BILATERAL ACTIONS OF THE RETICULOSPINAL TRACT IN THE MONKEY
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the
Graduate School of the Ohio State University
By
Adam G. Davidson
* * * * *
The Ohio State University
2004
Dissertation Committee Approved by
Dr. John A. Buford, Adviser
Dr. Georgia A. Bishop ______Adviser Dr. Jacqueline Bresnahan Neuroscience Graduate Studies Program
Copyright by
Adam G. Davidson
2004
ii
ABSTRACT
The motor output of the primate pontomedullary reticular formation (PMRF) was
investigated with spike- and stimulus triggered averaging (SpikeTA, StimulusTA) in
monkeys during reaching. The first study employed StimulusTA for ipsilateral arm and
shoulder muscles and bilateral trapezius muscles. The second study used StimulusTA and
SpikeTA for 24 muscles. Muscles studied on the ipsilateral (i) and contralateral (c) side
were extensor carpi ulnaris (ECU), flexor carpi radialis (FCR), brachioradialis (Brac),
biceps (Bic), triceps-long head (TrLo), triceps-lateral head (TrLa), anterior deltoid
(ADlt), posterior deltoid (PDlt), latissimus dorsi (Lat), pectoralis major (PMj), middle
trapezius (MTr), and upper trapezius (UTr). Average onset was significantly earlier for
post-stimulus facilitation (PStF) than for post-stimulus suppression (PStS). The average
duration of PStS was longer than the average duration of PStF, and response magnitude
was significantly larger for PStF. A clear pattern of PStF and PStS was observed among
all muscles. In the ipsilateral arm, flexors were facilitated and extensors were suppressed.
In the contralateral arm, extensors were facilitated and flexors were suppressed. Shoulder girdle muscles demonstrated similar proportions for responses: iUTr, cMTr, and cPMj were suppressed; cUTr , iMTr, and iPMj were facilitated. Lat was the only muscle to demonstrate the same response (PStS) bilaterally. Reciprocal, cofacilitation, and cosuppression responses were observed between anatomical antagonists within a limb
iii and bilateral homologues at individual stimulus sites. These responses were usually reciprocal and matched the most prevalent response for each member of the pair. Overall,
9 of 368 cells produced post-spike effects (PSpEs). 8 of the 11 total PSpEs were post spike facilitation (PSpF). 4 PSpEs matched the corresponding PStE obtained at the same site. Overall, StimulusTA and SpikeTA of bilateral arm and shoulder muscles revealed motor patterns that implicate the reticulospinal tract in the control of voluntary reaching movements and the coordination of bilateral movements.
iv
Dedicated to my wife, Kristen
v
ACKNOWLEDGMENTS
First, I wish to thank my advisor, John Buford, PhD, for his guidance and support
throughout this dissertation and during the past four years of graduate school. I also wish
to thank Stephanie Moran for all of her technical assistance in the lab. Thanks to Dr. S. T.
Sakai for assistance with the anatomical investigations performed for these studies. I
would like to thank my committee members, Georgia Bishop, PhD and Jacqueline
Bresnahan, PhD, for helping me with this dissertation and other endeavors during my
graduate career. I would also like to thank other Neuroscience faculty who have helped
me along the way, especially Dr. M. Scott Herness for his encouragement and guidance.
Thank you to Mom and Dad for your support through the years. To my brother
Brian, thanks for setting the example for me. Finally, to Kristen, thank you for your love
and support. I could not have done it without you.
The experiments presented in this dissertation were supported by NIH grant R01
NS37822. Chapter 2 is included in this dissertation with permission from the American
Physiological Society (cited in reference list). Portions of figure 3.12 appear with permission from John Wiley & Sons Inc. All other portions of this dissertation are
copyrighted.
vi
VITA
August 7, 1977...…………………….Born – Statesville, North Carolina
2000 – 2001………………………….Interdisciplinary Graduate Program Fellow, The Ohio State University
2001 – present……………………….Graduate Research Assistant, The Ohio State University
PUBLICATIONS
Davidson, A. G., & Buford, J. A. (2004). Motor output to arm and shoulder muscles from the PMRF of the monkey as revealed by stimulus triggered averaging. Journal of Neurophysiology. 10.1152/jn.00083.2003.
Buford, J. A. & Davidson, A. G. (2004). Movement related and preparatory activity in the reticulospinal system of the monkey. Experimental Brain Research. 10.1007/s00221-004-1956-4.
