Topography of Striate-Extrastriate Connections in ... · Topography of Striate-Extrastriate...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 592426 9 pageshttpdxdoiorg1011552013592426

Research ArticleTopography of Striate-Extrastriate Connections inNeonatally Enucleated Rats

Robyn J Laing1 Jurate Lasiene2 and Jaime F Olavarria1

1 Department of Psychology and Behavior and Neuroscience Program University of Washington PO Box 351525Seattle WA 98195-1525 USA

2 Laboratory for Motor Neuron Disease RIKEN Brain Science Institute 2-1 Hirosawa Wako-Shi Saitama Prefecture 351-0198 Japan

Correspondence should be addressed to Jaime F Olavarria jaimeuwedu

Received 29 April 2013 Revised 15 August 2013 Accepted 29 August 2013

Academic Editor Eduardo Puelles

Copyright copy 2013 Robyn J Laing et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

It is known that retinal input is necessary for the normal development of striate cortex and its corticocortical connections butthere is little information on the role that retinal input plays in the development of retinotopically organized connections betweenV1 and surrounding visual areas In nearly all lateral extrastriate areas the anatomical and physiological representation of thenasotemporal axis of the visual fieldmirrors the representation of this axis inV1 To determinewhether themediolateral topographyof striate-extrastriate projections is preserved in neonatally enucleated rats we analyzed the patterns of projections resulting fromtracer injections placed at different sites along the mediolateral axis of V1 We found that the correlation between the distance frominjection sites to the lateral border of V1 and the distance of the labeling patterns in area 18a was strong in controls andmuchweakerin enucleates Data from pairs of injections in the same animal revealed that the separation of area 18a projection fields for a givenseparation of injection sites was more variable in enucleated than in control rats Our analysis of single and double tracer injectionssuggests that neonatal bilateral enucleation weakens but not completely abolishes the mediolateral topography in area 18a

1 Introduction

Visual information is processed along ascending neuronalpathways that maintain the point-to-point topography of theretinofugal projections Much work has focused on under-standing how topography is established in the projectionsfrom ganglion cells to the superior colliculus and thalamus[1ndash3] but less is known about how higher-order pathwaysdevelop In particular there is little information aboutthe mechanisms guiding the development of retinotopi-cally organized connections known as topographic mapsbetween primary visual cortex (V1 striate cortex area 17) andhigher extrastriate visual areas Recent studies support theidea that development of cortical areas and their organizedinterconnections may depend on a combination of activity-independent cortical factors such as genetically determinedguidance labels and activity-dependent mechanisms drivenby sensory input [4ndash8]

During development interhemispheric callosal connec-tions in V1 are established between opposite cortical loci

that are retinotopically matched (ie they represent the samevisual coordinates [9ndash11]) Olavarria and colleagues proposedthat the temporal retina through a system of bilateralprojections promotes the stabilization of callosal linkagesthat while retinotopically corresponding are arranged ina nonmirror symmetric pattern with respect to the brainmidline [9 10 12] However in the absence of retinal inputcallosal connections are established between opposite corticalloci that aremirror-symmetricwith respect to themidline [1213] This reversal in the callosal map induced by enucleationprovides evidence that retinal input can specify corticaltopography However it remains unclear whether or notretinal input plays a major role in the establishment of topo-graphically organized intrahemispheric connections betweenV1 and surrounding extrastriate visual areas Data supportinga role for retinal input come froma recent study inmice show-ing that both neonatal enucleation and anophthalmia inducetopographic anomalies in striate-extrastriate projections [14]

Numerous anatomical and physiological studies in nor-mal rats have shown that V1 is surrounded by at least 10

2 BioMed Research International

extrastriate visual areas [15 16] which have been namedaccording to their location relative to V1 (see Figure 2(a))Each of these areas contains a representation of the oppositevisual hemifield and receives direct retinotopically orga-nized projections from V1 (see [17 18] for reviews) More-over callosal connections form a dense band that straddlesthe V118a border and a series of ring-like callosal bands thatseparate adjoining extrastriate visual areas in area 18a [19ndash23] (Figure 2(a)) These callosal rings provide fixed referencelandmarks for locating and identifying the various visualareas in area 18a of normal rats

The pattern of striate-extrastriate projections in normalrats is remarkably consistent from animal to animal [17 19]Moreover tracer injections placed into different loci in V1produce essentially the same extrastriate projection patternsexcept that the projection fields translocate locally accordingto the retinotopic map in each extrastriate visual area Thishas made it possible to obtain information about the topog-raphy of striate-extrastriate connections in normal rats usingsingle tracer injections into V1 [19] In contrast to normalrats we recently reported that neonatal enucleation induceshighly irregular and variable patterns of striate-extrastriateand callosal projections [23] However this previous studybased on the analysis of projection patterns produced bysingle restricted injections of anatomical tracers into V1 didnot report the effects of enucleation on the topography ofthese projections In the present study we have addressed thisissue by analyzing data from single tracer injections into V1as well as connection patterns resulting from pairs of discretetracer injections placed at different locations inV1 of the sameanimal

2 Materials and Methods

Our study is based on data obtained from a total of 24 Long-Evans pigmented rats Pregnant animals were monitoredseveral times daily and the births of the litters were deter-mined to within 12 hours Sixteen rat pups were anesthetizedwith isoflurane (2ndash4 in air) and binocularly enucleatedwithin 24 hours of birth (BE0) After recovering from theanesthesia pups were returned to their dams In addition8 rats were used for analyzing striate-extrastriate projectionsin normal adult animals All surgical procedures were per-formed according to protocols approved by the InstitutionalAnimal Care and Use Committee (IACUC) at the Universityof Washington

