University of Calgary

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Graduate Studies Legacy Theses

2001

Role of synaptic zinc in neocortical development and

plasticity

Brown, Craig E.

Brown, C. E. (2001). Role of synaptic zinc in neocortical development and plasticity (Unpublished

master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/22956

http://hdl.handle.net/1880/40790

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THE UNIVERSITY OF CALGARY

ROLE OF SYNAPTIC ZINC IN NEOCORTICAL DEVELOPMENT AND PLASTICITY

Craig E. Brown

A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE

DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF PSYCHOLOGY

CALGARY, ALBERTA

January. 200 1

O Craig E. Brown

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ABSTRACT

In the present study we examined deveIopmenta1 and experience-dependent

changes in the distribution of zinc containing axon terminals in the primary

somatosensory cortex of mice. In normal mice, Levels of zinc staining undergo dynamic

redisuibutions both across and within cortical layers during the first two weeks of

postnatal development. In layer IV. zinc staining appeared to respect barrel

compartments. localized initially to barrel hollows (PD 5-10) and later to barrel septae

(PD 15 onward). However. the barrel-specific compartmentation of zinc-ergic terminds

was absent in mice that exhibit abnormal patterns of thaIamocortical connectivity (ie.

MAG.4 KO mice). Furthermore. we examined the extent to which corticaI levels of

synaptic zinc were regulated by tactile experience. Our results show that levels of

synaptic zinc increased rapidly after whisker removal and these increases persist until the

corresponding whisker regrew. Taken together. these results demonstrate that zinc-ergic

innervation of the cortex is dynamically regulated throughout postnatal development and

may contribute to functional and mucturaI changes in the deveioping and adult

neocortex.

ACKNOWLEDGMENTS

I am deeply indebted and gratell for the support that each member of my family has provided. Mom, Dad, Glen, Sarah, Lloyd, Alice, George and Grace, thank-you.

t would like to thank, my co-supervisors Richard Dyck and Bob S ~ b u r y for their guidance and support during my tenure as a masters student.

I would also like thank: Cam Teskey, Brian Bland, Susan Graham, Rod Cooper, Jos Eggermont. Sheldon Roth. Amy, Janine. Kennedy, Isaac, Sammy, Nik, Monfils. Kellee, Brandi. Gavin. Janay, Christina, Adrian, Jayne, Scotty VH, Berg, Dobber. Brodie. Nan, Deenie. Wanvok. Fedorchuk, Jen Smith, Jennie B, Bomb, Phaedra, Shannon, Ross. Trout. Mare. Neal Melvin. Brooklyn and Charlotte.

TABLE OF CONTENTS

. . APPROVAL PAGE .................................................................................. 11 ... ABSTRACT ......................................................................................... -111

ACKNOWLEDGEMENTS ......................................................................... iv TABLE OF CONTENTS ............................................................................ v LIST OF FIGURES .................................................................................. vi . . LIST OF ABBREVIATIONS ...................................................................... vii

1 . General Introduction .................................................................... 1 I . I Historical foundation ....................................................... 1

............................... 1.3 Organization of rodent trigeminal system 3 ................................... 1.2.1 Anatomical organization 3

1.2.2 Functional organization ..................................... 9 1.3 Devefopment of thalamocortical and intracortical projections in the

S I cortex .................................................................... 10 1.4 Developmental and adult plasticity of S 1 cortex ...................... 11

.................... 1.5 Locus of experience-dependent corticai pIasticity 14 .................. 1.6 Mechanisms of deveIopmenta1 and adult pIasticity 15

............................... 1.6.1 Physiological mechanisms 15 1.6.2 Structural mechanisms .................................... 20

1.7 Molecular correlates of cortical plasticity .............................. 21 3' ............ 1.8 Why study zinc in cortical development and plasticity ..., 3

............ 1.8.1 Presence of zinc in central nervous system 23 1.8.2 Post-synaptic effects of zinc .............................. 25

.................. 1.8.4 Effect of zinc on neurotrophic factors 27 1.9 Rationale .................................................................... 28

2 . Ontogenic Distribution of Synaptic Zinc in S1 Cortex of C3H and Monoamine Oxidase-A Knockout Mice ............................................... 30

2.1 Introduction ................................................................ 30 2.2 Methods ..................................................................... 33

.......................... 2.2.1 Animals and tissue preparation 33 ............................................ 2.2.2 Histochemistry -33

................................................ 2.2.3 Data analysis 34 2.3 Results ...................................................................... 36

2.3.1 Zinc staining in S 1 cortex of WT mice ................. 36 2.3.2 Zinc staining in S 1 cortex of MAO-A KO mice ..... -39 2.3.3 Barrel-field patterning in WT mice ..................... 44 2.3.4 Absence of cytoarchitechtonic barreIs in MAO-A KO mice ............................................................... -47

2.4 Discussion .................................................................. 51 ........ 2.4.1 Source of zinc-ergic innervation in S 1 cortex -51

2.4.2 Synaptic zinc in normal S 1 development: Functional . - *

mpltcanons ....................................................... 52

2.4.3 Disrupted zinc-ergic innervation of layer IV: Potentiai ....................................................... mec hanisms 54

2.4.4 Role of 5-HT in maturation of zinc-ergic terminais .. 57

3 . Experience-dependent changes in levels of synaptic zinc in S1 cortex of the ................................................................................. adult mouse 60 ................................................................ 3.1 introduction 60

.................................................................... 3.2 Methods 63 ........................... 3.2.1 Animals and treatment groups 63

3 2.2 Analysis of zinc staining intensity ....................... 64 ...................................... 3.2.3 Correlation anaiysis -66

....................................................................... 3.3 Resul ts 67 3.3.1 Distribution of synaptic zinc in barre[-field of control

............................................................... mice -67 3.3 2 Whisker plucking increases levels of synaptic zinc in

............................................................... row C 70 3.3.3 Other patterns of whisker pIucking ..................... 75 3.3.4 Regression anaiysis of whisker regrowth and levels of . * ..................................................... synaptic zmc -78 3.3 -5 Are increases in zinc staining in row C absolute or

........................................................... relative? -81 ................................................................. 3.4 Discussion -83

.................................................. J . Conclusions and Future Directions 87

5 . References ................................................................................. 94

LIST OF FIGURES

Trigeminal system in rodents ......................................................... 5

Terminology of whisker to barrel pathway ....................................... 8

........... Laminar distribution of synaptic zinc in WT and MAO-A KO mice 38

...... Optical density profiles of cortical lamina in WT and MAO-A KO mice 43

Zinc staining of layer IV in WT mice ............................................... 46

Zinc staining of layer IV of MAO-A KO mice .................................... 50

......................... Distribution of synaptic zinc in layer IV of control mice 69

Zinc staining in layer IV after whisker plucking ................................... 72

....................... Temporal changes in zinc staining after whisker plucking 74

..................... &41terations in zinc staining 24 hours after whisker plucking 77

............ Relationship between levels of synaptic zinc and whisker regowth 80

LIST OF ABBREVIATIONS

Anatomy Terms:

PoM = posterior medial nucleus of the thalamus S 1 = primary somatesensory cortex V 1 = primary visual cortex VPm = ventral posteromediaI nucleus of the thalamus

measurement Terms:

hrs = hours kg = kilogram M = molar mg = milligram rnl = milliliter mm = millirnetre ms = milliseconds S.E.1M. = standard error of measurement pg = microgram pm = micrometer yM = micromolar

Other Terms:

2-DG = 2-deoxyglucose 5-HT = 5-hydroxytryptamine or serotonin AchE = acety lcholinesterase AMPA = alpha-amino-3-hydroxy-5methyI-J isoxazole proprionic acid BDNF = brain-derived neurotrophic factor Can/IKII = calcium-calmodulin dependent kinase tI CO = cytochrome oxidase GABA = gamma-aminobutyric acid GAD = glutarnic acid decarboxylase GAP43 = growth associated protein43 GluR = glutamate receptor subunit ION = &orbital nerve LTD = long-term depression LTP = long-term potentiation MAO-A = monoamine oxidase-A MAO-A KO = rnonoarnine oxidase-A knockout NEp = norepinephrine NGF = nerve growth factor NMDA = N-Methyl-D-Aspartate

NT-3 = neurotrophin-3 PD = posmataI day PKC = protein kinase C TTX = tetrodotoxin VDCC = voltage gated calcium channel WT = wild-type

Chapter 1: General Introduction

1.1 Historical foundation

A fundamental challenge of neuroscience is to determine the epigenetic

mechanisms that regulate the development and reorganization of highly ordered sets of

itvonal connections that are characteristic of the mammalian central nervous system.

Initially. these circuits emerge through genetically programmed signals that guide avonal

outgrowth and targeting. However these early connections are typically d i f i e and

imprecise. To establish adult patterns of connectivity and function. subsequent processes

of synapse elimination. refinement and remodelling are enacted upon the initial structure

(Shatz 1990: Katz and Shatz. 1996). It is generally assumed that the bases for these

synaptic processes are provided by the level and pattern of neuronai activity ofthe cells

involved.

Historically. sensory-driven neuronal activity (which is a subset of general

neurond activity) has been viewed as the strongest force in guiding circuit formation and

plasticity (ie. the ability to change) in the cortex (for review see Katz and Shatz. 1996).

The reason for this is plain enough: as the external world influences the brain by means

of action potentials and synaptic potentials. it follows that neural activity must be the

chief cause of changes wrought by experience (Purves et al., 1994). These experience-

dependent changes (i.e. experience-dependent plasticity) include not onIy external events.

but also internal events such as the effects of hormones, brain injury, aging and even

thoughts (Kolb. 1995). However. for the purpose of simpIicity. the term experience-

dependent plasticity will be used in this manuscript to refer to changes that are brought

about by sensory experience or changes in sensory experience.

7

The pioneering work of Hubel and Wiesel(1963) and their innumerable foUow-

ups has shown that manipulations of afferent sensory-driven neuronal activity,

particularly during early development, can dramatically alter the structural and functional

organization ofthe cerebral cortex. For example, blockade of neuronal activity in the

retina (via tetrodotoxin) or occlusion of one eye (monocular deprivation) during early

postnatal life. causes neurons in deprived regions of the primary visual (VI) cortex to

become more responsive to stimdation of the non-deprived eye (Wiesel and Hubel,

1965a b: Chapman et al.. 1986). Comparably. several studies have shown that the size of

tonotopic maps in the auditory cortex (Knudsen. 1998: Pantev et al.. 1998) and

somatotopic maps in the somatosensoy cortex (for reviews see Kossut. 1992: O'Leary et

al.. 1994). are prohundly affected by early sensory experience.

Activity- or experience-dependent mechanisms also appear to play a crucial role

in the re-amngement of synaptic connections in the mature cortex. In adult primates,

manipulations of somatosensory information via peripheral nerve transection or digit

amputation. induce topographic reorganizations in which the cortical location of the non-

deprived digit. shifts over. and expands into the cortical representation of the de-

afferented digit (Kaas. 1991). Sirnilariy, changing motor experience by training animals

on a motor learning task. produces use-dependent alterations in the topographic

representation of the trained limb within the motor cortex (Kleim et al-, 1998; Nudo et d..

1996). Taken together. these findings demonstrate the rernarkabIe capacity of the

mammalian cerebral cortex to modify its function and structure, and more importantly, to

adapt to a continuously changing environment throughout postnatal Life.

3

Although it is certain that the cortex has the ability to change, it is uncertain how

these changes come about. The intent of the present set of experiments was to W e r

characterize the molecular mechanisms that may participate in these changes. More

specifically. we exploited the whisker to cortical barrel system in mice to investigate the

role of zinc-ergic neurons in the development and plasticity of the primary somatosensory

(S 1) cortex. Thus. the remainder of this introduction wiI1 discuss: a) the organization and

development of the S 1 cortex. b) plasticity and mechanisms of reorganization in neonatal

and adult mice. and c) why zinc-ergic neurons may contribute to these processes.

1.2 Organization of the rodent trigeminal system

1.2.1 =Inaromical organization

The presence of visible c)rtoarchitecconic lanharks in the S 1 cortex of rodents

make it an invaluable model system for studying the development and plasticity of

sensory cortical maps (for review see Woolsey and Van der Loos. 1970: Killackey et al..

1980). Within each level of the ascending trigeminal system. are clear representations of

the facial whiskers that include barrelettes in the trigemha1 nuclei. barreloids in the

thalamus and barrels in the S I cortex (see Fig. I). Sensory information arising From the

whiskers (also called vibrissae) is first transmitted along primary uigeminal fierents that

terminate within three trigeminal subnuclei: the nucleus principalis. nucleus interpolaris

and nucleus caudalis (Durham and Woolsey, 1984; Ma, 199 I). Second order neurons in

the trigeminal nuclei then relay somatosensory information to the medial division of the

ventroposterior nucleus of the thalamus (Wrn). VPrn afferent5 then project to layer IV of

the S 1 cortex where they innervate neuronal aggregates known as "barrels". Thus, each

Figure 1: The trigeminal system of rodents (adapted &om O'Leary et al.. 1994). The

pattern of the facial whisker pad is represented at each level of the trigeminal neurrtxis:

barreleues in the trigeminal nuclei. barreloids in the VPm of the thalamus and bands in

the posteromedial barrel subfield (PMBSF) of the primary somatosensory cortex.

cortical barrel corresponds topologically to a specific vibrissa on the contrdateral hce.

To exemplify this one-to-one correspondance and to elaborate on the terminology used to

describe the whisker to barrel pathway. I have provided a schematic illustration of the

association between the whisker pad and the barrel-tield in Figure 2. In the layer IV

barrel-field of Sl cortex. there are tive rows of barrels (barrel rows A. B. C. D and E).

Within each row. there are a specific number of barrels that are referred to as the arcs (eg.

the second barrel in row D is called the D2 barrel or row D. arc 2). From Figure 2, it can

be noted that spatial organization of the barrel-field is identical to the distribution of

whiskers on the face.

The barrel-tield in layer IV of the S1 cortex is comprised of barrel hollow and

septal components. Barrel hollows are cell sparse regions that are rich in neuropil (i-e.

mons and dendrites) and contain the terminal arbors of VPm giutmateqic afferents. The

primary targets of these fibers within the barrel hollow. are the glutarnatergic spiny

stellate neurons. while a smaller hct ion (roughly 20%) terminate upon aspinous.

GABAergic interneurons (White et al.. 1984: Lubke et al.. 2000). Encapsulating the

barrel hollow is a cell dense region termed the "septa". Whereas barrel hollows receive

information through the VPm. cells that comprise the barrel septa receive afterent

projections From the posterior medial nucleus of the thaIamus (PoM; Koralek et al..

1988). In addition to receiving thaIamic input. these septal neurons appear to participate

in intracortical sensory processing as they send and receive ipsi- and coneralateral-

corticocortical projections (Kim and Ebner. 1999). Intracortical Iabeling studies have

shown that the intrinsic projections arising from septd regions typically extend two to

Figure 2: [Ilusations indicating the terminology used in the description of the whisker

to barrel pathway (adapted from Welker et al.. 1992). Lefi, drawing of the whisker pad

with each circle representing a whisker follicle. Note there are five rows (aiigned

horizontally) of whiskers that are labeled A to E. Within each row there are a particular

number of whiskers. for example in row C there are 5 whiskers (solid line connects

follicles of row C). Note that in the vertical plane. the broken line connects the third

follicles of each row. together forming an arc. Greek letters denote whisker foIIicIes that

straddle the caudal end of each row of whiskers. Right, drawing of the barrei-held

reconstructed from sections cut tangential to the pial surface. through layer IV of S L

cortex. Note that the distribution. orientation, lettering and numbering is such that the

one-to-one topological relationship of each barrel with a particular whisker on the face is

readily apparent. Scale bars: left = 2 mrn: right = 500 pm.

