four days for [ 3 H]PDBu. The autoradiograms were analysed with computer densitometry as described in chapter one. In both cases, non-specific binding was less than 15% of the total binding as determined by adding 10 u.M of unlabelled IP3 or unlabelled PDBu (both from Sigma) into the incubation media. 55 RESULTS The kinetic characteristics of [3H]IP3 binding are shown in figure 24. Binding reached saturation above a concentration of 40 nM. An Eadie-Hofstee plot of the saturation data indicates that [3H]IP3 binds to a single class of sites with a Kd of 15.13+.1.28 nM and a Bmax of 2.38 +0.13 pmol/mg protein. This value is compatible with previous data from rat cerebella205. In newborn kittens, a low density of [3H]IP3 binding (Fig.25, 26) was found in the superficial layers of the cortex. In general, however, few binding sites for [3H]IP3were observed until about postnatal day 20. Between 20-30 days of age, the beginning of the critical period, the density of [3H]IP3 binding sites rapidly increases in the visual cortex and other areas of the brain, including the hippocampus. Around the peak of the critical period (P30-40), the superficial cortical layers and layer IV were densely labelled, while layers V and VI showed the lowest density of silver grains. The density of the binding in the cortex was reduced in adulthood. Unlike IP3 receptors, the [3H]PDBu-binding site, labelling PKC, was highly expressed in newborn kitten cortex and altered its distribution in early development to arrive at its mature cortical laminar pattern by postnatal day 30-40. The binding for [ 3 H]PDBu increased during the critical period and then reduced in adulthood (Fig.25, 26). Strikingly, the laminar distribution of [3H]PDBu binding sites was quite different and actually somewhat complimentary to that of IP3 receptors in both developing and adult visual cortex. The lowest [3H]PDBu binding sites in animals older than P40 were found in layer IV, the site of the highest density of IP3 receptors. Similarly, the density of [3H]IP3 binding was low in the superficial and deeper layers where strong [3H]PDBu binding was present. Development of both [3H]IP3 and [3H]PDBu binding in the visual cortex were analysed quantitatively with computerized densitometry 56 (Fig. 26). The binding for [3H]PDBu increased within first 3-4 weeks of life whereas the increase in [ 3 H ] IP3 binding did not occur until postnatal day 20. However, binding for both of the ligands peaked in the middle of the critical period and then declined in adulthood. Expression of [ 3H]IP3 binding sites in the visual cortex at postnatal day 30-40 is about 9-fold that in newborn kittens, while binding of [ 3 H]PDBu only increased 1.6-fold from PI to the peak of critical period. In adult visual cortex, the total binding was reduced by _20% for [3H]PDBu and by _45% for [3H]IP3, compared to their maximal levels at P30-P40. In the hippocampus, the binding patterns of [ 3H]IP3 and [ 3H]PDBu were also distinct and largely complementary. Cresyl violet staining showed that IP3-associated binding was predominantly localized in pyramidal cell regions while [3H]PDBu binding showed a bi-laminar pattern in CA1 and CA2 (Fig. 25), concentrating in both pyramidal cell layers and in the dendritic arbor zone. An extreme example was seen in the dentate gyrus. At postnatal day 40, the IP3 receptors were concentrated in the granule cell layer of the dentate gyrus. In contrast, [3H]PDBu-associated grains were almost absent in this zone but instead strongly labelled the dendritic region of the granule cells. Developmentally, IP3 receptors in the hippocampus also show a different profile from that of PKC. The [ 3H]IP3 binding was apparent around postnatal day 30-40 in CA1, CA2 and the dentate but not in CA3. The density of silver grains in the latter region gradually increased subsequently. By comparison, substantial densities of [3H]PDBu binding were found in CA1-CA2 areas in kittens as young as PI. Binding levels of [ 3 H ] IP3 and [3H]PDBu also appeared to be regulated by different factors in developing visual cortex (Fig. 27). In agreement with PKC immunocytochemical results, the binding for [ 3H]PDBu was elevated in a visual cortical region that had been surgically isolated from its neuronal connections at postnatal day 24, 57 or in the hemisphere ipsilateral to a removal of the L G N , and this effect was only observed in animals operated at a young age. However, no matter when the manipulations were done, no effect could be found on [ 3H]IP3 binding in the operated animals. 58 DISCUSSION These results show that, although expressions of both IP3R.S and PKC in the visual cortex peak during the critical period, there are striking disparities. In both visual cortex and hippocampus, IP3R and PKC differ from each other in both location and developmental profiles. Moreover, in the visual cortex at least, they are regulated by different mechanisms. The mismatched locations of [ 3 H ] I P 3 and [ 3H]PDBu binding revealed by autoradiography can be explained in two alternative ways: IP3Rs and PKC may be in different populations of neuronal cells; or, alternatively, they may be present in distinct subcellular locations, such as somata versus processes, in the same cells. In the visual cortex, the first possibility seems more likely, the staining pattern of PKC immunoreaction in adult cat visual cortex described in chapter two is very similar to [3H]PDBu binding pattern and PKC immunopositive cells were found principally in the superficial and deep layers but not in layer IV (see chapter two). It is still possible, however, that a PKC-negative somata in layer IV may have PKC-positive processes in superficial or deep layers. The argument that IP3Rs and PKC may be located in different populations of cells is also supported by the report from Worley et al.206 who showed that, in the external plexiform layer of the olfactory bulb and substantial gelatinosa, the density of [3H]PDBu binding sites is 100-fold higher than that of [3H]IP3206. However, in the hippocampus, the second possibility may be more likely, that the two second messenger receptors are in the same cells but are at different intracellular locations. [3H]PDBu labelled both somata regions and dendritic zones in both. CA1-2 and the dentate gyrus of the adult hippocampus, while [3H]IP3 only bound to somata regions. The [3H]PDBu-labelled PKC in the dendritic zone is probably localized in the dendrites of the pyramidal neurons of CA1-2 and dentate granule cells but not in 59 a x o n a l t e r m i n a l s of input f i b e r s . This is e v i d e n c e d by i m m u n o c y t o c h e m i c a l o b s e r v a t i o n s in cat h i p p o c a m p u s (my unpublished data indicating labelling in cells, rather than terminals) and by quinolinic acid lesions in rat hippocampus, which deplete a major portion of PDBu-binding204. Therefore, while both PKC and IP3Rs are colocalized in somata of hippocampal neurons, only PKC is concentrated in the dendrites, suggesting a mismatch between the two second messenger targets at the subcellular level. Expression of both IP3RS and of PKC peak during the cr i t i c a l p eriod in the visual cortex, indicating that they both play roles in development of normal cortical function. Considering that the visual input f r o m the lateral geniculate nucleus is b e l i e v e d to be glutamatergic and mainly terminates in c o r t i c a l layer IV, the f o l l o w i n g c o i n c i d e n c e s may be si g n i f i c a n t : 1) IP3RS are also concentrated in layer IV; 2) the general development profile of IP3Rs agrees with that of ibotenate-sensitive glutamate receptor-regulated