21 Tracer Injections In both control and enucleated ani-mals striate-extrastriate projections were revealed followingrestricted tracer injections into striate cortex while thedistribution of callosal connections was demonstrated in thesame hemisphere following multiple injections of a differenttracer in the opposite hemisphere Anatomical experimentsin all normally reared and enucleated rats took place whenthe animals were at least 1 month old Tracer injections weremade under isoflurane anesthesia (2ndash4 in air) To studythe topography of striate-extrastriate connections animalsreceived restricted injections of various tracers into differ-ent places of striate cortex including biotinylated dextran

amine (BDA 10 in DW Molecular Probes Eugene ORUSA) which is predominantly transported anterogradelyhorseradish peroxidase (HRP Sigma Co 25 in saline)which is transported both anterogradely and retrogradelyand fluorescent tracers (rhodamine beads RB or greenbeads GB LumaFluor Naples FL USA concentrated stocksolution) which are transported retrogradely Small volumes(005ndash01120583L) of these tracers were injected into striate cortexof the right hemisphere (approx 29ndash42mm from the mid-line 01ndash20mm anterior to the lambda suture) In all casesanalyzed the tracer injections were restricted to grey matterThe size of single injections of BDA and HRP was estimatedas described previously [23] High levels of fluorescence fromGB andRB injectionswere typically restricted to the injectionsites because diffusion of these tracers is low To reveal theoverall pattern of callosal connections multiple injections(12ndash15 injections total volume approx 40 120583L) of either HRPor bisBenzimide (BB Sigma Co 10 in DW) were placedover visual cortex of the left hemisphere [24] All tracers werepressure-injected through glassmicropipettes (50ndash100 120583mtipdiameter) Data from some cases injected with BDA werepresented in Laing et al [23]

22 Histochemical Processing After a survival period of 2days the animalswere deeply anesthetizedwith pentobarbitalsodium (100mgkg ip) and perfused through the heartwith 09 saline followed by 4 paraformaldehyde in 01Mphosphate buffer (PB pH 74) After the brain was removedfrom the skull the cortical mantle to be analyzed was sepa-rated from the brainstem flattened between glass slides andsectioned tangentially (60120583m thick sections) as describedpreviously [23] The flattening procedure was done withgreat care to ensure that both striate and extrastriate corticeswere contained in the tangential sections The thalamus wascut into 60120583m thick coronal sections If fluorescence wascombined with either BDA or HRP patterns were analyzedin alternate series of sections Sections in the series examinedonly for fluorescence were mounted on slides and analyzedunder epifluorescence without further processing Howeversections in the series processed for BDA or HRP were alsooften analyzed for epifluorescence because the fluorescentlabeling in these sections was similar to that in sections notprocessed for these tracers This allowed a direct correlationof the spatial location of both fluorescent and nonfluorescentlabeling in the same section BDA labeling was revealed usingthe standard avidin-biotin-peroxidase protocol (VectastainElite ABC kit Vector Laboratories Burlingame CA USA)and 001H

2O2in 005 331015840-diaminobenzidine with cobalt

or nickel intensification sections were then mounted dehy-drated defatted and coverslipped HRP labelingwas revealedusing tetramethylbenzidine as the chromogen [25]

23 Data Acquisition and Analysis Digital images of theBDA- and HRP-labeling patterns in histological sectionswere obtained by scanning the sections at 2400 dpi usingan Epson 4990 scanner The distribution of cells labeledwith fluorescent tracers was analyzed using a microscopeequipped with a motorized stage (LEPCO) controlled by a

BioMed Research International 3

A

L

(a) (b)

LI = 052

C1C2

C3C4

(c)

(d) (e)

LI = 044

C1C2C3C4

(f)

Figure 1 Correlating the patterns of striate-extrastriate projections with the patterns of myelination and visual callosal connectionsprocedure for calculating LI (a) Myelin pattern from a rat (BEM-2) enucleated at birth Black arrows indicate borders of V1 and area 18aA auditory cortex Sml somatosensory cortex White arrow indicates BDA injection site Neighboring dark spots correspond to injectionsof other tracers (b) Callosal pattern revealed following multiple injections of HRP in the contralateral hemisphere in the same enucleatedrat Dark areas correspond to dense accumulations of retrogradely labeled somas and anterogradely labeled axons The profiles of V1 18aand auditory cortex were drawn from the myelin pattern in (a) Regions outlined in black represent BDA-labeled projection fields drawnfrom a section processed for BDA Black dot indicates the location of the BDA injection and the circular outline estimates the size of tracerdiffusion (see Section 2) (d) Pattern of BDA-labeled projections in another enucleated rat (caseM6e4) Grey dot and grey outline indicate theinjection and diffusion of BDA respectively Border of V1 outlined in black was determined from the callosal pattern (e) in the same case(e) Pattern of HRP-labeled callosal connections in the same enucleated rat Black lines in lateral extrastriate cortex outline the BDA-labeledprojections shown in (d) ((c) and (f)) Diagrams illustrating the compartments (C1ndashC4) in area 18a used in LI calculations The LI for eachcase is indicated Line passing through injection site was used to calculate the distance from the injection site to the lateral border of V1 Insetin (a) indicates orientation A anterior and L lateral Scale bars = 10mm

Dell XPS T500 computer and a graphic system (NeurolucidaMicroBrightField Williston VT USA) The borders of areas17 and 18a [26ndash28] were identified in the myelin pattern byscanning unstained tangential sections [23 29] (Figure 1(a))In both normal and enucleated rats area 18a is the target ofvirtually all projections from V1 to lateral extrastriate cortex[23] so in this report we will consider the terms ldquolateralextrastriate cortexrdquo and ldquoarea 18ardquo as synonymous Furtherinformation for identifying the location of the border of areas17 and 18a in control and enucleated rats came from analyzinglandmarks provided by the overall callosal pattern in visualcortex and the relation that these landmarks have with the

borders of areas 17 and 18a as revealed in the myelinationpatterns [23 24 30] (Figure 1)

The locations of the injection sites within striate cortexwere confirmed by analyzing the distribution of labeled fieldswithin the ipsilateral dorsal lateral geniculate nucleus of thethalamus (dLGN) [32ndash36]Thedistance between injections ofdifferent tracers ranged from 08 to 16mm These distanceswere judged adequate for studying the topography of striateprojections because the injections produced separate labelingfields in the dLGN (Figures 2(b) and 2(c)) Tangential sectionsthroughout the depth of the cortex were analyzed to ensurethat injections were restricted to grey matter