9

three barrels' distance along the row of the whisker representation and produce terminals

preferentially in other septa1 regions (Kim and Ebner, 1999).

1.12 F~nctional organization

Complementing the one-to-one anatomical relationship behveen the barrel-field

and the whisker pad are strong functional relationships between each barrel and a

particular whisker on the contralateral face. Early electrophysiological studies

demonstrated that neurons within a barrel column respond preferentially to stimulation of

the "principal" whisker (Simons. 1978). For exarnple. it was shown that neurons in the

D2 barrel column are excited powerfully ( 1-2 spikes per stimulus) and quickly (6- to I0

ms latency) to stimulation of the D2 whisker. These response biases to principal whisker

stimulation are presumed to reflect direct. monosynaptic inputs from the VPm of the

thalamus (Armstrong-James et al.. 1991). However. more recent evidence has shown that

stimulation of whiskers surrounding ("surround" whiskers) the principal whisker can also

evoke neuronal responses within a barrel column (Armstrong-James and Fox. 1987). For

example. detlections of neighboring whiskers (eg. Dl, D3, C2 or E2) excites neurons in

barrel D2 less strongly (1 spike every second or third simulus) and at a longer latency (>

10 rns). than principal whisker deflections. In this case, the slower and weaker responses

of D2 neurons to surround whisker stimulation are generated by a separate pathway: fiom

intncortical inputs tiom surrounding barrel columns (Armstrong-James et al.. 1991).

Thus. neurons within a barrel column process sensory information primarily fiom the

principal whisker and to a lesser extent from m o u n d whiskers through monosynaptic

thaiamocortical inputs and polysynaptic intracortical inputs, respectively.

10

1.3 Development of thalamocortical and intracortical projections in the S1 cortex

To establish the highly organized c ytoarchitec ture that is characteristic of the

barrel cortex requires a sequence of precisely timed and highly ordered events that begins

prenatally and ends in early postnatal development. In the S 1 cortex of mice and rats.

cortical barrels first become evident with histochemical markers (eg. Nissl. cytochrome

oxidase stains) about 4 days after birth (Rice and Van der Loos. 1977). For several days

preceding this. immature thalarnocortical afferents originating from the VPm course

radially through the white matter and into developing layers V and VI ofthe conical plate

(Senft and Woolsey. 1991; Agmon et d.. 1993: Schlagger and O'Leary. 1994: Catalano et

al.. 1996). By postnatal day (PD 2). these auons form two tiers of terminations. one at the

border between layer V and VT and another sparse termination into layer IV (Agmon et

al.. 1993). At this age. thalarnocortical afferents exhibit a rudimentary form of whisker-

related clustering that has been demonstrated with both acetylcholinesterase

histochemistry (Schlagger and O'Leary, 1994) and fluorescent tract tracing (Erzurumlu

and Jhaveri. 1990). By PD 3. the upper tier of VPm a..onal arbors in layer IV have

attained their appropriate spatial ordering, characterized by a periodic clustering of

thalamocortical afferents within barrel hollow domains (Jhaveri et al.. 1991 : Agrnon et

al.. 1993; Catalano et al.. 1996). Thereafter. the density of thalarnocortical arbors in both

the deep (layer V-VI) and upper (layer M tiers of termination. increases substantially

and reaches an adult distribution midway thr~ugh the second postnatal week (Agmon et

al.. 1993: Catalano et al.. t996).

The development of intracorticaI projectioas within the Sf cortex also follo~vs a

highly ordered sequence of events that occurs contemporaneously with thalarnocortica1

11

development. Studies employing anterograde and retrograde labeling have shown that the

barrel-related pattern of intracortical projections within layer IV is established by PD 4

(McCasland et al.. 1992: Rhoades et al., 1996). Two to three days prior to this.

intracortical projections are diffusely distributed throughout Iayer [V and do not respect

any form of barrel compartmentation (Rhoades et al.. 1996). However. between PD 2 and

-I. the projections of intracortical neurons are progressively remodeled into discrete

laminar and coLumnar cortical compartments (Koralek et al.. 1990: Rhoades et al., 1996).

In layer IV. these processes selectively innervate septal regions between the zones of

clustered thalamocortical arborization (Rhoades et al.. 1996). In supra- and infragranular

layers. intracortical projections are much more broadly distributed. extending

horizontally. in some cases over several millimeters and vertically across all cortical

lamina (Rhoades et al.. 1996: Kim and Ebner. 1999: Kossut and Juliano. 1999).

1.4 Developmental and adult plasticity of Sl cortex

Similar to other sensory cortices. the emergence of topographic representations in

the S 1 cortex can be altered by manipulations of sensory experience during a critical

period of development. In 1973. Van der Loos and Woolsey reported that perinatal

cauterization of a row of whisker follicles significantly altered the cytoarchitechtonic

arrangement of layer IV barrels in the S I cortex. These alterations were manifested by an

expansion or "swelling'" of neighboring, non-deprived barrel rows, that filled-in part of

the cortical territory corresponding to the removed vibrissae. Subsequent studies

regarding the formation of the barrel-tield have showed that some rnanipdations of the

sensory periphery, such as idhorbital nerve transection (i-e. the major afferent nerve of

12

whiskers), disrupt the normal somatotopic organization of thalarnocortical afferents into

barrel-related clusters (Belford and KilIackey, 1980; Jensen and Killackey, 1987). while

other manipulations such as pharmacoIogical bIockade of neural activity (via tetrodotoxin

or TTX) do not (Chiaia et al.. 1992). Furthermore, it was noted from these studies that

deprivation-induced changes of layer IV cytoarchitecture. were evident only from birth

until around PD 4 (Belford and Killackey, 1980; Jeanmonod et al.. 1981). thus defining

the "sensitive or critical" period for which manipulations of peripheral inputs can shape

the anatomical structure of the S 1 cortex. Taken together. these reports demonstrate that

during early neonatal development. the formation of barrels during the critical period of

postnatal development is dependent on the integlty of input horn the vibrissae. but not

on normal hctional activity from that pathway.

Experiments using more innocuous forms of sensory deprivation (ie. whisker

trimming or plucking) in which peripheral sensory receptors are left intact. do not alter

the physical appearance of barrels during the critical period (Weller and Johnson. 1975).

Despite the unchanged appearance of corticd barreb. several investigations have

demonstrated that the functional properties of neurons in layer IV are dependent on

normal vibrissa-tactile experience (for review see Kossut. 1992). For example. chronic

trimming of whiskers during the f i t few weeks of postnatal Iife, alters the topographic

organization of whisker-related barreb in the S 1. These changes were manifested by

Iayer IV neurons corresponding to the clipped whisker. becoming more responsive to

stimulation of an adjacent, spared whisker (Simons and Land. 1987). Comparably, Fox

(1992. 1994) examined the response properties of neurons In cortical layers II, III and IV

in rats that had d l but the D 1 vibrissae removed (called univibrissa rearing), starting from

13

the day of birth (PD 0), or from PD2, PD4 or PD7. His results showed that the area of

cortex driven by stimulating the spared D 1 vibrissa was enlarged despite the normal

anatomical appearance of the barrel-field. Moreover, he observed that changes in the

receptive field properties of cortical neurons were variable. with layer IV neurons

becoming less plastic between PD 0 and PD4. while neurons in layers 11 and 111 were

plastic from PD 4 and beyond. These results reinforced the notion that the duration of

critical periods for experience-dependent cortical plasticity may differ. Thus. unlike

neurons in thalarnorecipient layer IV. cells in supra- and infi-agranular layers appear to

remain plastic well into postnatal Iife (O'Leary et al.. 1994).

Although it has been widely recognized that neonatal brains are particularly

susceptible to alterations of sensory experience. there is evidence indicating that synaptic

connections in the adult cerebral cortex are also capable of a considerable level of

malleability. Studies assessing the metabolic activity of cortical neurons using 2-

Deoxyglucose (Kossut, 1992a: iMeIzer and Smith, 1995) or repeated optical imaging

(Polley et al.. 1999) have observed a large-scale expansion of a single whisker's

functional representation after severaI weeks of plucking all but one whisker. Recent

electrophysiological studies have shown functional reorganizations in the somatotopic

representation of the S 1, within hours (Ebner and Rema, 1999) to days (Diamond et d..

1993: Glazewski and Fox. 1996) &er whisker trimming. To induce these rapid changes.

Diamond et al.. (1993.1994) altered sensory experience by a brief period of 'whisker

pairing" in which two whiskers (eg. D 1 and D2) were left intact while all neighboring

whiskers were trimmed to within 2 mm of the face. Normally, neurons in the S1 cortex

respond preferentially to stimulation of their principal whisker. However. when recording

in the D3, barrel column just hours after whisker pairing, neurons became more

responsive to stimulation of the adjacent "paired" whisker (ie. Dl), while becoming less

responsive to stimulation of an adjacent (ie. D3) trimmed whisker. These results implied

that imbalances in whisker activity (ie. via whisker pairing) cause a strengthening of

synaptic connections in adjacent non-deprived barrel columns. while connections in

deprived barrel columns. are weakened.

1.5 Locus o f experience-dependent cortical plasticity

As alluded to earlier. neonatal or sensitive period plasticity in the S1 cortex is

typically characterized by changes in subcortical (i.e. thaiamocortical) inputs (Schlaggar

and O'Leary. 1991), whereas more adult forms of plasticity are characterized by

hct ional changes in intracortical circuitry that are mapped out onto the existing barrel

cytoarchitecture (KOSSUL I992b). However. it is important to keep in mind that the site of

plastic changes. is often dependent on the type of manipulation that is employed. For

example in neonatal rats and mice. permanent disruptions of the ascending ttigeminal

system via nerve transection or whisker follicie cautery (Van der Loos and Woolsey,

1973: Jeanmonod et al.. 198 1: Jensen and Killackey, 1987). alters the barrel-like

clustering of fierents from the thalamus. whereas more innocuous manipulations such as

whisker dipping. are associated with changes in the distribution of intracorticd circuits

(Keller and Carlson, 1999). There are severd lines of evidence to suggest that in the

adult the locus of experiencedependent plasticity is cortical. First, deprivation-induced

changes in the bct ional properties of neurons in the cortex, occur mainIy in supra-and

infia-granular layers (whose major source of synaptic input is intracorticaI), and less so in

15

layer IV where much of the afferent input is from subcortical structures (Donoghue,

1995). Second. electrophysiological studies have shown that whisker pairing or

univibrissa rearing (Fox, 1992; Diamond et al.. 1993; Diamond et al., 1994; Fox, 19941,

does not affect the short latency ( 4 0 rns) responses of cortical neurons. which

presumably reflect monosynaptic inputs from the thalamus. but rather. affects cortical

responses in epochs after the first 10 ms, suggesting that intracortical. heterosynaptic

inputs have been modified. Furthermore, several recent studies have shown that the

redistribution of receptive field properties in S 1 neurons after whisker pairing or

univibrissa rearing is not accompanied by changes in subcortical receptive fields

(Glazewski et al.. 1998; Wallace and Fox. 1999). However. in cases where peripheral

nerve conduction is prevented. either permanently by whisker cauterization or transiently

by application of a locaI anesthetic. the receptive tieid properties of neurons in

subcortical structures change immediately and thus. may contribute to experience-

dependent changes in the cortex (Faggin et al.. 1997: Parker and Dostrovsky, 1999).

1.6 Mechanisms of cortical plasticity

1.6.1 Physiological mechanisms

The mechanisms by which sensory experience modifies neuronal circuits during

the critical period of development and later on in adult life. are unclear. As described in

the preceding section, disruptions of the ascending trigeminal system during the critical

period can alter the gross somatotopic organization of VPm fierents to the S I cortex. By

contrast, more innocuous manipulations (whisker trimming or plucking) of sensory

experience during the critical period and beyond, cause reorganizations in the receptive

16

field properties of S I neurons. However, despite differences in the form of plasticity that

is expressed. it is generally believed that activitydependent processes of synapse

elimination, refinement and remodeling, are a common mechanism underlying these

plastic changes.

One mechanism that captures the eiements of these processes. was originally

postulated by Donald Hebb (1949) who stated that when one axon is near enough to

excite or repeatedlylpersistently take part in firing another neuron, some change takes

place in one or both cells such that the efficacy between these neurons is altered. This

hypothesis revolutionized the thinking about the neural mechanisms of plasticity and

provided a model (i.e. Hebbian-based synaptic piasticity) through which empirical

investigations could assess the extent to which neuronal activity. in particular that driven

by sensory experience could modifj the functional circuitry of the brain (Bear et d..

1987: Kirkwood et al.. 1996). A major frnding supporting Hebb's idea was that the

efficacy of synaptic connections could be strengthened or weakened for long periods of

time following certain protocols of neuronal stimulation (Bliss and Lomo. 1972; Bear et

al.. 1987). These long-term changes were referred to as long-term potentiation (LTP)

when synaptic transmission was strengthened (Bliss and Lomo. 1972), and Long-term

depression (LTD) when synaptic transmission was weakened (Bear and Malenka. 1994).

At the synaptic level. the induction and maintenance of LTP and LTD-like processes

involves a multitude of synaptic factors. Of these. it has been shown that LTP and LTD

are in part. dependent on glutamate release and activation of its receptors (in particdar

NMDA receptors), postsynaptic calcium entry and the generation of diffusible

intracellular messengers (for review see Bear and Malenka, 1994). At a higher level of

neuronal organization, LTP and LTD-like mechanisms follow Hebbian-based rules that

are thought to govern experience-dependent changes in the brain (Bear et al.. 1987: Fox.

2000). These Hebbian-based rules state that: a) the direction of change in the efficacy of

synaptic inputs. is dependent on their correlation with postsynaptic firing or with the

activity of other inputs (Buonomano and Merzenich. 1998; Van Rossurn et al.. 2000) and

b) changes in synaptic weighting should provide competition between hctionally

related neuronal inputs such that stronger synapses are maintained. while weaker inputs

are eliminated.

The appeal of LTP and LTD-like mechanisms in explaining critical period

plasticity is threefold. First. thalamocortical afferents in V1 cortex. and to some extent in

the S 1. appear to compete for targets in the cortex (Schlagger and 0' Leary. 1991 : Katz

and Shatz 1996). Consequently. a number of these afferent projections are retained while

many are eliminated (Shatz. 1990). This competition between functionally related inputs

can be explained through LTP and LTD-Iike processes such that more active circuits are

strengthened or retained by correlated pre- and postsynaptic activity while less active

circuits whose pre- and postsynaptic activity are uncorrelated. are weakened or

eliminated (Bear et al.. 1987: Feldrnan et ai., 1999). Second. LTP and LTD in

thalamocortical synapses in vifro. can be induced only during the period of deveiopment

that corresponds to the critical period of experience-dependent plasticity, in vivo (Crair

and Malenka. 1995; Feldrnan et al., 1998). Finally, some pharmacological or genetic

manipulations that block or inactivate receptors (ie. NMDA glutamate receptor) that are

necessary for the induction of certain f o m of LTP and LTD (Artola and Singer. 1983,

also disrupt the somatotopic patterning and topographic retinement of thalamocortical

18

afferents during the critical period (Schlaggar et al., 1993; Fox et al., 1996; Iwasato et al.,

1997: I~vasato et al.. 2000).

