A Disynaptic Relay from Superior Colliculus to Dorsal Stream Visual Cortex in Macaque Monkey

Neuron

Article A Disynaptic Relay from Superior Colliculus to Dorsal Stream Visual Cortex in Macaque Monkey David C. Lyon,1,2 Jonathan J. Nassi,1,3 and Edward M. Callaway1,* 1Systems

Neurobiology Laboratories, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA address: Department of Anatomy & Neurobiology, School of Medicine, University of California, Irvine, 364 Med Surge II, Irvine, CA 92697, USA 3Present address: Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA *Correspondence: [email protected] DOI 10.1016/j.neuron.2010.01.003 2Present

SUMMARY

The superior colliculus (SC) is the first station in a subcortical relay of retinal information to extrastriate visual cortex. Ascending SC projections pass through pulvinar and LGN on their way to cortex, but it is not clear how many synapses are required to complete these circuits or which cortical areas are involved. To examine this relay directly, we injected transynaptic rabies virus into several extrastriate visual areas. We observed disynaptically labeled cells in superficial, retino-recipient SC layers from injections in dorsal stream areas MT and V3, but not the earliest extrastriate area, V2, nor ventral stream area V4. This robust SC-dorsal stream pathway is most likely relayed through the inferior pulvinar and can provide magnocellular-like sensory inputs necessary for motion perception and the computation of orienting movements. Furthermore, by circumventing primary visual cortex, this pathway may also underlie the remaining visual capacities associated with blindsight.

INTRODUCTION The superior colliculus (SC), a major subcortical nucleus of the primate visual system, not only integrates top-down inputs from higher visual areas important for the planning and execution of eye movements (Wurtz and Albano, 1980; Schiller, 1984; Sparks, 1986; McPeek and Keller, 2004), it also transmits feedforward sensory information from the retina (Schiller and Malpeli, 1977; Perry and Cowey, 1984; Crook et al., 2008) to these same higher visual areas (see Kaas and Huerta, 1988; Shipp, 2004). Though extrastriate cortical areas project directly to the superficial layers of the SC where retinal projections also terminate (Fries, 1984; Kaas and Huerta, 1988; Lock et al., 2003; Shipp, 2004; Collins et al., 2005), SC does not project directly to cortex. Instead, SC projections are relayed through the pulvinar and, to a lesser extent, through the lateral geniculate nucleus (LGN; Harting et al., 1978; Benevento and Standage, 1983; Huerta and Harting, 1983; Stepniewska et al., 1999, 2000).

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Determining the nature of SC projections relayed to cortex is crucial to our understanding of the visual behaviors and percepts in which the SC plays a role. Feedforward retinal projections through SC to cortex involve parasol cells in the retina (Schiller and Malpeli, 1977; Perry and Cowey, 1984; Crook et al., 2008) and magnocellular-like cells in the SC (Schiller and Malpeli, 1977; Kaas and Huerta, 1988; Crook et al., 2008), implicating this pathway in the fast transmission of low contrast, quickly moving stimuli to dorsal stream visual cortex. Nevertheless, the speed with which this information arrives into cortex via the SC remains unclear as it is not known how many synapses in the thalamus are required to complete this circuit or whether ascending circuits may be more direct depending on the targeted cortical area. It has also been suggested that the phenomenon of blindsight, which exhibits some of these same visual properties, might be mediated, at least in part, by SC input to dorsal stream visual cortex, particularly area MT, via the pulvinar (Rodman et al., 1990; Stoerig and Cowey, 2007; Tamietto et al., 2010). However, indirect anatomical investigations have suggested that area MT does not receive major inputs from the SC through pulvinar (Stepniewska et al., 1999, 2000). Conversely, the available anatomical evidence suggests that areas other than MT, some of which are in the ventral stream do receive inputs from SC through the pulvinar (see Kaas and Lyon, 2007). To more adequately examine the multisynaptic SC projections to cortex anatomically we used rabies virus which acts as a timedependent transynaptic retrograde tracer (see Ugolini, 1995; Kelly and Strick, 2000, 2003; Nassi et al., 2006). By restricting the post injection survival time to 3 days we allowed retrograde transport to travel a maximum of two synapses. In doing so we were able to directly determine whether injections into several cortical visual areas, V2, V3, V4, and MT, result in retrograde disynaptic label of SC neurons through an intermediate thalamic nucleus, either the pulvinar or LGN. Furthermore, we addressed whether disynaptic inputs from the SC preferentially target either dorsal or ventral stream areas, by injecting dorsal stream areas MT and V3, and ventral stream area V4. We found disynaptically labeled neurons in the superficial layers of SC following rabies virus injections into MT and V3, but not areas V2 or V4. This finding shows for the first time that not only do higher order dorsal stream areas in fact receive disynaptic input from the SC, but early extrastriate area V2 and ventral stream area V4 do not. Through further anatomical analysis we found that this disynaptic pathway from SC to MT and V3

Neuron Superior Colliculus Cortical Relay

is retinotopically organized and predominantly relayed through the inferior pulvinar rather than the LGN. We suggest that this SC-dorsal stream pathway represents a direct, fast circuit for the relay of magno-like sensory input that can be used for early motion detection and the early stages of other functions shared by the dorsal stream and SC such as the planning of orienting movements (Galletti and Battaglini, 1989; Krauzlis, 2004; McPeek and Keller, 2004; Ungerleider and Pasternak, 2004; Campos et al., 2006). Furthermore, these observations provide evidence for a circuit that can relay visual information to dorsal stream visual areas independent of primary visual cortex and thus could mediate some aspects of blindsight.

