Subcortical Connections to Human Amygdala and Changes following Destruction of the Visual Cortex

29 July 2021

AperTO - Archivio Istituzionale Open Access dell'Università di Torino

Original Citation:

Subcortical Connections to Human Amygdala and Changes following Destruction of the Visual Cortex

Published version:

DOI:10.1016/j.cub.2012.06.006

Terms of use:

Open Access

(Article begins on next page)

Anyone can freely access the full text of works made available as "Open Access". Works made available under a

Creative Commons license can be used according to the terms and conditions of said license. Use of all other works

requires consent of the right holder (author or publisher) if not exempted from copyright protection by the applicable law.

Availability:

This is the author's manuscript

This full text was downloaded from iris - AperTO: https://iris.unito.it/

iris - AperTO

University of Turin’s Institutional Research Information System and Open Access Institutional Repository

This Accepted Author Manuscript (AAM) is copyrighted and published by Elsevier. It is

posted here by agreement between Elsevier and the University of Turin. Changes resulting

from the publishing process - such as editing, corrections, structural formatting, and other

quality control mechanisms - may not be reflected in this version of the text. The definitive

version of the text was subsequently published in CURRENT BIOLOGY, 22 (15), 2012,

10.1016/j.cub.2012.06.006.

You may download, copy and otherwise use the AAM for non-commercial purposes

provided that your license is limited by the following restrictions:

(1) You may use this AAM for non-commercial purposes only under the terms of the

CC-BY-NC-ND license.

(2) The integrity of the work and identification of the author, copyright owner, and

publisher must be preserved in any copy.

(3) You must attribute this AAM in the following format: Creative Commons BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/deed.en),

10.1016/j.cub.2012.06.006

The publisher's version is available at:

http://linkinghub.elsevier.com/retrieve/pii/S0960982212006513

When citing, please refer to the published version.

Link to this full text:

Subcortical Connections to Human

Amygdala and Changes

following Destruction of the Visual Cortex

Lawrence W eiskrantz, and Rainer Goebel

Summary

N onconscious [1 -6 ], rapid [7, 8], o r coarse [9] visual pro ­ cessing o f em otional s tim u li induces fu n ctio n a l a ctivity in a subcortical pathw ay to the am ygdala in vo lvin g th e su p ­ erio r c o llic u lu s and pulvinar. Despite evidence in lower mam mals [10, 11] and nonhum an prim ates [12], it rem ains speculative w hether anatom ical connections between these structures exist in th e human brain [13-15]. It is also unknow n w hether de stru ctio n of the visual cortex, w hich provides a m ajor in p u t to the amygdala, induces m odifica­ tio n s in anatom ical connections along th is subcortical pathway. We used d iffu s io n te n so r im aging to investigate in v ivo anatom ical connections between human amygdala and su b co rtica l visual structures in ten age-matched co n tro ls and in one patient w ith early unilateral destruction o f th e visual cortex. We fo u n d fib e r con n e ctio n s between pulvin a r and amygdala and also between s u p e rio r c o llic u lu s and am ygdala via the pu lvin a r in th e c o n tro ls as well as in the patient. Destruction o f th e visual cortex led to q ualitative and qu antitative m o d ifica tio n s along th e pathways connecting these three structures and the changes were confined to the p atient’s damaged hemisphere. The present fin d in g s th u s show extensive neural p la sticity in the anatom ical connections between subcortical visual stru ctu re s o f old evolutionary o rig in involved in th e processing of em otional stim u li.

Results

We investigated whether an anatomical pathway to the amyg­ dala (AMG), involving the superior colliculus (SC), and the visual pulvinar (Pulv), can be reconstructed in humans. This study provided an opportunity to address the controversial question as to whether functional activity previously reported along this subcortical pathway during nonconscious percep­ tion of emotional stimuli is also supported by anatomical connections among the implicated regions [14, 15]. In addi­ tion, we investigated whether the ability to process unseen emotional stimuli, often persisting in patients with damage

to the primary visual cortex (VI), may reflect compensatory mechanisms enhancing subcortical and VI-independent connections to the AMG.

We first reconstructed th e fib e r tracts connecting the ipsilat- eral SC and Pulv in each hemisphere separately and we followed their possible continuation to/from other cortical and subcortical areas. Then, we investigated w ith the same procedure the fibers connecting the Pulv and AMG. Finally, we traced fiber tracts supposed to selectively connect these three structures together, thereby forming a direct SC-Pulv- AMG pathway akin to the disynaptic pathway recently re­ ported in nonhuman primates [12].

Fiber tracts reconstructed in the brain of neurologically intact subjects were compared with those found in patient G.Y. with unilateral destruction of his left VI suffered at the age of 7 years, as the result of a traum atic brain injury. This offered the unique opportunity to study possible changes in anatomical connectivity following early loss of VI in a patient whose residual visual ability to process emotions noncon- sciously has been carefully documented and related to func­ tional activity in the SC-Pulv-AMG pathway [2, 5, 16]. Fiber extensions to/from areas not targeted a priori were retained fo r further comparison with those found in either hemisphere of G.Y. only if these fiber tracts could be reconstructed in at least six of the ten control subjects.

SC-Pulv Pathway

Controls

In controls, the fiber bundles connecting the SC and Pulv were highly symmetrical in the tw o hemispheres and extended to/from a number of other cortical and subcortical areas, prin­ cipally within the same hemisphere (Figure 1 A). Fibers passing through the left SC and Pulv continued ipsilaterally to/from striate (VI) and extrastriate visual areas (V2/3, V4, MT/V5), tem poral pole, posterior parietal cortex, primary m otor cortex, frontal eye fields, dorsal prefrontal cortex, and orbitofrontal cortex. Subcortically, other fiber bundles were traced to/from the ipsilateral AMG, caudate, and brainstem, including periac- queductal gray. A subset of fibers crossed the midline at the level of the intercollicular commissure and continued to the contralateral (right) SC and Pulv. Fibers connecting the right SC and Pulv showed exactly the same cortical and subcortical projections reconstructed in the left hemisphere (LH). Note that all reconstructed tracts have been previously described in histological studies on nonhuman primates or in vivo d iffu ­ sion tensor imaging (DTI) investigations in human subjects (see Table S4 available online). This illustrates the reliability of our tractography method and the high consistency with existing axonal connections.