Numerous studies have sho\vn that sensory deprivation initiated after the critical

period. induces rapid changes in the bct ional organization of the S 1 cortex (for review

see Kossut, 199%). It has been hypothesized that the major mechanism by which these

changes can occur (ie. that occur within hours to days after sensory deprivation), are by

changes in the synaptic efficacy of existing cortical circuits through LTP and LTD-like

processes. The appeak of LTP and LTD as processes that mediate experience-dependent

plasticity is in part due to the fact that LTP and LTD are widely available to sensory

pathways (Donoghue. 1995). For example, Castro-Alamancos et a1.. (1995) showed that

LTP and LTD can be induced in vertical intracortical connections (i.e. neurons that

project from layer IV into cortical layers 111111) by high and low frequency stimulation of

layer IV. respectively. Furthering these observations. Feldman (2000) demonstrated that

the expression of LTP and LTD at these vertically projecting synapses were dependent on

the timing and correlation between pre- and post-synaptic activity. Thus, when whiskers

are stimulated (or spared). layer IV neurons rapidly and synchronously activate neurons

in layers IIAII. thus correlating the two inputs and enabling synaptic potentiation (Fox.

3000). Conversely when whiskers are removed, synaptic activity in connections from

layer IV onto Iayer IVIII neurons becomes asynchronous. leading to uncorrelated activity

between pre- and post-synaptic targets. Hence in this circumstance, uncorrelated pre- and

post-synaptic activity leads to the depression of responses in Iayer IVlH neurons (Fox et

al.. 2000). With respect to LTP and LTD-like processes as a potential mechanism for

cortical plasticity. these findings proved that: a) LTP and LTD could be induced in

19

vertical inputs to layer IHII neurons, which has been hypothesized to be a primary

substrate for experience-dependent changes in the cortex (Cynader. 2000; Feldman, 2000:

Fox. 2000) and b) the Hebbian-based rules that LTP and LTD adhere to (ie. regarding

correlated activity). can explain the potentiation andlor depression of synapses that has

been observed in whisker pairing and univibrissa rearing paradigms of plasticity (Fox.

1992; Diamond et al.. 1993; Fox. 1994; Glazewski and Fox. 1996).

If LTP and LTD-like processes are important in the generation of experience-

dependent plasticity. then pharmacological agents that block these processes should dso

inhibit conical reorganizations. Two studies from Jablonska et al(1995; 1999) showed

that the NMDA glutamate receptor. which is important For LTP and LTD induction

( h o l a and Singer. 1987) is necessary for topographic changes in the cortex. Using 3-DG

autoradiography in the first study (1995). they showed that increases in the bctional

representation of cortical barrels in row C after removing all but row C whiskers, were

attenuated by subdural implants of an NMDA receptor antagonist. In the second

experiment. a learning-based paradigm was employed in which stimulation of a row of

vibrissae was paired with a taii shock. Normally. this type of pairing produces an

enlargement in the hctionaI representation of the row of whiskers that was stimulated.

However. if NMDA receptors were blocked, the expansion of the cortical representation

in the trained row of whiskers. was reduced by haIE Perhaps the most compelIing

demonstration of NMDA involvement in cortical plasticity arises from Rema et al.?

( 1998) who examined the receptive field properties of neurons in the D2 barrel column

after a brief period of Dl and D3 whisker pairing (for review see section 1.4). Their

results showed that the response biases fiom D2 neurons to the intact D 1 whisker. typical

20

of S 1 neurons after whisker pairing, were completely suppressed by NMDA receptor

inactivation. Taken together. these findings provide direct evidence of NMDA receptor

invoivement in cortical plasticity, and suggest that NMDAdependent LTP and LTD.

may contribute to this phenomena.

1.6.2 Srnrcrzcral mechanisms

A number of studies have demonstrated that sensory denervation (infraorbital

nerve transection) or deprivation (whisker removal) induces structural or morphological

changes that occur in parallel with physiological changes. As described previously.

manipulations of afferent sensory input early in postnatal life disrupts thalamocortical

and intracortical innervation of S1 cortex (Van der Loos and Woolsey. 1973: McCasland

st al.. 1992: Rhoades et al.. 1996; Keller and Carlson. 1999). The arrangement of

dendrites in cortical neurons is also profoundly affected by changes in sensory

experience. Harris and Woolsey (198 1) showed that after neonatal whisker follicle

ablation. neurons in deprived cortical areas reoriented their dendritic processes into

adjacent non-deprived areas. More recently. a report by Lendvai et al.. (2000)

demonstrated that whisker trimming in young rats, reduced the protrusive motility (idout

movements) of immature dendritic spines of layer Y3 pyramidal neurons in the deprived

region of the ban-el cortex. However. rather unexpectedly. whisker trimming had no

effect on the density. length and shape of dendritic spines in these affected regions. In the

adult S I cortex. it now appears that intracortical auons. which terminate upon these

dendrites. can over time. extend from deprived into non-deprived cortical zones.

Evidence of these structural changes has been most clearly documented in the V1, in

2 1

which focal retinal fesions have been shown to greatly increase the sprouting and

branching of intracortical avons into de-afferented regions of the cortex (Darian-Smith

and Gilbert. 1994, 1995). With respect to the barrel cortex, peripheral-induced changes in

cortical connectivity have not been well characterized. However. new evidence from

Kassut and Juliano (1999) suggests that whisker removal sipiftcantly alters the length of

monal projections within spared barrel columns: such that micrainjections of anterograde

and retrograde tracers into spared barrel columns. labeled avons extending for

significantly greater distances and labeled ceII bodies situated considerably Further than

aRer injections into deprived or control barrels. To conclude. these studies indicate that

experience-dependent changes in cortical topography. are mediated not only by

firnctional changes in synaptic cornmunication. but also by changes in the morphological

arrangement of dendrites and a~onrtl projections.

1.7 Molecular correlates of cortical ptasticity

In the visuaI cortex of neonatal kittens. deveIoprnental changes in the distribution

of certain neurotransmitters and receptors have been correlated with the critical period of

ocular dominance and orientation column plasticity. Of these. it has been shown that

binding Ievels of serotonin (Dyck and Cynader. 1993), NMDA (Gordon et al., 1996;

Chen et al.. 2000). and cholinergic muscarinic (Liu et al., 1994) receptors are highest

during peak periods of plasticity. In addition, high Ievels of zinc (Dyck et at.! I993),

serotonin (5-W. Gu et ai.. 1990) and norepinephrine (NEp: Liu and Cynader, 1994) have

been observed within layer IV during the critical period for plasticity in kinen V1.

. b o n g the neurotransmitter receptor binding patterns that have been investigated during

the critical period for barrel formation (ie. PD 0-4), only metabotropic glutamate

receptors (Blue et al.. 1997) and 5-HTlB receptors (Bennett-Clarke et al.. 1993)

exhibited high binding densities during the critical period. Other studies have reported

increased levels of AChE (Krisa 1987). 5-HT (Fujimiya et al.. 1986; D'Arnato et al..

1987). NEp (Lidov et al.. 1978). growth-associated protein (GAP43: Erzunrmlu et al..

1990) and brain-derived neurotrophic factor or BDNF (Singh et al., 1997) staining during

the first few days of postnatal development. Interestingly, despite the conspicuous

presence of these neuroactive molccu1es during the critical period. it appears that only

genetic or pharmacological manipulations of 5-HT (Cases et al.. 1995: Cases et al.. 1996: C

Vitalis et al.. 1998) or GAP43 (Maier et al., 1999) and NMDA receptors (Iwasato et al.,

1997) disrupt the development and topographic organization of cortical barrels.

As described earlier. neonatal whisker trimming or plucking initiated during the

first few weeks of postnatal development. but ajier the critical period of barrel formation.

can produce robust plastic changes in the response properties of S 1 neurons (Simons and

Land. 1987: Fox 1992.1994). Corresponding with these changes. are dynamic

redistributions in neurotransmitter receptor binding patterns. In particular. it has been

shown that NMDA (Glazewski et al.. 1993, AMPA (Blue and Johnston, 1995: Brennan

et al.. 1997), GABAA (Golshani et al.. 1997) and Pnoradrenergic (Kossut, 1992) receptor

binding levels are maximal only afier the critical period for barrel formation. In these

instances. receptor levels are low during the first postnatal week. rise in subsequent

weeks, and reach adult values by PD 21. In addition to these receptors. several caicium

binding proteins such as pacvalbumh (Sanchez et al., I992), calbindin (Alcantara et aI..

1993) and calretinin (Melvin and Dyck, 1999) are differentially expressed across and

within cortical laminae after the critical period, and reach adult patterns by the end of the

third postnatal week.

Investigations of plasticity in the adult cortex, have demonstrated that changes in

afferent sensory-driven neuronal activity via whisker plucking or cauterization. can

dynamically regulate a number of inhibitory and excitatory receptors and enzymes. For

example. whisker removal decreases GABA-A (Skangiel-Krarnska et al.. 1994; Fuchs

and Salazar. 1998). receptor binding in deprived areas of the barrel field, while

expression of the AMPA receptor subunit Glum. is increased in barrels associated with

the spared whiskers (Gierdalski et al.. 1999). Deprivation-induced changes have also

been identified using immunohistochemical markers for glutarnic acid decarbo.uylase

(GAD: Welker et al.. 1989: Akhtar and Land. 1991) and GAP43 (Dm-Meynell et al..

1992). whose level of expression is reduced in barrels corresponding to the trimmed

whiskers. By contrast. whisker deprivation appears to have no short-term effects on

elutamate irnrnunoreactivity (Kossut. 1992) or NMDA receptor binding (Glazewski et al.. - 1995).

1.8 Why study zinc in cortical development and plasticity?

I .8.1 Presence of zinc in the central nervous system

Zinc is the second most abundant trace element in the brain (after iron) with about

10 pg of zinc per gram of wet tissue (Frederickson, 1989). The majority of zinc in the

brain (approximately 90%) is bound up in metalloproteins and zinc-finger transcription

factors. in wbich zinc is used for the catalysis and structural stability of enzymes and is

34

necessary for DNA replication and transcription (Huang, 1997). The remaining LO% of

zinc in the brain constitutes a chelatable pool of zinc that can be visualized

histochemicaIly in axon terminals or cell bodies with the Timm sulfide-silver method

( 1 958) or the Danscher selenium-silver method (Danscher. 1982). The histochemically

detectable pool of zinc has been found to be restricted to presynaptic vesicles within the

avon terminals of zinc-containing (i.e. zinc-ergic) neurons (Frederickson. 1989). These

zinc-containing neurons are considered to be a subclass of glutamatergic neurons as they

contain clear. round vesicles. form asymmetric (Gray's type I) synaptic contacts and are

enriched in glutamate (Beaulieu et al.. 1992: Dyck et al.. 1993). Furthermore. there is

accumulating evidence suggesting that zinc acts as a neurotransmitter: namely it is

contained within synaptic vesicles and is released in a calcium and impulse-dependent

manner (Assaf and Chung. 1984). there are zinc-specific transporters responsible for its

uptake into the presynaptic terminal (Wenzel et al.. 1997). and finally. zinc can modulate

(and possibly mediate) synaptic transmission by binding to zinc-specific binding sites on

receptors of a number of ligand gated ion channels (Smart et al.. 1994).

Studies regarding the anatomical distribution of synaptic zinc have found that

high levels of histochemically-reactive zinc are associated with some of the most plastic

regions of the adult mammalian brain (Dyck et al., 1993: Czupryn and Skangiel-

Krarnska 1997). In particuiar, zinc-containing axon terminals are abundant in the mossy

fibers of the hippocampus and in layers I, MI1 and V of the neocortex (Frederickson and

Moncrieff. 1994; Czupryn and Skangiel-Kramska. 1997). Within the cerebral cortex. the

majority of zinc-containing avon terminals arise horn pyramidal-shaped cell bodies that

are located predominately in cortical layers LI. III and VI (Garrett and SIomianka 1992;

Dyck and O'Leary, 1995). These zinc-containing neurons form a vast intracortical

network. reciprocally interconnecting ipsi- and contralateral cortical domains

(Frederickson and Moncrieff. 1994).

1.8.2 Posr-synap f ic efecrs of zinc

An increasing number of studies over recent years have provided evidence that

synaptically-released zinc may contribute to activity- and experience-dependent forms of

cortical plasticity (eg. LTP and LTD). by virtue of its modulatory effects on voltage and

ligand-gated ion channels. calcium binding proteins and neurotrophic factors

(Frederickson. 1989: Smart et ai.. 1994). First. given that zinc is colocalized within a

subset of glutamatergic neurons (Beaulieu et al.. 1992) and released in a calcium and

impulse-dependent manner ( h s a f and Chung. 1983). the most prominent effects of

synaptic zinc may be upon glutamate receptors. Several authors have reported that

physiological concentrations of zinc (I 0- 100 pM) in hippocampal and neocortical slice

preparations. potently inhibits the activation of NMDA glutamate receptors (Westbrook

and Mayer. 1987: Christine and Choi. 1990: Vogt et al., 2000). Two mechanisms have

been suggested through which zinc can inhibit NMDA receptor function: one

independent of membrane voltage and affecting channel opening probability, and the

other a voltage dependent fast channei block (Westbrook and Mayer, 1987: Christine and

Choi. 1990). ConverseIy. zinc appears to mildly potentiate excitatory responses mediated

by the AMPA-type glutamate receptor (Xie and Smart. 1994), although the mechanisms

by which zinc enhances AMPA responses are unknown.

26

If synaptically-released zinc diffuses outside of the excitatory synaptic cleft, it is

possible that zinc may interact with nearby GABAergic (Westbrook and Mayer, 1987),

cholinergic (Palma et al.? 1998) and serotonergic (Hubbard and Lumrnis, 2000) synapses.

Zinc inhibits GABA receptor activation in neurons isolated from the thalamus and cortex

(Gibbs et al.. 2000), as well as GABA* responses in hippocampal neurons (Westbrook

and Mayer. 1987: Smart et al. 1994). In addition to G W A receptors, the alpha 7

nicotinic (Palma et d.. 1998) and 5-HT 3A (Hubbard and Lummis. 2000) receptor

subtype have zinc specific binding sites that, when activated. facilitate agonist-induced

currents in these ligand-gated channels.

There appear to be several routes through which synaptically-released zinc can

enter the post-synaptic neuron and potentially modulate its hct ion. One route through

which zinc can enter post-synaptic terminals is via voltage gated calcium channels

(VDCC: Wang and Quastel. 1990). Support For this contention stems from observations

that zinc attenuates current through VDCC (Sandow and Bien. 1962) and calcium

channel blockers such as nimodipine. attenuate K* -stimulated neuronal zinc influx (Choi

and Koh. 1998). Another potential route of zinc entry into neurons is through NMDA and

a subset of A i i A receptor gated channels. Choi and Koh (1994) demonstrated that zinc-

induced death of cortical neurons (ie. via toxic zinc entry into neurons) was reduced by

competitive NMDA receptor antagonists. In addition, zinc can also pass through a small

subset of AMPA receptor-gated channels that are highly permeable to calcium.

Supporting this. Y i et al.. (1995; 1998) showed that kainite-stimulated intracellular zinc

accumdation into cortical neurons, identified a common set of neurons that were

characterized by the expression of calcium permeable AMPAIkainate channels. And

27

finally. unpublished observations fiorn Choi and collegues (see Choi and Koh, 1998)

suggests that emcellular zinc can enter cortical neurons through a sodium-zinc

exchanger.