T V1

U

N

L

A

LU

RL

NT

N

L

U

T

N T

U

LNT

U

L

L

N

U

T

N T

L

18aAL

LM

LI

LL

PL

P

(a)

Control

V1

AL

LM

LI LLPL

P

LGN

019 045

(b)

BE0

V1

LGN

028 038

(c)

Figure 2 Topography of striate-extrastriate connections in normal and enucleated rats (a)Diagramof the distribution of callosal connectionsand visual areas in lateral striate cortex of normal ratsThe border of V1 is outlined in black callosal pattern is indicated in grey while acallosalareas are indicated in white The diagram summarizes previous studies (see references in text) of the overall topographic organization of V1and some visual areas in lateral extrastriate cortex (area 18a) Cortical regions representing upper lower nasal and temporal portions of thevisual field are indicated by U L N and T respectively RL rostrolateral AL anterolateral LM lateromedial LI laterointermediate LLlaterolateral PL posterolateral and P posterior Modified from [31] (b) Data from a normal rat (case C21) that received injections of GB(green dot) and RB (red dot) in V1 Cells labeled with either RB or GB are indicated by small red or green dots respectively Approximatelocation of the border of visual areas in area 18a is indicated by thin black lines (c) Data from an enucleated rat (case M4e9) that receivedinjections of RB (red dot) and BDA (black dot) in V1 BDA-labeled fields are indicated in black The laterality index for each tracer injectionin (b) and (c) is indicated Insets in (b) and (c) show labeled fields in the dLGN Callosal patterns as well as striate projection fields in medialextrastriate cortex are not represented in (b) and (c) Inset in (a) indicates orientation A anterior L lateral Scale bars = 10mm

Using Adobe Photoshop CS2 (Adobe Systems) digitizedimages of both anatomical tracer andmyelin labeling patternsfrom the same animal were carefully aligned with each otherusing the border ofV1 the edges of the sections blood vesselsand other fiducial marks Cells labeled retrogradely by theinjections of RB and GB were represented by red and greendots respectively Callosal patterns labeled with BB wererepresented by outlining the areas containing dense accumu-lations of labeled cells To illustrate the patterns of callosalconnections labeled with HRP or striate projections resultingfrom restricted injections of HRP or BDA thresholdedversions of these patterns were prepared after first applying amedian filter to reduce noise followed by a high-pass filter toremove gradual changes in staining density across the entiredigital image The same filter parameters and thresholdinglevels were applied to all control and enucleated animalsand thresholded versions were visually inspected to confirmthat they accurately represented the labeling pattern observedin the tissue sections Figures were prepared using AdobePhotoshop CS2 and all imaging processing used includingcontrast enhancement and intensity level adjustments wereapplied to the entire images

231 Quantitative Analysis We analyzed the distributionof labeled fields in lateral extrastriate cortex resulting frominjections placed at different mediolateral locations in V1The distance of the injection sites from the lateral borderof V1 was expressed as the percentage of the width of V1

measured along a line passing through the injection site andperpendicular to the V118a border (Figures 1(c) and 1(f))To evaluate the overall distribution of the labeling pattern inarea 18a with respect to the lateral border of V1 we dividedlateral extrastriate cortex into four compartments (C) ofequal width and numbered C1 to C4 from medial to lateralThese compartments were drawn parallel to the lateral borderof V1 and to each other (Figures 1(c) and 1(f)) For eachtracer injection we calculated a laterality index (LI) using thefollowing formula

LI = [(C4 minus C1) + 23 (C3 minus C2) + 119873]2119873

(1)

where C119899 is the number of pixels within compartment 119899 and119873 is the total number of labeled pixels in area 18a This indexranges from 00 to 10 values indicating that 100 of thelabeling produced by a particular injection in V1 is containedwithin C1 or C4 respectively Changes in LIs produced bypairs of tracer injections into V1 of the same animal wereevaluated statistically using paired t-tests

3 Results

The diagram in Figure 2(a) based on numerous anatomicaland physiological studies in normal rats (see referencesabove) illustrates the arrangement of visual areas in lateralextrastriate cortex their internal topography and their rela-tionship to the overall pattern of callosal connections This


Control

R2 = 081y = 056x + 003

Late

ralit

y in

dex

1009080706050403020100

Location of injection ( of V1 width)0 10 20 30 40 50 60 70 80 90 100

(a)

BE0

Late

ralit

y in

dex

1009080706050403020100


R2 = 036y = 043x + 022

(b)

Control

Late

ralit

y in

dex

1009080706050403020100


BDARB

GB

(c)

BE0

Late

ralit

y in

dex

10

09

08

07

06

05

04

03

02

01

00


BDARB

GBHRP

(d)

Figure 3 Laterality indices for normal and enucleated rats ((a) and (b)) Scatterplots correlating the LI values calculated for single injectionswith the distance of the injection sites to the lateral border of V1 expressed as a percentage of the width of V1 (see Figures 1(c) and 1(f)) Blackred green and brown dots represent injections of BDA RB GB and HRP respectively ((c) and (d)) Comparing the separation of labelingpatterns in area 18a produced by injections of different tracers into different regions of V1 in the same animal Black lines connect data fromthe pair of injections in the same animal

diagram shows that with the exception of the small area LIthe maps in all lateral extrastriate areas are mirror images ofthe map in V1 along the mediolateral axis (from temporalto nasal visual field representation) In contrast the maps ofthe anteroposterior axis in V1 (from lower to upper visualfield representation) differ among the areas while the mapsin areas AL P LI and LL show the same polarity as the mapin V1 the polarity is inverted in areas AL and PL

As it is difficult to identify with certainty specific extras-triate areas in enucleated rats due to marked abnormalitiesin striate-extrastriate and callosal connections [23 37] wedid not pursue the study of the effect of enucleation alongthe anteroposterior axis Instead we focused our attention onstudyingwhether or not displacements of the injections along

the mediolateral axis of V1 in enucleated rats lead to mirror-image displacements of the overall labeling patterns in lateralextrastriate cortex To this end we correlated the distancesfrom the injection sites to the V118a border with the LIcalculated for each labeling pattern in area 18a of control andenucleated rats (see Section 2)