PI turnover48_both peak at the height of the critical period. These c o i n c i d e n c e s imply that IP3 may be one of the crucial elements d e t e r m i n i n g the selective synaptic f o r m a t i o n between c o r t i c a l neurons in layer IV and glutamatergic terminals carrying v i s u a l information from the LGN. As the IP3 binding remains in the cortex after cortical isolation, these receptors probably are postsynaptically located. Hence, i f ibotenic a c i d receptor-mediated PI turnover indeed plays a role in the synaptic formation during the c r i t i c a l period, this process should mainly occur in cortical neurons but not in LGN terminals. The low number of IP3 receptors in neonatal kittens, contrasted with the relative high level of PKC suggests that IP3 is not as important in early cortical development. Possibly, the PIP2 second messenger system is not yet f u l l y f u n ctional i n newborn kitten cortex. Meanwhile, PKC may selectively act within other signal transduction pathways at these ages since PKC a c t i v i t y : can be 60 regulated by many factors beside DG53, such as the arachidonic acid c a s c a d e 137. The fact that IP3Rs are not well colocalized with PKC in some neuronal cells in adult brain raises the possibility that I P 3 and DG are asynchronously activated by two populations of receptors at different sites on the cell surface. There is some evidence indicating that hydrolysis of phospholipid f o l l o w i n g stimulation of muscarinic cholinergic receptors may not always cause activation of PKC in rat hippocampus208. I should point out that in some other cortical areas, such as area 21a and the cingulate gyrus, IP3 receptors and PKC were w e l l c o l o c a l i z e d (data not shown). Further investigations are necessary to d i s c o v e r the circumstances under which the two limbs of this important second messenger system exist in synchrony or in an inverse relationship. 61 Chapter Six GENERAL CONCLUSIONS AND DISCUSSION In the present work, two Ca + +-related second messenger systems, C a 2 + / C A M - K II and IP3 /DG, were studied in developing visual cortex. This study allows comparisons of development and localization in two dimensions. First, those features of some cell surface receptors (first messengers), M l and a l , can be compared with those of the second messenger elements, C A M - K II, PKC and IP3Rs. Second, as already done to some extent in previous chapters, the development profiles and localizations of the various neurotransmitter receptors and the second messenger elements can be compared one with another. Based on such comparable analyses of the results described in previous chapters, the following conclusions can be drawn: 1) In adult visual cortex, although the neurotransmitter receptors, a l and M l are colocalized in superficial and deep layers, localization of the second messenger receptors, PKC and IP3Rs, do not show good coincidence, especially in layer IV. 2) The elements in recognized signal transduction pathways do not develop synchronously in the cortex but are expressed in a specific sequence. 3) The development of neurotransmitter receptors is regulated by afferent neuronal activity. In addition, the development of some second messenger elements, such as PKC and C A M - K II, is also use-dependent. 4) While neurotransmitter receptors can be located within both intracortical cells and on the terminals of extracortical afferent fibers, all three second messenger elements, PKC, IP3R and C A M - K II, are mainly localized in intrinsic cortical cells. 62 GENERAL DISCUSSION Technical considerations 1. Immunocytochemistry: The antibodies used in the investigation of PKC and CAM-K II were provided by other laboratories and they were all originally against rat brain antigens. Since PKC and CAM-K II have never been purified from cat brain, it was impossible to determine the affinities of these antibodies to cat brain antigens although western blots of the antibodies to cat brain homogenate might be helpful to roughly test the specificity of these antibodies. However, treatment of brain tissue for immunocytochemistry, such as perfusion and fixation, may have great effects on the affinity and specificity of an antibody to its antigen. The two antibodies used in the present study were relatively insensitive to a PB buffer containing 4% paraformaldehyde. However, PKC polyclonal antibodies were more tolerant to perfusion of 4% paraformaldehyde/0.1% glutaraldehyde while the immunoreaction of CAM-K II antibody was weakened by the same concentration of glutaraldehyde in cats (but not in kittens). In order to obtain maximal preservation of both the antigen and ultrastructure for electron microscopic observation, EDC-PFA perfusion was used as described in chapter four. Although EDC-PFA perfusion did not alter lamination of the immunostaining of CAM-K II, possible changes in immunoreaction at ultrastructural level must be considered especially when the results were compared with that of PKC immunocytochemistry. Fortunately, there is not much likelihood that this was the case since the results of CAM-K II immuno-electron microscopy with EDC-PFA did not differ from that of some kittens perfused with 4% paraformaldehyde/0.1% glutaraldehyde. In general, any fixation procedure may cause translocation of some antigens and in addition, DAB reaction may nonspecifically coat postsynaptic membrane and some intracellular organelles, caution should be taken to interpret the manifested 63 locations of immunoreaction product. 2. Autoradiography a) Characterization and incubation condition For economic reason, full binding characterization of some radioactive ligands [3H]prazosin, [3H]rauwolscine, and [3H]pirenzepine were not performed in homogenized cat visual cortex. However, the concentration of each tritiated ligand was determined in preliminary experiments to obtain maximal ratio of specific/nonspecific binding for autoradiography. In addition, incubation time for each ligand was chosen, based on a time course obtained from these preliminary experiments, to ensure that binding reaches equilibrium. However, since these preliminary experiments were normally conducted in adult tissue, the chosen incubation conditions may not be optimal for tissues at different ages. This variable should be taken account when one compares autoradiograms of ligand binding in visual cortex at different ages. 3.Sample size and quantitative analysis Because of limited source of kittens, there were often not enough animals were available at each age, especially for experiments of autoradiography. Small sample size may not seriously affect the qualitative conclusions of developmental changes drawn from the present study because 1) previous experience has shown that variation of laminar distribution of a given ligand binding is usually small between different individual animals at same ages, 2) at some key stages of development, such as P30-40 (peak of the critical period) and P90 (end of the critical period), results of autoradiograms in normal animals were similar to control side of operated animals at same ages, bring the total number of animals in each age group up to 3-4 animals. Major caution should be taken for quantitative analysis of autoradiograms with densitometry. Absolute bound radioactivity 64 levels, therefore optical densities, do vary among individual animals, among different sections from the same animals and among experiments performed at different times. Thus, the developmental profiles (e.g., Fig.3, 6, etc.) taken from a few animals at each age should only reflect tendencies of development of given binding sites. In