Direct projections from the dorsal premotor cortex to the superior colliculus in the macaque (macaca...

Direct projections from the dorsal premotor cortex to the superior colliculus in the macaque

(Macaca mulatta)

Claudia Distler1, Klaus-Peter Hoffmann

1, 2, 3

1 Zoology & Neurobiology

2 Animal Physiology, 3 Dept. of Neuroscience

Ruhr-University Bochum

Germany

Abbreviated title: Direct projections of PMd to superior colliculus

Keywords: dorsal premotor - corticotectal – eye-hand coordination – sensorimotor – reaching

- AB_2336827 - AB_2315331

Support: This study was supported by grant Ho 450/25 from the Deutsche

Forschungsgemeinschaft and a ZEN grant from the Hertie Stiftung to KPH.

Corresponding author: Claudia Distler, Allgemeine Zoologie und Neurobiologie, Ruhr-

University Bochum, Universitaetsstr. 150, ND 7/26, 44780 Bochum, Germany, email:

[email protected], phone 49-234-3224365

John Wiley & Sons

Research Article The Journal of Comparative NeurologyResearch in Systems Neuroscience

DOI 10.1002/cne.23794

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an‘Accepted Article’, doi: 10.1002/cne.23794© 2015 Wiley Periodicals, Inc.Received: Jan 21, 2015; Revised: Apr 11, 2015; Accepted: Apr 15, 2015

This article is protected by copyright. All rights reserved.

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Abstract

The dorsal premotor cortex (PMd) is part of the cortical network for arm movements during

reach-related behaviour. We here investigate the neuronal projections from PMd to the

midbrain superior colliculus (SC) that also contains reach-related neurons in order to

investigate how the SC integrates into a cortico-subcortical network responsible for initiation

and modulation of goal-directed arm movements. Using anterograde transport of neuronal

tracers we found that PMd projects most strongly to the deep layers of the lateral part of the

SC and the underlying reticular formation corresponding to locations where reach-related

neurons have been recorded, and from where descending tectofugal projections arise. A

somewhat weaker projection targets the intermediate layers of the SC. By contrast, terminals

originating from prearcuate area 8 mainly project to the intermediate layers of the SC. Thus,

this projection pattern strengthens the view that different compartments in the SC are involved

in the control of gaze and in the control or modulation of reaching movements. The PMD-SC

projection assists the participation of the SC in the skeletomotor system and provides PMd

with a parallel path to elicit forelimb movements.

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Introduction

Reaching and grasping are everyday activities for all dexterous mammals including man.

Research during the last decades in primates revealed a widespread network of brain areas

involved in various aspects of these actions. In cerebral cortex, the main players are primary

motor cortex (M1), premotor cortex (PM), and posterior parietal cortex (area 5, AIP and

MIP), with with their cortical input from visual, somatosensory, and prefrontal cortex (e.g.

Johnson et al., 1996; Hoshi and Tanji, 2007; Padberg et al., 2007; Kaas et al., 2012, 2013 and

references therein). Based on electrophysiological recordings, microstimulation, and

anatomical investigations all these areas display topographic organization which, however, is

somewhat crude in premotor and posterior parietal cortex. This led to the concept of

functional zones interconnected within and between relevant cortical areas which would then

together control the various sectors, joint angles, orientation, etc. of the forelimb during

certain movements (e.g. Seelke et al., 2012; Kaas et al., 2012, 2013).

On functional and anatomical grounds, premotor cortex (Brodman area 6) can be divided in

dorsal (PMd, F2, F7) and ventral (PMv, F4, F5) compartments (Matelli et al., 1985). Area

PMv (F5) is mainly connected with the ventral portion of the dorsolateral prefrontal cortex

(DLPC) and with the inferior parietal lobule, especially area AIP and PF (e.g. Luppino et al.,

1999; Hoshi and Tanji, 2007; Borra et al., 2008; Gharbawie et al., 2011). Functionally, PMv

is involved in the preparation and execution of arm and hand movements related to grasping

and contains not only neurons related to the subject’s own actions but to actions of others

(mirror neurons) (e.g. Rizzolatti et al., 1988; Kakei et al., 2001; Ochiai et al., 2005; Raos et

al., 2006; Hoshi and Tanji, 2007; Theys et al., 2012; Lehmann and Scherberger, 2013; Bonini

et al., 2014).

By contrast, PMd is connected with dorsal DLPC and the dorsal parietal lobule including area

5 and MIP. Namely the caudal part of PMd (F2 dimple region and F2 ventrorostral of Matelli

et al., 1998; cPMd of Ghosh and Gattera, 1995) receives its main input from parietal areas

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PEc and PEip (F2 dimple), and MIP and V6A (F2 ventrorostral), respectively, as well as from

frontal areas SMA, M1, and the cingulate motor areas (Ghosh and Gattera, 1995; Matelli et

al., 1998). It is also involved in the preparation and execution of arm movements but shows

preparatory activity and codes for direction, speed and amplitude of the arm movement during

reaching and eye-hand coordination (e.g. Barbas and Pandya, 1987; Colby et al., 1988; Fu et

al., 1995; Tanné et al., 1995; Johnson et al., 1996; Matelli et al., 1998; Messier and Kalaska,

2000; Luppino et al., 2003; Churchland et al., 2006; Pesaran et al., 2006, 2010; Hoshi and

Tanji, 2007; Yamagata et al., 2012).