A normalized measure of fiber density has been recently introduced by Yo and colleagues [17] and considered to be a reliable indicator of connection strength between tw o neural structures. In fact, this measure takes into account the anatomical differences between regions and subjects by w eighting the number of reconstructed fibers found to connect tw o or more areas fo r the size of these structures and fo r the overall number of fibers passing through these regions when considered individually. We therefore quantified fiber density

^

. >J

t - ^ '- :

.k,

n

F ig u re 1. R e c o n s tru c te d F ib e r T r a c ts C o n n e c tin g th e S C w ith th e P u lv

(A) F ib e r tra c ts c o n n e c tin g th e S C (y ello w ) w ith th e P u lv (a zu re) a n d th e ir c o n tin u a tio n s to o th e r c o rtic a l a n d s u b c o rtic a l a re a s d is p la y e d s e p a r a te ly fo r th e LH a n d R H (le ft a n d rig ht p a n e l, re s p e c tiv e ly ) o f o n e re p re s e n ta tiv e a g e -m a tc h e d c o n tro l s u b je c t (s e e a ls o F ig u re S 2 a n d T a b le S2).

(B) T h e s a m e fib e r tra c ts re c o n s tru c te d in p a tie n t G .Y . F ib e r tra c ts e x te n d in g to d iffe re n t c o rtic a l a n d s u b c o rtic a l a r e a s a re c o d e d in d iffe re n t c o lo rs . T h e A M G is v is ib le (re d) a s w e ll a s th e lesio n to left V I in G .Y . (tra n s p a re n t gray).

A b b re v ia tio n s : C a u d , c a u d a te ; C C , c o rp u s c a llo s u m ; d P F C , d o rs a l p re fro n ta l c o rte x ; FE F, fro n ta l e y e field s; M l , p r im a ry m o to r c o rte x ; O F C , o rb ito fro n ta l c o rte x ; P A G , p e ria c q u e d u c ta l gray; P P , p o s te rio r p a rie ta l c o rte x ; T P , te m p o ra l p ole.

between ipsilateral SC and Pulv by normalizing the absolute quantity of connecting fibers fo r the size and fiber density of these tw o structures in each hemisphere and subject individu­ ally. Statistical comparisons revealed no significant difference between SC-Pulv connection strength in the LH (mean connection strength = 0.64 ± 0.12 SEM) and right hemisphere (RH) (0.75 ± 0.08) [paired sample t test: t(9) = 0.91, p = 0.39] (Figure S4).

Patient G.Y.

In the (intact) RH of G.Y., the fibers connecting the SC and Pulv extended to/from the same ipsilateral cortical and subcortical areas also identified in age-matched controls but failed to connect w ith the dorsal prefrontal cortex (Figure 1B). In addi­ tion, G.Y. showed prom inent bilateral tracts that crossed the intercollicular commissure as well as the corpus callosum and continued to the left extrastriate areas (V2/V3 and MT/V5), tem poral pole, posterior parietal cortex, SC, and Pulv. In the (damaged) LH, fibers passing through the SC and Pulv pro­ jected ipsilaterally to the extrastriate visual areas (V2/3, V4, and MT/V5), tem poral pole, posterior parietal cortex, and

AMG, therefore failing to connect with anterior areas in the frontal and prefrontal cortex identified in controls. Contralat­ eral connections reached the right SC, Pulv, caudate, and frontal eye fields passing through the intercollicular com m is­ sure. In G.Y., the connection strength between left SC and Pulv (0.298) was reduced to the 36% of the connection between the same structures in his (intact) RH (0.83) and to the 46% of the mean connection strength in the LH of controls. Statistical comparison of connection strength in the right SC-Pulv pathways between G.Y. and controls showed no significant difference [one sample t test: t(9) = 1.12, p = 0.3], whereas the same comparison in the (damaged) LH revealed a significant effect [t(9) = 2.93, p = 0.02].

Pulv-AMG Pathway

Controls

Reconstructed fibers connecting the left Pulv and AMG in the controls extended to/from the ipsilateral temporal pole, dorsal prefrontal cortex, orbitofrontal cortex, caudate, and SC (Fig­ ure 2A). Fibers connecting the right Pulv and AMG continued

F ig u re 2. R e c o n s tru c te d F ib e r T r a c ts C o n n e c tin g th e P u lv w ith th e A M G

(A) F ib e r tra c ts c o n n e c tin g th e P u lv (a zu re ) w ith th e A M G (re d) a n d th e ir c o n tin u a tio n to o th e r c o rtic a l a n d s u b c o rtic a l a re a s d is p la y e d s e p a ra te ly fo r th e LH a n d R H o f o n e re p re s e n ta tiv e a g e -m a tc h e d c o n tro l s u b je c t (s e e a ls o F ig u re S 3 a n d T a b le S3).

(B) T h e s a m e fib e r tra c ts re c o n s tru c te d in p a tie n t G .Y . F ib e r tra c ts e x te n d in g to d iffe re n t c o rtic a l a n d s u b c o rtic a l a r e a s a re c o d e d in d iffe re n t c o lo rs . T h e S C is a ls o v is ib le (y ello w ). T h e w h ite b o x e s d is p la y th e s a m e fib e rs s e e n in th e m a in fig u re b u t fro m th e o p p o s ite p e rs p e c tiv e (i.e., a n te ro m e d ia l v ie w in s te a d of p o s te ro la te ra l v ie w ) a n d lim ite d to th e s p a c e b e tw e e n th e P u lv a n d A M G . A b b re v ia tio n s : A M G -IP , fib e r tra c ts c o n n e c tin g th e A M G w ith in fe ro la te ra l p o rtio n s o f th e Pulv; A M G -m P , fib e r tra c ts c o n n e c tin g th e A M G w ith d o rs o m e d ia l p o rtio n s o f th e Pulv.