addition, values of bound radioactivity did not directly indicate total number of binding sites since concentrations of ligands used were below saturation. As the isotope used in the study was 3 H , a weak (3 ray source, the factor of quenching also has to be taken into consideration. It has been well established that the white matter absorbs more p* rays emitted by 3 H than the grey matter, due to its greater density. Particularly, as quenching is dependent on the degree of myelination which increases during maturation of the cerebral cortex. Although the time course of myelination process in cat visual cortex is not clear, the manifested decreases in binding of ligands at late ages could be partially due to an increased quenching of the radioactivity. Myelination caused quenching may be also partly responsible for apparent reduction of radioactivity in layer IV during late development since this layer shows the heaviest myelination in adult visual cortex. Therefore, the magnitudes of decreased binding sites estimated by densitometry measurement could be overestimated and meanwhile, the increased binding density during early development could be underestimated because of increased quench effect. However, it is unlikely that all the decreased densities of silver grains in the visual cortex at late ages were due to quench effect. This is supported by several peaces of evidence: 1) densities of silver grains for [3H]rauwolscine in hippocampus and entorhinal cortex were actually increased in adult animals; 2) binding assay of [ 3 H ] I P 3 with homogenized visual cortex also showed a decreased Bmax in an adult animal compared with that in a P30 kitten (data not shown); 3) in the case of [3H]PDBu binding, immunoreactivity of the 65 PKC antibodies also reduced at late ages and laminar pattern of PKC immunoreactive cells resembled that of [3H]PDBu binding, especially in adult animals. Finally, as all the ligands used in the present study were tritium-labelled and they were applied on the same tissue, the quench factor should be the same, which allows the comparisons Explanations and speculations 1. Why mismatch? As shown in figure 28, the laminar distribution of IP3Rs differs from that of other three elements involved in PI turnover, in both neonatal and adult animals. In the neonatal cortex, while both PKC and M l receptors are colocalized in the middle layers, the level of IP3Rs is very low (Fig. 28 and chapter 4), with the main concentrations being instead in superficial layers. In adulthood, the IP3Rs are found mainly in the middle cortical layer while the highest densities of PKC and the two surface receptors M l and a l are in the superficial layers. It is not uncommon that there are mismatches in locations of molecules that are expected to be functionally coupled in the central nervous system?6. One may argue that the mismatches in locations of IP3R and M l / a l receptors may not be surprising, as PIP2 turnover can be triggered by activation of a number of other receptors as well as M l and al39,47,57,72,lll,124,191. If this were the case, however, a logical deduction would be that most phospholipid related cell surface receptors would be located in the middle cortical layer where IP3 receptors are concentrated and the M l / a l receptors would only be exceptions. Although the laminar distributions of many receptors are still unknown, receptors such as 5 - H T i c , 5-HT2 and CCK, that are known to be also linked to PI turnover have been studied in our group and found to be only transiently expressed in layer IV at young ages, low concentrations 66 in the middle layer are found in adulthood. It is also difficult to understand, on this interpretation, why PKC, which may have a broader spectrum for its endogenous activators 5 3 , is colocalized with M l / a l receptors but not with IP3R in adult visual cortex. The question becomes more puzzling with the evidence that high levels of inositol phosphates can be induced by stimulation of M l receptor in newborn kitten cortex48 and rat pups6 although we have found few IP3R sites at that age. Why is IP3 generated when its receptors are not there? One possibility is that the receptor stimulation-generated IP3 does not always act as a second messenger but simply as a precursor of inositol-1,3,4,5-phosphate (IP4) under certain circumstances (such as in newborn cortex). This hypothesis is based on an emerging understanding of the metabolic pathway of IP3. It can be either rapidly metabolized to inositol-1,4-phosphate or further phosphorylated to IP48,16,46,88,89,92. Accumulating evidence shows that IP4 may also play a role in Ca 2 + -related signal transduction as another intracellular C a 2 + modulator. Indeed, several groups have reported a specific binding site for IP4 22,187. in adrenal microsomes, IP4 stimulates calcium release in an I P 3 receptor-independent manner50 . in identified neurons of Aplysia, intracellularly injected IP4 causes an inward cation current which lasts 30-60si59. These results suggest that an elevation of intracellular IP4 may regulate excitability of neurons. It is thus possible that, in newborn visual cortex, or in some cell populations of adult brain, when the IP3Rs is not fully expressed, IP4 as a metabolic product of IP3 plays a major role. In the neonatal cortex, stimulation of M l receptors would result in increased IP3, which would then be rapidly transformed to IP4 to regulate the general excitability of the cells by altering ionic currents across the plasma membrane. To test this hypothesis, an investigation of the postnatal development of the IP4 receptor and inositol-1,4,5-trisphosphate 3-kinase, the enzyme that phosphorylates IP3 to IP4, would be desirable. 67 An alternative explanation is that some cell surface receptors are coupled with other phospholipids instead of PIP2. In platelets and vascular smooth muscle, hormone stimulated inositol lipid metabolism may use PIP2 at the beginning and then switch over to hydrolyse phosphatidylinositol (PI) or phosphatidylinositol-4-phosphate (PIP), which release DG and either inositol or inositol-1-phosphate (IP 1), but not I P 3 1 4 . This pathway therefore biases towards the DG/PKC limb and away from the I P 3 / C a 2 + branch of this second messenger system. As mentioned in chapter 4, both M l and a l receptors may also couple to phospholipase D or phospholipase A 2 5 , 1 1 4 . The former hydrolyses phosphatidylcholine to produce DG without IP3 and the latter generates arachidonic acid. Both of these substances are activators of PKC 3 1 , 1 5 8 . On the other hand, it should be pointed out that, despite the difference in the overall distributions, a certain proportion of IP3R s are still colocalized with PKC and M l / a l receptors in the visual cortex. The extent of the overlap is maximal during the critical period (Fig. 28). The I P 3 / C a + + branch of the PI turnover therefore probably still takes part in the signal transduction in developing cortical cells to some extent and may be particularly important during the critical period. 