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The ontogeny and functional distribution of novel, neurochemically-defined columns in mammalian visual… Dyck, Richard H. 1993

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THE ONTOGENY AND FUNCTIONAL DISTRIBUTION OF NOVEL,NEUROCHEMICALLY-DEFINED COLUMNS IN MAMMALIAN VISUAL CORTEXbyRICHARD HENRY DYCKB.Sc., The University of Lethbridge, 1981M.Sc., The University of British Columbia, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESNEUROSCIENCEWe accept this thesis as conforming to the required standardTHE UNIVERSITY OF BRITISH COLUMBIAMarch 1993© Richard Henry Dyck, 1993In presenting this thesis in partial fulfillment of the requirement for an advanced degree at TheUniversity of British Columbia, I agree that the Library shall make it freely available forreference and study. I further agree that permission for extensive copying of this thesis forscholarly purposes may be granted by the Head of my Department or by his or herrepresentatives. It is understood that copying or publication of this thesis for financial gain shallnot be allowed without my written permission.Graduate Program in NeuroscienceThe University of British Columbia2075 Wesbrook PlaceVancouver, CanadaV6T 1Z4Date: 9 March 1993ABSTRACTThe synaptic organization of the mammalian visual cortex is susceptible to activity- andexperience-dependent modifications during a distinct temporal window of development. In aneffort to understand the synaptic mechanisms responsible for initiating and maintaining this periodof plasticity, the studies presented in this thesis describe the ontogenic distribution of specificmolecular indices of serotonergic and glutamatergic neurotransmission in the developing kittenvisual cortex and evaluate their patterns of expression following manipulations of early visualexperience.The distributions of serotonin (5-HT) 1A, 1C, 2 and 3 receptor subtypes and the 5-HT uptakesite were assessed in postnatal cat visual cortex using in vitro autoradiographic methods. Each5-HT receptor subtype exhibited a unique temporal, regional and laminar pattern of expression.The density of 5-HTiA receptors was highest within superficial and deep cortical layers aroundpostnatal day (PD) 30, while 5-HTic and 5-HT2 receptors exhibited peak levels in middle corticallayers at PD50 and PD120, respectively. Between PD30 and PD90, 5-HTic & 5-HT2 receptorswere expressed at high levels within the same columnar compartments, but within differentgeniculate-recipient layers of area 17. These columns of 5-HT receptors measured about 400 [t,mwide with a centre-to-centre spacing of approximately 900 Inn.The ontogenic distribution of synaptic zinc (Zn), a vesicular component of a subset ofglutamatergic-neuron terminals, was also examined in the cat visual cortex. In the adult, intenseZn-staining was highest in superficial and deep layers. The relative absence of Zn in layer IVconspicuously distinguished visual cortical areas 17 and 18 from adjacent cortical regions. Theearliest Zn-positive staining in visual cortex was apparent by PD2 and was restricted to a thin layerat the bottom of the cortical plate. By PD 10, and continuing through PD20, synaptic zinc formed atrilaminar pattern of dense staining in areas 17 and 18, which included the top of layer I, and layersIII and V. The laminar pattern of synaptic zinc in visual cortex appeared mature by PD30, exceptthat the distribution of zinc in layer IV was not uniform. When examined in the tangential plane ati iPD50, Zn-rich patches were found to demarcate columnar compartments in layer IV of area 17.The columnar expression of Zn exhibited similar temporal and spatial characteristics as 5-HT1c/2receptor columns. When compared in adjacent sections at PD50, columns demarcated by 5-HTic/2receptors and Zn were found to be precisely coaligned.The input- and activity-dependence of the organization of Zn / 5-HT receptor columns wasaddressed by constraining cortical visual experience. Lesions produced in early development,which interfered with normal binocular input to visual cortex, had similar detrimental effects on thecolumnar distributions of 5-HT 1 ci 2 receptors and Zn, and indicated that the columnarcompartmentalization of these molecules was dependent on normal visual input and activity. Zn /5-HT receptor-rich columns were found to be precisely complementary to columns rich incytochrome oxidase (CO) and acetylcholinesterase (AChE), molecules which are known todescribe functional columnar compartments in primate visual cortex. Zn and CO were also found todescribe an interdigitated columnar mosaic in primate visual cortex, suggesting that these moleculesmight demarcate functionally homologous columnar compartments in the visual cortex of thesephylogenetically distinct species.The results of these experiments suggest that serotonergic and zinc-containing glutamatergicprocesses may play important roles in the postnatal development of mammalian visual cortex,particularly with regard to mechanisms of compartmentalization into functional columnar domainsiiiTABLE OF CONTENTSAbstract^ iiTable of Contents ivList of Tables vList of Figures^ viAcknowledgement ixForward x1^General Introduction^ 12 Ontogenic Expression of Serotonin Receptors in the Cat Visual Cortex^ 15Materials and Methods 16Results^ 20Discussion 55Conclusions 673 Ontogenic Distribution of Synaptic Zinc in The Cat Visual Cortex^68Materials and Methods^ 69Results^ 73Discussion 954 Activity-Dependent Columnar Expression of Serotonin Receptors and Zinc^ 101Materials and Methods^ 105Results^ 107Discussion 1285 Multiple Markers of a Columnar Mosaic in Visual Cortex of Cats and Primates 136Materials and Methods^ 137Results and Discussion 1416 General Discussion^ 160Summary 160Discussion 165Methodological Considerations^ 165Functional Considerations^ 169Conclusion^ 1777 References^ 178ivLIST OF TABLESTable 1. Summary of ligand binding procedures^  18Table 2. Summary of kittens used to assess input - and experience-dependent lesion effects ^ 106vLIST OF FIGURESFigure 1.1 The retinogeniculocortical pathway labeled transneuronally at PD50 in a normallyreared and a neonatally enucleated kitten^ 4Figure 2.1a The ontogenic expression of 5-HTiA, 5-HTic, 5-HT2 receptors in visual cortexbetween birth and PD40^ 23Figure 2.1b The ontogenic expression of 5-HTiA, 5-HTic, 5-HT2 receptors in visual cortexbetween PD75 and adult^ 24Figure 2.2 The ontogenic distribution of 5-HT1 A, 5-HT] . c, 5-HT2 receptors in lateralsuprasylvian cortex^ 26Figure 2.3 Ontogeny of 5-HT uptake sites in the cat visual cortex labeled with[ 3 H]cyanoimipramine^ 28Figure 2.4 Developmental changes in the expression of 5-HTiA, 5-HTic, 5-HT2 receptors and5-HTup in kitten visual cortex collapsed across regions and laminae^30Figure 2.5 Regional comparisons between 5-HTiA, 5-HTic, 5-HT2 receptors and the 5-HTUpsite in developing cat visual cortex, collapsed across laminae^33Figure 2.6 Bar graphs showing quantitative comparisons of the ontogeny of laminar and regionallevels of 5-HTiA, 5-HTic, 5-HT2, receptors and the 5-HTUp site^35Figure 2.7 Regional and laminar distributions of 5-HTiA, 5-HTic and 5-HT2 receptors in kittenvisual cortex at birth^ 42Figure 2.8 Complementary laminar distributions of 5-HTiA, 5-HTic and 5-HT2 receptorsduring postnatal development^ 44Figure 2.9 Comparison of the laminar and columnar distributions of 5-HTiA, 5-HTic, 5-HT2,receptors and the 5-HTUp site in PD50 visual cortex at higher magnification^46Figure 2.10 The complementary regional and laminar distributions of 5-HTLA, 5-HTiu and5-HT2 receptor subtypes in PD50 kitten visual cortex^50viFigure 2.11 Comparison of the overall distribution of 5-HTic and 5-HT2 receptors in parasagittalsections through PD50 kitten visual cortex^ 52Figure 2.12 Tangential distribution of 5-H1'1c-specific columns in PD50 kitten visual cortex . ^ 54Figure 3.1Figure 3.2Figure 3.3Figure 3.4Figure 3.5Figure 3.6Figure 3.7Figure 3.8Figure 4.1Synaptic distribution of vesicular zinc in kitten visual cortex^76The regional distribution of zinc in adult cat cortex^ 81The laminar distribution of zinc in area 17, 18 and 19 of adult visual cortex^83Zinc in the kitten visual cortex at birth^ 85The laminar distribution of zinc in area 17 of young kittens at high magnification^87Ontogeny of the laminar-specific distribution of zinc in kitten visual cortex^89The patchy distribution of zinc in a coronal section through area 17 at PD50 ^92The tangential distribution of zinc in PD50 and adult visual cortex^94Schematic overview of the procedures used for deprivation of visual input ^ 104Figure 4.2 Comparison of the effects of long- and short-term monocular deprivation withbinocular deprivation on the expression of 5-HTic receptors at PD50^ 112Figure 4.3 The effects of unilateral enucleation on the distribution of 5-HT1A, 5-HT lc, and5-HT2 receptors and zinc in adjacent frontal sections^ 114Figure 4.4 The effect sof unilateral enucleation on tangential distribution of 5-HTic receptorsand zinc in the visual cortex of kitten BK369^ 116Figure 4.5 The effects of unilateral enucleation on tangential distribution of 5-HTic receptorsand zinc in the visual cortex of kitten BK370^ 118Figure 4.6 The effects of dark-rearing on the expression of 5-HT A, 5-HT c and 5-HT2receptor subtypes^ 120Figure 4.7 The effects of unilateral optic tract transection on the columnar expression of 5-HTicreceptors and zinc^ 122Figure 4.8 The effects of tetrodotoxin- and lesion-induced inactivation of the lateral geniculatenucleus on the expression of 5-HTic and 5-HT2 receptors^ 125viiFigure 4.9 The effect of quinolinic acid-induced lesions on the expression of 5-HT 1A, 5-HriC,5-HT2 receptor subtypes and 5-HTuptake in area 17^ 127Figure 5.1 Coronal and tangential distributions of 5-HTic receptors, zinc, cytochrome oxidaseand acetylcholinesterase compared in PD50 kitten visual cortex^ 144Figure 5.2 The vertical alignment of zinc, 5-HTic and 5-HT2 columns at high magnification, inPD50 kitten visual cortex area 17^ 146Figure 5.3 Tangential distributions of zinc, acetylcholinesterase and cytochrome oxidase inserially adjacent sections from 3 different strata within visual cortex^ 148Figure 5.4 Complementary patchy distributions of zinc and acetylcholinesterase in adjacenttangential sections in kitten visual cortex^ 150Figure 5.5 The complementarity laminar and columnar distributions of cytochrome oxidase andzinc in kitten visual cortex^ 152Figure 5.6 The complementary laminar and columnar distributions of zinc and cytochromeoxidase in the primate striate cortex^ 155Figure 5.7 Overall distribution of ocular dominance columns and patchy zinc-staining in kittenvisual cortex^ 157Figure 5.8 The relationship between zinc-rich columns and ocular dominance columns in kittenvisual cortex^ 159Figure 6.1 Summary schematic illustrating the development of the neurochemically definedcolumnar mosaic in cat visual cortex^ 162Figure 6.2 Summary schematic illustrating the effect of input-deprivation on the columnardistribution of 5-HT receptors in PD50 kitten visual cortex^ 164viaACKNOWLEDGEMENTI am deeply indebted to many individuals for their contribution to the work which isrepresented by this manuscript.First and foremost, I thank each member of my family for their encouragement and support.I thank Max Cynader, whose provision of support and insight was exceeded only by hisdesire to behold revolutionary discoveries; and Rob Douglas, who was always around to provideassistance and advice.The adept assistance by Kate Anderchek, Virginia Booth, Barry Gibbs, Elaine Hutchinson,Bill Kiss and Jirin Tan was graciously provided, and gratefully accepted.Among all the individuals with whom I have had the pleasure to work with during my tenurein this lab, my collegial and scholarly interactions with Clermont Beaulieu, James Boyd, AviChaudhuri, Paul Finlayson, Debbie Giaschi, Qiang Gu, Franco Lepore, Yu Lin Liu, Glen Prusky,Stuart Marlin, Joanne Matsubara, Donald Mitchell, Nick Swindale and Gavin Thurston have beenextremely rewarding.The research presented here was supported by a postgraduate fellowship from the NationalSciences and Engineering Council and research grants from the Medical Research Council and theNetworks of Centres for Excellence of Canada.Dedicated to Janine and Matthew, whose invaluable contribution to this thesis,provided by their unconditional love and support, I could not possibly, fully repay.ixFORWARDThe experiments described in Chapter 2 have been accepted for publication in TheJournal of Neuroscience (1993).The authors, in order, are:Richard Dyck and Max Cynader.The results described in Chapter 3 have been published in The Journal of ComparativeNeurology (1993) 329: 53-67.The authors, in order, are:Richard Dyck, Clermont Beaulieu, and Max Cynader.The contribution of each author to this publication is:Beaulieu:^Electron microscopy represented by Figure 3.1Cynader:^Provision of laboratory, supplies and insight.Dyck:^The remainder.x1,When you have eliminated the impossible,whatever remains, however improbable,must be the truth.Sir Arthur Conan Doyle (1890)1GENERAL INTRODUCTIONThe development of the nervous system is characterized by an intricate balance of geneticand epigenetic phenomena. The culmination of an early progression of developmental processes,which include cellular proliferation, differentiation, migration, process outgrowth and cell death,is the establishment of selective neural connections which form the basis for informationprocessing in the nervous system. The precision with which synaptic connections are formed isnot prespecified, but gradually arises from a diffuse innervation which becomes patterned as aresult of the interactions among local and extrinsic elements.Studies of the mammalian visual system have provided considerable insight into ourunderstanding of the mechanisms involved in establishing specific synaptic arrangements duringdevelopment. The utility of this system is exemplified by the high degree of orderliness ofprojections from the two eyes through successive stages of visual information processing, andcorrespondingly, the relative ease with which one can modify visual input (Sherman and Spear,1982 for review). Transneuronal labeling of retinogeniculocortical connections followingintraocular injection of radiolabeled amino acids, reveals that visual information from the twoeyes is conveyed by the axons of retinal ganglion cells to eye-specific layers of the lateralgeniculate nucleus (LGN) on both sides of the brain (Hickey and Guillery, 1974; Fig. 1.1,normal). From here, LGN relay cells project to the visual cortex where they terminate in alaminar-specific manner, and are parcelled into alternating vertical compartments dominated byone eye or the other (Hubel and Wiesel, 1972; Shatz et al., 1977; Shatz and Stryker, 1978;Introduction 2Wiesel et al., 1974; Fig. 1.1, normal). The existence of so-called "ocular dominance columns",has been verified physiologically and anatomically in a wide range of mammalian species(reviewed in LeVay and Nelson, 1991).In contrast to the highly ordered arrangement of eye-specific inputs in the mature visualcortex, the initial innervation by LGN afferents to layer IV of visual cortex is characterized by anintermingling of left and right eye-specific terminals (Hubel et al., 1977; LeVay et al., 1978;Rakic, 1976). During early stages of development, geniculocortical inputs gradually becomesegregated into their respective ocular dominance columns. The process of segregation is speciesspecific, beginning prenatally in monkeys (Rakic, 1976), but not until the third postnatal week inthe kitten (LeVay et al., 1978), and appears adult-like in both species by the sixth postnatal week(Hubel et al., 1977; LeVay et al., 1978; LeVay et al., 1980; LeVay et al., 1981 see Fig. 1.1 forexample).Anatomical evidence of ocular dominance segregation is supported by studies utilizingphysiological recordings of neuronal responses consequent to visual stimulation. Following asimilar time course to that described for the anatomical segregation of geniculate inputs, neuronsin layer IV initially respond equally well to stimulation of either eye (Hubel and Wiesel, 1963;LeVay et al., 1980), but gradually become exclusively driven by only one eye or the other (Hubeland Wiesel, 1962; Hubel and Wiesel, 1968). The importance of this process of input-shaping, inthe present context is that, shortly after the period of segregation begins, and continuing througha discrete window of development, the functional organization of the visual cortex is modifiableby visual experience.The pioneering studies of Wiesel and Hubel provided evidence that the segregation ofvisual cortex into eye-specfic compartments was not predetermined, but arose from a competitiveinteraction between LGN afferents representing the two eyes (Wiesel and Hubel, 1965). Theydemonstrated that if a kitten was monocularly deprived of vision from birth until 3 months ofage, visual cortical neurons responded, almost exclusively, to stimulation of the eye which hadnot been deprived. In these animals, the cortical territory which normally became devoted to theIntroduction^ 3Figure 1.1. The retinogeniculocortical pathway for one eye, mappedautoradiographically, in normal and neonatally enucleated kittens. On the left, an intraocularinjection of [3H]proline in the left eye labels retinal ganglion cell terminals in the eye-specific,ipsilateral (Al) and contralateral (A) laminae in the lateral geniculate nucleus (LGN). From theseterminals, [3H]proline is transported transynaptically to LGN relay neurons, and along theiraxons, which innervate eye-specific columns in layer IV of the visual cortex. Unlabeled bandsrepresent space occupied by the non-injected eye.By contrast, on the right, the patterned distribution of eye-specific terminals is not presentin the visual cortex of a cat which was monocularly enucleated at birth. Here the geniculateterminals representing the visually-experienced eye diffusely innervate the entire extent of visualcortex of both hemispheres. Note also, the reduction in size of the geniculate laminaerepresenting the deprived eye in the enucleated animal.In both cases the visual cortex was opened and flattened, and sections were cut tangentialto the cortical surface so as to be able to view large portions of visual cortex at once. The largeislands lacking label represent cortical layers other than those receiving a direct geniculate input,which dip in and out of the plane of section. Scale bar = 3 mm.4Figure 1.1Introduction 5deprived eye, remained invaded by the non-deprived eye (Shatz and Stryker, 1978; Fig. 1.1). Theeffects of deprivation were not due to changes in the deprived eye itself, nor in the LGN(although neurons within the deprived laminae show morphological changes; Wiesel and Hubel,1963a; Fig. 1.1), but were specific to the visual cortex, at sites where converging binocularinputs compete for synaptic space (Wiesel and Hubel, 1963a). By contrast, visually evokedresponses of visual cortical neurons in animals which had been raised to the same age, butdeprived of visual input to both eyes, appeared largely normal (Wiesel and Hubel, 1965). Byvarying the age of onset, and the duration of monocular deprivation, it has been determined thatthe period of enhanced sensitivity to the effects of deprivation extends from 4 weeks to 13 weekspostnatally (Hubel and Wiesel, 1970), but lesser effects of deprivation are still evident up to 35weeks of age (Cynader et al., 1980; Jones et al., 1984). The effect of deprivation during thissensitive period persists for the life of the animal, while monocular deprivation in adult cats, forperiods as long as one year, has no effect on the responses of cortical cells to stimulation of oneeye or the other.In addition to providing information regarding the processes involved in determining thefunctional organisation of visual cortex, these studies had far-reaching implications regarding theetiology of, and therapeutic intervention for, clinical conditions such as amblyopia andstrabismus. These disorders, and others which result in reduced visual capability in one eyeduring childhood (e.g. cataracts, hyperopia or myopia), could result in permanent impairment ofvision from the affected eye, if not corrected at an early age.The underlying bases for many of the processes which influence the plasticity of synapticfunction in developing nervous systems, including the stabilization or elimination of synapses,are provided by the levels and patterns of activity of the cells involved. Activity-dependentmechanisms are largely responsible for synapse reorganization during the development of theretinotectal projection in fish and amphibians (Meyer, 1982; Reh and Constantine, 1985), as wellas in the innervation of skeletal muscles by motoneurons (Jansen and Fladby, 1990). Similarly, inthe visual system of mammals, the successful reorganization of LGN terminals, in their quest toIntroduction 6appropriate cortical territory, is dependent on the activity of cells in the LGN, as well as thoseintrinsic to the visual cortex. In blocking the electrical activity of retinal ganglion cells in botheyes of young kittens, by intraocular injections of tetrodotoxin (TTX), Stryker and Harris wereable to prevent completely the segregation of LGN axons in visual cortex into ocular dominancecolumns (Stryker and Harris, 1986). If electrical activity was reinstated in this preparation, bystimulation of the two optic nerves, ocular dominance columns formed normally, but only whenthe two optic nerves were stimulated asynchronously (Stryker and Strickland, 1984). The shift inocular dominance normally observed following monocular deprivation was also prevented ifneurons in the visual cortex were silenced by chronic infusion of TTX (Reiter et al., 1987), or ifinhibitory processes were activated by infusion of the GABA agonist muscimol (Reiter andStryker, 1988). Moreover, the effect of altering the activity of postsynaptic neurons relative toafferent input, by infusing glutamate into the cortex, also prevented the shift of ocular dominancefollowing monocular deprivation (Shaw and Cynader, 1984). Based on these data it is apparentthat the amount of activity in developing systems is less important than the pattern of activity,and furthermore, that the patterned activity of pre- and postsynaptic neurons must be correlated.The search for mechanisms responsible for the induction and termination of periods ofplasticity in the brain, described here for the visual cortex during development, has been elusive,despite the sheer volume of effort which has been devoted to this issue. However, it is obviousfrom the preceding discussion that, whatever mechanisms are postulated, they must considerprocesses by which information is communicated, and how this information is modified andrefined to produce meaningful neuronal interactions at the synaptic level. Synapticcommunication between cells in the nervous system occurs mostly via chemical messengers. Theprimary goal of the studies which will be described in this thesis, is to cast some light onpotential mechanisms of synaptic plasticity in the development of the nervous system, byexamining the expression of molecules which are involved in cell-to-cell communication in thedeveloping visual cortex. The bulk of this work entails a detailed study of the anatomicaldistribution of molecules associated with two important neurotransmitter systems which areIntroduction 7involved in the processing of visual information, namely, glutamatergic and serotonergicpathways. An understanding of the development of these systems, relative to that of thefunctional organization of visual cortex, will provide much needed information regarding theircontribution to visual information processing in general, and their particular role during differentperiods of visual cortical development.Serotoninergic SystemsThe participation of specific neurotransmitter systems in the development and plasticityof visual cortical connectivity has been the subject of intensive study for several decades. In fact,noradrenergic (Kasamatsu et al., 1979), cholinergic (Bear and Singer, 1986) and glutamatergic(Fox et al., 1990; Kleinschmidt et al., 1987) systems have all been proposed to be necessary formaintaining visual cortical plasticity in kittens during the critical period. Moreover, transient,region-specific increases in the distribution of neurotransmitter-specific afferents (Bear et al.,1985b; Dyck et al., 1993), and / or their receptors (Aoki et al., 1986; Bode-Greuel and Singer,1989; Cynader et al., 1991; Cynader et al., 1990; Kasamatsu and Shirokawa, 1985; Prusky andCynader, 1990) have been described to parallel the time course of the critical period and are thuseffectively positioned to direct specific developmental processes (discussed in Cynader et al.,1990; Cynader et al., 1991).Initial studies assessing the serotonergic innervation of cerebral cortex, using thefluorescent histochemical technique, indicated that it was sparse and restricted primarily to layerI (Fuxe et al., 1968). However, the development of sensitive biochemical and anatomicaltechniques revealed that the entire neocortical mantle was penetrated by fine, varicose, andhighly convoluted serotonin-containing fibers (Descarries et al., 1975; Gaudin-Chazal et al.,1979; Lidov et al., 1980; Morrison et al., 1982; Reader, 1980; Reader, 1981; Reader et al., 1979;Takeuchi and Sano, 1984), leading one researcher to remark that "the density and distribution ofthese fibers are such that they might innervate every neuron in the neocortex" (Morrison et al.,Introduction 81984). Foremost among these techniques was the development of the serotonin(5-hydroxytryptamine, 5-HT) immunocytochemical technique (Steinbusch et al., 1978), whichpermitted a more detailed study of the 5-HT innervation of neocortex than had been previouslypossible. Although serotonergic fibers are distributed throughout the nervous system, their cellbodies are restricted to a group of nuclei in the rostral brainstem collectively known as the raphenuclei. The majority of ascending serotonergic projections to the forebrain arise from twoprinciple nuclei, referred to as the dorsal raphe and median raphe (Azmitia, 1987; Jacobs andAzmitia, 1992 for reviews). These serotonergic nuclei give rise to morphologically distinct setsof axons that form separate pathways which innervate the neocortex in a highly topographicmanner (O'Hearn and Molliver, 1984). These patterns of innervation are region specific and alsolaminar specific. The laminar specificity of the serotonergic innervation of neocortex is moststriking in the primate visual cortex where the primary geniculate recipient layer (IVO) isdifferentiated by a distinctive decrease of density relative to more superficial and deep laminae(Kosofsky et al., 1984; Morrison et al., 1982). Detailed descriptions of the serotonergicinnervation in adult cortex of various mammalian species have been extensively documented (seeJacobs and Azmitia, 1992 for recent review), and are discussed further in Chapter 2.The possibility that 5-HT has specific trophic or growth related functions in earlydevelopment, distinctly different from its role in the adult nervous system, has long beenindicated (Baker and Quay, 1969; Lauder and Krebs, 1978; Vernadakis and Gibson, 1974). Inmammals, serotonergic cells in the raphe nuclei are among the very first to differentiate andinnervate subcortical target areas within the brainstem, the thalamus and the tectum (Lauder andKrebs, 1978; Lauder et al., 1982; Lidov and Molliver, 1982a; Lidov and Molliver, 1982b;Wallace and Lauder, 1983). In the neocortex, serotonergic fibers have been observed to arrivebefore birth and provide their extensive innervation postnatally (Lidov and Molliver, 1982a).Perhaps the most revealing evidence for an important role for serotonergic mechanisms inneocortical development was provided recently in an immunohistochemical and autoradiographicexamination of serotonergic fibers in the rat during the early postnatal period (D'Amato et al.,Introduction 91987). In this study D'Amato and his colleagues demonstrated a hyperinnervation of serotonergicfibers in all primary sensory cortices, which was present only during the first two weeks of life,during a discrete period of enhanced synaptogenesis (Blue and Parnavelas, 1983a; Blue andParnavelas, 1983b). In somatosensory cortex, axon terminals formed dense patches in layer IVdemarcating specialized cortical columns, called barrels, which each receive input from onefacial vibrissa. In subsequent studies in the rat visual cortex, the transient serotonergicinnervation was found to be localized to discrete columnar compartments as well (Nakazawa etal., 1992); however, no functional correlates have yet been elucidated. In two related carnivores,the cat and ferret, the ontogenic innervation of visual cortex by serotonergic fibers exhibitsdistinct laminar changes in the first few weeks of life, before finally attaining the adultinnervation pattern (Gu et al., 1990; Voigt and De Lima, 1991b). Studies such as these, whichdemonstrate specific temporal and regional patterns of innervation during development, provideevidence for a particular role for serotonin in the ontogeny of visual cortex.The cellular actions of 5-HT in the central nervous system are mediated by specific, high-affinity receptors. In the last decade, the number of different 5-HT receptor subtypes hasproliferated from 2, to at least 7 members, belonging to 3 major families (for recent reviews seePeroutka, 1990; Radja et al., 1991; Zifa and Fillion, 1992). The 5-HT1 and 5-HT2 familiesbelong to the G-protein-coupled receptor superfamily (Hartig, 1989) and were originallyidentified on the basis of their differential affinities for [ 3H]5-HT and [ 3H]spiperone (Pedigo etal., 1981; Peroutka and Snyder, 1979). The 5-HT1 and 5-HT2 families are further subdividedinto specific subtypes. The 5-HT1 family consists of 5-HT i A , 5-1 -a1B (Pedigo et al., 1981),5-HTip (Heuring and Peroutka, 1987) and 5-HTiE (Leonhardt et al., 1989) receptor subtypes.The 5-HT1B and 5-HT ID subtypes are considered to be homologous receptors in differentspecies (Heuring and Peroutka, 1987). The 5-HT lc receptor subtype (Pazos et al., 1984a;Yagaloff and Hartig, 1985), initially classified as a 5-HT1 receptor, is now considered to belongtogether with the classical 5-HT2 receptor subtype, based on their similar molecular structure,second-messenger coupling, and pharmacological properties (Hartig, 1989; Hoyer, 1988;Introduction 10Sanders-Bush, 1988a), as members of the 5-HT2 family. The last major family of serotoninreceptors, called 5-HT3, belong to the ligand-gated ion channel superfamily (Derkach et al.,1989; Kilpatrick et al., 1987), and preliminary reports indicate that multiple subtypes may exist(Richardson and Engel, 1986), but their characterization has not yet been detailed.Although limited information regarding the distribution of some of the 5-HT receptorsubtypes is available for the visual cortex of the adult mammalian visual cortex (rat: Pazos et al.,1985; Pazos and Palacios, 1985; human: Hoyer et al., 1986a; Hoyer et al., 1986b; Pazos et al.,1987a; Pazos et al., 1987b; and non-human primate: Lidow et al., 1989; Parkinson et al., 1989;Rakic et al., 1988), surprisingly little is known about the distribution of the different receptorsresponsible for the transduction of the 5-HT signal during development.The studies described in Chapter 2 address the contribution by serotonergicneurotransmission, to the development of visual cortex, with the first examination of thecomparative distribution of the different receptors subtypes responsible for 5-HT signaltransduction in the visual