FIELD OF STUDY
Major Field: Neuroscience
vii TABLE OF CONTENTS
Page
Abstract……………………………………………………………………...……………iii
Dedication…..……………………………………………………………………………..v
Acknowledgments...……………………………………………………………………...vi
Vita………………………………………………………………………………….…...vii
List of Tables……………………………………………………………………………...x
List of Figures …………………………………………………………………………....xi
Chapters:
1. Introduction…..……………………………………………………………………1
1.1 Anatomy of the reticulospinal tracts……………………….………………….2 1.2 Studies of reticulospinal tract function……………………………………...... 3 Reticulospinal functions in the primate…………………………………...7 1.3 Patterns and organization of reticulospinal motor outputs……………………9 1.4 Mechanisms of reticulospinal actions in the spinal cord………………….…11 1.5 Spike and stimulus triggered averaging………..…………………………….16
2. Motor outputs from the primate reticular formation to arm and shoulder muscles as revealed by stimulus-triggered averaging ……....………...………………….20
2.1. Abstract….………………...…………………………….…………………..20 2.2. Introduction….…………………….…………………….…………………..21 2.3. Methods…….……………………….……………….....……………………24
viii 2.4. Results…………………………………………………………………...... 31 2.5. Discussion…………………….……………………………………………..39
3. Bilateral actions of the reticulospinal tract on arm and shoulder muscles in the monkey: Spike and stimulus triggered averaging ...………...………………….62
3.1. Abstract….………..…………………………….…………………..62 3.1. Introduction….………………………………….…………………..63 3.2. Methods…….……………………………….....……………………67 3.3. Results…………………………………………………………...... 75 3.4. Discussion…………………………………………………………...91
4. Conclusion……………….…………………...………………………………...125
List of References……………………………………………..……………………….128
ix LIST OF TABLES
Table Page
2.1 Onset latency and duration of post-stimulus events..……………………………51
3.1 Location of EMG implants …..……………………………..………………….106
3.2 Average onset of PStEs…. …..……………………………..…………….…….107
3.3 Average duration of PStEs…. …..…………………………..………………….108
3.4 Average MPC of PStEs…. …..……………………………..……………….….109
3.5 Average SDPk of PStEs…. …..………...…………………..…………….…….110
x
LIST OF FIGURES
Figure Page
2.1 Representative sample of EMG during task performance……………………….52
2.2 Anatomical location of effective stimulus sites for ipsilateral upper trapezius………………………………………………………………………….53
2.3 Detection of post-stimulus effects...……………….…………………………….54
2.4 Histograms of onset latency and mean percent change………………………….55
2.5 Histograms of post-stimulus effects by onset latency for each muscle………….56
2.6 Proportions of post-stimulus facilitation and post-stimulus suppression events by muscle……...... 57
2.7 A typical stimulus-triggered average…………………………………………….58
2.8 Additional examples of stimulus-triggered averages…………………………….59
2.9 Large amplitude, short latency response in ipsilateral upper trapezius………….60
2.10 Bilateral responses in upper trapezius……………………………………………61
3.1 Diagram of the behavioral task…………………………………………………112
3.2 Spike and stimulus triggered averaging……………………………………...…113
3.3 Representative sample of EMG during task performance…………………...…114
3.4 Histogram of onset latencies…………………………….…………………...…115
3.5 Effectiveness of stimulation/ Proportion of post-stimulus
facilitation……………………………………………….…………………...…116
3.6 Representative stimulus-triggered average……..……….…………………...…117
xi 3.7 Upper trapezius post-stimulus facilitation…...………….…………………...…118
3.8 Concurrent reciprocal response………………………….…………………...…119
3.9 Serial reciprocal response………………………………………………………120
3.10 Representation of frequent responses…………………..………………………121
3.11 Comparison of similar post stimulus and post spike effects..………………..…122
3.12 Anatomical reconstruction of effective stimulus sites………………………….124
xii
CHAPTER 1
INTRODUCTION
A variety of functions have been attributed to the reticular formation including, but not limited to, REM sleep (Siegel et al., 1981;Siegel et al., 1979), arousal (Steriade,
1996;Steriade et al., 1986;Steriade, 1970;Steriade, 1995;Steriade et al., 1988), sensory modulation (Abols and Basbaum, 1981;Basbaum et al., 1986;Peterson et al., 1974), and the control of gaze (Berthoz and Grantyn, 1986;Grantyn et al., 2004;Grantyn et al.,
1993;Grantyn et al., 1987;Grantyn and Berthoz, 1987;Olivier et al., 1993). In addition to these functions, the reticular formation has been implicated in the control of axial and limb muscles for posture and locomotion (Drew and Rossignol, 1984;Drew and
Rossignol, 1985;Drew et al., 1986;Drew and Rossignol, 1987;Drew et al., 2004;Schepens and Drew, 2003b), as well as voluntary reaching movements (Buford and Anderson,
1996;Ruffo and Buford, 1997). The exact role the reticulospinal system plays in motor control is not known. In fact, of the four major descending motor systems (the corticospinal, rubrospinal, vestibulospinal, and reticulospinal tract (RST)), the least is known about the reticulospinal system. The majority of what is known about the RST has
1 come from experiments in the cat; there have been few experiments examining the motor output of the RST in the non-human primate.
This chapter presents a review of studies that have examined the motor functions
of the reticular formation in the cat and monkey. Most studies of motor control in the
reticular formation have focused on the pontine and medullary reticular formation. In the
current literature and in this dissertation, this region is referred to as the pontomedullary
reticular formation (PMRF). The first section of this review presents a brief discussion of
the pattern of RST projections and the reticular nuclei from which they originate. This is
followed by a review of some functional studies of the PMRF and the mechanisms in
which the RST acts on spinal cord motoneurons and interneurons. A brief discussion of
the methods of spike and stimulus triggered averaging, the methods of investigation
employed in this study, is also presented.
1.1 Anatomy of the Reticulospinal Tracts
Anatomical and physiological experiments in the cat have shown that the RST can
be divided into two distinct pathways, the medial and lateral reticulospinal tracts (MRST,
LRST) (Brodal, 1957). In the cat, the MRST originates primarily from the nucleus
reticularis pontis caudalis (NRPc), the nucleus reticularis pontis oralis (NRPo), and the
dorsorostral region of nucleus reticularis gigantocellualris (NRGc). The NRPo is situated
in the rostral pons and gradually transitions caudally into the NRPc, which is located
rostral to the abducens nucleus. The NRGc occupies the medial two-thirds of the reticular
formation from a region dorsal to the rostral half of the inferior olive, up to the level of
the facial motor nucleus. The MRST descends ipsilaterally in the medial longitudinal
2 fasciculus (MLF), and in a region adjacent to the MLF, and continues into the
ventromedial funiculus of the spinal cord. In the cat, the MRST descends uncrossed
throughout the length of the spinal cord and terminates in Rexed’s laminae VI – IX of
cervical, thoracic, and lumber levels of the spinal cord (Petras, 1967;Nyberg-Hansen,
1965).