to/from the same areas in the RH. Noteworthy, the AMG appears to be connected with tw o different subregions of the ipsilateral Pulv, the dorsomedial and the inferolateral portions. This anatomical segregation is in keeping with tracer studies in nonhuman primates, indicating tw o efferent con­ nections from the Pulv to the AMG that parallel functional differences between Pulv subregions [12, 18]. The dorsom e­ dial portions include multimodal neurons preferentially con­ nected to the AMG and to frontal areas [19]. Note, in fact, that reconstructed fibers reaching frontal areas passed through this Pulv division. Conversely, inferolateral Pulv portions are predom inately visual and are bidirectionally con­ nected w ith striate and extrastriate visual areas [18]. These latter Pulv subregions receive direct afferents from the SC and from the retina and are functionally homologous to the Pulv regions recently found to send efferents to the AMG and involved in the disynaptic SC-Pulv-AMG pathway in

nonhuman primates [12]. There was no significant difference in the Pulv-AMG connection strength between the LH (0.02 ± 0.008) and RH (0.031 ± 0.014) of controls [t(9) = 0.756, p = 0.469].

Patient G.Y.

In G.Y.’s (intact) RH, the fibers connecting the right Pulv and AMG extended to/from the same ipsilateral areas and were qualitatively similar to the fibers reconstructed in controls, including the presence of tw o subset of fibers to/from different Pulv divisions (Figure 2B). Quantitatively, the connection strength between right Pulv and AMG (0.021) was similar to that of controls, with no significant difference [t(9) = 0.72, p = 0.49]. In contrast, in the (damaged) LH, the fibers passing through the left Pulv and AMG continued to ipsilateral extras- triate areas V2/V3, tem poral pole, SC, and brainstem. There­ fore, fibers connecting left Pulv and AMG apparently did not extend to frontal areas but rather to posterior visual areas.

F ig u re 3. R e c o n s tru c te d F ib e r T r a c ts C o n s titu tin g th e D ire c t S C -P u lv -A M G P a th w a y

(A) F ib e r tra c ts c o n s titu tin g th e d ire c t S C -P u lv -A M G p a th w a y in th e LH (g reen ) a n d R H (b lu e) o f o n e re p re s e n ta tiv e a g e -m a tc h e d c o n tro l s u b je c t. (B) T h e s a m e p a th w a y in p a tie n t G .Y . N o te th a t th e P u lv is m a d e tra n s p a r e n t in o r d e r to d is p la y th a t th e fib e rs run in te rru p te d ly b e tw e e n th e S C a n d A M G p a s s in g th ro u g h th e in fe ro la te ra l v isu al p o rtio n s o f th e P u lv (s e e a ls o M o v ie S I ) .

The connection between left Pulv and AMG of G.Y. (0.058) was almost 3-fold stronger (272%) than that found in his RH and in the LH of controls (287%), a difference that was highly significant [t(9) = 5.01, p < 0.001].

SC-Pulv-AMG Pathway

Controls

We were able to trace fibers that selectively and uninterrupt­ edly connect the ipsilateral SC, Pulv, and AMG in both hemi­ spheres of the controls (Figure 3A). Consistent with previous histological evidence in nonhuman primates, the fibers to / from the SC crossed the inferolateral visual portions of the Pulv before connecting with the AMG. It is, however, beyond the limits of DTI to assess whether these fibers make a synaptic relay in the visual Pulv. In both cases, the reconstructed fibers did not extend to/from any other cortical or subcortical struc­ ture, including the nonvisual subregions of the Pulv or extras- triate visual areas. There was no significant difference in the connection strength between LH (0.015 ± 0.009) and RH (0.25 ± 0 .1 1 ) [t(9) = 2.147, p = 0.49].

Patient G.Y.

The same SC-Pulv-AMG pathway was reconstructed in LH and RH of G.Y. (Figure 3B). Also in this case, the fibers did not extend to/from other brain areas. Quantitative analysis showed that there was almost a 10-fold increase (995%) in the strength of the connection in the (damaged) LH (0.24) of G.Y. compared to that estimated in his (intact) RH (0.024), and almost a 16-fold increase (1571%) compared to the mean connection strength in the LH of controls. The connec­ tion strength in the SC-Pulv-AMG pathway of G.Y. differed significantly from that estimated in controls only in the (damaged) LH [f(9) = 23.85, p < 0.0001].

Fractional A n iso tro p y A nalysis

For each hemisphere and pathway investigated, we indepen­ dently extracted fractional anisotropy (FA) values from each voxel spatially located where the tractography algorithm re­ constructed a fiber (Figure 4). FA values characterize the d iffu ­ sion properties in each voxel [20], thereby providing a measure of anisotropy that is unaffected by the specific tractography

Pu lv-A M G 0.35 0.31 0.27 0.23 0.19 S C -P u lv T---0.39 0.37 0.3S 0.33 0.31 0.29 ____________ I________ LH RH

* ' M ean ± 0 .95 confidence limits In controls • Patient G Y

technique used. Results based on FA values paralleled those obtained on connection strength values. In fact, there was no significant difference between G.Y. and controls on FA values recorded in voxels along the SC-Pulv pathway in the RH [t(9) = 0.71, p = 0.5], whereas the difference was significant in the LH [t(9) = 2.31, p = 0.046]. In the Pulv-AMG pathway, there was a significant increase of FA in the (damaged) LH of G.Y. compared to controls [t(9) = 2.89, p = 0.018], but not in the (intact) RH [t(9) = 0.53, p = 0.61]. Finally, FA values in the SC-Pulv-AMG pathway were significantly higher in G.Y.’s (damaged) LH compared to controls [t(6) = 4.56, p = 0.004], but not in the (intact) RH [t(7) = 2.1, p = 0.07].

D iscussion

In the present study, we characterized in vivo the anatomical connectivity between the SC, Pulv, and AMG and its changes following early loss of V I. Our results provide unequivocal evidence of qualitative and quantitative m odifications along the pathways connecting these three structures. These changes consist in the strengthening of several fiber tracts already existing in the intact brain as well as in the formation of seemingly new tracts not otherwise present.