cortex of the cat during postnatal development.Zinc-Containing SytemsThe possibility that glutamatergic neurotransmission plays an important role inmechanisms of visual information processing is obvious, considering that communication fromLGN neurons to the visual cortex is conveyed by an excitatory neurotransmitter, which isthought to be glutamate (Hagihara et al., 1988; Hicks et al., 1985; Tamura et al., 1990; Tsumotoet al., 1986), and that glutamatergic receptors are developmentally regulated and differentiallyexpressed in visual cortex (Bode-Greuel and Singer, 1989; Cynader et al., 1991; Fox et al., 1991;Fox et al., 1989; Fox et al., 1990).The mammalian telencephalon contains a class of neurons which can be histochemicallydifferentiated by virtue of a chelatable pool of zinc (Zn2+; Danscher et al., 1985; Fredericksonand Danscher, 1988). Although the methodology for selectively staining this pool of zinc hadIntroduction 11been reported four decades ago (Mager et al., 1953; Timm, 1958), the significance of a stainwhich only detected 10% of zinc in the brain was questioned by biochemists and was therefore,largely ignored. The subsequent demonstration that the "Timm-stain" was able to demarcatehippocampal subfields, clearly and beautifully, stimulated renewed interest in its application inthe brain (Danscher and Zimmer, 1978; Haug, 1967). The histochemically detectable pool of zinchas since been found to be restricted to presynaptic vesicles within the axon terminals of zinc-containing neurons (Friedman and Price, 1984; Haug, 1967; Ibata and Otsuka, 1969; Perez-Clausen and Danscher, 1985b). These zinc-containing neurons are considered, on anatomicalbases, to represent a specific subclass of glutamatergic-aspartergic neurons (Storm-Mathisen etal., 1983); namely they contain clear, round vesicles, form asymmetric (Gray's Type I) synapticcontacts (Haug, 1967), and are highly enriched with glutamate (Beaulieu et al., 1992; Martinez-Guijarro et al., 1991). Potassium-evoked and spontaneous release of zinc (Aniksztejn et al.,1987; Assaf and Chung, 1984; Charton et al., 1985; Howell et al., 1984) into the extracellularspace (Perez-Clausell and Danscher, 1986) has been clearly demonstrated in the hippocampus,and, at high rates of neuronal firing, the extracellular concentration of zinc has been estimated toattain 300[EM (Assaf and Chung, 1984; Chung et al., 1986).Recent ultrastructural evidence indicates that synaptic zinc is enhanced within asubpopulation of glutamatergic terminals in the cat and kitten visual cortex (Beaulieu et al.,1991; Beaulieu et al., 1992). However, the specific physiological function of released zinc hasnot been established. There is increasing evidence that zinc is co-released with glutamate andmay regulate neurotransmission by modulating receptor-mediated events. Exogenous zinc hasbeen demonstrated to affect binding of specific ligands to opiate (Stengaard-Pedersen et al.,1981b) and glutamate receptors (Slevin et al., 1986; Wolf and Schmidt, 1983) and it affectsneuronal activity by modulating GABAergic (Westbrook and Mayer, 1987) and both NMDA-and non NMDA-activated glutamatergic neurotransmission (Christine and Choi, 1990; Mayerand Vyklicky, 1989; Peters et al., 1987; Westbrook and Mayer, 1987), indicating that zinc couldparticipate in a modulatory capacity in neural communication.Introduction 12A particular role for glutamatergic neurotransmission in the plasticity of developingvisual cortex has recently been hypothesized (see Constantine-Paton et al., 1990; Rauschecker,1991 for reviews). As mentioned earlier in this discussion, the processes involved indevelopmental plasticity of synaptic connections in the visual cortex are experience-dependentand, furthermore, require the correlated participation of afferent inputs and intrinsic corticalneurons. The assumption that correlated activity of both presynaptic and postsynaptic neuronswas necessary for the activity-dependent stabilization of synaptic connections, and thatasynchronous activity between pre- and postsynaptic cells weakened their connections, was firstpostulated by D. 0. Hebb (1949). Much of the data gathered during the succeeding years, whichestablish potential mechanisms for the Hebbian rule, have been derived from studies of long termpotentiation (LTP), first described in the hippocampus (see Bliss and Lynch, 1988 for review).However, the mechanisms involved in LTP induction could be similar for other activity-dependent synapse stabilization mechanisms, particularly those occuring during development ofthe visual system. In hippocampal LTP, an increase in synaptic efficacy which is inducedfollowing a strong activation of convergent inputs (Bliss and Lynch, 1988), has beendemonstrated to depend on the presynaptic release of glutamate and, in most circumstances, theactivation of postsynaptic NMDA receptors (see Bliss and Lynch, 1988 for review). Zinc hasrecently been speculated as contributing to the induction of long-term potentiation (LTP), byvirtue of its morphological and physiological relationship with NMDA-activated glutamatergicneurotransmission (Weiss et al., 1989), however, direct evidence is not available. More recently,the modulation of NMDA-activated processes has been demonstrated to alter the processes ofexperience-dependent synaptic modifications in visual systems as diverse as kittens (Bear et al.,1990) and frogs (for review see Constantine-Paton et al., 1990).Developmental changes in the distribution of synaptic zinc have been demonstrated inthe rat olfactory cortex (Friedman and Price, 1984), striatum (Vincent and Semba, 1989) andamygdala (Mizukawa et al., 1989). However, the distribution and ultrastructural localization ofzinc in adult and developing cerebral neocortices of the rat, or other species, have not beenIntroduction 13reported. Several groups have demonstrated general increases in the total amount andconcentration of zinc per gram of wet weight brain tissue during the life span of humans (Volk etal., 1974) and rats (Crawford and Connor, 1972), but these do not differentiate synaptic fromnon-synaptic sources. The hippocampal mossy fibers, which contain the highest density ofsynaptic zinc in the brain, are the most well characterized of the zinc-containing neuronalsystems. In the rat (Zimmer and Haug, 1978) and cat (Frederickson et al., 1981) developmentalgradients in the distribution of histochemically localizable zinc in the hippocampal mossy-fiberregion have been reported to reflect developmental synaptogenetic gradients and may be relatedto synaptic maturity.In order to provide support for a particular role for synaptic zinc in neocorticaldevelopment and function, the results of the first ontogenic study of the ultrastructural andregional distributions of synaptic zinc in the cat visual cortex during postnatal develoment aredescribed in Chapter 3.Introduction^ 14RationaleThere is significant evidence indicating that serotonergic and zinc-containingneuromodulatory systems are intricately involved in specific roles during development of thenervous system. However, specific indices of their expression in neocortex during development,have not been described. The primary goal of the studies presented in this thesis was to provide adetailed anatomical description of the postnatal distributions of serotonergic receptors and ofzinc-containing fibers. Second, their contribution to mechanisms of activity-dependent plasticityin the developing visual cortex was assessed. Finally, the novel compartmentalized distributionsof serotonin receptors and synaptic zinc are compared to known indices of the functionalorganization of the visual cortex in cats and primates.152ONTOGENIC EXPRESSION OFSEROTONIN RECEPTORS IN THECAT VISUAL CORTEXThe synaptic organization of the visual cortex of mammals is particularly susceptible toexperience-dependent modifications during a distinct window of postnatal development. Althoughthe mechanisms underlying the formation and stabilization of synapses are not known, it isgenerally believed that the production of chemical messengers, at specific times duringdevelopment, might influence the synaptic organization of visual cortex. Indeed, manyneurotransmitter systems have been proposed to function in a unique capacity, during thedevelopment of the nervous system, in regulating neuronal differentiation, growth and synapticplasticity (for reviews see Lipton and Kater, 1989; Mattson, 1988; Whitaker-Azmitia, 1991).In this chapter are described the results of an autoradiographic study in cats, that comparesthe ontogenic distributions of 5-HT1A, 5-HTic, 5-HT2 and 5-HT3 receptors as well as the highaffinity 5-HT uptake site in visual cortical areas 17, 18, 19, and lateral suprasylvian cortex. Theunavailability of specific ligands and radioligands for 5-HT1B / D and 5-HT1E sites precluded anautoradiographic evaluation of their distribution at this time. The temporal and regional expressionpatterns of the 5-HT receptor subtypes that we did examine, strongly indicate that 5-HT receptorsare effectively positioned to mediate important functional processes at critical stages of visualcortical development.Ontogeny of Serotonin Receptors^ 16Materials and MethodsAnimalsThe normal distribution of 5-HT receptors was assessed in 24 cats at 10 ages betweenpostnatal day 0 (PDO, day of birth) and adulthood (> PD360). At least two animals were used atevery age described in the results, along with 4 adults and 8 kittens studied at PD50. The cats wereanaesthetized with an overdose of sodium pentobarbital and perfused through the ascending aortawith 50-200 ml 0.1M phosphate buffer (pH 7.4) containing 0.9% NaCl. The brain wasimmediately removed, frozen in isopentane at -50°C and then stored at -30°C prior to sectioning. Intwo PD50 animals, the visual cortex from one hemisphere was opened and flattened, prior tofreezing, to assess the overall distribution of 5-HT receptors in the tangential plane. The otherhemisphere was cut in the parasagittal plane. Serial sections were cut on a cryostat at -20°C at athickness of 20 !um, thaw-mounted onto gelatin-coated glass slides and stored at -20°C for notlonger than 4 weeks before processing for autoradiography.AutoradiographyAt each age 5-HT1A, 5-HTic, 5-HT2 receptors and the 5-HT uptake site (5-HTup) werelabelled in near adjacent sections using [ 3H]8-hydroxy-2(di-n-propyl-amino)tetralin ([ 3H]8-OH-DPAT; 169.9 Ci / mmol; New England Nuclear), [ 3H]mesulergine (78 Ci / mmol; Amersham),(±)-1-(2,5,-dimethoxy-4-[ 1251] iodophenyl)-2-aminopropane ([ 125I]DOT; 2200 Ci / mmol; NEN)and [ 3H[cyanoimipramine ([ 3H]CN-IMI; 83.6 Ci / mmol; New England Nuclear). In preliminarystudies we also assessed the utility of [ 3H]2,5-dimethoxy-4-bromoamphetamine ([ 3H]DOB; NEN)to label 5-HT2 receptors, and [3H]citalopram (NEN) to label 5-HTu p . Although all ligandsdemonstrated similar regional and temporal patterns of expression, [ 125 1[1)01 and [ 3 1-]CN-TM'proved superior, in terms of specificity and specific activity, in labeling 5-HT2 receptors and5-HTup , respectively, and the results presented in this study are based on the binding of theseligands. We also used [ 31-1[BRL43694, [3H]GR65630, or [ 3H]quipazine in binding studies toassess the distribution of 5-HT3 receptors during visual cortical development.Ontogeny of Serotonin Receptors^ 17Table 1. Summary of ligands and procedures used to label 5-HT1A, 5-HTic, 5-HT2 and5-HT3 receptors and the 5-HTup site in kitten visual cortex.Table 1. Summary of Incubation ParametersReceptor Subtype 5-HT1A 5-HT1C 5-HT2 5-HT3 5-HTupRadioligandconcentration[3H]8-OH-DPAT2.0 nM[3H]Mesulergine4.5 nM[125I]DOI0.5 nM[3H]BRL436942.0 nM[3H]CN-IMI0.3 nMCold Displacer 10 t.tM 5-HT 10 pM 5-HT 1 t.tM DOB 10 liM 5-HT 11.1M citalopramWash andIncubationBuffer170 mM Tris-HC1(pH 7.6);4 mM CaC12;10 [tM pargylline;0.01% ascorbate170 mM Tris-HC1(pH7.6);2 [tM spiperone;0.01% ascorbate50 mM Tris-HC1(pH 7.4);0.1 % BSA;0.01% ascorbate50 mM HEPES(pH 7.4)50 mM Tris-HC1(pH 7.4);5 mM KC12;120 mM NaC1Pre-IncubationWash30 min 30 min 30 min 15 min 30 minIncubation Time 60 min 120 min 60 min 60 min 24 hourPost-IncubationWash2 X 5 min 2 X 10 min 3 X 10 min 2 X 3 min 1 hourFilm Exposure 8 weeks 10 - 12 weeks 3 days 6 - 8 months 2 weeksDOB: 2,5 -dimethoxy-4-bromoamphetamine ; BSA: bovine serum albumin.18Ontogeny of Serotonin Receptors^ 19The binding procedures which were used, and the specificity of each radioligand forindividual 5-HT receptor subtypes, are based on ligand characterizations which have beendescribed previously in rat (Kilpatrick et al., 1987; Kilpatrick et al., 1988; Kovachich et al., 1988;McKenna et al., 1989; Nelson and Thomas, 1989; Pazos et al., 1985; Pazos and Palacios, 1985;Titeler et al., 1987; Titeler et al., 1985), primate (Lidow et al., 1989; Parkinson et al., 1989), andhuman brain (Hoyer et al., 1986a; Hoyer et al., 1986b; Pazos et al., 1987a; Pazos et al., 1987b).Any deviations from the published protocols are indicated by the specific incubation parametersused (buffers, incubation times, and ligand concentrations), outlined in Table 1, and brieflydescribed here. The frozen sections were thawed, washed for 30 min in buffer and then incubated,in the dark, in buffer containing the appropriate radioligand. Nonspecific binding was assessed innear adjacent sections by including an excess of non-labeled displacer in the incubation solution.Following incubation, sections were washed in buffer to remove unbound radioligand, brieflydipped in ice-cold distilled water, dried under a stream of cool air and then exposed againstHyperfilm- 3H along with either 3H- or 1251 - standards (Amersham). After developing theHyperfilm, selected sections were stained with cresyl violet to facilitate the identification ofcytoarchitectonic areas and cortical layers in relation to radioligand binding patterns.Quantitative DensitometryAutoradiographic images were captured digitally using a Macintosh llfx-based image analysissystem (Cohu 4915 CCD camera; Data Translation DT-2255 quick capture board) running Imagesoftware (NIH, v 1.45). This software allowed radioligand binding to be measured in calibratedunits of isotope concentration or moles of ligand (nCi / mg tissue; fmol / mg tissue), withinindividual cortical layers. Nissl stained sections were digitally superimposed uponautoradiographic images and individual regions and cortical layers were identified and measureddensitometrically.Ontogeny of Serotonin Receptors 2 0In the youngest animals (PDO), density measurements were obtained from 100 gm-wideregions, drawn within, and corresponding to, layers I, IV, V and VI as well as superficial (CPs),deep (CPd) cortical plate and white matter (Wm) from area 17, 18, 19 and lateral suprasylviancortex (LS). In animals older than PDO, the average density of 200 gm wide regions drawn fromlayers I, II, III, superficial IV (IVs), deep IV together with superficial V (IVd), VI, and Wm wasmeasured in each region. In all cases, four sections from each brain were used for thedetermination of specific ligand binding. Specific binding within an individual lamina or regionwas obtained by subtracting non-specific binding, measured in near-adjacent sections incubatedwith unlabeled displacer, from the total bound radioligand measured in each of the four sections.Several approaches were utilized to relate the pattern of ligand binding to laminar and regionalborders. Selected sections were counterstained with cresyl violet which were then used to establishlaminar borders in the various visual cortical areas based on cytoarchitectonic criteria established byOtsuka and Hassler (1962) in adult cats and Shatz and Luskin (1986) for early postnataldevelopment. Some sections were histochemically stained for cytochrome oxidase to demarcatelayer IV within areas 17 and 18 (Wong-Riley, 1979). The borders of visual cortical areas were alsodetermined according to the electrophysiologically defined maps of Tusa et al. (1978, 1979, 1980,1981) which describe the relationships of regional borders and gyral patterns. Additionalinformation was derived from comparisons with previous studies using autoradiographic markershaving laminar-specificity in area 17 / 18 of developing cat visual cortex (Prusky et al., 1987;Shaw et al., 1984; Shaw et al., 1986).ResultsThe photographs in Figure 2.1 illustrate the changing distributions of 5-HT ip (A-D, 5 -HT IC(A'-I') and 5-HT2 (A"-I") receptors in near-adjacent sections through visual cortical areas 17, 18,and 19 between birth and adulthood. Autoradiographic images showing the binding to thesereceptors in lateral suprasylvian cortex (LS) during develoment are shown in Figure 2.2.Ontogeny of Serotonin Receptors^ 21Photographs of representative sections which illustrate the binding of [41]-CN-IMI to 5-HTu psites, in PD20 (A), PD75 (B), and adult cat visual cortex (C), are presented in Figure 2.3. Allsections for each ligand were processed simultaneously, exposed against the same sheet of film,and were photographed and printed under identical conditions. Within these figures, the observablechanges in density across different regions and laminae reflect real changes in total boundradioligand.Regardless of the radioligand used, and even with 6 months film exposure, we were unableto detect specific binding to 5-HT3 receptors in the visual cortex at any age. However, very highconcentrations of 5-HT3 receptors (best labeled using [ 3H]BRL43694) were found in sectionsthrough the striatum and hippocampal formation of the same brains, indicating that this 5-HTreceptor subtype is not likely to be found in the visual cortex at detectable levels. The observationthat the density of 5-HT3 receptor binding sites is significantly less than that of any other 5-HTreceptor class, has been previously reported, and the authors also indicated that the utility oftritiated radioligands to visualize 5-HT3 sites in the brain would be limited (Radja et al., 1991).The photographic images generated from the autoradiograms represent the total binding(specific + nonspecific) of each radioligand. In the quantitative analyses described below, specificbinding was assessed densitometrically by subtracting nonspecific (ns) from total binding. Acrossall ages used in this study, specific binding accounted for greater than 88.6% of total binding of[3H]8-OH-DPAT (8.2% < ns < 11.4%) , > 75.5% of binding of [ 3H]mesulergine (11.1% < ns <24.5%), > 88.0% binding of [ 125I]DOI (8.9% < ns < 12.6%) , and > 84.0% of [ 3H]CN-IMI(8.6% < ns < 16.0%). In the following sections, the binding data are presented in units of fmol /mg tissue (wet weight) ± standard error of the mean.It is apparent from the autoradiograms in Figures 2.1, 2.2, and 2.3, that the specificexpression of each 5-HT receptor subtype is manifested by complex temporal, regional and laminarpatterns. In order to appreciate more fully this complexity, the autoradiographic data are brokendown in the following sections beginning with general temporal, regional and laminar analyses andending with an examination of specific laminar and infra-laminar distributions.Ontogeny of Serotonin Receptors^ 2 2Figure 2.1. Autoradiographic images demonstrating the total binding of [ 3H]8-OH-DPAT(A-I), [ 3H]mesulergine (A'-I'), and [ 125I]DOI (A"-I"), to 5-HT 1A, 1C and 2 receptors,respectively, in near-adjacent sections through visual cortical areas 17, 18, 19 and lateralsuprasylvian cortex (LS) during postnatal development (age, in postnatal days is indicated to left ofeach row). All the sections for each ligand were processed simultaneously, apposed to the samefilm and photographed under identical conditions. Regional and temporal changes in density thusreflect actual changes in level of binding for each radioligand. Regional boundaries are indicated bydotted lines in the left-hand panels.23Figure 2.1a24Figure 2.1bOntogeny of Serotonin Receptors^ 2 5Figure 2.2. Autoradiographic images demonstrating differential binding patterns of[ 3H]8-OHDPAT, [ 3H]mesulergine and [ 1251]D01 to 5-HT1A, 5-HTic and 5-1-1T2 in lateralsuprasylvian cortex (LS) at postnatal day 0, 10, 90 and in adult cat. The expression of 5-HT1A,and 5-HT2 receptors was very high in LS at ages beyond PD10 but 5-HTic receptors were presentonly at very low abundance at all ages.26Figure 2.2Ontogeny of Serotonin Receptors^ 27Figure 2.3. Autoradiographic images illustrating total binding of [3H]CN-IMI to 5-HTupsites in visual cortex of PD20 (A), PD75 (B) and adult (C) cats. 5-HTup sites exhibited a generalincrease in density with age but levels were essentially homogeneous across all cortical regions andlaminae throughout early development but exhibited a slight preference over superficial layers inthe adult.A•CFigure 2.328Ontogeny of Serotonin Receptors^ 2 9Figure 2.4. Age-related changes in the binding of [ 3H]8-OH-DPAT, [3H]mesulergine,[ 1251]D01 and [3H]CN-IMI to 5-HT 1A, 1C, 2 and Up receptors, respectively, in cat visual cortexare plotted as an average for all cortical regions and layers combined. The average density ofreceptor binding for each ligand, expressed as a proportion of maximal binding, was typically lowat birth, increased to a maximum, at a different ate for each binding site, and then declined to adultlevels. The peak binding levels of individual receptor subtypes exhibited surprisinglycomplementary temporal distributions with 5-HT1A receptors expressed earliest in development(PD10 - PD40) followed by 1C receptors (PD40 - PD75), and the 5-HT2 and 5-HTu p later indevelopment (PD75 - PD120).1[3H]8-OHDPAT [3H]Mesulergine1^1^1^1^1^1^1^1^1Up[311]CN-IMIOntogeny of Serotonin Receptors^ 301^1^1^1^1^1^1^1^15-1-1T/\61/\2[125IWO'0 ^0.210.8E 0.6r-1 0.4cto 0.21• 0.8P2• 0.6•c•4 0.4C/) 10 20 30 40 75 90 120Adult^0 10 20 30 40 75 90 120AdultAge (postnatal day)Figure 2.4Ontogeny of Serotonin Receptors^ 31Temporal Patterns of ExpressionFigure 2.4 demonstrates the effect of age on the binding of [ 3 f1]8 -OH-DPAT ,[3H]mesulergine, [ 125I]DOI, and [ 3H]CN-IMI to 5-HT1A, 5-HT]. c, 5-HT2 receptors and 5-HTuptake sites, respectively in postnatal cat visual cortex. These data represent the binding density ofeach receptor subtype averaged across areas 17, 18, 19 and LS collectively, and are plotted as aproportion of peak binding. In general, all receptor subtypes exhibited their lowest levels ofexpression at birth, increased with a different time course to temporally unique peak levels ofexpression, and then declined to adult levels. The 5-HT1A receptors exhibited maximal levels ofexpression between PD20 and PD30 but dropped to near-adult levels by PD75. 5-HTic receptorsalso exhibited a transient peak in expression, which occurred slightly later, between PD40 andPD75, but then declined, by adulthood, to levels similar to that seen in early postnataldevelopment. The expression of 5-HT2 receptors and 5-HTu p sites increased gradually throughearly postnatal development, with a very similar time course, to exhibit their highest levels betweenPD40 and PD75. In both cases the highest levels of expression were maintained beyond PD120,but the reduction to adult levels was much greater for the 5-HT2 subtype than for 5-HTu p sites.Regional and Laminar PatternsIn addition to displaying complementary temporal patterns of expression, individual 5-HTreceptor subtypes demonstrated striking regional and laminar complementarity in theirdistributions. Developmental changes in the levels of expression of each 5-HT receptor subtypewithin individual regions, averaged across laminae, are expressed in the three-dimensional plots inFigure 2.5. The binding data for each radioligand, within individual laminae of each visual corticalregion, are presented in Figure 2.6. Quantitative changes in regional and laminar expressiondescribed in the following sections in terms of fmol / mg tissue (wet weight), can also be followedqualitatively in the representative micrographs presented in Figures 2.1, 2.2 and 2.3.Ontogeny of Serotonin Receptors^ 32Figure 2.5. Three-dimensional plots demonstrating regional patterns in the distribution of5-HT 1A (A), 5-HT lc (B), 5-HT2 (C) 5-HTu p (D) receptors in postnatal cat visual cortical areas17, 18, 19 and lateral suprasylvian cortex (LS). A. The temporal pattern in the distribution of5HTiA receptors was similar across all visual cortical areas. From lowest levels at birth (PDO), thedensity of 5-HTIA receptors peaked between PD1O-PD30 and subsequently decreased to adultlevels. B. Receptor binding specific for 5-HT lc receptors was highest at PD40 and was essentiallyrestricted to area 17. C. 5-HT2 receptors demonstrated highest levels of expression in LS at allpostnatal ages beyond PD10 except for a striking decrease in level of expression at P40. In areas17, 18 and 19 peak levels were displayed between PD75 and PD120. D. Little regional disparitywas seen in the distribution of 5-HTup sites. 5-HTup sites demonstrated a gradual increase fromPDO to PD40 and then maintained this high level to adulthood. Note that the left abscissa in plots Cand D are reversed to make the relative binding in LS more visible than it would be otherwise.Ontogeny of Serotonin Receptors^ 335-HT 1 A13011090705030LS1301109070501718Area 19PP20^10PP40 P30P75P90P120AdultPO5-HTic30LS3024181718Area 19P90P120AdultP10 POP30 P205 P40HT2241812LS040 9,0  VO..mx■o%14 or^-,01.,.^f01°w 111„0019N4444° ,4011,w^P10Area is^ P30 P20^IN111---^P40P7517^P90Adult P12°19 '14111111101111hilbo^ 2 0 P 10Area is^P7517P40 P30Age (Postnatal Day)Adult P120P902045403530252015LS25-HTup/W-011110.11111 111140111111146.PO2014PO4540353025201510Figure 2.5Ontogeny of Serotonin Receptors^ 3 4Figure 2.6. The bar graphs illustrate temporal changes in specific binding of[3H]8-0H-DPAT (A), [ 3H]mesulergine (B), [ 1251]301 (C) and [31-1]CN-IMI (D) in individuallaminae within visual cortical areas 17, 18, 19 and LS. A detailed description of their expressionlevels and patterns can be found in the text.[3H]Mesulergine1601208040Ontogeny of Serotonin Receptors 35A [3H]8-OHDPAT80P120 Adult^PO P10 P20 P30 P40 P75 P90 P120 AdultE 400bACortical^rer (P01WM^SP 2 VNI 11 IV^CPd^CPs^I12511110130.rj 20CL)j:1,, 10481130201 04830-20-3020100Cortical Layer > POWM 0 VI 0 rvaiv tg: IVs Emmumi[311]CN-IMIPO P10 P20 P30 P40 P75 P90 P120 P10 P20 P30 P40 P75 P90 P120 AdultAgeFigure 2.6Ontogeny of Serotonin Receptors^ 3 65-HT1AThe binding of [ 3H]-8-0H-DPAT to 5-HT 1 A receptors was lowest at birth (PDO) withequally low levels distributed across all visual cortical areas (Fig. 2.5A, Fig. 2.6A; area 17, jt =27.68 ± 2.85; area 18, R = 29.06 ± 2.67; area 19, I( = 31.17 ± 2.70; LS, R = 28.39 ± 2.66). Themajority of 5-HT 1A receptors in all four regions were localized to a narrow band below the corticalplate (CP) which included layers V and VI and extended, only superficially, into the subplate (Fig.2.6A; Fig. 2.7A). The superficial layers, including the CP and layer 1, were equally labeled, atlower levels. A sharp increase in the level of binding was realized in all four regions between PD10and PD30 (Fig. 2.5A; Fig. 2.6A). This increase in binding density was greater in areas 17 and LS(x = 105.89 ± 6.96; 108.29 ± 7.26) than in areas 18 and 19 (x = 95.67 ± 6.13; 98.76 ± 6.22)and was manifested by increases in binding levels predominantly in supragranular (I-III) andinfragranular (V, VI) layers, although a significant increase was present in layer IV as well (Fig.6A). The increase in binding was clearly visual cortex specific, distinguishing these areas fromdirectly adjacent cortical regions (Fig. 2.1; Fig. 2.2). The level of 5-HT1A binding peaked at PD30with higher levels than those found at any other stage in development. The subsequent decline inexpression seen beyond PD40 followed a similar time course across the different cortical regions(Fig. 2.5A, Fig. 2.6A). By adulthood, in contrast, the visual cortex was conspicuously unlabeledrelative to adjacent non-visual areas. This regional disparity was due, primarily, to reducednumbers of 5-HT1A receptors in cortical layers III, IV, V and VI in areas 17 / 18 (x = 37.40 ±2.38; 30.44 ± 2.67), which distingushed them from the laterally adjacent area 19 ( x = 50.10 ±4.28) and the ventro-medial cingulate cortex (Fig. 2.1; Fig. 2.5). A very narrow band within layerV was labeled at intermediate levels (Fig. 2.1 adult). The level of binding in the subplate andsubcortical white matter was low at all ages (Fig. 2.1; Fig 2.6A).5-HT 1 C[ 3H]Mesulergine binding exhibited the greatest regional and laminar specificity of thesubtypes analyzed in visual cortex (Fig. 2.1, 2.11A). From Figures 2.1, 2.5B, and 2.6B, it isOntogeny of Serotonin Receptors 3 7apparent that 5-HTic binding sites exhibited their lowest levels of expression at birth within areas17 ( x = 9.26 ± 1.45), 18 (R = 13.22 ± 1.45), 19 ( x = 14.51 ± 1.45) and LS (x = 12.76 ± 1.45).Throughout development, the highest density of binding was limited exclusively to the middlecortical layers (Figs. 2.1, 2.6B). A denser band of receptors in the lowest portions of CP, amongcells destined for the base of layer IV (Shatz and Luskin, 1986), and upper layer V wasdistinguished from the equally dense binding in superficial and deep layers at birth (Fig. 2.1; Fig.2.7B). The number of receptors increased slowly with age in areas 18 (x = 15.98 ± 1.45), 19 (j -c. =17.24 ± 1.45) and LS (x = 17.01 ± 1.45) but doubled in area 17 ( )1 = 18.55 ± 1.45) by PD30(Fig. 2.5B). A significant increase in specific binding of [ 3H]mesulergine was observed at PD40which was caused, almost entirely, by an increase in area 17 (x = 31.03 ± 3.00; Fig. 2.5B).Between PD30 and PD75, the pattern of [ 3H]mesulergine binding was characterized by a denseband located in deep layer IV / superficial layer V, from which emerged columns of receptorsextending vertically into parts of layer III (Fig. 2.1, Fig. 2.6B). These high regional and laminarlevels in the expression of 5-HTic receptors were developmentally transient, peaking at PD40 andbecoming reduced through PD75 ( )1 = 13.02 ± 0.97) to near adult levels by PD90 ( R = 15.95 ±1.46). The reduction of columnar binding appeared gradual, remnants of 5-HT lc receptor columnswere present only at the layer III / IV border at PD90 (Fig. 2.1G'). At ages beyond PD120, thedistribution of 5HT1c receptors in visual cortex was adult-like, essentially homogenous across alllayers (Fig. 2.1, Fig. 2.6B).5-H T 2The patterns of [ 125I]DOI binding displayed the greatest complexity in regional (Fig. 2.1,2.5C, 2.11C) and laminar (Fig. 2.6C) distributions, of the four types of 5-HT binding sitesexamined. The highest levels of 5-HT2 receptors, visualized with [ 125I]DOI, distinguished LSfrom adjacent cortical areas between PD10 (x = 16.15 ± 2.06) and PD75 ( x = 17.11 ± 2.14).Except for a transient decrease at PD40 (x = 9.64 ± 1.05), their level of expression remained highthrough PD120 (x = 16.98 ± 2.17) after which they declined to adult levels ( x = 8.38 ± 1.42).Ontogeny of Serotonin Receptors^ 3 8The level of 5-HT2-specific binding in areas 17, 18 and 19 was lowest at birth ( R = 1.22 ± 0.23;1.91 ± .025; 2.14 ± 0.14) then increased to intermediate levels by PD40 ( R = 6.73 ± 0.72, 6.28 ±0.67; 7.07± 0.64). Although at very low levels, the expression of 5-HT2 receptors in lowercortical laminae appeared inhomogenous and patchy (Fig. 2.7C). Through early postnataldevelopment (PDO-PD40), the highest concentrations of 5-HT2 receptors in areas 17, 18 and 19were localized to the deeper cortical layers (IV, V, VI; Fig. 2.1, Fig. 2.6C). The highest laminar-specific density of receptors in area 17 / 18 was found in a band restricted to layer V/VI at birth(Fig. 2.7C) but highest in layer IV from PD10 - PD40 (Fig. 2.6C). A striking augmentation in thenumbers of 5-HT2 receptors in all visual cortical regions occurred between PD40 and PD75. Theyincreased to peak levels, to values which were the highest of all cortical regions, in areas 17 / 18 byPD75 ( R = 18.46 ± 1.79; 14.52 ± 1.95) and continuing through PD120 ( g = 23.25 ± 2.23; 18.22± 2.24; ), before declining to adult levels (g = 11.19 ± 1.52 / 7.47 ± 0.94). The laminar pattern ofexpression changed significantly over this period, becoming predominent in superficial layers. Inarea 17 and 18, the highest density of binding was always localized to a denser band withinsuperficial levels of layer IV (Fig. 2.6C). Among the different 5-HT subtypes, the 5-HT2 receptorswere found unusual in demonstrating high levels of expression in white matter (Fig. 2.1, Fig.2.6C). The binding in subcortical white matter was transient, reaching its highest levels at PD30within a narrow band directly subadjacent to layer VI (Fig. 2.1). This localization of 5-HT2receptors was observable through PD90 but was never seen in the adult.5-HTUpThe expression of 5-HTup sites closely matched the regional and temporal trends describedfor the 5-HT2 receptors (Fig. 2.5D); however, unlike 5-HT receptors subtypes, the distributions of5-HTup did not exhibit a great degree of laminar disparity (Fig. 2.3, Fig. 2.6D). In the later stagesof postnatal development there were discernably higher levels of expression in supragranularlayers, particularly in area 18 (Fig. 2.3, Fig. 2.6D). It is apparent from Figure 2.5D that, at earlypostnatal ages, the highest density of 5-HTup was localized to LS ( g = 21.58 ± 2.24) with theOntogeny of Serotonin Receptors^ 3 9lowest in area 19 (1 = 9.30 1.14) followed by area 17 ( = 9.98 ± 0.94) and area 18 ( =14.39 ± 1.11). The levels of expression increased progressively in all areas and by PD40 uptakesites were found at high levels, across all cortical areas (17, 44.61 2.46; 18, 38.40 ± 2.82; 19,40.47 2.12; LS, 39.37 2.47). These levels of expression were essentially maintainedthroughout the remainder of postnatal visual cortical development (Fig. 2.5D). The general trendfor all regions to exhibit decreases in receptor expression between PD 120 and adult was maintainedfor the 5-HTup site, but to a far lesser degree, in areas 17, 19 and LS (Fig. 2.5D). In fact, as isapparent in Figures 2.5D and 2.6D, the density of 5-HTu p sites increased slightly in area 18 fromPD120 ( = 43.48 ± 3.94) to adult levels (5Z = 46.66 3.94).Complementary Patterns of ExpressionA recurring theme in the developmental distribution of the multiple 5-HT receptor subtypeswas complementarity of laminar expression. This feature was found to be most conspicuous inarea 17. In Figure 2.8, the profile plots illustrate densitometric changes in binding of [ 3H]8-OH-DPAT, [3H]mesulergine, and [ 125I]DOI across cortical laminae within area 17 expressed as aproportion of peak binding for eight representative ages between PDO and adulthood. Thedistributions of 5-HTup sites did not exhibit significant laminar variation in area 17 and were notincluded to simplify visual comparisons. At PDO, only the 5-HT lc receptors exhibited a specificlaminar distribution (layer IV) which would be maintained through subsequent stages indevelopment. While 5-HT lc receptors were predominant deep within the cortical plate, amongprospective layer IV cells (Fig. 2.7B, Fig. 2.8A), the 5-HT1A (Fig. 2.7A) and 5-HT2 (Fig. 2.7C)receptors were concentrated within the same, deeper, infragranular laminae (Fig. 8A). Beginning atPD10 (Fig. 2.8B) and continuing throughout the remainder of postnatal development, the highestlevels of 5-HT1A receptors were concentrated in supragranular laminae (I-III). On the other hand,the highest levels of 5-HTic and 5-HT2 receptors were consolidated in layer IV. Between PD10(Fig. 2.8B) and PD20 (Fig. 2.8C) they were localized to the same lamina, but by PD40 (Fig.2.8D) their relative distributions were changed. 