Electrical microstimulation in PMd leads to movements of the upper limb but at significantly

higher thresholds than in M1 (Weinreich and Wise, 1982; Preuss et al., 1996; Fujii et al.,

2000; Raos et al., 2003). A systematic survey of the dorsal premotor cortex revealed a

topographic order of evoked movements with simple movements of the contralateral shoulder

mostly elicited from areas close to the precentral dimple, and simple movements of the

contralateral distal forelimb mostly elicited more laterally close to the spur of the arcuate. In

addition, neurons close to the dimple were mostly active during movements and

somatosensory stimulation of the arm whereas neurons close to the spur were active during

movement and somatosensory stimulation of the distal forelimb (Raos et al., 2003). About

half of the neurons were active during grasping, about 37% were active during reaching. This

forelimb-related region contains visual, somatosensory, purely motor, visually modulated and

visuomotor neurons (Fogassi et al., 1999; Raos et al., 2004). Electrical stimulation of PMd

resulted more often in suppression (50% of events) in upper limb muscles than stimulation of

SMA or M1 (20% of events) (Montgomery et al., 2013). With longer stimulation periods

(500ms) complex and more naturalistic movements of the arm could be evoked from PMd

(Graziano et al., 2002).

In recent years, reach related activity has also been identified in neurons of the intermediate

and deep layers of the midbrain superior colliculus (SC). These reach neurons are active

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before and during arm movements, and their activity is correlated with the direction of arm

movements (Kutz et al., 1997) and/or with the electromyogram of proximal shoulder and arm

muscles (Werner, 1993; Werner et al., 1997a, b; Stuphorn et al., 1999, 2000). Furthermore,

microstimulation at recording sites of these reach neurons elicits arm movements (Philipp and

Hoffmann, 2014).

The SC receives input from multiple cortical areas. The superficial layers are targeted mainly

by early visual areas whereas intermediate and deep layers receive input from posterior

parietal cortex including the lateral intraparietal area (LIP), forelimb representation of areas 2,

4, and 6, FEF, PMv, and prefrontal cortex (Finlay et al., 1976; Kuenzle et al., 1976; Leichnetz

et al., 1981; Fries, 1984, 1985; Komatsu and Suzuki, 1985; Lynch et al., 1985; Lock et al.,

2003; Borra et al., 2014; Cerkevich et al., 2014).

Only in some instances have these anatomical connections been characterized physiologically.

The corticotectal projection originating from primary visual cortex seems to be involved in

the control of intracollicular visual processing (Finlay et al., 1976). The FEF projection to the

SC mainly carries saccade-related and visual information about the central visual field

(Segraves and Goldberg, 1987). By contrast, tectal projections from LIP carry mostly visual

saccade-related information from the peripheral field (Paré and Wurtz, 1997; Gaymard et al.,

2003). On the other hand, projections from the dorsolateral prefrontal cortex transmit task

selective signals to the SC, i.e. neuronal signals selective for antisaccades that inhibit

reflexive prosaccades (Johnston and Everling, 2006). To date, only little data for the

premotor-tectal projections are available. Electrical microstimulation revealed excitatory as

well as inhibitory influences of PMd on forelimb muscles. We presume that at least part of the

PMd output is mediated indirectly, e.g. via subcortical nuclei including the SC. In a

preliminary study, antidromically identified premotor-tectal neurons were active during reach

movements. In some of these cells the reach activity varied with the direction of gaze which

corresponds to the firing properties of a subset of SC reach neurons (Stuphorn et al., 1996).

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In the present study we investigate the direct projections of PMd to the SC to reveal the

spatial location of anterogradely labelled terminals relative to recording sites of reach related

activity in the SC. This study is part of a project to further characterize the information

transmitted from PMd to the SC in the cortico-subcortical network for reaching

Materials and Methods

All experiments were approved by the local authorities (Regierungspräsidium Arnsberg, now

LANUV) and ethics committee, and were performed in accordance with the Deutsche

Tierschutzgesetz of 7.26.2002, the European Communities Council Directive RL 2010/63/EC,

and NIH guidelines for care and use of animals for experimental procedures.

Animals

Four adult male rhesus monkeys (Macaca mulatta) were used in the present investigation. All

animals had participated in electrophysiological experiments prior to the tracer injections

(Stuphorn et al., 2000; Kruse and Hoffmann, 2002; Kruse et al., 2002; Philipp and Hoffmann,

2014).

Surgery

After premedication with atropine sulphate (0.04mg/kg) the animals were initially

anaesthetized with ketamine hydrochloride (10mg/kg i.m., Braun, Melsungen). After local

anaesthesia with 10% xylocain (Astra Zeneka, Wedel) they were intubated through the mouth,

an intravenous catheter was introduced into the saphenous vein and, after additional local

anaesthesia with bupivacain hydrochloride 0.5% (Bupivacain®

), the animals were placed into

a stereotactic apparatus. Throughout the experiment the animals were artificially ventilated

with nitrous oxide: oxygen as 3:1 containing 0.3% - 1% halothane as needed. Deep analgesia

was ensured by intravenous bolus and continuous application of fentanyl

(3µg/kg/h,

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Janssen). Heart rate, SPO2, blood pressure, body temperature, and endtidal CO2 were

monitored constantly and kept at physiological levels. The corneae were protected with

contact lenses.