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In separate cortical hemispheres, we made injections of the CVS-11 strain of rabies virus into extrastriate visual areas V2, V3, V4, and MT and allowed a 72 hr survival period for disynaptic retrograde transport (see Experimental Procedures for details). Large numbers of rabies labeled neurons were found in the stratum griseum superficiale (SGS) of the SC following virus injections into MT (two hemispheres; Figures 1–3) and V3 (two hemispheres; Figures 2 and 4), but no labeling was observed in the SC after injections into V2 (five hemispheres; Figures 2 and 3) and V4 (three hemispheres; Figures 2 and 5). These disynaptic connection patterns, supported by further analyses described below, reveal that only extrastriate cortical areas associated primarily with the dorsal stream receive strong disynaptic projections from the SC. Images in Figure 1 clearly show labeled cells stained for the rabies nucleocapsid protein in the superficial layers of the SC following MT injections. The distribution of rabies labeled cells across the full extent of the SC from the same case is shown in reconstructions in Figure 3A (right side). Because SC does not project directly to cortex, these cells must have been labeled transynaptically. The 72 hr survival time insures that the rabies virus could have only transported across one additional synapse (see Ugolini, 1995; Kelly and Strick, 2000, 2003; Nassi et al., 2006); thus, the observed cells in SC were labeled disynaptically. Disynaptically labeled cells in the SC were found following MT injections in a second case as well (Figure 3F–3H). For both MT cases the interpolated number of labeled cells (see Experimental Procedures) in the SC was fairly high, totaling 1074 cells in one case (JNM1) and 864 cells in the other (JNM10-R; Figure 2A). Furthermore, in both cases thousands of disynaptically labeled cells were found in the thalamic reticular nucleus (TRN; Figure 2B; for examples see section 716 in Figure 3 and Figure 6D). The TRN is a structure that does not project directly to cortex but provides direct inhibitory inputs to nearby thalamic nuclei such as the pulvinar and LGN (see Kaas and Huerta, 1988; Pinault, 2004; McAlonan et al., 2008). For our purposes, the TRN serves as a good internal control for evaluating the effectiveness of tracer uptake at our injection sites and the quality of disynaptic transport. In addition to injections in MT, injections in V3 also labeled large numbers of cells in the SC (Figure 4). Multiple V3 injections were made in both hemispheres of one monkey (JNM4; for details see Experimental Procedures). The number of labeled

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Figure 1. Retrogradely Labeled Neurons in the Superior Colliculus following Injections of Rabies Virus in Cortical Area MT Digitally photographed images of a single section of the superior colliculus (SC) stained for the nuleocapsid protein of the rabies virus and for cytochrome oxidase (CO). (A) Darker CO staining distinguishes the upper layer 2a from layer 2b. Layer 3 lies just below layer 2a and is slightly darker for CO. The region inside the hatched box in (A) is shown at higher magnification in (B). (B) Neurons infected by the rabies virus (black) are present in both subdivisions of layer 2. (C) At higher magnification, it is evident that more neurons are present in layer 2b, and this is supported by our detailed reconstructions (Figures 3 and 4; also see Figure 2B). Scale bars, 250 mm.

cells from V3 injections in the left hemisphere (678 cells) was similar to those found for both MT cases (see Figure 2A). The V3 injections in the right hemisphere, however, resulted in only sparse SC label. Importantly, mono and disynaptically labeled cells in the pulvinar and TRN from this case, though still substantial, were fewer in number, indicating that the injections in the right hemisphere may have been located in a module of V3

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Figure 2. Comparison of Disynaptic SC Projections to Different Cortical Areas (A) Large numbers of neurons in the SC were labeled following injections into two MT hemispheres (yellow) and one V3 hemisphere (red). A very small number of neurons were found in a second V3 case (second red bar) and two V4 cases (blue). In five separate V2 cases and one other V4 case, no rabies-infected neurons were found in the SC. (B) In every case (indicated by white circles), a thousand or more neurons were labeled in the thalamic reticular nucleus (TRN), confirming that disynaptic transport was not compromised. Like the SC, cells in the TRN do not project directly to cortex, and could only have been labeled disynaptically through adjacent thalamic nuclei such as the pulvinar and LGN. (A and B) For all cases, the total numbers of cells found in every sixth section were multiplied by 6 in order to interpolate the number of cells expected to be present in the entire SC and the region of TRN covering the pulvinar. (C) Greater than 80% of the SC labeled cells following the two MT (yellow) and two V3 (red) injections were in layer 2b. Laminar positions of rabies labeled cells in the SC were determined based on CO stains (see Figure 1) and averaged across the two MT and two V3 cases. (D) Comparison of the retinotopic pattern of connections in V1 and SC following injections into MT (JNM1, orange; JNM10, yellow) and V3 (JNM4L, red). The estimated retinotopic pattern of rabies labeled cells in the SC (right) is similar to the pattern observed in V1 (left), indicating that the disynaptic SCdorsal stream relay is tightly organized. (Left) The images from a representative brain show a medial view of the calcarine sulcus that is partially unfolded (far left) and a dorsal view of the posterior end of the cortex (middle). Vertical