5-HTic receptors were maintained in deeper strataOntogeny of Serotonin Receptors 4 0of layer IV, but the 5-HT2 receptors were now concentrated in upper layer IV. This relationshipwas maintained beyond PD75 (Fig. 2.8E), as long as both receptor subtypes were present. ByPD120 the distribution of 5-HTic receptors was essentially homogeneous across different corticallaminae but the highest density of 5-HT2 receptors was evenly distributed through layers I-IV (Fig.2.8F). The strong overall tendancy in all the panels of Fig. 2.8, is for the concentration of eachbinding site to peak in different layers and at different cortical depths. The complementarity in thelaminar binding profile is most pronounced during intermediate ages examined (Fig. 2.8B - E).Ontogeny of Serotonin Receptors^ 41Figure 2.7. Photomicrographs showing the laminar distribution of 5-HT IA (A), 5-HTic(B) and 5-HT2 (C) in near-adjacent sections from kitten visual cortex (Area 17 and 18) at birth(PDO). Autoradiographic images are inset within a photograph of the same section stained withcresyl violet in order to establish laminar boundaries. Peak expression of 5-HT1A (A) and 5-HT2(C) receptors was limited to a single band within infragranular cortical layers (V - VI). 5-HTicreceptors were also limited to a single band (B) but this band was positioned more superficially,extending from upper layer V to deeper levels of the cortical plate (Cp). Scale bar = 500 pm.42Figure 2.7Ontogeny of Serotonin Receptors^ 4 3Figure 2.8. These densitometric profile plots illustrate the changing levels of 5-HT I A,5-HT lc and 5-HT2 receptors as a function of postnatal development. For each age, specificbinding density is displayed as a proportion of peak binding. The positions of cortical laminae areindicated by Roman numerals (I-VI); cp = cortical plate. 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0'•••••c71 0.8=cle= 0.6sl•S• 0.4•=1,RI■sCTS41.) 0.2rg I IV0.0^i^1 1^II II I^I^I V^ 1 VI I^i0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.20.2^0.4^0.6^0.8^1.0^1.2^1.4PD201.0PD750. .0^1[11[11111^I^I^0.00.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4^0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2I^III^IV^V^VI• ........ - 5HT1A.- 5HT1C■v_......•• • • 5HT2III^IV^V VIOntogeny of Serotonin Receptors^ 44Distance (mm)Figure 2.8Ontogeny of Serotonin Receptors^ 4 5Figure 2.9. Higher magnification photomicrographs demonstrating laminar andintralaminar relationships between the distributions of 5-HT1A (A), 5-HT lc (B) and 5-HT2 (C)receptors and 5-HTup (E) sites in near-adjacent sections from PD50 kitten visual cortex (Area 17).5-HT1A receptors were densely distributed throughout supragranular (I-III) and to a lesser degreein infragranular cortical laminae (V-VI). The highest densities of 5-HTic and 5-HT2 receptorswere found in middle layers. In addition to displaying a laminar complementarity with 5-HT1Areceptors, 5-HT lc and 5-HT2 receptors demonstrated an intralaminar complementarity with thehighest density of 5-HTic receptors located within deeper levels of layer IV and 5-HT2 receptorspredominant in superficial layer IV. Moreover, both receptor subtypes exhibited a patchyappearance within layer IV. These patches were —400 jim in diameter and spaced —900 Jim apartand were localized to the same vertical column (B and C, arrows). The columnar expression of5-HTic and 5-HT2 receptors also extended into the deeper portions of layer III. In addition to peaklevels in superficial strata of layer IV, 5-HT2 receptors exhibited intermediate levels of expressionin layers I-III, lower layer IV and subcortical white matter (wm). Panel E shows that thedistribution of 5-HTup sites was essentially uniform across all cortical laminae. The borders ofcortical laminae (I-VI) and subcortical white matter (wm) are indicated at the edge of each panelwith reference to the Nissl-stained section (D). Figures B & C are from serially adjacent sections.The distance of each section from that in A is indicated (in gm) in the lower left of each figure. Dand E are from the same section, which was Nissl-stained following autoradiographic processing.Scale bar = 1.0 mm.46Figure 2.9Ontogeny of Serotonin Receptors^ 47Columnar Distribution of 5-HT ReceptorsBetween PD30 and PD90, 5-HT lc and 5-HT2 receptors were augmented in a periodicmanner within layer IV of area 17. Although the patterned expression of these receptors was firstapparent at PD30, it was weak and not always readily demonstrable until after PD40, at ages whenboth receptor subtypes were found in particularly high abundance in area 17. Because of thisdevelopmental profile, we chose to concentrate on PD50 kittens to study the columnar distributionsof 5-HTic and 5-HT2 receptor subtypes.Columns of 5-HTic receptors emerged from a dense band of 5-HTic receptors at the layerIVN interface and extended radially, through the entire extent of layer IV, and up into the deepestlevels of layer III (Fig. 2.10B, Fig. 2.11A, B). This distinct distribution of 5-HT lc receptors wasstrictly limited to area 17 (Fig. 2.11B). The patterned distribution of 5-HT2 receptors resembledbeady excrescences radiating from a dense band, rather than columns (Fig. 2.10C, Fig. 2.11C).This band was consolidated within upper strata of layer IV (Fig. 2.10C) in area 17 and area 18(Fig. 2.11A, C). However, the bead-like pattern of expression was not apparent in area 18.The spatial relationship between the patchy 5-HT lc and 5-HT2 receptor distributions isapparent in Figure 2.10 (C, D) and at higher magnification, in the serially adjacent sections inFigure 2.9 (B, C). The patches of increased 5-HT102 receptor density were found to be preciselyin register, within the same vertical column (Fig. 2.9B, 2.9C; arrows). Although the highestdensity of each of these 5-HT receptor subtypes was centred within different substrata of layer IV,their patchy pattern of expression was precisely aligned, where they overlapped in upper layer IV(Fig. 2.9B, 2.9C; Fig. 2.10C, 2.10D arrow heads).The overall columnar distributions of 5-HT receptors in area 17 are best appreciated inparasagittal (Fig. 2.11) and tangential (Fig. 2.12) sections through layer IV of a PD50 kitten. Thepatchy distribution of 5-HTic receptors at this age was strictly limited, in the neocortex, to visualcortical area 17 (Fig. 2.11A, 2.12A). In sections tangential to the unfolded cortical surface (Fig.2.12), patches of 5-HTic receptors, with an average diameter of approximately 400 p.m (I( = 418Ontogeny of Serotonin Receptors 4 8± 18), were found distributed throughout area 17 with an average centre-to-centre spacing ofapproximately 900 gm (5( = 909 ± 211). In addition to distinctive distributions in visual cortices(Fig. 2.11D), high levels of 5-HT2 receptors were observed across a number of different cortical(e.g. FCtx, Cing, Hpc) and subcortical regions in the kitten brain (Fig. 2.11C). Measurements ofarea 17 from the two hemispheres which were opened and flattened indicate that the surface area ofarea 17 in PD50 kittens is approximately 312 mm 2 (306, 318) which is similar to values previouslypublished in the adult (Olavarria and Van Sluyters, 1985; Tusa et al., 1978; Van Essen andMaunsell, 1980). The total number of 5-HT lc-rich patches within area 17 of each hemisphere was296 and 315, respectively, which suggests a packing density of approximately 1 patch / mm 2 .These patches did not appear to be aligned along any particular axis nor was their periodicitysignificantly different across area 17.Ontogeny of Serotonin Receptors^ 4 9Figure 2.10. Autoradiographic images showing the regional and laminar complementarityof 5-HT1A (B), 5-HTic (C) and 5-HT2 (D) receptors in near-adjacent sections from PD50 visualcortex. The profile plot in A, generated from a densitometric slice through cortical areas 17 and 18at the level of the black arrows in B, compares laminar changes in binding density among the threereceptor subtypes. 5-HT1A receptors were most highly concentrated in supra-granular layers andof intermediate density within infragranular layers. A high density of 5-HT2 receptors wasdemonstrated in supragranular and granular layers of both area 17 and 18, with a band of peakbinding limited to superficial levels of layer IV. Peak expression of 5-HT1C receptors waslocalized to the deeper half of layer IV, and was limited to area 17. Both 5-HTic and 5-HT2receptors subtypes exhibited a patchy distribution, but within different strata of layer IV, in thesame vertical column (filled arrow heads indicate coincident columnar localizations). The bordersof areas 17 and 18 are indicated by the open arrows. Scale bar = 2.0 mm.50A4.35.30^0.2^0.4^0.6^0.8Relative DensityFigure 2.10Ontogeny of Serotonin Receptors^ 51Figure 2.11. The distribution of 5-HTic receptors, labeled with [ 3H]mesulergine (A), and5-HT2 receptors, labeled with [ 1251]1)01 (B) in adjacent, parasagittal sections through PD50 kittenbrain. Each of these subtypes of the 5-HT2 receptor family exhibited distinctly different regionaland laminar localizations. In area 17, high levels of both 5-HTic and 5-HT2 receptors exhibited aperiodic distribution. However, while the highest density of 5-HTic receptors was limited tovisual cortical area 17 at this age, 5-HT2 receptors were more broadly distributed within othercortical (e.g. FCtx, Cing, Hpc) and subcortical regions. The black arrow indicates the anteriorextent of visual cortical area 17. Scale bars = 3 mm. (choroid plexus, CP; corpus callosum, cc;suprasplenial sulcus, sspl).52Figure 2.11Ontogeny of Serotonin Receptors^ 53Figure 2.12. The tangential distribution of 5-HTic receptors in visual cortex at PD50, insections taken at 400, 640, and 1000 gm (top, middle, bottom panels, respectively) from thesurface of a flattened right hemisphere. The overall patchy distribution of 5-HTic receptors in theneocortex was limited to visual cortical area 17. Patches of 5-HT lc receptors averaged 400 pm indiameter and were separated by an average centre-to-centre spacing of 900 gm. It is apparent fromthese sections, that columns of 5-HTic receptors extended from deeper levels of layer III (top)through the entire extent of layer IV and disappeared in a dense band of binding at the layer IV / Vborder (bottom). The dotted line indicates the approximate position of the area 17 / 18 border.Scale bars = 10 mm and 2 mm (M, ventro-medial; L, dorso-lateral; A, anterior; P, posterior).rigure 2.1254Ontogeny of Serotonin Receptors^ 55DiscussionThe results of this study reveal that 4 serotonin receptor subtypes are expressed in unique andcomplementary temporal, regional, laminar, and intra-laminar patterns in developing cat visualcortex. 5-HTiA receptors reached peak levels between PD10 and PD30 and were concentrated insuperficial (MIT) and in deep (V, VI) laminae of all visual cortical areas. The 5-HTic and 5-HT2receptor subtypes displayed their highest levels between PD4O-PD75 and PD75-PD120,respectively, within different strata of layer IV. While 5-HT lc receptors were restricted to area 17,5-HT2 receptors were highly expressed in area 17, 18 and lateral suprasylvian cortex. Although5-HTup sites exhibited significant increases in level of expression throughout postnataldevelopment, they did not demonstrate notable regional or laminar disparity until adulthood.A striking result of these studies was the finding that peak densities of 5-HTic and 5-HT2receptors demonstrated transient columnar distributions within layer IV of area 17 in developingvisual cortex. Additionally, their differential intra-laminar and regional distributions offered novelevidence regarding a potential relationship of their columnar organization in visual cortex tofunctional afferent pathways. These results are discussed, in the following sections, with regard tocomparative 5-HT receptor-specific binding studies and the functional anatomy of the developingvisual cortex.Columnar SegregationPerhaps the most significant implication which emerges from the results of this study relatesto the transient columnar expression of 5-HT lc and 5-HT2 receptors in area 17 and, consequently,the potential role they may play in the formation of functional cortical columns, such as thoserelated to ocular dominance, orientation, or some other columnar property of visual cortex duringdevelopment.Ontogeny of Serotonin Receptors 5 6Other columnar markers. Despite abundant physiological evidence for the existence offunctionally similar columnar compartments among primates and carnivores, endogenousanatomical markers of any columnar organization in non-primate striate cortex had not beendescribed (see LeVay and Nelson, 1991 for review). The discovery that cytochrome oxidase-blobsare present in the visual cortex of adult cats (Murphy et al., 1990) and ferrets (Cresho et al., 1992),combined with evidence of a columnar distribution of 5'-nucleotidase (Schoen et al., 1990),cytochrome oxidase (Dyck and Cynader, 1992; Dyck and Cynader, submitted),acetylcholinesterase (Dyck and Cynader, 1992; Dyck and Cynader, submitted), serotonin receptors(Dyck and Cynader, 1990a; Dyck and Cynader, 1990b; Dyck and Cynader, submitted) andsynaptic zinc (Dyck et al., 1993; Dyck and Cynader, 1992; Dyck and Cynader, 1989) in youngkittens, indicates that this is no longer the case.The distribution of synaptic zinc (Zn), a vesicular component of a subset of glutamatergicterminals in cat visual cortex (Beaulieu et al., 1991; Beaulieu et al., 1992), is enriched in columnswithin layer IV of area 17 (Dyck et al., 1993; see Chapter 3). When compared in serially adjacentsections, Zn-rich columns are precisely coaligned with the 5-HT lc receptor columns describedhere (Dyck and Cynader, 1992; Dyck and Cynader, submitted; see Chapter 5). Moreover, we haverecently demonstrated that CO blobs in kitten visual cortex, are coaligned with patches of increasedstaining for acetylcholinesterase (AChE), and that the distribution of these CO / AChE blobs isprecisely complementary to 5-HT receptor / Zn columns (Dyck and Cynader, 1992; Dyck andCynader, submitted; see Chapter 5).The distribution of numerous columnar markers, and their relationships to CO-blob orinterblob zones, and to the functional organization of visual cortex, have been extensively studiedin primate striate cortex (see LeVay and Nelson, 1991 for review), but functional correlates ofcortical columns in the cat have not yet been described. We have recently demonstrated that Zn isalso distributed inhomogenously in primate striate cortex in a manner which is, both tangentiallyand laminarly, precisely complementary to CO (Dyck et al., 1993; Dyck and Cynader, submitted).In combination with the demonstration that the serotonergic innervation of striate cortex is moreOntogeny of Serotonin Receptors 5 7abundant outside of CO-blobs (Hendrickson, 1985), these studies indicate the possibility that aphylogenetic conservation of columnar markers in striate cortex may also reflect parallel functionalcompartmentalizations. A key question concerns the nature and properties of the columnarsystem(s) demarcated by the 5-HT receptors. In primates the CO blobs have been associated withseveral distinct functional and anatomical properties, including colour specificity, oculardominance, zones of low orientation selectivity, and differential inputs and output connectivity(Livingstone and Hubel, 1984a; Livingstone and Hubel, 1984b). Since the 5-HT receptor columnsreported here are precisely complementary to the CO blobs of the cat cortex, some of the samefunctional properties may be defined by the 5-HT system in development. Of the various featuresin primate, colour specificity is the least likely candidate to be defined by 5-HT receptors in cats.This is because cats have rather poor colour vision (Daw, 1973), and also because of evidencefrom nocturnal primates which indicates the presence of CO blobs, even in the absence of colourselectivity (Condo and Casagrande, 1990).Ocular dominance. In old world monkeys, CO blobs are in the centers of oculardominance columns and the same relationship may apply in cats. The temporal characteristics ofthe patchy distribution of 5-HT 1 c and 5-HT2 receptors parallel the time course for thedevelopmental plasticity of ocular dominance columns. The first signs of 5-HTic columns areevident around PD30, after the eye-specific geniculate afferents begin to segregate in layer IV(LeVay et al., 1978), and their columnar expression is highest through the developmental periodduring which ocular dominance columns are most sensitive to manipulations of visual input(Cynader et al., 1980; Hubel and Wiesel, 1962; Jones et al., 1984; Olson and Freeman, 1980).However, the spatial distributions of 5-HT receptor columns are not consistent with a directrelationship to ocular dominance. Anatomical and physiological studies indicate that oculardominance columns are spaced about 0.5 mm centre-to-centre, only one-half that of 5-HT receptorcolumns (Anderson et al., 1988; LeVay et al., 1978; LOwel and Singer, 1987; Shatz et al., 1977;Shatz and Stryker, 1978); a result which is substantiated by quantitative data showing that eachOntogeny of Serotonin Receptors 5 8cortical hemisphere contains approximately 600 ocular dominance columns (Anderson et al.,1988). Instead we estimate that each visual cortex contains around 300 5-HT receptor columns.Moreover, 5-HT 1 c receptors are only found in area 17, while ocular dominance columns arerepresented in areas 17 and 18. Finally, we have shown by direct comparison, in the same corticalsection, that the distributions of synaptic zinc-rich-columns, which are coaligned with 5-HTreceptor columns in area 17, are not explicitly related to ocular dominance columns labeledtransneuronally by intraocular injections of [ 3H]proline (Dyck and Cynader, 1992; Dyck andCynader, submitted).Orientation. In terms of their average centre-to-centre spacing, 5-HT receptor columnsappear to be more closely related to the orientation column system in cats (Hubel and Wiesel, 1962;LOwel et al., 1988). Metabolic mapping studies of the orientation column system in cat visualcortex using [ 14C]deoxyglucose, describe a regular system of parallel bands whose trajectory isorthogonal to the area 17 / 18 border (LOwel et al., 1987; Swindale et al., 1987). We do not findthat 5-HT receptor columns are found in bands, nor do they appear oriented along any preferredaxis (see Fig. 12). However, previous studies have indicated singularities in the cat and monkeycortical orientation maps, zones where different orientation bands coalesce and which containbroadly tuned neurons (Blasdel and Salama, 1986; Bonhoeffer and Grinvald, 1991; Swindale etal., 1987). These singularities are thought to be associated with CO-blobs in monkeys (Blasdel,1992) and may well have the same association in cat cortex. Since the 5-HT receptor columns arecomplementary to the CO blobs, these results imply that 5-HT receptor columns may distinguishneurons in the visual cortex which may show a relatively high degree of orientation tuning.Parallel processing streams. In the primate, the segregation of cortical compartmentsinto CO blobs and interblobs, has been functionally related to the hierarchical processing of colourand form perception which are, furthermore, related to similarly segregated geniculocorticalpathways (Hubel and Livingstone, 1987; Livingstone and Hubel, 1987a; Livingstone and Hubel,Ontogeny of Serotonin Receptors 5 91987b; but see Condo and Casagrande, 1990; Lachica and Casagrande, 1992). The lateralgeniculate innervation of cat visual cortex arises from three major classes of neurons (X-, Y-, andW-cells), which also form functionally, and anatomically separable processing streams (for reviewsee Sherman, 1985). The differential distributions of 5-HTic receptors appear to mirror the X-cellinnervation of visual cortex, while the 5-HT2 distribution appears to reflect non-X-cellgeniculocortical projections. Like the peak distributions of 5-HTic receptors, terminals of all X-cell axons are restricted to area 17, predominantly within deeper levels of layer IV, with lesserprojections to lower layer III and layer VI (Ferster and LeVay, 1978; Humphrey et al., 1985).Areas 18, 19 and LS, which receive no X-cell input, also do not contain high levels of[3H]mesulergine binding. Similar to the distribution of 5-HT2 receptors, Y- / W-cell projectionsarborize within upper levels of layer IV and lower layer III of areas 17 and 18 (Friedlander andMartin, 1989; Humphrey et al., 1985). Finally, high levels of 5-HT2, but not 5-HTic receptors,transiently demarcate the lateral suprasylvian cortex, which receives the majority of its geniculateinput from several classes of Y-cells in the C-laminae (Berson, 1985), and only a transient andsparse innervation from cells in the A laminae (Tong et al., 1991). Furthermore, the developmentof visual response properties of the suprasylvian cortex occurs between PD9 and PD15 (McCall etal., 1988; Price et al., 1988) at an age when 5-HT2 receptors exhibit a striking increase in theirlevels of expression.Corticocortical projections. Corticocortical connections among pyramidal neurons inlayers II/ III and V in the visual cortex of primates and non-primates are periodically distributed at1 mm intervals in the tangential plane (see Katz and Callaway, 1992 for review). When related toCO blobs in primate visual cortex, intrinsic cortical connections appear to be made reciprocallyamong CO-rich patches or CO-poor regions, but not between them (Livingstone and Hubel,1984b). Preliminary studies indicate that this relationship among CO blobs holds true forcorticocortical connections in cat visual cortex as well (Boyd and Matsubara, 1992). This patchypattern of intrinsic connections emerges early in development from an immature, homogenousOntogeny of Serotonin Receptors^ 6 0distribution (Callaway and Katz, 1990; Luhmann et al., 1986; Price, 1986), by a process of axonelimination, which is activity-dependent (Callaway and Katz, 1991; LOwel and Singer, 1992).Based on the interrelationship between the patchy distribution of 5-HT receptors and CO blobs incat cortex, it is possible that the transient columnar distribution of 5-HT receptors is related to theprocess of refining cortical interconnections during development. This is substantiated by theobservation that the critical period for the refinement of corticocortical projections ends by 14weeks (Dalva et al., 1992), at the same time that 5-HT receptor patches disappear.Comparative Studies5-HT1 Receptors. Among the numerous 5-HT receptor subtypes, the 5-HT 1 A subtypehas been the most intensively studied, primarily as a result of the development of the highlyselective radioligand [ 3H]8-OH-DPAT (Gozlan et al., 1983). Its specificity has been demonstratedto be identical in the neocortex of species as diverse as rat, pig and human (Hoyer et al., 1985;Hoyer et al., 1986a). Using [ 3H]8-OH-DPAT we have shown that 5 -HTIA receptor sites arehighly regulated in the cat visual cortex during development. Consistent with our results, levels of5-HT IA receptors in the cortex of developing rats (Daval et al., 1987) and humans (Bar-Peled etal., 1991) exhibit transient, 3- to 4-fold higher densities than in the adult. In the rat, levelsincreased markedly during the first 3 weeks after birth. This may be compared to the cat visualcortex, where the highest levels of 5 -HTIA receptors were demonstrated between 2 - 5 weekspostnatally. In human brain, the highest densities of 5-HTiA receptors were found in the fetalneocortex, between the 16th and 22nd weeks of gestation (Bar-Peled et al., 1991). Unlike the rat,where levels remain essentially stable through the remainder of the animals' lifespan (Daval et al.,1987; Gozlan et al., 1990), our observations of a progressive reduction of [ 3H]8-0H-DPATbinding density are consistent with results obtained in the aging human cortex (Cross et al., 1988;Dillon et al., 1991; Middlemiss et al., 1986).Ontogeny of Serotonin Receptors^ 61The ontogenetic expression of 5-HT receptors in cat visual cortex, analysed with [ 3}1]5-HT,is virtually identical to that of 5-HTiA receptors, suggesting that the 5-HTiA subtype may be thepredominant subtype of the 5-HT1 family found in cat visual cortex. During postnatal developmentof the kitten visual cortex, levels of [ 3H]5-HT binding in tissue homogenates increased from lowlevels at birth, to a peak at 4 weeks of age, and then subsequently declined to adult levels (Jonssonand Kasamatsu, 1983). In rat visual cortex, by contrast, the transient increase of [41]5-HTbinding was apparent during the first postnatal week followed by a progressive decline withincreasing age (Uzbekov et al., 1979; Zifa et al., 1988; but see Uphouse and Bondy, 1981; Zilleset al., 1985). Although providing circumstantial support, the usefulness of [ 3H]5-HT in studies of5-HT receptor subtypes is obviously constrained by its demonstrated lack of specificity.The temporal and laminar patterns of the distribution of 5-HTiA receptors did not varysignificantly between the different visual cortical regions examined. Except for the early postnatalperiod, 5-HTiA receptors were highest in supragranular cortical layers, lowest in middle layers,and attained intermediate levels in infragranular layers. Comparable laminar and ontogeneticanalyses of the distribution of 5-HTIA receptors in visual cortex of other species have not yet beenreported. Unlike the cat, the highest levels of [3H]8-OH-DPAT binding in the rat are localized todeeper cortical layers (Marcinkiewicz et al., 1984; Pazos and Palacios, 1985). On the other hand,the laminar pattern of 5-HT1A receptors among non-striate neocortical regions appears similarbetween adult cats and humans, but a detailed description of human visual cortex findings was notpresented (Dillon et al., 1991; Pazos et al., 1991; Pazos et al., 1987a). The laminar pattern of 5-HTiA receptors, which differentiates striate from the immediately adjacent, extrastriate visualcortical areas in the adult macaque, appears identical to the results presented here (Parkinson et al.,1989). In the cat, this distinct pattern develops between PD120 and adulthood as a result of alaminar- and region-specific reduction in [ 3E1J8-0H-DPAT binding. Developmental studies in thestriate cortex of primates are required to evaluate possible ontogenetic similarities between thesespecies.Ontogeny of Serotonin Receptors 6 2The resolution of in vitro autoradiography is not sufficient to visualize the cell bodies ofneurons or to determine the cellular localization of receptors. However, indirect evidence providedby neuron-specific lesions indicates that the 5-HTIA receptors are localized entirely on neuronsintrinsic to the kitten visual cortex (unpublished results). The regional colocalizations of 5-HT1AmRNA, assessed with in situ hybridization, combined with in vitro autoradiography, in the adultrat brain, support the idea that 5-HT1A receptors have a predominantly somatodendritic location(Chalmers and Watson, 1991; Pompeiano et al., 1992).5-HT2 Receptors. The mature distributions of 5-HT lc and 5-HT2 receptor subtypes havebeen extensively studied in the rodent, porcine, and primate brain (Blue et al., 1988; Gross-Isseroff et al., 1990; Hoyer et al., 1986b; Lidow et al., 1989; McKenna et al., 1989; Pazos et al.,1985; Pazos et al., 1991; Pazos et al., 1984b; Pazos and Palacios, 1985; Pazos et al., 1987a;Pazos et al., 1987b; Rakic et al., 1988). Consistent with the results presented here, 5-HT] . cbinding sites were reported to be present at low levels, with a slight preference in the middle layersreported in the adult human and rat occipital cortex (Hoyer et al., 1986b; Pazos et al., 1991; Pazosand Palacios, 1985; Pazos et al., 1987a). The localization of mRNA encoding the 5-HTic receptorin adult rodents, has confirmed and extended these results (Hoffman and Mezey, 1989; Mengod etal., 1990a; Molineaux et al., 1989).Possibly due to the greater availability of 5-HT2-specific ligands, the