Fiber tracts connecting the SC and Pulv in the (intact) RH of G.Y. were of similar strength to those reconstructed in age- matched controls. However, the contralateral projections to / from posterior regions of the (damaged) LH were considerably more prom inent than those found in control subjects. The homologous SC-Pulv pathway in the (damaged) LH of G.Y. was reduced in strength, failed to extend to frontal areas iden­ tified in controls and showed more connections to/from contra­ lateral posterior areas in the RH. These findings are consistent with recent evidence in G.Y. and other patients with cortical blindness showing that VI damage increases interhemispheric connections between homologous cortical areas as well as between subcortical and cortical visual structures in the o ppo­ site hemispheres with respect to the predominant ipsilateral connections found in controls [21, 22]. The reduced input to frontal areas in the damaged hemisphere is also remarkably consistent w ith prior functional results in cortically blind

S C -P u lv-A M G F ig u re 4. F ra c tio n a l A n is o tro p y V a lu e s in th e C o n tro ls a n d in P a tie n t G .Y .

M e a n fra c tio n a l a n is o tro p y v a lu e s ( ± 0 . 9 5 c o n fi­ d e n c e lim its) f o r e a c h p a th w a y a n d h e m is p h e re in th e te n a g e -m a tc h e d c o n tro ls a n d in p a tie n t G .Y .

patients [23] as well as in healthy sub­ jects [24] showing that activation in the prefrontal cortex (in particular Brod- mann area 46) is selectively associated to conscious stimulus perception.

The Pulv-AMG pathway in the (intact) RH of G.Y. was qualitatively and quanti­ tatively comparable to the homologous pathway in the RH of controls. This shows that changes in connectivity as well as formation of interhemispheric tracts are not unspecific consequences of VI damage, which instead affects selectively several pathways while leaving others substantially unchanged. Conversely, there was a strengthening of Pulv-AMG connec­ tions in the (damaged) LH of G.Y. compared to controls, and fiber tracts extended more posteriorly to visual areas than anteriorly to frontal regions.

Lastly, we traced the direct SC-Pulv-AMG pathway, there­ fore extending to the living human brain anatomical findings hitherto described only with the use of histological methods in nonhuman primates [12]. Nevertheless, independent confir­ mation with tracer methods in human postm ortem studies is needed to consider this evidence to be conclusive. This pathway displays specific anatomical characteristics that cannot be considered the simple summation of the connec­ tivity properties found individually in the SC-Pulv and in the Pulv-AMG pathways. In fact, when the analysis was con­ strained to the fibers selectively connecting these three structures, only a subset of fiber bundles was reconstructed among those found to connect SC-Pulv and Pulv-AMG. This subset of fibers passed through the visual subregion of the Pulv and did not extend to/from other cortical or subcortical structures. Moreover, the same pathway was also recon­ structed in both hemispheres of G.Y., but the strength of the connection was higher in his (damaged) LH compared to controls, therefore strongly suggesting that it conveys VI -independent visual information.

The existence of continuous fiber tracts connecting the SC, Pulv, and AMG does not necessarily im ply that these pathways are functionally relevant in underlying residual processing of emotions in the absence of VI and of visual awareness. More­ over, there is a m ultitude of VI -independent pathways under­ lying different aspects of nonconscious perception [25], and there are many alternative anatomical routes possibly relaying visual information to the AMG from extrastriate visual cortex [14]. These alternative pathways presumably coexist w ith the subcortical SC-Pulv-AMG route and could contribute to the nonconscious perception of emotions [26]. However, it is straightforward to relate the anatomical results reported here to the functional evidence repeatedly showing activation of the SC, Pulv, and AMG when emotional stimuli are projected in the blind visual field of G.Y. [2, 5, 16]. Moreover, the same structures are also activated in neurologically intact subjects

during early stages of emotion processing [7-9] or w lien nonconscious perception of emotions is induced by experi­ mental procedures such as visual masking [1, 3, 6, 27], flash suppression [28], or binocular rivalry [29] (see also [15] for a recent review). In particular, functional neuroimaging fin d ­ ings in G.Y. [2, 5] and healthy subjects [6, 30] have shown that nonconscious processing of emotional stimuli often results in the reduction or lack of cortical activity, especially in frontal areas, which is nevertheless normally present during conscious perception of the same stimuli. This is in keeping with the present anatomical data showing that the fiber tracts constituting the direct SC-Pulv-AMG pathway in both healthy controls and patient G.Y. do not continue to/from other cortical areas, in contrast to what was found fo r the SC-Pulv or Pulv-AMG pathways. Therefore, conscious and nonconscious perception of emotions seem to be mediated by neural systems that are partially segregated at the anatomical as well as functional level, and that undergo considerable neural plasticity and m acroscopic anatomical reorganization following early destruction of V I .

L im itations o f DTI

There are several methodological limitations to the conclu­ sions that can be drawn from DTI results. First, it is not possible to establish the directionality of a pathway and to differentiate between anterograde, retrograde, or bidirectional connections between tw o or more structures. Our main hypothesis was based on feedforward connections from the SC to the AMG with a synaptic relay in the visual Pulv [12], but this possibility cannot find direct support in our data and remains speculative at present.

Second, DTI cannot support claims about whether a recon­ structed fiber tract that passes through one or more gray matter structures is indicative of a monosynaptic or polysyn­ aptic axonal pathway. In fact, DTI does not actually detect neurons or axons but only units of volumes and reconstructed fiber bundles, each of them containing thousands of neurons and axons. Again, w e note that our results of uninterrupted streamlines connecting the SC with the AMG passing through the inferolateral visual portions of the Pulv are consistent with histological findings of a disynaptic pathway [12]. In fact, because of the spatial continuity between the (supposed) afferent fibers from SC to the inferolateral Pulv and the efferent fibers that (after the synaptic relay) should originate from the same inferolateral Pulv to reach AMG, the tractography was expected to find an uninterrupted fiber bundle, which is exactly what was observed.

A third limitation concerns the interpretation of the presence or absence of a given pathway and the risks of false positives. There is convincing evidence that in coherent w hite matter regions, the direction of diffusion corresponds to the principal axis of fiber direction [31, 32] and that tractography in such regions matches well with human postm ortem data [33]. In regions of fiber crossing, or of degeneration, however, the relationship between DTI-reconstructed fiber bundles and the underlying axonal architecture is not yet fully understood. Therefore, our evidence about the existence of a direct SC-Pulv-AMG pathway or about connections between visual Pulv and AMG in the human brain requires independent confirm ation with tracer methods in postm ortem studies.