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that receives less dense inputs from the pulvinar or, alternatively, that rabies virus transport was simply less effective (see Figure 2B for quantification of TRN label; for examples see right side of thalamic sections in Figure 4). Our rabies injections into MT and V3 clearly establish a disynaptic projection from SC (see summary diagram in Figure 7). However, despite indirect evidence in earlier reports that V2 and V4 are likely to receive SC relays through the pulvinar (Stepniewska et al., 1999, 2000; see Kaas and Lyon, 2007) our rabies injections in these cortical areas give little to no indication that such a pathway exists in disynaptic form. For example, in a total of five hemispheres we made over 30 separate rabies virus injections into V2 with none resulting in any labeled SC cells (see Figure 2A). Furthermore, as shown in Figure 3A, though rabies labeled cells were present in the right SC following multiple injections into MT of the right hemisphere, no labeled cells were present in the left SC following multiple injections into V2 of the left hemisphere. Importantly, using label in the TRN (see inset in Figure 3A) as an internal control for evaluating the effectiveness of tracer uptake at our injection sites and subsequent disynaptic transport, we find for all V2 injection cases that the number of TRN labeled neurons is quite comparable to TRN label following our V3 injections (see Figure 2B). Examples of the amount of TRN label following V2 injections in one case (JNM10-L) are shown on the left side of sections 740 and 716 (Figure 3A). Figure 3I shows additional examples of the amount of disynaptic TRN label following V2 injections in both hemispheres of a second case (JNM13). Overall, 3000–5000 cells were labeled in each of these three hemispheres (Figure 2B). As another indication that our injection sites were effective, in all V2 injected hemispheres, large numbers of monosynaptic and, likely, disynaptically labeled cells were found in the pulvinar (Figure 3A, left side of sections 740 and 716; Figure 3I). Motivated by the results described above, we made injections into V4 in order to determine if the existence of disynaptic input from SC to a given cortical area depends mainly on the area’s level in the visual cortical hierarchy, or on its relationship to dorsal and ventral stream visual cortex. Our results indicate the latter to be true, as multiple rabies injections in ventral stream area V4 did not yield labeled SC cells in two cases (Figure 5A and Figure 5E, right hemisphere) and only very sparse labeling in the SC of a third case (Figure 5E, left hemisphere; see also Figure 2A). The presence of a handful of labeled cells (eight cells in two coronal SC sections separated by 300 mm) in one of three V4 cases may indicate that one of our injection sites included a V4 (squares) and horizontal (circles) meridians are marked. Azimuths 1 , 5 , 10 , 20 , and 40 are shown, along with iso-azimuth contours (black lines). Upper (+) and lower ( ) visual fields are indicated. The V1 retinotopic summary has been adapted from Lyon and Kaas (2002) and is an extension of data shown previously in Nassi et al. (2006). It is based on retinotopic maps from Van Essen et al. (1984). V1 (primary visual cortex), IPS (intraparietal sulcus), STS (superior temporal sulcus), LuS (lunate sulcus), CalS (calcarine sulcus), POS (parietal occipital sulcus), OTS (occipital temporal sulcus). Scale bar, 5 mm. (Right) Summary of the retinotopic locations of rabies-labeled cells from the MT and V3 injections shown on a flattened dorsal view of the right superior colliculus. The SC retinotopic organization is based on Cynader and Berman (1972). Scale bar, 1 mm. Anterior, A; medial, M; posterior, P; lateral, L.