2. Sequential programmed development of cortex. A major cause of mismatches discussed above is the asynchrony in ontogeny of the receptors and their normal second messenger targets in the developing cortex. Figure 29 compares the development of the four elements of the PI turnover system (Ml, a l , PKC, IP3R) in the cortex. In superficial layers, PKC and a l receptors show relative high levels of expression at birth while M l and IP3 receptors display a delay of 10-20 days before onset of increases. In the middle layer, there is a striking difference in the levels of PKC 68 and IP3R in first 2-3 weeks. The PKC level is above 80% of its maximal at this age, while only 10 to 20 percent of IP3Rs have been expressed. However, in the next ten days, the number of IP3R dramatically increases and reaches the maximal level at the same time as PKC. As for cell surface receptors, the number of M l receptors increases more rapidly than that of the a l receptors in the first two weeks and arrives at its peak at least three weeks earlier than alpha-1 receptor. Unlike the superficial and middle layers, development of the four receptors in the deep layers is fairly synchronous, although PKC still shows the highest level at birth and peaks earlier than the others. It is apparent, therefore, that both cell surface and intracellular receptors develop at their own specific pace even though they work within the same signal transduction pathway. In Table 3, several neurotransmitter receptors studied in this group are listed to show the sequence at which their expressions peak in layer IV of the developing visual cortex. Table 3 Sequential development of cell surface receptors Receptors 5-HTia M2 Ml/pl/p2 5-HTic/2 a 1/2 G A B A A nAChR Ageofmaximal P w 4 P w 5 P w 6 PwlO Pwll P w l 4 Adult expression in layer IV What does this sequence mean to the maturation of the cortex? Selective formation of synapses in the visual cortex during cortical development is a complicated process. Besides direct visual stimulation via the geniculate input, many other systems have been suggested to be involved, including spontaneous activity of cortical neurons, activity of extraocular muscles and the ascending reticular activating system, etc.(review see60). As these inputs are mainly mediated by released neurotransmitters or modulators, corresponding receptors and second messenger systems can be considered as effectors of these inputs. Considering that subcortical 69 structures normally mature in advance of the cortex*4?, the sequential development of different types of cell surface receptors may, at least partly, reflect the sequence in which the different systems influence the developing cortex. For example, in first two weeks of life, Ml receptors may be actively involved in the early development of cortex since at this age, cholinergic terminals show high levels of acetylcholine45-148, M l receptors are highly expressed and the muscarinic-related PI turnover is already functional4 8. Meanwhile, alpha adrenoceptors in the subplate at this stage may be ^ important for interaction between growing-in fibers and subplate cells i 6 5 . Furthermore, since PKC levels are much higher than those of IP3R's in neonatal cortex, the early cholinergic and adrenergic stimulated intracellular signal transduction may be biased toward DG/PKC pathway and IP3R related C a + + release may not play a crucial role at this stage. However, intracellular calcium levels may still be elevated by stimulation of M l / a l receptors since the accelerated PI turnover can generate IP4 from IP3 to increase C a + + influx. Voltage-dependent calcium channels labelled with [3H]PN-200 are also highly expressed in layer IV at this early stage42. The increased calcium level may further activate CAM-K II which is highly expressed by the second week in layer IV. How is the sequential development of these receptors related with the morphological and physiological development of the visual cortex? Table 4 summarizes some developmental events in the visual cortex. The postnatal development of the visual cortex can be defined by two major stages, pre-critical and critical periods. During the pre-critical period (postnatal week 1-2 (Pwl-Pw2)), the cortical lamination is still undergoing final organization, axons from both extracortical sources and intracortical neurons are still growing, and dendrites of pyramidal cells in layers II, III and V are poorly developed 1 3 9 . At this stage, the development of the visual cortex is 70 probably mainly controlled by genetic factors (natural eye opening is around P8 in kittens) although spontaneous activity of the cortex is also important. The cortex at this age is undergoing a preparation for the critical period when selective synaptic connection, modification and stabilization occur. At this time, consistent with the morphological development, both PKC and CAM-K II, the final targets of calcium-related signal transduction pathways, are enriched in growth cone-like terminals and dendrites (see chapter II and III). High immunoreactivity of PKC is also present in the cortical plate in which cells are still migrating to form superficial layers at this early stage of development. During the critical period (Pw3-Pwl2), the number of synapses in the visual cortex increases dramatically in the first few weeks and declines by Pwl038,202. Meanwhile, afferent terminals from LGN are segregating to form specific connections with cortical neurons according to activity driven by either of the two eyes85,86,i09. in addition, intracortical neuronal circuit is also built up to form specific "patches". As consequences of synapse formation and neuronal circuit rewiring, cells show more specific response to their preferred visual stimuli and adult-like physiological properties of the visual cortex emerge. At this developmental stage, the external visual environment has the greatest influence on the cortex. This influence is imposed upon the visual cortex through several pathways (not only retino-geniculo-cortical pathway) in the nervous system by a c t i v a t i n g v a r i o u s n e u r o t r a n s m i t t e r sy Stems9-67,109,151,152,167,168,174,181. 71 Table 4 Morphological and physiological development of cat visual cortex Developmental stage  Laminar formation Pre-critical period (Postnatal week 1-2) Layer V-VI: matured Layer IV: formed (by PW1) Layer ll-lll: forming (PW1-PW4) subplate: thick Critical period (Pw3-12) all layers are matured subplate disappears by Pw9 highest by Pw6 then declines Synaptogenesis low density of synapses in grey matter (7.5% of adulthood at PD1), appearance of dendritic spines (PD7-10), LGN projection axons continue to develop and the terminals are segregated to form terminals form a continous band ocular dominance columns in layer IV Physiological properties conduction velocity: slow orientation selectivity: present receptive field: poor direction selectivity: poor ocular dominance: poor velocity selectivity: low speed increases rapidly in Pw2-6 increase of high-selective cells starting to develop by Pw3 matured by Pw4 formed by Pw6-8 increase of high speed -responding cells during Pw4 References:38,io9,i47 i In accordance with the extensive involvement of multiple systems in cortical development, most cell surface receptors are maximally expressed during the critical period. In addition, second messenger receptors are also present at maximal concentrations in the cortex (Fig. 29). For instance, it is during this stage that IP3 shows its densest binding to receptors, suggesting that the IP3-sensitive intracellular calcium pools may be actively involved. More importantly, distributions of various receptors show the maximal overlap at this age (Fig.28). Furthermore, PKC and CAM-K II are 72 concentrated in both pre- and postsynaptic membranes, synaptic vesicles and cytoskeletal elements in terminals. Presumably, phosphorylation of the corresponding proteins plays important roles in regulating neurotransmitter release, postsynaptic responses, dendritic spine and synapse modification, etc. However, within the critical period, the time at which receptors achieve their maximal expression varies. M l and 5-HTia receptors reach their peaks about one month earlier