Once inside the post-synaptic neuron. zinc can interact with calcium-binding

protein kinases that mediate long-term changes in synaptic efficacy associated with LTP

and LTD. For example. it has been reported that zinc modulates calcium/calmodulin-

dependent protein kinase 11 (CaMKII) activity (Weinberger and Rostas, 199 1; Lengyel et

al.. 2000). which is an enzyme that has been shown to piay an imponant role in the

induction of LTP/LTD (Malenka et al.. 1989: Mulkey et al.. 1993) and barrel cortex

plasticity (Glazewski et ai.. 2000). Other studies have sho\in that zinc can activate the

phospholipase C-protein kinase C (PKC) signal transduction cascade (Baba et d.. 199 1)

or inhibit Src tyrosine kinase-dependent activation of NMDA receptor channels (Zheng et

al.. 1998). In addition to these enzymes. several calcium binding proteins such as

calcyclin and parvalbumin are also zinc binding, and require zinc to exert their biological

effects (Permiakov et al.. 1988: FiIipek et al.. 1990; Kordowska et al., 1998). However.

despite the multitude of proteins that are regulated by zinc. it is unclear if any of these

zinc-protein interactions are involved in synaptic plasticity.

1.8.3 Effect oj+zinc on nerirotrophic factors

The establishment during early development, and subsequent refinement of

synaptic connections that occurs throughout postnatal life, is partially dependent on a

group of molecules that mediate trophic interactions between individual cetlular

components (Dyck et aI.. 1993). In particular. zinc appears to pIay an integral role in

28

regulating the b c t i o n of nerve growth factor (NGF). Young and Koroly (1980)

demonstrated that zinc acts as a control ion that keeps NGF in its inactive monomeric

form until it recognizes its naturally occurring substrate. A recent study from Ross et al..

(1997) has furthered these observations showing that zinc alters the conformation of

NGF. rendering it unable to bind to its native receptors (ie. p75 or tyrosine kinase A

receptors). or activate signal transduction pathways. At present. it is uncertain whether

zinc may have similar effects on other members of the NGF family such as BDNF or NT-

3. Nevertheless. these studies provide suggestive evidence that zinc is capable of

modulating neurotrophic factors that may be important for the devetopment and plasticity

of cortical circuits.

1.9 Rationale

It is generally believed that activity-dependent processes (such as LTP and LTD)

euide the formation. elimination and reorganization of cortical synaptic connections b

throughout postnatal development. However. despite this assumption. the moIecuIar

mechanisms that mediate these synaptic changes have remained elusive. The primary

coal of the studies described in this manuscript is to shed light on some of the potential - subceIlular mechanisms of synaptic plasticity in the developing and mature cerebral

cortex. In this regard. we chose to examine the role of neurons that contain and release

zinc from their axon terminals. As described in the preceeding sections. synaptically-

released zinc has the potential to profoundly affect synaptic communication by virtue of

its modulatory effects on excitatory and inhibitory neurotransmission, intracelldar

messenger proteins (eg. CaMKII, PKC) and neurotrophic factors.

29

As a first step in implicating zinc-ergic circuits in corticaI plasticity, we have

provided a detailed anatomical description of the postnatai distributions of zinc-

containing fibers in the S 1 cortex of wild-type mice. Following this, we then sought to

determine some of the developmental factors that specifv zinc-ergic innervation of the SI

cortex. In particular. developmental gradients in the laminar and columnar distribution of

zinc-containing axon terminals were assessed in mice that exhibit abnormal patterns of

synaptic connectivity in S 1 cortex. And finaIly. to implicate zinc-ergic processes in

experience-dependent forms of synaptic plasticity that occur in adulthood. we examined

the distribution of zinc-containing fibers in S 1 cortex of adult mice that have undergone

various periods of sensory (i-e. tactile) deprivation. Thus, the primary objective of these

studies was to characterize the posmatd distribution of zinc-containing fibers in S 1

cortex and to correlate changes in the distribution of hese fibers with plastic events that

occur in developing and mature brains,

30

Chapter 2: Ontogenic Distribution of Synaptic Zinc in S1

Cortex of C3H and Monoamine Oxidase-A Knockout Mice

2.1 Introduction

Developmental changes in the laminar and columnar organization of

neurotransmitter-specific afferents and receptors have been shown to occur during

particular windows of development and appear to play an integral role in guiding the

establishment and refinement of developing neocortical connections (Dyck and Cynader.

1993a. b: Cases et al.. 1996: Kojic et d.. 2000). [n particular, it has been proposed that a

specific population of cortical neurons that contain and release zinc may contribute to

activity-dependent mechanisms of corticai column formation and plasticity. by virtue of

zinc's modulatory effects on excitatory and inhibitory neurotranmission (Dyck et al..

1993: Smart et al.. 1994). Evidence supporting this contention arises from ontogenic

studies in the hippocampus that have revealed age-dependent and lamina-specific

changes in the distribution of zinc-ergic terminals that parallel periods of intense

synaptogenesis and cytoarc hitectonic differentiation (Zirnmer and Haug, 1978).

Moreover. studies characterizing developmental changes in the distribution of zinc-

containing axon terminals in the feline visual cortex. have found that synaptic zinc is

periodically distributed within layer IV and transiently delineates columnar

compartments during the criticd period for ocular dominance and orientation column

plasticity (Dyck and Cynader. 1993a). Comparably, a study by Czupryn and Skangiel-

Kramska (1997) showed that in the developing S1 cortex of mice, staining for synaptic

zinc transiently demarcates discrete laminar and columnar compartments during the first

3 1

1 to 2 weeks of postnatal development. However, the data reported in this study appeared

to be somewhat incongruent with our preliminary observations regarding the postnatal

distribution of synaptic zinc in S 1 cortex of mice and rats. As a first step in implicating

zinc-ergic circuits in cortical development and plasticity, we attempted to replicate and

refine Czupryn and Skangiel-Kramska's (1997) observations by describing ontogenic

changes in the distribution of zinc-containing axon terminals in the S1 cortex of wild-

type (WT) mice.

Once the postnatal distribution of zinc-containing axon terminals in S 1 cortex was

clearly and accurately characterized. we then sought to determine some of the

developmental factors that regulate zinc-ergic innervation of the S1 cortex. Fox example.

it is uncertain whether intracortical. zinc-ergic innervation of the barrel-field in layer IV.

is specified by. and follows the segregation of thalamocortical afferents into whisker-

specific domains. A report From Rhoades et al.. (1996) demonstrated that neonatal

manipulations of cortical activity (ie. infraorbital nerve transection on PD 0), that disrupt

the topographic organization of thalamocortical afferents. also profoundly affect the

distribution of intracortical projections. However. manipulations that alter

thalamocortical afferent activity (ie. l-R implants) but not the organization of

thalarnocortical afferent auons. have no effect on the vibrissae-related pattern of

intracorticai projections. Contrasting with these results, Keller and Carlson (1999) have

reported that neonataI whisker trimming signif~cantly alters the development of

intracorticai co~ec t ions in granular (layer IV) and non-granular (layers I* II/rII. V. W)

cortical layers. while having no effect on thalamocortical innervation in S1. The

3 2

discrepancies in the conclusions drawn fiom these two studies exemplify how poorly

understood organized patterns of intracortical connectivity are established.

Another approach in determining whether the development of intracortical

circuits is specified by thalamic innervation is to examine genetically altered mice that do

not exhibit the barrel-specific clustering of thalamocortical afTerents. characteristic of

normal mice. One transgenic mouse Iine that expresses these features (or the lack thereof)

is a C3H strain that lacks the gene encoding for monoamine oxidase-A (MAO-A).

Because MAO-A is an enzyme required for the breakdown of monomines. there is a

700-900% and 35-70% increase in the amount of 5-HT and Nep respectively, during the

tirst postnatal week (Cases et al.. 1995). Corresponding with the excessively high levels

of 5-HT and Nep are disruptions in the barrel-like clustering of thalmoconical axons

within their respective barrel-field targets (Cases et al.. 1995. 1996). Furthermore, these

disruptions appeared to be directly related to excessive amounts of 5-HT as

pharmacological inhibitors of 5-HT synthesis restored .the normal pattern of cortical

barrels in MAO-A knockout (MAO-A KO) mice. whereas inhibition of catecholamine

synthesis did not. With respect to the present study. we exploited the barrel-less

phenotype of MAO-A KO mice to determine if the development of intracortical zinc-

ergic projections in the S I cortex is dependent upon the barrel-related patterning of

thalamocortical afferents. Thus. using a modiication of the selenium-histochemical

method (Danscher. 1982). we describe the laminar and columnar distribution of zinc-

containing axon terminals in S I cortex of WT controls and MAO-A KO mice. across

postnatal deveIopment.

2.2 Methods

2.2.1 =Inimals and tissue preparation

Monoarnine oxidase-A knockout (MAO-A KO) mice with a C3WHeJ genetic

background (Tg8 strain; Cases et d.. 1995. 1996) and wild-type (WT) C3WHeJ mice. 3

to >60 (adult) days of age were used to study the distribution of zinc-containing ZKon

terminals. .4t least three mice at each age (PD 3.5.6,8. 10. 15.28 and >60) were used.

All experiments were conducted under the guidelines of the Canadian Council on Animal

Care.

Histochemical localization of vesicular-bound zinc was assessed by using the

selenium method developed by Danscher ( 1982). Mice were injected i.p. with 15 m a g

sodium selenite ( 5 mglml). dissolved in 0.9% physiological saline. After 60-minutes. the

mice were killed with an overdose of sodium pentobarbital (65 mgkg) and the brains

were removed and bisected. To visualize the barreI-field of layer TV. some cortical

hemispheres were prepared tbr tangentid sections (i.e. horizontal to the pial surface) by

separating the cortex from the underlying subcortical structures and flattening it between

two glass slides. Thereafter, the tissue was immediately frozen in crushed dry ice and

stored at -30°C. Sagittal or tangential sections were cut at 20-prn on a cryostat and thaw

mounted onto gelatin-coated glass slides. The sections were then stored at -30°C in

preparation for histochernica1 staining.

2.2.2 Histochemistry

Brain sections were thawed and briefly allowed to dry at room temperature. ftvcd

in a descending series of ethanol (95%, 15 minutes; 70%, 2 minutes; 50%, 2 minutes),

34

hydrated and then dipped in a 0.5% gelatin solution. Selenium-bound zinc was visualized

on slides by physical development in 250 ml of developer containing 50% Gum arabic

(I00 rnl), 2.0 M sodium citrate buffer (25 ml), 0.5 M hydroquinone (30 ml), 37 mM

silver lactate (30 ml) and distilled water (70ml). Sections were incubated in darkness at

room temperature for 90-180 minutes, depending on the age of the animal.

After staining, - the slides were washed in running water for 20 minutes at 40°C.

then rinsed in distilled water for (2 .u 2 rninutes), and immersed in 5% sodium

thiosulphate solution for 12 minutes. Slides were then postfixed in 70% ethanol (EtOH)

for at least 30 minutes. then dehydrated in 95% EtOH for 5 minutes and 100% EtOH for

10 minutes. cleared in xylene, and coverslipped with Permount.

2. L 3 Duru ..lncllysis

Video images were obtained (Panasonic BD400 CCD camera; Data Transtation

Dt-2255 quick capture board) using a Macintosh OS 8.6-based image analysis system

with running MH Image software. To create photographic montages of the laminar and

columnar distribution of zinc-containing &.on terminals in S 1 cortex. representative

images fiom saggital and tangential sections of S 1 cortex were imported into Adobe

Photoshop 5.0 (Adobe Systems. San Jose. CA) in which minimal adjustments of contrast

and brightness were made. In addition, to relate the pattern of zinc staining to laminar

borders. near adjacent saggital sections were stained with cresyl violet.

In order to assess the relative level of zinc staining across S 1 cortical laminae.

optical density profiles were constructed fiom representative sagittal sections in which no

adjustments of brightness and contract were made. Each optical density profile was

35

generated by sampling a 1200 pm by 1500 pm area of S1 cortex. The values obtained for

each profile were expressed as a percentage of the maxima1 staining intensity observed

within each section (see Figure 3). However, because staining times were variable across

age and strain groups. densitometric data displayed in Figure 1 were used only to

establish within-animal differences in staining intensity across cortical layers.

2.3 Results

2.3.1 Zinc staining in SI cortex of WT mice

The earliest indication of histochemicaily reactive zinc in the S I cortex of WT

mice was evident at PD 3. Laminar profiles of Sl cortex at this age revealed a

homogenous pattern of staining that extended throughout the subplate region until the

lower portion of the marginal zone (data not shown). After PD 3. dynamic changes in the

patterning of vesicular-bound zinc became evident across and within cortical layers. The

distribution of synaptic zinc in WT riice of different ages. and corresponding optical

d e n s i ~ profiles of the reIarive changes in staining intensity are shown in Figure :A. C. D,

E. At PD 5. zinc staining in WT mice was characterized by higher Ievels of shining in

layers IV. V and VI. than in superficial layers (Fig 3A). Twenty-four hours later (PD 6).

WT mice expressed subtle changes in the laminar distribution of synaptic zinc. These

changes were manifested by a heterogeneous increase in zinc staining specifically within

iayer IV. Although not shown in Figure 3. the emergence of barrels at this age can best be

observed in tangential sections of layer IV (see Fig. SC, D).

At PD 8. the highest level of zinc staining was localized to the lower portion of

layer IV (Fig. 3C). Optical density analysis cofirmed this observation showing a peak

staining intensity within the Iohver portion of layer [V that was juxtaposed by moderate

staining in upper iayer IV and emgranular (non-layer [V) layers (Fig. 3C). Furthermore.

within lower layer [V. zinc staining was distributed in a periodic manner, delineating

barrei compartments with higher levels of staining in barreI hollows than septa (Fig. 3C).

From PD 8 to I 0. there was a noticeabIe increase in the Ievei of zinc staining across all

cortical layers (Fig. 3 E). At this age (i.e. PD 1 O), zinc staining appeared denser and more

Figure 3: Development of the laminar distribution of histochemically-reactive zinc in

sagittal sections through somatosensory cortex of WT (A, C, E, G) and MAO-A KO (B,

D, F, H) mice at PD 5 (A@) 8 (C,D), 10 (E,F) and 15 (G,H). Adjacent Nissl-stained

sections are inset to the right in each panel to facilitate the identification of cortical

laminae. Plots inset in the lower left of each panel. profile the relative density of zinc

staining across cortical laminae at each age. In WT mice. zinc staining is moderate in

layers IV. V and lower layer VI at PD 5 (A). From PD 5 to 10. zinc staining increases in

supra- and infragranuIar layers and heterogeneously in layer IV. demarcating barrel

compartments by staining more intensely in barrel hollows (C,E). By PD 15. the laminar

pattern of synaptic zinc is mature. characterized by higher levels of staining in layers I. 11.

111 and V. and lower levels in layers IV and VI (G). Barrel compartments are still defined

by differential levels of zinc staining in the mature cortex. however, high levels of zinc

staining are limited to the interbarrel septae. In MAOko mice. zinc staining is initially

highest in layer IV at PD 5 (B). By PD 8, the distribution and levels of zinc have attained

the mature appearance with layers [. 11. [I1 and V staining at highest levels. (D). Zinc

staining did not appear to respect barrel compartments in MAO-A KO mice at any age

examined (D,F,H). Scale bar = 300 pm.

3 9

uniformly distributed across cortical laminae than in eariier ages. In layer IV, zinc

staining was characterized by dense staining within barrel hotlows. separated by lightly

stained inter-barrel septa (Fig. 3E). At PD 15, the laminar differentiation of zinc staining

became clearly apparent (Fig. 3G). This laminar pattern was typified by dense staining in

layers I. 11.111 and V. interdigitated with lower levels of staining in layers IV and VI (Fig.

33). Compared to younger ages. the pattern of zinc staining within layer IV was reversed

at PD 15. This reversal was manifested by more intense zinc staining in barrel septa than

in barrel hollows (Fig. 3G). From PD 15 to adulthood (i.e. PD 60). the inter-laminar

distribution of synaptic zinc remained relatively unchanged. with the exception of a slight

increase in staining in Iayer VI. and was considered to have attained its mature

distribution.

