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Examining the effects of early-life seizures on neuronal dendrite growth in vivo using a novel experimental… Hewapathirane, Daminda Sesath 2009

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EXAMINING THE EFFECTS OF EARLY-LIFE SEIZURES ON NEURONAL DENDRITE GROWTH IN VIVO USING A NOVEL EXPERIMENTAL MODEL SYSTEM   by    Daminda Sesath Hewapathirane  M.Sc., University of Toronto, 2004 Honours B.Sc., University of Toronto, 2000     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies   (Neuroscience)    THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  November 2009  ? Daminda Sesath Hewapathirane, 2009 - ii -  ABSTRACT The effects of highly prevalent early-life seizures on neuronal activity-dependent developmental programs within the immature brain remain unclear. To address this issue, the present work examined the acute and persistent effects of early-life seizures on neuronal dendritogenesis, a key activity-dependent component of neural circuit development. A novel experimental model system of early-life seizures, based on the albino Xenopus laevis tadpole, was developed for these studies. The transparency of this organism allows in vivo imaging of neuronal growth and activity within the intact developing brain. Additionally, immobilization of tadpoles using reversible paralytics and immersion in agar, for electrophysiological or imaging experiments, allows examination of seizure activity and seizure-induced effects on neuronal growth for the first time within the unanaesthetized and awake brain. Chemoconvulsant-induced seizures in tadpoles were extensively characterized using behavioural assessment, measures of cell death, and in vivo examination of neural activity during seizures through electrophysiological recordings and imaging of intracellular calcium dynamics. Rapid and long-interval time-lapse in vivo two-photon imaging of individual fluorescently labelled growing optic tectal neurons within the intact tadpole brain revealed that seizures inhibit dendritic arbor growth, that these effects are mediated cell-autonomously by excessive AMPA-receptor mediated excitatory activity, and that a single seizure episode persistently stunts subsequent arbor growth. Reduced dendritic growth is a result of decreased branch elongation, increased branch elimination, and loss of dendritic filopodia. Seizures also persistently reduced the density of immunostained excitatory synaptic markers within the tectal neuropil. Rapid time lapse imaging at 5 minute intervals for 5 hours reveals selective effects on filopodial growth dynamics, characterized by rapid increase in the rate of elimination of pre-existing filopodia within minutes of seizure onset, followed by hyper-stabilization of filopodia generated during seizures. These data suggest that seizures interfere with neural circuit development by acutely destabilizing filopodia present prior to seizure induction and hyper-stabilizing filopodia formed during seizures, leading to a persistent inhibition of continued arbor elaboration and growth. This is the first examination of the effects of common early-life seizures on dendritic morphogenesis within the intact and awake brain, and these findings identify a potential morphological correlate of persistent seizure-induced neural dysfunction. - 180 -  TABLE OF CONTENTS       1. GENERAL INTRODUCTION ............................................................................ 1 1.1 Seizures ? an overview ................................................................................ 1 1.1.1 Definitions ............................................................................................ 1 1.1.2 Epilepsy ................................................................................................ 3 1.1.3 Provoked seizures ................................................................................ 5 1.2 Early-life seizures ......................................................................................... 6 1.2.1 High incidence of seizures in the immature brain ................................. 6 1.2.2 Why is the immature brain more prone to seizures? ............................ 7 1.3 Are seizures damaging to the developing brain? ...................................... 9 1.3.1 Brain dysfunction associated with early-life seizures: clinical findings 12 1.3.2 Developmental animal models of seizures and epilepsy .................... 15 1.3.3 Effects of early-life seizures on brain function: experimental data ...... 18 1.4 How do early-life seizures induce persistent brain dysfunction? .......... 24 1.5 Do early-life seizures alter dendritic morphogenesis? ............................ 26 1.5.1 Growth of dendrites during normal brain development ....................... 27 1.5.2 Effects of early-life seizures on dendritic growth and morphology ...... 33 ABSTRACT ............................................................................................................ ii TABLE OF CONTENTS ......................................................................................... iii LIST OF TABLES ................................................................................................. vii LIST OF FIGURES .............................................................................................. viii LIST OF ABBREVIATIONS .................................................................................... x CKNOWLEDGEMENTS ...................................................................................... xi DEDICATION ....................................................................................................... xii CO-AUTHORSHIP STATEMENT ......................................................................... xiii  - iii - - 181 -  1.6 The albino Xenopus laevis tadpole as a model of early brain development ............................................................................................... 39 1.7 Research hypotheses and aims ................................................................ 44 1.8 References .................................................................................................. 46 2. DEVELOPMENT OF A NOVEL IN VIVO MODEL OF EARLY-LIFE SEIZURES BASED ON THE XENOPUS LAEVIS TADPOLE ........................................... 65 2.1 Introduction ................................................................................................. 65 2.2 Methods ....................................................................................................... 69 2.2.1 Animals .............................................................................................. 69 2.2.2 Seizure induction and behavioural observations ................................ 69 2.2.3 In vivo electrophysiological recordings ............................................... 70 2.2.4 Calcium indicator loading ................................................................... 71 2.2.5 In vivo two-photon fluorescence imaging of calcium dynamics .......... 72 2.2.6 Fluorescence imaging data analysis .................................................. 73 2.2.7 Analysis of seizure-related cell death within the tadpole brain ........... 74 2.3 Results ........................................................................................................ 76 2.3.1 Characterization of drug-induced behavioural seizures...................... 76 2.3.2 Extracellular electrophysiological recordings ...................................... 80 2.3.3 Imaging neuronal calcium activity during seizures ............................. 82 2.3.4 Assays of seizure-induced cell death ................................................. 86 2.4 Discussion .................................................................................................. 88 2.5 References .................................................................................................. 95 3. PROTOCOL: SINGLE CELL ELECTROPORATION IN VIVO WITHIN THE INTACT DEVELOPING BRAIN .................................................................... 100 3.1 Introduction ............................................................................................... 100 3.2 Protocol ..................................................................................................... 101 3.2.1 Equipment set-up ............................................................................. 101   - iv - - 182 -  3.2.2 Fabrication of micropipettes ............................................................. 102 3.2.3 Single-cell electroporation protocol .................................................. 104 3.2.4 Screening for successfully electroporated cells ................................ 110 3.3 Discussion ................................................................................................ 112 3.4 References ................................................................................................ 113 4. EARLY-LIFE SEIZURES INHIBIT DENDRITOGENESIS IN VIVO ............... 114 4.1 Introduction ............................................................................................... 114 4.2 Methods ..................................................................................................... 118 4.2.1 Animals and seizure induction .......................................................... 118 4.2.2 In vivo single-cell electroporation for fluorescent labeling and targeted transfection of developing neurons .................................................. 118 4.2.3 In vivo two-photon fluorescence imaging of dendritic growth ........... 120 4.2.4 Dendrite morphometric analysis ....................................................... 121 4.2.5 Immunohistochemistry of synaptic markers ..................................... 121 4.2.6 Statistical analyses ........................................................................... 123 4.3 Results ...................................................................................................... 124 4.3.1 Seizures inhibit dendritic growth ....................................................... 124 4.3.2 Seizure-induced inhibition of dendritic growth is due to excessive AMPA receptor activation ................................................................. 127 4.3.3 Seizures increase rates of filopodial and branch elimination ............ 128 4.3.4 Seizures induce rapid elimination and subsequent stabilization of specific filopodial sub-populations .................................................... 132 4.3.5 Reduced motility and restricted exploratory behaviour of filopodia during seizures ................................................................................. 135 4.3.6 Seizure-induced inhibition of dendritic growth is persistent .............. 137 4.3.7 Seizures persistently decrease excitatory synapse densities ........... 140 4.4 Discussion ................................................................................................ 142   - v - - 183 -  4.5 References ................................................................................................ 148 5. GENERAL DISCUSSION .............................................................................. 152 5.1 Summary of findings ................................................................................ 152 5.2 The albino Xenopus laevis tadpole as a novel model of developmental seizures ..................................................................................................... 155 5.2.1 Is the Xenopus laevis tadpole a valid seizure model? ...................... 155 5.2.2 Advantages and limitations .............................................................. 157 5.2.3 Significance ...................................................................................... 159 5.3 Early-life seizures interfere with neuronal dendritogenesis ................. 160 5.3.1 A model relating seizure-induced neural circuit dysfunction to altered dendritogenesis ................................................................................ 161 5.3.2 Additional factors potentially contributing to circuit dysfunction ........ 163 5.3.3 Significance ...................................................................................... 164 5.4 Future directions ...................................................................................... 165 5.4.1 Direct extensions of present work .................................................... 165 5.4.2 Examining the molecular mechanisms underlying seizure-induced effects on dendritogenesis ............................................................... 165 5.4.3 Examining the effects of developmental seizures on synaptogenesis ......................................................................................................... 166 5.4.4 Examining the effects of developmental seizures on neural circuit maturation, function, and plasticity ................................................... 167 5.5 Overall conclusions.................................................................................. 170 5.6 References ................................................................................................ 171 APPENDIX: ANIMAL CARE CERTIFICATES .................................................. 176      - vi - - vii -  LIST OF TABLES Table 1.1: Comparative stages of brain development in humans and rats  .............. 19   Table 1.2: Select ed studies examining the eff ects of earl y -life seizures in                    developing rats  ........................................................................................... 20  Table 2.1: Behavioural seizure stages in Xenopus laevis tadpol es  ......................... 77                     - viii -  LIST OF FIGURES Figure 1.1: Inhibition predominat es over ex citation in the immat ure brain  ............... 9   Figure 1.2: Dendrite grow t h in vivo ............................................................................ 29   Figure 1.3  Synaptot ropic dendrite grow t h  ................................................................ 31  Figure 1.4: Retinotect al system of Xenopus laevis tadpol es  .................................... 40  Figure 2.1: Charact erization of chemoconvulsant -induced behavioural seizu re                     activity in Xenopus laevis tadpol es  ......................................................... 77  Figure 2.2: In vivo extracell ul ar field recordings of elect rographic seizures in                     immobilized unanaesthetized tadpol es  ................................................... 81   Figure 2.3: In vivo tw o -photon imaging of neuronal cal cium dynamics during                     seizures within the unanaest hetized brain  .............................................. 82   Figure 2.4: Single -cel l analysis of neuronal cal cium dynamics during seizures,                            imaged in vivo within the unanaesthetized brain  .................................... 8 5  Figure 2 .5: Examination of cell deat h within the tadpol e brain fol l owing prolonged                     seizure activity  .......................................................................................... 8 7 Figure 3.1: Equipment set -up for singl e -cel l elect roporation  ................................. 102  Figure 3.2: Single cell elect roporation micropipet t e geomet ry  .............................. 103  Figure 3.3: Single cell elect roporation micropipet t e positioning in the albino                     Xenopus laevis tadpol e brain  ................................................................. 107  Figure 3.4: Examples of labell ed neurons imaged within the live tadpole                         brain  ......................................................................................................... 111  Figure 3.5: Example of in vivo imaging of intracel l ul ar synapse d yna mics  .......... 112  Figure 4.1: Developmental seizures inhibit dendrite grow t h through excessive                     AMPA rece ptor activation  ....................................................................... 126  Figure 4.2: Rapid time -lapse imaging of dendrite grow th during seizures in the                     intact and unanaest hetized brain  ........................................................... 130  Figure 4.3: Seizures rapidly eliminate existing and hyper -stabilize new filopodia                     during seizures  ........................................................................................ 134  Figure 4.4: 3D filopodia tracking reveals complex effect s of seizures o n filopodial                     motility and explorat ory behaviour  ........................................................ 136 - ix -   Figure 4.5: Seizures persistent l y stunt furt her arbor grow th and elaboration                     in vivo ....................................................................................................... 139   Figure 4.6: Seizures persistent l y reduce syna pse density  ..................................... 141                          - x -  LIST OF ABBREVIATIONS 4-AP    4-aminopyridine AMPA  ?-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate CaMKII  calcium/calmodulin -dependent protein kinase II EEG  electroencephalogram GABA  ?-aminobutyric acid (f)(E)GFP  (farnesylated) (enhanced) green fluorescent protein  FS  febrile seizures IQ   intelligence quotient MS-222  3-aminobenzoic acid ethyl ester  NA   numerical aperture NMDA  N-methyl-D-aspartate OGB1 -AM  Oregon Green 488 BAPTA -1, AM P  postnatal day PB  phosphate buffer PBS  phosphate buffered saline PDS   paroxysmal depolarization shift PF   post-fertilization  PI   propidium iodide PTZ    pentylenetetrazol RT   room temperature SCE   single-cell electroporation SE  status epilepticus TUNEL  terminal uridine deoxynucleotidyl transferase dUTP nick end labeling  - xi -  ACKNOWLEDGEMENTS Being a founding member of a new research laboratory as well as independently initiating a new direction of research within the Haas lab proved to be both an extremely challenging yet remarkably valuable learning experience.  I extend my genuine  appreciation to my colleagues in the Haas laboratory? Simon Chen, Clayton Cheng, Blair Duncan, Derek Dunfield, Sharmin Hossain, Xuefeng Liu, Kaspar Podgorski  and Wesley Yen ? and my friends, in particular Kathryn Hum, Geeth Gunawardana, and Richard Liu. Your friendship, support, and words of advice truly helped me overcome many of the challenges I faced during the course of my thesis work.  I also extend sincere thanks to my supervisor, Dr Kurt Haas, and the members of my supervisory committee?Drs. Cathy Rankin, Lynn Raymond, and Y u Tian Wang?for their support, time, and guidance.  I am truly grateful for the financial support that I received through graduate scholarships from the Savoy Foundation of Canada, the Scottish Rite Charitable Foundation of Canada, the British Columbia Innovation Council, and the University of British Columbia.  Lastly, I would like to thank my family . This would not have been possible without your unyielding encouragement and support. I am eternally indebted to you for the many sacrifices you have made.            - xii -   DEDICATION    for my Parents,  ...the hands that shield the flame.              - xiii -  CO-AUTHORSHIP STATEMENT  Chapter 2  For the work described in Chapter 2, I conceived of and designed the experiments, analyzed the data, prepared the figures and wrote the manuscript. While the majority of the experimental work was performed by myself, Derek Dunfield assisted with in vivo imaging of neural calcium dynamics, Simon Chen assisted with the electrophysiology experiments, and Wesley Yen assisted with behavioural experiments. Dr. Kurt Haas provided advice on experimental design and helped edit the manuscript.  Chapte r 3 For experimental protocol described Chapter 3, I wrote the manuscript text and prepared the figures.  Dr. Kurt Haas helped edit the manuscript.   Chapter 4 The experiments in  Chapter 3 were conceived of and designed by me. I conducted all of the experimental work and data analysis, prepared the figures, and wrote the manuscript. Simon Chen, Wesley Yen and Shay Neufeld provided assistance in the software-based manual tracking and measurement of dendritic filopodia, and Parisa Karimi Tari assisted with immunohistochemistry . Dr. Kurt Haas provided advice on experimental design and helped edit the manuscript.  - 1 -  1.     GENERAL INTRODUCTION  1.1 Seizures ? an overview  1.1.1 Definitions  An epileptic seizure is a state of abnormal and excessive neuronal discharge within the brain, typically involving synchronized bursts of neural activity within large populations of neurons (Penfield and Jasper, 1954) . Seizures are self-sustained and usually self-limiting (Burnham, 2002b) .   Clinically, the primary diagnostic tool in defining a seizure event is the electroencephalogram (EEG) (Lee, 1995) . Seizures are marked by the DSSHDUDQFH RI FKDUDFWHULVWLF ?VSLNH? RU ?VSLNH-and-ZDYH? ((* SDWWHUQV WKHelectrographic seizure), representing the electric fields created by large numbers of neurons firing in synchrony (Westbrook, 2000) . The initiation of a seizure ?DSURFHVVWHUPHG?ictogenesis?IURPWKHODWLQictus  referring in this context to a seizure?results from the stereotypic and synchronized electrical response called the paroxysmal depolarization shift (PDS), within a population of neurons comprising the seizure focus. The PDS consists of a sudden, large (20 -40 mV), long -lasting (50 -200 ms) depolarization, which triggers a train of action potentials at the peak of the PDS. The CHAPTER 1 - 2 -  simultaneous discharge of a large number of neurons during a PDS manifests as DV\QFKURQRXV?LQWHULFWDO?VSLNHLQ((*UHFRUGLQJV7KH3'6LVIROORZHGE\Dlarge after-hyperpolarization, which serves to terminate the PDS and the associated volley of action potentials. If, however, excitation becomes excessive, or inhibition is substantially reduced, the PDS can lead to an ictal discharge consisting of sustained neural firing. If the paroxysmal ictal burst discharges subsequently overcomes the powerful inhibition surrounding the seizure focus, excitation propagates into the surrounding circuitry leading to seizure spread along established neural pathways. The mechanisms of seizure termination are not well understood, but may include re-establishment of normal ionic gradients, release of neuromodulators, and the restoration of normal excitatory and inhibitory synaptic transmission and ionic currents. The period following seizure termination is often accompanied by an increase in inhibitory drive, a phenomenon termed post-ictal depression (Westbrook, 2000; Stafstrom, 2004) .  As defined by the Commission on Classification and Terminology of the International League Against Epilepsy (CCTILAE), seizures can be classified into two main categories?partial or generalized?according  to the source of the seizure onset within the brain (CCTILAE, 1981) . Partial seizures involve only a part of the brain, while generalized seizures appear to involve the entire brain. It is possible for a partial seizure to spread to other structures and to eventually develop into a generalized VHL]XUHWKURXJKDSURFHVVUHIHUUHGWRDV?VHFRQGDU\JHQHUDOL]DWLRQ?(CCTILAE, 1981) . - 3 -  Seizure episodes are commonly accompanied by a visible motor manifestation, referred to as the behavioural or clinical seizure, usually reflecting seizure activity within a region of the brain controlling motor functions. However, when seizure activity occurs within brain regions without direct motor outputs, a behavioural component does not necessarily accompany the electrographic seizure (Saint -Hilaire, 1995) . While seizure durations are commonly within the order of seconds to minutes, occasionally seizures fail to self-terminate within significantly extended time periods. Instances of unremitting seizure activity are referred to as sta tus epilep ticus (SE), a life -threatening condition that is most often defined as continuous seizure activity lasting more than 30 minutes (Haut et al., 2004) . SE is most often seen in patients with epilepsy, however, in a notable proportion of cases (10 -20%), SE occurs as the first known seizure  (Engel Jr. and Pedley, 1998) . T he prevalence of SE within the general population is estimated to lie between 1 in 400 and 1 in 1000 (Holmes and Dichter, 2008) .    1.1.2 Epileps y  Epilepsy is a highly prevalent serious neurological disorder characterized by the presence of spontaneous (unprovoked) and recurrent seizures, affe cting an estimated 1-2% of the general population (Hauser et al., 1991; Annegers, 1994; Berg et al., 1996) . In most patients seizure onset occurs in childhood, often - 4 -  before the age of 15 years (Burnham, 2002b) . It follows, therefore, that epilepsy is the most common neurological disorder in children (Ekinci et al., 2009) . Epileptic patients are thought to have a lower seizure threshold than non-epileptics, where endogenous brain activity is occasionally able to trigger a seizure (Scher, 1997) .  The causes of epilepsy differ widely  (Penfield and Jasper, 1954) . In some cases, structural brain abnormalities exist, including, but not limited to, neoplasms, vascular malformations, and scar tissue from stroke, birth trauma, or brain injury (Kim, 2001) . Such cases are referred to aV?V\PSWRPDWLF?HSLOHSVLHVreferring to the existence of an identifiable cause. In the majority of cases (~60 -70%) , however, there is no identifiable brain abnormality (Guberman and Br uni, 1999; Burnham, 2002b) 6XFKFDVHVDUHUHIHUUHGWRDV?LGLRSDWKLF?HSLOHSVLHV(Penfield and Jasper, 1954) , and are believed to largely result from genetic factors (Burnham, 2002b) .  Currently, pharmacological treatment is the first line of therapy for seizure control (Burnham, 1998) .  Unfortunately, seizures are fully controlled in on ly approximately 60% of the patients taking anti -seizure medications (Lothman et al., 1991; Burnham, 2002a; Sander, 2003) . Of the remaining 40%, approximately half (i.e., 20% of the epileptic population) are partially responsive to drug treatment.  The remaining 20% of the patients are highl y resistant to drug treatment, and are VDLGWRKDYH?UHIUDFWRU\?RU?LQWUDFWDEOH?VHL]XUHV(Shorvon, 1996; Guberman and Bruni, 1999) .  Non -drug therapies are often attempted to treat patients with intractable epilepsy, including brain surgery to resect seizure foci, the ketogenic - 5 -  diet, and vagal/deep brain stimulation  (Burnham, 2002a) .  However, the overall effectiveness of these approaches, as with drug therapy, is lower than would be desired.   1.1.3 Provoked seizures  All brains can be made to seize, provided a sufficient epileptogenic stimulus is applied. Accordingly, seizures can be induced, within non-epileptic brains, by the application of excessive electrical stimulation, exposure to chemical agents that directly/indirectly increase excitatory neural activity, or in response to brain trauma, among numerous other seizure-inducing stimuli (Engel Jr. and Starkman, 1994) . Individuals suffering from provoked seizures, however, are not considered epileptic since such seizures are not spontaneous. Additionally, a sizeable proportion of the general public (roughly 2-5%) will experience one unprovoked seizure during their lives but will have no further seizures (Hauser et al., 1990; Shinnar et al., 1990; Lothman et al., 1991) , however, such patients are not considered epileptic since seizures are not recurrent.   - 6 -  1.2 Earl y -life seizur es  1.2.1 High incidenc e of seizures in the imma ture brain  Seizures are most commonly observed during the first few years of life, with peak incidence in the first year (Hauser, 1995) , and sta tu s epilep ticus  is also more common in children that in adults (Holmes and Ben -Ari, 1998) . In the majority of individuals who develop epilepsy, the onset of sp ontaneous seizures occurs during childhood (Hauser, 1995; Cowan, 2002) . Neonates and infants are also at an increased risk of experiencing seizures provoked by pro-convulsant stimuli such as fever, hypoxia, drugs or trauma (Jensen and Baram, 2000) . Indeed, seizures induced by hypoxic-ischemic encephalopathy are the most prevalent seizure type in neonates (Rakhade and Jensen, 2009) . In addition to brain insults, several neonatal disorders often present initially with seizures, including congenital brain disorders, inborn errors of metabolism, and genetic disorders. It is estimated that approximately 5-8% of children will experience at least one seizure during their lifetime (Burnham, 2002b) . While the high incidence of provoked early-life seizures is partly due to these causative factors, and an increased likelihood of experiencing seizure-promoting brain insults, substantial experimental evidence demonstrates that the immature brain exhibits a reduced seizure threshold compared to the mature brain (Haas et al., 1990; Sperber et al., 1991; Moshe, 1993; Jensen, 1995; Ben -Ari and Holmes, 2006) . For instance, high fever, which rarely induces seizures in - 7 -  adults, causes seizures in ~5% of children worldwide (referred to as febrile seizures), and constitutes the most common seizure type in childhood (Chungath and Shorvon, 2008; Rakhade and Jensen, 2009) . In addition, febrile sta tu s epilep ticus  occurs in approximately 5% children experiencing febrile seizures (~1 in 400 of all children) (Holmes and Dichter, 2008) . A recent prospective multi-centre clinical study of febrile sta tu s epilepticus  cases reported a mean seizure duration of 68 minutes, with 24% of children having seizure durations of over 2 hours (Shinnar et al., 2008) .  