The LRST originates in the medullary reticular formation (Brodal 1957; Torvik
and Brodal 1957). In the cat, the principal origins of the LRST are the ventrocaudal
region of NRGc and the nucleus reticularis ventralis (NRVl). The LRST descends
primarily in the ipsilateral ventrolateral funiculus, although a smaller portion of the LRST
descends in the contralateral ventrolateral funiculus. The LRST projects predominately to
the cervical segments of the spinal cord, but sends axons to all levels of the spinal cord.
In the cat, terminals of the LRST reach the ventral and proximal regions of the lateral
horn and terminate in Rexed’s laminae VI - IX (Petras, 1967;Nyberg-Hansen, 1965).
1.2 Studies of Reticulospinal Tract Function
Previous hypotheses of RST function were based on the cellular organization of
the reticular formation. Anatomical descriptions of the reticular formation portrayed the
organization as indistinct, with a large number of afferent and efferent projections
forming numerous synapses (Brodal 1957). According to earlier hypotheses, this
disordered network of synapses was not capable of specific control of individual muscles, or even individual body segments, such as the head or a limb. As a result, the reticular formation was believed to have a global effect over somatic musculature.
3 Conclusions drawn from early electrophysiological experiments supported the
hypothesis of global motor control. Rhines and Magoun (1946) reported that stimulation
of the rostrodorsal region of the medial reticular formation in the decerebrate cat and
monkey facilitated cortically evoked movements. In these experiments, long stimulus
trains were applied to the reticular formation of the pons and midbrain using stimulus
currents as high as 1 to 2 v. The same stimulation methods applied to the caudoventral
region of the medial reticular formation in the cat was reported to produce the opposite
effect, suppression of cortically evoked responses (Magoun and Rhines 1946; Magoun
1944). Stimulation in this same region also eliminated the blink reflex, the patellar reflex, and decerebrate rigidity of all four limbs. At some of these stimulation sites, the forearm flexor reflex was suppressed concurrently with the patellar and blink reflexes; however, not every site in Magoun’s inhibitory region was effective at inhibiting the forearm flexor reflex.
Evidence contradicting descriptions of the global motor effects elicited from the
PMRF surfaced not long after Magoun’s reports were published. Among the first reports to contradict the findings of Magoun and Rhines were those of Sprague and Chambers
(1954). Sprague and Chambers investigated the motor output of the mPMRF by eliminating descending fibers passing through the mPMRF and applying stimulus trains with current levels slightly above threshold. Unlike Magoun and Rhines, Sprague and
Chambers observed a variety of movement patterns following stimulation. Most notably, excitation or suppression of either flexors or extensors could be observed at a single site and were often reciprocal in nature. The authors emphasized the presence of bilateral responses, with the most common response consisting of ipsilateral limb flexion,
4 contralateral limb extension, and turning of the head towards the ipsilateral limb.
Movements were not limited to multi-segmented or bilateral responses; movement of a single limb or the head could be observed at some sites. Generalized excitation or suppression was observed only with stronger stimulus currents, although reciprocal responses were evoked with lower, threshold level currents at these sites. Nevertheless,
Sprague and Chambers demonstrated that the motor output of the PMRF was capable of patterned or reciprocal movements and was not limited to generalized changes in muscle tone or posture.
In a later review, Magoun (1967) addressed the differences observed with stimulation in the PMRF reported by other investigators and in his own reports. In this discussion, Magoun noted that globalized responses are more often reported from anaesthetized, decerebrate cats, while reciprocal responses were more common with stimulation in the awake, intact animal; a point that was also made by Sprague and
Chambers. The PMRF receives afferents from several regions of the nervous system, predominately from the motor and premotor areas of the cortex (Keizer and Kuypers,
1984;Keizer and Kuypers, 1989;Sprague and Chambers, 1953;Brodal et al., 1967;ROSSI and Brodal, 1956), the cerebellum (Sprague and Chambers, 1953), and the superior colliculus (Werner et al., 1997a;Kawamura et al., 1974). Considering the inputs to the reticular formation, it is possible that PMRF output is altered in preparations where these inputs are removed. In a recent review of descending motor control, Canedo (1997) points out that the responses produced with stimulation at a particular site in the PMRF may not equate to the output that can be produced by a single reticulospinal neuron when afferents from other regions of the nervous system summate on that neuron. This may be
5 especially true in decerebrate preparations where cortical and tectal input may be removed entirely.
As discussed by Drew and Rossignol (1990b), it is not certain whether or not the state of the preparation does play such a role in the motor responses of the PMRF.
Sprague and Chambers investigations were conducted in both the intact and decerebrate preparations, with stimulation producing similar results in both preparations. On the other hand, generalized inhibition has been observed bilaterally with stimulation in intact, unanaesthetized cats (Lai and Siegel, 1988). Thus, the type of preparation or the behavioral state of the preparation can not fully explain the differences that are observed among these reports.
The differences of generalized or reciprocal responses likely reflect the differences in the stimulation methods employed. Today, stimulation methods utilizing intense currents (up to and exceeding 1 v) are rarely employed in the neurophysiological study of motor systems. Extreme intensities are effective in activating the targeted neurons or axons, though it is difficult to control the degree of current spread with these currents, especially when they are applied in long durations. It is generally accepted that this type of stimulation is difficult to localize to the specific area of interest. In contrast, the low intensity currents utilized by Sprague and Chambers were probably limited to reticular neurons located within a small radius of the stimulating electrode. This is consistent with recent experiments in the reticular formation of the cat that have estimated the current spread of a 35 µA stimulus train to be less than 0.5 mm (Drew and
Rossignol, 1990a).