As expected in control rats we observed a high corre-lation (1198772 = 081) between the distance of injection sitesfrom the lateral border of V1 and the LIs of the resultinglabeling patterns in area 18a (Figure 3(a) 14 injection sitesfrom 8 rats) This figure shows that as the distance of theinjections from the lateral border of V1 increased the overalllabeling patterns moved more laterally in lateral extrastriatecortex resulting in higher LIs A positive although weaker


correlation (1198772 = 036) was observed in enucleated rats(Figure 3(b) 24 injections from 16 rats) These results areconsistent with the high variability of striate-extrastriateprojections in enucleated rats [23] and suggest that in spiteof this variability the overall labeling patterns in area 18a ofenucleated rats tend tomove laterally when the injection sitesare displaced medially in V1

Due to the high animal-to-animal variability of striate-extrastriate projection patterns in BE0 rats it is typically notpossible to transform the pattern in one animal into that ofanother simply by displacing the fields in a consistentmanneras is often possible in normal animals [23] This variabilitymay to some extent obscure the mediolateral topography inarea 18a of BE0 rats when the data from different animals arepooled together as in Figure 3(b) To examine this possibilitywe compared the changes in LIs resulting from pairs of injec-tions placed at different locations in V1 of the same animals

Figure 2(b) presents the results from a control rat thatreceived restricted injections of GB and RB at differentdistances from the lateral border of V1 The arrangement ofthe injection sites (red dot = RB green dot = GB) permitsanalyzing the mapping along the mediolateral axis in V1In lateral extrastriate cortex red and green dots representindividual cells retrogradely labeled with either RB or GBrespectively Figure 2(b) shows that when the injection siteis displaced from lateral (green dot) to medial (red dot) inV1 the labeled fields in extrastriate cortex move in a mirror-image fashion with respect to the border of V1 as predictedfrom the mapping summarized in Figure 2(a) Thus the cellslabeled by theGB injections accumulate inmedial portions oflateral extrastriate cortex near the lateral border of V1 whilethe cells labeled by the RB injection accumulatemore laterallyin extrastriate cortex Consistent with these observations theLI associated with the GB injection (019) is smaller than thatassociated with the RB injection (045)

Results from a double-injection experiment in a BE0 ratare shown in Figure 2(c) This animal received an injectionof RB close to the lateral V1 border and an injection ofBDA furthermedially Both injections produced labeled fieldsdistributed over broad regions in lateral extrastriate cortexand the overall arrangement of labeled fields was the samefor both tracers The LI calculated for the distribution of RB-labeled cells (028) is smaller than that for the BDA-labeledpattern (038) suggesting that the mediolateral displacementof the overall pattern of labeling in area 18a mirrors thedisplacement of the injection sites in V1 with respect to thelateral border of V1

Analysis of a pair of injections in control rats (Figure 3(c))reveals a significant increase of the LIs (119875 lt 001) calculatedfor the medial injections (119872 = 049 SD = 014) comparedto the LIs calculated for the lateral injections (119872 = 027 SD= 012) (119899 = 5 paired t-test) In BE0 rats (Figure 3(d)) theLIs calculated for the medial injections (119872 = 050 SD =015) were also significantly larger (119875 lt 001) than the LIcalculated for the lateral injections (119872 = 032 SD = 010)(119899 = 8 paired t-test) However comparison of the slopes ofthe lines connecting each pair in the data sets shows that theslopes tend to be more variable in BE0 rats than in control(slopes range from 049 to 139 in control rats and from

minus023 to 141 in BE0 rats) These data suggest that in spiteof abnormalities in the projection patterns [23] the basicmirror-image topography of area 18a is largely preserved inBE0 rats They also suggest that for a given separation of theinjection site in V1 there is more variability in the separationof extrastriate labeled fields in BE0 than in control rats

In the dLGN we observed that restricted injectionsin striate cortex produced separate labeled fields in thedLGN (insets in Figures 2(b) and 2(c)) confirming that theareas of effective tracer uptake associated with the corticaltracer injections did not overlap significantly Comparedwithnormal rats the labeled fields in the dLGN of enucleatedrats appeared larger relative to the size of the dLGN possiblybecause neonatal enucleation reduces the size of both striatecortex [23] and dLGN [36 38] Our observations in thedLGN are consistent with previous studies indicating thatthe geniculocortical pathway in enucleated rats and otheranimals maintains the gross topography observed in normalanimals [33ndash36]

4 Discussion

A recent study in adult rats neonatally enucleated at birthshowed that single tracer injection into different regions of V1produce anomalous and highly variable patterns of striate-extrastriate and callosal projections [23] This previous studynoted that it was typically not possible to transform thepattern in one animal into that of another simply by dis-placing the fields in a consistent manner These results are inagreement with a recent study of the effects of enucleationand anophthalmia on the distribution of striate-extrastriateprojections in mice [14] In contrast in normal animalsthe arrangement as well as the retinotopic organization ofextrastriate areas is highly consistent from animal to animal[17 19] Although the distributions of callosal and striate-extrastriate projections in enucleated rats have an overallresemblance to those in normal rats [23] the anomalies andvariability in these projections impede the identification ofspecific visual areas in extrastriate cortex with certainty Itis therefore difficult to analyze the effect of enucleationon the topography of individual visual areas Here wetook advantage of the fact that the representation of thenasotemporal axis of the visual field in nearly all lateralextrastriate visual areasmirrors the representation of this axisin V1 with respect to the lateral V1 borderWe were interestedin determining whether the basic mediolateral topographyin the striate-extrastriate projections was preserved in spiteof abnormalities in the projection patterns Rather thananalyzing how injections into differentmediolateral locationsin V1 change the projections to specific extrastriate regionswe analyzed the displacements of the overall patterns oflabeling produced in area 18a by changes in injection sitesWe assessed these changes using a laterality index LI whichis based on the proportion of the total labeling pattern that iscontained in 4 equally spaced compartments oriented parallelto the lateral border of V1 The value of the LI increased ordecreased as the labeling tended to accumulate in lateral ormedial regions of area 18a respectively