Injections and histological procedures

In two of the animals, the injections were made through recording chambers that had

previously been implanted for the electrophysiological experiments to probe PMd for neurons

antidromically activated from SC. In the remaining two animals, the recording chamber was

removed to provide access to a larger cortical area. We used biotin dextrane (BDA) as an

anterograde tracer. In addition, in case A8 (prearcuate oculomotor area 8), during the final

electrophysiological experiment, we injected horseradish peroxidase conjugated to wheat

germ agglutinin (WGA-HRP) at the same location as the BDA injection (case A8-2). Table 1

summarizes the tracers, volumes, and survival times used in the present study.

After appropriate survival times, the animals received a lethal overdose of pentobarbital and

were perfused through the heart with 0.9% NaCl containing 0.1% procainhydrochloride

followed by paraformaldehyde-lysine-periodate with 4% paraformaldehyde. After appropriate

cryoprotection with 10% and 20% glycerol the brains were shock frozen and stored at -70°C

until processing.

The midbrains were cut at 40-50µm at a vibratome or a cryostat in a plane angled 45° from

posterior and thus approximately normal to the SC surface and parallel to the electrode

penetrations. Frontal or sagittal sections of the injected cortical hemispheres were cut at a

freezing microtome at 50µm. Neuronal transport of BDA was visualized with the avidin-

biotin method (ABC Elite, Vector 1:250, RRID: AB_2336827) with diaminobenzidine (DAB)

as chromogen enhanced with ammonium nickel sulphate (cases PMd1, PMd2, PMd3), or in

alternate sections with tetramethylbenzidine (TMB) as chromogen (case PMd3; after Ding

and Ellberger, 1995, van der Want, 1997). In case A8-2, WGA-HRP was visualized with

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TMB (after van der Want, 1997). A subset of midbrain sections was stained for AChE

histochemistry (Illing, 1988). Subsets of cortical sections were stained for Nissl, and

myeloarchitecture (Gallyas, 1979, as modified by Hess and Marker, 1983).

In a further subset of cortical sections, the neurofilament-based chemoarchitecture of cortical

areas was analyzed. In short, free-floating sections were washed in 0.1m Tris-buffered saline

(TBS), and treated for 30 min with 3%H2O2 and 0.2% Triton–X-100, respectively. Sections

were incubated in an antibody against non-phosphorylated neurofilament protein for 48h at

4°C under gentle agitation (SMI-32, 1:1000, Covance, cat# smi-32r; RRID: AB_2315331;

Hof and Morrison, 1995). Then sections were washed thoroughly in TBS, and incubated over

night in the biotinylated secondary antibody at 4°C (sheep anti mouse, 1:200, Amersham, GE

Healthcare Life Sciences, cat# RPN1001). After thorough washing, sections were incubated

in ABC Elite (Vector 1:250, RRID: AB_2336827) for 2 h at room temperature, and antigenic

sites were visualized with DAB. Controls omitting the primary antibody resulted in no

labelling.

Analysis

Labelled fibers and terminals in midbrain sections were drawn with camera lucida at a

microscope (ZEISS Axioplan) coupled to a reconstruction system (AccuStage MDPlot v5),

cortical connections were plotted with the reconstruction system. Cortical areas were

identified based on cyto-, myelo-, and neurofilament-based chemoarchitecture (e.g. Barbas

and Pandya, 1987; Blatt et al., 1990; Colby et al., 1993; Hof and Morrsion, 1995; Hof et al.,

1995; Preuss et al., 1997; Gabernet et al., 1999; Rozzi et al., 2006; Belmalih et al., 2007;

Takahara et al., 2012) largely following the criteria summarized and demonstrated by Lewis

and Van Essen (2000). The histological sections were photographed at a photomicroscope

(Zeiss Axioplan) equipped with a Canon EOS 600D camera, brightness and contrast were

adjusted with Adobe Photoshop (RRID: SciRes_000161, version 5.5).

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Results

The present study is based on three tracer injections into the dorsal premotor cortex. For

comparison, the prearcuate oculomotor cortex (area 8) was injected in an additional animal.

The location of the injection sites is presented on the reconstructions of the lateral view of the

left cortical hemispheres in Figure 1. All data are presented as left hemispheres to facilitate

comparison.

Case PMd1

In case PMd1, the tracer injections into PMd followed the electrophysiological recordings

from cortical neurons that projected to the SC as identified by antidromic stimulation. Three

tracer injections (green areas) were placed into the anterior part of PMd (F2) rostral to the

precentral dimple (pcd) (Fig. 1A) but clearly not encroaching on area 8 in the prearcuate

gyrus or FEF in the anterior bank of the arcuate sulcus. Examples of the anterogradely

labelled fibers and terminals are depicted in Fig. 2 B, C, the location of the labelled structures

in the SC and underlying mesencephalic reticular formation is given in Fig. 2A.