1.2.2 Why is the immatur e brain more prone to seizur es?  The underlying determinants of increased seizure susceptibility in the immature brain are not fully understood, but are thought to relate to the developmental imbalance between the maturation of excitatory and inhibitory circuits (Fig. 1.1) .  Since neuronal activity is critical for synaptogenesis and brain circuit development (Katz and Shatz, 1996) , excitation initially predominates over inhibition during neural circuit formation and refinement within the immature brain. It therefore follows that seizure incidence strongly correlates with the maturational stage of inhibitory circuitry within the brain (Haut et al., 2004; Rakhade and Jensen, 2009) .  Several features of the developing brain contribute to increased excitability, including: (i) immature neurons demonstrate relatively high input resistances, - 8 -  facilitating the generation of large changes in membrane potential, promoting action potential generation and increasing excitability; (ii) GABA ?-aminobutyric acid) acts as an excitatory neurotransmitter during early brain development (when acting through GABAA receptors), and reduced GABAergic inhibition during this period increases excitability and promotes synchronicity; (iii) both the prolong ed NMDA ( N-methyl-D-aspartate) receptor -mediated postsynaptic currents in immature neurons and the delayed maturation of metabotropic GABAB receptor-mediated inhibition results in a relative increase in excitatory neural activity; (iv) exuberant synaptic connections formed during early brain development, prior to circuit refinement, promote the formation of recurrent excitatory connections; and (v) the expression of both NMDA and AMPA ( ?-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) subtypes of ionotropic glutamate receptors reaches peak levels during the neonatal period in humans, before full maturation of GABAergic inhibition (Swann and Habli tz, 2000; Rakhade and Jensen, 2009) .        - 9 -             1.3 Are seizures damaging to the developing brain?  Early brain development involves a myriad of intricately coordinated, spatially and temporally defined programs culminating in the formation of complex neural circuits. Normal neural circuit development requires neuronal precursors to differentiate appropriately, migrate to correct locations, form dendrites and axons, express ion channels and other critical membrane and subcellular components, synthesize neurotransmitters and their receptors, form synapses with proper Figure 1.1: Excitation predominates over inhibition in the immature brain. Activation of GABA receptors is depolarizing in rats early in the first postnatal week and in humans up to and including the neonatal period. Functional inhibition, however, is gradually reached over development in rats and humans. Before full maturation of GABA-mediated inhibition, the NMDA and AMPA subtypes of glutamate receptors peak between the first and second postnatal weeks in rats and in the neonatal period in humans. Kai nate receptor binding is initially low and gradually rises to adult levels by the fourth postnatal week. AMPA, ?-amino-3-hydroxy-5-methyl-4-isoxazole propionate; GABA, ?-aminobutyric acid; NMDA, N -methyl-D-aspartate; P, postnatal day.  Reproduced from Rakha de and Jensen (2009) Nature Reviews Neurology 5, 380 - 391.  Reprinted by permission from Macmillan Publisher s Ltd: Nature Reviews Neurology, C opyright (2009).  - 10 -  targets, and create appropriate networks with which to establish correct firing patterns (Stafstrom, 2007) . During the earliest stages of nervous system development, the initial steps of neural circuit formation are mainly regulated by activity-independent molecular cues for pathfinding and target selection (Tessier -Lavigne and Goodman, 1996) . Spontaneous neural activity during this period of early brain development then transforms nascent neuronal networks into organized circuits and, subsequently, experience-dependent activity precisely refines and maintains patterns of connectivity to form circuits that subserve adult brain function (Catalano and Shatz, 1998; Crair et al., 1998; Penn et al., 1998) .  Given the exceptional complexity of these multiple steps, it is not surprising that pathological interference at different stages of development, due to genetic mutation or exogenous neurologic insult, can disrupt the normal sequence and result in abnormal circuit development and/or aberrant firing patterns, causing alterations in brain function (Ben -Ari and Holmes, 2006; Stafstrom, 2007) . Importantly, many fundamental processes involved in neural circuit development, including axonal (Ruthazer et al., 2003)  and dendritic (Sin et al., 2002)  arbor growth, as well as synapse formation and refinement (Cohen -Cory, 2002) ,  are dependent on specific patterns of neuronal activity in immature networks. Disruption of normal neural activity during the so-FDOOHG?FULWLFDOSHULRGV?in the developing visual, auditory or somatosensory systems, for instance through sensory deprivation or pharmacological treatment, modifies patterns of circuit organization which leads to long-term consequences on the functional properties - 11 -  of neural circuitry in sensory neocortex (Hubel and Wiesel, 1970; Hensch and Fagiolini, 2005) . The sensitivit y of the developing nervous system to abnormal neural activity during these critical periods of plasticity is further indicated by the observation that abnormal sensory experience in early life can also alter the capacity for neural plasticity in the adult visual and auditory systems (Kirkwood et al., 1996; Brainard and Knudsen, 1998) . These observations suggest that neural activity during development not only determines the organization of neural circuitry but may also influence the capacity for circuit plasticity later in life (Abraham and Bear, 1996; Feldman and Knudsen, 1998) . Unfortunately, the duration of heightened seizure susceptibility and seizure expression coincides with these critical periods of neural activity-dependent brain circuit development, raising the possibility that abnormal and excessive seizure-related neuronal activity may directly interfere with these developmental programs. Consequently, considerable attention has focused on whether seizures induce alterations in brain circuit development leading to persistent neural dysfunction, such as an increased susceptibility to psychiatric or neurological disorders, or subsequent seizures, later in life (Swann, 2004; Ben -Ari and Holmes, 2006) .   - 12 -  1.3.1 Brain d ys func tion associated with earl y -life seizures: c linica l  findings  Numerous large-scale, mainly retrospective, epidemiological studies have identified an increased association of early-life seizures with both neurological dysfunction and an increased susceptibility to subsequent seizures. Up to 27% of  infants with neonatal seizures?clinically defined as seizures occurring within the first 28 days after birth of a term neonate, or before 44 weeks of gestational age in a pre-term infant (Thibeault -Eybalin et al., 2009) ?develop epilepsy and/or cognitive and behavioural deficits later in life (Ronen et al., 2007) . Neonatal seizures are therefore considered a risk factor for childhood epilepsy, and in particular for refractory epilepsy (Ellenberg et al., 1984; Berg et al., 2003; Whitehead et al., 2006) . Additionally, seizures sustained in term neonates are associated with a high prevalence of abnormal neurodevelopment ( ~25% , Thibeault -Eybalin et al., 2009) . It is therefore not surprising that neonatal seizures continue to be a significant predictor of poor neurological outcome (Holmes and Ben-Ari, 1998) .  Febrile seizures (FS)  are the most commonly occurring seizures in children, with peak prevalence between the ages of 6 months and 5 years (Chungath and Shorvon, 2008) . 6KRUWIHEULOHVHL]XUHV?VLPSOH?)6KDYHEHHQassociated with a benign outcome without cognitive dysfunction (Verity et al., 1998)  or subsequent epilepsy (Shinnar, 1998) . Prolonged febrile seizures ?FRPSOH[?)6?PLQXWHVLQGXUDWLRQRQWKHRWKHUKDQGKDYHEHHQDVVRFLDWHG- 13 -  with subsequent brain dysfunction. In a recent review of 33 studies addressing the issue of seizures or epilepsy after febrile seizures, 16 reported an increased risk of a later afebrile seizure (see Chungath and Shorvon, 2008, and references therein). Based on the summated risk calculated from data obtained for all 33 of these studies, patients with a history of febrile seizures have a mean risk of 5.8% of later afebrile seizures, and a risk of between 2-7% of the development of subsequent epilepsy. Febrile status epilepticus, comprising ~5% of all instances of febrile seizures (Haut et al., 2004) , carries a substantially greater risk for the expression of subsequent afebrile seizures, ranging from 4-32% (Chungath and Shorvon, 2008) , representing a 3-6 fold greater risk than in general population children (Ellenberg and Nelson, 1981; Snyder et al., 1981) .   Numerous n eurological co-morbidities are also frequently observed in children with epilepsy, particularly prevalent in patients with intractable seizures. Children with epilepsy have an estimated risk of 21-60% for childhood psychopathology, significantly higher than the risk of 6.6% in general population children (Ott et al., 2001; Ott et al., 2003; Ekinci et al., 2009) . Neurological conditions having a significant association with childhood epilepsy include mental retardation (Singhi, 1992; Steffenburg et al., 1995) , attention-deficit/hyperactivity disorder (Dunn et al., 2003; Titus et al., 2008) , autism (Steffenburg et al., 2003) , developmental disabilities (Thibeault -Eybalin et al., 2009) , depression (Piazzini et al., 2001; Plioplys, 2003) , and anxiety (Williams et al., 2003) , with the latter two conditions being the most common (Ettinger et al., 1998; Pellock, 2004) . Further, - 14 -  studies of children with intractable seizures show a correlation between a low IQ (intelligence quotient) and duration of the seizure disorder (Burnham, 2002a) , and behavioural disturbances are up to 4.8-fold higher in children with epilepsy compared to general population children (Austin and Caplan, 2007) . Even children with normal IQs and well -controlled seizures are at a higher risk for learning disabilities (Bailet and Turk, 2000) .  Despite these reported links between early-life seizures and neurological dysfunction and/or an increased susceptibility for subsequent seizures, clinical studies have failed to completely resolve the pathogenic potential of seizures within the developing brain. Indeed, certain clinical studies have failed to demonstrate a significant increase in the risk of brain dysfunction following early-life seizures (Scher, 1997; Verity et al., 1998; Shinnar and Hauser, 2002) . When considering these divergent findings, it is important to highlight that a major challenge in clinical studies is establishing causality from correlative findings. Clinical studies are complicated by inherent variability of patients comprising study groups. Factors such as differences in the number, type and frequency of seizures;  age of seizure onset;  individual genetic susceptibilities;  underlying etiology;  medication;  and other environmental influences such as psycho-social stress invariably affect individual patient outcomes, thereby confounding across- and within-study comparisons (Holmes, 1991) . For instance, many commonly used anti-seizure medications are known to contribute to the expression of co-morbid psychiatric conditions, cognitive deficits, and/or - 15 -  behavioural problems associated with early-life seizures (Austin and Caplan, 2007) . Therefore, from clinical data i t has been difficult to resolve whether seizures directly induce the observed dysfunction (i.e., a causal link) or whether observed neurological dysfunction(s) arise from shared or unrelated pathological origins (i.e., a correlational link) (Tsopelas et al., 2001) .  In an effort to more directly address whether early-life seizures are damaging to the immature brain, this issue has been extensively examined using experimental animal models of seizures and epilepsy. Use of a nimal models offers an increased degree of control over variables commonly confounding clinical studies, greatly facilitates the ability to conduct longitudinal prospective investigations, and provides numerous sophisticated and sensitive measures of seizure effects on neuronal anatomy and function.   1.3.2 Develop ment al  animal mod els of seizures an d epileps y   A wide variety of developmental animal models of seizures and epilepsy exist, commonly utilizing rats as subjects . Animals models used in the study of HSLOHSV\DUHXVXDOO\GLYLGHGLQWR?VHL]XUH?PRGHOVDQG?HSLOHSV\?PRGHOV,Qseizure models, an animal with a normal brain produces a seizure due to the application of an exogenous stimulus, most often triggered by electrical stimulation (e.g., kindling, electroconvulsive shock, continuous hippocampal - 16 -  stimulation) or the localized/ systemic administration of chemoconvulsant drugs (e.g., kainic acid, pilocarpine, pentylenetetrazol or flurothyl) (Pitk?nen et al., 2006) .  High-frequency electrical stimulation readily induces seizures provided the VWLPXOXV LQWHQVLW\ LVDERYH WKH EUDLQ?V HQGRJHQRXV VHL]XUH WKUHVKROG ,Q WKHelectro-convulsive shock model, for instance, electrical stimulation is applied to the brain through external electrodes to elicit a generalized seizure. In the kindling model (Goddard, 1967; Goddard et al., 1969) , an unvarying localized stimulus (usually to a forebrain structure), which is initially sub -convulsive, upon repetition will eventually elicit progressively more severe behavioural convulsions, reflecting propagation of initially focal seizures to other brain regions (Hewapathirane and Burnham, 2005) . Kindling permanently renders the brain more susceptible t o seizure activity. Electrical stimulation is most often utilized for kindling, however, the repeated application of small amounts of pharmacological agents (e.g., carbachol, pentylenetetrazol, opioid peptides; administered either focally or systemically depending on the substance), also provides an effective kindling stimulus (Cain, 1992).  Prolonged kindling eventually leads to the development of spontaneous seizures (Cain, 1992) . Numerous chemoconvulsants are commonly used to initiate seizures, delivered either locally to a specific brain structure to induce focal seizures, or systemically to induce generalized seizures. Commonly used chemoconvulsants such as kainic acid, pilocarpine, and pentylenetetrazol are often delivered - 17 -  systemically to induce generalized sta tu s epilep ticus , but may also be applied to specific brain structures to induce prolonged partial seizures.  Flurothyl, on the other hand, is a volatile chemoconvulsant agent that is most often delivered via inhalation to induce brief seizures. While these agents act through differing cellular mechanisms, they share the basic property of increasing neural excitability either by directly enhancing neural excitation, or by reducing inhibition (dis -inhibition) (Pitk?nen et al., 2006) .  Physical conditions such as hyperthermia (Dube et al., 2006)  and hypoxia (Jensen et al., 1991)  can also be used to initiate seizure activity, most often used to model febrile and hypoxic seizures, respectively. In addition, certain genetic mutations (either spontaneous or experimentally manipulated) result in a  reduced threshold for provoked seizures (Garcia -Cairasco et al., 1993; Valjakka et al., 2000) , or in spontaneous recurrent seizure activity (Jobe et  al., 1995) .  In models of e pilep sy , animals are experimentally manipulated to produce seizures spontaneously. Common developmental models of epilepsy (i.e., spontaneous recurrent seizures) include genetic models, post-pilocarpine, post-kainate, post- tetanus toxin, and hyperthermic seizures in rats with induced focal cortical dysplasia.  The post -pilocarpine and post-kainate models are models of epileptogenesis following sta tu s epilep ticus , involving the initial induction of prolonged seizure activity ( ~3 0 to >150 min ), with spontaneous s eizures - 18 -  beginning after a latent period in the order of weeks (Cole et al., 2002) . In the tetanus toxin model, unilateral intra-hippocampal injection of tetanus toxin in immature rats during postnatal days 9-11 results in the appearance of repeated seizures that last for 5-7 days (beginning ~ 24-72 h after tetanus toxin injection ) (Lee et al., 1995) . Tetanus toxin induces seizures by interfering with the release of the inhibitory neurotransmitters GABA and glycine. A proportion of rats exhibit unprovoked behavioural seizures and epileptiform EEG activity as adults. Another model of spontaneous recurrent seizures is based on the induction of hyperthermic seizures (usually at postnatal day 10) in rats given a f ocal cortical lesion shortly after birth (postnatal day 1) (Scantlebury et al., 2005) . This model of febrile seizures in the predisposed brain leads to the subsequent appearance of spontaneous brief recurrent seizures within brain regions ipsilateral to the lesion site.  1.3.3 Effects of ea rl y -life seizu res on brain function: experimenta l data    To more directly address the question of whether early -life seizures lead to long-term neurological abnormalities or an increased risk for subsequent seizures, a large number of studies have been conducted using experimental animal models. Immature rats are most commonly utilized as subjects in these models. - 19 -  Table 1.1 shows the ages of rats where the stage of brain development is roughly equivalent to that of humans of different ages (Haut et al., 2004) .    In agreement with clinical findings reporting significant increases in the risk of brain dysfunction following early-life seizures, substantial experimental evidence reveals that seizures during development induce changes in behaviour, deficits in learning and memory, and an increased susceptibility to seizures in adulthood (see  Table 1.2 for a summary of results from 29 selected studies) . Importantly, the observed deficits are largely independent of the method of seizure induction, indicating a direct  effect of seizures.  Stage of human brain development Rough equivalent in rat development Fu ll - term newbo rn  P8 - 10                                Infant /t o dd ler  P12 - 1 8  Peripu b ert al child  P25 - 3 2  Onset of Pube rt y  P32 - 3 8  Ad u lt  >P60                                 (P=p o stn at al day)  Table 1.1: Co mpara tive stage s of brain developme nt in huma ns and rats.  - 20 -  Reference Method of seizure induction Age at seizure induction Age at testing Behavioural outcome Subsequent seizure susceptibility  Prolonged seizures: d e Feo et al., 198 6  (de Feo et al., 19 86)  Kain ic acid (KA) or pen ty len etetra zo l  P10 or P25  P45  Defi cits in acti ve avo id an ce beh aviou r (ShB) [on ly with KA ]  Not e xa min ed  Holme s et al., 1988  (Ho lme s et al., 198 8)  Kain ic acid  P24 - 2 8  P150  Defi cits in learn in g and me mo ry (WM, TM) and exp lora to ry ac tivity (OF); incr eas ed aggre s sion (HCIT)  Not exa min ed  Jen s en et al., 1992  (Jen sen et al., 199 2)  Hyp o xia  P5 or P10  P60 - 8 5  No difference in learning and memory (WM), exploratory activity (OF), or emotionality (H) ? Flu ro th y l seizu re susc ep tib ility [P10 ]  Thu rb er et al., 1992  (Th u rb er et al., 199 2)  Con tin u o u s hip p o campa l stimula tio n  P20, 30 or 60  P80  Impa ired learn in g (W M) [P 60 only ]  Not exa min ed  Sta fstro m et al., 1992 (Sta fstro m et al., 1992)  Kain ic acid  P5, 10, 20, 30 or 60  For 3 mon th s post -sta tu s epil ep ticu s  Not exa min ed  Spon ta n eo u s seizu re s se en in rat s giv en seizu re s on/aft er P20  Sta fstro m et al., 1993  (Stafstro m et al., 1993)  Kain ic acid  P5,  10, 20, 30 or 60  P80 (P120 for rats with seizu r e s on  P60)  Defi cits in learn in g and me mo ry (WM) [P 20, P30, P60 ] and exp lora to ry activity (OF) ; incr eased emotio n alit y (H) [P 30, P60 ]  Not exa min ed  Liu et al., 1994  (Liu et al., 1994)  Piloca rp in e  P25 or P45  P80  Defi cits in exp lora to ry activ ity (OF) an d in creased e motio n alit y (H)  [P45 ]; def icits in l ear n in g and me mo ry (WM),[ P25 and P45]  ?Flu ro th y l seizu re lat en cy [P45 ]  Table 1.2: Selected studies examining the effe c ts of earl y -life seizure s in developing rats  - 21 -  Reference Method of seizure induction Age at seizure induction Age at testing Behavioural outcome Subsequent seizure susceptibility Sark isian et al., 1997  (Sark isian et al., 1997)  Kain ic acid  P20 - 2 6  (4 episo d es of sta tu s epilep ticu s )  P60  No difference in learning and memory (WM) N o t exa min ed  Koh et al., 1999  (Koh et al., 1999)  Kain ic acid  P15 and P45  (2 episo d es of sta tu s epilep ticu s )  P50  Defi cits in learn in g and me m o ry (WM) [P 15 and P45]  Not exa min ed  Dub e et al., 2000  (Dub e et al., 2000 )  H yp erth ermia  P10 - 1 1  P90 - 1 00  Not exa min ed  ? T h re sh o ld for ka in ic acid - in d u ced seizu res  Ly n ch et al., 2000  (Ly n ch et al., 2000)  Kain ic acid  P1 - 24  P95  Defi cits in re fer en ce and work in g me mo ry (RAM)  ? Susceptibility to kindling Su tu la et al., 2000  (Sut u la et al., 2000)  Kain ic acid  P1 - 24  P95  Defi cits in re fer en ce and work in g me mo ry (RAM) ; increa sed an xiety (EPM); no change in exploratory activity (OF) Not exa min ed  Bru n so n et al., 2001  ( Brunso n et al., 2001)  Cortico tro p in - rel eas in g hormo n e  P10  3,6 or 10 mon th s of age  Defi cits in lear n in g and me mo ry (WM), wor sen in g with age  Not exa min ed  dos San to s et al., 2000  (dos Santos et a l., 2000)  Piloca rp in e  P7 - 9  (3 episo d es of st at u s epilep ticu s )  P60  Decr eased exp lora to ry activit y (OF); decrea sed anxiety (E PM); l ear n in g deficit s (ISD, S B)  Not exa min ed  Huan g et al., 2002  (Hu an g et al., 2002)  Pent y len etetra zo l  (5 se izu res )  P10 - 1 4  (1 se izu re/d ay )  P 35 and P60  Defi cits in learn in g ( WM)  No change in flurothyl seizure threshold N eill et al., 2005  (Neill et al., 200 5)  Piloca rp in e  P25 or P40  P105  Defi cits in aud ito ry locat ion discri min at ion  Not e xa min ed  Table 1.2 (continued)  - 22 -  Reference Method of seizure induction Age at seizure induction Age at testing Behavioural outcome Subsequent seizure susceptibility Koh 2004 (Koh et al., 2004)  Hyp o xia  (15 min)  P10  P14, 21, 28 or 45  Not exa min ed  ?L at en cy  to kain ic acid seizures (also ? seizure se verity )  Mika ti 2005 (M ika ti et al., 2005)  Hyp o xia  (~26 min )  P10  P81  In creas ed aggre ssion (H) and deficit s in memo ry (W M)  Not exa min ed  Dub e 2006  (Dub e et al., 2006)   Hyp erth ermia  (30 min)  P10  P90  Not exa min ed  S p o n ta n eo u s seizu re s  an d epilep tiform disch ar ge s obser v ed  Repeated brief seizures: Mo sh e and Alb ala , 1982  (Moshe and Alba la, 1982)  Kind ling  P10  P65 - 7 0  Not exa min ed  Kin d led rat pup s more easily re - kin d led as ad u lts  Holme s et al., 1993  (Ho lme s et al., 199 3)  Kin d ling  (16 s ta g e V seizu re s)  P20, 40 or 60  P80  In creas ed emotio n alit y [P2 0 and P40]  ?Flu ro th y l seizu re lat en cy [P40 and P60]  Lee et al., 199 5 (Le e et al., 1995)   Teta n u s toxin  (int ra - h ip p o campa l)  P 9 - 11  (~24 seizu r es/d ay over 1 we ek)  P 41 - 1 81  Not exa min ed   S pon ta n eo u s seizu re s  an d epilep tiform disch ar ge s obser v ed in ad u lth o o d  Neill et al., 1996  (Neill et al., 199 6)  Flu ro th y l  (15 seizu re s)  P15 - 1 9  (3 se izu res /d ay )  P53  Defi cits in learn in g ( WM) an d au d ito ry locat ion discrimin at ion  Not exa min ed  Holme s et al., 1998  (Ho lme s et al., 199 8)  Flur o th y l  (25 seizu re s)  P0 - 4  (5 se izu res /d ay )  P24 - 3 0 (beh av. te st s), P45 (s eizu re su sc ep .)  Defi cits in learn in g ( WM), dec reas ed exp lora to ry activity (OF)  ? P en ty len etetra zo l seizu re thre sh o ld  Huan g et al., 1999  (Hu an g et al., 1999)  Flu ro th y l  (50 seizu re s)  P0 - P 9  (5 se izu res /d ay )  P82  Defi cits in learn in g and me mo ry (WM)  ?Flu ro th y l seizu re th resh o ld  Table 1.2 (continue d)  - 23 -  Reference Method of seizure induction Age at seizure induction Age at testing Behavioural outcome Subsequent seizure susceptibility d e Rogalski Lan d ro t et al., 2001  (d e Rog a l ski La n dr ot e t al ., 20 0 1 )  Flu ro th y l  (55 seizu re s)  P0 - 11  (4 - 5 seizu r es/d ay )  P27  Defi cits in learn in g and me mo ry (WM)  Not exa min ed  Lee et al., 200 1  (Lee et al., 2001)  Teta n u s toxin  (int ra - h ip p o campa l)  P10  (~24 seizu r es/d ay over 1 we ek)  P57 - 6 1  Defi cits in learn in g ( WM)  Not exa min ed  Soga wa 200 1  (Sogawa et al., 20 01)  Flur o th y l  (45 seizu re s)  P0 - 8  (5 se izu res /d ay )  P20 or P35  Defi cits in learn in g and me mo ry (WM)  ? Flu ro th y l seizu re susc ep tib ility  Scan tlebu ry et al., 2005  (S cantlebury et al., 2005)  Hyperth ermia  (rat s gi ven foca l cort ical le sion on  P1)  P10  P100  Defi cits in learn in g and me mo ry (WM)  Spon ta n eo u s partia l seizu re s in adu lth o o d   Table 1.2 (continue d)  Abbr eviations:  P - postnatal age (days); KA - kainic acid; ShB - shuttle box; WM - water maze; TM - T -maze; OF - open field; HCIT - home cage intruder test; H - handling test; EPM - elevated plus maze; ISD - inhibitory step-down avoidance test; SB - Skinner box. - 24 -  Collectively, these findings suggest that early-life seizures can permanently alter neural function, resulting in persistent neurological deficits. Important questions arising from these observations are: (i) how do developmental seizures induce brain dys function , and (ii) can thera p eu tic strat egies be developed to reverse or attenuate the pathological effe cts of early - life seizu res ? Given that the cognitive and neurological deficits commonly associated with early-life seizures are often more detrimental tRDFKLOG?VRYHUDOOIXQFWLRQDQGTXDOLW\RIOLIHWKDQWKHseizures themselves (Stafstrom, 2002) , these research questions have significant clinical implications.  1.4 How do earl y -life seizur es induce  per sistent brain dysfunction?   Precisely how developmental seizures induce network hyperexcitability and/or alterations to endogenous network function and plasticity has been a topic of considerable research (Holopa inen, 2008; Rakhade and Jensen, 2009) . Although results from experimental work have established that early-life seizures induce abnormal network activity (Smith et al., 1998; Dube et al., 2000) , altered circuit plasticity (Jensen et al., 1998; Cornejo et al., 2007) , and deficits in normal functional response patterns of neurons (Chen et al., 1999; Karnam et al., 2009) , the processes underlying these detrimental alterations remain poorly understood. - 25 -  In part, this has been due to the lack of overt indications of seizure-induced damage within the immature brain.  