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Figure 3. Disynaptically Labeled Neurons in the SC following MT Injections, but Not V2 Injections (A) Reconstruction of the distribution of rabies labeled neurons (red dots) in several coronal sections of the SC and thalamus in case JNM10. The left side of each section shows rabies label resulting from multiple V2 injections in the left hemisphere, whereas the right side of each section shows labeled neurons resulting from MT injections in the right hemisphere. Sections are arranged from posterior (770) to anterior (716). The thin blue line in the SC designates the layer 2a-layer 2b border determined using CO stains (see Figure 1). The borders for the medial and caudal-lateral subdivisions of the inferior pulvinar (PIm and PIcl) and the ventral lateral subdivision of the lateral pulvinar (PLvl) were determined using CO architecture (bottom). Inset image shows a cluster of labeled neurons in the left thalamic reticular nucleus (TRN) of section 740. Medial pulvinar, PM; dorsomedial lateral pulvinar, PLdm. (B) Location of 1 V2 injection site (black oval) and distribution of locally labeled neurons (red dots) in the left hemisphere of JNM10, drawn from the image in (C). The blue line indicates layer 4 of V1. (C) A parasagittal section including a V2 injection site (indicated by black line) in the left hemisphere of JNM10. The section was stained for CO (orange) and the rabies nucleocapsid protein (black). The V1/V2 border is clearly visible about 1.5 mm posterior to the injection. Scale bar for (B) and (C), 1 mm. (D) Location of an MT injection and distribution of locally labeled neurons on the caudal bank of the superior temporal sulcus (STS) in the right hemisphere of JNM10, MT borders (blue lines) are indicated. (E) CO and rabies nucleocapsid stained coronal section used for the drawing in (D). Darker CO staining in the deeper layers was used to define MT borders. Scale bar for (D) and (E), 1 mm. (F) Reconstruction of the distribution of rabies labeled neurons in several parasagittal sections of the SC in case JNM1 (conventions as in A). Sections are arranged from medial (616) to lateral (596). (G) A parasagittal section through the STS showing the MT injection site in JNM1. The section was processed for the rabies nucleocapsid protein and shows a dense pattern of labeled cells (black) extending ventrally from the injection site. (H) An adjacent JNM1 section stained for myelin clearly shows that the injection site (also shown in G) was confined within the borders of MT. (I) Examples of the distribution of rabies labeled neurons in the pulvinar and TRN following bilateral V2 injections in case JNM13. Dorsal, D. Other conventions as in Figure 2.

compartment that receives sparse disynaptic projections from the SC, as compartmentalization within V4 has been well documented anatomically (DeYoe et al., 1994; Felleman et al., 1997; Xiao et al., 1999). Nevertheless, the number of labeled cells is

relatively weak compared to the large number following MT and V3 injections (see Figure 2A). In addition, as illustrated in Figures 5A and 5E, for all three V4 cases including the one with sparse SC label there was comparatively dense label in the

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Figure 4. Disynaptically Labeled Neurons in the SC following V3 Injections (Top) Reconstruction of the distribution of rabies labeled neurons in several coronal sections of the SC and a single section including the pulvinar and TRN in case JNM4. The left and right side of each section shows the positions of labeled neurons resulting from rabies injections into V3 of the left and right hemispheres, respectively. Sections are arranged from posterior (15) to anterior (69). (Bottom left) A drawing of the distribution of rabies labeled neurons resulting from an injection into the annectent gyrus (black line) approximately 1.5 mm anterior to the outer V2 border. (Bottom right) The inner and outer borders of V2 (red lines) were determined using myelin stains of adjacent sections. Lateral shell of the inferior pulvinar, PIL. Other conventions as in Figure 3.

TRN (Figure 2B) indicating that though only a handful of cells were found in SC of one case, for all three V4 hemispheres injections were effective and the disynaptic transport of the virus was not compromised. In addition, as seen following our V2 injections, monosynaptic and likely disynaptic transport to the pulvinar following these V4 injections was also quite dense (Figures 5A and 5E). Moreover, as shown in Figure 5D local monosynaptic and disynaptic connectivity near a V4 injection site just ventral to the tip of the inferior occipital sulcus (Figure 5B) was very robust. By comparison, locally labeled neurons following injections in V3 (Figure 4) and MT (Figure 3D) were much less numerous, but nevertheless labeled many neurons in the SC. With a disynaptic connection from the SC to dorsal stream cortical areas V3 and MT firmly established, we sought to

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Figure 5. Rabies Labeled Neurons following V4 Injections (A) Reconstruction of the distribution of rabies labeled neurons in three coronal sections of the thalamus in the left hemisphere of DLM1. (B) The distribution of labeled cells in A resulted from two injections placed just anterior to the lunate sulcus (LuS) and just ventral to the inferior occipital sulcus (IOS). Scale bar, 1 cm. (C) A coronal section through the more dorsal injection (indicated by a blue line in B) stained for CO and the rabies nucleocapsid protein. (D) Drawing of the local distribution of rabies labeled cells resulting from the injection shown in (C). Scale bar for (C) and (D), 1 mm. (E) Reconstruction of the distribution of rabies labeled neurons in the SC and thalamus in case DLM2. (Inset) The image shows a cluster of labeled neurons in the TRN of the right hemisphere of section 516. (Bottom) The locations of the V4 injections sites on the left and right hemispheres are indicated. Scale bar, 1 cm.