6 More recent experiments utilizing threshold level stimuli in the intact, awake cat have corroborated the findings of Sprague and Chambers (Drew and Rossignol,
1990b;Drew and Rossignol, 1990a). With short stimulus trains applied at juxtathreshold levels, Drew and Rossignol described movement patterns similar to those described by
Sprague and Chambers. The most common pattern observed by the authors was identical to Sprague and Chambers descriptions and included ipsilateral elbow flexion, contralateral elbow extension, and turning of the head to the stimulated site. Unlike
Sprague and Chambers, the authors reported that the majority of evoked movement patterns were multi-segmented and movement of a single limb was never observed; only head movement was observed in isolation. EMG responses to stimulation were also examined for the neck, forelimb and hind limb, and supported the investigators descriptions of visually observed movements. An analysis of EMG for these movements revealed that facilitation was the typical response for all muscles, and the only muscle that demonstrated suppressive effects was the ipsilateral elbow extensor, triceps.
Reticulospinal Function in the Primate
There are few studies that have examined the functional significance of the PMRF for motor control in the monkey. In a seminal pair of reports, Lawrence and Kuypers
(1968a;1968b) present a dichotomy of the neural control of movement by defining the concept of the medial and lateral motor systems. In these studies, the lateral and ventral regions of the spinal cord were lesioned and the behavioral deficits that followed were described. According to these descriptions, the lateral system, consisting of the corticospinal and rubrospinal tracts that descend in the lateral funiculus, is vital for the
7 control of skilled movements of the hands and fingers. The medial system is comprised of the descending motor tracts of the ventral funiculus: vestibulospinal, reticulospinal,
interstiospinal and tectospinal tracts. Disruption of the medial system resulted in deficits
of posture and balance, and the inability to climb freely within the cage. The monkeys
were capable of only gross reaching movements, but were unimpaired in their ability to
grasp and manipulate food with their hands and fingers. Despite that all the deficits
observed following lesions of the medial system can not be attributed to RST lesions, it is
likely that disruption of the RST was a major factor in the impairments observed.
In addition to the impairments reported following disruption of the medial system
in the monkey, evidence suggests the reticulospinal tract is involved in the performance
of voluntary reaching movements. In anatomical study, Keizer and Kuypers et al. (Keizer
and Kuypers, 1989) injected anterograde tracers into the cortex and retrograde tracers
into the cervical spinal cord. This revealed an abundance of corticoreticular projections
originating from the motor and pre-motor cortical areas that terminate in regions of the
PMRF where RS cells are located (Keizer and Kuypers, 1989). The idea that the
reticulospinal system is involved in the voluntary control of reaching is supported by
findings from our own lab that have demonstrated movement and preparatory related
activity in PMRF neurons in monkeys performing reaching movements (Buford and
Davidson 2004).
8 1.3 Patterns and Organization of Reticulospinal Motor Outputs
Several studies have attempted to categorize reticular neurons on the basis of their
behavioral related activity or efferent and afferent connections (Eccles et al.,
1975;Peterson et al., 1974;Siegel et al., 1983;Siegel and Tomaszewski, 1983;Siegel et al.,
1979;Siegel and McGinty, 1977;Suzuki and Siegel, 1985;Suzuki et al., 1989;Canedo and
Lamas, 1993;Yen and Blum, 1984). Other studies have attempted to define a somatotopic
organization of the motor output of the reticular formation (Eccles et al., 1976;Drew and
Rossignol, 1990b;Drew and Rossignol, 1990a;Peterson et al., 1979;Peterson, 1979;Ito et
al., 1970a). Peterson and colleagues have presented a somatotopic organization of
monosynaptic projections to axial and limb motoneurons in the cat (Peterson 1979;
Peterson et al. 1979). In a series of experiments, these investigators recorded intracellular
motoneuron potentials in the ipsilateral spinal cord in response to stimulation of the
PMRF in the decerebellate cat. In the somatotopic organization described by Peterson
and colleagues, excitatory monosynaptic projections to neck motoneurons (trapezius,
biventer cervicis, and splenius muscles) were located over the entire region of the PMRF,
including a region caudal to the abducens nucleus that was specific for projections for neck motoneurons. Back motoneurons were also excited monosynapticly from sites located over the extent of the PMRF, but from fewer sites than neck motoneurons.
Excitatory monosynaptic projections were less common for the limbs than for the neck and back, and were confined to an area rostral to the abducens nucleus to the dorsal half of the brainstem at the level of the rostral half of the inferior olive. Within this region, the investigators were unable to find any differences in the prevalence of hindlimb and forelimb projections, a finding that was confirmed by Habaguchi et al. (2002).
9 Polysynaptic excitation of all motoneurons was evoked from the entire region of the
PMRF.
Inhibitory projections to motoneurons were also described by Peterson and colleagues (Peterson et al. 1979). Polysynaptc inhibitory projections could be found for any motoneuron at any site in the PMRF. Slow rising, long duration inhibitory potentials, similar to those described by Magoun and Rhines, were observed in forelimb, hindlimb, back and neck motoneurons after simulating the caudal region of the medulla. Of all motoneurons studied, reticulospinal inhibition was found to be strongest and most prevalent in neck motoneurons. Inhibitory monosynaptic projections were found only for these motoneurons and were located in the most caudal portion of the medulla.
Additionally, polysynaptic inhibition of neck motoneurons was evoked from sites in the contralateral, as well as ipsilateral PMRF.