Early attempts to identify structural underpinnings of seizure-induced neural dysfunction utilized gross measures of damage after seizures, focusing primarily on neuronal loss. S ta tu s epilepticus  and recurrent brief seizures in adult rodents are associated with significant neuronal cell death?particularly within the hilar, CA1, and CA3 regions of the hippocampus (Haas et al., 2001b; Haut et al., 2004) . Seizure induced cell death is primarily caused by excessive excitatory neurotransmitter release that activates NMDA receptors  and voltage-activated calcium channels, thereby elevating intracellular calcium levels. High intracellular calcium concentration leads to generation of reactive oxygen species via activation of nitric oxide synthase, the uncoupling of oxidative phosphorylation in mitochondria, and the activation of a large range of enzymes (e.g., lipases, proteases, endonucleases, and other catabolic enzymes) that collectively have adverse consequences for cell function and viability (Olney, 1990; Me ldrum, 1991) . Remarkably, however, the immature brain has been shown to be highly resistant to seizure-induced cell death (Lynch et al., 2000; Haas et al., 2001b; Swann, 2004; Holmes, 2005) . This decreased vulnerab ility of young neurons appears to relate to the lower density of active synapses, lower cellular energy consumption, and the relative immaturity of biochemical signalling pathways that lead to cell death following insults (Holmes, 2004) . Persistent seizure induced effects in the immature brain, therefore, are likely induced by more subtle - 26 -  alterations to cellular and circuit structure and function, in the absence of cell death.  Given that early-life seizures occur during sensitive periods of brain development, it has been proposed seizures may induce persistent dysfunction by disrupting ongoing developmental programs such as cell division and migration; the sequential expression of intracellular component such as ion channels and neurotransmitter receptors; axonal and dendritic arborisation; and the formation and stabilization of synapses (Holmes and Ben -Ari, 1998; Ben -Ari and Holmes, 2006) . Seizures may disturb these processes through numerous potential mechanisms, including the induction of sub-lethal cellular injury, by interfering with neuronal activity-dependent developmental processes, or activating tissue-wide stress-responses (e.g., release of cytokines s uch as interleukins) (Holopainen, 2008) .  1.5 Do earl y -life seizur es alter dendritic morphogenesis?   The structural development of neuronal axonal and dendritic arbors is a key component of neural network formation and refinement, since arbor size and shape directly controls neural circuit connectivity, complexity, and function (H?usser et al., 2000; Wong and Ghosh, 2002; Cline and Haas, 2008) . However, while seizure-induced aberrant growth of axons has been widely studied, particularly those of hippocampal granule cells ( mossy fibres ) (Chakravarty et al., - 27 -  1997; Cavazos et al., 2003; Cornejo et al., 2007) , whether seizures alter dendrite structure has garnered considerably less attention.   In most central neurons, the vast majority of excitatory synaptic inputs terminate on the dendritic arbor. Dendrites allow neurons to integrate input from an array of synaptic inputs, and the specific branching pattern of dendrites limits the number and type of inputs that a neuron may receive. Dendritic morphology also influences action potential back-propagation, and how synaptic signals decay as they propagate towards the soma. Consequently, there is an important relationship between the dendritic morphology of a neuron and its function (Jan and Jan, 2003) . Therefore, determining whether and how seizures modify normal patterns of dendrite growth is of particular significance, since findings may reveal a novel and likely significant contributor to the expression of network hyperexcitability and altered circuit function and plasticity induced by early-life seizures.   1.5.1 Gro w th of dendrite s during norm al brain developme nt   The earliest studies of dendritic development were conducted over a century ago, during the time of Cajal and Golgi, using fixed tissue examinations at different stages of brain development (McAllister, 2000) . From these studies, it was concluded that dendritic growth occurred through a continuous and steady process of branch elongation and bifurcation. However, recent technical advances - 28 -  enabling in vivo imaging of dendritogenesis have revealed that dendritic growth is far more dynamic than initially thought. Experimental model organisms that have been pivotal in studies of dendritic growth are the transparent albino Xenopus laevis  tadpole and the zebrafish ( Danio rerio ), allowing repeated time -lapse imaging within the intact animal over periods of minutes to days (Cline, 2001; Wong and Ghosh, 2002) . The early stages of d endrite growth involves a combination of dendritic growth cone-mediated branch elongation and bifurcation, and the transformation of short thin protrusions called dendritic filopodia into nascent branches. Dendritic filopodia are short (typically <10?m in length), highly motile (often growing/shrinking by several ?m per minute), actin -rich protrusive structures that are the precursors of dendritic branches. Live  imaging studies in X enopus laevis  tadpoles and embryonic zebrafish have demonstrated that while most filopodia are eliminated, a small fraction are stabilized and eventually become stable dendritic branches (Jontes et al., 2000; Niell et al., 2004; Haas et al., 2006)   (Fig. 1.2).       - 29 -                  Fig ure 1.2: Dendrite growth in vivo.  ( a )  In vivo  time-lapse imaging of tectal neuronal dendrite growth reveals rapid structural rearrangement. ( b)  High density and rapid turnover of dendritic filopodia during arbor growth. Scale = 2?m ( c)  Overlay of colour-coded images captured hourly for 5 hours. ( d)   Daily imaging of a newly differentiated tectal neuron showing dramatic arborisation. ( e)  Dendrite growth involves an iterative sequence of selective filopodial and branch stabilization, followed by process elongation. Scale in a, c, d = 20?m   - 30 -  The rapid turnover and probing behaviour suggests that dendritic filopodia play a role sampling the local environment for appropriate presynaptic contact sites. Indeed, serial electron microscopy studies have shown that some filopodia have synapses at their tips, along their lengths, and at their bases (Fiala et al., 1998) . T hH?V\QDSWRWURSLFPRGHO?RIGHQGULWLFJURZWKSURSRVHVWKDWILORSRGLDOstabilization occurs upon the formation and maturation of a synapse (Vaughn et al., 1988; McAllister, 2000) . Compelling evidence supporting synaptotropic dendritogenesis comes from recent live imaging studies demonstrating a direct correlation between the formation of synapses and filopodial stabilization. In these VWXGLHVVWDELOL]HGILORSRGLDZHUHREVHUYHGWRVHUYHDV?DQFKRUSRLQWV?IURPZKLFKcontinued filopodial extension and branching occurs (Morgan and Wong, 2004; Niell et al., 2004)  (Fig. 1.3).  This mode of  dynamic structural rearrangement during arbor growth?with a significant proportion of processes being retracted?has been proposed to allow growing dendrites to rapidly remodel in order to optimize connectivity and subsequent growth/branching patterns (Cline and Haas, 2008) . This manner of growth would suggest that structures establishing functionally appropriate synaptic connections are retained and stabilized, while those either failing to contact a pre-synaptic partner or establishing an inappropriate synaptic connection are destabilized and eliminated.    - 31 -                  a  Fig ure 1.3: Synaptotropic dendrite growth.  ( a )  Dendrite growth occurs by an iterative sequence of selective filopodial stabilization and punctum formation. Still images from a time-lapse series, accompanied by a schematic rendering for clarity. Green represents PSD-95:GFP puncta (a post -synaptic marker) , red lines are newly formed (often transient) branches, and brown are persistent branches. Scale bar =  5? m. ( b )  Model of synaptotropic guidance of dendrite growth. A number of filopodia (solid red) extend from a dendritic branch. Those that encounter correct partners and form synaptic contacts (green dots) are stabilized as new branches (brown), whereas those that establish inappropriate contacts (blue dots) are retracted (dashed red). Successive rounds of selective stabilization result in arborization within a field of appropriate synaptic connections (dashed green region).  Reproduced from Niell et al. (2004)  Nature Neuroscience 7:254 - 260. Reprinted by per mission from Macmillan Publisher s Ltd: Nature Neuroscience , C opyright (200 4 ) .  b  - 32 -  Longer interval imaging over hours to days shows that overall dendritic arbor growth and elaboration occurs through the gradual accumulation of a relatively small population of newly added filopodia which become stabilized, and through the elongation of existing and newly stabilized structures. As neurons mature, the rates of dynamic growth processes decrease and are mainly restricted to the terminal branch tips, resulting in smaller changes in overall arbor shape and size during the latter stages of dendritogenesis (Cline, 2001) . Growing dendritic arbors exhibit a high density of filopodia, however, these structures are almost exclusively observed during early stages of neuronal morphogenesis (Wong and Ghosh, 2002) . In many sub-types of neurons, dendritic spines begin to appear during late stages of structural maturation, during which period remaining stabilized dendritic filopodia recede and draw their synapses onto the dendritic shaft, or transform into nascent dendritic spines (Boyer et al., 1998; Fiala et al., 1998) . Later, synapses on dendritic shafts and immature spines PDLQO\?VWXEE\?spines) decrease, and synapses on ?thin  ? and ?mushroom  ? dendritic spines emerge to become the dominant forms in the adult brain (Harris, 1999; Marrs et al., 2001) . In mature neurons lacking dendritic spines, the majority of synapses reside on dendritic shafts. The final shape and size of dendritic arbors results from interactions between cell type-specific developmental programs and local environmental cues. The co -ordinated variations in both intrinsic and extrinsic modulators are thought to give rise to the wide variety of neuronal dendritic phenotypes (McAllister, 2000) . - 33 -  Intrinsic factors are known to shape the basic geometry of the dendritic arbor, including how far dendritic branches extend, and how often they branch (Gao et al., 1999; Jan and Jan, 2003) . However, extrinsic cues?including synapse formation and neurotransmission?regulate dendritic patterning particularly in ways that are important for circuit formation and subsequent refinement (Rajan and Cline, 1998; Rajan et al., 1999; Sin et al., 2002; Haas et al., 2006) . This is further highlighted by observations that many extra- (McAlli ster, 2000; Jan and Jan, 2003)  and intra- (Chen and Ghosh, 2005)  cellular  signals controlling dendritogenesis are directly regulated or induced by synaptic activity.  Given the key regulatory role of neurotransmission in dendritogenesis, and the potentially deleterious effects of abnormal dendritic growth on subsequent circuit function, examining the potential effects of early-life seizures on dendritogenesis is highly warranted.    1.5.2 Effects of earl y -life seizures on dendritic gr ow th and morpholo g y  In human epilepsy a number of abnormalities in dendrites have been reported, primarily from pathological analysis of brain specimens resected as part of surgical treatment of patients with intractable epilepsy, from or near the region of the epileptic focus (most often in the neocortex or hippocampus)  (Swann et al., 2000) . Dendritic spine loss is commonly seen, which occurs in isolation or along - 34 -  with varicose swelling (beading) of dendritic branches  (DeMoor, 1898; Scheibel et al., 1974; Isokawa and Levesque, 1991; Belichenko and Dahlstrom, 1995; von Campe et al., 1997; Blumcke et al., 1999) . Other less common dendritic abnormalities have also been described in both neocortical and hippocampal epilepsy including changes in dendritic length, shape and branching patterns, as well as focal increases in dendritic spines (Belichenko et al., 1994; Multani et al., 1994; Isokawa, 1997; von Campe et al., 1997; Blumcke et al., 1999; Swann et al., 2000; Wong, 2005, 2008) .  Similar to clinical findings, dendritic atrophy, a loss of dendritic spines and appearance of varicose swelling on dendrites has been observed in mature rats subjected to seizures induced in vivo by various methods (Westrum et al., 1964; Evans et al., 1983; Sloviter, 1983; Altrup a nd Speckmann, 1988; Carpentier et al., 1991; Isokawa and Levesque, 1991; Isokawa, 1998; Di Rocco et al., 2001; Dashtipour et al., 2003; Mizrahi et al., 2004; Rensing et al., 2005; Ampuero et al., 2007; Zeng et al., 2007) , although aberrant dendrite sprouting and an increase in dendritic spines has also been occasionally reported (Danzer et al., 2002; Dashtipour et al., 2002) . However, given that seizures are most prevalent during periods of early brain development, when immature neurons elaborate dendritic arbors which undergo dramatic activity-dependent growth, the effects and consequences of seizures within the immature brain will likely be different than the adult state. - 35 -  To date, only three studies ?conducted by Swann and colleagues?have examined the effects of developmental seizures on dendritic structure. In the first of these studies (Jiang et al., 1998) , seizures were induced in immature rats  by intrahippocampal injection of tetanus toxin, which resulte d in brief and recurrent seizures lasting for 1 week after which animals were sacrificed. Examination of dye-filled neurons from fixed hippocampal tissue sections revealed a significant reduction in dendritic spine density within CA3C pyramidal neurons, particularly on terminal dendritic segments, which also displayed substantially smaller diameters. No significant differences were observed in total dendritic lengths or segment numbers.  In a subsequent study using hippocampal slice cultures obtained from young rats (Oliva et al., 2002) , bath application of kainic acid for 1 hour resulted in WKH DSSHDUDQFH RI ODUJH GHQGULWLF VZHOOLQJV RU ?EHDGV? D FKDUDFWHULVWLF RIexcitotoxic damage to dendrites). Beading was obser ved primarily in dendritic segments distal from the soma, and beading severity was stronger in CA3 pyramidal neurons compared to those in CA1. In a separate experiment designed to examine the effects of seizures on the dendritic arbors of inhibitory interneurons, hippocampal slice cultures were obtained from young mice. Kainic acid was iontophoretically applied to discrete dendritic regions within slice cultures. Dendritic beading was observed in inhibitory interneurons almost instantaneously upon application of kainic acid. Interestingly, beads often appeared in distal branches rather than sites proximal to the site of drug - 36 -  application, indicating selective vulnerability of distal dendritic segments to kainic acid-induced beading. Subsequently, beading would either reverse, or would progress more proximally and appeared to result in cell death. In spite of a transient loss of spines, no significant loss of spines was observed in neurons that recovered from beading.    In the most recent study (Nishimura et al., 2008) , hippocampal slices prepared from young transgenic mice, modified to fluorescently label CA1 pyramidal neurons, were cultured for 3 days in vitro , after which seizure activity was induced either by bath application of bicuculline (a GABA A receptor antagonist) or elevated extracellular K +  (to increase neural excitability) for an additional 4 days. Both treatments resulted in a significant reduction in total dendritic length, number of branch points, and average segment length. Dendritic morphometrics were also assessed after 1 or 2 days of bicuculline exposure. Results indicated that both total dendritic length and number of branch points were significantly reduced after the first day of bicuculline exposure, and failed to significantly change over the ensuing 3 days of continued drug exposure.  The results from these studies LQGLFDWH WKDW H[WHQGHG VHL]XUHV ?Kretard dendritic growth in vitro , while repeated brief seizures are associated with dendritic spine loss. While important, these findings have some limitations: (i) the use of fixed tissue methods (Jiang et al., 1998) , or  long-interval in vitro  examinations (Nishimura et al., 2008)  precludes visualization of seizure induced effects on dynamic yet functionally important growth events; and (ii) results - 37 -  obtained from in vitro studies (Oliva et al., 2002; Nishimura et al., 2008) , with compromised neural circuitry and artificial extracellular environments, are difficult to extrapolate to in vivo conditions. Seizures occurring in the intact organism have dramatic effects on ongoing physiological processes such as perfusion, oxygenation, glucose metabolism, acid-base regulation, and endocrine modulation among numerous other factors, some of which may have critical interactions with seizure expression and seizure-induced effects (Cole et al., 2002) .  Recent technological advances in cellular labelling and in vivo imaging techniques permits time-lapse imaging of dendritic morphogenesis within the living brain, allowing both rapid and protracted effects of experimental manipulations to be assessed longitudinally within the same neurons, while maintaining intact neural circuitry and extracellular environments. Using such preparations, live imaging studies have found that various manipulations, such as long-term potentiation and sensory deprivation induce rapid, but often transient, changes in dendritic structure over a time scale of minutes (Grutzendler et al., 2002; Trachtenberg et al., 2002; Holtmaat et al., 2005) ?raising the possibility that seizures may also induce dynamic and/or transient changes in dendrite morphogenesis that cannot be observed using traditional histology or long-interval time-lapse methods.  However, the application of these in vivo imaging techniques for the study of seizure-induced effects on neural development and circuit formation using - 38 -  conventional mammalian developmental seizure models is limited due to: (i) the requirement of anaesthetic agents during in vivo imaging of the brain during seizures, which disrupt and reduce neuronal excitability (Ishizawa, 2007) , and therefore would be expected to interfere with both seizure expression and activity-dependent dendritogenesis; (ii) the invasive surgical procedures required are impractical during periods of early brain development (Mizrahi et al., 2004; Rensing et al., 2005; Zeng et al., 2007) .  An attractive alternative approach is the development of non-mammalian in vivo model systems to examine the effects of seizures on brain development and function. Such model organisms enjoy  widespread use in other areas of neurodevelopmental research due to the many advantages they confer and the large number of specialized experimental techniques designed specifically for use in these animal species ( Haas et al., 2001a; Baraban, 2007) . Only recently have these advantages been exploited for the field of epilepsy research with the development of several new experimental models based on organisms such as zebrafish (Baraban et al., 2005) , the fruit fly Drosophila melanogaste r  (Titus et al., 1997; Ganetzky, 2000) , and the nematode C. elegans  (Dawe et al., 2001; Cockell et al., 2004) . Th erefore, for the present experiments we chose to develop a novel model of early-life seizures based on the albino X enopus laevis  tadpole, a vertebrate widely used in studies of dendritogenesis and other fundamental aspects of early brain development.   - 39 -  1.6 The albino Xenopus laevis tadpol e as a model of early brain development   The African clawed frog, X enopus laevis , has served as an important model system for studies of vertebrate ontogeny for over a century. The basis for much of our understanding of the early stages central nervous system development stem from studies conducted in X enopus la e vis , including seminal work conducted in the larval frog examining retinal ganglion cell axonal pathfinding, which provided critical evidence for the chemoaffinity model of axonal guidance (Sperry, 1963; Meyer, 1998) . The continued use of this organism as a convenient model system of brain development is due to evidence demonstrating that neuronal physiology and function, neuropharmacology, intra- and inter-cellular molecular signalling pathways, cellular growth and differentiation, and neural circuit development are all consistent with what is known of mammalian counterparts (Cline, 1991 ; Debski and Cline, 2002) .  X enopus tadpoles have proven particularly useful for the study of early development of vertebrate central neural circuits (Tao et al., 2001; Vislay -Meltzer et al., 2006; Aizenman and Clin e, 2007) . These studies have mainly focused on the development of the retinotectal system, in which the retinal ganglion cells of the eye send project ions to the contralateral optic tectum (superior colliculus in mammals), which is the primary visual area  in frogs and fish, creating a functional circuit capable of processing visual stimuli (Dong et al., 2009)  (Fig. 1.4).   - 40 -                  n a  b  c  d  Fig ure 1.4: Retinotectal system of Xenopus laevis tadpoles. ( a, b )  Stage 47 albino X enopus laevis  tadpole with brain outlined in (b). Scale bars = 2 mm . ( c)  Diagram of the Xenopus  visual circuit. Optic tectal neurons receive direct visual input from retinal ganglion cells of the eye. Reprinted from: Neuron, Vol. 58, Chiu SL, Chen CM and Cline HT. Insulin receptor signaling re gulates synapse number, dendritic plasticity, and circuit function in vivo, pp 708 - 719. Copyright (2008) , with per mission from Elsevier.  ( d)  Centre  - dorsal view of the intact albino tadpole brain. ot ? optic tectum; v ? ventricle, ob ? olfactory bulb, hb - hindbrain. Scale baU ?P Lef t  ? single optical plane of the optic tectum showing location of neuronal soma and neuropil (n). Right  - retinotopic organization of inputs in the optic tectum. Temporal and nasal  retina were labelled, respectively, with diI (red) and FITC -dextran (green). Scale bar = 20?m. Reproduced f rom: Insight s into activity - dependent map formation from the retinotectal syst em : a middle - of - the - br ain perspective, Rut ha zer ES and Cline HT. Journal of Neurobiology 59: 134 - 146. Copyright (2004); Reprinted with  per mission of John Wiley & Sons, Inc.   n c  - 41 -  As in mammalian sensory areas, the retinal ganglion cell axonal projection s terminate in an ordered, ?retinotopic  ? organization conserving retinal position to corresponding regions of the tectum. This retinotopic organization forms as a result of the combined influence of activity-independent mechanisms, based on position-dependent gradients of cell surface molecules on the RGC axons and tectal neurons (Sperry, 1963; Cline, 1991)  and subsequently,  activity-dependent mechanisms based on the highly correlated patterns of activity in neighbouring retinal ganglion cells (Mastronarde, 1989; Ruthazer et al., 2003) . Diffusible and cell surface cues are thought to guide retinal axons to target zones within the tectum, and this initially coarse arrangement of retinal axons is refined through a competition-based process, based on activity patterns in the retinal ganglion cells axons (Ruthazer et al., 2003) , ultimately forming  a precise point-to-point representation of the retinal surface on the tectal neuropil (Cline, 1991) . Similar to mammals, in X enopus , these developmental processes of neural circuit refinement encompass synapse formation and rearrangement as well as axonal and dendritic arbor remodelling (Ruthazer and Cline, 2004) .  The tadpole optic tectum is a particularly useful structure for study given its relatively large size, ordered cell body layers, its proliferative zone which continuously adds new neurons to the tectum, a completely isolated afferent projection from the eye, and its accessible dorsal position directly under the skin. Furthermore, tadpoles of naturally occurring albino X enopus laevis  are transparent, providing a clear window allowing direct in vivo imaging of the intact - 42 -  developing brain. Another important feature of this organism is the ability to immobilize tadpoles by use of reversible paralytics and immersion under a thin layer of in agar, thereby circumventing the requirement for anaesthetic agents during electrophysiological or imaging experiments. This represents a distinct advantage over traditional model species, given that anaesthetics interfere with neuronal transmission and therefore interfere with activity-dependent processes (Ishizawa, 2007) . Thus, the albino tadpole offers a remarkably accessible preparation for the study of early development of brain neural circuits, and provides significant technical advantages currently not available in mammalian systems.  The use of Xenopus laevis  tadpoles also confers numerous practical advantages over mammalian systems, in terms of rearing and husbandry as well as cost-effectiveness. Adult frogs can be induced to produce fertilized eggs throughout the year. Typically, a single mating pair will yield se veral hundred tadpoles, and adult frogs remain reproductively active for many years.  After fertilization, embryos rapidly progress through early developmental stages without parental care, and develop into freely swimming tadpoles in less than one week (Nieuwkoop and Faber, 1994) . Two recent major technological innovations have proven critical in establishing the X enopus  laevis  tadpole as a leading model for the study of early brain circuit development?two-photon fluorescence microscopy and single-cell electroporation. Two -photon microscopy allows imaging living tissue up to a depth - 43 -  of one millimetre, and provides deeper tissue penetration, more efficient light detection and reduced phototoxicity as compared to confocal microscopy (Denk et al., 1990; Garaschuk et al., 2006) . For these reasons, two-photon microscopy is currently the preferred imaging technique for most in vivo imaging preparations.  Single-cell electroporation (SCE), is a method first tested and described in the X enopus laevis  tadpole brain for restricted delivery of dyes, DNA, peptides, and other macromolecules to individual cells within intact tissue (Haas et al., 2001a; Haas et al., 2002; Hewapathirane and Haas, 2008)  (also see Chap te r 3 ) . The distinct advantage of this technique is that experimental manipulations ?such as genetic manipulations or pharmacological treatment?may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from global treatments. SCE can be used to label individual neurons, subcellular components, or intracellular events within the tadpole brain using fluorescent dyes or by transfecting cells with plasmid DNA encoding fluorescent proteins. Once fluorescently labelled, individual neurons imaged using in vivo two-photon microscopy provide high-resolution 3-dimensional images for accurate morphometric measurements of neuronal growth (Sin et al., 2002; Haas et al., 2006) . Repeated time-lapse imaging of the same neuron at intervals ranging from seconds to days allows accurate and comprehensive measurement of both dynamic and protracted growth events. SCE can be used to specifically target immature neurons within the well-defined proliferative zone in the caudo-medial tectum, allowing subsequent in vivo - 44 -  imaging of their growth. These methods hav e proven effective for imaging neuronal morphology in the developing brain to characterize growth behaviour and to identify molecular mechanisms of developmental plasticity (Wu and Cline, 1998; Cantallops et al., 200 0; Sin et al., 2002; Akerman and Cline, 2006; Haas et al., 2006; Chiu et al., 2008; Ewald et al., 2008; Liu et al., 2009) .   1.7 Research h ypot he ses and a ims  Th is work addresses the controversial and clinically important question of whether early-life seizures are harmful to the immature brain. Early-life seizures are exceptionally common, with an incidence of approximately 5-8% of the general population, yet whether these episodes of excessive and abnormal neural activity interfere with ongoing programs of activity-dependent brain development is unresolved.  Given that neuronal activity influences the growth and patterning of dendrites during development, and that dendritic structure directly relates to neural circuit function, assessing whether and how seizures may interfere with dendritogenesis is highly warranted. Indeed, reversing or attenuating the harmful effects of seizures on brain function represents a novel therapeutic approach to mitigate the co-morbidities of early-life seizures. Unfortunately, it is not known whether early-life seizures alter normal patterns of dendritogenesis in vivo. In part, this has been due to the lack of suitable experimental models allowing direct - 45 -  visualization of neuronal growth within the intact and unanaesthetized brain during seizures.  