Neuron Superior Colliculus Cortical Relay

determine whether the LGN or pulvinar most likely serves as the relay of this circuit. To do this, we determined the laminar location of disynaptically labeled cells in the SC (see Experimental Procedures). Existing evidence indicates that layer 2a of the SGS projects to interlaminar zones (i.e., intercalated or koniocellular layers) of the LGN, whereas layer 2b projects predominantly to the inferior pulvinar (Harting et al., 1978; Raczkowski and Diamond, 1978; Benevento and Standage, 1983; Huerta and Harting, 1983; also see Berson and McIlwain, 1982; Berson, 1987). As shown at low magnification in Figure 1A, layer 2 of the SGS can be subdivided into upper (layer 2a) and lower (layer 2b) zones based on higher levels of CO in layer 2a (see Kaas and Huerta, 1988). At greater magnifications (Figures 1B and 1C), rabies labeled neurons from MT injections are visible in both layers 2a and 2b. However, detailed reconstructions of cell body locations across a broader extent of the SC in the same MT case (JNM10; Figure 3A), a second MT case (JNM1; Figure 3F), and two V3 cases (Figure 4) showed that over 80% of the cells were located in lower layer 2b (see Figure 2B). Because the majority of our disynaptically labeled cells in the SC are in layer 2b, and layer 2b cells project predominantly to the inferior pulvinar (Benevento and Standage, 1983; Huerta and Harting, 1983), we conclude that SC projections to V3 and MT are primarily through the inferior pulvinar (see Figure 7). Since the inferior pulvinar (PI) can be subdivided into as many as five subregions (see Figure 7; Cusick et al., 1993; Gutierrez et al., 1995; Stepniewska and Kaas, 1997; Adams et al., 2000; Soares et al., 2001; Shipp, 2001; Kaas and Lyon, 2007; Lyon, 2007) we next sought to determine which of the PI subregions provide the relay to areas MT and V3. To do so, we plotted the locations of rabies labeled cells in the inferior pulvinar from all cortical injections and compared which inferior pulvinar subdivisions were labeled by the V3 and MT injections, but not the V2 and V4 injections (see Figures 3–6). Our results confirm published reports using monosynaptic retrograde tracers (Cusick et al., 1993; Stepniewska et al., 1999; Kaas and Lyon, 2007; also see Shipp, 2001) that two subdivisions of the inferior pulvinar, the medial (PIm) and the lateral shell (PIL) both project to MT and V3, but not to V2 and V4 (see Figure 7). For example, rabies labeled cells following MT (Figure 3, section 716 right side) and V3 (Figure 4, section 69 right side) injections were quite numerous in PIm, whereas label from V2 (Figure 3, section 716 left side) and V4 (Figure 5A, section 691; Figure 5E sections 516 and 528) injections were either very sparse or not present in PIm. In addition, Figure 6 shows that the strip of inferior pulvinar immediately adjacent to the posterior extent of the LGN, identified as PIL, is also labeled by MT (Figure 6D) and V3 (Figure 6C) injections but not from injections in V2 (Figure 5A) and V4 (Figure 6B). Thus, our results indicate two possible subdivisions in the pulvinar, PIm and PIL, that are both likely to provide the SC relay to dorsal stream areas MT and V3 (see summary diagram in Figure 7). DISCUSSION Using rabies virus as an anatomical method for tracing transynaptic connections, we have shown that higher-order dorsal stream areas of the visual cortex receive disynaptic projections

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Figure 6. Comparison of Label in the Lateral Shell Subdivision of the Inferior Pulvinar (PIL) following Rabies Injections into Four Different Visual Areas PIL was identified as a narrow strip of pulvinar just medial and posterior to the LGN. Locations of rabies labeled neurons in (A) a coronal section through the pulvinar in a V2 injected hemisphere (JNM10L); (B) a coronal section through the pulvinar in a V4 injected hemisphere (DLM2L); (C) a coronal section through the pulvinar in a V3 injected hemisphere (JNM4L); (D) a parasagittal section through the pulvinar in an MT injected hemisphere (JNM1). Thin blue lines outline lamina of the LGN. Other conventions as in Figure 3.

from the SC. Specifically, we found that areas V3 and MT can receive robust disynaptic projections. In contrast, the SC provides weak or no disynaptic projections to ventral stream area V4 and early extrastriate area V2. Thus, the robust disynaptic inputs from the SC to extrastriate visual cortex are exclusive to the dorsal stream. Our results are surprising because earlier reports using less direct tracing methods found little evidence for an inferior pulvinar relay from SC to area MT (Stepniewska et al., 1999, 2000). Those studies made injections of anterograde tracers in SC and retrograde tracers in MT. Although none of the tracers used could cross potential synapses in the pulvinar and directly demonstrate a relay between SC and cortex, it was thought that spatial overlap of labeled axon terminals and cell bodies in the pulvinar from both injection sites could be used as a good indicator of any such circuit. They found that SC projections were predominantly to inferior pulvinar nuclei that project to areas V2 (PIcl) and V4 (PIcl and PIcm), as well as to several dorsal stream visual areas other than MT (see Kaas and Lyon, 2007), with little to no labeled axon terminals found in PIm, the main inferior pulvinar nucleus projecting to MT. These results suggested that instead of area MT receiving disynaptic inputs from SC, areas V2 and V4 were the more likely candidates. However, by directly tracing the disynaptic projections to cortex we have shown the opposite of what was expected, MT (and V3) receives