2.3.2 Zinc staining in Si cortex of ;bLrlO-;l KO mice

Postnatal changes in the laminar distribution of synaptic zinc in MAO-A KO mice

and corresponding optical density profiles of zinc staining across corticaI laminae. are

shown in Figure 3B. D. E. F. At PD 3 (data not shown), zinc staining was sparsely

distributed across all cortical layers. By PD 5 the level of zinc staining throughout the S 1

cortex was reiatively homogenous. with the most intense staining observed in the lower

part of layer TV (Fig. 3B). This intenseIy stained region was characterized by a

continuous band of zinc staining that stretched across the horizontal extent of layer IV

(Fig. 3B). For the nelct 24 hours (i-e. PD 6), the laminar pattern of zinc staining remained

relatively stable and thus was not shown in Figure 3. However. by PD 8, zinc staining

was heterogeneously distributed across cortical layers, and appeared to define discrete

40

laminar boundaries (Fig. 3D). At this age, the laminar pattern of zinc staining appeared

mature (compare with WT mice at PD 15 in Fig. 3G) and was characterized by higher

levels of staining in layers I. II/III and V, with lower levels of staining in layers IV and

VI (Fig. 3D). Within layer IV. zinc staining was evenly and homogenously distributed

and did not e-uhibit any sign of barrel-like compartmentation typical of IVT mice. From

PD 8 onward. the level and pattern of zinc staining across and within cortical laminae

remained relatively constant. as the laminar profiles for MAO-A KO mice at PD 8 (Fig.

3D). 10 (Fig. 3F) and 15 (Fig. 3H) are virtrually indistinguishable.

The observation that zinc-ergic projections in S1 cortex of MAO-A KO mice

matured at a faster rate than that of their WT counterparts, prompted us to further

examine whether this "maturational effect" was in fact real. or rather was an artifact of

small sample sizes or reflected differences in tissue staining times between WT and

MAO-A KO mice. Moreover. due to age-related variability in staining time, it was

uncertain if the density of zinc staining in PD 8 mice was comparabie to the IeveI of

staining in adults. To address these concerns. sagittal sections obtained from six MAO-A

KO and six WT mice at PD 8.15 or 60 (2 mice per group). were used to compare age and

strain-related differences in staining for synaptic zinc. For each animal. care was taken to

ensure that sodium selenite injections were identical (ie. 15 mg/kg) across all animals. In

addition. all sections were stained in the same solution and staining tray. for the same

amount of time to establish between-group differences in staining intensity. After

staining. optical density profiles of cortical laminae in each animal were estabiished by

sampling a 1200 by 1500 pm area of S I. These values were then summed across the nvo

animals per group to create a mean opticaI density profile for each age group (see Fig. 3).

4 1

Qualitatively, the laminar pattern and intensity of zinc staining in both WT and

MAO-A KO mice. at all three ages (data not shown) were indistinguishable from the

photomicrographs shown in Figure 3. [t was also apparent from optical density profiles

shown in Figure 4. that the level and pattern of staining across cortical laminae for each

age group, were identical to those shown in Figure 3. In WT mice at PD 8, the level of

zinc staining across all cortical laminae was fairIy uniform with a peak staining intensity

in layer IV (Fig. 4). In cortical layers I. II/III and V. adult levels of staining were not

evident until PD 15. Optical density profiles for WT mice at PD 15 and 60 were similar.

both in the level and pattern of zinc staining across cortical laminae. In LMAO-A KO mice

at PD 8. optical density profiles revealed that zinc staining was highest in layers I. lI/III

and V. with lower levels of staining in layer IV and VI. This laminar pattern of zinc

staining (i.e. at PD 8) was comparable to optical density profiles generated For MAO-A

KO mice at PD 15 and 60. Furthermore. the intensity of zinc staining within each cortical

lamina at PD 8 was similar to that observed in PD 15 and 60 mice. Taken together. these

results provide convincing evidence that the pattern and level of zinc staining in MAO-

KO mice at PD 8, are mature. Furthermore. when comparing optical density profiles of

WT and MAO-A KO mice at PD 8. it appeared that the level of zinc staining in layer [V

for both strains was mature at PD 8 (Fig. 4). However. when comparing supra- and

inhgranular layers (i.e. layers I. IVIII. V. VI). the level and pattern of zinc staining is

mature in MAO-A KO mice at PD8. whereas in WT mice, zinc staining does appear to be

mature until PD 15 (Fig. 4). These results suggest that supra- and infragranular layers of

the S I cortex are where maturational differences between MAO-A KO and WT mice.

occur.

Figure 4: Opticd density profiles of cortical lamina in WT and MAO-A KO mice at PD

8 (solid black line). IS (dashed black line) and 60 (solid grey line). Optical densities of

zinc staining were expressed as an absolute value between 0 (white. i.e. no staining) and

156 (black), At PD 8 in WT mice. highest levels of staining were observed in iayer IV.

with less intense staining in supra- and inhagranular layers. However, by PD 15. Ievefs of

zinc staining in all cortical lamina, with the exception of layer VI. were comparable to

those observed at PD 60. and thus were considered to have achieved a mature

distribution. [n MAO-A KO mice. the level and pattern of zinc staining in cortical lamina

appeared mature at PD 8 as evinced by the likeness of optical density profiIes for all three

age groups. Furthermore. examination of optical dens@ profiles in WT and MAO-A KO

mice at PD 15 and 60. revealed a high degree of homology in the levei and pattern of zinc

staining across corticaI lamina

200 Wild-type

200 MAO-A KO

z. - .b - 8 **

I . " PD60 150 Etso- - .I . -~cft,zs~a cn - -+

b I s E .*'*' aJ \ a . C

0 1 2i3 4 5 6 0 Cortical layer Cortical layer

44

2.3.3 Burrel-field parrerning in WT mice

Tangential sections through the upper and lower levels of layer IV in WT mice at

selected ages of postnatal development. are pictured in Figure 5. At PD 3. a faint.

homogenous distribution of histochernicalIy-reactive zinc was present within the S 1 (data

not shown). At PD 5. barrel-like patterning was not yet observable in upper layer IV.

Despite the absence of barrels. the boundaries between the barrel-field and the adjacent

cortex were easily discernable (Fig. 5A). In deeper sections of layer IV. the fust signs of

barrel formation were evident (Fig. 5B). At this age. delineation of barrels could be

discerned by hint staining within barrel hoitows. encapsulated by septa that were devoid

of zinc (Fig. 5B). One day later (PD 6). the morphological cornpartmentation of barrels

was clearly apparent in WT mice (Fig 5C.D). Invalarninar analysis of layer IV revealed a

differential distribution of synapric zinc within the barrel-tield. In the upper regions of

layer IV (Fig. 5C). barrels were demarcated by lightly stained septa that encircled zinc-

poor barrel hollows. In deeper sections (Fig. 5D). this patterning was reversed with more

intense staining in barrel hollows. separated by faintly stained inter-barrel septa. Between

PD 6 and 10. the intensity of zinc staining within the barrel-field and surrounding

adjacent cortex increased. In the barrel-field of upper layer IV. zinc staining appeared to

be somewhat higher in barrel septae than hollows. Conversely in deeper sections of layer

[V. the barrel-field was punctuated by increased staining within barrel hollows.

surrounded by sparsely stained septa (Fig. 5F).

From postnatal day 10 to 15. substantial changes in the patterning of zinc were

observed (Fig. jE-H). By PD 15. barrel-field morphology was defined by intense staining

in barre1 septa. encompassed by lightly stained hoUows (Fig. 5G.H). Unlike younger WT

mice. this mosaic of zinc staining within the barrel-field was consistent throughout the

Figure 5: Development of zinc staining shown in tangential sections through upper (left

panels) and lower (right panels) levels of layer [V in WT mice. at PD 5 (A,B), 6 (C,D). 8

( E,F) and 15 (G,H). At PD 5. zinc staining in the barrel-field was relatively homogenous

(A) with some discernable signs of barrels in Iower layer IV (6). At PD 6 (C,D) and PD 8

(E,F) . barrel compartments became cleariy defined. In upper layer IV. zinc staining

increased preferentially uithin barrel septa (C,E) while staining in lower Iayer [V was

increased preferentially within barrel holiows (EJ). At PD 15 when zinc staining in the

barrel cortex was mature. zinc-ergic terminaIs were concentrated predominantIy in barrel

septa throughout layer IV (G,H). Scale bar = 500 pm.

47

upper and lower portions of layer IV (Fig. 5G,H). From PD 15 onward, barrel-field

patterning of vesicular-bound zinc remained relatively constant and thus. was considered

to have attained its mature distribution.

2.3.4 =I bsence of cytoarchitectonic barrels in M4O-A KO mice

Tangential sections through the upper and lower regions of layer IV in EVIAO-A

KO mice at selected ages of postnatal development. are depicted in Figure 6. Between 3

and 5 days of postnatal development. MAO-A KO mice displayed a faint and relatively

homogenous staining of the barrel-field (Fig. 6A.B) that was similar to their W

counterparts (Fig. 6A.B). However. at PD 6 when barrels were clearly evident in layer IV

of WT mice (Fig. 5C.D). MAO-A KO mice did not show the characteristic barrel-like

clustering of zinc-ergic projections within layer IV (Fig. 6C.D). Careful examination of

the upper portion of layer IV revealed very f i n t signs of inter-row septa (Fig. 6C) that

were not evident at later ages. [n deeper sections of layer IV. zinc staining of the barrel-

field was relatively uniform with the exception of a few darkly stained patches, which

may correspond to rudimentary barrel hollows (Fig. 6D). At PD 8. the density of

histochemicdly reactive zinc appeared to increase in a uniform fashion throughout the

tangential extent of the barrel-field and adjacent cortex (Fig. 6E.F). However. the

increase in barrel-field staining was transient as oider mice exhibited much lower

concentrations of synaptic zinc in the barrel-field than in surrounding cortex. Most

importantly. it was clearly evident from MAO-A KO mice at PD 8 (Fig. 6E.F). and later

stages of postnatal development (Fig. 6G.H), that zinc-ergic innervation of the S 1 did not

appear to respect barrel compartments (Fig. 6E.F). This finding was consistently

48

observed (in MAO-A KO mice 1 PD 8), and parallels previous descriptions of the barrel-

less organization of thalarnocortical fierents within layer IV (Cases et d. 1995, 1996)

Figure 6: Postnatal changes in the distribution of zinc-containing axon terminals in

tangential sections through upper (left panels) and lower (right panels) levels of layer IV

in LWO-A KO. At PD5 (A,B). barrel cortex can be distinguished by faint, homogenous

staining within the barrel-field. surrounded by adjacent cortical regions that are darkly

stained (A,B). .At PD6. an age when WT mice clearly reveal differential levels of staining

that are barrel-specific. zinc staining in MAO-A KO mice is homogenous (C,D). Subtle

signs of compartment-specitic staining may be discerned. such as inter-row septae in

upper layer IV (C) and darkly stained patches in lower layer [V (D) which may

correspond to barrel hollows. However. at PD 8 (E,F) and 15 (G,H). zinc-ergic

innervation of the barrel-field does not appear to respect barrel compartments. Scale bar =

500 pm.

5 1

2.4 Discussion

Zinc-selenide histochemistry was used to describe the laminar and tangential

distribution of zinc-ergic projections to the S I cortex of WT and MAO-A KO mice. In

normal WT mice. zinc staining was found to respect barrel compartments from PD 5

onward. and e.uhibited dynamic redistributions across and within cortical lamina during

the f is t two weeks of postnatal development. By contrast, in layer IV of W O - A KO

mice. zinc-ergic projections did not appear to be organized in barrel-specific

compartments. Moreover, although the lamina specific distribution of zinc-ergic

terminals in S 1 cortex of MAO-A KO mice was similar to that found in WT mice. it's

temporal development was significantly compressed. achieving a mature appearance by

PD 8. one week earlier than WT mice.

L 4.1 Sozcrce of zinc-ergic innervarion ofS1 correx

A hdamental assumption of the present study is that zinc-containing &yon

terminals are of corticocortical. and not thalamic origin. In the mammalian telencephalon.

a subset of glutarnatergic neurons sequester and release zinc within their terminal boutons

(Beaulieu et al.. 1992). Because thaIamocortical and corticocortical projections use

glutamate as their primary neurotransmitter. it is possible that some zinc-ergic projections

may arise fiom thalarnic cell bodies. However. there is considerable evidence to suggest

the contrary. First. there have been several studies that have utilized retrograde tracing

techniques to visualize zinc-containing somata. From these investigations. it was

demonstrated that zinc-rich neurons are located predominately in corticai laminae II. 111

and VI (Garrett and Slomianka, 1992: Garrett et al.. 1992; Dyck and 0' Leary, 1995).

These neurons are of pyramidal morphoIogy and send and receive ipsilateral and

52

transcallosal corticocortical projections (Dyck and O'Leary, 1995; Casanovas-Aguilar et

al., 1998). In addition. zinc-containing ceU bodies have been identified in subcortical

structures such as the hippocampus (Slomianka and Geneser. 1997)' amygdaia

(Frederickson and Moncrieff, 1994) and from the anterodorsal thalamus which sends

afferent projections to parahippocampal regions (Long and Frederickson. 1994).

However in light of these efforts. no studies to date have identified zinc-containing

neurons that project tiom the thalamus into ptimary sensory cortical areas (Frederickson

and Moncrieff. 1994). There is also correlational evidence suggesting that zinc-ergic

projections in the cortex are of cortical and not thalamic origin. For example. the

distribution of synaptic zinc is Iowest in thalmorecipient layer IV of primary sensory

cortices (Dyck ec al.. 1993: Czupryn and Skangiel-Kramska 1997). Within layer IV. zinc

is compartmentalized into cortical domains that show either high or low levels of

staining. In dternate sections. cortical regions that stain for cytochrome oxidase. which

presurnabIy demarcates the terminal projections of thaIamocortica1 afferents. are

precisely complementary to those regions that stain for zinc (Dyck et aI.. 1994). Thus.

despite receiving glutamatergic innentation From the thalamus. there has been no

evidence to suggest that these projections to the S 1. are zinc-ergic.

2-4.2 Svnaptic zinc in normal SI deve[opmen&: Fzmcrional implications

Consistent with previous observations in S1 cortex of wiId-type mice (Czupryn

and Skangiel-Kramska. 1997) and rats (Dyck and O'Learv, 1994): zinc staining appeared

to respect discrete laminar and columnar compartments. Our r e d s revealed that dnc

staining was distributed heterogeneously across the tangentid extent of layer IV,

demarcating barrel compartments with highest levels of staining localized initially, to

55

barrel hollows (From PD 5 to 10) and later to barrel septa (from PD 15 onward). Tke

transient periodic distribution of synaptic zinc within layer IV has been documented in

other species such as in the striate cortex of developing cats (Dyck et ai., 1993) and

monkeys (Dyck and Cynader. 1993). In these studies, it was hypothesized that synaptic

zinc may contribute to the mechanisms of ocular dominance andfor orientation column

Lbnnation during the critical period for use-dependent plasticity of the visual cortex. With

respect to the mouse S 1 cortex. our results. in addition to previous work by Czupryn and

Skangiel-Krarnska ( 1997). have shown that zinc staining of the barrel-field does not

occur until a$er the critical period for thalamocortical segregation (i.e PD 04). Thus. it is

unlikely that zinc-ergic circuits contribute to the initial formation of the barrel-field

cytoarchitecture.