In normal rats we found a strong correlation between themediolateral location of the injections and the correspondingLIs indicating that the sensitivity of this index is adequate fordetecting displacements of the labeling patterns produced byeven relatively small displacements of the injection sites Apositive correlation was also observed in enucleated rats butit was weaker than in normal rats Since this result was basedon the analysis of single injections from different animalsthe weaker correlation may simply reflect the variability ofprojection patterns across rats To examine this possibilitywe compared the separation of labeling patterns producedby injections of different tracers into different regions ofV1 in the same animal We found significant mirror-imageseparations of area 18a labeling patterns in both control andenucleated rats However our data suggest that the separationof labeling patterns in area 18a for a given separation of theV1 injections was somewhat more variable in enucleated ratsthan in control rats

Thus in enucleated rats we were able to detect mirror-image changes in the distribution of overall labeling patternsin area 18a in response to changes in themediolateral locationof tracer injections into V1 However these changes tended tobe more variable in enucleated than in control rats Togetherour analyses of single and double tracer injections suggestthat neonatal bilateral enucleation weakens but not com-pletely abolishes the mediolateral topography in area 18a

It is possible that the difference we found between normaland enucleated rats reflects differences in the injectionsplaced in the two groups of animals This is unlikely becausethe injections were small compared to the size of striatecortex arranged similarly along the mediolateral axis inboth groups and similar results were observed with differentcombination of tracers Moreover the separations betweenthe injection sites were judged to be adequate because theyproduced separate labeled fields in the dLGN

Previous studies have shown that neonatal bilateral enu-cleation reverses the topography of visual callosal connec-tions In normal rats callosal connections interlink oppositecortical loci that are retinotopically matched but locatedasymmetrically with respect to the brainmidline In contrastin neonatally enucleated rats callosal links are establishedbetween opposite cortical loci that are mirror symmetricwith respect to the midline [12 13] Moreover the callosaltopography in enucleated rats is less precise than in controlrats These results have led to the proposal that retinal inputguides the retinotopically precise ingrowth of callosal axonsin visual cortex whereas the cues that determine the mirror-symmetric callosal maps in enucleates exert only a weakcontrol on the topography of callosal fiber ingrowth [12 39]Thus under normal conditions these weaker cues would besuperseded by stronger influences of retinal origin leading toa nonsymmetric retinotopically matched callosal map

Our present results suggest that hierarchical topographiccues may also regulate the organization of striate-extrastriateconnections but in this case the maps that develop in bothnormal and enucleated rats show similar mediolateral topog-raphy rather than opposite topography as is the case withcallosal maps Although our data suggest that mediolateraltopography develops in area 18a under either retinally driven

cues or cues of central origin it is important to note thatretinal cues appear to be critical for the development ofconnections patterns that are highly consistent from animalto animalThus retinal inputmay not only specify the normalinternal topography of extrastriate visual areas but may alsohave an important role in the parcellation of extrastriatecortex into an array of visual areas that is remarkably constantin normal animals

How does retinal input influence the development ofstriate-extrastriate maps Projections from the dLGN areprimarily confined to striate cortex [40ndash42] so retinal inputfrom the dLGN could reach area 18a via the projections fromV1 However although enucleation reduces the size of boththe dLGN and V1 [23 36 38 43] it does not change thebasic topography of the dLGN projection to V1 [33 34 36]Retinal input can also reach area 18a via the lateroposteriornucleus of the thalamus (LP) [42 44] which receives directprojection from the superior colliculus [45] Thus enucle-ation could interfere with the layout of topographic cuesin area 18a by disrupting this alternative pathway IndeedNegyessy et al [46] reported that cortical projections fromLPwere abnormal in neonatally enucleated rats During normaldevelopment retinal input may regulate the arrangementand topography of extrastriate visual areas by specifyingthe normal distribution of cortical guidance labels througheither activity-dependent or activity-independent cues Forinstance interactions between gradients of EphAephrin-Acould guide the formation of topographically organized pro-jections from V1 to each extrastriate visual area in a mannersimilar as they guide the development of the thalamocorticalpathway [8] Central cues operating in the absence of retinalinput could specify reduced or distorted gradients leadingto the development of anomalous and variable topographicprojections between V1 and area 18a Detecting these gradi-ents and possible changes induced by neonatal enucleation orother manipulations will be specially challenging given thesmall size of the extrastriate visual areas Finally it shouldbe noted that whatever are the mechanisms by which retinalinput guides cortical topography they must exert their effectby postnatal day 6 (P6) because enucleation at P6 or laterno longer prevents the development of normal patterns ofstriate-extrastriate connections [23]

Acknowledgments

The authors thank A Bock for helping with some exper-iments This research was supported in part by a RoyaltyResearch Fund Award University of Washington to JaimeF Olavarria and Vision Training Grant no T32 EY7031 toRobyn J Laing

References

[1] D K Simon and D D M OrsquoLeary ldquoDevelopment of topo-graphic order in the mammalian retinocollicular projectionrdquoJournal of Neuroscience vol 12 no 4 pp 1212ndash1232 1992

[2] T McLaughlin C L Torborg M B Feller and D D MOrsquoLeary ldquoRetinotopic map refinement requires spontaneous


retinal waves during a brief critical period of developmentrdquoNeuron vol 40 no 6 pp 1147ndash1160 2003

[3] J G Flanagan ldquoNeural map specification by gradientsrdquo CurrentOpinion in Neurobiology vol 16 no 1 pp 59ndash66 2006

[4] L CKatz andC J Shatz ldquoSynaptic activity and the constructionof cortical circuitsrdquo Science vol 274 no 5290 pp 1133ndash11381996