Anterogradely labelled terminals (Fig. 2, red dots in Fig. 3) and fibers (Fig. 2, blue marks in

Fig. 3) were located in the intermediate and predominantly in the deep layers of the ipsilateral

SC throughout its entire anterior-posterior and medio-lateral extent (Fig. 3). As a label for the

intermediate layers we used AChE histochemistry (green structures in the left drawings of

Fig. 3), the other collicular layers were determined in neighbouring sections stained for

cytoarchitecture. The PMd terminals were located predominantly below the AChE rich

patches. In addition, labelled terminals were found in the mesencephalic reticular formation

close to the periaqueductal grey and in the pontine nuclei. To further characterize and

compare the injected area between cases we also analyzed the cortical connections revealed

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by the injections. We here largely follow the criteria and nomenclature of Lewis and Van

Essen (2000). Figure 4 demonstrates examples of the neurofilament-based cytoarchitecture of

various areas of interest. The border between area 4 and PMd was recognized by the larger

abundance of large layer V pyramidal cells and a decrease of immunoreactivity in

supragranular layers (Fig. 4A; Preuss et al., 1997; Gabernet et al., 1999). In parietal cortex

(Fig. 4B), areas LIP, VIP, MIP, and 5V could be identified (Hof and Morrison, 1995; Hof et

al., 1995; Lewis and van Essen, 2000). Intracortical projections were limited to few areas

largely confirming earlier studies (Johnson et al., 1996; Matelli et al., 1998; Leichnetz, 2001;

Luppino et al., 2003; Burman et al., 2014). In parietal cortex, retrogradely labelled cells were

mainly located in the medial bank of the intraparietal sulcus including areas MIP, VIP, and

ventral area 5, as well as in area V6A on the medial aspect of the parietal lobe. Frontally,

numerous retrogradely labelled neurons were present in the precentral motor cortex (area 4),

within dorsal area 6 anterior to the injection site and posterior in the spur of the arcuate

sulcus, and in area M2. Medially, fewer neurons were located in areas 23 and 24 in the

cingulate sulcus (Fig. 5).

Case PMd2

The tracer injection in case PMd2 was located more posterior than PMd1, between the

anterior half of pcd and the spur of the arcuate sulcus (Fig. 1B). Anterogradely labelled

terminals in the SC were exclusively located in the deep layers of the lateral SC far away from

the AChE patches. There was not a continuous band of labelled terminals from lateral to

medial as in case PMd1. Additionally, labelled terminals were found in the nucleus ruber and

widespread in the mesencephalic reticular formation (Fig. 6). Cortical projections were

restricted to the medial bank of the intraparietal sulcus including area MIP/ventral area 5, to

the precentral area 4, and to area 6V. In contrast to case PMd1, area M2 was far less heavily

labelled, area 23 and 24 on the medial aspect of the brain were largely devoid of label (Fig. 7).

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Case PMd3

Case PMd3 represents the caudal-most injection site in this study lying between the posterior

tip of the precentral dimple and the posterior tip of the spur of the arcuate (Fig. 1C). Examples

of the resulting anterograde labelling are shown in Fig. 2D-F. Labelled terminals were again

concentrated in the deep layers predominantly in the lateral SC and in the underlying

mesencephalic reticular formation (Fig. 8). In parietal cortex, retrogradely labelled neurons

were mostly found in the anterior part of the medial bank of the intraparietal sulcus (area 5V)

with additional label on the supraparietal gyrus (area 5), the infraparietal gyrus (area 7a), and

in the medial bank of the lateral fissure. Fewer neurons were labelled in the posterior bank of

the intraparietal sulcus, including area LIP. In frontal cortex, labelled cells were concentrated

in area 4 on the precentral gyrus, area 6V, and M2. Labelled cells in M2 were much denser on

the medial aspect of the brain than on the crown of the cortex (Fig. 9).

Case A8

As a control and for comparison, further injections were placed into the oculomotor area on

the prearcuate gyrus (area 8) in the representation of the peripheral visual field (Komatsu and

Suzuki, 1985) (Fig. 1D). In this case, in addition to the BDA injection, WGA-HRP was

injected at the same location resulting in a complete overlap of the two injection sites and the

resulting labelling. In contrast to the PMd projections, terminal labelling from both tracer

injections into area 8 was predominantly found in the intermediate layers of the medial SC,

the lateral SC was notably devoid of label (Fig. 10). Intracortical transport in this case was

rather limited. In frontal cortex, few patches of labelled neurons were located in area M2 and

area 6D (Fig. 11). Unfortunately, the posterior cortex was not available for analysis in this

case.

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Thus, the injection in PMd1 located in anterior PMd and possibly encroaching area 6Dr (F7;

Luppino et al., 2003) resulted in terminal labelling in the intermediate and deep layers of the

entire ipsilateral SC. After injections into more posterior parts of PMd solely the deep lateral

part of the SC was labelled. By contrast, output from our area 8 injection mainly terminated

more superficially compared to PMd in the intermediate layers sparing the most lateral SC.

Discussion

In our experiments we could show that area PMd (F2) directly projects moderately to the

intermediate and more strongly to the deep layers of the ipsilateral SC. These projections were

particularly heavy in the lateral part of the SC representing the ventral visual field, and

coincided with the location of reach-related neurons in the SC (Werner et al., 1997b) as well

as tectofugal neurons described in other studies (e.g. Castiglioni et al., 1978; Nudo et al.,

1993; Rathelot and Strick, personal communication).