Nevertheless, we adopted several strategies to minimize the risk of false positives. One is that we used determ inistic fiber tracking, which is less liable to false positives than probabilistic fiber tracking [17]. Moreover, errors and false positives in fiber

tracking increase w ith distance between the targeted regions [34], when fibers get closer to the cortex and/or to the cerebro­ spinal fluid (CSF) [35], and when fibers approach a lesion [21]. In the present case, we focused on relatively short fibers, located far from the cortex and from CSF and very distant from the lesion to V I . Moreover, fo r each pathway considered, we reversed seed and target regions, such that fibers were re­ constructed in both directions. This procedure has proved to be a good strategy to detect possible tractography biases, especially false positives [21]. Lastly, our choice to follow the continuation of reconstructed fiber tracts beyond the targeted regions offered a valuable opportunity to assess, in a data- driven fashion, the reliability of the tractography algorithm and its consistency w ith tracts certainly known to exist from previous postm ortem studies in humans. As summarized in Table S4, our results were in excellent agreement with previous studies and there were no false positives, as all reconstructed fiber bundles corresponded to existing axonal projections.

Because our tractography data remained highly consistent across subjects and hemispheres and all reconstructed fibers corresponded to existing axonal pathways, an interpretation of our novel findings in terms of false positives appears unlikely.

E x p e r im e n ta l P r o c e d u r e s

P a rtic ip a n ts

P a tie n t G .Y . is a w e ll-d o c u m e n te d m a le p a tie n t w ith rig h t h o m o n y m o u s h e m ia n o p ia a n d “ b lin d s ig h t.” G .Y . w a s a g e d 5 3 a t th e tim e o f te s tin g an d his v isu al s y s te m , in clu d in g his re s id u al a b ility to p ro c e s s e m o tio n s , has b e e n s tu d ie d e x te n s iv e ly in p re v io u s re p o rts using b e h a v io ra l [36] a n d p s y c h o p h y s io lo g ic a l m e th o d s [37], a s w e ll a s w ith n e u ro im a g in g te c h n iq u e s su c h a s f M R I [38, 3 9 ], M E G [40], a n d D T I [21].

T e n a g e -m a tc h e d h e a lth y m a le c o n tro ls w ith n o rm al o r c o r r e c te d to n o rm a l visio n a n d no p re v io u s h is to ry o f n e u ro lo g ic a l o r p s y c h ia tric d e fic its p a r tic ip a te d in th is s tu d y (s e e T a b le S I ) . In fo rm e d c o n s e n t w a s o b ta in e d fro m all p a rtic ip a n ts a n d th e s tu d y w a s a p p ro v e d b y th e lo cal E thic C o m m itte e in a c c o r d a n c e w ith th e e th ic a l s ta n d a rd s laid d o w n in th e D e c la ra tio n o f H e lsinki.

D T I D a ta A c q u is it io n a n d A n a ly s is

D T I c h a ra c te riz e s th e ra n d o m m ic ro s c o p ic m o tio n (d iffu sio n ) p ro p e rtie s of w a te r m o le c u le s , w h ic h c a n b e u s e d to re c o n s tru c t w h ite m a tte r fib e r b u n d le s in th e brain [41]. In fa c t, th e d iffu s iv ity is a n is o tro p ic in la rg e p a rts o f th e w h ite m a tte r, w ith th e p rin c ip a l d iffu s io n d ire c tio n c o rre s p o n d in g to t h e o r ie n ta tio n o f th e m a jo r fib e r tra c ts in e a c h v o x e l [20]. L o c al (v o x e l- w is e ) o r ie n ta tio n c a n th e n b e fo llo w e d th ro u g h s p a c e to tra c e d is ta n t c o n n e c tio n s b e tw e e n brain a re a s [32].

D iffu s io n -w e ig h te d M R I d a ta w e r e a c q u ire d a t M a a s tr ic h t U n ive rs ity, T h e N e th e rla n d s , on a 3 T S ie m e n s A lle g ra s y s te m (S ie m e n s , E rlan g en , G e rm a n y ). In th e s a m e s e s s io n , a T 1 -w e ig h te d a n a to m ic a l im a g e w a s a c q u ire d f o r re g is tra tio n a n d a n a to m ic a l lo c a liz a tio n p u rp o s e s . T h e t h r e e re g io n s o f in te re s t (R O Is ) (i.e., th e S C , P u lv, a n d A M G ) w e r e d e fin e d a n a to m ic a lly a n d in d iv id u a lly f o r e a c h h e m is p h e re a n d s u b je c t in T 1 -w e ig h te d stru c tu ra l im a g e s (s e e S u p p le m e n ta l In fo rm a tio n , F ig u re S I , a n d T a b le S I ) . D iffu s io n -w e ig h te d d a t a w e r e c o re g is te re d to th e a n a to m ic a l d a ta , a n d t h e d iffu sio n te n s o r w a s c a lc u la te d in e a c h v o x e l (2 m m ^) [41]. T h e R O Is w e r e s a m p le d w ith a d e n s e grid o f s e e d p o in t (3 4 3 s e e d s /v o x e l) to s ta rt f ib e r tra c k in g . T h e d a t a w e r e th e n e x p o r te d to an in -h o u s e d e v e lo p e d C /C + + to o l fo r d e te rm in is tic f ib e r tra c k in g . S tre a m lin e f ib e r tra c k in g used t h e local te n s o r a s a p ro je c tio n o p e ra to r [42] a n d w a s p e rfo rm e d fo rw a rd a n d b a c k w a r d b e tw e e n S C -P u lv , P u lv -A M G , a n d S C -P L V -A M G (i.e ., th e s e e d a n d ta r g e t R O Is w e re re v e rs e d ). A s e rie s o f sin g le s a m p le t-te s ts w e r e u s e d fo r s ta tis tic a l c o m p a ris o n s o f th e m e a n c o n n e c tio n s tre n g th in h e a lth y c o n tro ls w ith th e c o n n e c tio n s tre n g th in th e c o rre s p o n d in g p a th w a y o f G .Y ., w h e r e a s a se rie s o f p a ire d -s a m p le t te s ts w e r e used f o r s ta tis tic a l c o m p a ris o n o f th e m e a n c o n n e c tio n s tre n g th in th e s a m e p a th w a y b e tw e e n th e LH a n d R H o f co n tro ls . F o r d e ta ils on e x p e rim e n ta l p ro c e d u re s a n d d a ta a n a ly s is , s e e S u p p le m e n ta l In fo rm a tio n .