Therefore, the first major aim of this work (described in Chapter 2) was to develop a novel developme nta l seizure mode l s ys tem bas e d on the trans par e nt albino Xenopus laevis tad pole . While previous studies have demonstrated the ability to induce seizures in the adult frog using electrical or chemical stimuli (Servi and Strejckova, 1967; Bennett, 1972; Morrell and Tsuru, 1976) , seizure induction in tadpoles has not previously been reported. We hypothesized that the albino Xenopus laevis  tadpole can be validated as a suitable model of developmental seizures. Experiments were therefore designed to characterize chemoconvulsant-induced seizures in tadpoles to determine appropriateness as a new model of developmental seizures.   The second major aim of this work (described in Chapter 3 ) was to examine the effe c ts of developme nta l seizure s on both rapid and protra c ted dendritic grow th proc e ss e s in vivo , and whe the r obse rved change s w ere ass ociated with alt era tions in synaps e number s .  We hypothesized that developmental seizures would retard dendritic growth and lead to a reduction synapse numbers. 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DEVELOPMENT OF A NOVEL IN VIVO MODEL OF EARLY-LIFE SEIZURES BASED ON THE XENOPUS LAEVIS TADPOLE1  2.1 Introduction  The highest incidence of seizures occurs during the first years of life , and in patients who develop epilepsy, recurrent seizures often begin during childhood (Cowan, 2002; Hauser, 1995). Early brain development is marked by heightened neural plasticity and dynamic neural circuit formation involving ongoing activity-dependent neuronal growth, synaptogenesis and circuit refinement (Katz an d Shatz, 1996; Spitzer, 2006). While these ongoing maturational processes may underlie the immature brain's relatively low seizure threshold and promote seizure expression (Haas et al., 1990), it remains unclear whether seizures, in turn, interfere with activity-dependent maturation of neural circuits. Considerable debate has focused on whether seizures induce alterations in brain circuit development leading to persistent neural dysfunction or to an                                                           1 V ersions of this chapter have been published:   (i) Hewapathirane DS , Dunfield D, Yen W, Chen S, Haas K (2008)  In vivo  imaging of seizure activity in a novel developmental seizure model.  Exp erimental  Neuro logy,  2008 Jun;211(2):480 -8. [Article content reproduced here with permission from Elsevier, Copyright (2008)]   (ii) Hewapat hirane DS , Haas K  (200 9)  Xenopus laevis  tadpoles as a novel model of developmental seizures. In: Animal Models of Epilepsy: Met hods and Innovations ( Baraban SC, ed.), Humana Press: N ew Y ork, 2009, pp 45-58. [ Content reproduced here with permission from Springer Science & Business Media , Copyright (2009)]    CHAPTER 2 - 66 -  increased susceptibility to psychiatric or neurological disorders later in life (Ben -Ari and Holmes, 2006; Swann, 2004). While some clinical studies examining the long-term consequences of early-life seizures have reported an increased risk for the subsequent development of epilepsy, reduced cognitive function, or psychiatric illness (Cornaggia et al., 2006; Gaitatzis et al., 2004; Vestergaard et al., 2005; Vestergaard et al., 2007), others have found no such link (Scher, 1997; Shinnar and Hauser, 2002; Verity et al., 1998). Unfortunately, clinical studies are confounded by the inherent variability of patient populations, such as variation in number, type and frequency of seizures, age of seizure onset, individual genetic susceptibilities, underlying etiology, medication, and other environmental influences (Holmes,  1991; Scher, 1997). Furthermore, when seizures precede the onset of neurological dysfunction, it is difficult to determine whether seizures directly induce the observed dysfunction or whether both arise from shared pathological origins (Tsopelas et al., 2 001). It is also possible that gross measures currently employed to examine the long-term effects of seizures may be unable to detect subtle changes in developing neural circuits. Studies utilizing experimental animal models have been able to address these problems by offering increased control over experimental variables and more precise measures of seizure induced changes. In immature rodent models, animals experiencing early-life seizures demonstrate a remarkable resistance to seizure-induced neuronal death and reactive axonogenesis commonly seen in adult animals (Haas et al., 2001b; Sperber et al., 1991). However, developmental - 67 -  seizures have been shown to induce permanent changes in behaviour, learning and memory, and an increased susceptibility to seizures in adulthood (Holmes et al., 1988; Lynch et al., 2000). Given that experimentally induced seizures in immature animals permanently alter neural function, it is of critical importance to identify the precise molecular and structural alterations that underlie these changes. Recent advances in imaging methodologies coupled with improved fluorescent indicators of intracellular events has enabled the direct in vivo visualization of neuronal activity and morphological growth occurring during neuronal development (Garaschuk et al., 2006a; Niell and Smith, 2004; Sanchez et al., 2006; Sin et al., 2002; Haas et al., 2001a). However, the application of these techniques for the study of seizure-induced effects on neural development and circuit formation using conventional mammalian developmental seizure models is limited due to: (i) the requirement of an aesthetic agents during in vivo imaging of the brain during seizures, which disrupt and reduce neuronal excitability (Ishizawa, 2007); (ii) the invasive surgical proc edures required are impractical during periods of early brain development (Mizrahi et al., 2004; Rensing et al., 2005; Zeng et al., 2007). An alternative approach to overcome these limitations is the use of non-mammalian model organisms that are already in widespread use in neurodevelopment research (Baraban, 2007).  Here we describe a novel developmental seizure model system specifically designed to address questions of how seizures are initiated and propagate - 68 -  through immature brain circuits, and how seizures affect subtle yet critical developmental processes including neuronal growth, synaptogenesis and brain circuit formation. This model system is based on the transparent albino X enopus laevis  tadpole, a well-studied vertebrate used extensively for in vivo imaging of neuronal growth and synaptogenesis (Cantallops et al., 2000; Haas et al., 2006; Sanchez et al., 2006; Sin et al., 2002). The transparency of this organism allows direct in vivo imaging of the growth and activity of individual or large populations of neurons within the intact developing brain. Furthermore, immobilization of tadpoles by reversible paralytics and immersion in agar circumvents the need for anaesthetic agents during electrophysiological or imaging experiments. We have characterized experimentally induced seizures in tadpoles using behavioural assessment, measures of cell death, and in vivo examination of neural activity during seizures, using electrophysiological recordings and imaging of intracellular calcium dynamics within the intact unanaesthetized brain.     - 69 -  2.2 Methods   2.2.1 Animals  Freely swimming albino X enopus laevis  tadpoles were maintained in 10% Steinberg's solution (1? Steinberg's: 10 mM HEPES, 58 mM NaCl, 0.67 mM KCl, 0.34 mM Ca(NO 3) 2, 0.83 mM MgSO 4, pH 7.4), and housed at 22?C on a 12h light/dark cycle. Unless otherwise stated, experiments were performed on Stage 47 tadpoles (Nieuwkoop and Faber, 1994). All experimental procedures were conducted according to the guidelines of the Canadian Council on Animal Care, and were approved by the Animal Care Committee of the University of British Columbia's Faculty of Medicine.   2.2.2 Seizure induction and behaviour a l obs ervation s  Six chemoconvulsants: pentylenetetrazol (PTZ, Sigma -Aldrich, St. Louis, MO), bicuculline, picrotoxin, kainic acid, pilocarpine, and 4-aminopyridine (4 -AP), were tested for their ability to induce convulsive behaviour in freely swimming tadpoles when applied to the bath solution (all drugs obtained from Tocris, Ellisville, MO unless otherwise noted). For ea ch chemoconvulsant a range of doses was tested to measure the severity and latency of behavioural seizure induction, as well as toxicity (n = 20 tadpoles/dose). Tadpoles were monitored continuously for 30 min of baseline activity, 60 min of drug exposure, and for 60 - 70 -  min following drug washout. Twenty -four hours following drug exposure tadpoles were examined for survival and behavioural assessment. More detailed characterization of seizure behaviour was conducted using PTZ (15 mM) after determination that this drug and dose elicited rapid and consistent convulsive behaviour. Tadpoles (n = 20) were individually observed during 30 min of baseline, during 60 min of drug exposure, and for 2 h following washout of drug. Tadpoles were subsequently examined daily fo r 1 h over 5 days to assess behaviour and survival. To determine whether seizure susceptibility changes during the course of tadpole development, seizure responses in tadpoles of differing maturational stages were examined. Tadpoles of stage 29 ?30 (1d, 11 h post-fertilization (PF)), stage 35?36 (2d, 2 h PF), and stage 40 (2d, 18 h PF) were individually observed over a 30 min baseline period, during 60 min of PTZ exposure (15 mM), for 60 min of washout, and for brief periods over the following 5 days (n = 20 /group).   2.2.3 In vivo electr oph ysiological recor dings  Extracellular field recordings were taken from agar-immobilized tadpoles to examine neuronal population responses during convulsant-induced seizures. To circumvent the confounding effects of anaesthetic agents during recordings, tadpoles were paralyzed by bath application of the reversible paralytic agent pancuronium dibromide (10 ?M), and embedded under a thin layer of agarose (1%, prepared with 10% Steinberg's solution). Tadpoles were subsequently - 71 -  placed in a recording chamber and immersed in oxygenated 10% Steinberg's solution at room temperature (22 oC). Under visual guidance using an upright stereomicroscope, a glass extracellular recording microelectrode (10 ? 0?resistance, filled with 2 M NaCl) was inserted into the optic tectum. Neuronal activity was recorded using an Axiopatch 200B amplifier, digitized with a Digidata 1322A interface, and data stored on a PC computer running PClamp software (Axon instruments, Sunnyvale, CA). Baseline activity w as recorded for 30 min followed by application of PTZ (15 mM), 4 -aminopyridine (1 mM), kainic acid (0.25 mM), or pilocarpine (75 mM). Seizure activity was recorded for at least 1 h and, in some experiments, followed by bath application of the antiepileptic drug valproate (5 mM, Sigma -Aldrich).   2.2.4 Calcium indicator loading  The calcium -sensitive fluorescent indicator Oregon Green 488 BAPTA -1, AM (OGB1 -AM, Molecular Probes, Eugene, OR) was bulk loaded into neurons within the tadpole brain (Niell and Smith,  2005). OGB1 -AM was prepared at a concentration of 10 mM in DMSO with 20% pluronic acid (Molecular Probes) and 0.01% Fast Green dye directly prior to each imaging experiment. Under visual guidance using an upright stereomicroscope, a sharp glass pipette loaded with OGB1 -AM solution was inserted into the optic tectum of tadpoles anaesthetized with 0.02% 3 -aminobenzoic acid ethyl ester (MS222, Sigma -Aldrich). Dye solution was slowly perfused into the brain using low pressure settings on a Picospritzer III - 72 -  (Ge neral Valve Corporation, Fairfield, NJ). Tadpoles were subsequently returned to normal bath solution and allowed to recover from anaesthesia. Thirty minutes following OGB1 -AM loading, tadpoles were immobilized with pancuronium dibromide (10 ?M), embedded i n 1% agarose, and placed in an imaging chamber continuously perfused with oxygenated 10% Steinberg's solution at room temperature (22?C).   2.2.5 In vivo tw o -photon fluoresc ence imaging of cal cium d yna mics  The imaging chamber was mounted on the stage of a c ustom-built two-photon laser-scanning microscope, constructed from an Olympus FV300 confocal microscope (Olympus, Center Valley, PA) and a Chameleon XR laser light source (Coherent, Santa Clara, CA). Optical sections through the optic tectum were captured using a 60?, 1.1 NA, water immersion objective (Olympus), and images were recorded and processed using Fluoview software (Olympus). The optic tectum was imaged using a resolution of 640 ? 480 pixels and zoom factor of 1.5?, encompassing an area of 177 ? 13 ?PDOORZLQJVLPXOWDQHRXVLPDJLQJRIapproximately 50?100 neurons in a single X ?Y scan. Repeated X ?Y scans of a single optical section were taken at a rate of 1.3 s/frame using a wavelength of 910 nm to excite OGB1 -AM dye. Baseline spontaneous neural activity was recorded for 30 min PTZ (15 mM) was applied to the bath and seizure activity was recorded 60 min later, from the same optical section imaged during baseline. - 73 -  Throughout imaging, drift was minimized by comparing each image to a pre -baseline reference image and compensated for by manual repositioning.  2.2.6 Fluores cenc e imaging data anal ysis  Quantification of changes in intracellular calcium -sensitive dye fluorescence over time was conducted using Fluoview and Image J (NIH) software. Initially, the mean noise introduced by the imaging setup was determined by imaging an isolated volume of OGB1 -AM dye at a concentration similar to that typically loaded into cells. For each experiment, individual regions of interest (ROIs) were manually drawn over neur onal cell bodies in each optical section (n > 50 cells). Fluorescence data are presented as the relative  change in IOXRUHVFHQFH ?)) 'XULQJ EDVHOLQH UHFRUGLQJV DQ LQFUHDVH LQ LQWUDFHOOXODUIOXRUHVFHQFHZDVGHILQHGDVDFDOFLXPHYHQWLIWKH?))YDOXHZDVWZRVWDQGDUGdeviations above the mean noise of the imaging setup, with peak values defined as calcium spikes. Analysis of calcium event amplitudes during seizures indicated a distinct population of large amplitude events, which were distinguished from VPDOOHUHYHQWVVHHQGXULQJEDVHOLQHUHFRUGLQJVXVLQJD?))WKUHVKROGRIstandard deviations above the mean peak value of baseline spikes.  - 74 -  2.2.7 Anal ysis of seizure -related cell death within the tadpol e br ain  The effect of seizures on cell death within the tadpole brain was examined using two approaches. A fluorescein-based in situ terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to detect DNA fragmentation associated with apoptosis (Roche Diagnostics, Quebec, Canada). Tadpoles were exposed to PTZ (15 mM) for 4 h, followed by drug washout. Six, 12, 24 or 48 h following PTZ exposure, seizure tadpoles and yoked controls (n = 6/group) were anaesthetized with 0.02% MS -222, and the brains were removed. Dissected brains were immediately fixed by immersion in ice-cold 4% paraformaldehyde solution (prepared in phosphate bu ffered saline, PBS) for 15 ?20 min Brains were then rinsed with ice-cold PBS, and stored at 4?C. Subsequently, the TUNEL assay was conducted according to the manufacturer's guidelines on whole-mount brains. As a positive control, brains were treated with DN Ase I, to induce DNA strand breaks. Negative controls were processed without the terminal uridine deoxynucleotidyl transferase enzyme, which catalyzes the addition of the fluorescein-conjugated dUTP to DNA strand termini. Labelled  brains were examined using confocal microscopy, and the total number of TUNEL -positive cells within both optic tecta ZHUHFRXQWHGXVLQJVL]H!?Pdiameter) and morphological characteristics to differentiate between cellular nuclei and background labeling. The second approach used to assess cell death was propidium iodide (PI) incorporation, examined in vivo. The flu orescent nuclear stain PI is only incorporated into dead/dying cells with compromised - 75 -  membranes. PI (Sigma -Aldrich) was prepared in 10% Steinberg's solution (5 ?JPOZLWK)DVW*UHHQG\HDQGSUHVVXUHLQMHFWHGYROXPHaQOLQWRthe brain ventricle of acutely anaesthetized tadpoles. Twenty minutes later, tadpoles were re-anaesthetized and imaged using two-photon fluorescence microscopy using an excitation wavelength of 760 nm. The total number of PI -labelled nuclei within the optic tectum was counted. As a positive control, PI-loaded tadpoles were subjected to laser -micro-lesion targeting a single tectal cell, which rapidly induced membrane permeation and nuclear labeling lasting for at least 1 h. PI-incorporation was assayed immediately following a 1, 2 or 4 h exposure to PTZ (each group with yoked controls, n > 6/group), and 6, 12, 24 or 48 h following 4 h of PTZ exposure.             - 76 -  2.3 Results   2.3.1 Charact erization of drug -indu ced behaviour al seizur es  Under control conditions, normal tadpole behaviour is characterized by continuous slow swimming with brief pausing, while continuously maintaining an upright orientation. Each of the six chemoconvulsants tested consistently induced similar patterns of abnormal behaviour, which we have categorized into the following progressive classes (table 2.1) : (I) bouts of intermittent rapid swimming, involving swimming in tight circles, or sporadic rapid swimming with abrupt FKDQJHVLQGLUHFWLRQ?GDUWLQJ?,,behavioural arrest, or immobility, with periods of class I behaviour; (III) loss of postural control (on side or upside down), with periods of class I and II behaviour; (IV) repetitive side -to-side lateral movements of the head (without propulsion), with periods of class I ?III behaviour; (V) rapid, biODWHUDOO\DOWHUQDWLQJUHSHWLWLYHFRQWUDFWLRQVRIWKHD[LDOPXVFXODWXUH?&-shaped FRQWUDFWLRQ?)LJ2.1d), with periods of class I ?IV behaviour. Behavioural classes demonstrated dose-dependency ? i.e., the lowest effective doses elicited class I behaviour only, while higher doses elicited class V behaviour. Furthermore, for each behavioural class, the onset latency after drug application became more rapid with increasing doses. Once elicited, seizure behaviours were continuously observed for the duration of exposure to drug. Upon drug washout, abnormal behaviour ceased over the course of approximately 1 h. - 77 -                        Figure 2.1 : Charact erization of chemoconvulsant -induced behaviour al seizu re activity in Xenopus laevis tadpol es.   (a) Bars represent the latency for 50% of tadpoles to demonstrate class V seizure behaviour. Infinity symbols denote occasions where 50% of tadpoles failed to display class V behaviour within 60 min Tadpole survival  (denoted by lines) was assessed 24  h after drug exposure. (b) Detailed analysis of the time course of seizure behaviours induced following application of a single dose of pentylenetetrazol (PTZ, 15  mM) and recovery after drug washout. (c) Average latency to onset of class V seizures in response to application of PTZ (15  mM) in tadpoles of differing stages of development. Maturational state given in Nieuwkoop and Faber stages and corresponding age post-fertilization (PF) (d) Schematic drawing of a tadpole d isplaying a C-shaped contraction (class V behaviour) produced by pronounced unilateral contraction of the axial musculature.  Table 2.1: Behavioura l seizure stage s in Xenopus laevis tadpole s.  - 78 -  Class V behaviour (C -shaped contractions) was chosen as an indicator of severe seizure activity due to its consistent expression and the clonus-like convulsive nature of this motor pattern. The onset latency for class V behaviour and 24 h survival rate was determined for each chemoconvulsant tested, using a wide range of drug doses (Fig. 2.1a). The latency for class V behaviour demonstrated dose-dependence for each convulsant, with lower doses failing to elicit class V seizures in most tadpoles within the 60 min period of drug exposure, and higher doses eliciting class V behaviours more rapidly. The highest doses tested were also associated with significant levels of lethality. The following drug doses elicited behavioural seizures rapidly (within 20 min) with no, or minimal, lethality: pentylenetetrazol (PTZ) ? 10?50 mM; bicuculline ? 1?5 mM; picrotoxin ? 1 mM; kainic acid ? 0.25?1 mM; pilocarpine ? 75 mM; 4 -aminopyridine ? 0.5?2.5 mM. PTZ demonstrated a particularly wide range of doses that rapidly elicited class V behaviour in the absence of toxic effects. We therefore chose to further characterize PTZ -induced seizure activity in subsequent experiments, using a dose of 15 mM. Detailed analysis of the time-course of abnormal behaviours induced by PTZ (15 mM) showed a progressive onset of seizure motor patterns, culminating in continuous seizure behaviour within 10 min of drug application (Fig. 2.1b). All seizure behaviours gradually dissipated after 60?90 min following washout, after which normal swimming patterns resumed. PTZ -induced seizure behaviour could be rapidly terminated within 5?10 min of exposure to the anti-epileptic drugs - 79 -  valproate ( 5 mM), phenytoin (100  ? M) or diazepam (50 ? M) ( data not shown). Daily observation of tadpoles for 5 days after PTZ exposure indicated no long -term toxicity or lethality, and no seizure-related abnormal behaviour. Tadpoles of differing maturati onal stages were exposed to PTZ (15 mM) to examine variations in seizure susceptibility and seizure-related behaviour during development (Fig. 2.1c). The initial stages of development of X enopus laevis  occur rapidly, and basic motor patterns appear approximately 1 day post-fertilization (PF). At 1.5 days PF (Stage 29 ?30), tadpoles are mainly immobile with normal behavioural repertoire restricted to occasional bouts of rapid swimming elicited as a startle response. At this developmental stage, PTZ elicits ab normal behaviours including increased bouts of rapid swimming and C-shaped contractions with an average latency of approximately 20 min Stage 35?36 tadpoles (2 days, 2 h PF), a stage at which tadpoles are still largely immobile, demonstrated C-shaped contractions within 12 min of exposure to PTZ. Tadpoles begin to demonstrate continuous free swimming behaviour roughly at stage 40 (2 ?3 days PF), a period which also marks the beginning of visual responses due to innervation of the optic tectum by growing retinal ganglion cell axons. At this stage of development, PTZ elicits C -shaped contractions with a faster latency than earlier stages (approximately 10.5 min post-PTZ). Notably, seizure behaviours spanning class I-V were observed at this stage. These results demonstrate that as more robust motor behaviours emerge during tadpole - 80 -  development, including continuous swimming, the latency to behavioural seizure onset decreases and the complement of seizure behaviours increases (Fig. 2.1d).   2.3.2 Extrac ellular elect r oph ysiol ogical reco rdings  Extracellular field recordings from the intact unanaesthetized tadpole brain revealed characteristic seizure-like electrographic discharges following exposure to convulsants with distinct receptor actions, including PTZ, pilocarp ine, 4-aminopyridine and kainic acid (Fig. 2 .2). PTZ -induced seizure activity was characterized by the appearance of epileptiform discharges which gradually increased in magnitude following drug exposure, eventually developing into periodic high amplitude spikes (> 2 mV, 4.7 ? 0.64 spikes/ min; n = 8 recordings, Fig. 2.2a). Such high amplitude oscillatory activity was absent from baseline recordings prior to PTZ application or control recordings of similar durations, which consisted of irregular low-amplitude activity. A power spectral analysis revealed an increase in the power of events in the 0.01?1 Hz and 8?14 Hz range during seizures, compared to baseline recordings (Fig. 2.2b). Electrographic seizure activity gradually diminished to baseline after approximately 30?60 min following PTZ washout (data not shown), and could be readily suppressed by bath application of valproate (5 mM), with a latency of 5 ?10 min (Fig. 2.2c). Pilocarpine (75 mM) application induced spaced, periodic high -amplitude epileptiform spiking (0.3 mV, Fig. 2.2d), while 4 -aminopyridine (1 mM) elicited sustained epileptiform spiking with markedly higher frequency but with lower periodicity (Fig. 2.2e). - 81 -  Kainic acid elicited extended periods of sustained bursting, consisting of fast rhythmic spiking (Fig. 2.2f).                     Figure 2.2 : In vivo extracell ular field recordings of elect rographic seizures in immobilized unan a est hetized tadpol es.  (a) Baseli ne recordings from the tadpole optic tectum show low amplitude irregular neural activity. Pentylenetetrazol (PTZ, 15 mM) progressively elicited periodic high amplitude spiking, characteristic of epileptiform activity. Short durations of baseline and seizure activity are shown at an expanded time scale (b) Power spectrum analysis of the trace shown in (a), for 5 min periods during baseline and seizure. Note the seizure -associated increase in the power of events with low frequency (< 1 Hz), and in the 8 ?14 Hz range. (c) Application of the anti -epileptic drug valproate (5 mM) rapidly suppressed PTZ -induced epileptiform activity. (d ?f) Field recordings of abnormal high amplitude neural activity elicited by pilocarpine (75 mM, d), 4 -aminopyridine (1 mM, e), and k ainic acid (0.25 mM, f). Each convulsant elicited distinct patterns of epileptiform disharge.  - 82 -  2.3.3 Imaging neuro nal calcium activity during seizu res  Loading of the intact brain with a calcium -sensitive fluorescent indicator allowed direct and simultaneous imaging of activity from large populations of neurons within the unanaesthetized brain during seizures. Changes in fluorescence of neuronal soma correspond to changes in intracellular calcium levels, which serve as an indirect measure of neuronal activity (Smetters et al., 1.                  Figure 2. 3: In vivo tw o -photon imaging of neuronal cal cium dynamics during seizures within the unan a est hetized brain.  (a) Image of a stage 47 albino X enopus laev is  tadpole (brain outlined, scale bar = 2 mm). (b) Higher magnification image of the tadpole brain. (ob ? olfactory bulb; ot ? optic tectum, left tectum outlined with dotted line; v ? ventricle; hb ? hindbrain). Rectangle demarcates region of the optic tectum imaged fRUFDOFLXPG\QDPLFV6FDOHEDU ?PFConsecutive time-lapse images showing baseline neuronal calcium fluctuations, demonstrating lack of large calcium spike HYHQWV6FDOHEDU ?PG8SSHUSDQHO? Consecutive time-lapse images of neuronal calcium fluctuation during PTZ -induced seizures showing a wave of high amplitude calcium spikes progressing from rostral to caudal tectum. Lower panel ? Demarcation of cells in upper seizure panels that exhibited large calcium spikes. Cells first spiking in the first frame are outlined in black, in the second frame are shaded gray, and in the final frame are shaded black. (e) Number of simultaneously spiking cells increases with duration of PTZ exposure. Scale EDU ?P e - 83 -  levels, which serve as an indirect measure of neuronal activity (Smetters et al., 1999). Repeated time -lapse two-photon fluorescence microscopy captured a stable field of view encompassing approximately 50?100 neurons within the optic tectum. During baseline recordings, occasional low amplitude fluctuations in LQWUDFHOOXODU FDOFLXP IOXRUHVFHQFH ?))   ?0.24) of individual neurons occurred spontaneously and sporadically throughout the brain without discernable temporal or spatial pattern (Fig. 2.3c). Following exp osure to PTZ, repetitive waves of abnormal synchronous high amplitude calcium spikes were observed (3 ?4/ min?))!ZKLFKSURJUHVVHGDFURVVWKHWHFWXPPRYLQJLQDURVWUDO-to-caudal direction (Figs. 3d and e). Long -term time-lapse imaging revealed that the number of neurons recruited into these synchronized waves of activity increased with duration of PTZ exposure ( Fig. 2.3e). Such synchronized waves were observed throughout PTZ exposure for the duration of the recording events (up to 1.5 h).  Detailed quantification of calcium spikes occurring in 10 cells distributed across the entire extent of the tectum (spanning approximately 180  ?PZDVconducted ( Fig. 2.4a). In baseline recordings, an event was consid ered a calcium VSLNHLILWVSHDN?F / F  value was significantly larger than the mean noise value of the imaging setup ( Fig. 2.4b, small ticks). During the baseline recording, cells demonstrated occasional small amplitude calcium spikes (mean ?F / F  =  0.12 ?  0.01; 0.98  ?  0.19 spikes/ min/cell), with no detectable synchrony ( Fig. 2.4c). Calcium responses in the same cells, examined 1  h after PTZ - 84 -  exposure, revealed two distinct populations of calcium spike events. Calcium spikes of similar amplitude to baseline spikes were seen during seizures, however, these events occurred at a higher frequency (1.79  ?  0.12 spikes/ min/cell). Notably, large amplitude calcium spikes were dete cted that were VLJQLILFDQWO\ ODUJHU WKDQ EDVHOLQH VSLNHV PHDQ ?F / F  =  0.56 ?  0.01), which occurred at a high frequency of 3.6 ?  