Corticotectal projection from PMd

The existence of a direct connection between PMd and SC was suggested by earlier

retrograde tracing studies (Leichnetz et al., 1981; Fries, 1984, 1985; but see Lock et al.,

2003), however, the density of this projection seemed rather low. Our anterograde data

indicate that PMd projects primarily to the deep layers of the SC, below the AChE rich

patches in the intermediate layers. The most complete labelling was achieved by the anterior-

most injection (PMd1) which coincided with the area where neurons could be antidromically

activated from SC in related electrophysiological experiments. Based on the location and the

cortical connections, i.e. input from V6A and MIP in the parietal cortex and from cingulate

motor areas, case PMd1 includes the ventrorostral part of area F2 (Matelli et al., 1998;

Luppino et al., 2003). It also corresponds to the region from where simple and complex

movements of the distal forelimb can be elicited by intracortical microstimulation (Raos et al.,

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2003) and where grasp-related neurons are located (Raos et al., 2004). More posterior

injections mainly labelled the lateral part of the SC. Both PMd2 and PMd3 were located in the

region between the precentral dimple and the spur of the arcuate, with little or no input from

cingulate cortex (Matelli et al., 1998; Luppino et al., 2003). The core of the PMd2 injection

was closer to the dimple in the region where microstimulation leads to movements of the

proximal forelimb (Raos et al., 2003). PMd3 included the entire distance between dimple and

spur, thus including both proximal and distal forelimb regions (Raos et al., 2003). Therefore,

all our PMd injections were in the part of area F2 that controls arm reaching movements. The

differences in our SC labelling could well be due to topographical differences between the

injected regions, but could also be the result of differently sized injections. The tracer volume

injected and the resulting injection site was slightly larger in PMd1 than in PMd2 and PMd3

(Table 1, Fig. 1). Similarly, topographic differences have also been reported for the

projections from basal ganglia to rostral and caudal regions of F2 (Saga et al., 2011).

Interestingly, corticotectal projections from PMv (F5) and other areas of the cortical network

for grasping, e.g. ventral prefrontal areas 46 and 12, parietal area AIP, but also from saccade-

related area LIP also terminate in the deep and intermediate layers mainly of the lateral SC

(Lynch et al., 1985; Selemon and Goldman-Rakic, 1988; Borra et al., 2010, 2014). The same

SC region also receives direct input from orofacial primary motor cortex (Tokuno et al.,

1995). Injections into area 6DC (supplementary eye field) at the crown of the cerebral cortex

also revealed projections to the intermediate layers of the lateral SC (Shook et al., 1990) thus

putting this part of the SC in an even wider context.

Our control injection into prearcuate area 8 confirms findings of previous studies. It resulted

in labelling of the intermediate layers of the SC which was stronger in the medial than in the

lateral part of the SC which may be due to the topography in the corticotectal projection from

the prearcuate oculomotor cortex. Similar results were achieved by comparable injections in

other studies (Komatsu and Suzuki, 1985; Lynch et al., 1985; Stanton et al., 1988; Shook et

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al., 1990; Tokuno et al., 1995), the labelling of the entire SC shown by Leichnetz and

coworkers may again be due to the larger injection site (Leichnetz et al., 1981).

Unfortunately we could not analyze the cortical connections with the posterior cortex in our

area 8 case but labelling in the posterior bank of the intraparietal sulcus including LIP as well

as in visual and polymodal extrastriate areas has been demonstrated in other studies

(Leichnetz et al., 1981; Schall et al., 1985) which confirms that even our largest and most

anterior PMd injection shows an intracortical projection pattern distinct from that of

prearcuate oculomotor cortex.

Figure 12 summarizes the spatial relationship of cortical projections from PMd (red dots) and

area 8 (black dots) to the SC (this study) relative to the location of tectofugal neurons

retrogradely labelled from the cervical spinal cord (Castiglioni et al., 1978, green areas) or

transsynaptically from the spinodeltoid muscle (Rathelot and Strick, personal communication,

blue areas). For this chart the labelled SC regions from all our PMd cases were combined.

There is a striking overlap of input from PMd and the SC output to the cervical spinal cord

and the shoulder muscles demonstrating the putative neuronal substrate for modulating head

and arm movements via the SC.

Functional considerations

The optic tectum of non-mammalian vertebrates and the SC of mammals is traditionally

viewed as a key structure for orienting behaviour especially of body, head, eyes and pinnae

(e.g. Akert, 1949; Alstermark and Isa, 2012). With the discovery of arm-movement related

activity in the SC this scheme was extended to limb movements also in monkeys in agreement

with earlier studies in the cat (Courjon et al., 2004). Reach neurons are located in the

intermediate and deep layers predominantly in the lateral part of the monkey SC and in the

underlying mesencephalic reticular formation (Werner 1993; Werner et al., 1997a, b;

Stuphorn et al., 1999, 2000) and are active before and during reach movements mainly but not

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exclusively of the contralateral arm. They reside in a region where descending tectofugal

projections arise (e.g. Castiglioni et al. 1978; Grantyn and Grantyn 1982; Olivier et al. 1991;

Nudo et al. 1993; Rathelot and Strick, personal communication).