distribution of 5-HT2receptors has been more extensively studied, particularly in striate cortex. In cats, we found thatthe highest densities of 5-HT2 sites are equally distributed across I-IV in striate cortex, andpredominantly in superficial layers in extrastriate regions. In rats, the highest concentration of5-HT2 receptors was demonstrated in the middle cortical layers (III, IV and V), with highestconcentrations at the layer IV/V border (Blue et al., 1988; Mengod et al., 1990b; Pazos et al.,1985). In human and primate material, there are concentrations in layer III, IVa and IVc (Gross-Isseroff et al., 1990; Lidow et al., 1989; Parkinson et al., 1989; Pazos et al., 1991; Pazos et al.,1987b; Rakic et al., 1988). It is possible that these inconsistencies can be attributed to species-Ontogeny of Serotonin Receptors 6 3specific localization patterns. Alternatively, the majority of these studies used 5-HT2 antagonists(e.g. [3H]ketanserin, [ 125I]LSD), which have been shown to label more than one population of5-HT2 receptors (McKenna and Peroutka, 1989; Pierce and Peroutka, 1989) and/or differentialaffinity states of multiple receptor subtypes (Leonhardt and Titeler, 1989; Lyon et al., 1987).Very little is known of the ontogenesis of the 5-HT2 receptor family in other species orcortical areas. In this report we have demonstrated that the distributions, and levels, of 5-HTic and5-HT2 receptors are robustly regulated in the kitten visual cortex during postnatal development.The ontogenetic regulation of 5-HTic and 5-HT2 receptors has also been studied in rat and humanbrain, but have not been determined with regional specificity. In the whole rat brain, Roth et al.have reported that transient increases in 5-HTic and 5-HT2 sites during early development (Roth etal., 1991b), were accompanied by parallel increases in the corresponding mRNA (Roth et al.,1991a), which became markedly reduced with age. A marked reduction of 5-HT2 receptors inaged rats has also been reported by others (Battaglia et al., 1988; Gozlan et al., 1990). In addition,although studies of early development have not been reported, age-dependent reductions of 5-HT2receptors have been reported to occur in the human brain, beyond adolescence, during normalaging and in age-related disease (Biegon, 1991; Cross et al., 1988; Gross-Isseroff et al., 1990;Marcusson et al., 1984; Reynolds et al., 1984; Wong et al., 1984).The cellular localization of 5-HT lc and 5-HT2 receptors in the adult rodent brain has beeninferred, using in situ hybridization and lesion studies, to reside predominantly on non-serotonergic neurons which are intrinsic to the neocortex (Fischette et al., 1987; Hoffman andMezey, 1989; Mengod et al., 1990a; Mengod et al., 1990b; Molineaux et al., 1989). However,based on lesion studies, and the effects of input-manipulations reported previously (Dyck et al.,1991), it would appear that, at least during a distinct phase of postnatal development, 5-HT 1 creceptors are associated with axonal terminals of lateral geniculate neurons or possibly with glialcells. In addition, the transient, high levels of 5-HT2 receptors distributed within the subcorticalwhite matter suggests that a significant proportion of these binding sites are localized to glial cells.The resolution of the autoradiographic technique is not sufficiently fine to determine this withOntogeny of Serotonin Receptors^ 6 4certainty. A detailed analysis of these receptors using techniques providing cellular resolution arenecessary.As in other species (Hoyer et al., 1986b; Pazos et al., 1984a; Yagaloff and Hartig, 1985;Zilles et al., 1986), we also found very high concentrations of [ 3H]mesulergine binding sites in thechoroid plexus at all stages of development. Our results also indicate high levels of [ 125I]DOIbinding in the choroid plexus of the cat brain (see Fig. 2.11), which are consistent with previousfindings (McKenna et al., 1989).5-HT 3 Receptors. As mentioned in the results, the presence of 5-HT3 receptors in thedeveloping kitten visual cortex was not detected using currently available tritiated ligands.Consistent with binding studies in other species (Barnes et al., 1992; Kilpatrick et al., 1988;Kilpatrick et al., 1989; Waeber et al., 1989), we did find dense labeling in limbic structures,indicating that the lack of binding in visual cortical areas was not due to lack of sensitivity, or othermethodological issues. The development of radioligands with higher specific activity may help toallow autoradiographic localization of the low levels of 5-HT3 sites that the cortex may yetpossess.5-HT Uptake Site. A robust developmental regulation of 5-HTu p sites has beenpreviously described for the rat visual and somatosensory cortex (D'Amato et al., 1987). We alsofound that 5-HTup sites were developmentally regulated in the postnatal kitten visual cortex.Unlike the kitten, where we observe a gradual increase in expression until adulthood, which isessentially homogenous across cortical laminae, 5-HTu p sites in the developing rat are transientlyexpressed, during the early postnatal period, at very high levels within layer IV in primary sensoryareas (D'Amato et al., 1987). These 5-HTup sites are presumably localized on axon terminals ofserotonergic neurons which arise from the raphe nuclei (Bennett-Clarke et al., 1991; D'Amato etal., 1987). Apart from the apparent lack of a transient increase in expression during earlydevelopment, our descriptions of the regional and laminar distributions of 5-HTu p sites in the adultOntogeny of Serotonin Receptors 6 5cat are consistent with those found in human (Duncan et al., 1992), primate (Lidow et al., 1989)and rat (Duncan et al., 1992; Kovachich et al., 1988) visual cortices. In each of these studies, thedistribution of high affinity 5-HTu p sites was correlated with the density of serotonergicinnervation. The relationship of 5-HT receptor subtypes to serotonergic innervation of visualcortex is discussed in greater detail below.Relationship to Serotonergic InnervationAlthough the ontogeny of the serotonergic innervation of visual cortex has been investigatedin rats (Bennett-Clarke et al., 1991; D'Amato et al., 1987; Descarries et al., 1975; Lidov et al.,1980; Nakazawa et al., 1992; Papadopoulos et al., 1987), ferrets (Voigt and De Lima, 1991a;Voigt and De Lima, 1991b), cats (Gu et al., 1990; Mulligan and TOrk, 1988), and primates (deLima et al., 1988; Foote and Morrison, 1984; Kosofsky et al., 1984; Morrison et al., 1984;Morrison et al., 1982), corresponding data regarding 5-HT receptors during early postnataldevelopment are only available for the rat.In the rat, a transient hyperinnervation of primary sensory areas by raphe neurons (Bennett-Clarke et al., 1991; D'Amato et al., 1987; Nakazawa et al., 1992), is accompanied bycorresponding high levels of 5-HTup sites (D'Amato et al., 1987) and 5-HT1B receptors (Leslie etal., 1992). Somewhat similar to the rat, the expression of 5-HTu p sites in kitten visual cortexappears to be loosely correlated with its serotonergic innervation. In the second postnatal week (theearliest age studied), serotonin-immunoreactive fibers were equally and sparsely distributed acrosscortical laminae I - V (Gu et al., 1990), but by postnatal week 6 appeared adult-like, with fiberdensities greatest in superficial laminae, less dense in lamina V, and sparse in laminae IV and VI(Gu et al., 1990; Mulligan and TOrk, 1988). Among the different 5-HT receptor subtypesinvestigated, the laminar distribution of 5-HTiA receptors in the cat visual cortex most closelyresembled the pattern of its serotonergic innervation. Although their spatial distributions throughpostnatal development were similar, the marked increases of 5-HTiA receptors between PD20 andOntogeny of Serotonin Receptors 6 6PD40, followed by a reduction to adult levels, does not appear to be reflected by a correspondingchange in fiber density (Gu et al., 1990). Biochemical analyses indicate a marked increase in theendogenous concentrations of 5-HT in the developing kitten visual cortex between the third andfifth postnatal weeks (Jonsson and Kasamatsu, 1983), with a parallel transient increase throughoutthe brain (Daszuta et al., 1979), which is identical to the autoradiographic data, but in conflict withthe available immunocytochemical results (Gu et al., 1990). Furthermore, the ontogenesis ofserotonergic fibers in the ferret, a related carnivore whose cortical histogenesis is nearly identical tothat of the cat, demonstrates an increased density of serotonergic fibers during the equivalentpostnatal period (Voigt and De Lima, 1991b). These results suggest that this period of postnataldevelopment is potentially critical for the phenotypic maturation of serotonergic neurons.The relationship between 5-HT receptors, and particularly the 5-HTIA subtype, with theserotonergic innervation of visual cortex, appears more clearly defined in the adult cat.Immunocytochemical studies in the adult cat, indicate that the arbors of serotonergic axon terminalsare mostly restricted to the superficial laminae (I-III) and layer V (Gu et al., 1990; Mulligan andDirk, 1988). The predominantly superficial distribution of fibers is consistent with the highestlevels of the 5-HT1A, 5-HT2 and 5-HTup , but only the 5-HTjA receptor exhibits a higher level ofexpression specific to layer V. Although not reported in these studies, it would be interesting to seewhether the decreased density of 5-HT1 A receptors in area 17 and 18, relative to the directlyadjacent cortical regions, is reflected by a similarly sharp transition in the density of innervation byserotonergic fibers.The serotonergic innervation of the mammalian visual cortex arrives via two parallelascending projections, which are morphologically distinct, and originate from neurons in either thedorsal or median raphe nuclei (Molliver, 1987; Mulligan and Dirk, 1988). Their differentialdistributions and their relative contribution to the innervation of the visual cortex of cats duringdevelopment are not known, which precludes an analysis of their relative distribution to that of5-HT receptor subtypes. However, the laminar pattern of 5-HT2 receptors in the neocortex of ratshas been described to be associated with the fine axon projections which arise from the dorsalOntogeny of Serotonin Receptors 6 7raphe nucleus (Blue et al., 1988). Similarly, serotonin axons (Kosofsky and Molliver, 1987;Morrison et al., 1982) and synapses (de Lima et al., 1988) in primate striate cortex, are present inall cortical layers, but form especially prominent, dense bands of arborizing fibers from midlayerIII through Nal( (Kosofsky and Molliver, 1987; Morrison and Foote, 1986; Morrison et al.,1982), which is reflected by the distribution of 5-HT2 receptors (Lidow et al., 1989; Parkinson etal., 1989; Rakic et al., 1988). However, without the development of a method with improvedcellular resolution, which is compatible with double label studies (e.g. 5-HT receptor-specificantibodies), this relationship cannot be ascertained, nor extended to studies in the cat.The data presented here indicate that the regulation of serotonergic innervation and 5-HTreceptors are independently controlled during cortical development. Furthermore, the existence oftwo pharmacologically and anatomically separate ascending serotonergic systems, combined with amultiplicity of receptors to activate, provides a diverse range of responses which may be necessaryto guide the multiple processes involved in visual cortical development.ConclusionsIn the present study we have described the comparative distributions of the 5-HT 1A, 1C, 2and 3 receptor subtypes as well as the 5-HT uptake site, in the kitten visual cortex during postnataldevelopment. The temporal and spatial complementarity of their respective distributions indicatethat 5-HT might play multiple roles in different visual cortical regions and layers at different timesduring development. Furthermore, this first demonstration that specific 5-HT receptors aretransiently organized in columns of kitten visual cortex, argues for a specific role for 5-HT toparticipate in processes which determine the synaptic development and functional organization ofcolumnar systems in visual cortex. Investigations of the physiological consequences of activatingspecific 5-HT receptor subtypes in the visual cortex during postnatal development, together withanatomical studies to determine their precise cytological localization, are underway to address fullythis role for 5-HT.683ONTOGENIC DISTRIBUTION OFSYNAPTIC ZINC IN THE CATVISUAL CORTEXIt is apparent that the chelatable pool of zinc plays a complex role in the modulation ofneurotransmission in the adult brain (see Chapter 1). Its distribution in the adult neocortex (Haug,1967; Zilles et al., 1990) and hippocampus (Haug, 1967) is specific to neuronal terminals in alaminar-specific manner which might reflect an underlying functional, rather than just a chemical,specificity. Moreover, ontogenic studies of the hippocampal and striatal distribution revealdevelopmental gradients which appear to reflect underlying synaptogenic gradients (Crawford andConnor, 1972; Zimmer and Haug, 1978; Vincent and Semba, 1989). However, the ontogenicdistribution of zinc in the neocortex of mammals has not been examined.It has long been established that neurotransmission via the geniculocortical projection isconveyed by an excitatory amino acid (Hagihara et al., 1988; Tsumoto et al., 1986), and that it islikely to be aspartate or glutamate (Erdo and Wolff, 1990; Fosse et al., 1989; Miller et al., 1989;Tamura et al., 1990). However, the precise neurochemical identity has not been established byanatomical methods. We have determined, by ultrastructural colocalization, that glutamate-containing axonal terminals in the cat visual cortex are highly enriched with vesicle-bound zinc(Beaulieu et al., 1992). The histochemical localization of synaptic zinc may provide a procedure forexamining the development of, at least in part, the glutamatergic innervation of visual cortex. Ananatomical understanding of the ontogenic distribution of zinc-containing, glutamatergic terminalswould provide a starting point from which to determine their contribution to activity-dependentOntogeny of Synaptic Zinc^ 6 9mechanisms of plasticity during development. Using a modification of the selenium-histochemicaltechnique developed by Danscher (1982), the regional, laminar and ultrastructural distribution ofthe vesicle-bound, synaptic pool of zinc in the postnatal cat visual cortex are described.Materials and MethodsLight MicroscopyTissue preparation. The histochemical localization of zinc in the visual cortex of 28 catsranging in age from birth (postnatal day 0; PDO) to adulthood (> PD360) was assessed by amodification of the selenium method developed by Danscher (1982). We used three cats at PDOand PD10; two each at PD2, PD20, PD30 and PD40; six at PD50; one each at PD5 and PD75; andsix as adults. Under general anaesthesia (PDO - PD50, halothane to effect; > PD50, sodiumpentothal, 28 mg / kg i.v.) an intravenous injection of 20 mg / kg sodium selenite (20 mg / ml) wasadministered through the cephalic or saphenous vein at a rate of 1 ml / min. After a 15 - 20 minsurvival period the animals were perfused through the ascending aorta with 50 - 200 ml 0.1MSorenson's buffer (SB; pH 7.6). The brains were quickly removed, immediately frozen inisopentane (-70°C) and stored at -20°C. Coronal, cryostat sections (16 gm) were cut through thevisual cortex, thaw-mounted onto gelatin-coated glass slides and stored dessicated at -20°C inpreparation for histochemical staining. The brains of two PD50 and two adult animals werehemisected, the brainstem removed and the cortex unfolded and flattened according to a methodsimilar to that described by Olavarria and Van Sluyters (1985) in order to assess the tangentialpattern of zinc staining within individual visual cortical laminae. Fiduciary landmarks were created,by piercing the block with a 31 gauge needle, in order to facilitate alignment and reconstruction ofserial sections. Cryostat sections were cut at 20 - 30 gm, thaw-mounted on glass slides andprocessed in the same manner as the coronal sections.Ontogeny of Synaptic Zinc 7 0In order to assess and control for the degree to which non-specific staining might contribute tooptical density measurements, the brains of three additional animals (PD10, PD50 and adult),which had not received sodium selenite infusions, were processed identically to those of theexperimental groups.Histochemistry. All glassware was acid cleaned and solutions were prepared with deionizedwater to prevent contamination by exogenous zinc. In preparation for staining, the sections werethawed and allowed to dry at room temperature, fixed in a descending series of ethanol (95%, 15min; 70%, 2 min; 50%, 2 min), hydrated, and then dipped in 0.5% gelatin to prevent autocatalyticstaining.Zinc-selenide precipitate was visualized on slides by physical development in 200 ml of freshlyprepared developer containing 50% Gum arabic (120 ml), 2.0 M sodium citrate buffer (20 ml), 0.5M hydroquinone (30 ml) and 37 mM silver lactate (30 ml). In complete darkness, sections wereincubated in the developing solution at 26°C for 50 - 150 min, depending on the age of the animaland desired staining intensity. The histochemical detection of zinc in the PDO brain required adevelopment time of 150 min while animals of ages greater than PD50 required only 60 minutes inthe developer to achieve optimum staining intensity. These differences in developing timeprecluded a quantitative analysis of zinc distribution across different age groups and allowed us toonly make within group comparisons.After staining, slides were washed for 20 min in running tap water at 40°C to remove thegelatin coat, rinsed in distilled water (2 X 2 min), and stabilized in 5% thiosulphate for 12 min.Slides were postfixed in 70% ethanol (EtOH) for at least 30 min, dehydrated in 100% EtOH,cleared in xylene and coverslipped with Permount.Regional and Laminar Boundaries. Several approaches were utilized to relate the patternof zinc staining to laminar and regional borders. Selected sections were counterstained with cresylviolet which were then used to establish laminar borders in the various visual cortical areas basedon cytoarchitectonic criteria established by Otsuka and Hassler (1962) in adult cats and Shatz andLuskin (1986) for early postnatal development. The borders of visual cortical areas were alsoOntogeny of Synaptic Zinc 71determined according to the electrophysiologically defined maps of Tusa et al. (1978, 1979, 1980),which describe the relationships of regional borders and gyral patterns. Additional information wasderived from a number previous studies in which we utilized autoradiographic markers withlaminar-specificity or were found on lateral geniculate nucleus terminals in developing cat visualcortex (Prusky et al., 1987; Shaw et al., 1984; Shaw et al., 1986).Densitometric analysis and photography. Video images were captured and opticaldensity profiles across cortical layers of stained sections were generated on a Macintosh llfx-basedimage analysis system (Panasonic BD400 CCD camera; Data Translation DT-2255 quick captureboard) running Image software (NIH, v 1.42). Unless otherwise indicated, density profiles wereobtained across cortical laminae from area 17 at the level of the suprasplenial sulcus. Because thevariability in the developing time for the zinc staining process made it impossible to makequantitative comparisons across ages, the densitometric data were used only to establish within-animal differences in staining intensity between cortical areas and across cortical laminae.The reverse contrast photographic images of silver-stained brain sections were produced byprojecting the slide-mounted histological image from a photographic enlarger directly ontophotographic paper.Electron MicroscopyTissue Preparation. The electron microscopic localization of zinc in developing cat visualcortex was undertaken in 12 cats (PDO-1, n = 3; PD10-15, n = 2; PD30, n = 1; PD50-60, n = 2;adult, n = 4). The cats were prepared as in the light microscopic studies except that the SB washwas followed by perfusion with 200 - 500 ml SB containing 1.0 - 2.5% glutaraldehyde. The brainwas removed and the visual cortex (area 17 / 18) was blocked into 4 mm slabs which werepostfixed for 1 hour in the fixative. Tissue blocks were then washed for 15 min in several changesof 0.1M phosphate buffer (PB, pH 7.4). Vibratome sections were cut at 50 and 100 tm and thenstored, floating, in PB in preparation for the histochemical visualization of zinc.Ontogeny of Synaptic Zinc 7 2Histochemistry. Two different methods were employed to visualize the zinc-selenideprecipitate at the ultrastructural level in developing cat visual cortex. In the first, a series of sectionswas incubated, free-floating and in the dark, in the identical developer solution with developingtimes similar to those found optimal for light microscopic visualization. Developed sections werewashed twice, 5 min each, in PB prior to embedding.The zinc-selenide reaction product was also visualized in alternate series by silver enhancementusing the IntenSE M kit (Jannsen). Physical development by this method provided severaladvantages over traditional methods for zinc visualization; namely the tissue is processed forsignificantly shorter times in a solution that is light insensitive and of neutral pH (7.3). The tissuewas processed as directed in the accompanying brochure except that the sections were washed ineither distilled H2O, PB, or 0.1M citrate buffer (CB, pH 7.4) prior to development. Sections wereincubated for times ranging from 6 - 30 minutes to obtain optimal staining. After development, thesections were washed for 1 mM in each of 3 changes of the appropriate pre-incubation solution(dH2O, PB, or CB). Several sections were mounted on gelatin-coated slides, dehydrated inethanols and xylene, and coverslipped with Permount for light microscopic evaluation. Theremaining stained sections developed by either technique were prepared for embedding by washingin distilled H2O, PB, or CB and then postfixing for 30 min in 1.0% 0s04 dissolved in eitherdistilled H2O, PB, or CB. Following osmium fixation, the sections were again washed in distilledH2O, PB, or CB and then dehydrated in an ascending series of ethanol; 50% and 70% (5 mineach), 1.0% uranyl acetate in 70% (20 min), 90% and 95% (5 min each), 100% (2 x 10 min). Thedehydrated sections were immersed in two changes of propylene oxide (10 min each) andembedded between glass slides in Durcupan ACM resin (Fluka) which was allowed to polymerizeat 56°C for 2 days. Individual blocks representing supragranular, granular and infragranularcortical laminae were then microdissected and re-embedded.Ultrathin sections of silver interference colour were cut on an ultramicrotome Ultracut E,Reichert) and mounted on Pioloform coated, single slot copper grids. Some sections were contrastenhanced with a dilute solution of lead citrate. Grids were viewed under a Phillips model 410Ontogeny of Synaptic Zinc^ 7 3electron microscope and photographs of zinc-containing profiles were taken at magnificationsranging from 7,700X to 16,500X.ResultsTechnical Considerations and Controls. Our modification to the route of administrationof sodium selenite from that initially described by Danscher (1982) was not found to reduce thesensitivity of the selenite histochemical technique for visualizing zinc. Intravenous administrationof sodium selenite following induction of general anaesthesia required shorter survival periods thanwere necessary with i.p. or i m administration while maintenance of anaesthesia for the durationof the survival period eliminated the behavioural stress of selenium toxicity we had previouslynoted in the awake animal. These modifications were also found to lead to greater consistency ofthe histochemical results both among and within the various age groups studied.The ultrastructural distribution of zinc in the developing cat visual cortex was assessed andcompared by physical development and IntenSE M silver intensification (Jannsen). At the lightmicroscopic level, no obvious qualitative or quantitative differences were observed between thetwo methods. However, the Jannsen method resulted in much better preservation of the tissuewhen viewed at the ultrastructural level. This was particularly evident when citrate- or phosphate-buffered washes were applied rather than distilled H20. Physical development according to theoriginal method did, however, result in silver particles within positive structures whose sizestended to be more uniform. Similar observations regarding these two methods have been recentlymade by Stierhof et al (1991).Tissue sections from the visual cortex of control animals which were not injected with sodiumselenite, but were otherwise identically processed, were found to be devoid of silver reactionproduct regardless of the age of the animal or the development procedure used. In addition,densitometric measurements from these sections did not reveal any variation across corticallaminae. Neither intrinsic lamina-specific differences in optical density or non-specific backgroundOntogeny of Synaptic Zinc 7 4staining contributed to the densitometric measurements reported in this study. The laminar stainingobserved in the developing visual cortex thus reflects the intrinsic zinc distribution and is not aresult of artifactual, nonspecific staining.Ultrastructural Distribution of ZincAt the ultrastructural level, almost all of the histochemical reaction product was concentratedover synaptic vesicles regardless of the animal's age (Fig. 3.1). In the younger animals (<PD30)silver particles were, infrequently, observed outside the axon terminal. In these cases, they wereusually associated with either multivesicular bodies (Fig. 3.1B, open arrow) or with microtubules(not illustrated).When the synaptic contact of the positive terminal was cut at the appropriate angle, it alwayshad an asymmetric appearance (Fig. 3.1, large arrows). This distinct distribution confirmspreviously published results (Faber et al., 1989; Frederickson and Danscher, 1988; Haug, 1967;Perez-Clausell and Danscher, 1985a), and is consistent with the observation that zinc-containingvesicles are colocalized with glutamate in presynaptic terminals (Beaulieu et al., 1992). In Figure3.1D, the zinc-positive asymmetric synapse faces a symmetric, non-reactive contact (small arrow)on the same post-synaptic dendritic spine. At all ages, the majority of positive synapses areapposed to dendritic spines (Fig. 3.1A, B, D) and to a lesser extent onto small, distal dendriticshafts (Fig. 3.1C). Dendritic shafts were identified by the presence of mitochondria andmicrotubules. In contrast, dendritic spines were distinguished by their small diameter, absence ofmitochondria and microtubules, and the occasional appearance of a spine apparatus. No zinc-positive contacts were found on proximal dendrites or cell somata.The differential laminar distribution of zinc, which we describe at the light microscopic level, isreflected, at the ultrastructural level, by an apparent increase both in the total number of positiveterminals as well as in the density or number of zinc-positive particles within each terminal.However, a complete analysis is underway to test this observation critically.Ontogeny of Synaptic Zinc^ 75Figure 3.1. These electron microscographs demonstrate the ultrastructural localization of zincin visual cortex of kittens at postnatal day 1 (A), 10 (B), 30 (C) and in adult cat (D). At all agesstudied, histochemically visualized zinc was found over synaptic vesicles. These zinc-containingterminals always made an asymmetric synaptic contact on postsynaptic elements (large, solidarrows) which were most often found to be spines, based on their size and lack of microtubules.The zinc-containing vesicles were never found in terminals making symmetric synapses (D, smallarrow). In younger animals (Fig. 1A) the contact was often opaque and less well defined. Onlyoccasionally, and more often in young animals, when the zinc was visualized outside the axonterminal, it was either associated with microtubules or multivesicular bodies (B, open arrow).Scale bar in A, B, C = 0.125 iim and in D = 0.25 jam.76Figure 3.1Ontogeny of Synaptic Zinc^ 77Regional and Laminar Distribution of ZincAdult visual cortex. The distribution of histochemically reactive zinc in a representativecoronal section through one hemisphere of the adult cat brain is pictured in reverse contrast inFigure 3.2. Intense, zinc-positive staining (white) was essentially limited to hippocampus (Hpc)and cerebral cortex. Representative diencephalic (lateral geniculate nucleus, Lgn; medial geniculatenucleus, Mgn) and brainstem nuclei ( superior colliculus, Sc) found in this plane of section arerelatively unstained, as are fiber tracts (corpus callosum, cc).In the adult cat cerebral cortex, the typical laminar pattern of synaptic zinc was characterized byintense staining in layers I, II, III and V while layer VI was moderately stained and layer IV wasonly very lightly stained. Several borders between adjacent cortical areas were revealed by zincstaining although a general pattern of zinc staining throughout the cortex is evident (e.g. lateralsuprasylvian cortex, PMLS, PLLS). Areas 17 and 18 of visual cortex (boundaries indicated byarrows in Figure 3.2) were distinctly differentiated from adjacent cortical area 19 laterally andcingulate cortex ventro-medially by a conspicuous lack of staining in layer IV. Unlike areas 17 &18, the lateral boundary of visual cortical area 19 was not distinguished from adjacent corticalregions by zinc histochemistry.Figure 3.3 is a higher power view which displays the laminar pattern of synaptic zincdifferentiating adult visual cortical areas 19 (Fig. 3.3A, left), 18 (Fig. 3.3A, right), and 17 (Fig.3.3B). The distinct 19 / 18 border is indicated with an arrow in Figure 3.3A. The tissue-section inFigure 3.3A was counterstained with cresyl violet to demonstrate that, when compared to Figure3.3B, zinc-barren profiles in the zinc-stained tissue represent unstained neuronal somata. Opticaldensity profiles plotted in Figure 3.3C delineate the relative changes in staining intensity acrosslayers and between the three visual cortical areas. In each cortical area, layers I, II, III and Vclearly contain the highest concentrations of histochemically-reactive zinc while in layer VI, thestaining intensity is reduced by about 20%. The major difference in staining intensity betweenadjacent visual cortical areas is that layer IV in area 19 stains as darkly as layer VI while in area 17 /Ontogeny of Synaptic Zinc 7818, layer IV contains only slightly more zinc than the subcortical white matter, only 30% of that inlayers I-III & V. No staining was evident in the visual cortex of animals processed without sodiumselenite treatment. The laminar staining difference thus reflects the intrinsic zinc distribution and isnot a result of artifactual, nonspecific staining.Developing visual cortex. At birth (postnatal day 0, PDO; Fig. 3.4), visual cortical areas17 and 18 were distinguished from adjacent areas 19 and cingulate cortex (Cg) by the near-absenceof histochemically-reactive zinc. The cingulate cortex and area 19 contained a single lamina of zinc-positive staining deep within the cortical plate (CP) and layer V. The most intensely staining area inthe brain at birth was the region of mossy fiber terminals which originate from the granule cells ofthe hippocampal dentate gyrus (Hpc). At this age, and at all other ages studied, the histochemicalreaction product was not seen in cell bodies but