S u p p le m e n ta l In fo rm a tio n

S u p p le m e n ta l In fo rm a tio n in c lu d e s fo u r fig u re s , fo u r ta b le s . S u p p le m e n ta l E x p e rim e n ta l P ro c e d u re s , a n d o n e m o v ie a n d c a n b e fo u n d w ith th is a rtic le o n lin e a t d o i:1 0 .1 0 1 6 /j.c u b .2 0 1 2 .0 6 .0 0 6 .

A c k n o w le d g m e n t s

B .d .G a n d M .T . w e r e s u p p o rte d b y th e p ro je c t “T A N G O - E m o t io n a l in te ra c ­ tio n g r o u n d e d in re a lis tic c o n te x t” u n d e r th e F u tu re a n d E m e rg in g T e c h n o l­ o g ie s (F E T ) p ro g ra m fro m th e E u ro p e a n C o m m is s io n (F P 7 -IC T -2 4 9 8 5 8 ). R .G . w a s s u p p o r te d b y th e E u ro p e a n R e s e a rc h C o u n c il u n d e r th e E u ro p e a n U n io n ’s S e v e n th F ra m e w o r k P ro g ra m m e (2 6 3 4 7 2 ). R e c e iv e d : J a n u a r y 17, 2 0 1 2 R e v is e d : M a y 1 6 , 2 0 1 2 A c c e p te d : J u n e 6, 2 0 1 2 P u b lis h e d online: J u n e 2 8 , 2 0 1 2 R e fe re n c e s 1. L id d e ll, B .J ., B ro w n , K .J ., K e m p , A .H ., B a rto n , M .J ., D a s, P ., P e d u to , A., G o rd o n , E., a n d W illia m s , L .M . (2 0 0 5 ). A d ire c t b r a in s te m -a m y g d a la - c o rtic a l ‘a la r m ’ s y s te m fo r s u b lim in a l sig n a ls o f fe a r. N e u ro im a g e 24, 2 3 5 -2 4 3 .

2. M o rris , J .S ., D e G e ld e r, B ., W e is k r a n tz , L., a n d D o la n , R .J . (2 0 0 1 ). D iffe re n tia l e x tra g e n ic u lo s tria te a n d a m y g d a la r e s p o n s e s to p re s e n ­ ta tio n o f e m o tio n a l fa c e s in a c o rtic a lly b lin d fie ld . B rain 124,1 2 4 1 -1 2 5 2 . 3. M o rris , J .S .,O h m a n , A .,a n d D o lan , R .J . (1 9 9 9 ). A s u b c o rtic a l p a th w a y to

th e rig ht a m y g d a la m e d ia tin g “ u n s e e n ” fe a r. P ro c . N a tl. A c a d . S c i. U S A 9 6 , 1 6 8 0 -1 6 8 5 .

4. P e g n a , A .J ., K h a te b , A ., L a z e y ra s , F., a n d S e g h ie r, M .L . (2 005). D is c rim in a tin g e m o tio n a l fa c e s w ith o u t p rim a ry v isu al c o rtic e s invo lves th e rig ht a m y g d a la . N a t. N e u ro s c i. 8 , 2 4 - 2 5 .

5. V a n d e n S to c k , J ., T a m ie tto , M ., S o rg e r, B., P ic h ó n , S ., G ré z e s , J ., and d e G e ld e r, B. (2 0 1 1 ). C o rtic o -s u b c o rtic a l v is u a l, s o m a to s e n s o ry , and m o to r a c tiv a tio n s fo r p e rc e iv in g d y n a m ic w h o le -b o d y e m o tio n a l e x p re s s io n s w ith a n d w ith o u t s tria te c o rte x ( V I) . P ro c . N a tl. A c a d . S ci. U S A 70S, 1 6 1 8 8 - 1 6 1 9 3 .

6. W h a le n , P .J ., R a u c h , S .L ., E tc o ff, N . L , M c ln e rn e y , S .C ., L ee , M .B ., and J e n ik e , M .A . (1 9 9 8 ). M a s k e d p re s e n ta tio n s o f e m o tio n a l fa c ia l e x p re s ­ s io n s m o d u la te a m y g d a la a c tiv ity w ith o u t e x p lic it k n o w le d g e . J . N e u ro s c i. 18, 4 1 1 ^ 1 8 .

7. G a rrid o , M . I., B a rn e s , G .R ., S a h a n i, M ., a n d D o la n , R .J . (2 0 1 2 ). F u n c tio n a l e v id e n c e f o r a d u a l ro u te to a m y g d a la . C u rr. B iol. 22,1 2 9 -1 3 4 . 8. Luo , Q ., H o lro y d , T ., J o n e s , M ., H e n d le r, T ., a n d B lair, J . (2 0 0 7 ). N e u ral

d y n a m ic s f o r fa c ia l th r e a t p ro c e s s in g a s re v e a le d b y g a m m a b an d sy n c h ro n iz a tio n using M E G . N e u ro im a g e 34, 8 3 9 -8 4 7 .

9. V u ille u m ie r, P ., A rm o n y , J .L ., D river, J ., a n d D o la n , R .J . (2 0 0 3 ). D is tin c t s p a tia l fre q u e n c y s e n s itiv itie s f o r p ro c e s s in g fa c e s a n d e m o tio n a l e x p re s s io n s . N a t. N e u ro s c i. 6, 6 2 4 -6 3 1 .

10. D o ro n , N .N ., a n d L e d o u x , J .E . (1 9 9 9 ). O rg a n iz a tio n o f p ro je c tio n s to th e late ra l a m y g d a la fro m a u d ito ry a n d v isu al a re a s o f th e th a la m u s in th e rat. J . C o m p . N e u ro l. 412, 3 8 3 -4 0 9 .