0.21 spikes/ min/cell ( Fig. 2.4c, large ticks). Analysis of the number of calcium sSLNHVZLWKGLIIHUHQW?F / F  values, in baseline compared to seizure, clearly shows a distinct population high amplitude spikes VHHQRQO\GXULQJVHL]XUHV?F / F  >  0.24, Fig. 2.4e). A striking feature of these large seizure-related calcium spikes was the degree of synchrony exhibited across the brain region examined ( Figs. 4c, d, and f). An analysis of temporal correlation between calcium spikes in cells throughout the tectum ( Fig. 2.4f) clearly shows that only seizure-associated large amplitude calcium spikes are highly synchronized, while small calcium spikes, both in baseline and during seizures, are not synchronized in neurons across the brain. Fig. 2.4d shows an enlargement of synchronized calcium spikes plotted in Fig.2. 4e, demonstrating the wave-like progression of spiking from rostral cells (cells 1 ?7, Fig. 2.4a) to caudal cells (cells 8 ?10).      - 85 -                        Figure 2.4: Single -cel l anal ysis of neuronal cal cium dynamics during seizures, imaged in vivo within the unan a est hetized brain.  (a) Location of 10 cells within the optic tectum selected for detailed quantificati on of calcium dynamics. Rostral tectum towards top of image. n ? neuropil; v ? YHQWULFOH6FDOHEDU ?PEUpper trace ? &KDQJHLQIOXRUHVFHQFH?))RYHUWLPHIURPDQLQGLYLGXDOFHOOGXULQJEDVHOLQHDQGsubsequent PTZ -induced seizure. Lower trace ? Calcium spikes could be distinguished into small (short ticks) and large amplitude events (large ticks). (c) Plot of activity during baseline and seizure for 10 cells demarcated in (a). Grey vertical lines highlight synchrony of large seizure -related calcium spikes. (d) An individual seizure -evoked wave of calcium activity presented at a magnified temporal scale, demonstrates rostral-to-caudal progression of activity. (e) Histogram of calcium spike amplitudes versus frequency. Note the increase of low ampl itude spikes and emergence of a distinct category of high amplitude spikes during seizures. (f) Plot of the degree of synchronicity of the different classes of calcium spikes. For each calcium spike (set to t = 0), the number of cells showing a spike of the same class within a time window of ? 6.5 s was determined. Large seizure -induced calcium spikes demonstrated high incidence of synchronicity, while small amplitude spikes were not synchronous across tectal cells during baseline or seizures. - 86 -  2.3.4 Assa ys of seizure -induced cell death  Two assays were performed to detect cell death within the tadpole brain at  multiple time points following prolonged PTZ seizures (Fig. 2.5). As expected, a small number of TUNEL -positive and PI-positive cells were observed throughout control brains, indicating low levels of continuous cell loss associated with normal development (Figs. 5 a, d, and g). Prolonged seizures (4 h) failed to induce an increase in cellular apoptosis within the brain, as measured by TUNEL labeling over an extended period spanning 6 h to 2 days post-seizure (Figs. 5 b, e, and i). A slight yet significant increase in PI labelled cells, indicative of aberrant cell membrane permeability most often associated with necrotic cell death but occasionally associated with apoptotic death, was detected immediately following 4 h of PTZ seizures (Fig. 2.5j), but no sig nificant differences were seen at later time points (Figs. 5h and k), or after shorter durations of seizure activity (Fig. 2.5j). Notably, the observed change involved an increase on the order of a few cells per brain, and only after 4 h of seizure activity, underscoring the marked resistance of the immature tadpole brain to seizure-induced cell death.       - 87 -                        Figure 2.5: Examination of cel l deat h within the tadpol e brain fol l owing prol onged seizure activity.  (a, b) Representative confocal fluorescence images of whole -mount tadpole brains assayed for apoptosis using the TUNEL technique (a ? control; b ? seizure). Detailed q uantification of TUNEL positive cells within the optic tectum (boxed region in a) was performed using higher magnification. 6FDOHEDU ?PF'1$VH-treated positive control showing extensive TUNEL labeling. (d) High-magnification image of control optic tectum treated with TUNEL assay. (e) TUNEL labeling within tectum 24 h following a 4 h-long PTZ -induced seizure. For c?HVFDOHEDU ?PI&HOOdeath assayed in vivo with propidium iodide (PI). Targeted laser microlesions to kill individual cells ZLWKLQWKHRSWLFWHFWXPXVHGDVDSRVLWLYHFRQWURO6FDOHEDU ?PJ3,LQFRUSRUDWLRQDVVD\RIcontrol tectum, and (h) 6 h following a 4 h -long PTZ -induced seizure. For g and h, scale bar = 50 ?PL4XDQWLILFDWLRQRI781(/SRVLWLYHDSRSWRWLFFHOOVwithin the optic tectum at various time-points following a 4 h-long PTZ -induced seizure. (j) Counts of PI -positive cells in optic tectum following increasing exposure to PTZ (15 mM). * indicates significance (p < 0.05). (k) No significant change in PI-positive cell counts at different times following 4 h-long PTZ seizures.  - 88 -  2.4 Discussion  The present study describes a novel model of developmental seizures, based on the albino Xenopus laevis  tadpole, which offers distinct advantages for direct examination of seizure activity and seizure-related effects on neural circuits during critical periods of brain formation. We have characterized the ability of several common chemoconvulsants to reliably elicit seizure behaviour in freely swimming tadpoles in a dose-dependent manner. Our findings that similar seizure behaviourVZHUHHOLFLWHGE\GUXJVZLWKGLVWLQFWPHFKDQLVPVRIDFWLRQLQFOXGLQJ?-aminobutyric acid (GABA) receptor antagonists (PTZ, picrotoxin and  bicuculline), a kainate receptor agonist (kainic acid), a muscarinic receptor agonist (pilocarpine), and a potassium channel blocker (4 -aminopyridine), indicate that these behaviours are seizure-related and not direct effects of each drug on motor programs. The induction of increasingly severe, repetitive and stereotyped behaviours in tadpoles is similar to the progressive nature of behavioural seizure stages in zebrafish (Baraban et al., 2005) and rodent models (Racine, 1972; Velisek et al., 1995), corres ponding to the propagation of seizure activity to brain regions controlling different motor patterns. In the tadpole, stereotyped seizure behaviours reflected interference with normal motor patterns, such as prolonged periods of immobility with loss of postural control, or abnormal tonic or clonic motor movements including head bobbing, rapid swimming, and strong unilateral contractions of the axial musculature producing C-shaped contractions. Rapid swimming and C-shaped contractions may reflect propagation of seizure activity - 89 -  WRQHXUDOFLUFXLWVXQGHUO\LQJQRUPDOVZLPPLQJDQGWKH?&-VWDUW?NLQHPDWLFSDWWHUQnormally observed in tadpoles during the initiation of turns and during rapid escape (Hoff and Wassersug, 2000).   Extracellular electrophysiological field recordings in immobilized unanaesthetized tadpoles confirmed that PTZ, pilocarpine, 4-aminopyridine, and kainic acid induce electrographic seizure activity characterized by rhythmic high-amplitude epileptiform discharges. Power spectrum analysis of electrographic recordings during PTZ exposure compared to baseline demonstrates that PTZ evoked activity exhibits similar spike frequency and spectral characteristics to those recorded in other in vivo and in vitro  seizure models (Leweke et al., 1990; Mirski et al., 2003; Piredda et al., 1986), supporting that these are seizure events.  Dose-response studies found PTZ to be particularly well suited as an experimental chemoconvulsant for tadpoles due to the wide range of doses that rapidly and reliably elicited severe seizure behaviour without toxicity or lethality. A developmental profile of susceptibility to PTZ induced seizures was performed on tadpoles ranging from Stage 29?30 to Stage 47, a period ranging from approximately 1 to 5 days post-fertilization. Thes e stages were chosen since they span phases of initial motor behaviour and critical stages of early brain development. At Stage 29?30 motor behaviour is rudimentary, largely limited to reflexive escape responses. Stage 39?40 represents a major step in brai n maturation when retinal ganglion cell axons first reach and initiate synaptogenesis in the optic tectum. Over the next 2 to 3 days from Stages 40 to 47 the brain - 90 -  continues to grow through continuous neurogenesis, neurons undergo morphologic maturation, and neurons wire into functional networks. In the retinotectal system, neuronal activity driven by visual stimulation mediates competition-based axonal pruning, dendritic arbor growth, and synaptogenesis to optimize tectal neuron receptive field properties. These same events of neuronal and neural circuit development occur within the mammalian brain at both embryonic and early postnatal periods depending on species and exact brain region. Our findings that even the earliest tadpole stages to exhibit motor behaviours responded to convulsants with seizure-like motor patterns suggests that even simple neural circuits may support seizure-like recurrent aberrant neuronal discharge. Although all stages exhibited seizure-like convulsive behaviours, the latency to initiation of these behaviours decreased as the tadpole brain matured, suggesting that the establishment of more refined and functional networks may better support seizure activity. Cell death assays following seizures in tadpoles revealed a minimal increase in cells exhibiting propidium iodide incorporation, representing only a few cells per brain and only immediately following 4 h seizures. Both PI-incorporation and TUNEL assays revealed an endogenous low rate of cell loss during brain development, which appears neither enhanced nor restricted by prolonged seizure activity. The absence of marked cell loss following prolonged seizures supports previous findings of the remarkable resistance of the immature brain to seizure-induced neuronal death (Haas et. al.,  2001b).  - 91 -  Calcium imaging offers an effective method to examine neural activity during seizures in a large population of neurons within the brain, with single cell resolution. While baseline intracellular calcium spikes were of small amplitude and not synchronized between cells, PTZ -induced seizures elicited high amplitude calcium fluctuations, which were highly synchronized across cells. These high amplitude spikes propagated as waves of neural activity through the optic tectum in a rostral-to-caudal manner. Previous studies have directly associated somatic calcium spikes to neuronal discharge, demonstrating that intracellular calcium spike amplitude directly correlates to the number of action potentials (Badea et al., 2001; Ramdya et al., 2006; Smetters et al., 1999). Therefore, the synchronized high amplitude calcium spikes observed during seizures in tadpoles indicates an increased rate of neuronal firing and an increase in neuronal synchronicity throughout the brain region sampled. Collectively, these results establish that the tadpole brain can support seizure activity and that the characteristics of induced seizures are similar to those of existing in vivo experimental seizure models. The albino tadpole's transparency provides a clear window for in vivo imaging of the developing brain with two-photon or confocal microscopy, which has been extensively employed for direct time-lapse imaging of growth of individual neurons (Cantallops et al., 2000; Haas et al., 2006; Sanchez et al., 2006; Sin et al., 2002). The tadpole optic tectum has been widely utilized in studies of neuronal development because this brain region exhibits ongoing - 92 -  neurogenesis, neuronal axon and dendrite growth, synaptogenesis, and refinement of neural circuits over a protracted period. The  accessible and well ordered retinal projection to the tectum has also led the tadpole optic tectum to be a leading model for the study of the development of topographic wiring of distant interconnected neuronal tissues. The wealth of previous studies on t his system therefore offers a solid base from which to investigate the effects of seizures on both individual neuronal structure, as well as functional brain circuit formation. Study of the optic tectum in tadpoles may also have direct relevance to seizure studies in higher organisms given that the mammalian counterpart, the superior colliculus, is implicated in initiation of audiogenic seizures (Garcia -Cairasco et al., 1993). Furthermore, it is becoming clear that many developmental processes underlying brain circuit formation are evolutionarily conserved between frogs and mammals. Similar programs of glutamatergic synapse maturation (Wu et al., 1996; Ewald et al., 2008), transition from depolarizing to hyperpolarizing GABAA potentials (Akerman and Cline, 2 006), and activity -dependent circuit refinement (Engert et al., 2002) suggest that the study of seizure activity and seizure -related effects in the tadpole is relevant to developmental seizures in higher organisms. The recent development of new calcium and  voltage sensitive dyes, and genetically-encoded indicators or regulators of neural activity are well suited for direct visualization of seizure activity within the intact brain (Garaschuk et al., 2006a; Garaschuk et al., 2006b; Knopfel et al., 2006). Simu ltaneous imaging of activity in large populations of neurons, with single neuron resolution, provides a - 93 -  platform for study of the initiation and propagation of seizure activity through immature brain circuits, as well as possible changes in network structure and function induced by seizures. An important quality of the X enopus  tadpole seizure model is the ability to directly examine brain function in vivo under unanaesthetized conditions during seizures, avoiding potentially confounding effects of anaesthetics on neuronal activity (Ishizawa, 2007). Such imaging can be used to measure development of neural circuit function, such as retinotectal receptive field properties, and the acute and long-term effects of seizures on these network functions. Another distinct advantage of this system is the ease of seizure induction and inhibition by bath application of drugs to freely swimming tadpoles, after which drugs can be readily washed out. Since tadpoles are highly cost-effective, housed in small volumes of solution, and can be reared in multi-well plastic dishes, these organisms are ideally suited for large-scale drug screens and could potentially be used in screens for novel anti-epileptic drugs. Another important seizure model based on zebrafish has recently been described (Baraban et al., 2005; Baraban, 2007). The z ebrafish shares many of the positive attributes of the X enopus  system, with the added advantage of well-developed genetic tools. However, acute and targeted control over gene expression is relatively easy in X enopus  (Haas et al., 2001a; Haas et al., 2002) and X enopus  has an advantage of a more protracted developmental period of active brain growth, particularly useful for studies of neuron growth and network formation. - 94 -  The high incidence of seizures du ring development in humans and the unknown potential for these common seizures to disrupt normal maturation of developing brain circuits warrants the creation of suitable maturational stage-specific developmental seizure model systems. The X enopus  tadpole brain at the maturational states described here (Stages 39 ?47) offers an accessible model of early brain development corresponding to embryonic through early postnatal periods in humans, including critical processes of neurogenesis, neuronal morphological growth, and activity-dependent synaptogenesis for construction of functional neural networks. The X enopus  tadpole seizure model is a powerful new system that allows direct observation of seizure activity, as well as the acute and long-term effects of seizures on neuronal growth and brain circuit formation.          - 95 -  2.5 References  Akerman, C.J., Cline, H.T., 2006. Depolarizing GABAergic conductances regulate the balance of excitation to inhibition in the developing retinotectal circuit in vivo. J Neurosci 26 , 5117-5130.  Badea, T., Goldberg, J., Mao, B., Yuste, R., 2001. Calcium imaging of epileptiform events with single-cell resolution. J Neurobiol 48, 215 -227.  Baraban, S.C., 2007. Emerging epilepsy models: insights from mice, flies, worms and fish. Curr Op in Neurol 20, 164 -168.  Baraban, S.C., Taylor, M.R., Castro, P.A., Baier, H., 2005. Pentylenetetrazole induced changes in zebrafish behaviour, neural activity and c-fos expression. Neuroscience 131, 759 -768.  Ben-Ari, Y., Holmes, G.L., 2006. Effects of sei zures on developmental processes in the immature brain. Lancet Neurol 5, 1055 -1063.  Cantallops, I., Haas, K., Cline, H.T., 2000. Postsynaptic CPG15 promotes synaptic maturation and presynaptic axon arbor elaboration in vivo. Nat Neurosci 3, 1004-1011.  Cornaggia, C.M., Beghi, M., Provenzi, M., Beghi, E., 2006. Correlation between cognition and behaviour in epilepsy. Epilepsia 47 Suppl 2, 34-39.  Cowan, L.D., 2002. The epidemiology of the epilepsies in children. Ment Retard Dev Disabil Res Rev 8, 171-181.  Engert, F., Tao, H.W., Zhang, L.I., Poo, M.M., 2002. Moving visual stimuli rapidly induce direction sensitivity of developing tectal neurons. Nature 419, 470 -475.  Ewald, R.C., Van Keuren -Jensen, K.R., Aizenman, C.D., Cline, H.T., 2008. Roles of NR2A and N R2B in the development of dendritic arbor morphology in vivo. J Neurosci 28, 850 -61.  Gaitatzis, A., Trimble, M.R., Sander, J.W., 2004. The psychiatric comorbidity of epilepsy. Acta Neurol Scand 110, 207 -220.  Garaschuk, O., Milos, R.I., Grienberger, C., M arandi, N., Adelsberger, H., Konnerth, A., 2006a. Optical monitoring of brain function in vivo: from neurons to networks. Pflugers Arch 453, 385-396.  - 96 -  Garaschuk, O., Milos, R.I., Konnerth, A., 2006b. Targeted bulk -loading of fluorescent indicators for two-photon brain imaging in vivo. Nat Protoc 1, 380 -386.  Garcia-Cairasco, N., Terra, V.C., Doretto, M.C., 1993. Midbrain substrates of audiogenic seizures in rats. Behav Brain Res 58, 57-67.  Haas, K., Jensen, K., Sin, W. -C., Foa, L., Cline, H.T., 2002. Targe ted electroporation in X enopus  tadpoles in vivo - from single cells to the entire brain. Differentiation 70, 148-154.  Haas, K., Li, J., Cline, H.T., 2006. AMPA receptors regulate experience -dependent dendritic arbor growth in vivo. Proc Natl Acad Sci U S A 103, 12127-12131.  Haas, K., Sin, W.C., Javaherian, A., Li, Z., Cline, H.T., 2001. Single -cell electroporation for gene transfer in vivo. Neuron 29, 583 -91.  Haas, K.Z., Sperber, E.F., Moshe, S.L., 1990. Kindling in developing animals: expression of severe seizures and enhanced development of bilateral foci. Brain Res Dev Brain Res 56, 275-280.  Haas, K.Z., Sperber, E.F., Opanashuk, L.A., Stanton, P.K., Moshe, S.L., 2001. Resistance of immature hippocampus to morphologic and physiologic alterations following status epilepticus or kindling. Hippocampus 11, 615-625.  Hauser, W.A., 1995. Epidemiology of epilepsy in children. Neurosurg Clin N Am 6, 419-429.  Hoff, K., Wassersug, R., 2000. Tadpole Locomotion: Axial Movement and Tail Functions in a Largely Verte braeless Vertebrate. American Zoologist 40, 62 -76.  Holmes, G.L., 1991. The long -term effects of seizures on the developing brain: clinical and laboratory issues. Brain Dev 13, 393-409.  Holmes, G.L., Thompson, J.L., Marchi, T., Feldman, D.S., 1988. Behavioural effects of kainic acid administration on the immature brain. Epilepsia 29, 721-730.  Ishizawa, Y., 2007. Mechanisms of anesthetic actions and the brain. J Anesth 21, 187-199.  Katz, L.C., Shatz, C.J., 1996. Synaptic activity and the construction of c ortical circuits. Science 274, 1133-1138.  - 97 -  Knopfel, T., Diez -Garcia, J., Akemann, W., 2006. Optical probing of neuronal circuit dynamics: genetically encoded versus classical fluorescent sensors. Trends Neurosci 29, 160 -166.  Leweke, F.M., Louvel, J., Raus che, G., Heinemann, U., 1990. Effects of pentetrazol on neuronal activity and on extracellular calcium concentration in rat hippocampal slices. Epilepsy Res 6, 187-198.  Lynch, M., Sayin, U., Bownds, J., Janumpalli, S., Sutula, T., 2000. Long -term consequences of early postnatal seizures on hippocampal learning and plasticity. Eur J Neurosci 12, 2252 -2264.  Mirski, M.A., Tsai, Y.C., Rossell, L.A., Thakor, N.V., Sherman, D.L., 2003. Anterior thalamic mediation of experimental seizures: selective EEG spectral coherence. Epilepsia 44, 355-365.  Mizrahi, A., Crowley, J.C., Shtoyerman, E., Katz, L.C., 2004. High -resolution in vivo imaging of hippocampal dendrites and spines. J Neurosci 24, 3147 -3151.  Niell, C.M., Smith, S.J., 2004. Live optical imaging of nervou s system development. Annu Rev Physiol 66, 771-798.  Niell, C.M., Smith, S.J., 2005. Functional imaging reveals rapid development of visual response properties in the zebrafish tectum. Neuron 45, 941 -951.  Nieuwkoop, P.D., Faber, J., 1994. Normal table of X enopus laevis . Elsevier North Holland Publishing Company, Amsterdam.  Piredda, S., Yonekawa, W., Whittingham, T.S., Kupferberg, H.J., 1986. Enhanced bursting activity in the CA3 region of the mouse hippocampal slice without long-term potentiation in the dentate gyrus after systemic pentylenetetrazole kindling. Exp Neurol 94, 659 -669.  Racine, R.J., 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32, 281 -294.  Ramdya, P., Reiter, B., Engert, F., 2006. Reverse correlation of rapid calcium signals in the zebrafish optic tectum in vivo. J Neurosci Methods 157, 230 -237.  Rensing, N., Ouyang, Y., Yang, X.F., Yamada, K.A., Rothman, S.M., Wong, M., 2005. In vivo  imaging of dendritic spines during electrographic seizures. Ann Neurol 58, 888 -898.  - 98 -  Sanchez, A.L., Matthews, B.J., Meynard, M.M., Hu, B., Javed, S., Cohen Cory, S., 2006. BDNF increases synapse density in dendrites of developing tectal neurons in vivo. Development 133, 2477-2486.  Scher, M.S., 1997. Seizures in the newborn infant. Diagnosis, treatment, and outcome. Clin Perinatol 24, 735-772.  Shinnar, S., Hauser, W.A., 2002. Do occasional brief seizures cause detectable clinical consequences? Prog Brain Res 135, 221-235.  Sin, W.C., Haas, K., Ruthazer, E.S., Cline, H.T., 2002. Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases. Nature 419, 475 -480.  Smetters, D., Majewska, A., Yuste, R., 1999. Detecting action potentials in neuronal populations with calcium imaging. Methods 18, 215-221.  Sperber, E.F., Haas, K.Z., Stanton, P.K., Moshe, S.L., 1991. Resistance of the immature hippocampus to seizure-induced synaptic reorganization. Brain Res Dev Brain Res 60, 88-93.  Spitzer, N.C., 2006. Electrical activity  in early neuronal development. Nature 444, 707-712.  Swann, J.W., 2004. The effects of seizures on the connectivity and circuitry of the developing brain. Ment Retard Dev Disabil Res Rev 10, 96-100.  Tsopelas, N.D., Saintfort, R., Fricchione, G.L., 2001. The relationship of psychiatric illnesses and seizures. Curr Psychiatry Rep 3, 235-242.  Velisek, L., Veliskova, J., Moshe, S.L., 1995. Developmental seizure models. Ital J Neurol Sci 16, 127 -133.  Verity, C.M., Greenwood, R., Golding, J., 1998. Long -term intellectual and behavioural outcomes of children with febrile convulsions. N Engl J Med 338, 1723-1728.  Vestergaard, M., Pedersen, C.B., Christensen, J., Madsen, K.M., Olsen, J., Mortensen, P.B., 2005. Febrile seizures and risk of schizophrenia. Schizophr Res 73, 343-349.  Vestergaard, M., Pedersen, C.B., Sidenius, P., Olsen, J., Christensen, J., 2007. The long -term risk of epilepsy after febrile seizures in susceptible subgroups. Am J Epidemiol 165, 911 -918. - 99 -   Wu, G., Malinow, R., Cline, H.T., 1996. Matur ation of a central glutamatergic synapse. Science 274, 972-976.  Zeng, L.H., Xu, L., Rensing, N.R., Sinatra, P.M., Rothman, S.M., Wong, M., 2007. Kainate Seizures Cause Acute Dendritic Injury and Actin Depolymerization In vivo. J Neurosci 27, 11604 -11613                       - 100 -   3. PROTOCOL: SINGLE CELL ELECTROPORATION IN VIVO WITHIN THE INTACT DEVELOPING BRAIN2  3.1 Introduction  Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations (Haas et al., 2001; Haas et al., 2002; Bestman et al., 2006) . The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and e x vivo  preparations, we have optimized this technique for use in Xenopus laevis  tadpoles. Here, we detail the procedure for SCE of a fluorescent dye or plasmid                                                           2 A version of this chapter has been published:   Hewapathirane DS , Haas K (2008)  Single Cell Electroporation in vivo within the Intact Developing Brain. Journal of Visualized Experiments (17) pii: 705. [Article content reproduced here with permission from the Journal of Visualized Experiments, Copyright (2008)]    CHAPTER 3 - 101 -  DNA into neurons within the intact brain of the albino X enopus  tadpole, and discuss methods to optimize yield  3.2 Protocol  3.2.1 Equipme nt set -up  - The electrical equipment required for this technique are a stimul ator, an oscilloscope, and a micropipette holder fitted with a silver wire to be inserted into the micropipette. We typically use an Axon instruments Axoporator 800A stimulator, however other common stimulators such as the Grass instruments SD9 stimulators are also suitable for single-cell electroporation (Fig. 3.1) . The stimulator is connected to the headstage, which interfaces with the silver wire electrode to be placed inside the glass micropipette containing an internal conducting solution. The other le ad from the stimulator is connected to the oscilloscope input, which also receives input from the tadpole external ground. The tadpole external ground is simply a silver wire that remains in electrotonic contact with the tadpole during electroporation. The  circuit is complete once the micropipette is placed in contact with the tadpole.  - Single cell electroporation requires a microscope with a good working distance (at least 5cm), since this gives room to maneuver and allows the micropipette to be brought in at an angle of about 30-45 degrees.  - 102 -            3.2.2 Fabrication of micr opipette s  Preparation of appropriate micropipettes is a critical step in this protocol. The GLIILFXOW\OLHVLQEDODQFLQJDQDUURZWLSOHVVWKDQ?PLQGLDPHWHUZLWKRQHWKDWwill not easily break when puncturing through the tissue. We have also found that tips with a taper angle of greater than 10 degrees are preferable (Fig. 3.2) .  - 3UHSDUHDSXOOHGJODVVPLFURSLSHWWHZLWKDWLSGLDPHWHURIOHVVWKDQ?PDQGDtip taper angle of greater than 10 degrees.  Fig ure 3.1: Equipment set -up  f or singl e -cel l elect roporation  Wiring of the single-cell electroporation setup for the Axoporator 800A ( a)  and the Grass Stimulator SD9 ( b ) . a. ba. - 103 -  o Borosilicate glass with an internal filament with outer diameter of 1.5mm and inner diameter of 0.75mm is well suited for this purpose. ? The relatively thick glass provides a degree of rigidity to the tip.  ? The internal filament i s important in drawing back-filled fluid to the micropipette tip.  - Fabrication of suitable tips will often requires several modifications based on observed results. Different tissues may require a tip with a slightly different shape or tip diameter.           Figure 3.2: Single ce ll elect roporation  micropipet t e  geomet ry  Scanning electron microscopy images of a typical single cell electroporation micropipette, with ( a)  a tip diameter <1 mm and ( b )  tip cone angle >10  degrees.   a. b. - 104 -  3.2.3 Single -cell el ectrop oration protoco l  For new users it is advisable to begin with fluorescent dextran dyes since, unlike genetically encoded fluorescent proteins, they can be immediately seen under epifluorescence. Use of fluorescent dyes ena bles the user to directly see the location of the micropipette tip within the tissue, and upon electroporation gives instant feedback as to whether the equipment has been properly set up and whether the micropipette tip is appropriate.  - Fill micropipette with solution of compound to be electroporated either by using a loading syringe or by back-filling.  o If using plasmid DNA, ensure that samples are endotoxin -free, and at a concentration between 1-2?g/ ?l, prepared in water. o The concentration of fluorescent dextran dyes should be empirically determined. We often use fluorescent dyes diluted in water to approximately 300?M. o Typically, volumes of 0.5 -1 ?l are sufficient.  o Prior to loading, briefly centrifuge solutions at high speed to reduce the amount of particulate debris drawn into the micropipette, which may lead to clogging.  - 105 -  o Air bubbles within the tip often affect current flow and should be dislodged by vigorously flicking the micropipette prior to mounting on the headstage. - Insert micropipette into holder or headstage mounted on a 3-axis micromanipulator. Ensure that silver wire electrode is inserted into micropipette, and is in electrotonic contact with the internal solution.  - Anaesthetize tadpoles by immersion in 0.02% MS -222 (3 -aminobenzoic acid ethyl eVWHUSUHSDUHGLQWDGSROHQRUPDO6WHLQEHUJ?VUHDULQJVROXWLRQS+ o We normally use stage 44-48 albino X enopus  laevis tadpoles in our experiments. o Tadpoles are usually fully an aesthetized after approximately 5 minutes. o Several tadpoles may be anaesthetized simultaneously to reduce wait-time, however, avoid exposure to MS-222 for periods beyond 1 hour.   - Transfer a single an aesthetized tadpole to electroporation chamber using a plastic transfer micropipette.  o The electroporation chamber can be easily ma de in-house by carving a tadpole-shaped cavity in a small Sylgard? silicone block, and inserting a silver ground wire into the cavity such that it will be in electrotonic contact with the tadpole in the chamber.  - 106 -  - Position tadpole dorsal side up using a moistened paintbrush.   - Lower the micropipette until it is in contact with the skin, directly adjacent to the region of interest. o Targeting regions with densely packed cell bodies will greatly increase chances of successful electroporation. We typically target the tadpole optic tectum (Fig. 3.3) . o Bring the micropipette in at an angle of 30 to 45 degrees. Shallower trajectories make skin puncture more difficult, while steeper angles RIWHQREVWUXFWRQH?VYLHZ  - Continued lowering of the micropipette will initially dimple the skin, and then pass through into the underlying tissue. o Superficial cells are preferred for in vivo imaging, and can be targeted by ensuring that the micropipette tip is lowered slowly upon dimpling of skin.  - One should constantly monitor th e micropipette resistance once placed within the tissue. If using an Axoporator 800A (Axon Instruments), resistances of approximately 10-0?DUHRSWLPDO5HVLVWDQFHLVDJRRGLQGLFDWRURIZKHWKHUthe tip diameter is appropriate for single-cell electroporation.  - 107 -                       Figure 3.3: Single cell elect roporation  micropipet te  p ositioning in the albino Xenopus laevis tadpol e brain  ( a)  Diagram of tadpole and pipette positioning for in vivo electroporation of single neurons. ( b )  Dorsal view of the intact albino tadpole brain using bright field microscopy (rostral end towards top of image). ot  ? optic tectum; v ? ventricle. Rectangle delineates region of optic tectum typically targeted6FDOHEDU ?P ( c)  Bright-field microscopy image showing insertion of single-cell electroporation micropipette into tadpole brain. Scale bar = 2mm ( d )  Higher magnification image of ( c) . Scale bar ?P a. b. c. d. - 108 -  o High micropipette reVLVWDQFHV!0?DUHLQGLFDWLYHRIFORJJHGWLSVor micropipettes with tips that are too narrow. o /RZPLFURSLSHWWHUHVLVWDQFHV0?DUHLQGLFDWLYHRIDWLSGLDPHWHUthat is too large, often a result of a broken tip.  o If using a stimulator that does not provide a direct resistance measure, micropipette resistance can indirectly measured by observing the voltage pulses on the oscilloscope (discussed below).   - Apply voltage train.  o For plasmid DNA, pulse trains found to be most effective consist of nega tive  square wave pulses 1ms in duration, delivered at 300Hz with pulse train duration of 500ms.  ? The pulse voltage is determined empirically such that the amplitude of pulses measured on the oscilloscope is between DQG?$ o For fluorescent dextran dyes, positive  VTXDUHZDYHSXOVHVRI?Vduration are delivered at a frequency of 300Hz with pulse train duration of 10ms.  ? As with the protocol for DNA, the pulse voltage is determined empirically such that the amplitude of pulses measured on the RVFLOORVFRSHLVEHWZHHQDQG?$ - 109 -  ? Additional adjustments to the stimulus parameters can be made based on observing the results of electroporation under epifluorescence. ? If electroporated cells appear dim, pulse parameters should be gradually increased until cells appear brighter. ? On the other hand, if clusters of cells are labelled, pulse parameters should be reduced until single cells are labelled.  - Retract micropipette, re-insert into a different site within the tissue, and apply voltage train. o To improve yield, we typically electroporate multiple sites in each hemisphere of the tadpole optic tectum, with sufficient spacing to ensure that labelled cells do not overlap.  - Transfer the electroporated tadpole into a c RQWDLQHUZLWKQRUPDO6WHLQEHUJ?Vrearing solution. o Tadpoles will rapidly recover from an aesthesia within a few minutes.  - The same micropipette can be used for numerous tadpoles. It is important to constantly observe the amplitude of the pulses observed on the oscilloscope. - 110 -  o If the pulse amplitude remains low after significantly increasing the pulse intensity, this is often a sign of the tip being clogged, usually seen when electroporating many tadpoles in one sitting.  ? A method to dislodge a minor tip clog is to apply a single positive  pulse after inserting the micropipette into the tissue. ? If the clog cannot be dislodged, or clogging frequently recurs, replace the micropipette.  o If the pulse amplitude remains very large after significantly decreasing the pulse parameters, this is a sign that the tip has broken and needs to be replaced.  3.2.4 Screening for succ essfull y elec tropor ated cel ls  - After electroporation, tadpoles are screened for labelled cells using an upright epifluorescence microscope.  - In the case of fluorescent dyes, tadpoles may be screened after as little as 30 minutes post-electroporation. o We have found, however, that longer intervals are associated with lower background fluorescence levels.  - For genetically encoded fluorescent proteins, screening is typically conducted at least 12 hours post-electroporation. - 111 -  o Cells expressing fluorescent proteins will continue to get brighter with time, therefore dim cells may be re-screened after 12-24 hours.  - Tadpoles are an aesthetized as described above, placed in a Sylgard? chamber and coverslipped. Individual tadpoles are then examined under epifluorescence. o Note that excessive exposure to epifluorescence light will result in phototoxicity that may kill the labelled cell. Therefore, minimize the amount of time that cells are exposed to the fluorescent light.   - 5HWXUQWDGSROHVWRFRQWDLQHUZLWKQRUPDO6WHLQEHUJ?VUHDULQJVROXWLRQ         a. b. Figure 3.4: Examples of labell ed neurons imaged within the live tadpole brain  In vivo  two-photon microscopy imaging of ( a)  protracted and ( b )  rapid neuronal growth using repeated time-lapse imaging protocols. Here, neurons were fluorescently labelled either with the fluorescent dye Alexa Fluor 488 dextran 3000MW (a), or a plasmid encoding green fluorescent protein (b). Scale bars  = 20?m .  - 112 -  3.3 Discussion  Single-cell electroporation (SCE) is a powerful tool for determining gene function and performing targeted genetic manipulation. The transparency of the albino tadpole and the accessibility of the brain make this model system ideally suited for visualizing neuronal growth (Fig. 3.4) and intracellular events (Fig. 3.5) within a live organism. SCE allows the visualization of the growth of a single neuron, and to perform cell-autonomous manipulations within an otherwise unaltered brain. While this article details the procedure for single-cell electroporation in Xenopus  laevis tadpoles, this technique has been used in other organisms, and has also been used in hippocampal slices and dissociated cell cultures.        Figure 3 .5: Example of in vivo imaging of intracel l ul ar synapse dyna mics  Co-electroporation of two plasmid vectors encoding different proteins to concurrently visualize excitatory synapses (PSD -95::CFP) and cellular morphology (YFP) of an individual tadpole tectal neuron in vivo. Boxed region showed at higher magnification in lower panels. Scale bar: upper SDQHOV ?PORZHUSDQHOV ?P - 113 -  3.4 References  Bestman JE, Ewald RC, Chiu SL, Cline HT. In vivo single -cell electroporation for transfer of DNA and macromolecules. Nature Protocols. 20 06;1(3):1267 -72.  Dunfield D, Haas K. Single cell electroporation. In: Encyclopedia of Neuroscience (4th Ed.) Elsevier, Amsterdam. (in press)   Haas K, Jensen K, Sin WC, Foa L, Cline HT. Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain. Differentiation. 2002 Jun;70(4 -5):148 -54.  Haas K, Sin WC, Javaherian A, Li Z, Cline HT. Single -cell electroporation for gene transfer in vivo. Neuron. 2001 Mar;29(3):583 -91.                  - 114 -  4. EARLY-LIFE SEIZURES INHIBIT DENDRITOGENESIS IN VIVO3  4.1 Introduction  7KHLPPDWXUHEUDLQ?VLQKHUHQWHQKDQFHGVXVFHSWLELOLW\IRUVHL]XUHDFWLYLW\(Haas et al., 1990; Sperber et al., 1991; Haas et al., 1992), together with epileptogenic factors common in early life (Jensen and Baram, 2000) re sults in an exceptionally high prevalence of seizures during infancy and early childhood (Moshe, 1993; Ben -Ari and Holmes, 2006; Thibeault -Eybalin et al., 2009). This period of high seizure occurrence is concomitant with periods of heightened brain plasticity when precisely patterned neuronal activity directs both the structural and functional maturation of nascent neural circuits (Katz and Shatz, 1996). Whether early-life seizures disrupt or alter ongoing programs of activity-dependent circuit formation is of significant clinical relevance since the establishment of abnormal neural circuits may produce persistent deficits in cognitive function, and potentially predispose individuals to neurological disorders later in life (Stafstrom et al., 2000; Swann, 2004; Holmes, 2005). Indeed, clinical studies have established correlations between early-life seizures and persistent deficits in brain function                                                           3 A version of this chapter has been submitted for publication:   Hewapathirane DS , Chen S, Yen W, Karimi Tari P, Neufeld S, Haas K (200 9)  Developmental seizures inhibit dynamic dendritogenesis in vivo by excessive AMPA receptor-mediated transmission. CHAPTER 4 - 115 -  (Burnham, 2002; Austin and Caplan, 2007), as well as an increased susceptibility to subsequent seizures (Lee, 198 9; Vestergaard et al., 2007) and other neurological disorders (Gaitatzis et al., 2004; Vestergaard et al., 2005; Ekinci et al., 2009). Controlled, experimentally induced seizures in animal models have similarly found lasting deficits in learning and memory and cognitive tasks, and heightened susceptibility to further seizures, suggesting a direct causal effect of seizures rather than separate neuropathological origins (Holmes et al., 1988; Huang et al., 1999; Jensen and Baram, 2000; Lynch et al., 2000; Rutt en et al., 2002). Precisely how early -life seizures alter developing brain circuits to produce lasting functional deficits, however, remains poorly understood.  The structural development of neuronal dendritic arbors is an important component of neural network formation and refinement since dendritic arbor size and shape directly influences neural circuit connectivity, complexity, and function (H?usser et al., 2000; Wong and Ghosh, 2002; Cline and Haas, 2008). Direct in vivo time-lapse imaging of growing neurons has found that dendritogenesis is a highly dynamic process involving continuous extension and retraction of both branches and shorter filopodia, which are precursors to branches at this stage of maturation. The high motility and turnover of dendritic  structures is proposed to enable growing dendrites to explore the surrounding extracellular space for appropriate presynaptic partners, a theory supported by findings that glutamatergic transmission (Haas et al., 2006) and synapse formation (Niell et al.,  2004) promote stabilization of dendritic filopodia. Neuronal activity -dependent, - 116 -  synaptotropically-driven dendritogenesis therefore may direct arbor growth into regions of appropriate afferent innervation, thereby optimizing circuit morphology and connectivity to process specific afferent activity (Cline and Haas, 2008). Evidence for aberrant seizure activity during development influencing dendritic morphology has been found in in vitro  and post-mortem histology studies (Jiang et al., 1998; Swann et al., 2 000; Nishimura et al., 2008), yet it remains unclear whether and how early-life seizures directly interfere with ongoing dynamic patterns of dendrite growth.   Here, we utilize a newly developed model of early-life seizures based on the transparent albino Xenopus laevis  tadpole (Hewapathirane et al., 2008; Hewapathirane and Haas, 2009) and in vivo two-photon time-lapse imaging in combination with comprehensive 3D analysis of dynamic and protracted dendrite growth patterns to examine whether seizures alter dendritogenesis during critical periods of early brain development. By conducting prolonged rapid imaging over 5 hours using 5 minute intervals, encompassing periods before and during seizures, we have precisely characterized the subtle effects of seizures on dendritogenesis within the intact and awake developing brain, by tracking and analyzing all dendritic branches and filopodia over time. Longer -interval imaging over hours to days allowed identification of persistent effects on neural circuit structures. From these methods of direct imaging and comprehensive morphometric quantification, we have identified two distinct yet opposing effects of seizures: the rapid destabilization of dendritic processes existing prior to seizures and hyper-- 117 -  stabilization of those emerging during seizures. We find that overall, early-life seizures inhibit dendritic arbor growth resulting in persistently stunted morphological structures. Seizures also significantly decreased the density of immunostained excitatory synaptic markers, further establishing that early-life seizures lead to a lasting reduction in neural circuit complexity. Our findings identify morphological substrates potentially underlying persistent neural circuit dysfunction commonly associated with early-life seizures.               - 118 -  4.2 Methods   4.2.1 Animals and seizur e induction  Stage 47 albino X enopus laevis  tadpoles (Nieuwkoop and Faber, 1994) ZHUHPDLQWDLQHGLQ6WHLQEHUJ?VVROXWLRQ;6WHLQEHUJ?VP0+(3(6mM NaCl, 0.67 mM KCl, 0.34 mM Ca(NO 3) 2, 0.83 mM MgSO 4, pH 7.4), and housed at room temperature (22 oC) on a 12 h light/dark cycle. Seizures were induced by bath application of either pentylenetetrazol (15 mM, PTZ, Sigma -Aldrich), a non -competitive GABA receptor antagonist; pilocarpine (75 mM, Tocris , Ellisville, MO), a non -selective muscarinic receptor agonist; or 4 -aminopyridine (1 mM, Tocris), a potassium channel blocker. At these doses, each convulsant reliably induces behavioural and electrographic seizures in freely swimming, and in agarose-immobilized tadpoles (Hewapathirane et al., 2008). Drugs were dissolved in normal rearing medium adjusted to pH 7.4. All experimental procedures were approved by the Animal Care Committee of the University of British Columbia.  4.2.2 In vivo sing le -ce ll electr op oration for fluore scent labe ling and targeted trans fection of developing neu rons  Individual immature neurons within the tadpole optic tectum were fluorescently labelled using in vivo single-cell electroporation (Haas et al., 2001a; - 119 -  Hewapathirane and Haas, 2008) of the fluorescent dye Alexa Fluor 488, 3000MW (1mM in dH2O, Molecular Probes, Eugene, OR), or a plasmid vector encoding membrane-targeted, farnesylated enhanced green fluorescent protein (fEGFP, 2? g/ ? l endotoxin-free plasmid DNA in dH2O). For ele ctroporation, tadpoles were briefly anaesthetized by immersion in 0.02% 3 -aminobenzoic acid ethyl ester (MS -222, Sigma-Aldrich, St. Louis, MO). A pulled glass micropipette (tip diameter 0.6? m) filled with dye or plasmid DNA solution was inserted into the c ell-body layer of the optic tectum, and an Axoporator 800A (Axon Instruments, Union City, CA) was used to deliver brief trains of square wave pulses between a silver wire within the pipette and an external bath electrode. For plasmid DNA, pulse stimulus parameters were: intensity = 1.5 ? A, duration = 200 ? sec, frequency = 300 Hz, train duration = 300 msec. For Alexa Fluor 488 dye, stimulus parameters were: pulse intensity = 2 ? A; pulse duration = 1 msec; pulse frequency = 300  Hz; train duration = 20 msec. I QH[SHULPHQWVWHVWLQJWKHUROHRI?-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor -mediated neurotransmission in seizure-induced effects, a plasmid vector encoding the carboxy-terminal domain of the Xenopus laevis  AMPA receptor subunit GluR1 (residues 809 ?889; GluR1Ct) fused to the carboxy -terminal of EGFP was delivered by single-cell electroporation, and cells were imaged 48 h post-transfection (Haas et al., 2006).   - 120 -  4.2.3 In vivo tw o -photon fluoresc ence imaging of den dritic gro w th  For rapid time-lapse imaging of dynamic dendritic growth behaviour, tadpoles were initially paralyzed by bath application of the reversible paralytic pancuronium dibromide (2mM in normal rearing medium, Tocris, Ellisville, MO), embedded under a thin layer of agarose (1%, prepared in normal rearing medium), and immersed in an imaging chamber continuously perfused with oxygenated rearing medium (Hewapathirane et al., 2008). The imaging chamber was mounted on the stage of a custom-built two-photon laser-scanning microscope, constructed from an Olympus FV300 confocal system (Olympus, Center Valley, PA) with a Chameleon XR laser light source (Coherent, Santa Clara, CA). Using an excitation wavelength of 910 nm, stacks of images ( z -axis step size = 1.5 ? m) of labelled neurons were captured using an LUMFI 60X water immersion objective (Olympus, NA = 1.1) and images were recorded using Fluoview software (Olympus). In experiments employing imaging intervals of 4 or 24 hours, tadpoles were briefly anaesthetized with 0.02% MS -222, placed in a Sylgard (Dow Corning, Midland, MI) imaging chamber, coverslipped, and imaged. After imaging, tadpoles were immediately returned to their chambers where they fully recovered from anaesthesia within 5 min.   - 121 -  4.2.4 Dendrite morp hom e t ric an al ysis  Custom-designed software (based on a program created by Dr. Jamie Boyd, University of British Columbia), running on the IGOR Pro 6 platform (WaveMetrics, Portland, OR) was developed to comprehensively analyze both rapid and protracted dendritic growth events. Every dendritic filopodium and branch within a 3D stack encompassing the full dendritic arbor were identified, measured, and tracked through successive 5 min time points over the entire 5 h imaging period. In experiments with long-interval (4 and  24 h) time -lapse imaging paradigms, dendritic arbors were manually drawn using computer-assisted 3D rendering software (Neurolucida, MicroBrightField, Williston, VT).   4.2.5 Immunohistoc hemistr y of s yn aptic ma rkers  Fluorescence immunohistochemistry was performed on horizontal 20 ?m cryostat sections of whole tadpoles fixed with 4% paraformaldehyde (Sigma -Aldrich, diluted in 0.1M phosphate buffer, pH 7.4 (PB)). Experimental and control group tissue sections were yoked, with identical numbers of tissue sections in experimental and control groups mounted onto each microscope slide. Sections were pre-incubated in blocking solution consisting of 5% (v/v) normal goat serum in 0.1 M PB containing 1% Triton X -100 (Acros Organics, Geel, Belgium), for 1 h at room temperature (RT).  Sections were then rinsed and incubated for 24 h at 4o&ZLWKSULPDU\DQWLERGLHVWRERWKUDEELW?-SNAP -25 (Stressgen, Ann Arbor, MI), - 122 -  DQG PRXVH ?-PSD-95 (Millipore, Billerica, MA) diluted 1:400 and 1:200, respectively, in blocking solution. Sections were subsequently rinsed and incubated in the dark for 1 h at 57ZLWKVHFRQGDU\DQWLERGLHVJRDW?-rabbit Alexa- DQG JRDW ?-mouse Alexa-633 (Molecular Probes, each diluted 1:200 in blocking solution). Immunofluorescence within a randomly selected region of the tadpole optic tectal neuropil region was visualized and imaged with an Olympus FV1000 confocal system, using a PlanApo N 60X oil immersion objective (Olympus, 1.42 NA). Dual -channel images were obtained with sequential fluorophore excitation using the 543 nm and 633 nm laser lines, and emitted signal was filtered using a 560?600 nm bandpass filter and a 650 nm long-pass filter. Following acquisition, blinded images were subjected to background subtraction and immunostained puncta with sizes in the range of 0.2-3.0 ?m 2 (Ruthazer et al., 2006; Lim et al., 2008) were quantified and subjected to colocalization analysis. In order to assess whether colocalization of fluorescence signals was due to actual juxtaposition of pre - and post-synaptic elements as opposed to random colocalization of fluorescent puncta, analyses were repeated where one image was rotated by 90 degrees. In all experiments, rotated images showed a significant decrease in colocalization densities to ~10% those of original values. Image analysis was performed using NIH ImageJ software.    - 123 -  4.2.6 Stat istical an al yse s  Statistical tests were conducted using PASW Statistics 17 software (SPSS Inc., Chicago, IL). Multiple -way repeated measures ANOVA comparisons followed E\7XNH\?VSRVW-hoc analysis were used to examine both within- and between-group differences, unless otherwise noted. Within-group differences compared baseline values to subsequent measurements in individual subjects of a given experimental group, while between-group differences compared time-matched measurements between two or more experimental groups. Kolmogorov -Smirnov comparisons were used to compare differences between cumulative proportion datasets.               - 124 -  4.3 Results   4.3.1 Seizure s inhibit den dritic grow th  Single-cell electroporation was used to introduce the fluorescent dye Alexa Fluor 488 into individual immature neurons within the tadpole optic tectum, a midbrain structure corresponding to the mammalian superior colliculus. Dendritic growth of labelled neurons was imaged at 4 h intervals over 8 h, with the first 4 h used as an internal control of baseline growth for a subsequent 4 h period in which seizures were induced. All neurons were re-imaged 1 day following experiments to confirm cell viability following seizures. Three chemoconvulsants with distinct mechanisms of action were tested. Following imaging of baseline growth, freely-swimming tadpoles were exposed to PTZ (15  mM), pilocarpine (75 mM), or 4 -AP (1  mM) for 1 h (Hewapathirane et al., 2008). In this and all subsequent experiments, control tadpoles were exposed to vehicle solution composed of normal rearing medium. Dendritic arbor growth rates were similar during the first 4 h baseline period across groups, with neurons in all groups demonstrating ~20% increase in total arbor length, and ~13% increase in the combined number of branch and filopodial tips, relative to each individual QHXURQ?V LQLWLDO YDOXHV Figs. 4.1A and 4.1B). owever, neurons in tadpoles experiencing seizure activity lasting 1 h showed no increase in dendritic arbor size during the second 4 h period ( 4-AP: ?1.41 ? 5.45%, n=8 neurons; pilocarpine: - 125 -  0.00 ? 5.61%, n=8 neurons; PTZ: ?1.27 ? 8.55%, n=9 neurons; control: 17 ? 6.34% n=10 neurons), and an overall loss of branch and filopodial tips (4 -AP: ?8.79 ? 9.91%; pilocarpine:  ?7.00 ? 5.87%; PTZ: ?15.08 ?  12.32%; controls: 12.42 ? 7.99%). A separate group of tadpoles was exposed to PTZ (15mM) for 4 h to examine the effects of extended seizure activity on dendritic growth. Seizure activity lasting 4 h significantly reduced dendritic length, and numbers of dendritic processes ( ?21.64 ? 5.52% and ?19.58 ? 6.95%, respectively; n=8 neurons), GHFUHDVLQJWRYDOXHVEHORZHDFKLQGLYLGXDOQHXURQ?VLQLWLDOYDOXHV$OWKRXJKRUh seizures interfered with normal patterns of growth, dendritic beading was not observed, and all neurons were viable 24 h following seizures. These data demonstrate a duration-dependent effect of seizures on dendritogenesis in vivo, with 1 h of seizure activity retarding growth and 4 h of seizure activity inducing dendritic arbor atrophy. The  similar effects of seizures induced by chemoconvulsants with different mechanisms of action indicate that the observed effects are directly caused by seizures, as opposed to drug-specific effects not related to their seizure-inducing activity. For this reason, PTZ was used for seizure induction in subsequent experiments.      - 126 -                        Figure 4 .1: Development al seizures inhibit dendrite grow th through excessive AMPA recept or activation. Two -photon imaging of single growing neurons within the intact brain was used to examine dendritic arbor growth of individual fluorescently labelled neurons. (A) Arbor growth over the initial 4 h baseline period was compared to growth during the subsequent 4 h period in which seizures were induced. Pentylenetetrazol (PTZ) -induced seizures lasting 1 h inhibited further arbor growth, while prolonged seizures lasting 4 h induced marked branch retraction leading to arbor atrophy. Arrowheads denote axons. Scale bar = 20 ?m. (B)  Quantification of arbor growth ( l eft,  change in total dendritic length) and changes in dendrite tip number ( right , branch and filopodial tips) reveals that 1 h of seizure activity induced by either 4-aminopyridine (4 -AP, 1mM), pilocarpine (75mM), or PTZ (15mM) inhibits further arbor growth and leads to an overall loss of dendritic tips. 4 h of PTZ -induced seizures induced overall arbor DWURSK\Q?QHXURQVJURXS(C)  Single neurons with reduced AMPA receptor-mediated transmission (GluR1Ct -expressing neurons) within the otherwise unaltered brain continue to elaborate their dendritic arbors even after being subjected to 1 h of PTZ -induced seizure activity. Scale as in (A); arrowheads denote axons. (D) No significant differences in arbor growth ( l eft ) or tip number ( right ) indicated that reduced AMPA receptor-mediated transmission protects developing neurons from seizure-induced inhibition of dendritic growth (n=12 neurons/group). *p<0.05, multiple -way repeated measures ANOVA. Data presented as means ? SEM.   - 127 -  4.3.2 Seizure -induc ed inhibition of de nd ritic grow th is due to exces sive AMPA receptor activation  Previous studies have implicated excessive excitatory glutamatergic neurotransmission as a potential mediator of seizure-induced dendritic spine loss and arbor atrophy (Swann et al., 2000; Nishimura et al., 2008). However, studies involving systemic application of glutamate receptor antagonists are confounded by the potential of such agents to alter seizure expression. To investigate the role of AMPA receptors in seizure affects on dendritogenesis without altering seizure activity, we employed single-cell electroporation to selectively reduce synaptic AMPA receptor-mediated transmission only in the individual developing tectal neurons being imaged (Haas et al., 2006). This cell -autonomous genetic manipulation, within the otherwise unaltered brain, circumvents systemic and circuit-level effects of pharmacological approaches. Dendrite growth of individual transfected neurons was imaged at 4 h intervals over 8 h as above, with tadpoles in the seizure group being exposed to PTZ (15mM) for 1 h in the second 4 h period. No significant difference in arbor growth rates was obs erved between groups during the baseline 4 h period (~11% increase in arbor length and ~13% increase in dendrite tip number; n = 12 neurons/group; Figs. 4.1C and 4.1D). Strikingly, individual neurons with reduced AMPA transmission in tadpoles that experienced seizures also continued to grow at rates similar to controls (arbor growth: 10.05 ? 1.61%, dendritic tip number increase: 13.45 ? 1.68%; Figs. 4.1C and 4.1D). These results strongly implicate AMPA receptor -mediated excitatory - 128 -  input as a critical cell-autonomous mediator of seizure-induced effects on dendritic growth, rather than systemic factors.   4.3.3 Seizure s incr ease rates of filopodial an d branch elimination  The effects of ongoing seizures on dynamic dendritic growth were examined using rapid time-lapse imaging within intact and awake tadpoles. Anaesthetics were not used during continuous rapid time-lapse imaging experiments to circumvent potentially confounding effects of prolonged anaesthetic exposure. Images of single fEGFP labelled neurons were taken at 5 min intervals over 5 h, comprising of an initial 1 h baseline period followed by either 4 h of PTZ -induced seizures or 4 h exposure to control solution (n=5 neurons/group; Fig. 4.2A). Control neurons demonstrated linear and significant increases in total branch length, increasing after 5 h to 132.87 ? 