was limited to the neuropil.Qualitative developmental changes in the distribution of zinc in the visual cortex of postnatalcats ranging in ages from PD2 through PD75 are illustrated in Figure 3.5. Although these arerepresentative figures, the laminar patterns and intensity of staining in visual cortex did not differsubstantially between individuals within each age group. The first indication of zinc staining withinvisual cortical areas 17 / 18 appeared between PD2 (Fig. 3.5A) and PD5 (not shown) and wasmanifested as a single faint lamina. Analysis of area 17 in the PD2 kitten at higher magnification(Fig. 3.6A, 3.6B) revealed that the single lamina containing the densest concentration of synapticzinc represents the lower CP and upper layer V. The optical density plot (Fig. 3.5A) confirms apeak distribution in this lamina and shows that layers I, VI and upper CP were evenly and equallystained. The bottom of layer I was characteristically punctuated by an unstained compact zone ofcells at the top of CP.The laminar differentiation, which distinguished areas 17 and 18 from adjacent cortex, becamemore apparent at PD10 (Fig. 3.5B, arrows). While most other neocortical areas contained a singlelamina of zinc-positive staining, visual cortex began to manifest a trilaminar pattern. The highermagnification photomicrographs of area 17 at PD10 (Fig. 3.6C, D) demonstrate that the highestOntogeny of Synaptic Zinc 7 9concentrations of histochemically-reactive zinc were found in layers III, V and the top of layer I.The corresponding optical density profile (Fig. 3.5B) confirmed a lesser relative distribution ofzinc in layer IV compared to that stained in layers III and V. This trilaminar pattern was essentiallymaintained at PD20 with the exception that layers II and III became equally stained and a narrowgap of reduced staining distinguished layer I from layer II (Fig. 3.5C).A considerable increase in the amount of zinc in the visual cortex was apparent between PD20and PD30 (Fig. 3.5D). Only a qualitative statement, however, based on staining intensity anddifferences in developing time, can be made in this regard. The distribution of zinc in the cat visualcortex at this age appeared to have attained its adult pattern of distribution with layers I-III and Vstaining intensely, and layers IV and VI less stained. However, as revealed in the optical densityplot, the relative intensity of zinc staining in layer IV was still at a moderate level, only slightly lessthan that of layer VI. In addition, a thin zone of reduced staining at the top of layer II could still bedetected at this age, particularly along the medial bank of area 17. This feature disappeared in theolder kittens.After PD30, changes in the relative intensity and pattern of zinc staining appeared to beconfined to layer IV of areas 17 and 18. The density of zinc staining in layer IV declined at PD50to approximately 75% of that in the most intensely staining layers I-III and V (Fig. 3.5E). Therelative reduction from layer IV at this age, however, was not uniform and appeared patchy (Fig.3.5E; Fig. 3.7, arrows). This patchy appearance in layer IV was not readily apparent in coronalsections at the other ages studied, including PD30 (Fig. 3.5D) and PD75 (Fig. 3.5F). The stainingof synaptic zinc within layer IV became reduced, at PD75, to almost 50% of that found in layers I-III and V (Fig. 3.5F). The relative reduction of synaptic zinc from layer IV compared to that oflayers I-III and V in adult cortex (-30%, Fig. 3.3C), however, was still not attained at this, thelatest pre-adult age studied.Ontogeny of Synaptic Zinc^ 8 0Figure 3.2. Distribution of synaptic zinc in a frontal section through the cortex of the adultcat. Histochemically reactive zinc, demonstrated here in reverse phase (white), is predominant intelencephalic areas such as cerebral cortex and hippocampus (Hpc) but avoids diencephalic andmesencephalic structures and fiber tracts such as the corpus callosum (cc). In the cerebral cortex,zinc staining distinctly differentiates visual cortical areas 17 and 18 from the subadjacent cingulatecortex (CG) and lateral area 19 (boundaries indicated by solid arrows), by avoiding layer IV(bounded by open arrows). (Lgn, Lateral geniculate nucleus; Mgn, Medial geniculate nucleus; Sc,Superior colliculus; cc, corpus callosum; PMLS, postero-medial lateral suprasylvian cortex; PLLS,postero-lateral lateral suprasylvian cortex; Scale bar = 2.0 mm).81Figure 3.2Ontogeny of Synaptic Zinc^ 8 2Figure 3.3. Higher power view of areal and laminar distribution of zinc in adult cat visualcortex. (A) The boundary between area 19 on the left and area 18 on the right is indicated by thearrow. (B) The distribution of zinc across cortical layers (I-VI) in area 17 is similar to that in area18 but is distinguished from that in area 19 by a relative lack of zinc in layer IV. The section in Awas counterstained with cresyl violet to demonstrate that the zinc-barren "holes" in B representunstained cell somata for figs. 3A & B. (C) Optical density profiles taken across visual corticallayers indicate little difference in staining intensity between layers I-III, V or VI of the differentvisual cortical areas; but the intensity of staining in layer IV of areas 17 and 18 is 50% less thanthat in area 19, at levels near that found in the white matter. (Scale bar = 250 pm).8341, +4. .^‘)1.V 11'tArea 19604020WM^VI v w III I M VI V IV m l WM^VI V IV III ICortical Layer0Figure 3.3Ontogeny of Synaptic Zinc^ 8 4Figure 3.4. Staining of synaptic zinc, which appears in this reverse phase photograph aswhite, avoids visual cortical areas 17 & 18 (boundaries indicated by arrows) of kittens at birth, butis demonstrable in area 19, cingulate cortex (Cg) and hippocampus (Hpc). (Scale bar = 1.0 mm)85Figure 3.4Ontogeny of Synaptic Zinc^ 8 6Figure 3.5. Postnatal changes in the distribution of synaptic zinc in kitten visual cortex arepictured in reverse-phase. Profile plots (inset) represent optical density measurements acrosscortical laminae (I-VI) in area 17 at the level of the suprasplenial sulcus, and are included tofacilitate the interpretation of laminar descriptions here and in the text. The boundaries betweenareas 17 / 18 and adjacent cortex are indicated by arrows. Synaptic zinc in areas 17 / 18 appearedsoon after birth, just below CP, as a single, dense layer of staining (A). At PD10 (B), the highestconcentrations of synaptic zinc are found within laminae V, III and the top of layer I. By beginningto avoid layer IV, the distribution of synaptic zinc differentiates areas 17 / 18 from adjacent cortex.This trilaminar appearance is still evident at PD20 (C); however, layers II / III have become moreintensely stained. At PD30 (D) layers I, II, III and V were most densely stained while layers IVand VI contained relatively less, and equal, densities of synaptic zinc. Changes in zinc stainingbetween PD30 and adult were limited to layer IV of area 17 / 18. This was seen as a relativereduction of staining to about 75% of that seen in supra- and infragranular layers at PD50 (E)which decreases further to 60% at PD75 (F). (Scale bar = 2.0 mm).7Figure 3.5 Ontogeny of Synaptic Zinc^ 8 8Figure 3.6. Zinc-stained (A, C) and near adjacent, Nissl-stained sections (B, D) throughvisual cortical laminae (I-VI) of area 17 at PD2 (A, B) and PD10 (C, D). Between PD2 and PD5,synaptic zinc first appears, and is most dense in the neuropil just below the cortical plate (CP). ByPD10 synaptic zinc is localized primarily to layers III, V and the top of layer I. The staining of zincin layer IV is slightly less intense than that of layers III and V, giving the PD10 visual cortex atrilaminar appearance. (Scale bar = 500 gm).Ontogeny of Synaptic Zinc 9 0Patchy Distribution in Layer IV. In cutting sections parallel to the surface of unfoldedand flattened cortex, the patchy distribution of zinc staining in layer IV of the PD50 visual cortexbecame much more apparent (Fig. 3.8A, C, E). Athough an irregular pattern of staining in layer IVof adult visual cortex was not distinguishable in coronal sections (Fig. 3.1 & 3.2), sections cuttangential to the surface revealed a patchy pattern, similar to that of the younger animals, but ofmuch lower relative intensity and contrast (Fig. 3.8B, D, F). The periodic pattern of zinc-stainingin tangential sections, robust in 20 jim sections from PD50 cortex (Fig. 3.8C), was only clearlyapparent, in sections from adult cortex which were at least 30 p.m thick (Fig. 3.8D).In addition to clearly revealing the inherent patchy distribution of zinc in layer IV, sections cutin the tangential plane provided information regarding the periodicity and areal distribution of thezinc-rich patches in layer IV. From the light-field micrographs in Figure 3.8, at levels near the top(A, B), middle (C, D) and bottom (E, F) of layer IV, it was apparent that patches of darker stainingin area 17 extended through layer IV and appeared to form spotted irregular bands whose directionof elongation varied with location in the cortex. The pattern of zinc-rich zones in area 17 appearedperiodic, with patches being, on average, around 400 ium in diameter and spaced 900 gm apart.Moreover, the patchy staining was restricted to area 17, while in area 18, the staining appearedhomogeneous.Ontogeny of Synaptic Zinc^ 91Figure 3.7. In this coronal reverse-phase photograph of PD50 visual cortex, staining forsynaptic zinc appears patchy in layer IV of area 17. Zones of decreased staining in layer IV areindicated by arrows. (Scale bar = 1.0 mm)92Figure 3.7Ontogeny of Synaptic Zinc^ 9 3Figure 3.8. Zinc stained sections at three levels through layer IV from opened and flattenedcortex of a PD50 kitten (A, C, E) and an adult cat (B, D, F). At both ages, the columnar staining inlayer IV was manifested as patches of darker staining at approximately 0.9 mm intervals, organizedin spotty bands oriented mediolaterally or obliquely. The appearance of zinc patches was coincidentwith that of layer IV and limited to visual cortical area 17; in contrast, zinc staining of layer IV inarea 18 appeared homogeneous. The boundaries of areas 17 & 18 are indicated by the dotted linesin D and E. The patchy pattern of staining in layer IV of the adult visual cortex was only visible in30 gm thick sections and was of much lower contrast and intensity than that of the younger animal.The position of fiduciary landmarks, used to facilitiate section alignment, are indicated by arrows.(A, anterior; L, lateral; M, medial; P, posterior; III, IV, V indicate cortical laminae; Scale bar = 4.0mm)94Figure 3.8Ontogeny of Synaptic Zinc^ 9 5DiscussionIn the adult cat, visual cortical areas 17 and 18 were conspicuously distinguished by thereduced staining density of zinc in layer IV. Layers I, II, III and V exhibited intense staining whilelayer VI was moderately stained. The pattern of staining in the laterally-adjacent visual cortical area19 more closely resembled that of most other neocortical areas; layers I, II, III and V were mostintensely staining, while layers IV and VI appeared equally and moderately stained.At birth (PDO) visual cortical areas 17 and 18 were distinguished from adjacent cortex by acomplete absence of staining for synaptic zinc. Soon after birth synaptic zinc in the visual cortexwas localized to a single lamina within and just below the cortical plate. By PD10, zinc-positivestaining was visible at the top of layer I and throughout layers III and V, while it was reduced inlayer IV. The first indication of a differentiation of area 17 and 18 from adjacent cortical areas by arelative reduction of zinc in layer IV was apparent at this age. At PD30 the distribution of synapticzinc appeared to attain its adult distribution, except that the relative density of zinc in layer IV wasalmost as high as in layer VI. The relative reduction of synaptic zinc from layer IV represented theonly observable, qualitative change in distribution from this age through adulthood. The reductionfrom layer IV was uneven and demonstrated a banded, patchy pattern in area 17.The laminar localization of zinc predominantly to layers I, II, III and V in the neocortex of adultmammals has been described for several other species (see Frederickson, 1989 for survey).Developmental changes in the distribution of histochemically reactive zinc have been demonstratedin the developing rat olfactory cortex (Friedman and Price, 1984), striatum (Vincent and Semba,1989) and amygdala (Mizukawa et al., 1989); however, the distribution and ultrastructurallocalization of zinc in the adult and developing cerebral neocortices have not been reportedpreviously. Several groups have demonstrated general increases in the total amount andconcentration of zinc per gram of wet weight brain tissue during the life span of humans (Volk etal., 1974) and rats (Crawford and Connor, 1972), but these do not differentiate synaptic from non-synaptic sources. The hippocampal mossy fibers, which contain the highest density of synapticOntogeny of Synaptic Zinc 9 6zinc in the brain, are the best characterized of the zinc-containing neuronal systems. In the rat(Zimmer and Haug, 1978) and cat (Frederickson et al., 1981), developmental gradients in thedistribution of histochemically localizable zinc in the hippocampal mossy-fiber region have beenreported to reflect developmental synaptogenetic gradients and may be related to synaptic maturity.The vesicular localization of zinc within terminals of zinc-containing neurons in the adult ratbrain has been well established (Haug, 1967; Holm et al., 1988; Perez-Clausell and Danscher,1985a). We have confirmed this in the cat visual cortex, and, in addition, have demonstrated thathistochemically-reactive zinc is present over synaptic vesicles in a subset of axon terminals of zinc-containing neurons making asymmetric contacts with postsynaptic targets, throughout postnataldevelopment. Although a quantitative stereological analysis was not performed in these studies,age-related increases of synaptic zinc in the developing rat amygdala have been described as beingdue primarily to an increase in the number of zinc-positive particles within each terminal(Mizukawa et al., 1989). It is likely that developmental variations in zinc staining observed in thecat visual cortex at the light microscopic level can be accounted for similarly.The vast majority of cortical zinc-containing terminals in the adult rat appears to be from localcortical interneurons rather than from long projection neurons (Hill and Frederickson, 1988). Thisappears true of cat visual cortex as well, since lesions which interrupt ascending brainstem,callosal, or geniculate afferents to visual cortex in the adult cat are unsuccessful in altering theoverall laminar pattern of zinc staining (Dyck and Cynader, unpublished data). However, in lightof the results presented here, an assessment of the effect of manipulations of input and/or visualexperience on the expression of zinc-rich patches in layer IV should prove interesting (Chapter 4).The postnatal changes in zinc staining in kitten visual cortex appear to reflect known anatomicaland functional developmental landmarks. In supragranular layers, the cells which will inhabitlayers II and III are still migrating at birth, and do not assume their final positions until about 3weeks postnatal (Shatz and Luskin, 1986). Functional corticocortical synapses are established insupragranular layers around PD30 (Toyama and Komatsu, 1987), at the age when the mature,patchy distribution of local cortical connections becomes defined (Callaway and Katz, 1990), andOntogeny of Synaptic Zinc 9 7the essentially mature pattern and density of zinc staining in the superficial layers are established.In layer IV, the first signs of the geniculocortical projection can be detected shortly after birth(Shatz and Luskin, 1986). Around 3 weeks postnatal, these inputs begin to segregate into oculardominance columns within layer IV (LeVay et al., 1978). This segregation, which is input- andactivity-dependent, is complete by eight or nine weeks of age. The transient, patchy staining ofzinc in layer IV suggests that synaptic zinc may contribute in the mechanisms of column formation,such as ocular dominance or orientation, in the visual cortex during the critical period. Only twoother endogenous molecules have, to date, been shown to be expressed transiently in patchesduring the critical period for cat visual cortical development (Dyck and Cynader, 1990a; SchOen etal., 1990; but see Chapter 5). When compared in adjacent sections, the transient columnardistribution of serotonin receptors localized with autoradiographic techniques, is coextensive withzinc positive patches (Dyck and Cynader, 1992; Chapter 5). In addition, the CNS glycoprotein5'-nucleotidase, which is a major component of myelin (Cammer et al., 1980), and regulated byzinc (Mallol and Bozal, 1983), is transiently localized to ocular dominance columns with the sameperiodicity and laminar distribution as that described for zinc patches (Schoen et al., 1990).Finally, histochemical zinc has been demonstrated to transiently demarcate barrels insomatosensory cortex of the developing rat (Akhtar and Land, 1990). Thus, the transient, patchystaining of zinc in area 17 further indicates a potential contribution of zinc-relatedneurotransmission to cortical column formation.Functional Considerations. The precise physiological role of a releasable pool of zinc inthe adult central nervous system has not yet been established. However, zinc has been implicatedin regulating excitatory and inhibitory neurotransmission via interaction with opiatergic (Stengaard-Pedersen, 1982; Stengaard-Pedersen et al., 1981a; Stengaard-Pedersen et al., 198 1 b; Stengaard-Pedersen et al., 1983), GABAergic (Legendre and Westbrook, 1991; Smart and Constanti, 1990;Smart et al., 1991; Westbrook and Mayer, 1987) glutamatergic (Christine and Choi, 1990;Legendre and Westbrook, 1990; Peters et al., 1987; Westbrook and Mayer, 1987), serotonergicOntogeny of Synaptic Zinc 9 8(Peters et al., 1988) and glycinergic (Yeh et al., 1990) receptors. The Zn-sensitivity of opiatereceptors has prompted the suggestion that Zn may act as the endogenous ligand at 6t2 sites in therat hippocampus. Recent evidence suggests that, in the developing brain, endogenous zincmodulates GABAergic neurotransmission by acting on both GABAA (Smart and Constanti, 1990;Smart et al., 1991) and GABAB (Xie and Smart, 1991) receptor subtypes. In these and otherstudies (Draguhn et al., 1990), the sensitivity of GABA receptors to zinc is described as decreasingwith age and to be receptor subunit specific, suggesting a specific role for synaptically releasedzinc in modulating inhibitory processes in development.An important characteristic of the cat visual cortex which has been extensively studied is themarked modifiability of neuronal responsiveness by visual experience early in development. Thephenomenon of long-term potentiation (LTP), a possible mechanism responsible for synapticmalleability and best characterized in the hippocampus (Bliss and Lynch, 1988), has also beendescribed in both adult (Artola and Singer, 1987) and developing visual cortex (Connors and Bear,1988; Komatsu et al., 1981). In the adult visual cortex, Toyama and Komatsu found that synapticpotentiation was greater in the superficial layers, which receive corticocortical synapses, than inlayer IV, which is the recipient of geniculocortical synapses (Toyama and Komatsu, 1987). Oculardominance plasticity is predominantly localized to layer IV in the young kitten but in the zinc-richlayers II, III, V and VI in older kittens (Fox et al., 1989); these layers also contain the highestabundance of NMDA receptors (Fox et al., 1989). Although excitatory neurotransmission from thelateral geniculate to layer IV of cat visual cortex is mediated by both NMDA and non-NMDAreceptors (Tsumoto et al., 1986), the induction of LTP requires the activation of postsynapticNMDA receptors, presumably by glutamate. Some additional factor, perhaps zinc, co-releasedwith glutamate may be essential (Kauer et al., 1988; Weiss et al., 1989), or zinc could simplypotentiate LTP induction by the blockade of GABAergic inhibition (Westbrook and Mayer, 1987).In the adult visual cortex, the induction of LTP requires activation of NMDA receptors with theconcomitant reduction of GABAergic inhibition (Artola and Singer, 1987), a phenomonenonOntogeny of Synaptic Zinc^ 9 9which has been well characterized in the hippocampus (Douglas et al., 1982; Wigstriim andGustafsson, 1983).The transduction of extracellular signals to functional messages within the cell is thought to belinked to controlled fluctuations in the levels of the intracellular messenger calcium. As a divalentcation, zinc can replace or modify the effects of calcium at specific intracellular binding sites andthereby further influence signal transduction pathways (Csermely et al., 1989; Csermely et al.,1988). Several calcium binding proteins whose functions have been reported as significantregulators of intracellular calcium, such as calcyclin (Filipek et al., 1990), calmodulin (Baudier etal., 1983) and S100 (Baudier, 1988; Baudier and Gerard, 1986; Baudier and Gerard, 1983;Baudier et al., 1986; Baudier et al., 1984; Baudier et al., 1983; Baudier et al., 1985), actually bindzinc with higher affinity than calcium, or alter their affinity to calcium in the presence of zinc. Theconcentrations of these and several metallothionin-like zinc binding proteins appear to bedevelopmentally regulated, implicating important zinc-protein and zinc-calcium interactions in thedevelopment and the maturation of the brain (Ebadi, 1986; Ebadi and Hama, 1986). Calcium ionsalso mediate the activation of one branch of the phospholipase C - protein kinase C (PKC) signaltransduction cascade (Nishizuka, 1986). Zinc has been shown to be able to enter postsynapticterminals via voltage-gated Ca2+ channels (Wang and Quastel, 1990). Once inside, zinc has theability to activate PKC, either directly (Murakami et al., 1987), or by regulating the interaction ofphorbol dibutyrate with PKC (Forbes et al., 1990) to induce PKC translocation to membranes(Csermely et al., 1988). In the developing cat visual cortex, PKC is transiently expressed inpresynaptic terminals of layer IV at a time when the concentration of zinc is highest in layer IV and,furthermore, is found predominantly in superficial and deep layers in the adult cortex (Jia et al.,1990). Zinc has also been demonstrated to produce a strong blockade of Ca2+ current throughdihydropyridine-sensitive Ca2+ channels (Winegar and Lansman, 1990). This effect appears to bechannel-selective (Biisselberg et al., 1992). In the cat visual cortex, the expression of these sites,visualized by in vitro autoradiography using PN200, is limited to layer IV at birth but is high inOntogeny of Synaptic Zinc^ 1 00superficial and deep layers after P20 (Cynader et al., 1990), following a similar distribution andtime course as the distribution of synaptic zinc.The establishment during development, and maintainance in maturity, of the synaptic form ofthe CNS is to some degree dependent on a group of molecules which mediate trophic interactionsbetween individual cellular components. Zinc plays an integral role in the function of one of thebest characterized of such molecules, namely the nerve growth factor (NGF). Zinc ions areinvolved in the activation process of the gamma-subunit esteropeptidase, and that activationinvolves removal of zinc ion from the native 7S NGF (Pattison and Dunn, 1976a; Pattison andDunn, 1976b). The NGF proprotein contains and is stabilized by zinc ions; removal of zinc fromthe 7S component of NGF alone, without dissociation, is sufficient to allow expression of itsesteropeptidase activity (Greene and Shooter, 1980; Pattison and Dunn, 1976a; Pattison and Dunn,1976b). In this sense, zinc serves to act as a control ion which keeps the NGF protein in aninactive form (the zymogen) until it recognizes its naturally occurring substrate (Young andKoroly, 1980). A role for zinc as a critical factor in the regulation of NGF-mediated trophicphenomena in the hippocampal formation has been reported , but such a role has not yet beenrecognized in the cerebral cortex. Whether zinc plays an analogous role for other members of theNGF family, such as BDNF and NT-3, remains unknown.Conclusions. Patterns in the distribution of synaptic zinc appear to be developmentallyregulated and reflect the process of synaptic maturation of a subset of glutamate-containing neuronswhich project to the developing visual cortex in a laminar- and region-specific manner.Furthermore, the patchy distribution of synaptic zinc in layer IV of area 17 suggests that zinc maycontribute to the mechanisms of column formation during the critical period in development withinwhich the visual cortex exhibits use-dependent plasticity. This anatomical description of theontogenic localization of zinc and the redistributions involved in attaining the adult pattern placeimportant constraints on the precise role of zinc-containing neurons in the processes of visualcortical development and plasticity. These possibilities are further addressed in Chapter 4.1014ACTIVITY-DEPENDENT COLUMNAREXPRESSION OF SEROTONIN RECEPTORSAND ZINCThe anatomical description, in Chapters 2 and 3, of distributions of serotonin (5-HT)receptors and zinc (Zn) which are transiently expressed in columns at high levels, are suggestive oftheir participation in processes whereby particular columnar domains are defined. Moreover, thecolumnar expression of these molecules was found to be highest during the period within whichthe visual cortex exhibits an enhanced sensitivity to activity-dependent modifications of itsorganization. The decline in sensitivity to visual deprivation closely parallels the reduction in levelsof the columnar expression of these molecules. As outlined in Chapter 1, the segregation of lateralgeniculate axons into ocular dominance columns, is susceptibile to modifications of visual input-and experience. If 5-HT receptors and Zn participate in activity-dependent mechanisms of columnformation, such as those for ocular dominance, then their expression in visual cortex should, likethese columnar systems, be constrained by early visual experience. This hypothesis is addressedby the studies described in this chapter.The deprivation procedures were divided into two general categories; first, those whichmodified visual experience, but left visual pathways intact; and second, those which interruptedvisual input, by inactivating successive levels of the visual processing pathway. Figure 4.1outlines, in schematic form, the different procedures utilized to determine the effects ofmanipulating visual experience or input, on the expression of 5-HT receptors and Zn. Thedependence of 5-HT receptor and Zn expression, on normal binocular visual experience wasActivity-Dependent Expression 10 2assessed by subjecting kittens to: 1) a short period of monocular deprivation (MD) by eyelid suture(Fig. 4.1B), 2) long-term MD either by eyelid suture (Fig. 4.1B) or by unilateral enucleation (Fig.4.1C), or 3) binocular deprivation, by rearing kittens from birth in complete darkness (Fig. 4.1D).The role of binocular visual input in the normal development of the neurochemically definedcolumns was determined by selectively eliminating the participation of different visual processingpathways by: 1) unilateral enucleation (Fig. 4.1C), 2) unilateral optic tract section (Fig. 4.1E), 3)LGN aspiration or inactivation of intrinsic neurons with tetrodotoxin (Fig. 4.1F). The effects ofrestricting cortical visual experience on the columnar expression of 5-HT1c/2 receptors and Zn wasassessed in these kittens at the age during which they display the highest levels of their column-specific expression, around PD50. If related to ocular dominance, for example, then modificationsin the columnar appearance of these molecules might be expected to vary from normal (Fig. 4.1A),in the same manner ocular dominance columns are affected (cortical schematics, Fig. 4.1B-F).If any of these molecules are on cells which receive direct input from lateral geniculateneurons or alternatively, on geniculate terminals, then their participation in processes involved incolumnar segregation might be substantiated. Although the columnar expression of 5 -HT1c/2receptor subtypes in geniculate recipient laminae support this possibility, direct evidence is notavailable due to the poor resolution of the autoradiographic method. In a preliminary assessment,the cytological localization of 5-HT receptor subtypes to cells intrinsic to visual cortex or onafferent axon terminals, was inferred from the effects of neuron-specific lesions produced by aninfusion of quinolinic acid. The utility of this toxin, which is reported to be selective to neurons,while leaving glial cells and the fibers and axon terminals of neurons outside the lesion zone intact(Schwarcz et al., 1983), has been determined previously for other neurotransmitter systems(Prusky et al., 1988; Shaw et al., 1989).Activity-Dependent Expression^ 103Figure 4.1. Schematic diagram illustrating the manipulations of visual input andexperience imposed on kittens. The outcome of the columnar expression of 5-HT receptors andZn, following each of these manipulations, is schematically predicted for visual cortical area 17, bythe effects of monocular deprivation (B, C), binocular deprivation (D), optic tract section (E) andLGN lesions (F); based on the assumption that their distributions are affected the same as those ofeye-specific inputs into ocular dominance columns. A. The normal columnar projection of red andgreen eye-specific afferents through the lateral geniculate nucleus to their respective corticalcompartments in the visual cortex is shown. Note that there is normally a contralateral bias(exaggerated here) of the representation of each eye in the visual cortex. B.The elimination ofpatterned visual input from one eye during early development, induced by eyelid suture, results ina reduction of cortical space innervated by and influenced by the deprived eye. C. Corticalrepresentation is exclusively maintained by the remaining eye following early enucleation. D. Thedeprivation of any photic stimulation during the animals' lifetime, results in a reduction of thenormal segregation of ocular dominance columns (exaggerated here, for effect). E. Limiting visualcortical input to that generated spontaneously by neurons in the lateral geniculate nucleus, wasassessed by completely depriving geniculate neurons of visual input, by early optic tracttransection. F. The contribution of the lateral geniculate nucleus, to the columnarcompartmentalization of these markers in visual cortex, was assessed by eliminating the influenceof even spontaneous activity from neurons in the lateral geniculate.LateralGenicul aleNucleusLateralGeniculaleNucleusLateralGeniculaleNucleusEnucleationSitatiMUNAMMVIEfaeMEALTORISIMICI^MEMAIMIFZIEKW.A^ I - 3• .cilliMiSIONOMMISISHARNEW4giniftlidMonoculareprivation• ,^v ............