Recent microstimulation experiments revealed that arm movements can indeed be elicited by

microstimulation at recording sites of collicular reach neurons (Philipp and Hoffmann, 2014),

and that ongoing reach movements can be perturbed by simultaneous SC stimulation in the

cat (Courjon et al., 2004). Another subpopulation of SC neurons have a clear somatosensory

component as they are active when the hand touches and pushes a target but are inactive

during the reach phase (Nagy et al., 2006). They were also located in the intermediate and

deep layers of the SC, between 1.2–4mm below the collicular surface (median = 3.1mm); i.e.,

in the same depths as the reach neurons (median = 2.8mm, range: 1.4–4mm). The coincidence

of the anatomical location of SC reach and somatosensory-motor neurons with the cortical

afferents originating from dorsal premotor (this study) and ventral premotor cortex and related

cortical areas (e.g. Borra et al. 2014) suggests that the SC is strongly involved in arm and

hand movements in addition to the well known gaze movements. The convergence of the

reach-to-grasp system (i.e. PMv) and the reach-to-destination system (PMd) onto the same

regions close to the gaze system in the SC puts this structure in a unique position to modulate

reach and grasp movements in various behavioural contexts involving eye-hand coordination.

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Acknowledgements

We thank E. Brockmann, S. Dobers, H. Korbmacher and G. Reuter for expert technical

assistance. We are also grateful to H. Lübbert for lab space.

Conflict of interest:

The authors state no conflict of interest.

Role of authors:

All authors had full access to all the data in the study and take responsibility for the integrity

of the data and the accuracy of the data analysis. Study concept and design: KPH and CD,

acquisition of data: KPH and CD, analysis and interpretation of data: CD and KPH, drafting

of the manuscript: CD and KPH.

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Legends

Figure 1

Reconstructions of the left hemispheres of cases PMd1 (A), PMd2 (B), PMd3 (C), and A8

(D). The core of the tracer injections is shown in solid green, the diffusion area in stippled

green. For abbreviations see list. Scale bars represent 5mm.

Figure 2

Examples of anterogradely labelled fibers and terminals after BDA injections into PMd in

case PMd1 (A-C) and case PMd3 (D-F). Grey dots in A mark labelled terminals, black lines

in A mark labelled fibers The regions where the microphotographs shown in B-C were taken

are marked in the reconstruction of the midbrain section (A). D: darkfield photomicrograph of

labelled fibers and terminals in the lateral SC of PMd3 visualized with TMB as a chromogen.

The single asterisk marks the approximate location of the label shown in E, the double

asterisk marks the approximate location of F. E, F: Fibers and terminals in the deep lateral SC

of PMd3. In B-D and E-F, labelled structures were visualized with enhanced DAB as a

chromogen. Scale bars represent 500µm for D, and 50µm for B, C, E, and F.

Figure 3

Camera lucida drawings of serial sections through the midbrain of case PMd1. Sections are

arranged from posterior (upper left) to anterior (lower right). Intersection distance is 480µm

from the third section onwards. Sections in the left row present the anatomical areas as seen in

Nissl-stained sections, green dashed lines indicate AChE rich areas in the intermediate layers

of the SC and the underlying midbrain. Anterogradely labelled fibers are shown in blue,

labelled terminals in red. In this case almost the entire anterior-posterior and the entire medio-

lateral extent of the SC contains labelled terminals. Additional label is found in the pontine

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nuclei and in multiple locations within the mesencephalic reticular formation. For

abbreviations see list. Scale bar represents 2mm

Figure 4

Neurofilament protein-based chemoarchitecture used to distinguish motor (area 4) and

premotor (area 6) cortex (A), and intraparietal cortical areas LIP, VIP, and MIP (B) in case

PMd1. Transition zones between neighbouring areas are marked by black bars. Scale bars

represent 1mm.

Figure 5

Frontal sections through the left cerebral cortex of case PMd1 exemplifying the main

intracortical projections resulting from the tracer injection into PMd. The sections are

arranged from anterior (upper left) to posterior (lower right), their positions in the brain are

indicated in the inset. Dash-dot lines mark the border between the grey and the white matter,

areal borders determined from SMI-32 immunohistochemistry, myelo- or cytoarchitecture are

marked by arrows and/or by black bars underlying the transition zones. Each red dot

represents a retrogradely labelled neuron. Blue lines indicate the location of anterogradely

labelled fibers and terminals. The injection site is marked solid green, the diffusion area

stippled green. For abbreviations see list. Scale bar represents 5mm.

Figure 6

Camera lucida drawings of serial sections through the midbrain of case PMd2. Intersection

distance is 800µm. For other conventions see Fig. 3. For abbreviations see list. Scale bar

represents 2mm. The injection in PMd2 resulted in anterograde labelling restricted to the deep

layers of the lateral part of the SC, to the nucleus ruber, and to multiple sites within the

mesencephalic reticular formation.

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Figure 7

Coronal sections through frontal cortex and sagittal sections through the parietal cortex of the

left cerebral cortex of case PMd2 demonstrating the intracortical projections resulting from

the tracer injection into PMd. The sections are arranged from anterior (left, A) to posterior

(right, C), and from lateral (D) to medial (E), respectively. Their positions in the brain are

indicated on the insets, shaded areas show the extent of the anterior and posterior block of

tissue. For other conventions see Fig. 5. For abbreviations see list. Scale bar represents 5mm.

Figure 8

Camera lucida drawings of serial sections through the midbrain of case PMd3. Intersection

distance is 320µm. For other conventions see Fig. 3. For abbreviations see list. Scale bar

represents 2mm. Anterogradely labelled terminals are present mainly in the deep layers of the

lateral SC and in the underlying mesencephalic reticular formation.