11. L e D o u x , J .E . (1 9 9 6 ). T h e E m o tio n a l B rain (N e w Y o rk : S im o n & Sc h u s te r). 12. D a y -B r o w n , J .D ., W e i, H ., C h o m s u n g , R .D ., P e try , H .M ., a n d B ic k fo rd , M .E . (2 0 1 0 ). P u lv in a r p ro je c tio n s to th e s tria tu m a n d a m y g d a la in th e tre e s h re w . F ro n t N e u ro a n a t 4,143 .

13. C o w e y , A . (2 0 1 0 ). T h e b lin d s ig h t s a g a . E x p . B rain R e s. 200, 3 - 2 4 . 14. P e s s o a , L., a n d A d o lp h s , R . (2 0 1 1 ). E m o tio n p ro c e s s in g a n d th e a m y g ­

da la : fro m a ‘lo w r o a d ’ to ‘ m a n y r o a d s ’ o f e v a lu a tin g b io lo g ic a l s ig n ifi­ c a n c e . N a t. R e v . N e u ro s c i. 11, 7 7 3 -7 8 3 .

15. T a m ie tto , M ., a n d d e G e ld e r, B . (2 0 1 0 ). N e u ral b a s e s o f th e n o n -c o n s c io u s p e rc e p tio n o f e m o tio n a l sig n als. N a t. R ev. N e u ro s c i. 11, 6 9 7 -7 0 9 . 16. d e G e ld e r, B ., M o rris , J .S ., a n d D o la n , R .J . (2 0 0 5 ). U n c o n s c io u s f e a r

in flu e n c e s e m o tio n a l a w a re n e s s o f fa c e s a n d v o ic e s . P ro c . N a tl. A c a d . S c i. U S A 102, 1 8 6 8 2 - 1 8 6 8 7 .

17. Y o , T .S ., A n w a n d e r, A ., D e s c o te a u x , M ., F illard , P ., P o u p o n , C ., and K n ö s c h e , T .R . (2 0 0 9 ). Q u a n tify in g brain c o n n e c tiv ity : a c o m p a ra tiv e tra c to g r a p h y s tu d y . M e d Im a g e C o m p u t C o m p u t A s s is t In te rv 12, 8 8 6 -8 9 3 .

18. G rie v e , K .L ., A c u ñ a , C ., a n d C u d e iro , J . (2 0 0 0 ). T h e p rim a te p u lv in a r nuclei: visio n a n d a c tio n . T re n d s N e u ro s c i. 23, 3 5 - 3 9 .

19. J o n e s , E .G ., a n d B u rto n , H . (1 9 7 6 ). A p ro je c tio n fro m th e m e d ia l p u lv in a r t o th e a m y g d a la in p rim a te s . B rain R e s . 104,1 4 2 -1 4 7 .

2 0 . M o ri, S ., a n d v a n ZijI, P .O . (2 0 0 2 ). F ib e r tra c k in g : p rin c ip le s a n d s tr a te ­ g ie s - a te c h n ic a l re v ie w . N M R B io m e d . 15, 4 6 8 ^ 8 0 .

2 1 . B rid g e , H ., T h o m a s , O ., J b a b d i, S ., a n d C o w e y , A . (2 0 0 8 ). C h a n g e s in c o n n e c tiv ity a fte r v isu al c o rtic a l brain d a m a g e u n d e rlie a lte re d visual fu n c tio n . B rain 131, 1 4 3 3 -1 4 4 4 .

2 2 . Leh , S .E ., J o h a n s e n -B e r g , H ., a n d P tito , A . (2 0 0 6 ). U n c o n s c io u s vision: n e w in s ig h ts into th e n e u ro n a l c o rre la te o f b lin d s ig h t using diffu sio n tra c to g r a p h y . B rain 129,1 8 2 2 -1 8 3 2 .

2 3 . S a h ra ie , A ., W e is k r a n tz , L., B a rb u r, J .L ., S im m o n s , A ., W illia m s , S .C ., a n d B ra m m e r, M .J . (1 9 9 7 ). P a tte rn o f n eu ro n al a c tiv ity a s s o c ia te d w ith c o n s c io u s a n d u n c o n s c io u s p r o c e s s in g o f visual s ig n als. P ro c . N atl. A c a d . S ci. U S A 94, 9 4 0 6 -9 4 1 1 .

2 4 . Lau , H .C ., a n d P a s s in g h a m , R .E . (2 0 0 6 ). R e la tiv e b lin d s ig h t in norm al o b s e rv e rs a n d th e neu ra l c o rre la te o f v is u a l c o n s c io u s n e s s . P ro c . N a tl. A c a d . S ci. U S A 103, 1 8 7 6 3 - 1 8 7 6 8 . 2 5 . S c h m id , M .C ., M ro w k a , S .W ., T u rc h i, J ., S a u n d e rs , R .C ., W ilk e , M ., P e te rs , A .J ., Y e , F .Q ., a n d L e o p o ld , D .A . (2 0 1 0 ). B lin d s ig h t d e p e n d s on th e late ra l g e n ic u la te n u c le u s . N a tu re 4 6 6 , 3 7 3 -3 7 7 . 2 6 . d e G e ld e r, B ., v a n H o n k , J ., a n d T a m ie tto , M . (2 0 1 1 ). E m o tio n in th e b rain: o f lo w ro a d s , high ro a d s a n d ro a d s less tra v e lle d . N a t. R e v. N e u ro s c i. 12, 4 2 5 . 2 7 . W h a le n , P .J ., K a g a n , J ., C o o k , R .G ., D a vis , F .C ., K im , H ., P o lls, S ., M c L a re n , D .G ., S o m e rv ille , L .H ., M c L e a n , A .A ., M a x w e ll, J .S ., a n d J o h n s to n e , T . (2 0 0 4 ). H u m a n a m y g d a la re s p o n s iv ity to m a s k e d fearfu l e y e w h ite s . S c ie n c e 3 0 6 , 2 0 6 1 . 2 8 . V iz u e ta , N ., P a tric k , C .J ., J ia n g , Y ., T h o m a s , K .M ., a n d H e , S. (2 011). D is p o s itio n a l fe a r, n e g a tiv e a ffe c tiv ity , a n d n e u ro im a g in g r e s p o n s e to v is u a lly s u p p re s s e d e m o tio n a l fa c e s . N e u ro im a g e 59, 7 6 1 -7 7 1 . 2 9 . W illia m s , M .A ., M o rris , A .P ., M c G lo n e , F., A b b o tt, D .F ., a n d M a ttin g le y ,

J .B . (2 0 0 4 ). A m y g d a la re s p o n s e s to fe a rfu l a n d h a p p y fa c ia l e x ­ p re s s io n s u n d e r c o n d itio n s o f b in o c u la r s u p p re s s io n . J . N e u ro s c i.