than a 1/2 adrenoceptors and 5-HTic/2's (Table 3). It seems that different receptors may play their roles in turn during the critical period by sequential culmination of their maximal expressions. It is then reasonable to suggest that the early expressing receptors, such as M l , may be particularly involved in induction of the plasticity and those late expressed may be more important for maintenance or turning off of the plasticity or for final tuning of neuronal circuits. It is interesting to notice that several receptors studied are first maximally expressed in layer IV and the densities of these receptors are reduced later in this layer which often presents the lowest receptor densities in the adult cortex. This argument is supported by both immunocytochemical 192 and autoradiographic evidence although, as mentioned in "technical consideration", ' factor of quenching caused by increased myelination at late ages can not be ignored for autoradiographic results. Layer IV bears unique features of cortical development also in other aspects. As the gateway of the retino-geniculo pathway to the visual cortex, it shows the highest concentration of the geniculate input as defined by both physiological and anatomical evidence^,86,109. And, most interestingly, it displays the most narrow temporal window for influence of visual experience on its synaptic formation. In other words, the plasticity in layer IV is only displayed around Pw3-7 and after which this layer loses its susceptibility to environment influence within a few weeks; other layers, in contrast, especially the superficial ones maintain their 73 plasticity to a certain extent in aduithood58 , i 3 i . This transiently displayed plasticity in layer IV seems to correlate with transient expression of a number of receptors in the layer, further implicating the involvement of these receptors in cortical plasticity during development. 3. What decides the development of the receptors? As suggested above, cell surface receptors and second messenger targets develop in specific temporal and spatial patterns and this specific sequence of development of the molecules for signal transduction may result in a series of modifications of morphology and physiology in the developing cortex and lead to maturation of the cortex. The next obvious question is how this complex sequence is governed? What factor(s) decides this sequence? Results from surgically manipulated animals in the present work throw some light on this question. Manipulations performed in the present work were designed to abolish two main types of inputs: i) modulatory afferents from the basal forebrain and hindbrain, and ii) subcortical afferents from the thalamus. As shown in the previous chapters, concentrations of both the neurotransmitter receptors and protein kinases are more or less affected by removal of the subcortical inputs, particularly the LGN afferents, but not by abolition of modulatory inputs (by the front cut). In addition, removal of either type of input had no effect in adult cats. For the neurotransmitter receptors studied here and from previous work of our g r o u p s , isolation of the cortex from the subcortical inputs in young animals mainly results in either reduction in numbers of binding sites or blockade of the normal laminar redistribution process of the receptors. Unlike the cell surface receptors, concentrations of the two kinases, P K C and C A M - K II, are increased when the cortex is isolated from subcortical inputs. Furthermore, 74 these increases are not homogenous across cortical laminae, indicating that the manipulations have selective effects on certain populations of neurons. These results strongly indicate that: 1) cortical levels of many receptors (both cell surface and intracellular ones) are at least partly regulated by extracortical activity and are not solely genetically determined; 2) the effects are heterogeneously imposed on specific populations of cortical cells; and 3) the influence of input activity is age-dependent. Although the reduction in numbers of some receptors, such as nicotinic ACh receptors150 and probably certain population of a 2 adrenoceptors, can occur simply because these receptors are located on the terminals of subcortical afferents which degenerate after the surgery, it is almost completely unknown how the subcortical activity regulates cellular levels of other receptors and kinases. However, it is likely that the effects of deafferentation are due to reciprocal interactions between the cell surface receptors and kinases. It has been showed that phosphorylation of cell surface receptors is an important process to induce down-regulation of the receptors71. PKC has been found to be involved in down-regulation of both a-adrenergic and muscarinic cholinergic receptors35,96 , ioo , i07,i54. Although immunocytochemistry and autoradiography utilized in the present work do not necessarily reflect activity of the kinases, an increased PKC level suggests the possibility of an extensive down-regulation of cell surface receptors by PKC following the deafferentation. On the other hand, little is known about metabolism of PKC or CAM-K II and how extracellular signals affects cellular levels of the kinases (most work only measures changes in activity). However, some evidence shows that a second messenger may play a role in influencing the turnover of its target kinase. In several non-neural cell lines, prolonged high levels of cAMP results in enhanced 75 degradation of the catalytic subunit of the cAMP-dependent kinase 1 5 3 . This phenomenon seems similar to the case of cell surface receptors, which are down-regulated by prolonged agonist stimulation and up-regulated by denervation. It is thus conceivable that metabolism of PKC is up-regulated by a possibly declining DG level following the changes in M l or a l receptors as a consequence of reduced cortical activity. Similarly, CAM-K II turnover rate may be regulated by intracellular calcium levels, which may also decline after isolation of the cortex from subcortical inputs. As there is no apparent correlation between development of the kinases and any particular receptor, it is likely that changes in the kinase levels are due to the combined effect of alterations in many receptors. Regardless of the mechanisms involved, the increases in the levels of the kinases after cortical deafferentation indicate that, in normal young kitten cortex, the kinase levels are tonically down-regulated by subcortical inputs. This suggests that many proteins that are substrates for the kinases may tend to be in a dephosphorylated state in highly activated cortical regions. This is supported by the finding that MAP 2, a substrate of PKC, CAM-K II and cAMP-dependent kinase (PKA), is mostly phosphorylated in dark-reared kittens and, once the kittens are exposed to light, the MAP 2 becomes dephosphorylated and can then be easily phosphorylated by PKA in vitro4. Interestingly, dark-rearing does not have any effect on the phosphorylation status of MAP 2 in adult cats4. Similarly, cortical levels of PKC are not regulated by deafferentation in adulthood. A similar age-dependence is also seen in regulation of alpha adrenoceptors and other cell surface receptors 1 73. This age-dependent feature in activity-regulation means that the metabolism of these receptors and kinases are most susceptible to external environment influences in immature cortex. 