Figure 9

Frontal sections through the left cerebral cortex of case PMd3 showing the intracortical

projections resulting from the tracer injection into PMd. For other conventions see Fig. 5. For

abbreviations see list. Scale bar represents 5mm.

Figure 10

Camera lucida drawings of serial sections through the midbrain of case A8. The plots

demonstrate the WGA-HRP labelling. Intersection distance is 300µm. For other conventions

see Fig. 3. For abbreviations see list. Scale bar represents 2mm. In contrast to the PMd

injections, the area 8 injection resulted in terminal labelling predominantly but not exclusively

in the intermediate layers of the medial SC.

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Figure 11

Frontal sections through the left cerebral cortex of case A8 showing the intracortical

projections resulting from the tracer injection into area 8. For other conventions see Fig. 5.

For abbreviations see list. Scale bar represents 5mm.

Figure 12

Schematic summary of the relationship of the cortical input to the SC from PMd (red) and

prearcuate oculomotor cortex (area 8, black) with the location of tectofugal neurons projecting

to the cervical spinal cord (green, data taken from Castiglioni et al., 1979) and indirectly to

the shoulder muscles, e.g. spinodeltoid muscle (blue, Rathelot and Strick, personal

communication). Cortical input from PMd largely overlaps with the tectospinal output in the

deep layers of the SC.

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case tracer volume survival time

PMd1 15% BDA MW3000 in 0.1m

citrate/NaOH pH 3.3

4µl 2 weeks

PMd2 15% BDA MW3000 in 0.1m

citrate/NaOH pH 3.3

2µl 2 weeks

PMd3 20% BDA MW3000 in 0.9%

NaCl

3µl 2 weeks

A8 2.5% WGA-HRP in

KCl/Tris/DMSO

0.3µl 48h

A8-2 10% BDA MW10000 in 0.9%

NaCl

1µl

3 months

Table1: Summary of the data base, tracers used, volume injected, and the survival time in the

different cases

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List of abbreviations

A: aqueduct

A8: area 8

amt: anterior mediotemporal sulcus

ar: arcuate sulcus

BSC: brachium of the superior colliculus

ca: calcarine sulcus

ce: central sulcus

ci: cingulate sulcus

DMZ: densely myelinated zone of the medial superior temporal area

dpc: decussatio pedunculi cerebellaris

ec: ectocalcarine sulcus

FEF: frontal eye field

flm: fasciculus longitudinalis medialis

IC: inferior colliculus

io: infero-occipital sulcus

ip: intraparietal sulcus

la: lateral sulcus

LIP: lateral intraparietal area

ll: lemniscus lateralis

lu: lunate sulcus

M2: motor area 2

MIP: medial intraparietal area

MT: middle temporal area

NPo: n. pontis

NRm: n. reticularis mesencephali

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NRp: n. reticularis parvocellularis

NRpo: n. reticularis pontis oralis

NRu: n. ruber

NTr: n. trapezius

ot: occipito-temporal sulcus

p: principal sulcus

PAG: periaqueductal grey

pc: pedunculus cerebri

pca: pedunculus cerebellaris anterior

pcd: precentral dimple

po: parieto-occipital sulcus

Pt: pretectum

Pul: pulvinar

Rd: raphe dorsalis

SC: superior colliculus

SN: substantia nigra

SGI: stratum griseum intermedium of the SC

SGP: stratum griseum profundum of the SC

SGS: stratum griseum superficiale of the SC

st: superior temporal sulcus

VIP: ventral intraparietal area

V1: primary visual cortex

V4t: visual area 4 transitional zone

V6: visual area 6

V6A_ visual area 6A

III: n. oculomotorius

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IV: n. trochlearis

3a, 4, 5, 6, 7a, 8, 23, 24: Brodman areas 3a, 4, 5, 6, 7a, 8, 23, 24

5v: ventral area 5

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Figure 1 Reconstructions of the left hemispheres of cases PMd1 (A), PMd2 (B), PMd3 (C), and A8 (D). The core of the tracer injections is shown in solid green, the diffusion area in stippled green. For abbreviations see list. Scale

bars represent 5mm.

180x204mm (300 x 300 DPI)

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Figure 2 Examples of anterogradely labelled fibers and terminals after BDA injections into PMd in case PMd1 (A-C)

and case PMd3 (D-F). Grey dots in A mark labelled terminals, black lines in A mark labelled fibers The

regions where the microphotographs shown in B-C were taken are marked in the reconstruction of the midbrain section (A). D: darkfield photomicrograph of labelled fibers and terminals in the lateral SC of PMd3 visualized with TMB as a chromogen. The single asterisk marks the approximate location of the label shown in E, the double asterisk marks the approximate location of F. E, F: Fibers and terminals in the deep lateral SC of PMd3. In B-D and E-F, labelled structures were visualized with enhanced DAB as a chromogen. Scale

bars represent 500µm for D, and 50µm for B, C, E, and F.