[5] E MMiyashita-Lin R Hevner K MWassarman S Martinezand J L R Rubenstein ldquoEarly neocortical regionalization in theabsence of thalamic innervationrdquo Science vol 285 no 5429 pp906ndash909 1999

[6] P Vanderhaeghen Q Lu N Prakash et al ldquoA mapping labelrequired for normal scale of body representation in the cortexrdquoNature Neuroscience vol 3 no 4 pp 358ndash365 2000

[7] A Dufour J Seibt L Passante et al ldquoArea specificity andtopography of thalamocortical projections are controlled byephrinEph genesrdquo Neuron vol 39 no 3 pp 453ndash465 2003

[8] J Cang M Kaneko J Yamada G Woods M P Stryker andD A Feldheim ldquoEphrin-As guide the formation of functionalmaps in the visual cortexrdquo Neuron vol 48 no 4 pp 577ndash5892005

[9] J F Olavarria ldquoNon-mirror-symmetric patterns of callosallinkages in areas 17 and 18 in cat visual cortexrdquo Journal ofComparative Neurology vol 366 no 4 pp 643ndash655 1996

[10] J F Olavarria ldquoCallosal connections correlate preferentiallywith ipsilateral cortical domains in cat areas 17 and 18 and withcontralateral domains in the 1718 transition zonerdquo Journal ofComparative Neurology vol 433 no 4 pp 441ndash457 2001

[11] W H Bosking R Kretz M L Pucak and D FitzpatrickldquoFunctional specificity of callosal connections in tree shrewstriate cortexrdquo Journal of Neuroscience vol 20 no 6 pp 2346ndash2359 2000

[12] J F Olavarria and C P Li ldquoEffects of neonatal enucleation onthe organization of callosal linkages in striate cortex of the ratrdquoJournal of Comparative Neurology vol 361 no 1 pp 138ndash1511995

[13] J F Olavarria and R Hiroi ldquoRetinal influences specify cortico-cortical maps by postnatal day six in rats and micerdquo Journal ofComparative Neurology vol 459 no 2 pp 156ndash172 2003

[14] M E Laramee G Bronchti and D Boire ldquoPrimary visualcortex projections to extrastriate cortices in enucleated andanopthalmic micerdquo Brain Structure and Function 2013

[15] V M Montero H Bravo and V Fernandez ldquoStriate-peristriatecortico-cortical connections in the albino and gray ratrdquo BrainResearch vol 53 no 1 pp 202ndash207 1973

[16] V M Montero A Rojas and F Torrealba ldquoRetinotopic organi-zation of striate and peristriate visual cortex in the albino ratrdquoBrain Research vol 53 no 1 pp 197ndash201 1973

[17] V M Montero ldquoRetinotopy of cortical connections betweenthe striate cortex and extrastriate visual areas in the ratrdquoExperimental Brain Research vol 94 no 1 pp 1ndash15 1993

[18] Q Wang and A Burkhalter ldquoArea map of mouse visual cortexrdquoJournal of Comparative Neurology vol 502 no 3 pp 339ndash3572007

[19] J Olavarria and VMMontero ldquoRelation of callosal and striate-extrastriate cortical connections in the rat morphological defi-nition of extrastriate visual areasrdquo Experimental Brain Researchvol 54 no 2 pp 240ndash252 1984

[20] S G Espinoza and H C Thomas ldquoRetinotopic organization ofstriate and extrastriate visual cortex in the hooded ratrdquo BrainResearch vol 272 no 1 pp 137ndash144 1983

[21] H CThomas and S G Espinoza ldquoRelationships between inter-hemispheric cortical connections and visual areas in hoodedratsrdquo Brain Research vol 417 no 2 pp 214ndash224 1987

[22] T A Coogan and A Burkhalter ldquoHierarchical organization ofareas in rat visual cortexrdquo Journal of Neuroscience vol 13 no 9pp 3749ndash3772 1993

[23] R J Laing A S Bock J Lasiene and J F Olavarria ldquoRole ofretinal input on the development of striate-extrastriate patternsof connections in the ratrdquo Journal of Comparative Neurologyvol 520 no 14 pp 3256ndash3276 2012

[24] J Olavarria and R C van Sluyters ldquoOrganization and postnataldevelopment of callosal connections in the visual cortex of theratrdquo Journal of Comparative Neurology vol 239 no 1 pp 1ndash261985

[25] MMMesulam ldquoTetramethyl benzidine for horseradish perox-idase neurohistochemistry a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferentsand efferentsrdquo Journal of Histochemistry and Cytochemistry vol26 no 2 pp 106ndash117 1978

[26] W J S Krieg ldquoConnections of the cerebral cortex I The albinorat A Topography of the cortical areasrdquo Journal of ComparativeNeurology vol 84 no 2 pp 221ndash275 1946

[27] V S Caviness Jr ldquoArchitectonicmap of neocortex of the normalmouserdquo Journal of Comparative Neurology vol 164 no 2 pp247ndash263 1975

[28] K Zilles B Zilles and A Schleicher ldquoA quantitative approachto cytoarchitectonics VI The areal pattern of the cortex of thealbino ratrdquo Anatomy and Embryology vol 159 no 3 pp 335ndash360 1980

[29] C P Richter and C L Warner ldquoComparison ofWeigert stainedsections with unfixed unstained sections for study of myelinsheathsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 71 no 3 pp 598ndash601 1974

[30] J Olavarria R Malach and R C van Sluyters ldquoDevelopmentof visual callosal connections in neonatally enucleated ratsrdquoJournal of Comparative Neurology vol 260 no 3 pp 321ndash3481987

[31] J Olavarria and V M Montero ldquoOrganization of visual cortexin the mouse revealed by correlating callosal and striate-extrastriate connectionsrdquo Visual Neuroscience vol 3 no 1 pp59ndash69 1989

[32] V M Montero J F Brugge and R E Beitel ldquoRelation of thevisual field to the lateral geniculate body of the albino ratrdquoJournal of Neurophysiology vol 31 no 2 pp 221ndash236 1968