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Figure 7. Potential Pathways for Disynaptic Projections from the Superior Colliculus to MT and V3 Red arrows indicate the potential source for disynaptic projections from layer 2b of the superior colliculus to inferior pulvinar subdivisions, PIm and PIL, and then on to dorsal stream cortical areas, MT and V3. The thicker red arrow from PIm to MT reflects denser projections that we and others (Kaas and Lyon, 2007) have observed compared to projections from PIm to V3 and PIL to V3 and MT. Gray arrows from PIcl and PLvl represent additional pathways from pulvinar to extrastriate visual cortex, however these pathways are not likely to be the source of disynaptic projections from the SC to MT and V3. While we did observe substantial projections from PLvl to MT and V3, PLvl was also substantially labeled by V2 and V4 injections (thick gray arrows) that did not result in SC label. Furthermore, while we observed strong PIcl projections to V4, we found little evidence for PIcl projections to areas MT and V3. The summary of pulvinar subdivisions has been adapted from Lyon et al. (2003).

disynaptic SC inputs whereas areas V2 and V4 do not (see summary diagram in Figure 7). One reason why we may have failed to detect disynaptic inputs to V2 and V4 is that they might be relatively weak (i.e., involving fewer synaptic contacts which results in infection by fewer virions; see Ugolini, 1995) so that the 3 day survival period was not sufficient for detectable levels of rabies virus infection and transport to SC cells. Although the 3 day survival period that was used guarantees that all labeled cells must be either directly or disynaptically infected (trisynaptic label is avoided), it may not always assure labeling of weaker disynaptic connections (see Ugolini, 1995; Kelly and Strick, 2000, 2003). The possibility of rabies virus transporting more slowly across weaker

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connections is a limitation of the rabies tracing technique. Nevertheless, our results indicate that, even if disynaptic projections from SC to areas V2 and V4 are present, they are far less prominent than projections to MT and V3. Because the disynaptic projections to MT (and V3) were so robust it is unclear why these projections were not linked to PIm through less direct tracing studies. One possibility is that the SC projections to MT and V3 are not relayed through PIm, a major projection zone to MT and V3 (see Cusick et al., 1993; Adams et al., 2000; Shipp, 2001; Kaas and Lyon, 2007) but through a second inferior pulvinar subdivision, PIL (see Figures 6 and 7). In support of this, data from earlier reports show that regions corresponding to the location of PIL do project to MT (Cusick et al., 1993; Kaas and Lyon, 2007) and V3 (Shipp, 2001). Importantly, the PIL region also receives projections from SC (Stepniewska et al., 1999). In support of PIL projecting exclusively to dorsal stream areas, we have shown here that PIL is labeled from our rabies injections in MT and V3, but not V2 and V4 (Figure 6). However, it is worth noting that label in PIL from our MT and V3 injections is not as dense as it is in PIm, consistent with other studies using standard retrograde tracers (see Kaas and Lyon, 2007). As diagramed in Figure 7, while PIL could be a relay station for SC to areas V3 and MT, the existing evidence does not adequately rule out PIm. For example, in the Stepniewska et al. (1999, 2000) studies, while the majority of labeled axon terminals from an anterograde tracer injection in SC were outside of PIm, in some cases there was evidence of sparse labeling within PIm. While such sparse connections might appear too weak to give rise to the robust disynaptic label we observed, it is possible that the actual density of the projection to PIm is even higher than estimated. For example, the lack of reported axon terminations in PIm might arise from the fact that the brachium of the SC sends axons through a relatively large portion of PIm (see Figures 3–5); while many of these axons were labeled with anterograde tracer in studies that injected the SC (Stepniewska et al., 1999, 2000), they were not considered termination zones because they were considered to be within the brachium fiber tract. Because the transynaptic spread of rabies virus requires synaptic contacts, our results suggest that these axons might actually form synapses with PIm neurons. The functional significance of this relatively direct SC-cortical pathway could be fast transmission of feedforward retinal inputs directly to higher order dorsal stream areas. This pathway likely originates from parasol ganglion cells in the retina (Schiller and Malpeli, 1977; Perry and Cowey, 1984; Kaas and Huerta, 1988; Crook et al., 2008; also see Berson and McIlwain, 1982) and is relayed by magnocellular-like cells in layer 2b of the SC (see Kaas and Huerta, 1988), suggesting that the information mediated by this pathway may be particularly sensitive to lowcontrast, quickly moving stimuli and transmitted with high conduction velocities. Parasol ganglion cell projections can also be relayed to dorsal stream cortical areas through magnocells in the LGN that project first to V1 then to dorsal stream cortical areas (Hendrickson et al., 1978; Fitzpatrick et al., 1985; Burkhalter et al., 1986; Shipp and Zeki, 1989; Merigan and Maunsell, 1993; Yabuta et al., 2001; Sincich and Horton, 2003; Nassi and Callaway, 2006; Nassi et al., 2006). In contrast to