15.85% relative to initial values ( Fig. 4.2B). In contrast, while seizure neurons exhibited a similar rate of branch growth during the baseline period, the mean total branch length following seizure induction significantly decreased over the subsequent 4 h, with final lengths of 78.21? 8.25% relative to initial values ( Fig. 4.2B). A significant reduction in total branch number was also observed during seizures, whereas control neurons continued to add new branches over the entire course of imaging ( Fig. 4.2C). Seizure -related reduction in branch number was due to a significant increase in branch eliminations compared to control neurons ( Fig. 4.2D). However, neurons continued to add new branches during seizures ( Fig. 4.2E).   - 129 -                        Figure 4 .2: Rapid time -lapse imaging of dendrite grow t h during seizures in the intact and unanaesthetized brain . (A) 3D images of neurons were taken over 5 min intervals for 5 h, comprising of an initial 1 h baseline period followed by 4 h of PTZ -induced seizures. Representative images are colour coded to highlight dendritic segments that are added (green) and retracted (red) during each hourly period (arrowheads denote axons; scale bar = 20 ?m). Boxed regions are shown at higher magnification as insets, displaying overlays of 4 colour-coded images spaced by 15 min intervals (red, blue, green and cyan; overlap = white; scale bar for insets = 5 ?m). (B -E) Each dendritic branch was identified, measured and tracked individually over the entire period of imaging (n=5 neurons/group). Branch length (excluding constitue nt filopodia) significantly decreased during seizures, while control neuron branches exhibited elongation. Seizures also induced a significant reduction in branch number (C), due to a significant increase in branch elimination (D, cumulative branches lost/ hour epoch) compared to controls. Seizures did not significantly alter branch addition rates (E, cumulative branches added/hour epoch).  (F -J)  Seizures induced a significant decrease in total filopodia number, with values steadily decreasing over time (F). Filopodial density was not significantly decreased (G), presumably due to concomitant branch retraction. Seizures also significantly reduced the rate of filopodial addition, which continued to decline over time (H). Seizures did not alter the rate of filopodial retraction over the first hour post-induction, however, rates of elimination gradually declined over the subsequent 3 h to values significantly less than controls (I). The net loss of filopodia during seizures was found to be due to a relative increa se in filopodial retraction rates compared to addition rates within individual neurons, demonstrated by significant decrease in filopodial addition:elimination ratio during seizures (J). *p<0.05, multiple -way repeated measures ANOVA. Data presented as mean s ? SEM.   - 130 -                       - 131 -  Total filopodial numbers on each arbor were analyzed at 5 min intervals over the entire 5 h time-course of imaging as a measure of dynamic dendritic growth (a total of 3275 filopodia an alyzed in all neurons; n=5 neurons/group). Each dendritic filopodium on a developing dendritic arbor was identified, measured and tracked individually over time, using custom-designed software. Seizures induced a decrease in total filopodia number, reaching levels significantly less than control values 85 min following seizure induction, after which values continued to decline ( Fig. 4.2F). Notably, filopodial density did not significantly differ from controls, presumably due to the concomitant decrease in total branch length ( Fig. 4.2G). Filopodial addition and elimination rates were measured to determine which aspect of filopodial turnover contributed to the overall loss of filopodia during seizures. Rates of filopodial addition significantly decreased during the first hour of seizure activity, and continued to decrease over time to a final value of 47.32 ? 12.70%, relative to the baseline rate ( Fig. 4.2H). Seizures did not affect the rate of filopodial elimination over the first hour post-induction (114.24 ? 11.32 of baseline values), however, eliminations significantly declined over the following 3 h, reaching a final value of 60.98 ? 11.02 % relative to the baseline rate ( Fig. 4.2I). Although both the rates of additions and eliminations were significantly reduced during seizures when compared to control values, the rate of addition was reduced to a larger degree, resulting in an overall net loss of filopodia over time ( Fig. 4.2J). Collectively, these data indicate that seizure-induced inhibition of growth, and the eventual atrophy of developing - 132 -  dendritic arbors, is due to an inhibition of branch elongation, an increase in the rate of branch elimination relative to addition, and an overall loss of dendritic filopodia. While dendrites continue to exhibit growth behaviour during seizures, including addition of new branches and filopodia, larger increases in the rates of retraction result in an overall retardation of growth.    4.3.4 Seizure s indu ce ra pid elimination and subseq uent stabilization of specific filo p odial sub -pop ulations  Since filopodial stabilization is associated with synapse formation and maturation (Niell et al., 2004), and given that early -life seizures potentiate glutamatergic synapses (Rakhade et al., 2008), we examined whether excessive synchronous seizure activity interferes with normal patterns of filopodial stabilization. Analysis of the maintenance of filopodia present prior to seizure LQGXFWLRQ UHIHUUHG WR KHUH DV ?SUH-VHL]XUH? ILORSRGLD UHYHDOV D UDSLG ORVV RIfilopodia over the first 15 min following PTZ exposure ( Fig. 4.3A). Using electrophysiological recordings we have previously demonstrated that PTZ -induced seizures (15mM) typically initiate within 10 -15 min following drug application in agar-immobilized tadpoles (Hewapathirane, 200 8). The observed rapid destabilization of pre-existing filopodia therefore reflects events occurring within minutes of seizure onset. Over the subsequent 20 -170 min, the proportion of pre-seizure filopodia remaining was similar across groups, with values steadily declining over time. Notably, however, over the final 65 min period (175 -240 min - 133 -  post-seizure induction), seizure neurons retained significantly more pre -seizure filopodia than control neurons, indicating hyper-stabilization of a subpopulation of pre-seizure filopodia during seizures ( Fig. 4.3A, see inset). Conversely, filopodia generated during seizures were found to be hyper-stabilized when compared to control filopodia generated over the same period ( Fig. 4.3B). Analysis of the cumulative distribution of filopodial lifetimes demonstrates a significant population shift, with a larger proportion of filopodia displaying longer lifetimes in seizure neurons compared to controls ( Fig. 4.3C). Correspondingly, the mean lifetime of filopodia generated during seizures was significantly greater than control values ( Fig. 4.3D). Together, these results demonstrate that seizures induce two distinct, yet opposing, effects on filopodia: (i) destabilization occurring rapidly after seizure onset, and (ii) a hyper -stabilization effect on a subset of pre-seizure filopodia and those that are added during the seizure event.   - 134 -                Figure 4 .3: Seizures rapidly eliminate existing and hyper -stabilize new filopodia during seizures. Images of individual neurons were taken every 5 min for 5 h in vivo, within intact and unanaesthetized tadpoles. Each dendritic filopodium was identified, measured and tracked individually over time, spanning 1 h baseline followed by 4 h of pentylenetetrazol (PTZ) -induced seizures (n=3275 filopodia from 5 neurons/group). (A) Seizures rapidly induced filopodial destabilization, seen as an increase in the rate of elimination of filopodia present prior to seizure induction (pre -seizure filopodia), after which values matched those of controls until ~120 min post-seizure induction. Over the subsequent period (highlighted as inset with expanded y -axis), the proportion of remaining pre-seizure filopodia was significantly elevated in seizure neurons, indicative of a hyper-VWDELOL]DWLRQHIIHFWSXQSDLUHG6WXGHQW?VW-test. (B)  Cumulative plot of the proportion of filopodia generated after the baseline period which remained until the end of imaging. Filopodia generated during seizures were significantly hyper-stabilized, with a greater proportion of those lasting until the end of imaging being added at earlier time-points compared to controls (*p<0.001, t wo sample Kolmogorov -Smirnov (KS) comparison, D  =0.1590). (C -D)  Filopodia generated during seizures exhibited a significant shift towards longer lifetimes, compared to control neuron filopodia generated over the same time period (F; *p<0.001, two sample Ko lmogorov-Smirnov comparison, D  =0.3830), resulting in a significant increase in DYHUDJHOLIHWLPH*SXQSDLUHG6WXGHQW?VW-test). Data presented as means ? SEM.   - 135 -  4.3.5 Reduce d motilit y and restricted explorat or y behaviour  of filopodia during seizures  The highly dynamic growth behaviour of dendritic filopodia is proposed to mediate exploratory searching of local extracellular space for potential presynaptic partners. The effects of seizures on the dynamic 3D motility of filopodia were determined by comparing the change in length of each filopodium over eYHU\PLQLQWHUYDOPRWLOLW\ ?OHQJWK&KDQJHVLQILORSRGLDOOHQJWKVVKRZsimilar variability during the 1 h baseline period, whereas this variability steadily decreases after seizure induction, indicating an overall reduction in the degree of filopodial motility during seizures ( Fig. 4.4A). Quantification of these data revealed that seizures induced a significant reduction in absolute filopodial motility within the first hour of seizures, with values continuing to decline over time, while control filopodial motility remained stable over the entire 5 h ( Fig. 4.4B). Mean filopodial lengths were not significantly different between groups over the course of imaging (~2.25 ?m; Fig. 4.4C), indicating that seizure -induced reduction in filopodial motility was not an indirect result of a reduction in filopodial length. To ascertain whether seizures alter filopodial exploratory behaviour, we examined the variability of filopodial growth by calculating the absolute mean change in motility of each filopodium (where _?PRWLOLW\_ _PRWLOLW\t ? motilityt+1 | for two subsequent REVHUYDWLRQVWDQGW/DUJH_?PRWLOLW\_YDOXHVFRUUHVSRQGWRODUJHIOXFWXDWLRQVin the rate and/or direction of movement over successive observations, indicating heightened search-like growth behaviour. Plotted against mean motility, a - 136 -  measure of the overall direction and speed of movement over the entire lifetime of a filopodium, it is evident that during seizures filopodia are less variable in growth, suggesting a decrease in normal exploratory behaviours ( Fig. 4.4D). These results suggest that seizures limit the exploratory searching behaviour of filopodial during dendrite growth, potentially reducing the complexity or fidelity of circuits formed.                Figure 4.4: 3D filopodia tracking reveals complex effect s of seizures on filopodial motility and explorat ory behaviour. Individual dendritic filopodial lengths were measured every 5 min over the period spanning 1 h baseline followed by 4 h of pentylenetetrazol (PTZ) -induced seizures (n=3275 filopodia from 5 neurons/group; ). (A)  Overlay o f  >13000 3D length change measurements from all filopodia present over the imaging period reveals a reduction in the variance of motility as seizures progressed, consistent with reduced growth. (B -D)  Seizures induced a significant decrease in the absolute PRWLOLW\RIILORSRGLDPHDQ_?OHQJWK_FRPSDUHGWRcontrols (B; * p<0.05, multiple -way repeated measures ANOVA), without significantly altering mean filopodial lengths (C). Normal filopodial exploratory behaviour is characterized by large fluctuations in the rate and/or direction of movement over successive observations. We examined whether seizures interfere with the filopodial search behaviour by examining the variability in PRWLOLW\PHDQ _?PRWLOLW\_UHODWLYHWRWKHLU DYHUDJHFKDQJHLQ OHQJWKPHDQPotility). During seizures, filopodia displayed reduced variability in growth, suggesting a decrease in normal exploratory behaviours compared to controls (D, data points represent individual filopodia). Data in B and C presented as means ? SEM.   - 137 -  4.3.6 Seizure -induc ed inhibi tion of dend ritic grow th is persisten t  To determine whether the acute and short -term seizure effects on dendritic growth have persistent consequences on neuronal morphology, we imaged newly differentiated Alexa Fluor 488-labelled tectal neurons daily for 4 days, a period spanning tectal dendritic arbor maturation, a period after which arbors demonstrate no net changes in total dendritic length (Wu et al., 1999; Haas et al., 2006). Tadpoles were subjected to a single 1 h PTZ -induced seizure after acquisition of the day 2 image, an early time-point during dendritic maturation (n=14 neurons/group; Fig. 4.5A). Over the first 2 days, total dendritic arbor length and dendritic tip number increased at similar rates across groups (controls: 342.20 ? 49.42  ?m arbor g rowth and 30.96 ? 5.35 additional tips; seizure group: 387.25 ? 49.33 ?m arbor growth and 27.08 ? 4.47 additional tips, Figs. 4.5B and 4.5C). However, while control neuron arbors continued to grow over the following 2 days, exhibiting arbors 320.62 ? 64.01  ?m larger with 19.23 ? 3.53 additional dendritic tips, arbors of neurons experiencing seizures did not continue to grow over days 2 to 4, demonstrating significantly reduced dendritic arbor length and dendritic tip number on days 3 and 4 compared to age-matched control cells. Similarly, while control neurons significantly increased dendritic arbor field volume after day 2, field volumes of seizure neurons failed to significantly increase after day 2, with day 3 and 4 volumes remaining significantly lower than age-matched controls ( Fig. 4.5D). Dendritic complexity, as assessed using 3D Sholl analysis, showed similar increases across groups in the dendritic length added over the - 138 -  first 2 days of imaging, within the 3D space corresponding to a spherical shell 40-100?m from the cell body ( Fig. 4.5E). Seizure neurons showed no significant increase in complexity over the days subsequent to the seizure episode, contrasting with control neurons which significantly increased dendritic complexity within the 3D space 60-80?m radial to the cell body over the equivalent time period. These finding indicate that seizure -induced inhibition of dendrite growth persists well beyond the duration of the seizure event, resulting in persistently stunted arbors. - 139 -                 Figure 4.5: Seizures persistent l y stunt furt her arbor grow t h and elaboration in vivo. Individually labelled neurons were imaged daily within the intact brain over 4 days, a period spanning tectal neuron dendritic maturation. Tadpoles were subjected to a single 1 h pentylenetetrazol (PTZ) -induced seizure after acquisition of the day 2 image to examine long-term effects on dendritogenesis. (A)  Neurons experiencing a single seizure showed no significant increases in arbor size or complexity over the following 2 days. Scale bar = 20 ?m; arrowheads denote axons. (B -E)  A single seizure restricted subsequent increases in arbor size (B), dendritic tips (C; branches and filopodia), dendritic field volume (D), and branch complexity as assessed by 3D Sholl analysis (E), over th e subsequent 2 days post-seizure. n=14 neurons/group; *p<0.05, multiple-way repeated measures ANOVA. In D, markers indicate significant differences from day 2 values. Data presented as means ? SEM.   - 140 -  4.3.7 Seizure s per sisten t l y decr ease excitat or y s yn apse densities  Since dendritic size limits the membrane surface area available for synapse formation, reductions in dendritic arbor size and complexity likely also induce changes in synapse numbers. We examined whether seizures alter the density of excitatory synapses within the neuropil region of the optic tectum using immunohistochemical colocalization analysis of the pre- and post-synaptic markers SNAP -25 and PSD-95, respectively (Lim et al., 2008; Figs. 4.6A-C). To examine short-term effects, tadpoles were either exposed to PTZ for 1 h and sacrificed 3 h later, or exposed to PTZ for 4 h and sacrificed immediately. Persistent changes were examined in tadpoles exposed to PTZ for 1 h and sacrificed after either 24 or 48 h ( Fig. 4.6D). While the density of SNAP -25 puncta remained unchanged across all groups, a significant reduction in the density of both colocalized puncta and total PSD-95 puncta were observed after seizures, compared to controls ( Fig. 4.6E). Alth ough colocalized and total PSD-95 puncta densities significantly increased after intervals of 24 and 48 h post-seizure, suggesting partial recovery from the acute effects of seizures, values remained significantly lower than controls. Changes in punctum colocalization were solely due to changes in PSD-95 puncta number, since the relative proportion of PSD-95 puncta that were colocalized did not differ across groups ( Fig. 4.6F). Our findings of persistent reductions in excitatory synapse number are consistent with reductions in dendritic arbor size over the same time period. These findings - 141 -  further highlight the functional implications of seizure-induced inhibition of dendritogenesis on reduced neural circuit complexity.                       Figure 4 .6: Seizures persistent l y reduce syn apse density . (A)  Dorsal view of the albino Xenopus laevis  tadpole brain ( stage 47, rostral end towards top of image). ot  ? optic tectum; v ? YHQWULFOH6FDOHEDU ?P(B)  orizontal cryostat sections from fixed tadpoles were immunostained with antibodies against pre- and post-synaptic elements SNAP -25 (red) and PSD -95 (green), respectively . The dotted line demarcates border between tectal cell body layer ( cbl ) and neuropil ( n), relative to the ventricle ( v). For each section (n= 4 sections/tadpole; 3 -4 tadpoles per group), a random region of neuropil was analyzed (boxed). Scale bar = ?P(C)  Higher magnification images of immunolabelled tectal neuropil demonstrate that 4 h of PTZ seizures decreases the density of colocalized SNAP -25 and PSD-95 puncta (yellow). Scale bar = 2 ?P(D)  Experimental paradigm used to test both the acute and persistent effects of seizures on synapse numbers. (E)  Both total PSD-95 and colocalized punctum densities were significantly reduced after seizures. Densities slightly recovered with longer intervals post-seizure, however, values remained significantly lower than controls. SNAP -25 punctum densities remained unchanged across groups, indicating that changes in the numbers of colocalized puncta reflect changes in PSD-95 punctum numbers. (F)  The percentage of PSD -95 puncta that are colocalized remains invariant despite changes in total PSD-95 punctum densities across groups. Colour key for histograms as in D. *p<0.05, one -way ANOVA. Data presented as means ? SEM.   - 142 -  4.4 Discussion  Previous attempts to identify structural correlates of developmental seizure-induced neural circuit dysfunction have been limited, in part, by the inability to detect subtle alterations in neuronal morphogenesis due to the use of single time-point or long-interval time-lapse examinations (Jiang et al., 1998; Swann et al., 2000; Nishimura et al., 2008). By employing a developmental model system that allows direct in vivo imaging of neuronal growth before, during and after seizures, at both short and long intervals, we have comprehensively characterized the acute and persistent effects of seizures on the structural maturation of developing neurons. Furthermore, the examination of seizure-related effects on neuronal morphogenesis within the intact and unanaesthetized brain advances previous experimental approaches by maintaining intact central nervous system circuitry and physiologically relevant micro- and macro-cellular environments during seizures, while avoiding the confounding effects of prolonged anaesthetic exposure on neural growth, circuit function, and seizure expression. We find that early-life seizures induce a complex effect on dynamic growth behaviour which culminates in persistent stunting of dendritic arbor development.  Extended in vivo rapid time-lapse imaging at 5 min intervals over 5 h, followed by tracking and measurement of all dendritic filopodia and branches in 3D reveals bi-directional effects of seizures on dendritic growth dynamics. Seizures rapidly induced a significant reduction in the number of pre-existing dendritic filopodia, within minutes of seizure onset. In contrast, filopodia that arise - 143 -  during seizures exhibit hyper-stabilization, demonstrated by decreased motility and prolonged lifetimes. As seizures progressed, branch elongation decreased and rates of branch elimination increased. Together, these effects produce significant seizure-induced retardation of dendritic arbor growth, resulting in an overall reduction in arbor size. Critically, long-interval imaging over days reveals that the effects of individual seizures are persistent, resulting in stunting of subsequent arbor growth. Since X enopus  tectal neuronal dendritic arbors demonstrate no net changes in total dendritic length upon maturation, a period typically spanning 4 days (Wu et al., 1999; Haas et al., 2006) , we predict that the observed changes are persistent. Reduced arbor size and complexity likely has enduring functional consequences, given that dendritic size and shape influences dendritic integration of synaptic inputs, intrinsic excitability based on input resistance, and the number and types of pre-synaptic sites that may be contacted (H?usser et al., 2000; Wong and Ghosh, 2002; Cline and Haas, 2008). In support for such decreased circuit complexity, we find that seizures persistently decrease the density of excitatory synaptic markers, indicating a loss of excitatory synapses. In light of substantial evidence demonstrating that altered neural activity during critical periods of development modifies neural circuit development leading to lasting effects on subsequent circuit function and plasticity (Crowley and Katz, 2002; Hensch and Fagiolini, 2005; Taha and Stryker, 2005), our findings reveal a mechanism through which early-life seizures may induce lasting brain dysfunction. - 144 -   In order to identify possible mechanisms mediating seizure-induced alterations in dendritogenesis, we tested the effects of various chemoconvulsants working through distinct molecular targets. Since seizures induced by either enhancing glutamatergic or cholinergic transmission, inhibiting potassium channels, or inhibiting GABA receptors each produced similar effects on dendritogenesis, we conclude that altered growth results from seizure activity rather than drug-specific effects unrelated to seizure induction. Furthermore, we tested the role of excessive glutamatergic neurotransmission in seizure-induced inhibition of dendrite growth by selectively reducing AMPA receptor-mediated transmission in individual neurons within otherwise unaltered brains through the targeted expression of a peptide construct which interferes with AMPA receptor trafficking to synapses (Shi et al., 2001; Haas et al., 2006). We find that reduced AMPA receptor-mediated transmission within individual neurons protects those neurons from seizure-induced growth inhibition. These results support a direct role for excessive glutamatergic receptor activity on altered dendritic growth rather than secondary systemic factors, such as metabolic distress or abnormal extracellular pH and ion concentrations. Excessive AMPA receptor-mediated activity during seizures has been linked to memory deficits in immature rats subjected to prolonged seizures (Mikati et al., 1999), establishing a potential link between seizure-induced higher-order functional deficits and the dendritic structural abnormalities reported here.     - 145 -   It remains unclear how excessive glutamatergic transmission alters dendrite growth, yet two distinct processes may be involved: sub-lethal cellular injury induced by excitotoxici ty, and/or the pathological activation of endogenous activity-dependent, competition-based synaptotropic dendrite growth mechanisms. Excitotoxicity-induced neuronal death is a well-documented hallmark of prolonged seizures (Meldrum, 1991). While the immatu re brain demonstrates remarkable resistance to seizure-induced neuronal loss (Haas et al., 2001b; Thibeault -Eybalin et al., 2009), it is possible that seizures activate sub -lethal excitotoxic processes that interfere with normal cellular growth patterns. Sustained membrane depolarization during seizures facilitates activation of calcium permeable NMDA receptors and voltage -gated calcium channels which, in turn, lead to calcium-dependent release of intracellular calcium stores (Olney, 1990). Cumulatively, th ese events may induce focal or cell-wide increases in intracellular calcium concentrations sufficient to activate excitotoxicity-related pathways (Swann et al., 2000). However, such injury -related responses might not be expected to result in filopodial hyper-stabilization, but rather in an overall decrease in filopodial lifetimes. Further, seizures did not eliminate ongoing growth processes, since new branches and filopodia continue to be added during seizures, even after extended periods of seizure activity.   Alternatively, excessive synchronous excitatory neurotransmission during seizures may activate endogenous, competition-based programs of synaptic plasticity which underlie synaptotropic stabilization and elimination of nascent - 146 -  dendritic processes. Mounting evidence supports the synaptotropic model of dendritogenesis in which synapse formation and maturation, driven by activity-dependent competition between afferent inputs, confers morphologic stabilization to newly extended labile dendritic filopodia, while synapse loss is associated with process retraction (Vaughn et al., 1988; Niell et al., 2004; Cline and Haas, 2008). Indeed, experimental manipulations in individual X enopus  tadpole optic tectal neurons which promote maturation of synapses induce dendritic morphological stabilization (Wu and Cline, 1998), while those that reduce synapse maturation result in filopodial destabilization (Haas et al., 2006). Here, we propose that strong seizure-associated afferent input out-competes endogenous circuit activity, leading to synapse weakening and the morphological destabilization and elimination of dendritic process which were integrated into endogenous circuits. Existing or new synapses involved in circuits transmitting seizure activity, however, would be expected to strengthen, resulting in stabilization of the dendritic process within which they reside. This model is supported by evidence linking increases in seizure susceptibility following neonatal seizures to AMPA-receptor-mediated synaptic potentiation ( Rakhade et al., 2008), further implicating our observations of altered dendritic structure with negative sequelae of early-life seizures. Conducting comprehensive morphometric analysis of dynamic growth behaviour is necessary to detect and decipher the subtle, yet functionally significant changes to neural circuit structural formation caused by developmental - 147 -  seizures. Our results demonstrate two distinct consequences of early -life seizures on circuit function. First, the rapid destabilization of existing labile dendritic structures observed within minutes of seizure onset indicates a loss of morphologic processes formed and stabilized by pre-seizure endogenous activity. Seizure-induced loss of dendritic processes and associated synapses involved in normal circuit function may result in deficits in processing of endogenous activity. Secondly, seizures promote the hyper-stabilization of dendritic processes that arise during seizures, presumably by the stabilization and strengthening of synapses contained within these structures. The persistence of such structures and their constituent synapses suggests that seizures may hardwire seizure-associated circuits. These abnormal neural circuit structures may be more prone to support future seizure activity and/or under lie abnormal network activity. Thus, we find evidence for morphological substrates potentially underlying deficits in normal circuit function, and the possible origin of subsequent neuropathology later in life. While dendritic abnormalities have long been observed in patients with epilepsy (Wong, 2008), it has remained unclear whether these changes are directly induced by seizures. 