76 4. What is the significance of this temporal plasticity in metabolism of the elements involved in signal transduction? The susceptibility of the metabolism of cell surface receptors and coupled second messenger systems (and perhaps also plasma ion channel proteins), in my point of view, is an essential part of the biochemical basis underlying the plasticity manifested in the visual cortex at young age. In other words, the so-called "critical period" for the visual cortex is mainly determined by the period of time when the metabolism of these molecules is most susceptible to influence of the external world. Each receptor molecule (including ion channels) on the cytoplasmic membrane can be thought of as an interface of the cell with the outside world. Second messengers and their target kinases work like a multi-channel-amplifier within the cell. Each channel represents a second messenger system. The overall output of the amplifier, which controls intracellular reactions causing modifications in morphology and physiology of the cell in response to external stimulation, is determined by transfer functions of each of the channels and the overall input from the interfaces. The quantitative and qualitative combination of different types of receptors and their spatial distribution on the cell surface constitutes a specific conformation of the input to the amplifier. Apparently, any conformational change in the overall input (such as alteration in density of any particular type of receptor), or any modification in the transfer function (such as elevation in cellular level of any particular kinase) will alter the output of the amplifier, resulting in a change of the cellular response to a given stimulation. Since, as shown above, the conformation and transfer functions vary with age and, particularly, the alterations are dependent on the extracellular environment, the output of the amplifier changes as a function of both age and environmental influence. This leads to morphological 77 and physiological modifications of the cell during development and causes the manifestation of cortical susceptibility to the environment. It is also predictable that, if the conformation of cell surface receptors and the transfer functions of second messenger systems are no longer dependent on external environment, as shown in adult cortex, the cortical cells will lose a great deal of plasticity in response to changes of environment. This is exactly the case in the adult visual cortex. However, alterations in the conformation and transfer functions are not solely dependent on the external environment. The numbers and distributions of some cell surface receptors, such as a l (chapter one), are not or only slightly affected by alterations of cortical activity. In addition, IP3 receptors, as an element in our amplifier, also show no change after such manipulations. Hence, the overall output of the signal transduction pathways are undoubtedly also controlled by genetic factors. In summary, the present study chose some elements involved in calcium-dependent signal transduction pathways to examine the significance of signal transduction in cortical plasticity and development by investigating the postnatal ontogenesis, cortical distribution and influence of extracortical afferents on the development of these molecules. Of course, many other elements within signal transduction pathways, such as G proteins and membrane phospholipases, play equally important roles in the development of the cortex and they were not investigated in this study. The results obtained here indicate that elements of signal transduction, even though within the same pathway, do not develop synchronously and that the development of these elements in the visual cortex is regulated by both environmental and genetic factors. As a consequence of these developmental asynchronies, conformation of the interfaces, which are composed of different types of cell 78 surface receptors, and total output of second messenger systems vary with age. Regulated by the total output of these signal transduction pathways, individual cells, as well as the entire visual cortex, alter their behavior and their responses to external environment with age. As the output of signal transduction pathways is most susceptible to the environmental influence at certain developmental stages, the visual cortex is most plastic at this "critical period". 79 FIGURES: Figure 1, Autoradiograms of [3H]prazosin (al) and [3H]rauwolscine (a2) binding in developing visual cortex. The animal age (postnatal days) is given in the middle of each pair of adjacent sections. Note that the binding of both ligands demarcate the visual cortex (areas 17 and 18) from the subadjacent cingulate cortex and area 19 laterally in kittens between 10 and 40 days of age. The binding sites are concentrated in subplate/white matter at neonatal ages (P0-P10). The numbers beside the scale represent color coding of optical densities. Scale bar=6 mm Figure 2, Autoradiograms of [3H]pirenzepine binding for M l muscarinic cholinergic receptors in visual cortex of kittens of various ages. Conventions are the same as in Figure 1. See text for a detailed description. O.D., optical density. Scale bar=2 mm 80 Figure 3, Development of alpha adrenoceptors and M l cholinoceptors at different depths of the visual cortex. The autoradiograms in figure 1 and 2 were analysed using quantitative densitometry and the optical density was calibrated to radioactivity of bound ligands with tritiated standards. The cortical layers were determined by comparing the autoradiograms to the same sections stained with Cresyl violet. Each point in the curves represents an average of the measurements in at least four sections. | I 1 I 1 i 1 I alpha-1 receptors PSO POO P7S P120 Adult Postnatal Day alpha-2 receptors 120 100 -ao -60 -P30 POO P75 P120 Adult Postnatal Day M1 receptors Layer I III Layer IV Layer V VI P10 P20 P40 POO P1?0 Adull Postnatal Day 82 Figure 4, Autoradiograms of [ 3H]prazosin (alpha-1) and [ 3H]rauwolscine (alpha-2) binding in operated animals, a and b, L G N lesion combined with front cut at P14 with 4-weeks survival; c and d, L G N lesion at P l l with 10-weeks survival; e and f, optic tract lesion at P10 with 7-weeks survival; g and h, L G N lesion in adulthood with 10-weeks survival. O.D., optical density 83 84 Figure 5, Densitometric analysis of the ligand binding data of figure 4. The optical densities were calibrated as described in figure 3. Note that, after early L G N lesion and combined LGN/front cut surgeries, the reduction in the radioactivity is uniform in all cortical layers on the operated side except for subplate (labelled as SP) for a 1 receptors (a and c). It is however heterogeneous across the layers for a2 receptors (b and d) with the greatest decrease in layer IV. 85 86 Figure 6, Comparison of development of alpha-1, alpha-2 adrenoceptors and Ml cholinoceptors in various cortical laminae. Data are expressed as percentages of the maximal binding for each ligand in sections of the visual cortex at various ages. It is clear that the maximal binding in superficial layers for M l receptors is achieved around P60, which is later than in the middle and deep layers (around P40). The numbers of the two alpha adrenoceptor sites achieve their maxima in all laminae at the same age (around P75). Meanwhile, in the superficial layers, development of all the three ligand binding sites shows two stages in their rising phases with various latencies in first few weeks followed by a rapid increase. However, in the middle and deep layers, only a2 receptor sites show a 30-day latency in their development. Each point represents an average of measurements from at least four sections. 