[33] P Godement P Saillour and M Imbert ldquoThalamic afferentsto the visual cortex in congenitally anophthalmic micerdquo Neu-roscience Letters vol 13 no 3 pp 271ndash278 1979

[34] I R Kaiserman-Abramof A M Graybiel and W J H NautaldquoThe thalamic projection to cortical area 17 in a congenitallyanophthalmic mouse strainrdquo Neuroscience vol 5 no 1 pp 41ndash52 1980

[35] R W Guillery M Ombrellaro and A L LaMantia ldquoTheorganization of the lateral geniculate nucleus and of the genicu-locortical pathway that developswithout retinal afferentsrdquoBrainResearch vol 352 no 2 pp 221ndash233 1985

[36] S S Warton S E Dyson and A R Harvey ldquoVisual thalam-ocortical projections in normal and enucleated rats HRP andfluorescent dye studiesrdquo Experimental Neurology vol 100 no 1pp 23ndash39 1988

[37] H Bravo and O Inzunza ldquoEffect of pre- and postnatal retinaldeprivation on the striate-peristriate cortical connections in theratrdquo Biological Research vol 27 no 1 pp 73ndash77 1994


[38] D Heumann and R T RabinowiczTh ldquoPostnatal developmentof the dorsal lateral geniculate nucleus in the normal andenucleated albino mouserdquo Experimental Brain Research vol 38no 1 pp 75ndash85 1980

[39] J F Olavarria and P Safaeian ldquoDevelopment of callosal topog-raphy in visual cortex of normal and enucleated ratsrdquo Journal ofComparative Neurology vol 496 no 4 pp 495ndash512 2006

[40] C E Ribak and A Peters ldquoAn autoradiographic study of theprojections from the lateral geniculate body of the ratrdquo BrainResearch vol 92 no 3 pp 341ndash368 1975

[41] W Schober and E Winkelmann ldquoDie Geniculo-KortikaleProjektion bei Albinorattenrdquo Journal fur Hirnforschung vol 18no 1 pp 1ndash20 1977

[42] J Olavarria ldquoA horseradish peroxidase study of the projectionsfrom the latero-posterior nucleus to three lateral peristriateareas in the ratrdquo Brain Research vol 173 no 1 pp 137ndash141 1979

[43] S J Karlen and L Krubitzer ldquoEffects of bilateral enucleationon the size of visual and nonvisual areas of the brainrdquo CerebralCortex vol 19 no 6 pp 1360ndash1371 2009

[44] H C Hughes ldquoAnatomical and neurobehavioral investigationsconcerning the thalamo cortical organization of the ratrsquos visualsystemrdquo Journal of Comparative Neurology vol 175 no 3 pp311ndash335 1977

[45] E G JonesTheThalamus Plenum Press New York NY USA1985

[46] L Negyessy V Gal T Farkas and J Toldi ldquoCross-modalplasticity of the corticothalamic circuits in rats enucleated onthe first postnatal dayrdquo European Journal of Neuroscience vol12 no 5 pp 1654ndash1668 2000


extrastriate visual areas [15 16] which have been namedaccording to their location relative to V1 (see Figure 2(a))Each of these areas contains a representation of the oppositevisual hemifield and receives direct retinotopically orga-nized projections from V1 (see [17 18] for reviews) More-over callosal connections form a dense band that straddlesthe V118a border and a series of ring-like callosal bands thatseparate adjoining extrastriate visual areas in area 18a [19ndash23] (Figure 2(a)) These callosal rings provide fixed referencelandmarks for locating and identifying the various visualareas in area 18a of normal rats

The pattern of striate-extrastriate projections in normalrats is remarkably consistent from animal to animal [17 19]Moreover tracer injections placed into different loci in V1produce essentially the same extrastriate projection patternsexcept that the projection fields translocate locally accordingto the retinotopic map in each extrastriate visual area Thishas made it possible to obtain information about the topog-raphy of striate-extrastriate connections in normal rats usingsingle tracer injections into V1 [19] In contrast to normalrats we recently reported that neonatal enucleation induceshighly irregular and variable patterns of striate-extrastriateand callosal projections [23] However this previous studybased on the analysis of projection patterns produced bysingle restricted injections of anatomical tracers into V1 didnot report the effects of enucleation on the topography ofthese projections In the present study we have addressed thisissue by analyzing data from single tracer injections into V1as well as connection patterns resulting from pairs of discretetracer injections placed at different locations inV1 of the sameanimal

2 Materials and Methods

Our study is based on data obtained from a total of 24 Long-Evans pigmented rats Pregnant animals were monitoredseveral times daily and the births of the litters were deter-mined to within 12 hours Sixteen rat pups were anesthetizedwith isoflurane (2ndash4 in air) and binocularly enucleatedwithin 24 hours of birth (BE0) After recovering from theanesthesia pups were returned to their dams In addition8 rats were used for analyzing striate-extrastriate projectionsin normal adult animals All surgical procedures were per-formed according to protocols approved by the InstitutionalAnimal Care and Use Committee (IACUC) at the Universityof Washington

21 Tracer Injections In both control and enucleated ani-mals striate-extrastriate projections were revealed followingrestricted tracer injections into striate cortex while thedistribution of callosal connections was demonstrated in thesame hemisphere following multiple injections of a differenttracer in the opposite hemisphere Anatomical experimentsin all normally reared and enucleated rats took place whenthe animals were at least 1 month old Tracer injections weremade under isoflurane anesthesia (2ndash4 in air) To studythe topography of striate-extrastriate connections animalsreceived restricted injections of various tracers into differ-ent places of striate cortex including biotinylated dextran

amine (BDA 10 in DW Molecular Probes Eugene ORUSA) which is predominantly transported anterogradelyhorseradish peroxidase (HRP Sigma Co 25 in saline)which is transported both anterogradely and retrogradelyand fluorescent tracers (rhodamine beads RB or greenbeads GB LumaFluor Naples FL USA concentrated stocksolution) which are transported retrogradely Small volumes(005ndash01120583L) of these tracers were injected into striate cortexof the right hemisphere (approx 29ndash42mm from the mid-line 01ndash20mm anterior to the lambda suture) In all casesanalyzed the tracer injections were restricted to grey matterThe size of single injections of BDA and HRP was estimatedas described previously [23] High levels of fluorescence fromGB andRB injectionswere typically restricted to the injectionsites because diffusion of these tracers is low To reveal theoverall pattern of callosal connections multiple injections(12ndash15 injections total volume approx 40 120583L) of either HRPor bisBenzimide (BB Sigma Co 10 in DW) were placedover visual cortex of the left hemisphere [24] All tracers werepressure-injected through glassmicropipettes (50ndash100 120583mtipdiameter) Data from some cases injected with BDA werepresented in Laing et al [23]