These results present some evidence that supports Magoun and Rhines descriptions of an inhibitory region in the medulla. On the other hand, this area was not entirely inhibitory since monosynaptic excitation of neck and back motoneurons could be observed at stimulation sites within this same region. Also in the decerebrate cat,
Habaguchi et al. (2001) found similar mixed effects on hindlimb motoneurons from the caudal medulla that included EPSPs and IPSPs in flexor and extensor motoneurons, although IPSPs were more prevalent.
Other studies have examined the organization of reticulospinal output on a behavioral level. Drew and Rossignol (1990 a,b) examined the somatotopic organization of movements evoked with stimulus trains in the medullary reticular formation of the awake, intact cat. In a pair of companion reports, the authors describe a
10 rostrocaudal/dorsoventral organization of sites evoking movement. Neck movements
were more commonly evoked from caudal regions, hindlimb movements were confined
to the rostroventral medulla, and forelimb movements were typically evoked from
regions in between. The authors emphasized this organization was not specific and some
overlap existed between these regions; head and forelimb movements were evoked over the entire medullary reticular formation. In a bilateral comparison, contralateral limb movements were more frequently evoked from ventral regions and ipsilateral limb
movements were more frequently evoked from dorsal regions.
Some studies have examined movement resulting from reticular formation stimulation in the monkey (Quessy and Freedman 2004). Cowie and Robinson
(1994;1994) explored the PMRF of awake, intact rhesus monkeys to investigate the
control of head movement. Using repetitive microstimulation, the authors described a
region that corresponds to the NRGc that was effective in producing head movement. In
addition to head movement, the authors also reported the observation of neck, back,
shoulder, and arm movement. Sites producing movement were found to have a
dorsoventral organization, with head and neck movements found more ventral, arm
movements more dorsal, and shoulder back movements found in between. These
movements, however, were never quantified with EMG.
1.4 Mechanisms of Reticulospinal Actions in the Spinal Cord
It is possible that the fast, monosynaptic type responses observed with PMRF
stimulation are a result of current spread to other descending motor systems, such as the
pyramidal tract. Since the presence of corticospinal tract (CST) collaterals in the PMRF
11 has been well documented (Kably and Drew, 1998), it is possible the PMRF stimulation
merely excites these collaterals and eventually the CST. To determine if this was indeed
the case, Shapovalov (1971b;1973) attempted to characterize reticulomotoneuronal (RM,
monosynaptic projections from the PMRF) to α lumbar motoneurons in the monkey. For
these experiments, single pulse and repetitive stimulation were applied to the PMRF, and
the resulting EPSPs in lumbar motoneurons were quantified with intracellular recordings.
Large magnitude, short latency monosynaptic responses were observed in these
motoneurons with latencies in the range of 0.5 to 0.8 ms. The pyramidal tract was then
lesioned caudal to the medulla in some subjects and the precentral gyrus was destroyed in
others. Disruption of the corticospinal tract or the motor cortex had no effect on the
magnitude, sign, or latency of α lumbar motoneuron response to PMRF stimulation.
Shapovalov concluded that the monosynaptic effects observed following PMRF
stimulation were not a result of the activation of the corticospinal tract or collaterals of
the corticospinal tract. A comparison of RM and corticomotneuronal (CM) projections
also revealed that RM projections were weaker than and not as common as CM projections.
Shapovalov (Shapovalov et al., 1971a;Shapovalov et al., 1967;Shapovalov et al.,
1971b) conducted similar experiments in the cat to determine if the rubrospinal or
vestibulospinal tracts contributed to the responses observed with stimulation in the
PMRF. In these experiments, the responses of lumbar α motoneuron to PMRF
stimulation in cats with chronic lateral vestibular nucleus or red nucleus lesions were
compared to those in the intact cat. It was discovered that the motoneuron responses were
12 identical in all three cases. Furthermore, it was concluded that these effects were indeed mediated by the RST.
Even though excitatory and inhibitory monosynaptic projections have been well documented in the ventral reticulospinal pathways (Jankowska et al., 1968), some evidence indicates that reticulospinal actions are mostly indirect and mediated by interneurons (Lloyd 1941; Engberg et al. 1965; Drew and Rossignol 1990 a,b). In the deceberate cat, it was shown that descending projections from the PMRF also act on interneuronal pathways in the dorsolateral region of the spinal cord (Engberg et al.,
1968a;Engberg et al., 1968b). Specifically, it was demonstrated that the RST exerts effects exclusively on the Ib inhibitory and flexor reflex afferent (FRA) pathways. After sectioning the ventral half of the spinal cord rostral to the region studied, it was shown that PMRF stimulation attenuates EPSPs and IPSPs in interneurons excited by flexor reflex afferents, and reduces Ib inhibitory responses on interneurons and hindlimb motoneurons. More recently, Takakusaki et al. (2001) sought to determine if reticulospinal inhibition was mediated indirectly through interneuronal pathways, or acted directly on motoneurons. The efforts of these investigators revealed that inhibitory reticulospinal actions were parallel, acting both indirectly on interneuron populations and directly on motoneurons. These authors found that actions of FRA interneurons were depressed with stimulation of ventral reticulospinal pathways, which is similar to the reports from Engberg and colleagues for the dorsal reticulospinal system. In contrast to
Engberg and colleagues, some Ib interneurons were found to be excited with reticular formation stimulation. It was concluded by Takakusaki et al. that inhibitory FRA and Ib interneurons likely mediate reticulospinal inhibition of motoneurons.