,i\fter the establishment of thalamocortical a..ons within their respective banel-

field domains. there is a marked increase in the number of excitatory and inhibitory

synapses in the S 1 cortex from PD 4 to 16 (De Felipe et al.. 1997). Concomitant with

these synaptogenic events are the stabilization and/or elimination of new synapses (De

Felipe et a1.. 1997). The results of present study have shown that after the critical period.

zinc staining exhibits dynamic redistributions across and within cortical lamina. until a

mature. stable distribution was achieved at PD 15. Developmental _mdients in the

distribution of synaptic zinc have been reported in the hippocampus of rodents (Zimrner

and Haug, 1978) and kittens (Frederickson et al.. 1981), and may reflect the process of

maturation of glutamatergic terminals. Conversely, developmental variations in the Ievel

of synaptic zinc may play an active role in the stabilization or elimination of new

synapses. Previous work has shown that sensory-driven neuronal activity is especially

important for these processes to occur, as neonatal whisker trimming alters the response

54

properties of cortical neurons (Simons and Land, 1987), and disrupts the anatomical

arrangement of developing synaptic connections (KeUer and Carlson, 1999; Lendvai et

al.. 2000). These observations regarding the importance of neuronal activity in the fine

tuning of cortical connections and response properties. in conjunction with the notion that

synaptic zinc acts as a modulator of synaptic transmission and is regulated by tactile

experience (see Chapter 3), suggest that synaptically-released zinc may provide the

substrate for experience-dependent modifications of developing cortical synaptic

connections. However in light of this evidence, no studies to date have examined the

functional importance of zinc-ergic circuits in neocorticd deveIopment,

2.4.3 Disrupted zinc-ergic innenqarion oj-layer W: Porenrial mechanisms

Previous studies employing pharmacological inhibition (Vitalis et al.. 1998) or

genetic deletion (Cases st al.. 1996; Upton et al.. 1999) of MAO-A. have demonstrated - that the normal patterning of afferent projections in both the somatosensory

(trigeminothalarnic and thalamocortical) visual (retinogeniculate) systems. is disrupted.

In the present study. we have observed that intracortical zinc-ergic innervation of the

barrel cortex in b W - A KO mice. is also disrupted. Examination of these mice revealed

that zinc-containing axon terminals were distributed homogenousiy across the tangential

extent of layer IV and did not show barrel-like clustering, characteristic of WT mice.

An important question raised by our results concerns whether the topogaphic

arrangements of zinc-ergic fibers in layer IV are dependent on the initial establishment of

thalarnocorticai afferents within their respective banel-field domains. Indeed. there are

several lines of evidence to suggest that thalamic afferents b c t the barrel-related

pattern of cortical neurons and their projections. First, the cortical bane1 pattern of zinc-

55

ergic projections was not visible until PD 5, which is 1 to 2 days after thalamocortical

fibers have manifested a whisker pad motif (Senft and Woolsey, 1991; Agmon et al.,

1993). Second. early postnatal lesions of the whisker pad or the thalamus. that prevent

the segregation of thalamocortical fibers within their respective barrel-field targets. also

disrupt the topographic organization of S 1 cortical neurons. And finally, thalamocortical

disruptions in MAO-A KO mice have been shown to prevent the clustering of layer IV

granular neurons or glial e.vtracelIular matrix molecules within barrel septa domains

(Cases ct al.. 1996). Thus. the undifferentiated distribution of zinc-ergic terminals in layer

IV of MAO-A KO mice may reflect a primary abnormality in the topographic ordering of

thalamoconical arbors. Alternatively, because Pc,iAO-A KO mice exhibit seven to nine

fold increases in levels of 5-HT during early postnatal development (Cases et al.. 1995,

1996). it is possible that 5-HT may acr directly upon. and disrupt the compmmentation of

zinc-ergic terminals within layer IV. For example. several authors have suggested that

excessive amounts of 5-HT may inhibit activity-dependent mechanisms potentially

responsible for the segregation of thdamocortical and intracortical axons into barrel-

specific compartments (Rhoades et al.. 1994; Cases et al., 1996: Vitalis et al.. 1998). In

particular. recent evidence has provided evidence that excessive serotonergic stimulation

of 5-HTI B receptors prevent the clustered organization of thalamocortical afferents in S 1

(Young-Davies et al.. 2000) and retinogeniculate projections in the visual system (I. Seif.

personal communication). In addition. there is also correlative evidence of 5-HT1B

receptor involvement in thalamocortical development. For example, 5-HTI B receptors

are transiently expressed on the terminal arbors of thalamocortical axons during the

critical period for barrel formation (Bennett-Clarke et al., 1993). Moreover,

electrophysiological recordings have shown that activation of the 5-HTlB receptor.

56

suppresses glutamate release and mediates strong inhibitory effects on excitatory

thalamocortical transmission (Mooney et al., 1994; Rhoades et ai., 1994). Thus, excessive

stimulation of serotonergic receptors (in particular the 5-HTlB) may disrupt activity-

dependent mechanisms of thalamocortical segregation into whisker-specific domains.

Whether a similar process is involved in the topographic ordering of layer IV intracortical

projections. will be the subject of hmher investigation.

-4n alternate expianation regarding the disrupted topographic organization of zinc-

ergic projections in Iayer [V. is that excessive amounts of 5-HT may disrupt the growth

of developing intracortical ~xonal connections. Studies investigating the effects of 5-HT

on neurite outgrowth. in vitro. have yielded equivocal results. For example, addition of 5-

HT to culture mediums containing Helisoma neurons. causes an immediate cessation of

neurite elongation (Haydon et al.. 1984) and depletion of 5-HT in embryonic Helisoma

neurons increases dendritic growth of 5-HT target cells (Goldberg et al.. 1991).

Comparably. stimulation of 5-HT1A receptors decreases dendritic branching and total

neuritic length in embryonic cultured cortical neurons (Sikich et d.. 1990). Contrasting

with these results. elevating 5-HT levels in tissue cultures have dso been shown to

potentiate neuronal growth. In particular, addition of 5-HT (Lieske et al., 1999) or 5-

HTlB agonists (Lotto et al.. 1999) to thalarnic neurons in vitro, produced a significant

increase in neurite outgrowth, length of the primary (longest) process growing out of the

cell body, and the total length of all processes. However. in Iight of these results, it is

uncertain whether the 5-KT modulates the growth and branching of intracortical axons.

Future investigations will be required to determine if disruptions in the topographical

organization of intracortical projections in PUIAO-A KO mice, are related to excessive

serotonergic moduIation of axonal growth and branching.

2.4 4 Role of j-HT in rhe marurarion of zinc-ergic terminals

Postnatal changes in the distribution of synaptic zinc have been suggested to reflect

the process of synaptic manuation of glutamatergic terminals in the mouse

somatosensory (Cmpryn and Skangiel-Krarnska, 1997) and cat visual cortex (Dyck et al..

1993). In sagittai sections of WT S 1 cortex, the laminar distribution of zinc-ergic

terminals appeared mature at PD 15. and was characterized by high levers of zinc staining

in layers I. 11.111 and V. interdigitated with moderate staining in layer [V and VI. In

MAO-A KO mice, a similar pattern of synaptic zinc was observed. except that a mature

distribution was attained one week earlier, at PD 8. These observations imply that high

levels of 5-HT in MAO-A KO mice. hasten the development and maturation of

intracortical circuits. at least those having a zinc-ergic phenotype.

In the rodent and feline cerebral cortex, 5-HTI and 5-HT2 receptors are over-

expressed and heterogeneously distributed across cortical lamina during early postnatal

development (Whitaker-Azmitia et al., 1990: Dyck and Cynader. 1993b: Morilak et al..

1993). These studies. in addition to the precocious development of serotonergic afferents

to many target regions in the cortex. suggest that 5-HT may act as a regulator of neuronal

development (for review see Whitaker-Azmitia et al.. 1990). In support of this

conrention. studies have shown that early depletion of 5-HT delays the emergence of

thaIarnocorticaI periphery-related patterns (Blue et al.. 1991) and prolongs the time-

course of development of cortical layers (Osterheid-Haas and Hornung, 1996). Taken

together. these studies imply that manipulations of endogenous Ieveis of 5-HT may have

a global. non-selective effect on cortical development. However. preliminary

observations from our laboratory have shown that developmental gradients in the

58

distribution of calretinin-positive neurons and astrocytes, are unaffected by high levels of

5-HT in MAO-A KO mouse pups. Thus, in Iight of the results described in the present

study. it would appear that the maturation of specific subpopulations of cells (eg. dnc-

ergic neurons) in the cortex. are influenced by high levels of 5-HT during early postnatal

development. whereas others (eg. calretinin-positive neurons and astrocytes) are not.

Future experiments wiIl be conducted to determine if these maturational differences are

mediated through excessive stimulation of particular 5-HT receptor sub-types.

59

Preface to Chapter 3

In Chapter 2, we have shown that the pattern of zinc staining in S 1 cortex is layer-

specific and describes functional columnar compartments in layer IV that are refined in a

column-specific manner during development. Moreover, the columnar expression of

synaptic zinc within layer [V coincides with the period of postnatal development in

which newly established synaptic co~ect ions are reorganized and refined into discrete

hct ional columnar units (Fox et al.. 1996). From these results, we have suggested that

developmentally regulated gradients in the distribution of synaptic zinc may contribute to

activity-dependent modifications in the synaptic organization of the developing cerebral

cortex. However. if zinc-ergic circuits are important in mediating plastic changes in the

developing brain. then they are also likely to be important for changes that occur in

adulthood. To address this possibility. Chapter j will focus on zinc-ergic involvement in

activity- and experience-dependent forms of synaptic plasticity in the adult S 1 cortex

Chapter 3: Experience-dependent Changes in Levels of

Synaptic Zinc in S1 Cortex of the Adult Mouse

3.1 Introduction

The synaptic organization of the adult cerebral cortex is continuously modified by

sensory experience. In the visual cortex, electrophysiological studies have demonstrated

that manipulations of visual experience produce immediate reorganizations in receptive

tield size and cortical topography (Gilbert and Wiesel. 1992; Trachtenberg et al., 2000).

Comparably. digit amputation (Merzenich et al.. 1984: Garraghty and Kaas. 199 1) in

non-human primates. or removal of facial whiskers in rodents (Diamond et al.. 1993:

.Armstrong-James et al.. 1994). initiates a sequence of events in which the topographical

representations of deprived and non-deprived regions of the somatosensory cortex are

reorganized. These events are characterized initially. by the redistribution of receptive

field properties in deprived and non-deprived cortical areas (Merzenich et al.. 1984;

Diamond st al.. 1993; Glazewski. 1998) that is followed by anatomical changes in the

neuronal circuitry of cortical (Kossut and Juliano. 1999) and subcortical regions

(Garraghty and Kaas. 199 1 ; Sengelaub et al.. 1997).

The mechanisms underlying experience-dependent changes in the adult cerebral

cortex are at present. uncertain. Nevertheless, it is generally agreed upon that these

experiencedependent modifications are mediated by rapid changes in the synaptic

eficacy of existing cortical connections, through LTP or LTD-like processes (Donoghue.

1995). In particular, recent studies have implicated synaptic zinc as contributing to

activity-dependent mechanisms of cortical plasticity, such as LTP and LTD, by virtue of

its potent ability to modulate glummatergic neurotransrnission (Westbrook and Mayer.

1987: Christine and Choi. 1990: Dyck et al., 1993; Smart et al.. 1994; Vogt et d.. 2000).

Moreover. anatomical studies have shown that developmental gradients in the laminar

and columnar distribution of synaptic zinc. correlate with periods of development in

which the visual cortex is particularly sensitive to activity- and experience-dependent

modifications of its organization (Dyck et al,. 1993; Dyck and Cynader. 1993b).

To investigate the role of synaptic zinc in cortical plasticity, we examined

changes in the barrel-specific distribution of zinc-containing axon terminals in the S I

cortex of adult mice at different time points following whisker plucking. This form of

sensory deprivation was selected for several reasons. First. unlike peripheral lesions.

nerve transection. or TTX treatments. whisker plucking does not induce any permanent

structural changes (i.e. degeneration) to the whisker follicle or to the ascending pathways

of the trigerninal system (Li et al.. 1995). Therefore, because of the more innocuous

nature of whisker plucking, Functional changes in the cortex are more likely reflect

alterations in tactile experience or whisker use, rather than gross alterations in afferent

ncuronal activity or degeneration of ascending nerves. Second, for assessing the short-

term etyects of sensory deprivation (i.e. within a couple of hours). whisker plucking is

desirable because of the rapidity and ease through which whiskers can be removed.

Finally. due to the fact that whiskers can regrow after plucking, we were able to correlate

changes in levels of synaptic zinc with changes in whisker length. Thus, in the remainder

of this chapter, we characterized: a) experiencedependent changes in cortical levels of

synaptic zinc and the time-course for which these changes persisted and b) the extent to

which deprivation-induced changes in synaptic zinc could be attributed to alterations in

the use of the deprived whisker.

3.2 Methods

3.2. I =Inimals and trearment groups

Forty maIe CDI mice, between 60 and 65 days of age were used to study the

effects of whisker p l u c b g on levels of synaptic zinc in the adult barrel cortex. All

animals were obtained From the University of Calgary Breeding Colony and maintained

on standard laboratory diet and water ad libitum. Animals were group housed in clear

plastic cages on a 12:12-hour 1ight:dark cycle. The procedures used for tissue preparation

and histochemical staining of synaptic zinc are described in detaiI in Chapter 2.

To investigate the role of synaptic zinc in experience-dependent plasticity. we

examined the distribution of zinc-containing axon terminals in the S1 cortex of adult

mice at different t h e points following whisker plucking. Thirty-five mice underwent the

removal of the first tive vibrissae in row C (whiskers C 1 to CS). Following this. mice

were then assigned to one of 7 experimental groups (ie. 3.6, 12,24 hrs. and 1.2.3 week

groups) to determine the time-course of deprivation-induced changes in zinc staining in

row C. Additionally. 5 mice had whiskers in rows A. B. C or had all but the C2 (row C.

arc 2) whisker removed and were sacrificed 24 hours later. The control group consisted

of mice that had either whiskers (from row C) on one side of the face removed. or had no

whiskers removed. These two conditions were included to properly control for the

possibility that unilateral whisker plucking may affect levels of synaptic zinc in the

ipsilateral hemisphere. Our pilot data indicated that the level of zinc staining in row C

between unoperated and unilaterally plucked controls, was not significantIy different (t =

0.4: p > 0.6) and thus data from both groups were pooled together. Furthermore. because

levels of zinc staining did not change in S 1 cortex ipsilateral to the plucked whiskers,

each hemisphere was considered independent (n = I ) and all mice in the experimental

groups received bilateral whisker plucking. To remove whiskers, mice were Iightly

anesthetized with halothane and gently restrained while vibrissae were plucked with

surgical tweezers. Once the vibrissae were removed, mice were placed back into their

home cages until the time of sacrifice*

3.22 =Inalysis oj'zinc staining inrensity

To quantib Levels of zinc staining in barrel hoIlows of control and whisker-

deprived mice. six-20 ym tangential sections from layer fV in each hemisphere. were

examined under a Iight microscope (Zeiss. A~ioskop 2) equipped with 1.15 x objective

Irns. Images were then captured and digitized onto a monitor (Panasonic BWOO CCD

camera: Data Translation Dt-2255 quick capture board). wherein the grey scales were

andyzed with a Macintosh OS 8.6-based image analysis system with running MH Image

s o h a r e . Zinc staining intensities were determined by assigning a numerical vdue

b e m e n 0 (white) and 255 (black) to each pixel according to its grey scale. To create

figures (see Figs. 7.4.8 and 10). some of the images were then imported into Adobe

Photoshop 5.0 (Adobe Systems. San Jose. CA) in which minima1 adjustments of contrast

and brightness were made.