181x182mm (300 x 300 DPI)

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Figure 3 Camera lucida drawings of serial sections through the midbrain of case PMd1. Sections are arranged from

posterior (upper left) to anterior (lower right). Intersection distance is 480µm from the third section

onwards. Sections in the left row present the anatomical areas as seen in Nissl-stained sections, green dashed lines indicate AChE rich areas in the intermediate layers of the SC and the underlying midbrain. Anterogradely labelled fibers are shown in blue, labelled terminals in red. In this case almost the entire

anterior-posterior and the entire medio-lateral extent of the SC contains labelled terminals. Additional label is found in the pontine nuclei and in multiple locations within the mesencephalic reticular formation. For

abbreviations see list. Scale bar represents 2mm

180x275mm (300 x 300 DPI)

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Figure 4 Neurofilament protein-based chemoarchitecture used to distinguish motor (area 4) and premotor (area 6) cortex (A), and intraparietal cortical areas LIP, VIP, and MIP (B) in case PMd1. Transition zones between

neighbouring areas are marked by black bars. Scale bars represent 1mm.

117x152mm (300 x 300 DPI)

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Figure 5 Frontal sections through the left cerebral cortex of case PMd1 exemplifying the main intracortical projections resulting from the tracer injection into PMd. The sections are arranged from anterior (upper left) to posterior

(lower right), their positions in the brain are indicated in the inset. Dash-dot lines mark the border between the grey and the white matter, areal borders determined from SMI-32 immunohistochemistry, myelo- or cytoarchitecture are marked by arrows and/or by black bars underlying the transition zones. Each red dot represents a retrogradely labelled neuron. Blue lines indicate the location of anterogradely labelled fibers and terminals. The injection site is marked solid green, the diffusion area stippled green. For abbreviations

see list. Scale bar represents 5mm.

178x140mm (300 x 300 DPI)

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Figure 6 Camera lucida drawings of serial sections through the midbrain of case PMd2. Intersection distance is

800µm. For other conventions see Fig. 3. For abbreviations see list. Scale bar represents 2mm. The injection

in PMd2 resulted in anterograde labelling restricted to the deep layers of the lateral part of the SC, to the nucleus ruber, and to multiple sites within the mesencephalic reticular formation.

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Figure 7 Coronal sections through frontal cortex and sagittal sections through the parietal cortex of the left cerebral cortex of case PMd2 demonstrating the intracortical projections resulting from the tracer injection into PMd. The sections are arranged from anterior (left, A) to posterior (right, C), and from lateral (D) to medial (E), respectively. Their positions in the brain are indicated on the insets, shaded areas show the extent of the anterior and posterior block of tissue. For other conventions see Fig. 5. For abbreviations see list. Scale bar

represents 5mm.

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Figure 8 Camera lucida drawings of serial sections through the midbrain of case PMd3. Intersection distance is

320µm. For other conventions see Fig. 3. For abbreviations see list. Scale bar represents 2mm.

Anterogradely labelled terminals are present mainly in the deep layers of the lateral SC and in the underlying mesencephalic reticular formation.

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Figure 9 Frontal sections through the left cerebral cortex of case PMd3 showing the intracortical projections resulting from the tracer injection into PMd. For other conventions see Fig. 5. For abbreviations see list. Scale bar

represents 5mm.

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Figure 10 Camera lucida drawings of serial sections through the midbrain of case A8. The plots demonstrate the WGA-HRP labelling. Intersection distance is 300µm. For other conventions see Fig. 3. For abbreviations see list.

Scale bar represents 2mm. In contrast to the PMd injections, the area 8 injection resulted in terminal labelling predominantly but not exclusively in the intermediate layers of the medial SC.

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Figure 11 Frontal sections through the left cerebral cortex of case A8 showing the intracortical projections resulting from the tracer injection into area 8. For other conventions see Fig. 5. For abbreviations see list. Scale bar

represents 5mm.

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Figure 12 Schematic summary of the relationship of the cortical input to the SC from PMd (red) and prearcuate

oculomotor cortex (area 8, black) with the location of tectofugal neurons projecting to the cervical spinal

cord (green, data taken from Castiglioni et al., 1979) and indirectly to the shoulder muscles, e.g. spinodeltoid muscle (blue, Rathelot and Strick, personal communication). Cortical input from PMd largely

overlaps with the tectospinal output in the deep layers of the SC.

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The dorsal premotor cortex (PMd) is part of the cortical network for arm movements during reach-related behaviour. We here investigate the neuronal projections from PMd to the midbrain superior colliculus (SC)

that also contains reach-related neurons in order to integrate the SC into a cortico-subcortical network

responsible for initiation and modulation of goal-directed arm movements. Using anterograde transport of neuronal tracers we found that PMd projects most strongly to the deep layers of the lateral part of the SC and the underlying reticular formation corresponding to locations where reach-related neurons have been recorded, and from where descending tectofugal projections arise. This projection pattern supports the

involvement of the SC in the skeletomotor system and provides PMd with a further path to elicit forelimb movements.

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The dorsal premotor cortex (PMd) is part of the cortical network for arm movements during

reach-related behaviour. We here investigate the neuronal projections from PMd to the

midbrain superior colliculus (SC) that also contains reach-related neurons in order to integrate

the SC into a cortico-subcortical network responsible for initiation and modulation of goal-

directed arm movements. Using anterograde transport of neuronal tracers we found that PMd

projects most strongly to the deep layers of the lateral part of the SC and the underlying

reticular formation corresponding to locations where reach-related neurons have been

recorded, and from where descending tectofugal projections arise. This projection pattern

supports the involvement of the SC in the skeletomotor system and provides PMd with a

further path to elicit forelimb movements.

Page 49 of 49

John Wiley & Sons



Direct projections from the dorsal premotor cortex to the superior colliculus in the macaque (macaca...

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