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this more traditionally described pathway, the pathway through the SC and pulvinar does not involve the LGN or V1, and may forgo some of the perceptual aspects of visual processing in V1 to instead emphasize fast transfer of information for early detection of motion and the early planning of orienting eye gaze, features shared by the SC and dorsal stream cortex (Galletti and Battaglini, 1989; Krauzlis, 2004; McPeek and Keller, 2004; Ungerleider and Pasternak, 2004; Campos et al., 2006). In addition, bypassing V1 suggests that this pathway is likely to be responsible for carrying visual information that underlies at least some aspects of blindsight, particularly those aspects that appear to be associated with dorsal stream visual areas (Rodman et al., 1990; Stoerig and Cowey, 2007). Finally, if these pathways are indeed the substrate that mediates blindsight, this suggests that they are not able to independently mediate conscious visual awareness. This is consistent with the hypothesis that visual perception, ‘‘the conscious experience of seeing’’ (Milner and Goodale, 2008), is mediated by the ventral stream and not the dorsal stream (Goodale and Milner, 1992).

either just ventral (DLM1; Figure 5B) or dorsal to the inferior occipital sulcus (DLM2; see bottom left and right of Figure 5E). These locations for V4 were deemed as the most reliable based on agreement from several publications (Gattass et al., 1988; Nakamura et al., 1993; Felleman et al., 1997; Stepniewska et al., 2005). Two glass microelectrode (tip diameter, 30 mm) penetrations spaced mediolaterally 3 mm were made in each hemisphere and 0.3 ml of virus was injected at three depths spaced 0.5mm (0.4, 0.8, and 1.2 mm deep) using a Picospritzer. Rabies Virus Strain and Speed of Transport A 3 day survival time following injections of the CVS-11 strain of rabies virus into cortical areas V2, V3, V4, and MT allowed for disynaptic retrograde transport. As we have noted in previous publications (Nassi et al., 2006; Nassi and Callaway, 2006), different preparations of rabies virus, including those of the same strain, can spread through the nervous system at different rates, mainly dependent on the passage history of the virus (Morimoto et al., 1998; Kelly and Strick, 2000). The particular preparation of the CVS-11 rabies virus that we used was obtained from Dr. Donald Lodmell, Rocky Mountain Laboratory (Hamilton, MT), and was passaged four times in mouse brain, followed by five times in cultured chicken embryo-related cells. Based on similarities of our own control experiments (Nassi et al., 2006) and published results of others (Kelly and Strick, 2003), injecting the same strain of CVS-11 into motor cortex (M1) of the macaque monkey we confirmed that the virus we injected into MT, V4, V3, and V2 would travel only disynaptically within the 3 days.

EXPERIMENTAL PROCEDURES Surgical Procedures Seven adult male macaque monkeys (JNM1, JNM4, JNM7, JNM10, JNM13, DLM1, DLM2) were used, following procedures approved by the Salk Institute Animal Care and Use Committee. In addition, all procedures using rabies virus were conducted using biosafety level 2 precautions as described elsewhere (Kelly and Strick, 2000). As described elsewhere (Nassi and Callaway, 2006; Nassi et al., 2006), stereotaxic coordinates for the posterior bank of the superior temporal sulcus (STS) and the anectant gyrus were identified using a 1.5 Tesla Siemens Symphony MR scanner (UCSD Hillcrest Medical Center/Tenet Magnetic Resonance Institute) to guide injections into MT and V3, respectively. Two hemispheres were targeted for MT injections, JNM1 and JNM10R (the right hemisphere of JNM10). MT injections were made using Hamilton syringes with a 30 gauge needle. In monkey JNM1 three penetrations were aimed at the posterior bank of the STS. Approximately 0.4 ml of the challenge virus strain-11 (CVS-11) strain of rabies virus (see below for rabies virus strain and speed of transport; also see Nassi et al., 2006) was injected at each of four depths spaced 0.5 mm apart (0.5, 1.0, 1.5, and 2.0 mm deep). In monkey JNM10R, two penetrations were made into the posterior STS, and 0.4 ml of virus was injected at five equally spaced depths extending to a depth of 2 mm. See below for histological verification of injection sites. Bilateral V3 injections were made in monkey JNM4 using Hamilton syringes with a 30 gauge needle. In each hemisphere, two penetrations were aimed at the annectent gyrus, spaced 1 mm mediolaterally. Approximately 0.3 ml of virus was injected at each of three depths spaced by 0.5 mm (0.4, 0.8, and 1.2 mm deep). See below for verification of injection sites. V2 was targeted in five hemispheres of three monkeys, JNM10L (left hemisphere of JNM10) and bilaterally in JNM7 and JNM13. A total of 35 V2 locations were injected by using glass micropipettes (tip diameter 30 mm) and pressure applied by a Picospritzer. In monkey JNM10L, seven penetrations were made along the opercular surface, perpendicular to the cortical surface, just posterior to the lip of the lunate sulcus. In monkey JNM7, four penetrations in a similar location were made in each hemisphere. In all three hemispheres, penetrations were separated by 5 mm mediolaterally. Approximately 0.3 ml of virus was injected at each of three depths spaced 0.5 mm (0.4, 0.8, and 1.2 mm deep). In each hemisphere of monkey JNM13 ten penetrations were made similarly as in JNM10L, except with mediolateral separation between penetrations of 2 mm. V4 was injected in three hemispheres of two monkeys, DLM1L (the left hemisphere of DLM1) and bilaterally in DLM2. In all three hemispheres penetrations were targeted midway between the lunate and superior temporal sulci and