^e^e ......LateralGeniett I ateNucleus\kftx\\%\‘'w\r‘•NeLateralGen telltaleNucleusOptic Tract' etionLateralen kill aleNucleusLGIN Lesion'CrFigure 4.1Activity-Dependent Expression^ 105Materials and MethodsAnimals and Deprivation ProceduresTwenty-two kittens served as subjects in the various experiments described here. Asummary of the kittens and the procedures of visual input- / experience-deprivation are summarizedin Table 2 and Figure 4.1, respectively. The procedures used for perfusion, tissue preparation andfor the autoradiographic and histochemical staining techniques were described previously, in detailin Chapters 2 & 3.Monocular Deprivation. Monocular visual deprivation was induced either by eyelidsuture (MD) or by enucleation (ME). Eyelid suture was performed under halothane anaesthesia (toeffect) either near the age of eye-opening (n=4) or 1 week prior to perfusion (n=2). All animalswere checked daily to assure the integrity of sutures. For ME, kittens (n=6) were anaesthetizedwith halothane, the extraocular muscles and optic nerve were transected, and the entire globe wasexcised. The eyelids were sewn together and the animals were reared normally.Binocular Deprivation. Two kittens were raised in complete darkness from birth untilPD51 (raised by Dr. D. Mitchell, Dalhousie University). On the day of perfusion, one kitten wasbrought into the light and allowed 2 hours of unrestricted vision prior to perfusion. The other wasanaesthetized while still in the dark and its eyes were kept covered until the brain was removed.Optic Tract. Kittens (n=2) were anaesthetized with halothane, the optic tract wasaccessed using the trans-buccal approach (Lepore et al., 1983), and then transected unilaterallyunder direct visual guidance (performed by Dr. F. Lepore, University de Montreal).Activity-Dependent Expression^ 106Table 2. Summary of Animals and Manipulation of Visual ExperienceCat Procedure Age at Procedure Age at Perfusion MarkerBK209 Right MD PD49 PD55 1A, 1C, 2, ZnK91-12 Right MD PD40 PD52 1A, 1C, 2, ZnMDZn Right MD PD8 PD50 1A, 1C, ZnK91-21 Right MD PD8 PD50 1A, 1C, 2, ZnK92-6 Right MD PD10 PD50 1A, 1C, 2, ZnK91-29 Right MD PD5 PD50 1A, 1C, 2, ZnK91-0 Right ME PD5 PD50 1A, 1C, ZnK91-17 Right ME PD3 PD52 1A, 1C, ZnK91-27 Left ME* PD8 PD50 1C, ZnBK369 Left ME* PD2 PD56 1C, ZnBK370 Left ME* PD2 PD56 1C, ZnBK431 Left ME* PD5; PD53** PD66 Zn, ProlineC502 Dark Rear PDO PD51 1A, 1C, 2C503 Dark Rear PDO PD51 + 2 hr*** 1A, 1C, 2OPZn Left Optic Tract PD10 PD50 1A, 1C, ZnBK240 Left Optic Tract PD20 PD51 1A, 1C, ZnRC77 TTX Right LGN PD23 PD37 1C, 2RC78 TTX Left LGN PD23 PD37 1C, 2RC79 TTX Right LGN PD23 PD37 1C, 2RC68 Asp Right LGN PD40 PD47 1CUC561 QA PD58 PD65 1A, 1C, 2, UpUC567 QA PD57 PD64 1A, 1C, 2, Up*Tangential sections **Intraocular [ 3H]proline injection ***2 hr visual exposureI A, IC, 2, Up: Autoradiography for 5-HT I A, 1C, 2 receptor subtypes, and 5-HT uptake site respectively.Zn, zinc histochemistry; PD, postnatal day; QA, quinolinic acid; TTX, tetrodotoxin; Enuc, enucleation;MD, monocular deprivation by eyelid suture; Asp, lesion by aspiration.Activity-Dependent Expression 107Lateral Geniculate Nucleus. One kitten was anaesthetized with halothane and the rightLGN was accessed and aspirated via a lateral approach using visual and stereotactic guidance(performed by Dr. M. Cynader). The other three kittens had a cannula implanted in the LGN (A,3.5 mm; L, 8.0 mm, H, 11.0 mm) which was attached to an osmotic minipump (Alzet 2002), andtetrodotoxin (TTX, 100 tM in ACSF) infused at a rate of 0.5 tl / hr for 2 weeks (performed byDr. Qiang Gu, University of British Columbia).Quinolinic Acid. Two kittens were anaesthetized with halothane to effect and small holeswere drilled through the skull at three antero-posterior levels overlying area 17 in one hemisphere.One microliter of quinolinic acid dissolved in saline (pH 7.4; 0.3 p,M / ge was infused, over 5min, through a 30 gauge cannula lowered 1.2 mm below the cortical surface at each site. Theanimals survived 7 days prior to perfusion.ResultsThe effects of visual deprivation were most clearly reflected by changes in the distributionof the 5-HTic receptor subtype. The normal, characteristic pattern of [ 3H]mesulergine binding atPD50 consisted of a continuous band of dense binding at the base of layer IV. From this band,periodic, radially oriented columns of receptors emerged and extended through layer IV into thelower part of layer III. This pattern became progressively more distinct with age, and peaked atPD50. At this age, the greatest density of 5-HTic receptors in neocortex was strictly limited tolayers III/IV of visual cortical area 17 (see Figs. 2.1, 2.9, and 2.10, Chapter 2). When comparedto the patterns observed following the varous manipulations in Figure 4.2, it is apparent that theincidence and contrast of the columns of [ 3H]mesulergine binding in visual cortex was unalteredby a short period of MD (Fig. 4.2A). An extended period of MD resulted in a reduction of bindingin the columns perforating layer IV of both hemispheres (Fig. 4.2B). The normal organization of5-HTic-rich columns in layer IV of area 17 exhibited an even greater reduction, bilaterally, withlong-term ME (Fig. 4.2C). The complete restriction of visual information imposed by dark-rearingActivity-Dependent Expression 1 0 8kittens from birth to PD51, prevented the segregation of 5-HTic receptors into columns in layer IVof visual cortex (Fig. 4.2D). Instead, a thicker than normal, dense band of binding, extendedthroughout layer IV of area 17 in these kittens. This pattern of binding was unchanged even when2 hours of visual exposure was given prior to perfusion. These results indicate the importance ofvisual exposure in general and of binocular vision in particular, in the formation of 5-HT 1 creceptor columns during development.In none of the manipulations of visual experience, did the pattern or levels of 5-HT 1 Areceptors appear different than normal. The effects of restricting both visual experience and input,were non-existent or negligible (compare Figs. 4.3A, 4.6B; with Figs. 2.1, 2.10 in Chapter 2). Bycontrast, the effects of visual deprivation on the columnar patterns of 5-HT2 receptors and Zn,although less well-defined than for 5-HTic receptors, appeared to be similarly affected. Theeffects of the various manipulations on the distribution of Zn, and 5-HT1A and 5-HT2 receptorsubtypes are described in the following sections, and are compared and contrasted to the effectsobserved on 5-HTic receptor expression.Unilateral Enucleation. Long-term ME resulted in effects on Zn and 5-HT j c/2receptors which were apparent even in sections cut in the frontal plane (Fig. 4.3). The bindingpattern of [ 125I]DOI to 5-HT2 receptors (Fig. 4.3C), was distinctly different than that of kittensraised normally to PD50 (Figs. 2.1, 2.10; Chapter 2). The densest binding was expressed at thelayer III / IV border, as was normally observed; however, the periodic pattern of binding was notclearly apparent (Fig. 4.3C). In addition, a reduced density of binding within this lamina in area18, combined with a relative increase in the levels of binding within lower strata of layer IV clearlydemarcated area 17 from area 18 (compare to adjacent section labeled with [3H]mesulergine, Fig.4.3D). Similarly, the distribution of Zn in layer IV of kittens raised to PD50 with only one eye(Fig. 4.3B), appeared more homogenous than that observed normally (see Fig. 3.8, Chapter 3).Although apparent in the frontal plane, the effects were more clearly seen in sections through layerIV which were cut tangential to the cortical surface (Fig. 4.4, Fig. 4.5). These figures demonstrateActivity-Dependent Expression 1 0 9the distribution of 5-HTic receptors and Zn in near adjacent sections, from two kittens (who werealso littermates) raised from PD2 to PD56 with only the left eye removed. In both kittens there wasa marked decrease in the level and contrast of 5-HTic receptors and Zn expressed in patches inarea 17 bilaterally. This reduction appeared to be represented by a decrease in the level of contrastbetween patch and interpatch regions, and also by a reduction in the size of the patches (compare toFig. 2.12 for 5-HTic and Fig. 3.8 for Zn). However, the total number of 5-HTic-rich columnsdid not appear to be different from normal (enucleates (n=4), x = 292; control (n=2), x = 305.5).The effect of early enucleation was not identical in each animal examined. The resultswhich are depicted for three different animals in Figures 4.3-4.5 (K91-0, Fig. 4.3D; BK369, Fig.4.4; BK370, Fig. 4.5), are representative of the range of effects observed, among all the kittensstudied thus far. A bilateral asymmetry in the residual levels of the columnar expression of both Znand 5-HTic receptors was observed in only 2 of the 6 animals (K91-0, Fig. 4.3D ; BK369, Fig.4.4). This disparity could not be attributed to whether the deprived eye was ipsilateral orcontralateral. Furthermore, although the column-specific labeling for both Zn and 5-HT1 creceptors was attenuated in all animals, the effects of enucleation appeared to exert a greater effecton the expression of Zn patches (Fig. 4.5).Dark-Rearing. As described above, and depicted in Figure 4.6, the effect of binoculardeprivation by dark-rearing, on the distribution of 5-HTic receptors was marked (Fig. 4.6C, C').This was also the only manipulation which produced an effect on the normal pattern of 5-HT1 Areceptors (Fig. 4.6B). During early postnatal development (PD 10 - PD40, but not beyond PD40;Fig. 2.1B-F & 2.10B, Chapter 2), 5-HT1A receptors demarcate visual cortical areas 17, 18 and 19from adjacent cortical regions by relatively increased levels of expression. In Fig. 4.6B, thedistribution of 5-HT1A receptors clearly demarcate area 17 from adjacent regions. However, dark-rearing did not change the complementary laminar-specificity exhibited by the relative distributionsof each 5-HT receptor subtype described in Chapter 2 (compare Fig. 4.6A with Fig. 2.10A).Similar to the changes observed in the 5-HTic receptors, the dense band of binding at the layer IIIActivity-Dependent Expression 110/ IV border, characteristic of 5-HT2 receptors at this age, appeared more homogenous. The effectof binocular deprivation, by dark rearing, on Zn-staining in visual cortex was not examinedbecause neither of the two animals studied thus far was treated with sodium selenite prior toperfusion.Optic Tract Lesion. The sections in Figure 4.7, through the visual cortex of twodifferent kittens (kitten OPZn, A-D; kitten BK240, E, F), are representative of the effects ofunilateral optic tract transection on the distribution of 5-HTic receptors (Fig. 4.7A, D, E) and Zn(Fig. 4.7B, D, F). The overall effect of transecting the optic tract, in both animals, was to reducethe columnar expression of 5-HTic receptors in the ipsilateral hemisphere (hemisphere on rightside of panel). Columns of 5-HTic receptors in layer IV, emerging from the dense band at thelayer IV / V border, were clearly present in both hemispheres of kitten OPZn (Fig. 4.7 A, C), butthey were greatly reduced in the ipsilateral hemisphere, particularly in the anterior-most sections ofarea 17 (Fig. 4.7C, right hemisphere). In kitten BK240 (Fig. 4.7E, F), the normal columnarappearance of 5-HTic receptors in the left hemisphere of panel E, was contrasted by the completelack of columnar segregation in layer IV on the lesion side (right hemisphere). In addition todiffering in their age at time of surgery (see Table 2), the two kittens differed in the extent of theirlesions; the transection was incomplete in kitten OPZn (Fig. 4.7A-D), but complete in BK240(Fig. 4.7E, F). In the near-adjacent sections stained for Zn, changes in the frequency of Zn-positive columns appeared to mirror those of 5-HTic columns, but not nearly as clearly (Fig. 4.7B, D, F). The effect of unilateral optic tract transection on the expression of 5-HT2 receptors hasnot yet been examined.Activity-Dependent Expression^ 111Figure 4.2. The effect of manipulating visual experience on the subsequent columnardevelopment of 5-HT 1 c binding sites in visual cortex was assessed in kittens who weremonocularly deprived for one week by eyelid suture (A); from the time of eye-opening by eyelidsuture (B) or unilateral enucleation (C); or were dark-reared from birth (D). All animals surviveduntil PD50. [3H]Mesulergine-labeled columns were still present, with normal contrast, in area 17of kittens monocularly-deprived by eyelid suture for a short duration (A) but were somewhatreduced following long-term deprivation (B). The expression of 5-HTic columns in layers III / IVof area 17 was significantly reduced in both hemispheres of the unilaterally enucleated kittens, butthe lamina-specific binding at the bottom of layer IV appeared unaffected (C). The segregation of5-HTic receptors into columns was prevented in kittens who were raised in the dark from birth(D). Scale bar = 2 mm.112Figure 4.2Activity-Dependent Expression^ 113Figure 4.3. The effect of long-term monocular deprivation by enucleation, on thedistribution of 5-HT1A (A), Zn (B), 5-HT2 (C), and 5-HTic (D) in near-adjacent sections from thevisual cortex of kitten K91-0 (sections A, C, D are serially adjacent). A. The laminar- andregional-specificity of 5-HTiA receptors was left unchanged following unilateral enucleation fromPD5 (see FIg. 10). B. Although not as clear as in tangential sections (see Fig. 24; Fig. 25), thedistribution of Zn in layer IV of area 17 appeared more homogenous than in the normal PD50visual cortex (see Chapter 2). C. A reduction in the binding of [ 1251]DOI to 5-HT2 receptors,along the dense band at the layer III / IV border in area 18 relative to area 17, combined withincreased levels of expression in lower strata of layer IV, distinguished area 17 from area 18 in theunilaterally enucleated kitten. This pattern was not present in normal animals of the same age. D.The expression of 5-HTic columns in layers III / IV of area 17 was significantly reduced in bothhemispheres of the unilaterally enucleated kittens, but the lamina-specific binding at the bottom oflayer IV appeared unaffected . Scale bar = 2 mm.114Figure 4.3Activity-Dependent Expression^ 115Figure 4.4. The effect of enucleation of the left eye at PD2 on the tangential distributionof 5-HTic receptors (top) and synaptic zinc (bottom) in the right (R) and left (L) hemispheres ofkitten BK369 at PD 56. The columnar expression of both 5-HT1 c receptors and Zn wassignificantly reduced relative to normal PD50 visual cortex (Chapters 2, 3). This reduction, wasreflected in a reduced diameter, and contrast, of 5-HTic columns, because the absolute number ofcolumns did not vary significantly from normal. In this animal, the attenuation of column-specificlabeling for both Zn and 5-HT lc receptors was bilaterally asymmetric, with losses greater in thehemisphere ipsilateral to the enucleated eye. Scale bars = 10 X 2 mm.116Figure 4.4Activity-Dependent Expression^ 117Figure 4.5. The effect of enucleation of the left eye at PD2 on the tangential distributionof 5-HTic receptors (top) and synaptic zinc (bottom) in the right (R) and left (L) hemispheres ofkitten BK370 at PD 56. The columnar expression of both 5-HT1 c receptors and Zn wassignificantly reduced relative to normal PD50 visual cortex (Chapters 2, 3). This reduction, wasreflected in the diameter, and contrast of 5-HTic columns, because the absolute number ofcolumns did not vary significantly from normal. In this animal, a greater effect was observed onthe column-specific expression of Zn, where patches of increased Zn-staining in area 17 wererarely observed. Scale bars = 10 X 2 mm.118Figure 4.5Activity-Dependent Expression^ 119Figure 4.6. The effect of dark-rearing on the expression of 5-HTiA (B), 5-HTic (C, C')and 5-HT2 (D, D') receptors, in serially adjacent sections (B, C', D'; C, D), at two caudo-rostrallevels (C, D). The relative laminar distribution of 5-HT receptors was not affected by dark-rearing.The characteristic complementarity of peak expression levels in area 17 were not different fromnormal (see Fig. 2.10A). However, compared to normal, the columnar segregation of either5-HT lc or 2 receptors was not apparent. Instead, each exhibited virtually homogenous levelsthroughout their respective laminar strata. Moreover, the expression of 5-HT1 A receptors wasabnormally high in area 17, relative to adjacent visual cortex, compared to that seen normally at thisage (compare with Fig. 2.1E, F & 2.10B) Scale bar = 2 mm.120Figure 4.6Activity-Dependent Expression^ 121Figure 4.7. The effect of a unilateral optic tract section on the expression of 5-HT 1 creceptors (A, C, E) and synaptic zinc (B, D, F) in two animals (OPZn, A-D; BK240, E, F).Although the optic tract section in the kitten OPZn (A-D) was incomplete, a marked reduction in thecolumnar expression of both Zn and 5-HTic receptors was apparent, particularly at rostal-mostportions of area 17 (C-D). With a complete transection (E, F) the columnar segregation of 5-Hricreceptors (E) and Zn (F) was completely repressed. Scale bar = 2 mm.122Figure 4.7Activity-Dependent Expression 12 3Lateral Geniculate Nucleus. Intrageniculate infusions of TTX compromised both thelevels and patterns of 5-HTic and 5-HT2 receptor binding in kitten visual cortex (Fig. 4.8). In allcases, the effect on binding appeared to be limited to the visual cortex ipsilateral to the infusedLGN. A reduction in the laminar-specific binding of [ 125 1]DOI to 5-HT2 (A), and of[3H]mesulergine to 5-HTic receptors (B), in kitten RC78 was virtually complete, except for asmall region of 5-HT lc receptors in the ventral-most portion of area 17 (B, arrow). Similarly, in adifferent animal (RC77), a distinct border in the reduction of 5-HT2 receptors, suggests thatalterations in binding levels are directly related to the extent of LGN inactivated. These results weresubstantiated by a complete reduction of the laminar-specific binding pattern of 5-HTic receptorsobserved following removal of the LGN by aspiration (Fig. 4.8D).Quinolinic Acid. The effect of neuron-specific lesions in visual cortical area 17, on thedistribution of 5-HT receptor subtypes at PD50, is shown in Figure 4.9. The differential effect ofquinolinic acid lesions on the levels of expression suggests that these receptors might be localizedto different cytological compartments within area 17. The virtual elimination of all 5-HT1 Areceptors within the lesion zone suggests that these receptors are localized on neurons intrinsic toarea 17 (Fig. 4.9A). However, the 40% reductions in the binding of [ 3H]CN-IMI and [ 125I]DOI to5-HTup (Fig. 4.9C) and 5-HT2 receptors (Fig. 4.9D), suggest that they are, in part, localized toextrinsic elements or glial cells. Although the expression of 5-HT2 receptors was reduced tobackground levels in superficial and deep cortical laminae, 60% remained within the dense band atthe layer III / IV border, perhaps an indication of expression on two different cytological elements.On the other hand, the expression of 5-HTic receptors, although slightly reduced (< 10%), is stillexpressed in columns, in laminar-specific manner, potentially indicating a localization on afferentfibers (Fig. 4.9B).Activity-Dependent Expression^ 124Figure 4.8. The effect of unilateral LGN aspiration (D) or inactivation by TTX (A-C), onthe expression of 5-HTic (B, D) and 5-HT2 (A, C) receptors. A reduction in the laminar-specificbinding of 5-HTic and 5-HT2 receptors was observed in all animals, in the hemisphere ipsilateralto the TTX infusions and lesion. The binding indicated by the arrows in B and C, observed in twodifferent animals (RC78, A, B; RC77, C), probably reflects residual activity of 5-HT2 (C) and5-HTic (B) receptors in the LGN resulting from incomplete TTX infusion. Residual binding of5-HT receptors was not observed in any portion of the visual cortex of kitten RC68, wherecomplete LGN aspiration was confirmed (D). Scale bar = 2 mm.125Figure 4.8Activity-Dependent Expression^ 12 6Figure 4.9. Effect of quinolinic acid lesion on binding of [ 3 H]8-OH-DPAT (A),[3H]mesulergine (B), [ 1251]1)01 (C) and [3H]CN-IMI (D) in near adjacent sections through area 17of PD50 visual cortex. The effect of the lesion on levels of 5-HT receptors, compared with thesham injected hemisphere, is reflected in each panel by the density plots. The outline of eachdensitometric slice used to generate these profiles is indicated, in each panel, by the rectangle. Thelimited effect of a quinolinic acid lesion on the binding of [ 3H]mesulergine in area 17 suggests thatthe 5-HT lc receptors are located on terminals of neurons projecting to area 17, or on glial cells. Onthe other hand, the complete reduction in binding of 5-HTiA receptors, implies that these receptorsare strictly localized to cortical cells intrinsic to area 17. The significant reduction in the binding of[ 1251]D01 and [3H]CN-IMI within the lesion site suggest that 5-HT2 receptors and 5-HTu p sitesare also, at least partially, localized to elements intrinsic to cortex. Arrows indicate borders of theprimary necrotic zone. Scale bar = 2.0 mm.I2 9.8p0.60.4rts 0.26.0^5.0^4.0^3.0^2.1^1.1^0. 1Distafice (mm)0.sAi 0.6!,." 0.4tt,* 11.27,4zg6.0^5,0^4.0^3,0^2.1^1.1^0.1„Distance(nun) ,MINN=111•11101•10118••••• 0.6-0.4-=as 0.24+ 0 -5.0 4.0^3.9^2.1^1.1^0.11Distance (mm)Activity-Dependent Expression^ 128DiscussionThe results of the various experiments described in this chapter indicate that the normallevels of expression, and the distributions, of several neuroactive molecules which demarcate aparticular columnar organization of visual cortex, are constrained by early visual experience. Thehigh levels of expression of Zn, 5-HTic and 5-HT2 receptors in columns in geniculate recipientlaminae of the developing visual cortex, described in Chapters 2 & 3, were suggestive of thepossibility that these molecules could be regulated by visual experience. The effects of alteringvisual experience during early development on an animals' visual abilities in later life are profound.Anatomical changes to the visual pathway which underlie the obvious behavioural deficits havebeen extensively examined (for reviews see Movshon and Van Sluyters, 1981; Sherman andSpear, 1982). It is highly likely that they also represent the basis for the changes in 5-HT receptorexpression and Zn distribution that have been described here.As indicated in Chapter 1, abnormalities produced by monocular deprivation (MD) havebeen proposed to result from a competitive disadvantage placed on the deprived eye, by the non-deprived eye, in acquiring cortical territory (Wiesel and Hubel, 1965). After several weeks ofmonocular deprivation, the majority of cells in the visual cortex are responsive to visual stimulationonly through the eye which had been left open (Wiesel and Hubel, 1963b). The locus of effect isnot in the eye, or in the lateral geniculate nucleus, but in the visual cortex, at the site wherebinocular inputs converge (reviewed in Movshon and Van Sluyters, 1981; but see Sherman andSpear, 1982, and discussion below). The predominant anatomical effect seen in the cortex is thatthe normal geniculocortical projection is physically rearranged as a consequence of MD, with acorresponding shift in ocular dominance represented by a reduction of afferents representing thedeprived eye and a corresponding increase in cortical space occupied by the nondeprived eye(Shatz and Stryker, 1978). Although responses of visual cortical neurons to visual stimulation aredriven almost exclusively by the non-deprived eye, a significant visual cortical projection ofgeniculate axons representing the deprived eye is still present (Garey and Blakemore, 1977; LinActivity-Dependent Expression 129and Sherman, 1978; Shatz and Stryker, 1978; Spear and Ganz, 1975). Similar to that seen forocular dominance, the columnar distributions of 5-HT receptors and Zn also appear to reflect thelevel of competitive activity arising binocularly. The levels of Zn and 5-HT 1 02 receptors incolumnar compartments were unaffected by short periods of monocular deprivation and were onlyslightly reduced following eyelid suture before eye-opening. By contrast, a much greater, bilateralreduction in the degree of columnar expression of both 5-HTic receptors and zinc was apparent asa result of unilateral enucleation prior to eye-opening. In one kitten showing no evidence ofresidual Zn columns, the organization of non-deprived eye inputs were homogenously distributedthroughout the visual cortex bilaterally (Fig. 1.1). Thus, a reflection of the severity with whichcompetitive interactions are reduced appears to be mirrored in the residual columnar expression ofthese molecules. Recently Bliss-Tieman has shown that deprived geniculocortical cells make fewerand abnormal synapses in layer IV of visual cortex, but similar to the expression of 5-HTic andZn described here, they are restricted to ocular dominance columns which are faint and usually failto extend into extragranular layers (Bliss-Tieman, 1991). These results substantiate transneuronaldata which indicate that the distribution of deprived eye terminals appears more widespread inlower layer IV (Shatz and Stryker, 1978). These data, combined with the results of monoculardeprivation presented here, indicate that diffuse visual information, provided through the suturedeyelid (Spear et al., 1978), appears sufficient to drive the columnar segregation of 5-HT receptorsand Zn. Moreover, the observation that columnar segregation continues, albeit abnormally, withretinal input from only one eye, indicates that spontaneous activity in the deprived laminae of thelateral geniculate itself might be sufficient to organize cortical comparments based on binocularcompetition.Many lines of evidence indicate that the effects of visual deprivation on the LGN areminimal; however, a selective vulnerability of geniculocortical Y-cell pathways to both monocularand binocular deprivation has been reported (Kratz et al., 1979; Sherman et al., 1972; reviewed inSherman and Spear, 1982). Based on pharmacological studies, this physiological phenomenon hasbeen attributed to the formation of abnormal excitatory synaptic connections. When inhibitoryActivity-Dependent Expression 130neurotransmission is antagonised in visual cortex by infusion of bicuculline, the ability of thedeprived eye to drive cortical cells is reversibly restored (Burchfiel and Duffy, 1981; Duffy et al.,1976; Sillito et al., 1981). These data are supported by the observation that early MD produces areduction in intracortical inhibition (Tsumoto and Suda, 1981; Wilson and Sherman, 1976),attributed to a loss of Y-cells, which are thought to mediate such inhibition in normal cats (Singeret al., 1976; Tsumoto, 1978; see also discussion of Y-cells below). That many cortical cells wouldreceive functional excitatory synaptic contacts from a greater proportion of X-cells, might provide afunctional basis for the homogeneity of Zn-containing fibers in layer IV observed to resultfollowing enucleation (Figs. 4.3-4.5). Based on the precise colocalization of Zn columns, with theX-like distribution of 5-HT 1 c receptor columns (Dyck and Cynader, 1992; Dyck and Cynader,submitted; see Chapter 5), Zn-containing glutamatergic terminals and X-cell terminals may be oneand the same. Although the results provided by physiological recording are apparently confoundedby problems of sampling error, a large number of anatomical reports are indicative of a selectivereduction of Y-like cells in the LGN and, correspondingly, Y-cell terminals in the visual cortex(reviewed in Sherman and Spear, 1982). Much of this support stems from the reduced incidence ofY-cells labeled retrogradely from tracer injections in area 18. The geniculate projection to area 18arises exclusively from Y-cells in the A lamina, whereas area 17 receives mixed X- and Y- inputs,albeit to different laminar strata (see Discussion, Chapter 2). Furthermore, the expression ofCAT301, a Y-cell specific antigen, is significantly reduced with monocular and binoculardeprivation (Sur et al., 1988). Some support for this hypothesis is provided in this study by theselective abnormal reduction of 5-HT2 receptors in area 18 following early unilateral enucleation(Fig. 4.3B). The data presented in Chapter 2, although inferential, indicate that 5-HTic and 5-HT2receptors likely differentiate geniculocortical X- and Y-pathways, respectively. Further support forthese inferences might be provided by assessing the effect of deprivation on the transientexpression of 5-HT2 receptors expression in the lateral suprasylvian cortex, where receptive fieldsof neurons are abnormal (Spear and Tong, 1980), and which receives exclusive input from Y- andpossibly W-cells (Berson, 1985).Activity-Dependent Expression 131The normal laminar-specific expression of 5-HT1c/2 receptors prevailed following theelimination of all photic stimulation during development but their segregation into normal columnarcompartments was prevented. The segregation of geniculate terminals into ocular dominancecolumns in the visual cortex does develop, to a limited extent, in dark-reared and binocularly lid-sutured cats (Stryker and Harris, 1986; Swindale, 1981). Considering that ocular dominancecolumns do not segregate when the retinal ganglion cells have been silenced by intraocular infusionof TTX (Stryker and Harris, 1986), it is apparent that spontaneous activity of retinal ganglion cellsprovides a sufficiently patterned input to permit some aspects of the activity-dependent molding ofneural connectivity recognized in visual cortex. However sufficient spontaneous activity is for thesegregation of ocular domains, it does not appear to be adequate for the columnar segregation of5-HT receptors.The predominant effect of dark-rearing in the kitten visual cortex is the prolongation of thecritical period during which functional changes can be induced by monocular deprivation (Cynaderand Mitchell, 1980; Mower et al., 1981). This delay in maturation is reflected by numerousphysiological (reviewed in Sherman and Spear, 1982) and morphometric parameters (O'Kusky,1985; Takacs et al., 1992). The present results, where we describe the altered distribution of 5-HTreceptors after 7 weeks of dark-rearing, confirm and extend those described previously (Mower,1991). There, the effects of dark-rearing cats to an age of 4 - 5 months were described by anincrease in the number of receptors labeled with [ 3 H]5-HT, which was specific to thesupragranular and infragranular laminae of visual cortex (visual cortical regions were notdifferentiated, nor were temporal data presented). In animals raised in parallel,immunocytochemical results demonstrated that this increase in binding was not reflected by anincrease in serotonergic fiber density (Mower, 1991). Due to the nonselectivity of [ 3H]5-HT,Mower was not able to discriminate the effect of dark-rearing on individual receptor subtypes.However, together with the results described thus far in this thesis, it is apparent that the delay inmaturation of the visual cortex is reflected by a parallel delay in the temporal patterns of each of the5-HT receptors examined. The high density of 5-HTiA receptors in area 17 relative to adjacentActivity-Dependent Expression 132visual areas after 7 weeks of dark-rearing, is similar to that seen at earlier stages of developmentbut not normally at PD50 (see Fig. 2.1A-E), and probably represent the majority of receptorsdescribed by Mower. Similarly, the homogenous distributions of 5-HTic and 5-HT2 receptors aresimilar to those found in younger kittens, where levels of binding were homogenous in layer IV(Fig. 2.1A'-E'; Fig. 2.1A"-E"). On the basis of studies indicating the recovery of orientationselectivity and ocular dominance column formation (Cynader and Mitchell, 1980; Swindale, 1988),these results might predict that the process of 5-HT1c/2 receptor segregation would proceed withsubsequent exposure to light.The effects of dark-rearing were not evaluated with respect to the normal columnardistribution of synaptic zinc, but several indices of glutamatergic neurotransmission have beenfound to be dramatically altered in kitten visual cortex as a result of dark-rearing. Physiologicalevidence for a delay in the developmental decrease in visual cortical NMDA-receptor efficacy inlayers IV, V, and VI has been reported for kittens raised to PD43-49 in the dark (Fox et al., 1991).The normal