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Pediatr Neurol 40:175 -180. Vaughn JE, Barber RP, Sims TJ (1988) Dendritic development and preferential growth into synaptogenic fields: a quantitative study of Golgi-impregnated spinal motor neurons. Synapse 2:69-78. Vestergaard M, Pedersen CB, Sidenius P, Olsen J, Christensen J (2007) The long-term risk of epilepsy after febrile seizures in susceptible subgroups. Am J Epidemiol 165:911-918. Vesterg aard M, Pedersen CB, Christensen J, Madsen KM, Olsen J, Mortensen PB (2005) Febrile seizures and risk of schizophrenia. Schizophr Res 73:343 -349. Wong M (2008) Stabilizing dendritic structure as a novel therapeutic approach for epilepsy. Expert Rev Neuroth er 8:907-915. Wong RO, Ghosh A (2002) Activity -dependent regulation of dendritic growth and patterning. Nat Rev Neurosci 3:803 -812. Wu GY, Cline HT (1998) Stabilization of dendritic arbor structure in vivo by CaMKII. Science 279:222 -226. Wu GY, Zou DJ, Raj an I, Cline H (1999) Dendritic dynamics in vivo change during neuronal maturation. J Neurosci 19:4472 -4483.           - 152 -  5.        GENERAL DISCUSSION  5.1 Summar y of findings  It remains unclear whether and how early-life seizures affect the structural development of neural circuits, possibly contributing to the observed long-term functional consequences of developmental seizures. The overall aim of the present work, therefore, was to examine the effects of early-life seizures on a critical aspect of neural circuit development?dendritogenesis. In order to comprehensively characterize both the dynamic and persistent effects of seizures on the dendritic growth, we developed an experimental model system which allows direct in vivo imaging of dendritic growth before, during and after seizures, at both short and long intervals, within the intact and awake brain. Therefore, the initial experiments of the present work focussed on the characterization of a novel experimental model of developmental seizures based on the transparent albino X enopus laevis  tadpole. Pentylenetetrazol (PTZ), kainic acid, bicuculline, picrotoxin, 4 -aminopyridine, and pilocarpine were tested for their ability to induce behavioural seizures in freely swimming tadpoles when bath applied. All six chemoconvulsants consistently induced similar patterns of abnormal behaviour in a dose-dependent manner, characterized by convulsive clonus-like motor patterns with periods of behavioural arrest. Extracellular electrophysiological field CHAPTER 5 - 153 -  recordings demonstrated rhythmic synchronous epileptiform electrographic responses induced by convulsants irrespective of mechanism of action that could be terminated by the anti-epileptic drug valproate. PTZ -induced seizures were further characterized using in vivo two-photon fluorescence imaging of neuronal calcium dynamics, within the intact and unanaesthetized tadpole brain. Imaging of calcium dynamics during PTZ -induced seizures revealed waves of neural activity propagating through large populations of neurons within the brain. Analysis of single-cell and network ensemble responses demonstrated distinct synchronized high-amplitude calcium spikes not observed under baseline conditions. Further, prolonged seizures failed to induce marked neuronal death within the brain, detected by cellular propidium iodide incorporation in vivo or TUNEL labelling.  This model system has distinct advantages for controlled seizure induction, and direct visualization of both seizure activity and seizure-induced effects on individual developing neurons within the intact unanaesthetized brain, d u ring seizu res . Notably, the fundamental characteristics of induced seizures in the X enopus laevis  tadpole were found to be remarkably similar to those seen in commonly utilized mammalian developmental seizure models, and we therefore chose to use this model system to examine the effects of early-life seizures on dendritogenesis.  In vivo  two-photon fluorescence imaging of individual developing tadpole optic tectal neurons revealed that seizures inhibit overall dendritic arbor growth due to decreased branch elongation, increased branch elimination, and loss of - 154 -  dendritic filopodia. These effects were found to be cell -autonomously mediated by excessive AMPA-receptor mediated excitatory activity. Rapid, 5 minute interval time-lapse imaging over 5 hours, followed by comprehensive 3-dimensional analysis of the growth behaviour of all dendritic filopodia and branches present on developing dendritic arbors found that seizures induce complex effects on dynamic growth. Seizures promoted rapid destabilization and retraction of filopodia generated prior to seizure onset, and conversely hyper-stabilized those generated during seizure episodes, demonstrated by reduced filopodial motility and prolonged lifetimes. Longer -interval imaging over hours to days revealed that a single seizure episode persistently stunted subsequent arbor growth, indicating that short-term growth changes have lasting effects on overall arbor growth patterns. Seizure-induced stunting of dendritogenesis was also associated with a loss of excitatory synapses, evident from a reduced density of immuno-stained excitatory synaptic markers within the tectal neuropil.  This study is the first to examine the effects of developmental seizures on dendritogenesis within the intact and awake developing brain. Importantly, the use of a powerful new in vivo experimental seizure model system allowed the identification morphological substrates potentially underlying persistent neural circuit dysfunction commonly associated with early-life seizures.  - 155 -  5.2 The albino Xenopus laevis tadpole as a novel model of developmental seizur es   5.2.1 Is the Xenopus laevis tadpol e a valid seizure model?  ,QFRQVLGHULQJWKH?YDOLGLW\?RIH[SHULPHQWDOPRGHOVRIVHL]XUHVDQGRUepilepsy, numerous factors are often considered. Perhaps the most critical requirement of a seizure model is the observation of electrographic seizures ? periods of neural hyperexcitability marked most often by repetitive spikes or spike-wave activity corresponding to synchronized neuronal discharge (Penfield and Jasper, 1954; Burnham, 2002) . Each of the six chemoconvulsants tested elicited repetitive complex spiking patterns, recorded as population responses, with characteristics remarkably similar to chemoconvulsant-induced seizures in mammals (Velisek et al., 1995) . Imaging of intracellular calcium dynamics in vivo revealed repetitive waves of synchronized activity propagating across a large population of neurons during seizures, further supporting our electrophysiological observations. As expected, induced seizures appear to be generalized, similar to seizures induced by systemic administration of chemoconvulsants in other in vivo experimental preparations (Pitk?nen et al., 2006) .  Another defining feature of seizures is the associated behavioural manifestation. The range of seizure -related behaviours in experimental animal models is not expected to fully reproduce human behavioural phenotypes, - 156 -  however, several basic unifying characteristics are often observed. Generalized convulsive seizures are most often associated with tonic and/or clonic movements (Pitk?nen et al., 2006) . At sufficiently high concentrations, each of the convulsants tested reliably elicited similar patterns of repetitive stereotyped convulsive behaviour with clear tonic and clonic components. Additionally, the progressive stages of seizure-related behaviours observed is similar to progressive seizure stages described in other seizure model systems (Racine, 1972; Baraban et al., 2005) , corresponding to the propagation of seizure activity to brain regions controlling different motor patterns (Hewapathirane and Burnham, 2005) .  Another key element in validation of experimental seizure models is the demonstrated responsiveness to anticonvulsant drugs. PTZ -induced seizures in tadpoles were suppressed by a variety of common anticonvulsants, including valproate, diazepam, and phenytoin, consistent with observations made in several mammalian models of generalized convulsive seizures. Notably, these drugs have been widely used clinically to control generalized convulsive seizures as well as convulsive status epilepticus (Burnham, 1998) . Based on these collective observations, we propose that chemoconvulsant-induced seizures in the X enopus laevis  tadpole constitutes a valid experimental model of acute generalized convulsive seizures within the immature brain.  - 157 -  5.2.2 Advanta ges and l imitations  The number of experimental model systems of seizures and epilepsy h as grown exponentially, paralleling the development of novel experimental techniques allowing more sophisticated examination of seizures and their consequences. The albino Xenopus laevis  tadpole presents several distinct advantages which are of significant benefit to the study of developmental seizures and the effects of such seizures on brain structure and function.  An important quality of this system is the ability to record neuronal activity from the intact developing brain under unanaesthetized conditions during seizures, circumventing numerous confounding effects of anaesthetic agents on neuronal function (Ishizawa, 2007) . Since the albino tadpole is transparent, this system is well suited for the examination of seizure activity in hundreds of neurons with single-cell resolution, using calcium-sensitive fluorescent indicators (Dunfield and Haas, 2009) . Use of this technique enables, to our knowledge for the first time, the study of seizure initiation and propagation through developing neuronal circuits within the intact unanaesthetized brain. Potential applications of this technique in this system also extend to addressing long standing questions related to the immediate- and long-term effects of seizures on developing neural circuits and network function.  Additionally, tadpoles are ideally suited for large-scale experiments since they are easily reared, are highly cost- effective, may be housed in small volumes of solution, and seizures can be reliably induced and/or inhibited by bath application of relatively small amounts of drug.  - 158 -  A valuable new seizure model based on the larval zebrafish Danio rerio  has recently been developed (Baraban et al., 2005) , which shares several of the previously discussed advantages of the tadpole model. Zebrafish also provide the additional advantage of genetic tractability and refined genetic tools (Baraban, 2007) . However, specialized techniques for acute and targeted genetic manipulation have been developed specifically for X enopus  tadpoles (Haas et al., 2002; Hewapathirane and Haas, 2008) . Further, X enopus laevis  tadpoles undergo a more protracted developmental period compared to zebrafish (Nieuwkoop and Faber, 1994) , making this organism particularly useful in studies of neuronal growth and network formation during development. T he tadpole model, at the present state of development, is a model of acute seizures, and is not a model of epilepsy or of epileptogenesis. To this end, it will be interesting to determine whether tadpoles can be chemically kindled, or if seizures are facilitated, or spontaneously generated, following one or more episodes of sta tu s epilep ticus . Further, systemic chemoconvulsant application is not suited for studies of partial seizures or seizures triggered from specified sites within the brain. Future work will be required to determine whether partial seizures can be triggered, for instance, by direct drug injection into specifi c brain regions. Finally, the high-level organization of the amphibian brain shows some differences with that of mammals. Therefore the study of seizure effects on specific brain regions subserving higher cognitive functions, or on defined neural circuits, is limited to those that are common between the amphibian and mammalian brain in - 159 -  order for findings to be of relevance to the human condition. Notably, however, the mammalian optic tectum (th e superior colliculus) has been implicated in the initiation of some seizure types in mammals (Garcia -Cairasco et al., 1993) . In recognizing the limitations of this tadpole seizure model, it is important to note that several exceptionally useful models of seizures and epilepsy already exist (Pitk?nen et al., 2006) , which are better suited to address these specific issues.  5.2.3 Significance  The high degree of similarity between the fundamental neurobiology of X enopus  with higher vertebrates, together with distinct experimental and practical advantages, have established the albino tadpole as an important in vivo model of early brain development. The X enopus laevis  tadpole has been widely used in studies of activity-dependent neuronal growth, synaptogenesis, neuronal plasticity and circuit formation?issues of particular importance with respect to epilepsy research. Use of this newly developed seizure model system in epilepsy research holds much promise to address long-standing issues related to the effects of seizures on brain development (Holmes, 2008; Hewapathirane and Haas, 2009) , and by complementing and contributing to work conducted in mammalian model systems, may prove useful in the development of novel therapeutic strategies to suppress seizures and their adverse effects on brain function.  - 160 -  5.3 Earl y -life seizur es inter fer e with neur onal dendritogenesis   Overall, we find that early -life seizures inhibit normal patterns of dendrite growth, and that extended seizure durations result in dendritic arbor atrophy. These findings co nfirm a recent in vitro  study using cultures mouse hippocampal VOLFHVZKHUHSURORQJHGFKHPRFRQYXOVDQWH[SRVXUH?KRXUVZDVDVVRFLDWHGwith significantly retarded dendritic growth (Nishimura et al., 2008) . In addition to conducting the present experiments in vivo, and moreover within the intact and awake developing brain, our findings further extend earlier observations by describing seizure-induced alterations to dynamic dendritic growth events. We also demonstrate that a single seizure episode persistently stunts subsequent arbor elaboration and induces a persistent reduction of excitatory synapses.  In human epilepsy, reductions in dendritic spine number and the appearance of dendritic varicosities have been frequently reported; however, reduced dendritic branching complexity has also been documented (Scheibel et al., 1974; Multani et al., 1994) . While these studies examined mature neurons within resected human epileptic brain tissue, whether these abnormalities stem from disrupted dendritogenesis induced by early-life seizures is an interesting possibility raised by our findings.  Our results also implicate excessive AMPA receptor -mediated neurotransmission as a critical mediator of seizure-induced effects on dendritic growth. We propose that the observed seizure-induced effects are primarily caused by excessive glutamatergic neurotransmission, and not significantly - 161 -  influenced by tissue-wide or systemic factors. This assertion is supported by findings that NMDA receptor blockade protects from seizure -induced retardation of dendritic growth in vitro  (Nishimura et al., 2008) , given that AMPA receptor-mediated membrane depolarization facilitates NMDA receptor activation.  Further work is necessary to elucidate the specific molecular mechanisms underlying the observed alterations to dendrite growth. Results from such work will shed light on whether excessive glutamatergic neurotransmission disrupt dendritogenesis through injury -related cellular processes and/or the disruption of normal developmental programs.   5.3.1 A model rel ating seizure -induce d neu ral circuit d ys fun ction to altered dendritogen esis  As discussed earlier, substantial evidence suggests that early-life seizures induce persistent functional deficits and an increased susceptibility to further seizures.  Based on our findings we propose a model linking altered dendritogenesis to these functional outcomes. The proposed model is based on the suggestion that excessive neuronal activity during seizures disrupts endogenous, competition-based programs of synaptic plasticity (Swann et al., 2000)  which underlie synaptotropic stabilization and elimination of nascent dendritic processes (Vaughn et al., 1988; Cline and Haas, 2008) .  Based on the synaptotropic theory of dendritogenesis and canonical Hebbian competition-based rules of synapse maintenance and elimination (Penn - 162 -  and Shatz, 1999; Sanes and Lichtman, 1999) , synapses?and the dendritic structures within which they reside?will be stabilized and maintained if these connections transmit strong, correlated, physiologically-relevant afferent input. Non -coincident or weak synaptic connections are weakened and eventually eliminated.   We find that rapidly upon seizure onset, a significant proportion of pre-existing dendritic filopodia are eliminated, and over longer intervals existing dendritic branches also begin to retract. We hypothesize that this structural remodelling reflects excessive seizure-related neurotransmission out-competing endogenous circuit activity, resulting in the weakening and elimination of synapses subserving physiological functions. In this manner, seizures would be expected to induce deficits in endogenous circuit connectivity, leading to functional deficits. Additionally, reduced arbor size and complexity, together with a loss of excitatory synapses would also be expected to compromise normal circuit connectivity and function.  Our results also indicate that seizures hyper -stabilize both filopodia generated during seizures and a proportion of pre-existing dendritic filopodia. Within the proposed framework, filopodial hyper-stabilization would indicate that their constituent synapses were recruited into ictal circuitry and that strong, seizure-related neural activity selectively strengthened and stabilized these synaptic connections. The selective maintenance of synaptic connections that are DFWLYHGXULQJVHL]XUHVZRXOGEHH[SHFWHGWRHVWDEOLVK?VHL]XUHFLUFXLWV?RUFLUFXLWV- 163 -  that are optimized to transmit seizure-like neural activity. Therefore, this model offers one explanation as to how dendritic spine loss or arbor atrophy may act to both facilitate hyperexcitability and produce deficits in normal function. If such reorganization of neural circuitry occurs during critical periods of network plasticity, it is conceivable that early-life seizures may induce persistent functional deficits and an increased susceptibility to further seizures through such mechanisms.   5.3.2 Additiona l fact ors potentiall y cont ributing to circuit d ys fun ction  While the focus of the present work was to examine the effects of seizures on dendritogenesis, numerous other factors may also contribute to altered connectivity leading to long-term circuit dysfunction. These include, but are not limited to: (i) aberrant axonal sprouting; (ii) altered receptor/ ion channel expression and cellular distribution; (iii) altered neurogenesis; (iv) delayed maturation of GABA responses from depolarizing to hyperpolarizing; (v) altered levels of neurotrophic factors and their receptors; (vi) inflammatory responses; (vii) ne uronal death; and (viii) gliosis (Lado et al., 2000; Holmes, 2004; Holopainen, 2008; Rakhade and Jensen, 2009) . Additional work is necessary to determine to what extent dendritic abnormalities contribute to seizure-induced neural circuit dysfunction, and whether seizure-induced dendritic remodelling is related to these factors.  - 164 -  5.3.3 Significance  This work addresses a controversial and clinically important question of whether seizures are damaging to the immature brain. Our results show that early-life seizures acutely and persistently retard normal dendrite growth patterns in vivo, concomitant with a loss of excitatory synapses?indicating that seizures sustained early in life can induce persistent alterations to neural circuit structure, and likely function. Using custom designed comprehensive 3D analysis tools we find that seizures differentially influence the stability of specific populations of dendritic processes. Overall, our results predict a deficit of normal neural circuit function and persistent abnormal dysfunction potentially leading to further seizures or other neurological disorders. The findings of this work are of significance since they provide morphological evidence for persistent cognitive and neurological dysfunction commonly observed in human patients who have experienced early-life seizures.  - 165 -  5.4 Future directions   5.4.1 Direct extensions of presen t w ork  Interesting experimental questions extending from the present work include: (i) do brief seizur es (individual or repeated), or seizures induced by different means (e.g. electrical stimulation or hyperthermia) also alter dendritogenesis? (ii) does dendrite stunting persist for longer periods of time? (iii) are neurons at specific maturational states more prone to seizure-induced effects?   5.4.2 Examining the molecular mecha nisms underl ying seizure -induced effect s on dendritog enesis  An important focus of future work will be to determine the specific molecular mechanisms downstream of AMPA receptor activation, which result in altered dendrite growth. It is conceivable that seizure-related excessive neural activity, transmitted via AMPA receptors, may over-activate endogenous molecular pathways that govern normal dendritic growth, or alter signals that maintain/disrupt dendritic structural integrity. A number of activity -dependent intracellular signals have been shown to regulate dendritic growth, including:  (i) calcium/calmodulin -dependent protein kinase II (Wu an d Cline, 1998; Zou and - 166 -  Cline, 1999) ; (ii) protein kinase M zeta (Liu et al., 2009) ; (iii) molecular motors and microtubule-associated proteins (Matus, 1988; Sharp et al., 1997) ; (iv) Rho-family GTP -ases (Sin et al., 2002; Van Aelst and Cline, 2004; Benarroch, 2007) ; (v) caspases (Williams et al., 2006) ; and (vi) numerous genes regulating specific aspects of dendritic growth and patterning, many of which are expressed in an activity-dependent manner (Ne divi et al., 1998; Gao et al., 1999; Jan and Jan, 2003) .  Targeted experimental approaches such as single -cell electroporation to selectively alter signalling within individual neurons, will be invaluable for future work aimed at dissecting the roles of these and other signalling molecules in the regulation of seizure-induced effects on neuronal growth.   5.4.3 Examining the effects of developmenta l seizure s on s ynapto gene sis   It is widely recognized that aberrant synapse formation or alterations in normal patterns of synapse pruning and/or stabilization can lead to the formation of abnormal neural circuits (Katz and Shatz, 1996) 7KH?V\QDSWRWURSLFWKHRU\?RIdendritogenesis suggests a direct correlation between dendrite growth and retraction and synaptic formation and loss, respectively (Vaughn et al., 1988; Niell et al., 2004) . Based on our findings, it would therefore be expected that seizures will directly interfere with synapse formation and maintenance, and such changes will be persistent. Thus, an important direction for future research will be to - 167 -  examine the effects of developmental seizures on synapse dynamics, using the X enopus  tadpole seizure model.  For these experiments, single-cell electroporation may be used to co-express a fluorescently-tagged postsynaptic marker (e.g. PSD -95) along with a soluble fluorophore of a different colour to visualize neuronal morphology (see Fig. 3.5).  Labelled neurons imaged in vivo, before and during PTZ -induced seizures, would allow measures such as changes in the rates of synapse formation and elimination, synapse lifetimes, and fluorescence intensities (an indirect assay of stability) to be acquired. Results would determine whether pre-existing synapses are lost during seizures in vivo, and whether the formation and/or stabilization of new synapses during seizures differs from basal levels.  Since the formation and selective maintenance of synapses is critical mediator of correct neural circuit formation and function, such studies will shed light on a potential mechanism through which early-life seizures induce persistent neural dysfunction. Furthermore, results from these experiments will complement the present studies on dendritogenesis, potentially providing a synaptogenic mechanism for seizure-induced effects on dendrite growth.  5.4.4 Examining the eff ects of developm ental seizures on neura l circuit matura tion, function, and pl asticity  Results from previous work indicate that seizures alter basal neural activity and modify plasticity of neural circuits (McNamara, 1994; Sutula et al., 2000; - 168 -  Holmes and Ben-Ari, 2001; Sutula, 2004; Swann, 2004) . Building on the present work, an important direction for future research will be to investigate precisely how seizures affect circuit properties and plasticity in the developing brain during critical periods of network formation.  In vivo  imaging of neural calcium responses in the X enopus  tadpole retino-tectal system would be ideally suited for such experiments. This system offers several advantages over commonly used electrophysiological approaches, including the ability to conduct network-level examinations with single-cell resolution, within an intact and awake organism. Additionally, the tadpole retino-tectal system enables examination of tectal circuit activity and plasticity in response to physiologically-relevant input, through the presentation of visual stimulation while simultaneously recording neural calcium responses from optic tectum (Dunfield and Haas, 2009) . Since the retino-tectal system of the tadpole is continuously undergoing neural activity-dependent circuit development, indicated by activity-dependent receptive field refinement (Gaze et al., 1974; Sakaguchi and Murphey, 1985; Zhang et al., 1998; Tao et al., 2001) , this system allows direct examinations of whether seizures interfere with the refinement of neuronal response properties, or result in altered neural plasticity, during sensitive periods of neural circuit formation and refinement.  Results from such studies will provide direct evidence of how seizures impact brain circuit development and plasticity, setting the stage for subsequent studies to elucidate the molecular, structural and synaptic mechanisms through - 169 -  which these changes arise. An exciting additional dimension for subsequent experiments would be to utilize newly developed optic tectum-dependent visual-cue based avoidance learning assays in freely-swimming tadpoles (Dong et al., 2009) . Such work will provide important insight into how observed tectal circuit dysfunction manifests as altered behavioural patterns, having implications for higher-order functional deficits seen in human epilepsy.  - 170 -  5.5 Overall concl usi ons  The Xenopus  tadpole seizure model is a powerful new experimental system, conferring the ability to directly image neuronal structure and circuit function within the intact and awake brain, while seizures are ongoing. The present research represents the first in vivo examination of the effects of seizures on dendrite growth during brain development, and our findings demonstrate that seizures directly interfere with normal patterns of dendritogenesis. Future directions include examination of the molecular and synaptic mechanisms underlying circuit dysfunction, as well as examining how alterations to circuit activity and plasticity affect behaviour. This model system is poised to allow clear structure-function relationships to be established, providing insights into the mechanisms underlying seizure-induced neural dysfunction.  The impact of early -life seizures on neural circuit formation and function is poorly understood, and the results from the present experiments together with subsequent work will further our understanding on this important issue. The long -term application of this work will be in the development of novel neuroprotective strategies to counter seizure-induced neural dysfunction in clinical populations. - 171 -  5.6 References  Baraban SC (2007) Emerging epilepsy models: insights from mice, flies, worms and fish. Curr Opin Neurol 20:164 -168. Baraban SC, Taylor MR, Castro PA, Baier H (2005) Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 131:759-768. 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J Neurosci 19:8909 -8918.                 - 176 -  APPENDIX:  ANIMAL CARE CERTIFICATES  APPENDIX April 17, 2009 - 177 -   - 178 -   - 179 -                            

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