87 Layers I-III ! T 1 1 1 1 1 1 1 • 1 1 10 20 30 40 60 75 120 Adult Postnatal day Layer IV 1 10 20 30 40 60 75 120 Adult Postnatal day Layers V-VI T 1 1 1— 1 1 1 1 1 1 1 10 20 30 40 60 75 120 Adult Postnatal day 82 Figure 7, Comparison of laminar distribution of the three receptors in developing visual cortex. In order to fit the binding density of the three ligands into the same scale of the ordinate, radioactivity of each of the bound ligands is normalized as a ratio of the maximal value in the sections at each age. Note that, except for postnatal day 1, the distribution of M l overlaps that of alpha receptors although the identical laminar patterns of Ml and a l receptors are only seen in adulthood. The two alpha receptors are colocalized in most layers. However, at P30, despite a colocalization in the middle layer, the binding of [3H]prazosin and [3H]rauwolscine peaked in very different layers (the outmost for a2 and the deepest for al) . 89 90 Figs. 8: Light micrographs of immunostaining for protein kinase C in area 17 (a) and area 18 (b) of kitten visual cortex at various ages. Cortical layers were determined by examining the morphology of the c e l l s 1 0 . Notice the pronounced decrease in immunoreactivity in both areas during development. The immunostaining was reduced first in layer IV and then at later ages in other layers. See text for detailed description. 1), postnatal day 1; 2), P10; 3) P 20; 4) P 40; 5) P 90. Scale bars=100 um. 91 5a-5b II/III 4b ' , " • ' • « . ' • • V . ' v. \ \ ' - . / • 4 i • » ^ « « IV ?2.2 Figure 9: Area 17 (top) and area 18 (bottom) of the visual cortex stained with the polyclonal antibodies at day 10. The overall immunoreactivity in area 18 is remarkably stronger than area 17. The laminar patterns, however, are similar in both areas. Many horizontally-oriented cells can be seen in the white matter. Scale bar=200 u.m. Figure 10: High magnification of immunostaining in area 17 of the visual cortex at day 40. Pyramidal cells in layers II, III and V were densely stained. Stained long apical dendrites originate from pyramidal cells in layer V, bifurcate and terminate in various superficial cortical layers. Arrows show such a dendrite bifurcating in layer II/III and terminating in layer II; black arrow heads show another one bifurcating at the border of layers III/IV and terminating in layer III. Empty arrow heads show some faintly stained fibers with varicosities in layers IV, V and VI but not in the superficial layers. Scale bar=100 (im. Figure 11: Bundles of fibers with PKC immunoreactivity in upper layer VI of area 17 at day 40. These fibers possess numerous varicosities (arrows) which show high levels of PKC immunoreactivity. Both pyramidal and nonpyramidal cells can be seen to be PKC positive. Scale bar=20 pm. 93 Figure 12: Cytoplasm of a P K C immunoreactive cell from a postnatal day 10 kitten visual cortex. Note that the end-product can be found throughout the cytoplasm and that the membranes of the cell organelles are immunoreactive. Another feature of the staining is that the cytoplasmic membranes are stained (arrowheads). This staining is more obvious between the cytoplasm and the nucleoplasm, possibly because of the better preservation of these membranes. This cell body staining pattern was observed at every age studied. No striking differences were found during postnatal development, m, mitochondria; er, endoplasmic reticulum. Scale bar= 1 pm 9 5 Figure 13: Electron micrographs of immunoreactive profiles taken from the visual cortex of a postnatal day 30 kitten. A) An immunoreactive vesicle-containing profile (+) makes a "perforated" synaptic contact (arrows) with an immunonegative dendritic spine (-). This synapse is classified as asymmetric because of the presence of the postsynaptic opacity. Note that another immunonegative vesicle-containing profile (-) is in close apposition to the same postsynaptic target. Scale bar= 0.20 um B ) An immunopositive vesicle-containing profile (+) makes a synaptic contact (arrows) on a small immunonegative dendritic profile (-). Note also the well-defined postsynaptic opacity. Scale bar= 0.20 pm C) An immunoreactive dendritic profile (+) which receives a synaptic contact (arrows) from a immunonegative vesicles-containing profile (-). Note that microtubules, plasma membranes and portions of the mitochondria membranes are stained (arrowheads). Scale bar= 0.20 pm 97 Figure 14: Distribution of P K C immunoreactive structures in a postnatal day 4 kitten visual cortex. A ,B ,C) Electron micrographs from serial sections through a structure that was highly immunoreactive (+) for the antibody against PKC . This profile contains mitochondria (small arrows) and microtubules but does not have any synaptic vesicles and does not make any synaptic contacts. The structure sends prolongations (large arrows) through the neuropil. These profiles could be found only in very young animals. Scale bars for all three pictures= 1pm D) Electron micrograph of a profile lightly immunoreactive (+) for P K C . This micrograph has been taken from the same ultrathin section as that shown in A). Note the immunoreaction is weaker than the profile shown in A) and that this profile is smaller in size. Because of the presence of the microtubules and of mitochondria, I classified this profile as a dendrite. Scale bar= 0.25 pm E) High power light micrograph of layer V in the visual cortex of a postnatal day 4 kitten. A few large pyramidal cells in layer V shown in the middle part of the picture are PKC-positive. Note the highly immunoreactive puncta marked with arrowheads. Scale bar=15 pm 99 /oo Figure 15: PKC immunoreactivity in the visual cortex surgically isolated at day 14. The immunoreaction was processed after perfusion at postnatal day 90. Square area in figure a) is shown as figure b) with high magnification. Immunoreactivity in layers II/III and V/VI is much higher in isolated region (right side of b) than corresponding layers in the neighboring control areas and in the contralateral unoperated hemisphere (not shown). Notice that the staining in layer IV is not much different between the isolated region and control areas. In a), Scale bar=300 pm; in b), Scale bar=200 pm. 101 ! ° 2 Figure 16 Immunostaining of C A M - K II antibody in tissues perfused with two different protocols, EDC-PFA (upper panel) and P F A only (lower panel). Immunoreaction with EDA-PFA perfusion tends to be stronger and more cells were stained. Scale bar=350 p m . 103 Figure 17, CAM-K II in cat visual cortex, a) Neurons with strong immunoreactivity are concentrated in layers II, III, VI and lower layer IV. Many dendritic fibers are present at the top of layer II. The strong, but diffuse immunostaining in layer I may partly represent an edge artifact. The strongest CAM-K II immunoreactivity is present in nonpyramidal cells in lower layer IV. These cells compose a thin lamina with a thickness of 3 or 4 cell-layers. Scale bar= 200 pm; b) High magnification of nonpyramidal cells in lower layer IV. Scale bar= 70 pm. 105 Fig 18, CAM-K II immunoreactivity in kitten visual cortex (area 17) at various ages. The numbers at upper right corner represent the age at perfusion, a) the pattern of the day 1-4 age group, in which cells in superficial laminae show unclear outlines and the pyramidal cells in layer V are stained with the antibody. Scale bar= 50 pm; b) another pattern in the same group, in which the morphology of the cells in the superficial layers seems more mature. Some large pyramidal cells in layer V are still densely stained. In general, the immunoreaction in tissues of this age group is relatively weaker than that at later ages. Scale bar= 50pm; c) day 14: Many neurons show strong immunoreactivity in both somata and fibers. These neurons, both pyramidal