distribution of NMDA-receptors labeled with [ 1251]MK-801 progresses from ahomogenous distribution across laminae during the first few postnatal weeks, but by PD50 ispredominant in supragranular layers. Ligand binding data from the same kittens as those reportedin this chapter, indicate that glutamatergic receptors labeled with [ 1251]MK-801 are homogenouslydistributed across all laminae (Cynader et al., 1991). Thus, the similar effect of dark-rearing, todelay the redistribution of 5-HT and glutamatergic receptors, might also be imposed on thecolumnar segregation of glutamatergic, zinc-containing fibers. This remains to be seen.The effects of early optic tract transections on the columnar expression of 5-HT receptorshave been determined only from two animals. Even though they might be considered preliminary,as such, the effects observed on the expression of 5-HTIC/2 receptors contribute significantly to anunderstanding of the observed effects of visual deprivation. The consequences of an optic tractsection could be considered to be analogous to dark rearing one cortical hemisphere, except withrespect to the contribution of spontaneous retinal ganglion cell input. The observation that somecolumnar segregation of 5-HT 102 receptors occurred in the animal with an incomplete lesion ofActivity-Dependent Expression 133the optic tract, compared to no apparent segregation in the animal with a complete optic tracttransection, provides evidence that columnar segregation in the cortex requires normal binocularinput. Residual columnar expression of 5-HT receptors was higher in posterior portions of thevisual cortex (Fig. 4.7A), which might be correlated with the specificity of the optic tract fibers cut;however, the high degree of retinotopic order found in the LGN and visual cortex is not reflectedby a similar organization in the pathway of fibers in the optic tract (Horton et al., 1979).From the results of direct manipulation of geniculate input, it is apparent that thecontribution of spontaneous activity by neurons of the deafferented lateral geniculate nucleus wassufficient for the laminar-specific expression of 5-HT1c/2 receptors, but not for their segregationinto columnar compartments. The selective reduction of 5-HT1c/2 receptors in layer IV of visualcortex following LGN inactivation, either by lesion or TTX infusion, indicates that their laminar-specific expression at this age is dependent on the integrity of the LGN. Evaluation of the lateralgeniculate nucleus, in Nissl-stained sections near the infusion site, indicates that neurons near theinfusion site are not pyknotic, suggesting that the effects of TTX were not due to neurotoxicity.The question arises as to whether 5-HTic and 5-HT2 receptors are localized directly on LGNterminals, or are highly regulated in an activity-dependent manner, on cells directly under theinfluence of geniculate input. The lack of a significant effect of quinolinic acid lesions on thelaminar-specific distribution of 5-HTic receptors, contrasted by a complete reduction of 5-HT1Areceptors, are highly indicative of the cytological distribution of 5-HT 1 c receptors on afferentfibers, compared to a possible localization of 5-HT 1 A receptors on intrinsic neurons. Althoughthere is evidence for 5-HT receptors on cortical astrocytes, they are believed to belong to the5-HT1 family (Whitaker-Azmitia, 1988; Whitaker-Azmitia and Azmitia, 1986). The significantreduction in the levels of high affinity 5-HT uptake sites following quinolinic acid lesions isdisconcerting because reuptake mechanisms for 5-HT are thought to be localized predominantly onthe same nerve terminals from which it is released. However significant evidence exists for highaffinity uptake of 5-HT into cortical astrocytes (Kimelberg, 1988; Kimelberg and Katz, 1985). Thereduction of uptake sites by lesions induced by quinolinic acid puts into question the interpretabilityActivity-Dependent Expression 134of the varied effects on the expression of the other receptor subtypes observed here. However,limited evidence indicates that quinolinic acid, a tryptophan metabolite which is endogenous to thecortex, and whose levels are developmentally regulated (Moroni et al., 1984), has a neurotoxicaction on serotonergic terminals in the hippocampus (El Defrawy et al., 1986). If serotonergicterminals in the kitten visual cortex are damaged by quinolinic acid, then the reduction of 5-HT2receptors and 5-HTup sites by almost 40%, indicates their representation on serotonergicterminals. The remaining 5-HTu p sites could be on glial cells (Kimelberg, 1988; Kimelberg andKatz, 1985), and the residual 5-HT2 receptors could either be on glial cells or afferent fibers.Further investigation is required to address this question.The majority of studies assessing the consequences of visual deprivation have emphasizedthe effects imposed on ocular dominance column formation, although detrimental effects on othercolumnar features of visual cortex have also been described. The arrangement of orientationcolumns is similar to ocular dominance in that they are likely to be determined by the arrangementof geniculocortical afferent inputs (Stryker et al., 1990; Chapman et al., 1991). However, theircolumnar segregation is impaired by binocular (suture or dark-rearing), but not monocularrestriction of visual experience (Blakemore and Price, 1987; Cynader et al., 1980 but see Fregnacet al., 1981).The differential effects of monocular, versus binocular, deprivation are very similar tothose observed in the organization of horizontal connections during development. Althoughdisputed, there is some evidence that columnar compartments, defined by patchy horizontalconnections in supragranular layers, link zones of like orientation selectivity (Gilbert and Wiesel,1989; but see Matsubara et al., 1985; Matsubara et al., 1987). The secondary disruption of activityin supragranular layers of the visual cortex, resulting from reduced synaptic input from the poorlydriven cortical cells in layer IV (Bliss-Tieman, 1991), would also be expected to affect theorganization of intracortical patches. Interestingly, these patches have a periodicity similar to thatdescribed by 5-HT receptors and Zn (see Katz and Callaway, 1992 for review). The formation ofthe patchy pattern of intracortical connections arises from an activity-dependent, selective axonActivity-Dependent Expression 135retraction during early postnatal development (Callaway and Katz, 1990; Liibke and Albus, 1992;Luhmann et al., 1986; Price, 1986). Similar to the effects of dark-rearing described here, where5-HT1c/2 receptors fail to segregate, the effects of binocular lid suture prior to eye-opening, resultsin the maintenance of an homogenous distribution of intracortical projections (LOwel and Singer,1992). In addition, the critical period for the effects of deprivation extends to about 14 weeks ofage (Dalva et al., 1992), when the 5-HT receptor columns are no longer present (see Chapter 2).The results of this study clearly indicate that the columnar expression of 5-HT receptorsand Zn are dependent on normal binocular visual experience and activity. However, arguments foran implicit relationship between the columnar system(s) described by these molecules, and those ofeither ocular dominance columns, orientation columns and/or patchy horizontal connections couldbe made based on the results of the various deprivation paradigms employed here.The experiments in Chapter 5 attempt to address the possibility of explicit functionalrelationships using double label procedures to determine whether the columnar systems describedthus far are related to the geniculocortical ocular dominance columns which have been intensivelystudied.1365MULTIPLE MARKERS OF ACOLUMNAR MOSAIC IN VISUALCORTEX OF CATS AND PRIMATESThe columnar arrangement of functionally similar elements has long been considered to bea fundamental principle of neocortical organization (Lorente de NO, 1949; Mountcastle, 1978). Thedemonstration that cytochrome oxidase-enriched blobs overlie eye-specific columns in primatevisual cortex (Horton and Hedley-White, 1984) has proven to be invaluable in providing ananatomical reference point from which theories regarding the functional compartmentalization ofmammalian visual cortex have evolved (reviewed in LeVay and Nelson, 1991). Several othermetabolic enzymes (Horton, 1984; Sandell, 1986) as well as a variety of molecules which areassociated with the function of GABA-ergic (Hendrickson, 1985; Hendrickson et al., 1981),peptidergic (Kuljis and Rakic, 1990), and cholinergic (Graybiel and Ragsdale, 1982; Horton,1984) neurotransmission, among others (see LeVay and Nelson, 1991), have been found to beselectively localized within blobs, or to the interblob region, in primate visual cortex. These resultssuggest a potential relationship between neurotransmitter systems, and functionally specializedcompartments in visual cortex. Cytochrome oxidase-blobs were initially reported to be unique toprimates (Horton, 1984; Horton and Hubel, 1981), leading to hypotheses which constrainedfunctional relationships of these anatomically-defined compartments to features of visualprocessing also unique to primates, such as colour (Livingstone and Hubel, 1984a). Despitenumerous attempts, anatomical evidence for the periodic distribution of any endogenous moleculein the visual cortex of non-primates had not been provided until recently (Schoen et al., 1990).Columnar Mosaic 137Over a decade after having been discovered in primates, preliminary reports indicate that a blob-likepattern of cytochrome oxidase-staining is present in the visual cortex of adult carnivores (Cresho etal., 1992; Murphy et al., 1990). These data, in combination with the data presented thus far in thisbody of work, indicate that anatomical correlates for the columnar organization of visual cortexmight not be species-unique, and, furthermore, provide the first evidence that functional correlatesof the columnar distribution of neuroactive molecules might be conserved.As a first step toward understanding the functional relationships of Zn and 5-HT receptorcolumns, the first series of experiments described in this chapter establish that cytochrome oxidase(CO) and acetylcholinesterase (AChE; its anatomical relationship with CO in primate visual cortexis known) are present in patches in developing cat visual cortex. Their relationship to the columnardistributions of Zn and 5-HT receptors described in Chapters 2 and 3 are also established.Secondly, the columnar distributions of Zn, 5-HT, CO and AChE are compared to the distributionof ocular dominance columns, visualized by transneuronal autoradiography, in kitten visual cortex.Finally, the possibility that the distribution of these neuroactive molecules describe corticalcompartments which are functionally conserved across species is addressed by comparing thedistribution of Zn to CO in the striate cortex of monkeys.Materials and MethodsAnimals and Surgical ProceduresCats. Ten kittens between the ages of 50 and 60 postnatal days were anaesthetized to effectwith halothane, the saphenous vein was cannulated and an intravenous injection of sodium selenite(10 mg / kg) was administered at a rate of 1 ml / min. Following a 15 - 20 min survival period theanimals were killed with an overdose of sodium pentobarbital and perfused through the ascendingaorta with 100 ml of 0.1M Sorenson's buffer. The brain was quickly removed and the visualcortex from each hemisphere was blocked, opened, flattened between glass slides and then quicklyColumnar Mosaic 13 8frozen on a bed of dry ice. Prior to cutting sections, fiduciary landmarks were placed using a 30gauge needle, at approximately 10 mm intervals, to facilitate section alignment. Serial sectionswere cut at a thickness of 25 i_tm on a cryostat at -20°C, thaw-mounted on gelatin-coated glassslides and stored at -20°C.Monkeys. Three adult vervet monkeys (Cercopithicus aethiops) were heavily sedated withketamine and slowly administered an intravenous injection of sodium selenite (10 mg / ml; 10 mg /kg). After 15 minutes, the monkeys were killed with an overdose of sodium pentobarbital andperfused transcardially with Sorenson's buffer (0.1M, pH 7.4). The brain was quickly removedand the visual operculum from one hemisphere was dissected and flattened between glass slides inorder to cut sections tangential to the cortical surface. The other hemisphere was left intact forcoronal sections. The tissue blocks were quickly frozen as previously described. Prior tosectioning, pin holes were placed in the flattened hemisphere to provide fiduciary landmarks.Sections were cut on a cryostat (-20°C), at a thickness of 25 gm, and thaw-mounted on gelatin-coated slides.Transneuronal Autoradiography. Transneuronal labeling of eye-specific inputs tovisual cortex was performed in 8 kittens (PD45 - PD53) by intra-ocular injection of [3H]proline(Wiesel et al., 1974) . Under halothane anaesthesia 2.0 - 2.5 mCi of [ 3H]proline (Amersham), in20 gl of saline, was injected slowly (15 min) into one eye through a 27 gauge cannula whose tipwas positioned near the optic disc via a lateral approach. An equivalent volume of saline wasinjected into the other eye to control for possible asymmetric effects on the ocular dominancedistribution. The animals survived 8 - 13 days and were then perfused as above. In two of theanimals, the brain was frozen intact for horizontal sections to be cut. In the remaining kittens, thevisual cortex from both hemispheres was prepared for tangential sections as described in Chapter2.Columnar Mosaic^ 139Autoradiography and StainingFor comparisons among the different columnar markers, slide-mounted sections weredivided into sets, comprising near-adjacent sections, which were processed for either 5-HTicreceptor autoradiography, or zinc, acetylcholinesterase or cytochrome oxidase histochemistry.Sections from proline injected animals were divided into 4 sets, which were either: 1) stained forsynaptic zinc, 2) stained for synaptic zinc and then apposed to Hyperfilm (4 weeks), 3) apposed toHyperfilm (4 weeks) then stained for zinc or, 4) apposed to Hyperfilm (4 weeks), unprocessed. Inthis way, the distribution of eye-specific label could be directly compared to the distribution of zincpatches in near-adjacent, directly adjacent or the same sections. Naturally, the last provided the bestresults.Serotonin Receptor Autoradiography. 5-HT 1 c and 5-HT2 receptors were visualizedusing methods identical to those described in Chapter 2.Zinc Histochemistry. The distribution of synaptic zinc in sections from kitten andmonkey visual cortex was assessed using the methods described in Chapter 3.Acetylcholinesterase Histochemistry. Sections intended for the detection ofacetylcholinesterase (AChE) were fixed in 4% paraformaldehyde in PB for 10 min. The stainingprocedure was as described by Karnovsky and Roots (1964) (Karnovsky and Roots, 1964). Thefixed sections were preincubated for 30 min in PB containing 30 j.tMtetraisopropylpyrophosphoramide (iso-OMPA) to inhibit butyrylcholinesterase activity. The AChE-positive reaction was visualized following incubation for 6 - 8 hours (at room temperature) in 300ml of a 50 mM Tris-maleic buffer (pH 6.0) containing 150 mg acetylthiocholine iodide, 441 mgsodium citrate, 225 mg cupric sulphate and 49 mg potassium ferricyanide. The slides were rinsedin distilled water, allowed to air dry overnight, cleared in xylene, and coverslipped with Permount.Columnar Mosaic 14 0Cytochrome Oxidase Histochemistry. The slide-mounted cryostat sections wereprocessed within 24 hours of sectioning in order to minimize enzyme degradation. Prior tostaining, the sections were fixed for 5 min in 4% paraformaldehyde in 0.1M phosphate buffer (PB,pH 7.4). Two different staining methods were used with equivalent results; however, the cobaltenhanced version proved superior for visualizing cytochrome oxidase enriched patches.The first method was described by Horton (1984) and is based on a modification of aprocedure developed by Wong-Riley (1979). Following fixation, the slides were rinsed twice for 5minutes in PB and then immediately incubated at 37°C, for 2 - 8 hours, in a solution consisting of50 mg DAB, 30 mg cytochrome C (Type III, Sigma) and 20 mg catalase (Sigma) in 100 ml PB.After incubation, the slides were rinsed in buffer, dehydrated in an ascending series of alcohol,cleared in xylene and coverslipped with Permount.The second method utilized the same histochemical principles but enhanced the visibility ofthe reaction product, and the contrast between differentially stained regions, by heavy metalintensification (Silverman and Tootell, 1987). Following fixation, the sections were transferredthrough 4 serial changes of PB containing 10% sucrose and then placed for 10 min into a 0.05 MTris-HC1 buffer (pH 7.6) containing 275 mg cobalt chloride / 1 buffer, 10% sucrose and 0.5%dimethylsulfoxide (DMSO). Following a brief rinse in PB (1 min), the sections were incubated inthe reaction medium made up of 50 mg DAB, 15 mg cytochrome C, 5 g sucrose, 10 mg catalaseand 0.25 ml DMSO in 100 ml PB (bubbled with oxygen @ 37°C). The reaction time varied from 2- 3 hours. Following incubation, the slides were rinsed in buffer, dehydrated in an ascendingseries of alcohol, cleared in xylene and coverslipped with Permount. For either method, controlsections incubated in the presence of sodium cyanide (0.01M) showed complete inhibition ofreaction product formation.Image Analysis. Histochemically stained sections and autoradiographic images weredigitally captured using a Cohu CCD camera (4915) and a Data Translation (DT-2255) framegrabber card installed in a Macintosh IIfx computer running Image (NIH, v 1.47) software. SerialColumnar Mosaic 141sections were aligned using an Image module programmed in house, which allowed precisealignment of successive images on line. The relationship of the autoradiographic and histochemicalfeatures in sections of kitten visual cortex was determined by arithmetically blending, ortransparently superimposing, digital images using Adobe Photoshop (v 2.1, Adobe Systems Inc.).The captured and aligned figures were lettered and labeled using Canvas (v 3.1, Deneba Software)and hard copies were digitally processed by printing onto black and white negative film (IlfordFP4) using a slide processor, and then conventionally printed onto photographic paper (eg. Figs.5.3 - 5.7).Results and DiscussionThe laminar and tangential distributions of 5-HTic receptors (A, B), Zn (C, D), CO (E, F)and AChE (G, H) are compared in Figure 5.1 in frontal (A, C, E, G), and tangential sections (B,D, F, H), through the visual cortex of PD50 kittens. It is clear from this figure that the columnardistribution of each of these molecules was more readily apparent in the tangential plane than in thefrontal plane. For CO, and particularly AChE, the patterned appearance is virtually invisible in thefrontal plane.The characteristics of the periodic pattern of 5-HT lc receptors in layer IV of area 17, weredescribed in Chapter 2. The distribution of Zn, which also exhibited column-specific labeling inlayer IV of area 17, was described in Chapter 3. In comparing their columnar distribution indirectly adjacent sections (Fig.5.1A, C; Fig. 5.3A, B), these molecules were found to be preciselyoverlapping, and localized to the same column (note arrows).The distribution of these markers was compared with that of CO and AChE in seriallyadjacent sections (Fig. 5.3). As in the adult cat (Murphy et al., 1990), the patchy distribution ofCO in PD50 kittens was not readily apparent in coronal sections (Fig. 5.1E), but was clear in thetangential plane (Fig. 5.1F; Fig. 5.3A'-C'). We also found AChE to exhibit a periodic pattern intangential sections (Fig. 5.1H, Fig. 5.3A"-C"), which was not apparent in the coronal plane (Fig.Columnar Mosaic 14 25.1G). AChE-rich patches were limited to a thin band at the layer III / IV border (Fig. 5.1H, Fig.5.2 A"-C"), which were aligned with CO blobs (Fig. 5.3), but complementary to Zn / 5-HTiccolumns (Fig. 5.4). The complementary relationship between CO / AChE-enriched blobs and theZn / 5-HTic patches in layer IV can be seen in Figure 5.5 at higher magnification. Here, digitallycaptured images showing the patchy distribution of Zn (Fig. 5.5A) and CO (Fig. 5.5B) are shownindividually, and then superimposed and summed in Figures 5.5C and 5.5D. The homogeneousimage resulting from the summation of carefully aligned Zn and CO stained sections (Fig. 5.5C),contrasted with the reappearance of the distinct periodicity when the overlying image was shifted tothe right by a distance equal to one-half of the average patch spacing (450 vim, see arrows) andthen summed (Fig. 5.5D), indicating that these two systems were precisely complementary in thetangential domain.The distributions of several enzymes and other molecules associated with the functions ofneurotransmitters have been shown to be either aligned with, or complementary to, CO-blobs inprimate visual cortex (see LeVay and Nelson, 1991 for review). These data, and those reportedhere, suggest that column-specific molecules might demarcate common functional domains in thevisual cortex of species as phylogenetically diverse as carnivores and primates. Indeed, we foundthat the laminar-specific distribution of Zn in striate cortex of adult primates (Cercopithicusaethiops) was exactly complementary to that of CO (CO, Fig. 5.6A, C, F; Zn, Fig. 5.6B, D, G),just as it was in the cat. In the tangential plane of section, CO-rich blobs (Fig. 5.6A) were clearlyshown to be interdigitated within a Zn-stained matrix (Fig. 5.6B) in layers II and III. This precisecomplementarity was evident, in the frontal plane, between laminae as well (Fig. 5.6C, 5.6D; Fig.5.6F, 5.6G). Preliminary results indicate that this complementary relationship between Zn and COpatches in vervet monkeys is present as early as 4 weeks of age (Dyck and Cynader, unpublishedresults). The distribution of serotonin receptors in the visual cortex of developing primates has notyet been examined, although previous studies have shown the distribution of serotonergic afferentsto be more abundant in interblob zones (Hendrickson, 1985).Columnar Mosaic^ 143Figure 5.1. Multiplicity of markers for columnar domains in PD50 kitten visual cortex. Thecolumnar distributions of 5-HTic receptors (A, B) and synaptic zinc (C, D) in layer IV of area 17were apparent even in the frontal plane (A, C). The registration of several columns is apparent inthe frontal plane (arrow heads). The distributions of cytochrome oxidase (E, F) andacetylcholinesterase (G, H), which are also cytochemical markers of the columnar architecture inprimate striate cortex, did not appear patchy in frontal sections (E, G), but clearly demarcatedcolumnar domains in the tangential plane (F, H). The most marked columnar distribution of eachof the four markers was found in upper layer IV; however, their individual distributions werecontained within distinct layer III / IV substrata. Nevertheless, their organization into patches ofincreased density all exhibited the same periodicity in area 17 (900-1000 gm, centre-to-centre).Scale bars = 3.0 mm.144Figure 5.1Columnar Mosaic^ 145Figure 5.2. Compared in serially adjacent sections, patches of Zn (A), and those of5-HT lc (B) and 5-HT2 (C) receptors were found localized to the same vertical columns in layer IVof area 17 (note arrows). The patchy distributions of Zn and 5-HT2 receptors were greater withinthe more superficial strata of layer IV. Levels of 5-HTic receptors were highest as they emergedfrom the dense band at the layer IV / V border, and became less dense as they extended through theentire extent of layer IV into layer III. Note that panel A is slightly shifted to the right, relative to Band C. Scale bar = 1 mmFsgure 5.2146Columnar Mosaic^ 147Figure 5.3. The distribution of Zn (A-C), CO (A'-C'), and AChE (A"-C") in serialsections from levels near the top (A, A', A"), middle (B, B', B") and bottom (C, C', C") of layerIV in PD50 kitten visual cortex. Patches of Zn and CO were predominent in upper strata of layerIV and waned in deeper strata, although Zn was found at deeper levels than CO. AChE-richpatches, on the other hand, were limited to a thin region at the layer III / IV border. Note that noneof these molecules exhibited a periodic pattern in area 18. The area 17 & 18 borders are indicatedby the white dotted lines in A-C. The positions of selected laminae are indicated by Romannumerals in A'-C'. Fiduciary landmarks are indicated by the black arrows in A"-C". Scale bar = 3MM.lere#110".gkeirt'. -11r-.N4 4ta*•-•tColumnar Mosaic^ 14 9Figure 5.4. The relationship between patches of Zn (A) and AChE (B) in adjacent,tangential sections through the visual cortex of PD50 kitten. The negative correlation observedbetween Zn- and AChE-rich patches are indicated in several places by the black arrows. Selectedlaminae are indicated by Roman numerals. White arrows indicate fiduciary landmarks. (M, ventro-medial; P, posterior; L, dorso-lateral; A, anterior) Scale bar = 5 mm.150Figure 5.4Columnar Mosaic^ 151Figure 5.5. High magnification digital images of cytochrome oxidase (A) and zinc (B)staining in serially adjacent sections through area 17 of PD50 cat visual cortex. The periodic,patchy distributions of both cytochrome oxidase and synaptic zinc are evident in layer IV. Whenthe two sections were superimposed and summed, the precise laminar and columnarcomplementarity of these two markers was manifested by the disappearance of the patchy pattern inlayer IV(C). Only the microtome blade scratches showed an increased density following theirspatial summation (C, diagonal streaks). When the overlying cytochrome oxidase image wasshifted to the right by 450 µm, and then summed (D), the patchy pattern reappeared, indicating thatsynaptic zinc and cytochrome oxidase are distributed with precise laminar and intra-laminarcomplementarity. White arrows indicate fiduciary landmarks used for section alignment. Thedotted lines and Roman numerals in C outline and indicate laminar boundaries. Scale bar = 1 mm.152Figure 5.5Columnar Mosaic 153In primates, the CO-rich zones are associated with: (1) the centers of eye dominancecolumns, (2) areas of high colour selectivity, (3) zones of broad orientation selectivity, and (4)preferential input from various processing streams originating in the lateral geniculate nucleus(LeVay and Nelson, 1991; Livingstone and Hubel, 1988). To begin to examine the cross-speciesgenerality of these functional relationships, we compared the distribution of Zn patches with oculardominance (OD) columns in kitten cortex. Figures 5.7 and 5.8 illustrate the tangential distributionof Zn patches (Fig. 5.7A', B'; Fig. 5.8C, D), compared with ipsilateral eye-specific patcheslabeled autoradiographically (Fig. 5.7A, B; Fig. 5.8A, B), in the same sections through layer IV ofkitten visual cortex. At higher magnification in Figure 5.8C, the white, dotted lines indicate theoutline of the OD pattern superimposed on the Zn-stained section, and the outlines of Zn-richpatches are indicated on the OD map of Figure 5.8B. Although some overlap between the twopatchy patterns was evident, they were not explicitly aligned with one another.These findings demonstrate that the relationship between the CO/AChE and 5-HT receptor/Znsystem and eye-specific innervation observed in primates, is not obligatory in cats. Likewise, theassociation with colour is unlikely to be obligatory, given the cat's poor colour vision capacities(Daw, 1973), and the presence of CO-blobs in primates without colour vision (Condo andCasagrande, 1990). Our findings are consistent with at least two of the remaining functionalinterpretations. (1) Previous studies have indicated singularities in the cat and monkey corticalorientation maps, zones where different orientation bands coalesce and which contain broadlytuned neurons (Blasdel and Salama, 1986; Bonhoeffer and Grinvald, 1991; Swindale et al., 1987).These singularities are thought to be associated with CO blobs in monkeys (Blasdel, 1992) andmay well have the same association in cat cortex. (2) In primates, the CO-blob / interblob systemhas been reported to contain geniculate inputs representing different processing streams (LeVayand Nelson, 1991). This is consistent with the findings described in Chapter 2 which indicate, onthe basis of spatial characteristics of 5-HT receptor expression, that 5-HTic and 5-HT2 receptorsare individually related to different, parallel processing streams. The precise relationship and theextent to which functional homology is maintained among species, await further examination.Columnar Mosaic^ 154Figure 5.6. The distribution of cytochrome oxidase (CO; A, C, F) and synaptic zinc (Zn;B, D, G) in striate cortex of an adult Vervet monkey. The precise complementarity of CO-blobswithin a Zn-stained matrix in laminae II / III of V1 was clearly seen in the near-adjacent sectionswhich were cut tangential to the cortical surface (A, B). The complementary relationship wasmaintained in lamina IVa, but in this layer, Zn-stained blobs were, instead, surrounded by a COmatrix. The periodic patterns of CO and Zn were not robust in coronal sections (C, D, F, G; notearrows), but these panels demonstrate that the laminar-specific distributions of these two moleculesalso exhibited a high degree of complementarity in V1 and V2 (C, D). In contrast with CO (A, C,F), the highest levels of Zn (B, D, G) were found in layers I, IVb and VI, while layers II / III andV were moderately stained. Nissl-stained sections (E) were used to establish laminar boundaries.The white arrows in A & B indicate fiduciary landmarks, used to facilitate section alignment.Roman numerals in B & E indicate the relative locations of cortical laminae. Scale bars in A - D =2.0 mm; in E-G = 500 ium.D•^.,7.-E,^711 F••„:.^'^-• `.1A-0;.017^• ,^• t,• Lr;^'^ frtt,,ii.';'.,, • *,114.415`!* '^' ..ii - - -.,,- ..4.0c4( 4ifili,. •^, lx14;44, , - ..-”•• • '-'o.'t^, ,;,•,s., •$^•t _^• '155Figure 5.6Columnar Mosaic^ 156Figure 5.7. The overall distribution of eye-specific "columns" (A, B), labeled using[3H]proline injected in the ipsilateral eye, are compared with Zn-rich patches (A', B') in adjacentsections (A-A'; B-B') at two depths from the cortical surface (700 & 800 1.1.m) through visualcortical areas 17 and 18. These figures clearly indicate that the spatial patterns, of their respectivepatchy distributions, is very similar in extent. Note that the ocular dominance bands double theirspacing in area 18 and are clearly oriented perpendicular to the 17 / 18 border. There is a weakindication that Zn may similarly change its pattern in area 18. Common fiduciary landmarks areindicated by the arrows. Medial is down. Scale bars = 10 X 2 mm.A,^. A • 18I^Alt taw ..^,— — - - ''' ...^■ \4% t\ ...\^I.4 4 1%4L.C.: 1 \ 4% 4^,,,I rot IF 4. 4^\\ Ido ikatp.. ..„..