In control mice. IeveIs of zinc staining for each individud barrel row (ie. mws A

through E), were measured by sampling the mining intensity of each row and comparing

that value to the average shining intensity of the remaking four rows. We refer to the

difference in staining intensities between one row (eg. row A) and the remaining four

rows (eg. average of rows B. C, D, E), as the percent difference in staining intensity for a

particular row (in this case row A). and calculated it according to the following formula:

(staining intensity of row A) ---------------------*-------------- x 100 (staining intensity of rows B + C + D + E 14)

= percent difference score in staining intensity for row A

First, percent difference scores were obtained for each row in each of the six

seriai sections (per hemisphere). These percent difference scores (for each row) were then

averaged across six serial tangential sections to obtain a mean percent difference score

for each row in each hemisphere. This procedure was done for each control hemisphere

In = 10) to create a single mean percent difference score for each row (see Figure 7B).

In order to determine the effects of whisker plucking (i.e. row C) on levels of

zinc staining in row C, we compared the staining intensity of row C in plucked mice to

the staining intensity of row C in non-deprived control mice. First, in deprived mice, a

percent difference score in staining intensity was calculated (using the above formula) for

row C in each of the six tangential sections (in one hemisphere). From this, we calculated

the mean percent difference score for row C in each of the deprived hemispheres. The

mean values for each hemisphere were then summed together to mate a single mean

percent difference score for each experimentd group (see Fig. 8). The mean percent

difference scores for row C in each of the seven experimentd groups (ie. 3,6, 12,24 hrs.

1.2 . 3 week p u p s ) were then compared to the mean percent difference score obtained

for row C in control mice (see Fig. 6B), using a one-tailed Student's t-test. The

Bonferroni adjustment was used to set the significance level at p < 0.007. A11 values are

expressed as the mean k S.E.M.

3.2.3 Conelation analysis

Our piiot studies indicated that increased levels of zinc staining in deprived barrel

holIows were associated with the Iength of regrown whiskers. To examine the correlation

between whisker regrowth and levels of zinc staining, we first measured the length of C3

whiskers from animals sacrificed one. two or three weeks after row C whiskers (i.e. C 1 to

C5 whiskers) were removed. The length of regrown C2. whiskers were expressed as a

percentage of the normal length of C2 whiskers {mean of 20 rnm), which was determined

horn unoperated control mice (n = 8). Thereafter. we assessed the relative staining

intensity of the C2 barrel hollow in the hemisphere contnlated to the regrown C2

whisker. This was done by calculating a percent difference score in which the staining

intensity of the deprived C2 barrel hollow was compared to the average staining

intensities of adjacent non-deprived A2. B2. D2 and E2 barre1 hollows. Percent

difference scores tbr C 2 were obtained from six serial tangential sections in each

hemisphere. These values were then summed together to create a mean percent difference

score for C2 in each hemisphere (n = 20). We then calculated a Pearson r correiation co-

efficient for the length of each regrown C2 whisker with the percent difference score

obtained for the corresponding C 2 barrel holIow. Note: percent difference score for C2

barrel hollows in deprived mice were compared then to percent difference scores of C2

barrel holiows in controi mice (which is 0 in Fig. 10) and plotted as the percent increase

in staining intensity (fiom conuoi levels).

3.3 Results

3.3.1 Distribution of synaptic zinc in the barrel--eld of control mice

The patterned distribution of histochemicalIy-reactive zinc into barrel-like

structures. is readily apparent in tangentiai sections from layer IV (Fig. 7A). In normal

adult mice, zinc staining of the barrel-field was periodically distributed, characterized by

regions containing low levels of staining that were separated from one another by more

darkly stained inter-barrel septae. As has been previously described (Czupryn and

Skangiel-Kramska. 1997; Land and Akhtar. 1999). these zinc-poor regions within the

barrel-field correspond to barrel hollows and are predominantiy innervated by VPm

afferents horn the thalamus. By contrast, cortical regions that stain heavily for synaptic

zinc such as the interbarrel septat. are principally innervated by intracortical and PoM

thalamocorticd projections (Kim and Ebner: 1999). In Figure 7A. five rows of barrels

can be discerned that are aligned in a posterior to anterior manner and are Iertered From A

to E. with row A closest to the midIine and row E being the most lateral. Qualitatively.

the intensity of zinc staining in barrel hollows of each row appeared somewhat

homogenous. although outer rows A and E periodicalIy appeared darker than inner rows

B, C and D. Our quantitative analysis of zinc staining (Fig. 7B) supported this contention

showing that the relative amount of zinc staining in outer rows A and E (percent

difference scores of 5.8% and 1.9%. respectively) was higher than middle rows B, C and

D. In particular. the percent difference scores of rows C and D were much tower than

adjacent rows k B and D. These low Ievels of staining were reflected by negative optical

density scores, which are produced when the optical density of a particular row

Figure 7: Distribution of histochemicat~y-reactive zinc in corticd Layer IV of control

mice. A: shows zinc staining in a tangential section through layer IV of S 1 cortex. There

are five rows (A - E) of whisker-related cortical barrels that delineate the location of the

posteromedid bmel subfield in the S 1 cortex. Barrel compartments were characterized

by high levels of zinc staining in inter-barrel septae and iow levels of staining in barrel

hollows. Scale bar = 500 pm. B: Mean ( k S.E.M) zinc staining intensity for each barrel

row. Notice that barrei rows A, 8 and E had positive percent difference scores showing

that on average. the staining intensity of that particular row was greater than the mean

mining intensity of adjacent rows. By contrast. negative percent difference scores were

obtained for rows C and D indicating that the level of zinc staining in these rows tended

to be less than rows A. 0 and E.

.

A B C D E ;

Barrel Row

(eg. row C) is lower than the average optical density of adjacent barrel rows (eg. rows -4.

B. D. E).

3.3.2 Whisker plucking increases levels of synaptic zinc in row C

In control mice. the relative optical density of zinc staining within row C was

approximately 3.6% less than adjacent rows (see Fig. 78). However. within hours after

the removal of row C whiskers. levels of zinc staining increased in barrel hollows

corresponding to the plucked whiskers (see arrows in Fig. 8). At 3 hours, there appeared

to be a very subtle increase in the [eve[ of zinc staining in row C (Fig. 8A). This increase

in staining was 5.8% above controI IeveIs (Fig. 9). but did not reach statistical

significance ( t = 2.96: p = -008). Six hours after whisker removal (Fig. 9). levels of zinc

staining within row C had significantly increased (average increase = 5.2%: t = 3.5: p <

.OM) relative to control levels. At this survival time. a dark band of zinc staining could be

discerned within row C (Fig. 88). Within twelve hours. increased zinc staining within

row C was clearly evident (Fig. 8C). As Figure 9 ilIustrates, the level of zinc staining

within deprived row C was 16.0% above controI levels (t = 9.8: p < -000 1). Twenty-four

hours to 1 week following whisker removal. levels of zinc staining continued to be much

higher within row C than adjacent nondeprived rows (Fig. 8D,E). Quantitatively. the

optical density of zinc staining in the 24-hour and 1 week groups. was 13.8% (t = 8.1; p <

.000 1) and 16.9% (t = 7.6; p < -000 1) respectively, higher than control levels. With

longer survival times. levels of zinc staining gradually declined in deprived barrel

hollows and appeared normal by 2 weeks after whisker removal (Fig. 8 0 . At this time,

Figure 8: Changes in the distribution of synaptic zinc in tangential sections through layer

IV at 3 hrs (A). 6 hrs (B), 12 hrs (C). 24 hrs (D). 1 week (E) and 2 weeks (F) following

removal of row C whiskers. Between 3 (A) and 6 (B) hours after whisker removal. subtle

increases in zinc staining of row C were apparent (indicated by white arrows). However

at 12 (C). 34 (D) hours. and 1 week (E) after whisker removal. levels of zinc staining in

the deprived barrels (row C) were clearly higher than adjacent non-deprived barrel rows.

Two weeks (F) after whiskers were removed. levels of zinc staining in row C returned to

normal (compare with Figure 7A). Scale bar = 500 pm.

Figure 9: Increase of zinc staining in barrel row C at different survival times following

removal of row C whiskers. Relative to control mice. IeveIs of zinc staining increased

si-enificantly at 6. 12.24 hours and 1 week after whisker plucking. However with longer

survival times (ie. 2 and 3 weeks). the level of zinc staining in row C was not

significantly different from baseline levels. *p < .005: **p < .000 1.

zinc staining in row C had increased only 1.7% (Fig. 9) and was not signif~cantly

different from controls (t = 0.8: p = 22). Similarly. no qualitative or quantitative

differences (see Fig. 9) were observed three weeks after whisker plucking (t = 1.17; p =

.13).

3.3.3 Other patterns oj'whisker plucking

Previous work has shown that the degree and form of whisker deprivation

plasticity is highly dependent on the exact pattern of whisker deprivation (Wallace and

Fox. 1999). To determine if the spatial extent of deprivation induced changes in zinc

staining would be affected by different patterns of plucking, we removed whiskers from

rows A. B and C or had all whiskers removed except the C2 whisker. and allowed

animals to survive for 24 hours. Removal of whiskers kom rows A, B and C resulted in a

robust increase in the density of histochemically-reactive zinc in bane1 hollows

associated with the plucked whiskers (Fig. IOA). Similarly. removal of all whiskers

except the C2 whisker. also markedly increased the density of zinc staining within

deprived barrel hollows (Fig. IOB). It was evident from both types of manipulations that

increases in zinc staining were confined exclusively to the deprived barrel hollow.

To further characterize activity-dependent changes in zinc staining, high

magnification photomicrographs were taken from a non-deprived barrel hollow (ie. barrel

C2 in Fig. 10B). and compared with an adjacent deprived barrel hollow (ie. barrel D2 in

Fig. 100). Examination of these photomicrographs (Figs. 10C. D) revealed that zinc

histochemical reaction product consisted of numerous black punctae that occured either

in small singular spots or in larger irregular clusters. In the nondeprived C2 barrel

Figure 10: Alterations in zinc staining 24 hours after whisker plucking. Tangential

sections through lamina [V of S I cortex in mice that had whiskers from rows A. 5 and C

removed (A) or had ali but the C2 whisker removed (B). Zinc staining in conical barrels

associated with plucked whiskers (see white arrows in A and B) appeared much darker

than that observed for nondeprived barrels (see black arrows in A and B). Scale bar =

500 pm tbr A and B. Higher magnification photomicrographs of a non-deprived (taken

tkom C2 barrel. see black arrow in B) and deprived (taken From D2 barrel. see white

arrow in B) barrel hollow are shown in C and D. Zinc stained punctae in both non-

deprived (C) and deprived (D) barrel hollows occurred either in small singular spots or

larger irregularly shaped clusters. Note that in the deprived barrel hollow (D), zinc

stained punctae appeared much more numerous and more densely clustered than that

observed in the non-deprived barrei hollow (C). Scale bar = 20 pm for C and D.

hollow (Fig. IOC), the majority of zinc stained punctae occurred singly, interspersed with

a few more largely shaped clusters. Contrasting with this. zinc stained punctae in the

hollow (Fig. 10C). the majority of zinc stained punctae occurred singly. interspersed with

a few more largely shaped clusters. Contrasting with this. zinc stained punctae in the

deprived D2 barrel hollow (Fig. IOD) appeared much more numerous and more densely

clustered than the non-deprived barrel hollow (Fig. 10C). Comparably. zinc stained

punctae in other deprived barre1 holIows were distributed in a manner similar (data not

shown) to that observed in Figure 10D. These results indicate that higher levels of zinc

staining in deprived barrel hollows are likely related to an increase in the density of zinc

stained punctae within those particular hollows.

3.3.4 Regression analysis of whisker regrowih and levels of synaptic zinc

The observation that longer survival periods (ie. 2-3 week groups in Fig. 8) were

associated with normal levels of zinc staining in deprived barrel hollows. prompted us to

examine the relationship between whisker regrowth and levels of zinc staining in

deprived barrel hollows. The results of our correlation analysis (see Fig. 1 1) indicated a

highly significant (p < -000 11, linear relationship between levels of zinc staining in

deprived barrel hollows and the length of regrown whiskers. More specifically, when

whiskers that were a small fraction of their normal Iength were associated with increased

levels of zinc staining, while more l l l y regrown whiskers were associated with more

normal levels of zinc staining. Furthermore, increased levels of zinc staining appeared to

be directly related to the regrowth of the whisker, rather than to the length of survival

Figure 1 1 : Scatterplot showing the linear correlation between increased levels of zinc

staining in the deprived C2 barrel hollo\v and the length of the regrown C 2 whisker. This

significant correlation (R' = -66; p < .001) demonstrates that higher levels of zinc staining

in deprived barrel hollows are associated with shorter whiskers while hlly regrown

whiskers are associated with a return of zinc staining to normal levels.

20 40 60 80

Regrowth of C2 whisker (%)

time following whisker removal. Supporting this assertion was the observation that some

mice From the one week group. exhibited greater regrowth and had smaller increases in

zinc staining (eg. between 0 and 10%). than animals fiom the 3 week group who had less

regrowth but had much higher levels of zinc staining (between 10 and 20%). Thus, our

resuIts demonstrate that levels of zinc staining in deprived barrel hollows are directly

proportional to the degree of regrowth in whiskers.

3 .; .5 .-Ire increases in zinc sraining in rorv C absoltcre or relative?

Recent evidence has shown that whisker removal disinhibits neurons in cortical

barrels adjacent to the deprived barrel column (Kelly et al.. 1999). This finding implies

that if levels of zinc staining are related to changes in neuronal activity. then it is

possible that concomitant with increased staining in row C. were reduced levels of

staining in adjacent non-deprived rows. To determine if our relative measure accurately

quantified changes in zinc staining, five mice had row C whiskers from one side of the

face removed, and were sacrificed 24 hours later. Thereafter, tangential sections from

both hemispheres were cut and then stained for equal amounts of time. In doing this, we

were able to directly compare the staining intensity of each barrel row in the ipsilateraI

hemisphere versus those obtained for each row in the hemisphere contralatemi to the

pIucked side. Using a two-tailed, one-sample t-test with a bonferroni adjustment (alpha

level = -0 1). our results showed that the level of zinc staining for each of the non-

deprived barrel rows (ie. rows A, B, D. E) in the ipsilateral control hemisphere. were not

significandy different fiom the level of staining in non-deprived rows in the hemisphere

contralateral to the plucked whiskers (row A: t = -2.83, p = -05; row B: t = -.76, p = -50;

row D: t = .16, p = .88; row E: t = .93, p = -41). Furthermore when examining row C, we

observed that the level of zinc staining was significantly higher (t = 4.75. p < .O1) in row

C for the deprived hemisphere (approx. 15% increase), than row C in the control

hemisphere. These results not only validate our use of a percent difference score (ie. in

Fig. 9) to assess changes in zinc staining, but also show that higher levels of staining in

row C, reflect an absolute, rather than a relative increase in staining.

3.4 Discussion

Zinc-selenide histochemistry was used to visualize zinc-containing axon terminals

in layer IV of the adult mouse S 1 cortex, and to determine the extent to which levels of

synaptic zinc are regulated by tactiie experience. In the barrel-field of normal mice, zinc

staining demarcates barrel compartments with high levels of staining in the inter-barrel

septae and low levels of staining in b m I holiows. However when vibrissae were

removed, the intensity of zinc staining within deprived barrel hollows increased

significantly. This increase was evident three hours after whisker removal and persisted

for one to two weeks. During this time, whiskers on the contralateral face began to

regrow and corresponding with this, Ievels of synaptic zinc gradually declined in

deprived barrel hollows and appeared to reach baseline levels by 2 weeks after whisker

plucking. Furthermore, this normalization of zinc staining in deprived barrel hollows was

highly correlated with the length of regrown whiskers in an inverse, linear fashion.