13 A question of particular interest is whether or not the reticulospinal system exerts a differential control over flexors and extensors. Previous studies in the monkey have shown that the red nucleus is preferential for the facilitation of both proximal and distal extensors (Belhaj-Saif et al., 1998;Cheney, 1980;Cheney et al., 1991b;Cheney and Fetz,
1984;Mewes and Cheney, 1991;Mewes and Cheney, 1994), while the motor cortex is more effective on extensors distally and flexors proximally (Cheney et al., 1985;Cheney and Fetz, 1985;Cheney et al., 1991a;Cheney, 1985;Cheney et al., 1982;Fetz and Cheney,
1980;Fetz et al., 1976;Kasser and Cheney, 1985;McKiernan et al., 1998;Park et al.,
2001). It has been reported that reticular formation stimulation in the decerebrate cat attenuates hindlimb flexor reflex activation of flexor motoneurons (Llinas and Terzuolo,
1965), and reduces decerebrate rigidity in extensor motoneurons (Llinas and Terzuolo,
1964). Also in the decerebrate cat, Lund and Pompeiano (1968) discovered the opposite pattern by recording monosynaptic EPSPS in hindlimb flexor and extensor motoneurons.
In the decerebellate cat, Grillner and Lund (1966) found reticular formation stimulation produced monosynaptic excitation in hindlimb flexor and extensor motoneurons, but excitation was more common for flexors. Peterson and colleagues (Peterson 1979;
Peterson et al. 1979), as well as Wilson and Yoshida (1969), were unable to confirm this finding and found monosynaptic excitation to be just as common in flexor and extensor motoneurons. By examining EMG responses in the intact cat, Drew and Rossignol (1990 b) were also unable to support this finding and found that inhibition of any muscle was rare, which was observed only in the ipsilateral extensor, triceps. Similar to Sprague and
Chambers (1954), Drew and Rossignol also found that excitation of flexors were more common with medial stimulation sites, where extensor excitation was more common
14 laterally. The authors attributed the prevalence of extensor excitation in the lateral
regions of the reticular formation to the presence of descending fibers from the lateral
vestibular nucleus.
The focus of a recent series of reports by Jankowska and collegues were the
crossed reticulospinal projections to contralateral motoneurons in the hindlimb of the cat
(Bannatyne et al., 2003;Jankowska et al., 2003;Krutki et al., 2003). Jankowska et al.
(2003) investigated the mechanisms by which descending reticulospinal projections may
influence contralateral hindlimb motoneurons. The authors presented several pathways to
describe this interaction, the most direct pathways consisting of monosynpatic projections
from the ipsilateral PMRF to contralateral motoneurons or branches of ipsilaterally
descending fibers that cross over the midline at the segmental level of the motoneuron.
These branching patterns have been demonstrated in previous reports (Peterson et al.,
1975); however, they are not the only pathways by which contralateral motoneurons are
influenced by the ipsilateral PMRF. When Jankowska and colleagues sectioned the
contralateral spinal cord at rostral lumbar regions, responses in contralateral motoneurons
were not eliminated and it was concluded that these responses were mediated through
interneurons. In one pathway, contralateral motoneurons could be targeted by
contralateral interneurons that receive projections that cross the midline from the
ipsilateral cord. In a second pathway, contralateral motoneurons could be contacted by
commissural interneurons from the ipsilateral spinal cord. Exactly which pathway may
mediate these polysynaptic, bilateral effects is not understood.
15 1.5 Spike and Stimulus Triggered Averaging
The methods of spike and stimulus triggered averaging (SpikeTA, StimulusTA) were introduced over twenty years ago (Cheney and Fetz, 1985;Fetz and Cheney, 1980).
SpikeTA and StimulusTA have been employed in numerous experiments to study the motor cortex (Botteron and Cheney, 1989;Cheney et al., 1985;Fetz and Cheney, 1980), supplementary motor cortex (Hummelsheim et al., 1986), sensory cortex (Widener and
Cheney, 1997), red nucleus (Belhaj-Saif et al., 1998;Cheney, 1980;Cheney et al., 1991b), and in the dorsal root ganglia (Flament et al., 1992) and interneurons of the spinal cord
(Fetz et al., 1996). With these methods, it is possible to correlate the activity of a single neuron, or the electrical activation of a very small region of cells, with changes in EMG.
Prior to the development of these methods, it was difficult to study the motor output of a single cell, and the effects of electrical stimulation were measurable only with stimulus intensities much larger than what are used for StimulusTA. In SpikeTA and StimulusTA, the changes in the membrane potential of a muscle are correlated with a neuron or a small electrical stimulus by using the action potential or stimulus as an averaging trigger for
EMG. This is accomplished by compiling peri-spike or peri-stimulus averages from EMG recorded concurrently with the neuron or stimulus. Since the responses in the averages are often small and unnoticeable with one or two occurrences, several hundred
(StimulusTA) or several thousand (SpikeTA) triggers are typically used. For StimulusTA, stimulus intensity is kept to a level below threshold to ensure EMG changes recorded with stimulation are not the result of spatial summation and movement is not evoked.
These stimuli are also applied at a low frequency to prevent temporal summation.
16 SpikeTA and StimulusTA are generally more effective when there are fewer
synapses between the neuron (or stimulus site) and the motoneuron. Since StimulusTA
examines the output of all neurons at a stimulus site and SpikeTA examines the effect of
a single neuron, StimulusTA can reveal mono-, di- or polysynaptic connections, while
spike triggered averaging typically reveals monosynaptic, and sometimes disynaptic,
connections (Fetz and Cheney 1980; Cheney and Fetz 1985). The magnitude of responses
resulting from each of these methods may also differ, since the output of more than one
neuron is more likely to produce stronger responses in a muscle than a single neuron.
When used together, these methods can help reveal the long pathways and connections that underlie a descending motor system.