24, 2 8 9 8 -2 9 0 4 .

3 0 . A n d e rs o n , A .K ., C h ris to ff, K., P a n itz, D ., D e R o s a , E ., a n d G a b rie li, J .D . (2 0 0 3 ). N e u ral c o rre la te s o f th e a u to m a tic p ro c e s s in g o f th r e a t fa c ia l s ig n als. J. N e u ro s c i. 2 3, 5 6 2 7 -5 6 3 3 .

3 1 . Lin, C .P ., T s e n g , W .Y ., C h e n g , H .C ., a n d C h e n , J .H . (2 0 0 1 ). V a lid a tio n of d iffu s io n te n s o r m a g n e tic r e s o n a n c e a x o n a l fib e r im a g in g w ith re g is ­ t e r e d m a n g a n e s e -e n h a n c e d o p tic tra c ts . N e u ro im a g e 14,1 0 3 5 -1 0 4 7 . 3 2 . T h ie b a u t d e S c h o tte n , M ., F fy tc h e , D .H ., B izzi, A ., D e ll’A c q u a , F., Allin,

M ., W a ls h e , M ., M u rra y , R ., W illia m s , S .C ., M u rp h y , D .G ., a n d C a ta n i, M . (2 0 1 1 ). A tla s in g lo c a tio n , a s y m m e tr y a n d in te r-s u b je c t v a ria b ility of w h ite m a tte r tra c ts in th e h u m a n brain w ith M R d iffu s io n tra c to g ra p h y . N e u ro im a g e 54, 4 9 - 5 9 .

3 3 . S tie ltje s , B., K a u fm a n n , W .E ., v a n ZijI, P .O ., F re d e ric k s e n , K., P e a rls o n , G .D ., S o la iy a p p a n , M ., a n d M o ri, S. (2 0 0 1 ). D iffu s io n te n s o r im a g in g a n d a x o n a l tra c k in g in th e h u m a n b ra in s te m . N e u ro im a g e 14, 7 2 3 -7 3 5 . 3 4 . L a z a r, M . (2 0 1 0 ). M a p p in g brain a n a to m ic a l c o n n e c tiv ity usin g w h ite

m a tte r tra c to g r a p h y . N M R B io m e d . 23, 8 2 1 -8 3 5 .

3 5 . J o n e s , D .K . (2 0 0 8 ). S tu d y in g c o n n e c tio n s in th e living h u m a n brain w ith d iffu s io n M R I. C o rte x 44, 9 3 6 -9 5 2 .

3 6 . d e G e ld e r, B., V ro o m e n , J ., P o u rto is , G ., a n d W e is k ra n tz , L. (1 9 9 9 ). N o n - c o n s c io u s re c o g n itio n o f a ffe c t in th e a b s e n c e o f s tria te c o rte x . N e u ro re p o rt 10, 3 7 5 9 -3 7 6 3 .

3 7 . T a m ie tto , M ., C a s te lli, L., V ig h e tti, S ., P e ro z z o , P ., G e m in ia n i, G ., W e is k r a n tz , L., a n d d e G e ld e r, B. (2 0 0 9 ). U n s e e n fa c ia l a n d b o d ily e x p re s s io n s trig g e r fa s t e m o tio n a l re a c tio n s . P ro c . N a tl. A c a d . S ci. U S A 7 0 6 , 1 7 6 6 1 - 1 7 6 6 6 .

3 8 . G o e b e l, R ., M u c k li, L., Z a n e lla , F.E ., S in g er, W ., a n d S to e rig , P . (2 001). S u s ta in e d e x tra s tria te c o rtic a l a c tiv a tio n w ith o u t v isu al a w a re n e s s re­ v e a le d b y fM R I s tu d ie s o f h e m ia n o p ic p a tie n ts . V isio n R e s . 41, 1 4 5 9 - 1 4 7 4 . 3 9 . T a m ie tto , M ., C a u d a , F., C o ra z z in i, L.L., S a v a z z i, S ., M a rz i, C .A ., G o e b e l, R ., W e is k r a n tz , L., a n d d e G e ld e r, B . (2 0 1 0 ). C o llic u la r visio n g u id e s n o n c o n s c io u s b e h a v io r. J . C o g n . N e u ro s c i. 22, 8 8 8 -9 0 2 . 4 0 . H o llid a y , I.E ., A n d e rs o n , S .J ., a n d H a rd in g , G .F . (1 997). M a g n e to e n c e p h a lo g r a p h ic e v id e n c e fo r n o n -g e n ic u lo s tria te visual in p u t to h u m a n c o rtic a l a re a V 5 . N e u ro p s y c h o lo g ia 35,1 1 3 9 -1 1 4 6 . 4 1 . B a s s e r, P .J ., M a ttie llo , J ., a n d L e B ih a n , D. (1 9 9 4 ). M R d iffu s io n te n s o r

s p e c tr o s c o p y a n d im a g in g . B io p h y s . J . 6 6 , 2 5 9 -2 6 7 .

4 2 . W e s tin , C .F ., M a ie r, S .E ., M a m a ta , H ., N a b a v i, A ., J o le s z , F .A ., a n d K ikinis, R. (2 0 0 2 ). P ro c e s s in g a n d v is u a liz a tio n fo r d iffu s io n te n s o r M R I. M e d . Im a g e A n a l. 6, 9 3 - 1 0 8 .