and nonpyramidal and of various sizes, are distributed over all cortical layers with the highest density in layers II-IV. d) day 24: Many immunopositive neurons with numerous densely stained particles can be seen at this age. These particles are concentrated in layers II-IV. Note that both large cells in upper layer IV and small cells in lower layer IV are stained. Large pyramidal cells in layer V are still strongly immunoreactive, while staining in cells of layer VI has started to fade, e) day 40: While numerous neurons in the superficial layers are CAM-K II immunopositive, cells in layers V and VI show weak immunoreactivity. Note that the staining of some large pyramidal cells in layer III is weaker than that of nonpyramidal cells and small cells in lower layer IV are less densely stained than the large ones in upper layer IV. f) day 90: The strong immunopositive cells are localized in three bands, namely layer II/III, lower layer IV and layer VI. Scale bars=100 pm 107 Figure 19, High magnification photomicrograph taken from an area in upper layer IV of the animal at 24 days of age. Numerous puncta are densely stained. Many puncta are shown to be expanded terminals of weakly stained fibers (thin arrows). They are presumably growth cones. Thick arrows show some puncta approaching cell bodies. Scale bar= 100pm Figure 20, CAM-K II immunoreactivity in an animal with an early LGN lesion, a) the control hemisphere, b) the operated hemisphere. Note that there are more immunopositive cells in all layers but layer V on the operated side. Scale bar=100pm 109 I vo Figure 2 1 : Cytoplasm of CAM-KII immunoreactive neurons from an adult cat (left) and a postnatal day 4 kitten (right) visual cortex. Note that the end-product can be found throughout the cytoplasm and that the membranes of the endoplasmic reticulum (er) and of the mitochondria (m) are immunoreactive in both adult and young animals. However, no obvious immunoreactivity can be found inside these cell organelles or on the cell membranes, m, mitochondria; er, endoplasmic reticulum. Scale bar= 1 pm 111 M2 Figure 22: Electron micrographs of post-synaptic CAM-K II immunoreactive profiles taken from an adult cat (A and B) and a postnatal day 4 kitten (C) visual cortex. A: An immunonegative vesicle-containing profile makes a synaptic contact ( upper large arrow) with an immunopositive dendritic shaft. The end-product forms a dense and wide aggregate adjacent to the post-synaptic opacity: Compare the size of the post-synaptic opacity of immunonegative dendrites (lower large arrows). Microtubules are also immunoreactive (small arrows). Scale bar= 0.50 pm B: An immunopositive dendrite(*) cut transversely. Note that the immunoreactivity is concentrated in microtubules (small arrows) and in the membranes of the mitochondrion (arrowheads). Scale bar=0.5 pm. C: An immunonegative vesicle-containing profile makes a synaptic contact (arrow) on an immunopositive dendritic profile. Note that the immunoreaction end-product in the profiles is more uniformly distributed than in A. Scale bar= 0.50 pm 113 Figure 23: Electron micrographs of presynaptic CAM-K II immunoreactive profiles taken from an adult cat (A) and a postnatal day 4 kitten (B) visual cortex. A: A vesicle-containing profile immunoreactive for CAM-K II (+) makes a synaptic contact (large arrow) on an immunonegative post-synaptic element. The synapse is classified as asymmetrical because the postsynaptic membrane has a well-defined postsynaptic opacity. The end product is concentrated mainly on the membranes of the synaptic vesicles (small arrows) and mitochondria. Note the small immunonegative vesicle-containing profile(-) located close to the CAM-KII terminal. Scale bar= 0.25 pm B: A large immunopositive nerve growth cone was found in the subplate region and in the layer VI. It contains many vacuoles and has a bulbous ending attached to a tail containing microfilaments. Scale bar= 0.5 pm. 115 lib Figure 24, Characterization of [ 3H]IP3 binding in cat visual cortex. Saturation curve of the binding with Eadie-Hofstee plot (insert). Each point represents an average of three measurements. 117 4 8000 -\ pH]IP3(nM) 118 Figure 25, Color coded optical density of autoradiography of [ 3H ] IP3 and [ 3 H]PDBu binding in adjacent sections of the visual cortex and hippocampus in developing kitten brain (left panel). Optical density of the area between the two lines was analysed for radioactivity, after calibration with 3H-standards (Amersham).- The results are shown in right panel. In both the visual cortex and hippocampus, binding of tritiated IP3 and PDBu (nCi/mg protein) peak at different laminar locations. At P40, the binding of [ 3 H]PDBu peaks in dendritic regions (arrows) of the dentate gyrus and that of [ 3 H ] IP3 peaks in the layer of granule cell somata. D, dentate gyrus. O.D., optical density. 119 I'liinmii I'IIIII'I t'liii-iiiii, h'niii'i 120 Figure 26, Development of [3H]IP3 and [3H]PDBu binding sites in visual cortex. A) Comparison of the binding among different cortical layers. Development of IP3Rs shows a delay at early ages when [3H]PDBu binding is increasing, although at different rates in the various cortical layers. B) Comparison of binding of the two ligands in developing cortex. Data are expressed as the percentages of the maximal binding at all ages studied. High levels of [3H]PDBu binding sites exist at age PI while few IP3R sites are present until postnatal day 20. PKC reaches its maximal levels earlier than IP3R, except in layer IV where they peak at the same age. 121 122 Figure 27 Autoradiography of [ 3H]IP3 and [3H]PDBu binding in undercut visual cortex (left) and the results of densitometry (right). The isolated zones are indicated by arrows. The binding of [ 3H]IP3 shows little difference between the undercut zone and the control hemisphere (1.873±0.026 nCi/mg and 1.897+0.091 nCi/mg protein, respectively), while the binding for [3H]PDBu in the isolated region (211.092+12.044 nCi/mg) is more than twice (t=8.548, p<0.0001) that of the control side (105.237+2.879 nCi/mg). 123 O.I). 124 Figure 28, Colocalization of receptors for PI turnover in developing visual cortex. The density of each ligand in cortical layers is expressed as a percentage of the maximal binding density across all cortical laminae. Notice the distributions of a l and IP3R completely differ from that of Ml and PKC in newborn cortex and a high degree of overlap are seen during the critical period. In adults, despite the colocalization of M l , al and PKC, lamination of IP3R is still distinct. Figure 29, Comparison of development of receptors for PI turnover in the visual cortex. Data for each autoradiographic are expressed in percentages of the maximal binding density among animals with various ages. Each point represents an average of at least four measurements. See text for details. 125 Postnatal Day 1 60 40 4 20 J Mill IV V VI S P Cortical Layer Postnatal Day 30-40 II III IV V VI Cortical Layer Adult <rt NAR MIAChH PKC IPjR I II III IV V Cortical Layer L aye r s I - III Laye r IV Laye rs V - VI ' 10 20 30 40 60 75 120 Postnatal Day 1 10 20 30 40 60 75 120 Postnatal Day 1 10 20 30 40 60 75 120 Adult Postnatal Day 126 ACKN0WLELX3EMENT I thank Dr. F. Huang and Dr. M. Kennedy for providing the P K C and C A M K II antibodies. I also gratefully acknowledge my supervisor, Dr. M. Cynader, for his many helpful and stimulating suggestions and for the lesioned animals operated by him. Special thanks to Dr. C . Beaulieu for his great assistance in E M observation. This project was supported by M R C grant No. 127 BIBLIOGRAPHY 1. 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