Histology After 3 days survival time, the animals were sacrificed, perfused with fixative, and their brains removed. Sections were cut at 40 mm on a freezing microtome parasagitally (JNM1) or coronally (all other cases). Injection sites in MT and V3 were verified through cytochrome oxidase (CO; Wong-Riley, 1979) and myelin stains (Gallyas, 1979) of cortical sections involving the STS (for MT) and the anectant gyrus (for V3; see Nassi and Callaway, 2006; also see Lyon and Kaas, 2002). As covered in more detail elsewhere (Nassi and Callaway, 2006; Nassi et al., 2006), anatomical features of MT (Van Essen et al., 1981; Tootell et al., 1985), such as heavy and uniform myelination in deep layers, darker CO staining, and relative position on the posterior bank of the STS, were used to verify that injections were confined to area MT (see Figures 3E and 3H). Anatomical features of V3, such as heavy and uniform myelination in the deeper layers (Van Essen et al., 1986) and distance from the V1/V2 border (Brewer et al., 2002; Lyon and Kaas, 2002) were used to verify that injections were confined to V3 (see Figures 4A and 4B). Injection sites for V2 were verified histologically using cytochrome and myeloarchitecture as described in Nassi and Callaway (2006). V4 injections (see Figure 3B and bottom of Figure 3E) were targeted based on in vivo identification of the anterior bank of the lunate sulcus and the anterodorsal most extent of the inferior occipital sulcus (Gattass et al., 1988; Nakamura et al., 1993; Felleman et al., 1997; Stepniewska et al., 2005) and verified histologically in CO stained cortical sections (see Figures 3C and 3D). Every sixth subcortical section that included the SC and pulvinar were processed for the rabies nucleocapsid (see Nassi et al., 2006) and CO. SC lamination was determined using CO architecture (see Figure 1; Kaas and Huerta, 1988). The borders for the medial and caudal-lateral subdivisions of the inferior pulvinar (PIm and PIcl) and the ventral lateral subdivision of the lateral pulvinar (PLvl), were determined through CO stains (see bottom of Figure 3A). Locations of other pulvinar subdivisions were estimated. Criteria used to delineate pulvinar regions were based on Cusick et al. (1993), Gutierrez et al. (1995), Stepniewska and Kaas (1997), Adams et al. (2000), and Kaas and Lyon (2007). Data Analysis Positions of cortical, thalamic and collicular neuronal cell bodies labeled with rabies virus were plotted using Neurolucida software (MicroBrightField, Williston, VT). Reconstructions of the laminar boundaries of the SC and subdivisions of the pulvinar were aided by CO staining of the same sections that were stained for rabies nucleocapsid (see Figure 1 and bottom of Figure 3A for an examples of sections processed for both rabies and CO). For each case, every sixth section of the SC and thalamus was examined for rabies labeled neurons. Because our sampling rate for SC and TRN label was 1/6,

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we multiplied the number of cells found in each region by 6. For example, from the MT injections in case JNM10 we found 179 labeled neurons by sampling every sixth SC section, multiplying by 6 we get 1074 neurons (see Figure 2A).

Fries, W. (1984). Cortical projections to the superior colliculus in the macaque monkey: a retrograde study using horseradish peroxidase. J. Comp. Neurol. 230, 55–76.

ACKNOWLEDGMENTS

Galletti, C., and Battaglini, P.P. (1989). Gaze-dependent visual neurons in area V3A of monkey prestriate cortex. J. Neurosci. 9, 1112–1125.

We are grateful for support from the National Institutes of Health. We thank Dr. Donald Lodmell for providing the CVS-11 strain of rabies virus and antibodies to the rabies nucleocapsid, Cecelia Kemper for technical assistance with structural MRIs, Mauricio De La Parra for assistance with animal care and surgical procedures, Drs. Roberta Kelly and Ian Wickersham for technical advice and helpful discussions, and Dr. Rich Krauzlis for helpful comments on this manuscript. Accepted: January 5, 2010 Published: January 27, 2010

Gallyas, F. (1979). Silver staining of myelin by means of physical development. Neurol. Res. 1, 203–209. Gattass, R., Sousa, A.P., and Gross, C.G. (1988). Visuotopic organization and extent of V3 and V4 of the macaque. J. Neurosci. 8, 1831–1845. Goodale, M.A., and Milner, A.D. (1992). Separate visual pathways for perception and action. Trends Neurosci. 15, 20–25. Gutierrez, C., Yaun, A., and Cusick, C.G. (1995). Neurochemical subdivisions of the inferior pulvinar in macaque monkeys. J. Comp. Neurol. 363, 545–562.

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