The experiments presented in this dissertation were conducted in an attempt to improve the understanding of the motor functions of the PMRF in the non-human primate. In particular, the focus of this dissertation was to examine the bilateral influence of the RST on muscles of the wrist, elbow, and shoulder in awake, behaving monkeys trained to perform a reaching task. This examination was conducted in two experiments, each utilizing different methods that examine correlations between the activity of a single neuron or electrical stimulus with muscular activity. The methods of SpikeTA and
StimulusTA were used to quantify the bilateral motor output of the reticular formation.
The first emphasis was to determine if the pattern of bilateral movements evoked from the cat PMRF described by Sprague and Chambers and Drew and Rossignol can also be observed in the monkey. This question was examined using both SpikeTA and
StimulusTA. Included in this examination was an analysis of proportions of facilitation and suppression, amplitude of responses, and onset latency and duration of responses for
17 StimulusTA. A similar analysis was conducted for SpikeTA, though the primary goal of
SpikeTA was to determine if a single neuron demonstrated wide ranging, bilateral effects,
or if the output of a single PMRF neuron is limited to one or two muscles. A secondary
emphasis was to determine the organization of the PMRF on the basis of motor output.
Additionally, the results yielded from each method were compared for similarities or
dissimilarities in patterns of output. Differences or similarities in the results of SpikeTA
and StimulusTA may help indicate patterns of organization within the PMRF by
revealing differences in the output of a single neurons and groups of PMRF neurons
located within a small region.
The study presented in Chapter 2 has recently been published (Davidson and
Buford 2004). The results of this study indicate that the motor outputs of the PMRF are
involved in the control of proximal arm and shoulder muscles, which is sometimes
organized in a reciprocal pattern for antagonists around a joint. A similar pattern could be
observed in the upper trapezius, where the ipsilateral and contralateral muscles often
demonstrate opposite responses. In the second study presented in Chapter 3, the previous
findings of Sprague and Chambers (1954) and Drew and Rossignol (1990a) are
confirmed by the finding that, in the monkey, PMRF motor outputs are highly organized
in bilateral patterns. Stimulation in the PMRF generally resulted in bilateral responses
that were opposite for the same muscles studied on the contralateral and ipsilateral side,
or for flexors and extensors acting against one another at a single joint. The results of
SpikeTA in the monkey PMRF also corroborate other investigators reports in the cat
(Lloyd 1941; Drew and Rossignol 1990a,b), and suggest that PMRF control of movement
18 is primarily mediated by interneurons, rather than monosynaptic connections with motoneurons.
19
CHAPTER 2
MOTOR OUTPUTS FROM THE PRIMATE RETICULAR FORMATION TO
SHOULDER MUSCLES AS REVEALED BY
STIMULUS-TRIGGERED AVERAGING
2.1 Abstract
The motor output of the medial pontomedullary reticular formation (mPMRF)
was investigated using stimulus-triggered averaging (StimulusTA) of EMG responses
from proximal arm and shoulder muscles in awake, behaving monkeys (M. fascicularis).
Muscles studied on the side ipsilateral (i) to stimulation were biceps (iBic), triceps (iTri),
anterior deltoid (iADlt), posterior deltoid (iPDlt), and latissimus dorsi (iLat). The upper and middle trapezius was studied on the ipsilateral and contralateral (c) side (iUTr, cUTr, iMTr, cMTr). Of 133 sites tested, 97 (73%) produced a post stimulus effect (PStE) in one or more muscles; on average, 38% of the sampled muscles responded per effective site. For responses that were observed in the arm and shoulder, post-stimulus facilitation
(PStF) was prevalent for the flexors, iBic (8 responses, 100% PStF) and iADlt (13 responses, 77% PStF), and post-stimulus suppression (PStS) was prevalent for the
20 extensors, iTri (22 responses, 96% PStS) and iLat (16 responses, 81% PStS). For trapezius muscles, PStS of upper trapezius (iUTr, 49 responses, 73% PStS) and PStF of middle trapezius (iMTr, 22 responses, 64% PStF) were prevalent ipsilaterally, and PStS of middle trapezius (cMTr, 6 responses, 67% PStS) and PStF of upper trapezius (cUTr,
46 responses, 83% PStS) were prevalent contralaterally. Onset latencies were significantly earlier for PStF (7.0 ±2.2 ms) than for PStS (8.6 ±2.0 ms). At several sites, extremely strong PStF was evoked in iUTr, even though PStS was most common for this muscle. The anatomical antagonists iBic/iTri were affected reciprocally when both responded. The bilateral muscle pair iUTr/cUTr demonstrated various combinations of effects, but cUTr PStF with iUTr PStS was prevalent. Overall, the results are consistent with data from the cat and show that outputs from the mPMRF can facilitate or suppress activity in muscles involved in reaching; responses that would contribute to flexion of the ipsilateral arm were prevalent.
2.2 Introduction
The reticulospinal tract is one of the four major descending motor systems, but understanding of this system is limited. Early experiments in the medial pontomedullary reticular formation (mPMRF) of the cat reported that stimulation of the rostrodorsal region facilitated cortically-evoked facilitation of muscle tone, but stimulation of the caudoventral region suppressed cortically-evoked facilitation, suppressed segmental reflexes, and eliminated decerebrate rigidity (Magoun and Rhines, 1946;Rhines and
Magoun, 1946). Sprague and Chambers (1954) later demonstrated that these global
effects were elicited only by prolonged, intense stimulation. With stimulus current levels
21 just above threshold, Sprague and Chambers revealed more complex reticulospinal
actions. The most common response observed was a combination of ipsilateral forelimb
flexion, contralateral forelimb extension, and turning of the head to the stimulated side.
In some cases, movement was limited to a single body segment (forelimb, head, or
hindlimb). Thus, Sprague and Chambers found that outputs from the mPMRF can be more specific than those described by Magoun and Rhines.