In the primary visual cortex (Vl) of cats and monkeys, it has been demonstrated

that cortical zones deprived of visual experience for short periods of time, become more

responsive to stimulation of adjacent nondeprived visual inputs (Mioche and Singer.

1989: Gilbert and Wiesel, 1992; Trachtenberg et d., 2000). Similarly, plasticity can be

induced in the adult rat S 1 cortex by plucking all but one (univibrissa rearing) or two

(whisker pairing) whiskers (Diamond et al.. 1993: Glazewski, 1998). In non-deprived or

normal rats, neurons within a barrel column respond most vigorousiy to stimulation of

their "principal" whisker. However, within hours (ie. 12 to 24 hours after whisker

pairing) to days (7 days of univibrissae rearing) after whisker plucking, the intact

vibrissae became more strongiy represented (i.e. eIicits stronger neuronal responses) in

both deprived and non-deprived cortical barrel-columns (Glazewski et al., 1998; Ebner

and Rema, 1999). Furthermore, this redistribution of receptive field properties occurred

first within extragranular layers and only later in layer IV, suggesting that the initial

stages of cortical reorganization were driven by intracortical rather than thalamocortical

synapses (Fox. 1992; Diamond et al., 1994; Cynader. 2000; Trachtenberg et al.. 2000).

This phenomenon, in which the sensory system utilizes or "fills-in" deprived

cortical areas, implies a Hebbian-form of cortical plasticity in which the synaptic

connections of non-deprived cortical columns are strengthened while circuits in deprived

cortical columns are weakened (Bear et al., 1987). Accordingly, numerous studies have

examined the effects of sensory deprivation on molecules that may participate in

regulating the synaptic efficacy of intracortical circuits. Of these, it has been shown that

monocular lid suture or plucking of whiskers reduces the expression of glutamate, GAD,

and GMIA in cortical areas corresponding to the deprived input (Hendry and Jones.

1988: Welker et al.. 1989; Carder and Hendry, 1994). However, the time course of these

changes was on the order of days to weeks afier sensory deprivation, thus precluding

their involvement in the initiation of rapid cortical reorganizations.

In the present study we examined whether cortical levels of synaptic zinc were

regulated by sensory experience. Our data indicate that within 3 to 6 hours after whisker

plucking. levels of zinc in terminal boutons increased in cortical barreis associated with

the plucked whiskers. Furthermore, we observed with longer survival times that these

increases were directly proportional to the length of the regrown whisker. These

observations raise intriguing questions as to what may cause higher levels of zinc in axon

terminals and more importantly, do these increases have any functional consequences?

To answer the first question, increases in zinc staining may reflect an imbalance between

zinc release and uptake into axon terminals. Previous work on the kinetics of zinc

turnover and histochemical studies of zinc-containing axon terminals suggest that zinc is

released in an activity-dependent manner (Assaf and Chung, 1984). Once released,

synaptic concentrations of zinc are continuously regulated by transporters that facilitate

the accumulation of zinc back into the presynaptic termind (Palmiter et al., 1996; Wenzel

et al.. 1997). However. if the release of zinc is activity-dependent but the uptake is not.

then it would seem plausible that decreasing afferent activity by whisker plucking may

disrupt zinc homeostasis such that more zinc is taken up than is released. Future

experiments exploring this interaction between neuronal activity and the eficacy of zinc

transporters. will be necessary to resolve this ambiguity.

Increasing pre-synaptic Ievels of zinc may have functional implications. The

observation that levels of zinc staining increase within deprived barrel hollows suggests

that the post-synaptic targets of zinc-containing axon terminals are likely to be

glutamatergic spiny steIlate cells. which reside in the barrel hollows of layer [V

(FIeidervish et al., 1998 Feldmeyer et al., 1999; Lubke et al.. 2000). Recently, it has been

proposed that intracortical modulation of these vertical inputs may account for the rapid.

NMDAdependent depressiodpotentiation of neuronal responses that occurs in V1 and

S 1 during sensory deprivation (Cynader, 2000; Feldman, 2000). Although it is uncertain

whether intracortical zinc-ergic circuits regulate the synaptic transmission of these

connections. numerous authors have demonstrated that zinc is capable of moduiating a

variety of ligand-gated ion channels (Smart et al., 1994). In particular, physiological

concentrations of zinc have been shown to potently inhibit NMDA receptor h c t i o n

while mildly potentiating the activity of non-NMDA receptors (Peters et al., 1987;

Westbrook and Mayer, 1987; Smart et at., 1994). These effects on glutamate receptor

activation imply that zinc co-released with glutamate, may dynamically regulate post-

synaptic excitability by differentially activating NMDA receptor-gated channels relative

to AMPA-gated channels (Choi and Koh, 1998).

The idea that synaptic zinc acts as a reguIator of post-synaptic excitability is

intriguing considering that levels of zinc in the cortex change rapidly as a function of

sensory experience while levels of gtutamate do not (Carder and Hendry, 1994). In this

sense, it is possible that rapid changes in cortical levels of synaptic zinc, may alter the

strength of synaptic connections. For example. reducing afferent input to the cortex (ie.

via whisker plucking) may increase the ratio of zinc to glutamate in presynaptic

terminals. Thus. if the primary h c t i o n of zinc is to suppress NMDA receptor activation

(Peters et al., 1987; Christine and Choi, 1990), then increasing the presynaptic ratio of

zinc to glutamate may favor NMDA-dependent depression of cortical synapses.

Conversely. if increased whisker activity reduces the presynaptic ratio of zinc to

glutamate, then one might expect NMDA-dependent cortical synapses to be potentiated.

Experiments are currently underway to determine whether synaptic zinc is an active

contributor to activity- and NMDA-dependent potentiation and depression of cortical

synapses.

Chapter 4 Conclusions and Future Directions

The studies described in this thesis have provided important anatomical

information regarding the role of zinc-ergic processes in neocortical development and

plasticity. From these experiments. it was evident that developmental and adult changes

in the neocortical distribution of synaptic zinc, correlate spatially and temporally with

plastic events in the cortex. However. due to the enigmatic nature of synaptically-released

zinc. it is uncertain whether anatomical changes in synaptic zinc contribute to Functional

changes in the cerebral cortex. Thus, for the remainder of this chapter, the results of each

experiment will be summarized followed by a discussion on hture directions of

investigation.

In the S 1 cortex of normal mice. zinc staining e.xhibited dynamic redistributions

across cortical lamina during the first two weeks of postnatal development. Within layer

IV. levels of zinc staining appeared to respect barrel compartments, localized initially to

barrel hollows and later to barrel septa. As described in the discussion of chapter two. a

major question from these results pertains to whether developmental gradients in the

distribution of synaptic zinc, simply reflect gradients in the synaptic maturity of zinc-

ergic projections. or perhaps contribute to the bc t iond development and organization of

synaptic connections in the S 1 cortex. In regard to the former concern, it is possible that

transient changes in zinc staining within particular laminar (especialIy Iayer [V), may

reflect the elaboration and subsequent elimination of exuberant, intracortical axonal

projections. For example, Iky and Killttckey (I98 1,1982) have shown in the S I cortex of

rats, that a number of callosal projections, especially those originating from layer V, are

selectively eliminated during the first two weeks of postnatal development. Thus, in order

to enrich our understanding of developmental changes in the distribution of zinc-ergic

projections. it will first be necessary to characterize the neurons or cell bodies &om

which these projections arise. Once this has been accomplished, we can then address the

functional aspects of zinc-ergic innervation of the S 1 cortex. [n particular, do

manipulations of endogenous levels of synaptic zinc affect the organization and plasticity

of developing neocortical connections?

In addition to normal development, experiment 1 also dealt with some of the

developmental factors that regdate zinc-ergic innervation of S 1 cortex. Our results

indicated that thalamocortical disruptions present in MAO-A KO mice. also disrupt the

topographic organization of intracortical zinc-ergic projections within layer IV. These

disruptions were manifested by the absence of barrel-like compartments as zinc-ergic

terminals were homogenously distributed across Iayer IV. However. it is unctear from

these results whether a) disrupted zinc-ergic innervation is due to a primary disruption in

thalamocortical organization or is due to the direct action of 5-HT on corticocortical

projection neurons and b) zinc-ergic terminals exhibit some form of compartmentation

(i-e. are zinc-ergic terminals distributed diffusely and randomly across layer IV or are

they Iocalized within a cortical column, except without septa1 boundaries). To address the

former concern. some obvious directions for future investigations might be to manipulate

thalmocortical innervation patterns (i-e. neonatal whisker lesions or barrel-less mutant

mice such as GAP-43 KO mice, see Maier et ai.. 1999) to see if zinc-ergic innervation

patterns are affected accordingly. Additionally, it \dI be necessary to identify if, and

what types of 5-HT receptors are expressed on zinc-containing neurons- As a number 5-

HT receptor specific antibodies are becoming increasingly available, double-labeling

studies of 5-HT receptors and zinc-ergic neurons should prove interesting. With respect

to the latter concern regarding compartmentation of zinc-ergic projections in MAO-A KO

mice, experiments are currently underway assessing changes in zinc staining within layer

IV in MAO-A KO mice after whisker plucking. As experiment 2 has shown, plucking a

single whisker (eg. C2 whisker) in normal mice, leads to a selective increase in zinc

staining within the deprived barrel hollow. Thus, if plucking of a single whisker in MAO-

A KO mice leads to a column-specific increase in zinc staining, it would appear that zinc-

ergic terminals do in fact respect some form of compartmentation.

Perhaps the most intriguing and unanticipated finding of experiment I , was that

the lamina-specific distribution of zinc-ergic terminals in S1 cortex of MAO-A KO mice.

appeared to mature at a much faster rate than in their WT counterparts. Moreover, our

results indicated that a primary substrate of these maturational differences was in supra-

and ihgranular layers (see Chapter 2. Figure 3). To our knowledge, this is the fust

study in MAO-A KO mice to show abnormal cytoarchitecture in non-thalamorecipient

layers of the cortex. Yet, in spite of these findings, it is uncertain whether these

maturational differences are: a) mediated by increased levels of 5-HT or NEp, as both

neurotransmitters are elevated in MAO-A KO pups, and b) whether receptors for either of

these neurotransmitters are selectively expressed on developing zinc-ergic neurons. Thus,

in order to dleviate some of these questions, two approaches may be undertaken. The

first would be to "phenocopy" maturational differences in wild-type mice by selectively

increasing neonatal levels of either 5-HT or NEp. Second, if for example 5-KT is the

causative agent in these a b n o d t i e s (as is the case for thalamocorticaI disruptions, see

Cases et al., 1995, 1996), it would be intriguing to see if zinc-ergic projections mahue

normally in MAO-A KO mice that have received chronic &ions of a 5-HT receptor

antagonist (for example blocking the 5-HTIA or 5-HTlC receptors).

In chapter 3, the influence of sensory experience on levels of synaptic zinc in

layer IV of the adult S 1 cortex, were examined. From this, it was shown that within hours

after whisker plucking, Ievels of synaptic zinc significantly increased in deprived barrel

hollows and these increases persisted until the corresponding whisker regrew.

Furthermore. increased zinc staining in deprived barrels was directly proportional to the

length of regrown whiskers, One of the primary implications suggested by rapid,

experience-dependent changes in cortical levels of synaptic zinc, is the possibility that

synaptically-released zinc may contribute to hctional reorganizations in the adult

cerebral cortex. As described in the preceding discussion (see Chapter 3),

electrophysiological changes in the synaptic strength of cortical connections, are

observed within minutes to hours after manipulations of sensory experience (Calford and

Tweedale, 1988; Diamond et al., 1994). However, in light of these rapid, and well-

characterized physiological changes, very Iittie is known about the molecular

mechanisms that subserve these changes. Thus, an obvious experiment would be to

genetically or pharmacologicaliy alter cortical levels of synaptic zinc, and examine

experience-dependent reorganizations (i.e. via whisker pIucking) in receptive field size

and cortical topography.

The observation that tactile experience reguiates Ievels of synaptic zinc in the S1

cortex, suggests the possibility that zinc-ergic circuits in other cortical and subcortical

areas, may also be affected by changes in sensory experience. For example, in the aduIt

primate V I, deprivation of visual experience in one eye increases levels of synaptic zinc

in ocular dominance columns corresponding to the deprived eye (R. Dyck, personal

cornrnunication). With this in mind, it would be interesting to see if the experience-

dependent changes in the motor cortex (for example after motor training tasks), correlate

with changes in the histochemicai distribution of synaptic zinc. Alternatively, one might

examine experience-dependent changes in zinc staining in subcortical regions such as the

thalamus. In particular. the thalamic reticuIar nucleus would be an attractive site to

examine because it receives an abundance of corticothalamic projections from layer VI of

the S1 cortex, that are heavily enriched with zinc (Gibbs et al., 2000). In addition to

receiving reciprocal connections from the cortex, this region of the thalamus plays an

important role in modulating both normal and oscillatory thalamocortical activity in the

VPm (reviewed in Steriade and Llinas. 1988). Furthermore. in vitro studies have shorn

that physiological concentrations of zinc potently inhibit GABA-ergic neurotransmission

in reticular neurons (Gibbs et al.. 1000). Taken together, these studies suggest the

possibility that activity-dependent changes in zinc-ergic circuitry may contribute to, or at

least correlate with. plastic events in cortical and subcortical structures outside of the S I .

High levels of zinc staining in barrel hollows during early postnatal deveIopment

and after whisker plucking in adulthood, suggest a commonality in etiology and fitnction

among these two events. As has been described in Chapters 3 and 4, high levels of zinc

staining in barrel hollows during these two instances correlate with elevated periods of

synaptic plasticity. Accordingly, we have hypothesized that changing levels of synaptic

zinc may have a profound effect on activity and experience-dependent mechanism

involved in sculpting these synaptic rearrangements. However, in addition to addressing

these hctional considerations, it will also be necessary to determine what causes these

changes in zinc staining. From the results in chapter 3. in particular the highly correlated

relationship between levels of zinc staining and whisker regrowth, it would appear that

increased levels of staining in barrel hollows of adult mice, are directly related to

whisker-plucking induced changes in afferent sensory-driven neuronal activity. To

provide more conclusive support for this contention, experiments are currently in

preparation to examine the effect of increasing whisker activity on adult. cortical levels of

synaptic zinc. By contrast. much Iess is known about the factors that underlie

developmental shifts in the distribution of synaptic zinc within barrel hollows of neonatal

mice. One possibility is that. similar to sensory-deprived adult mice. increased zinc

staining may reflect reduced neuronal activity in ascending projections of the trigeminaI

system. This hypothesis could potentially be verified by examining cortical levels of zinc

staining after neonatal whisker stimulation (i.e. predicting that increased activity would

decrease levels of staining with barrel hollows). Alternatively, transient increases in zinc

staining of layer IV, may reflect the elaboration and elimination of exuberant intracortical

zinc-ergic projections. Intracortical labeling of zinc-ergic cell bodies and their projections

should provide some insight regarding this latter hypothesis.

In conclusion, the results described in this thesis provide anatomical evidence that

histochemically-identifiable pooh of synaptic zinc. delineate discrete laminar and

columnar compartments in the developing and adult S 1 cortex of mice. Furthermore. it

appears. at least in adult mice. that the level of synaptic zinc within these compartments

is rapidly and dynamically regdated by changes in sensory experience. Thus, a W e r

understanding of zinc-ergic neurons and synaptically-released zinc, via anatomical,

pharmacological, and physioiogical approaches, may shed newfound light on the

mechanisms that govern activity- and experience-dependent changes in the synaptic

organization of the developing and adult cerebral cortex.

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