22 Histochemical Processing After a survival period of 2days the animalswere deeply anesthetizedwith pentobarbitalsodium (100mgkg ip) and perfused through the heartwith 09 saline followed by 4 paraformaldehyde in 01Mphosphate buffer (PB pH 74) After the brain was removedfrom the skull the cortical mantle to be analyzed was sepa-rated from the brainstem flattened between glass slides andsectioned tangentially (60120583m thick sections) as describedpreviously [23] The flattening procedure was done withgreat care to ensure that both striate and extrastriate corticeswere contained in the tangential sections The thalamus wascut into 60120583m thick coronal sections If fluorescence wascombined with either BDA or HRP patterns were analyzedin alternate series of sections Sections in the series examinedonly for fluorescence were mounted on slides and analyzedunder epifluorescence without further processing Howeversections in the series processed for BDA or HRP were alsooften analyzed for epifluorescence because the fluorescentlabeling in these sections was similar to that in sections notprocessed for these tracers This allowed a direct correlationof the spatial location of both fluorescent and nonfluorescentlabeling in the same section BDA labeling was revealed usingthe standard avidin-biotin-peroxidase protocol (VectastainElite ABC kit Vector Laboratories Burlingame CA USA)and 001H

2O2in 005 331015840-diaminobenzidine with cobalt

or nickel intensification sections were then mounted dehy-drated defatted and coverslipped HRP labelingwas revealedusing tetramethylbenzidine as the chromogen [25]

23 Data Acquisition and Analysis Digital images of theBDA- and HRP-labeling patterns in histological sectionswere obtained by scanning the sections at 2400 dpi usingan Epson 4990 scanner The distribution of cells labeledwith fluorescent tracers was analyzed using a microscopeequipped with a motorized stage (LEPCO) controlled by a


A

L

(a) (b)

LI = 052

C1C2

C3C4

(c)

(d) (e)

LI = 044

C1C2C3C4

(f)






T V1

U

N

L

A

LU

RL

NT

N

L

U

T

N T

U

LNT

U

L

L

N

U

T

N T

L

18aAL

LM

LI

LL

PL

P

(a)

Control

V1

AL

LM

LI LLPL

P

LGN

019 045

(b)

BE0

V1

LGN

028 038

(c)






(1)


3 Results



Control

R2 = 081y = 056x + 003

Late

ralit

y in

dex

1009080706050403020100


(a)

BE0

Late

ralit

y in

dex

1009080706050403020100


R2 = 036y = 043x + 022

(b)

Control

Late

ralit

y in

dex

1009080706050403020100


BDARB

GB

(c)

BE0

Late

ralit

y in

dex

10

09

08

07

06

05

04

03

02

01

00


BDARB

GBHRP

(d)














4 Discussion










Acknowledgments


References



















































A

L

(a) (b)

LI = 052

C1C2

C3C4

(c)

(d) (e)

LI = 044

C1C2C3C4

(f)






T V1

U

N

L

A

LU

RL

NT

N

L

U

T

N T

U

LNT

U

L

L

N

U

T

N T

L

18aAL

LM

LI

LL

PL

P

(a)

Control

V1

AL

LM

LI LLPL

P

LGN

019 045

(b)

BE0

V1

LGN

028 038

(c)






(1)


3 Results



Control

R2 = 081y = 056x + 003

Late

ralit

y in

dex

1009080706050403020100


(a)

BE0

Late

ralit

y in

dex

1009080706050403020100


R2 = 036y = 043x + 022

(b)

Control

Late

ralit

y in

dex

1009080706050403020100


BDARB

GB

(c)

BE0

Late

ralit

y in

dex

10

09

08

07

06

05

04

03

02

01

00


BDARB

GBHRP

(d)














4 Discussion










Acknowledgments


References



















































T V1

U

N

L

A

LU

RL

NT

N

L

U

T

N T

U

LNT

U

L

L

N

U

T

N T

L

18aAL

LM

LI

LL

PL

P

(a)

Control

V1

AL

LM

LI LLPL

P

LGN

019 045

(b)

BE0

V1

LGN

028 038

(c)






(1)


3 Results



Control

R2 = 081y = 056x + 003

Late

ralit

y in

dex

1009080706050403020100


(a)

BE0

Late

ralit

y in

dex

1009080706050403020100


R2 = 036y = 043x + 022

(b)

Control

Late

ralit

y in

dex

1009080706050403020100


BDARB

GB

(c)

BE0

Late

ralit

y in

dex

10

09

08

07

06

05

04

03

02

01

00


BDARB

GBHRP

(d)














4 Discussion










Acknowledgments


References



















































Control

R2 = 081y = 056x + 003

Late

ralit

y in

dex

1009080706050403020100


(a)

BE0

Late

ralit

y in

dex

1009080706050403020100


R2 = 036y = 043x + 022

(b)

Control

Late

ralit

y in

dex

1009080706050403020100


BDARB

GB

(c)

BE0

Late

ralit

y in

dex

10

09

08

07

06

05

04

03

02

01

00


BDARB

GBHRP

(d)














4 Discussion










Acknowledgments


References


























































4 Discussion










Acknowledgments


References


























































Acknowledgments


References


















































Topography of Striate-Extrastriate Connections in ... · Topography of Striate-Extrastriate...

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Transcript of Topography of Striate-Extrastriate Connections in ... · Topography of Striate-Extrastriate...