* bi '• el!ft*^4,800 pm4146I•""dr40—700 pm417Columnar Mosaic^ 15 8Figure 5.8. The relationship between the neurochemically-defined columnar architecture inlayer IV of kitten visual cortex, and ocular dominance columns was determined in the same sectionstained first for synaptic zinc (C, D) and then processed for [ 3H]proline autoradiography (A, B).In C, the outline of the pattern of ipsilateral-eye projections is superimposed on the zinc patches inlayer IV, and in D the zinc patch outline is superimposed on the ocular dominance distribution inB. There does not appear to be an explicit relationship between these columnar systems. Fiduciarylandmarks used for image alignment are indicated by the arrows in A and D. Scale bar = 4 mm X 1MM.159Figure 5.81606GENERAL DISCUSSIONSummaryThe studies described in the previous four chapters have provided important anatomical andfunctional information regarding the expression of serotonin receptor subtypes and synaptic zinc inthe visual cortex of cats during postnatal development; and have specifically indicated that:1. The levels of serotonin 1A, 1C and 2 receptor subtypes and the high affinityserotonin transporter were developmentally regulated, with each receptorexhibiting unique temporal and spatial profiles in their levels of expression.2. The serotonin 1C and 2 receptor subtypes were transiently expressed at high levelsin the same cortical columns, but within different geniculate recipient sublaminae.3. Synaptic terminals containing vesicular zinc were developmentally regulated andhighly enriched in the same cortical columns and during the same temporalwindow described by serotonin receptor columns.4. The columnar expression of synaptic zinc and serotonin receptors required normalbinocular visual input and was activity-dependent.5. Cytochrome oxidase and acetylcholinesterase were localized to corticalcompartments which were precisely complementary to those demarcated bysynaptic zinc and serotonin receptors.6. The precise laminar and columnar complementary relationship of zinc and cytochromeoxidase observed in kitten visual cortex was also found in primate visual cortex.These points are summarized in schematic form in Figures 6.1 and 6.2.General Discussion^ 161Figure 6.1 Schematic summary diagram of the relative distribution of synaptic zinc,serotonin receptors, and acetylcholinesterase in the kitten visual cortex during postnataldevelopment. The interdigitated columnar mosaic formed by 5-HT receptors / zinc and CO / AChEis not present at birth but becomes prominent during a critical period of development during whichthe synaptic organization of the visual cortex is susceptible to activity- and experience-dependentmodifications. The columnar expression of 5-HT1c/2 receptors and AChE is transient andrestricted to this period of development. However, a remnant of the compartmentalizedorganization of CO and Zn is still present in the adult visual cortex.EARLY POSTNATALArea 17 Area 18IVV/V1Area 19CRITICAL PERIODV.^.. . . . . ,.^•^..„,• .• . • • z • • .• •AV^clAArea 17ADULTArea 18 Area 19IVaIV b/cVVIArea 17^ Area 18General Discussion^ 162MI 5- HT IC HiLoZincBis Zinc &2M12 5-Hl'1C •S-HT2MI Zinc..111 &5-HT 2CytochromeL-1 oxidase a CholinesteraseFigure 6.1General Discussion^ 16 3Figure 6.2 Schematic diagram summarizing the effects of manipulating visual input andexperience on the columnar expression of Zn / 5-HTic and 5-HT2 in the kitten visual cortex. Thecolumnar compartmentalization of zinc and 5-HTic&2 receptors is dependent on the integrity of thelateral geniculate nucleus and, in particular, normal binocular input during development. The effectof dark rearing and compromising the lateral geniculate input on the columnar distribution of zincwas not assessed and is, therefore not indicated.General Discussion^ 164MonocularNormal , privation, Dark RearingIVVVI5 HTIC OZn 111105-HT2 Optic TractSection LGN Lesion IV IVVVIV EMESEMSENVIFigure 6.2General Discussion^ 165DiscussionThe results of the experiments outlined here raise important issues with regard to themechanisms of activity-dependent plasticity in the nervous system. Many of the implications ofthese findings, with respect to the anatomical and functional development of kitten visual cortex,are discussed extensively within the individual chapters. As such, they will not be raised againhere. However, several methodological issues and specific functional implications which were notdealt with, or only briefly, warrant further review.Methodological ConsiderationsAutoradiographic procedures. The ligand concentrations and incubation parameterswhich were used to label the different 5-HT receptor subtypes were determined from previouslypublished studies, which had characterized these ligands in a number of different species, butpredominantly in adult animals. In addition, all of the binding procedures were carried out atconcentrations which were below the level of saturation for each receptor. As a consequence, onecan not be certain whether the variations in binding density that were observed reflect changes inreceptor number (Bmax), or in receptor affinity (Kd), or both. Studies in other species, whichhave characterized developmental changes in 5-HT receptor expression, have reported ontogeneticchanges in binding to be exclusively due to changes in their number, and not affinity (Biegon,1991; Gross-Isseroff et al., 1990). However, this cannot simply be assumed to be true in cats aswell. In fact, changes in the binding affinity (Kd) of a number of neurotransmitter receptors havebeen described in kitten visual cortex during postnatal development (Shaw et al., 1984; Shaw etal., 1985). Regardless of whether they reflect changes in affinity or number, the densitometricchanges in receptor binding that have been described here are likely to reflect specific indices ofreceptor sensitivity during development. However, a complete pharmacological characterization ofthese ligands at each age would be required to address these questions definatively.General Discussion 16 6The pharmacological and molecular characteristics of 5-HTic and 5-HT2 receptors are verysimilar, and several studies have indicated that currently available ligands lack subtype specificity(Closse, 1983; Glennon et al., 1992). However, the reported lack of specificity of[3H]mesulergine for 5-HTic receptors in rats (Closse, 1983; Pazos et al., 1988), differs fromstudies in human and porcine brain, where the binding of [ 3H]mesulergine was determined to be5-HTic-specific (Lyon and Titeler, 1988; Pazos et al., 1988; Pazos et al., 1984b). Several lines ofevidence indicate that the specificity of [3H]mesulergine and [1251]DOI binding for the 5-HTic and5-HT2 subtypes, respectively, holds true in cats as well. First, the distinctly different temporal andspatial distributions of [3H]mesulergine and [ 125I]DOI binding indicate that these ligands labeldifferent populations of receptors. The inclusion of spiperone, which has a 1000-fold loweraffinity to 5-HT lc than 5-HT2 receptors, in the incubation medium has been used to confer greaterspecificity of [3H]mesulergine for 5-HTic receptors. In the present studies, no differences werefound in the regional and temporal binding patterns exhibited by [ 3H]mesulergine binding in catvisual cortex with, or without, the inclusion of spiperone. Finally, the temporal and regionalpatterns in the expression of 5-HT2 sites were identical when either [ 125I]DOI or [3H]DOB wasused. These data do not, however, rule out the possibility that [ 3H]mesulergine and [ 1251]D01differentiate different affinity states of either the 5-HTic and/or 5-HT2 receptor, or the existence ofadditional subtypes within the 5-HT2 family (Hartig et al., 1990; Leonhardt and Titeler, 1989;Lyon and Titeler, 1988; Pierce and Peroutka, 1989; Teitler et al., 1990). Further pharmacologicalcharacterization, combined with molecular biological studies, are required to fully address theseissues.The differential absorption of tritium emissions in the brain, based upon tissue variations inmyelin content (Geary and Wooten, 1985; Herkenham and Sokoloff, 1984), could provide asource of variation to the laminar binding patterns observed in the autoradiographic studies carriedout in this thesis. The process of myelination in visual cortex of the kitten starts around 4 weekspostnatal (Looney and Elberger, 1986; Remahl and Hildebrand, 1990) and proceeds progressivelyin a deep-to-superficial laminar gradient, reaching adult levels around 12 weeks of age (Daw,General Discussion 16 71986). The highest levels of myelin are in layers IV-VI. The issue of quenching is not significantwith regard to [ 1251E1-labeled ligands, but the signal provided by the tritiated ligands used to detect5-HriA, 1C, 3 & Up binding, might have been attenuated, particularly at ages beyond 4 weeks ofage. This might be significant, particularly with regard to the columnar representation of 5-HTicreceptors, whose increasing numbers during this period of time, might have been underestimatedin these studies. It should be noted that, in autoradiographic analyses of major neurotransmitterreceptors in the primate visual cortex, where laminar variation in myelination is much more extremethan in the cat, the total effect of quenching was no greater than 20% (Lidow et al., 1989; Rakic etal., 1988).Histochemical procedures. It is estimated that 85-90% of the zinc in the brain is boundinto the tertiary structure of over 50 different zinc-containing enzymes (Wallwork, 1987). Thebasis of selectivity for the methods used here to indicate the remaining 10-15% of zinc, which islocalized to presynaptic vesicles of zinc-containing neurons, is conferred by the fact that themetabolic pool of zinc is bound within the tertiary structure of proteins and is, therefore,inaccessible to chelation by sulphide or selenite ions (Vallee, 1983; Vallee and Galdes, 1984).Although initial versions of the zinc histochemical method stained heavy metals in addition to zinc(Danscher et al., 1976; Haug, 1973), the selenium-histochemical method utilized in this study hasdemonstrated specificity for that portion of zinc in the brain which is localized to presynapticvesicles of zinc-containing neurons (Danscher et al., 1985; Frederickson, 1989; Frederickson andDanscher, 1988). However, if any portion of the staining presented here is due to labeling of otherheavy metal ions, then, based on the ultrastructural analyses which find histochemically-labeledsilver particles almost exclusively limited to vesicles within synaptic terminals, their role insynaptic modulation should be studied as well.One of the most significant methodological issues raised in these, and in other studieswhich have succeeded in demarcating columnar compartments in mammalian neocortex (e.g.primate, Tootell et al., 1988; cat, Murphy et al., 1990; ferret, Cresho et al., 1992; rat, NakazawaGeneral Discussion 168et al., 1992) is with regard to the plane within which the various markers are visualized, and thesensitivity and contrast of the staining method employed. Thus, by cutting tissue sections in sucha way as to maximize the view of the particular region of interest (eg. tangential to the corticalsurface for laminar and intralaminar analyses), the levels of acetylcholinesterase and cytochromeoxidase, whose developmental expression has been studied previously in kitten visual cortexwithout an indication of columnar compartmentalization (CO, Kageyama and Wong-Riley, 1986;Price, 1985; AChE, Bear et al., 1985a; Bear et al., 1985b), were found here to vary in a periodicmanner. The most robust markers of the columnar compartmentalization of visual cortex werethose demonstrated by zinc histochemistry and 5-HT receptor autoradiography. In initial studies,the periodic distributions of AChE and CO were not always readily seen, due to the limited contrastprovided by brown reaction products of unenhanced DAB or thiocholine, unless compared inadjacent sections using 5-HT autoradiography or zinc as a metric. When heavy metal enhancementtechniques were used (CO, Silverman and Tootell, 1987; AChE, Schatz et al., 1992; ourpreliminary results not reported here), the periodic patterns became easily and clearlydistinguishable.To achieve higher cytological resolution with the currently available anatomical markersutilized in these studies was not possible due, in part, to an incompatibility of paraformaldehydefixation with the procedures used to localize receptors and for zinc histochemistry. The detrimentaleffects of fixation on receptor integrity are obvious, but those on synaptic zinc are obscure.Because glutaraldehyde fixation has no effect on the integrity of zinc-staining, it is likely thatparaformaldehyde adversely affects the bond between zinc and selenite ions (Danscher andZimmer, 1978), which is a requirement of physical development for visualizing synaptic zinc.Serendipitously, by being unable to use paraformaldehyde in studies comparing the distribution ofzinc with the other molecules in adjacent sections, the column-specific cytochrome oxidase stainingwas found to be much more robust in unfixed tissue, as long as the tissue was flash-frozen,sectioned and stained within 24 hours or, alternatively, stored at -70°C until it was processed.General Discussion^ 16 9Functional ConsiderationsThe data presented in this thesis have advanced existing studies which have assessed theanatomical distributions and potential functional contribution of serotonin receptors, zinc,acetylcholinesterase and cytochrome oxidase to various aspects of information processing in themature and developing mammalian visual cortex. The primary goals of this thesis, as described inChapter 1, were to assess anatomical distributions during postnatal development, and to determinethe functional significance of serotonin receptors and synaptic zinc in the cat visual cortex. As faras these goals have been advanced, they still remain inadequately answered. Determination of theprecise cytological distributions of serotonin receptors and synaptic zinc, combined with anunderstanding of how their actions are transduced to signals which are developmentally andfunctionally meaningful, is crucial. The remainder of this discussion provides a preliminarydiscussion of these important issues, as well as directions for future studies.Where are these molecules ? Available evidence regarding the cytological expressionof the different 5-HT receptor subtypes on glial cells or neuronal somata, dendrites and axonterminals was provided in Chapters 2 and 4. A significant implication, provided by the results ofChapter 4, was that 5-HT1c/2 receptors might be localized on geniculocortical afferents to visualcortex. Similarly, inferential support for a similar source of zinc-containing fibers in layer IVwas provided by the data presented in Chapters 3 and 4. The idea that 5-HT receptors might belocalized on zinc-containing geniculocortical fibers, albeit speculative, is attractive.The obvious directions for the next generation of studies are to distinguish clearly theprecise location and source of 5-HT receptors and synaptic zinc with respect to afferent input to thevisual cortex at different ages. These studies would require using techniques which permit tract-tracing combined with receptor detection and / or histochemistry, with subcellular resolution.The distribution of cells intrinsic to the cortex which express 5-HT receptors could bedetected by immunocytochemistry or with in situ hybridization studies (ISH). The 5-HT ii, (AlbertGeneral Discussion 170et al., 1990; Fargin et al., 1988), 5-HT1B (Hamblin et al., 1992; Mochizuki et al., 1992), 5-HT1p(Weinshank et al., 1992), 5-HT1E (McAllister et al., 1992), 5-HT 1 c (Liibbert et al., 1987;Saltzman et al., 1991), and 5-HT2 (Chen et al., 1992; Julius et al., 1990; Pritchett et al., 1988)receptors have all been cloned and their nucleic acid sequences are published. The utility ofreceptor-specific antibodies to localize 5-HT receptors would be substantial, however, theproduction of antibodies against members of the G-protein-coupled receptor family has provenextremely difficult because of the tremendous levels of homology between receptors for differentneurotransmitters, even at the protein level (D. Julius; F. Liibbert, personal communication).Some, however, are becoming available. Preliminary studies with 5-HT 1 A receptor-specificantibodies provided by J. Raymond (Duke University) have confirmed the receptor binding resultsprovided here (Dyck, unpublished results). The use of antibodies against the other subtypes willprove useful, as they become available.One of the primary implications suggested by the unique temporal, regional and laminardistributions of the neuroanatomical markers utilized in the studies presented in this thesis, is thepossibility that these molecules differentiate functionally distinct compartments in the developingvisual cortex (see Chapters 2 and 5 for discussion). The functional nature of these new columnarsystems remains uncertain, but several lines of evidence indicate that they may be related to specificfeatures of visual information processing such as those described by geniculocortical processingstreams. These possibilities could be addressed with the application of methods which can identifythese functional domains in combination with receptor localization. For example, the temporallyand spatially unique distributions of 5-HTic and 5-HT2 receptors appear to differentiate theanatomically and functionally distinct X and Y geniculocortical pathways, respectively (see Chapter2). An evaluation of the distribution of Cat-301, a neuronal surface-associated proteoglycan whichdemarcates Y-like pathways in the cat and primate visual system (Hendry et al., 1988; Sur et al.,1988), combined with 5-HT receptor identification would provide support for this hypothesis.Although an anatomical marker of the X-cell pathway has not yet been found, individual X- and Y-cells in the lateral geniculate nucleus can be physiologically identified and labeled (Freund et al.,General Discussion^ 1711985; Friedlander et al., 1985) in a manner which might be compatible with methods of receptorcolocalization.The synthesis and release of transferrin from choroid epithelial cells (Aldred et al., 1987),which has been shown to have growth factor-like activity on CNS neurons (Beach et al., 1983), isregulated by 5-HTic receptors (Esterle and Sanders-Bush, 1992). Epithelial cells of the choroidplexus express the highest levels of 5-HT1 02 receptors in the kitten brain (see Chapter 2).Activation of 5-HTic receptors leads to an increase in the production of transferrin (Esterle andSanders-Bush, 1992), and would, thereby, provide a trophic influence on brain development,conveyed humorally by the CSF. The localization of 5-HT and transferrin receptors withcytological resolution might reveal other distinct cell populations which provide a significantcontribution to the processes involved in visual cortical development and plasticity.The discussion thus far, concerning the locus of expression of 5-HT receptors in the catbrain, has been biased by the assumption that most of their function would be conferred by theirlocation on neurons, glial cells or epithelial cells of the choroid plexus. However, the physiologicalcontrol of cerebral blood flow by serotonergic mechanisms is thought to be mediated by highaffinity serotonin receptors (see Edvinsson et al., 1991; Parsons, 1991 for reviews) and thecomplex action of 5-HT on cerebral blood vessels is possibly attributable to an heterogeneousoccupation by multiple subtypes. This possibility is suggested by the observation of differentvascular responses to varied pharmacological manipulations, but a thorough understanding islacking. Additional complexity is brought about by apparent species differences in the particularreceptor subtypes found on blood vessels. Pharmacological profiles indicate that cerebrovasculartone is mediated by 5-HT1A-like receptors in guniea pig and dog, 5-HT1B/1D-like receptors in cat ,and 5-HT2_iike receptors in the rat, dog and cat (for details see Bonvento et al., 1991). Thecontribution of vascular serotonin receptors to the binding levels of the various ligands examined inthe cat visual cortex is unknown, but could be addressed by studies utilizing anatomical methodsproviding greater cellular resolution.General Discussion 17 2The precise localization of the cell bodies of zinc-containing terminals in the visual cortexhas not yet been determined. Indirect evidence provided by lesion studies have indicated that themajor source of zinc-containing terminals arises from local projections neurons intrinsic to thecortex (see Discussion of Chapter 3); however, these studies were performed in adult animals. Thehigh levels of synaptic zinc found in columnar compartments within layer IV of younger kittensindicates the potential for an extrinsic source, perhaps even from neurons originating in the lateralgeniculate nucleus. It would be important to tackle this question, possibly with the judiciousapplication of specific lesion and tract-tracing studies combined with zinc histochemistry in youngkittens (c. PD50).What are they doing, wherever they are ? The existence of multiple receptorsresponsible for transducing the signal provided by 5-HT was apparent, in retrospect, from the firststudies which assessed the physiological consequences of applying 5-HT onto neurons in the catvisual cortex (Roberts and Straughan, 1967). Thus the effects of 5-HT were reported to bothinhibit and excite cortical neurons (Bassant et al., 1990; Roberts and Straughan, 1967), orremarkably, to change from one to the other in response to a change in their basal firing rate(Waterhouse et al., 1990). The cellular mechanisms of the inhibitory response are thought to bedue to the activation of a hyperpolarizing K+ current, resulting from the activation of 5-HT1 Areceptors. Excitation on the other hand, is thought to be mediated by the activation of 5-HT2receptors which reduce the two distinct K+ currents Imy and IM (Andrade and Nicoll, 1987;Colino and Halliwell, 1987; Davies et al., 1987; for reviews see Aghajanian et al., 1990; Andradeand Chaput, 1991; Bobker and Williams, 1990). The activation of these specific membranecurrents is mediated by two intracellular signalling cascades: the adenylyl cyclase / cyclic AMPpathway, and the phospholipase C / phosphoinosotide hydrolysis pathway (PI). The specificserotonin receptor subtypes examined in this thesis are cumulatively linked to both of thesetransduction systems. Studies examining 5-HT stimulated adenylyl cyclase in the mammalian brainindicate that the 5-HT1 A receptor mediates this response. Occupation of the receptor binding site ofGeneral Discussion^ 1735-HTic and 5-HT2 receptors results in the activation of phosphoinositide hydrolysis (reviewed inSanders-Bush, 1988a; Sanders-Bush, 1988b; Zifa and Fillion, 1992).All of the members of the 5-HT1 family, in addition to 5-HTiA receptor, are coupled to thecAMP / adenylyl cyclase signal transduction pathway. The potential significance of thisrelationship in the context of cortical development is embodied by their transient levels ofexpression during cortical development in both rats and cats. Thus, the transient expression of5-HT 1 A receptors, which was described in Chapter 2, is coincident with the peak period ofsynaptogenesis (PD8-37; Cragg, 1975), the establishment of functional corticocortical synapses(PD30; Toyama and Komatsu, 1987), and the age when the mature distribution of local corticalconnections in kittens becomes defined (Katz and Callaway, 1992). In the rat, a transientserotonergic hyperinnervation of rodent somatosensory and visual cortex (D'Amato et al., 1987;Nakazawa et al., 1992) is combined with a transient, increased expression of 5-HT1B receptors(Leslie et al., 1992) and is coincident with the period of increased growth and synaptogenesis in ratcortex (Blue and Parnavelas, 1983b). There is, therefore, a significant possibililty that 5-HT1-related stimulation of adenylyl cyclase might provide integral mechanisms for the neuronaldifferentiation, neuropil formation, and synaptogenesis induced in neocortical cells in vitro by theapplication of 5-HT (Chubakov et al., 1986).Additional support for a role for cAMP mediated 5-HT mechanisms in synaptic plasticityhas been provided by studies in invertebrate models. Facilitatory processes exerted by serotonin,such as those seen in LTP, which are governed by Hebbian rules (eg. concurrent pre-andpostsynaptic changes), have been demonstrated using the gill-withdrawal response in Aplysia. Theapplication of 5-HT to sensory neurons triggers two intracellular mechanisms which result inincreased presynaptic transmitter release. The first facilitates release by prolonging the actionpotential thereby allowing more Ca2+ entry, and a second improves the efficiency of Ca2+-dependent release processes, possibly by increasing the number of vesicles available for release orby an increased efficiency of release machinery. These actions are dependent on the stimulation ofthe adenylyl cyclase / cAMP transduction pathway. Long-term changes require protein synthesisGeneral Discussion 174which may be maintained by the persistent activation of cAMP-dependent protein kinases(reviewed in Carew, 1989; Goelet et al., 1986; Greenberg et al., 1987; Kandel, 1976). Similarserotonin-mediated facilitatory mechanisms could be acting at synapses in the kitten visual cortex.Several neural receptors, including both members of the 5-HT2 family, have mitogenicpotential and can induce a state of uncontrolled growth when transfected into fibroblasts, a featurewhich is correlated with the activation of the PI second messenger pathway (Hanley, 1989; Juliuset al., 1989). The transient distributions of 5-HT 1 c and 5-HT2 receptors, may have growthstimulating functions conferred by a linkage to PI; however, this requires further investigation.Nevertheless, it is apparent that 5-HT receptors, which are coupled to different effector systemscapable of supporting mechanisms of growth and plasticity, are found at specific loci of enhancedgrowth and synaptogenesis in the developing kitten visual cortex.Many different neurotransmitter / neuromodulator receptors have demonstrated laminar-specific distributions in the postnatal kitten visual cortex. It is highly likely that two, or more,different receptors coupled to the same, or different second messenger system(s) could be localizedon the same cells. Their synergistic activation of neurons during distinct temporal windows, withindifferent laminae and cortical regions may be necessary to mediate the long-term plastic changesobserved in developing visual cortex (see Cynader et al., 1989; Cynader et al., 1990; Cynader etal., 1991). Consistent with a particular role for 5-HT tc/2 receptors and the PI-signalling cascade incontributing to mechanisms of visual cortical plasticity is the observation that, among over 30receptor subtypes which have been examined in the developing visual cortex, those receptorswhich are differentially expressed in layer IV during the critical period, are either coupled toPI-turnover, or related to the mobilization of calcium or zinc (Cynader et al., 1994).The postsynaptic, molecular mechanisms which culminate in the expression of LTP involvean NMDA receptor-activated Ca2+-influx which stimulates the activity of protein kinases and afurther triggering of intracellular Ca2+ mobilization. The hydrolysis of phosphatidylinositolbisphosphate in the PI-signal transduction pathway, with the production of diacylglycerol (DAG),acts synergistically with Ca2+ to activate protein kinase C (PKC). The activation of PKC togetherGeneral Discussion^ 175with type II Ca2+ / calmodulin-dependent protein kinase (CaM) has been shown to be necessaryfor LTP induction in the hippocampus (see Bliss and Lynch, 1988; Kennedy, 1989; Madison etal., 1991 for reviews). In recent studies, 5-HT has been demonstrated to modulate excitatoryamino acid responses in cat neocortex (Nedergaard et al., 1987). A facilitatory action of 5-HT wasnot apparent alone, but only when combined with glutamate-receptor-specific agonists. Thisobservation is also supported by studies indicating that 5-HT is a direct positive modulator ofglutamate-binding to NMDA receptors (Mennini and Miari, 1991), suggesting that 5-HT mayparticipate in the modulation of LTP. Furthermore, the possibility that 5-HT102 receptors mightmediate PI-related effects in LTP-like processes in visual cortex has been proposed (reviewed inFields and Nelson, 1991), but remains to be investigated. The columnar colocalization of 5-HT102 receptors and synaptic zinc during the critical period, combined with the demonstratedability of Zn2+ to replace Ca2+, or to modify the response produced by Ca 2+ in many cellularresponses traditionally thought to be exclusively mediated by Ca 2+ (see Discussion Chapter 3),provide significant potential for 5-HT and zinc to contribute to the mechanisms of activity-dependent synaptic changes in visual cortex. Studies assessing the relative ability to induce LTPwithin 5-HT / Zn columns, as opposed to CO / AChE blobs, would provide an interestingperspective on this hypothesis.Many neurotransmitters perform special roles during early development, which are notnormally in their repertoire in the mature nervous system (see review by Lipton and Kater, 1989).Among them glutamatergic and serotonergic neurotransmitter systems have both been implicated inproviding trophic and growth cues for neurons and glial cells during development (reviewed inMattson, 1988; Whitaker-Azmitia, 1991). An interesting link between 5-HT and zinc has recentlysurfaced, based on the colocalization of zinc and 5-HT receptors, and their similar modulation incolumnar compartments in visual cortex by visual input and activity during sensitive periods ofvisual cortical development. Recently, cortical astrocytes have been demonstrated to providespecific trophic support for serotonergic neurons (Whitaker-Azmitia and Azmitia, 1989). Theneurotrophic factor has been identified as S 10013 (Azmitia et al., 1990), whose primary role in theGeneral Discussion^ 17 6nervous system was assumed to be conferred by its' Ca 2±-binding abiltiy (reviewed in Kligmanand Hilt, 1988; Van Eldik and Zimmer, 1988). S 10013 might be called, more appropriately, aZn2+-binding protein which binds more molecules of Zn2+ with higher affinity than Ca2 +(Baudier et al., 1986; Baudier et al., 1983). Moreover, the Ca 2+ and Zn2+ binding sites aredistinct, with Zn2+ binding increasing the affinity of Ca2+ for its binding site (Baudier, 1988;Baudier and Gerard, 1986; Baudier and Gerard, 1983). The expression of S 10013 in visual corticalastrocytes is developmentally regulated and highest levels are selectively, and transiently expressedin layer IV between 3 and 6 weeks postnatally (Dyck et al., 1993). At ages beyond PD50, thehighest levels of the S 10013 protein in visual cortex are limited to superficial and deep laminae,thereby avoiding layer IV and precisely mirroring the distribution of synaptic zinc described inChapter 3. An understanding of the specific functional relationships between 5-HT, S 10013, andzinc in visual cortex during development would be provided by colocalization studies and mightreveal an underlying cooperativity among these molecules which is necessary for maintainingplasticity in the developing visual cortex.The functional significance of a columnar compartmentalization of neuroactive molecules,such as zinc, 5-HT receptors, and acetylcholinesterase, in the developing visual cortex has beenextensively discussed in this manuscript; however, the manner in which these molecules mightparticipate in the processes of visual information processing is unknown. An understanding of theexplicit contribution of these molecules to the development of the mammalian visual cortex will notbe provided by anatomical or physiological studies alone, but will require an interdisciplinaryapproach, utilizing a combination of anatomical, molecular biological, pharmacological andphysiological approaches as has been eluded to in the foregoing discussion.General Discussion^ 177ConclusionThe experiments described in this thesis provide novel anatomical and functional indices ofthe development and plasticity of mammalian visual cortex. The disparate, yet complementarytemporal and spatial distributions of serotonin receptor subtypes and synaptic zinc indicate theirpotential for participating in diverse developmental processes. 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