As a descending system, the reticulospinal tracts are remarkable for the potential of individual neurons to have widespread effects. Projections from the mPMRF descend
throughout the length of the spinal cord in the medial and lateral regions of the ventral
funiculus (Kuypers, 1981). Terminals from single reticulospinal axons project to both
sides of the spinal cord at multiple segmental levels (Kuypers, 1981;Matsuyama et al.,
1997;Matsuyama et al., 1999;Peterson et al., 1975;Sasaki, 1997) and are concentrated in the ventromedial grey, but also reach the lateral horn and motoneurons of the neck and forelimb (Alstermark et al., 1987;Holstege and Kuypers, 1987;Iwamoto et al.,
1990;Sasaki, 1999). Electrophysiological methods have revealed excitatory monosynaptic projections to motoneurons of neck and axial muscles from the dorsocaudal and ventrorostral regions of the mPMRF (Peterson et al., 1975;Peterson et al., 1979;Peterson, 1979). Stimulation in the ventrorostral region has also produced monosynaptic excitation of flexor and extensor motoneurons, while polysynaptic excitation and inhibition was produced in the same motoneurons from the dorsocaudal regions (Peterson et al., 1979;Peterson, 1979). Inhibitory effects appear to rely on spinal interneurons (Takakusaki et al., 2001), and contralateral effects likely depend on
commissural interneurons as well as direct projections (Jankowska et al., 2003).
22 In intact, awake cats, Drew and Rossignol (Drew and Rossignol, 1990a) reported
that repetitive microstimulation produced movement patterns that most often included
flexion of the ipsilateral limb, extension of the contralateral limb, and turning of the head
to the ipsilateral side. These results were consistent with the findings of Sprague and
Chambers (Sprague and Chambers, 1954), except Drew and Rossignol (Drew and
Rossignol, 1990a) did not observe exclusive movement of a single body segment.
Drew’s work has demonstrated a central role for the reticulospinal system in the control
of locomotion in the cat (Drew et al., 1986;Drew, 1991;Matsuyama et al., 2004), as well
as postural support for reaching movements from a tripedal stance (Schepens and Drew,
2003a). This functional role is consistent with the anatomical and physiological evidence described above, indicating that reticulospinal outputs can concurrently affect axial muscles and proximal limb muscles in the forelimb and hindlimb.
Existing data from the primate indicates that, as in the cat, some reticulospinal projections are monosynaptic (Shapovalov, 1972). Reticulomotoneuronal projections, however, are less prevalent than corticomotoneuronal projections, and terminate mostly in laminae VII and VIII, instead of lamina IX. In the awake monkey, stimulation within and around nucleus reticularis gigantocellularis (NRGc) has produced head movement, as well as movement of the face, mouth, neck, shoulder, and arm (Cowie and Robinson,
1994). These findings were based solely on observation without EMG, and the effects for individual stimulus sites were not reported.
In the primate, the majority of cortical input to the mPMRF originates in the premotor cortex, supplementary motor area, and primary motor cortex (Keizer and
Kuypers, 1989). Considering the critical roles of these areas for the planning,
23 preparation, and execution of reaching (Tanji, 2001;Wise et al., 1997), one might expect
the reticulospinal system to play an important role in voluntary reaching. Such a role is supported by the presence of movement related discharge in mPMRF neurons during
reaching for the primate (Buford, 1996) and the cat (Schepens and Drew, 2003b). Areas of the mesencephalic reticular formation that project to the mPMRF have also been
shown to have neural activity related to reaching (Gibson et al., 1998;Werner et al.,
1997a). To begin to describe the role of the reticulospinal system for the control of
voluntary reaching movements in the primate, the present study employed the method of
stimulus triggered averaging (StimulusTA) to characterize the sign and distribution of
mPMRF outputs to axial, shoulder, and proximal arm muscles (Cheney and Fetz, 1985).
Portions of these results have been presented in abstracts (Davidson and Buford,
2001;2002).
2.3 Methods
Subjects, Task, and Surgery
The subjects were two male Macaca fascicularis monkeys (C and D) trained for a
separate study. In the context of an instructed delay task, the subjects performed planar reaching movements (5.08-cm displacement) from a central position to one of four peripheral targets (45°, 135°, 225°, 315° in Cartesian coordinates). A sip of flavored applesauce was the reward for each correct trial. The head was restrained for recording and stimulation to help maintain stable electrode positioning. Experimental procedures were approved by the ILACUC of the Ohio State University, and subject care was according to the NIH Guide for the Care and Use of Laboratory Animals.
24 Following training, a stainless steel recording chamber was implanted over a
craniotomy in the left parietal bone. The chamber’s axis was in the frontal plane, angled
10° to the left from the parasagittal plane to allow access to the right mPMRF, and aimed
for stereotaxic coordinates AP0, ML0, DV-12 (Horsley-Clark stereotaxic coordinates
(Szabo and Cowan, 1984). Surgery was performed under Isoflorane inhalation anesthesia with Ketamine HCl as a preanesthetic. Analgesics (buprenorphine, ibuprofen) and a
long-acting antibiotic (Baytril) were given following surgery.
EMG Implants
Electromyographic data were collected with acute percutaneous (first six averages
from subject D) and chronically-implanted pairs of Teflon-coated stainless steel wires.
Wire pairs were separated by approximately 5-mm and inserted into the muscle by a
hypodermic needle (Betts et al., 1976;Park et al., 2000). For the chronic implants, the
wires were led subcutaneously to a 17-pin plug (WPI #223-1617) mounted in the dental
acrylic of the cranial implant. The integrity of EMG implants was verified by stimulating
through the EMG wires, and by periodic testing of electrode impedances. Since
recordings were made from the right reticular formation, ipsilateral muscles were located
on the right side. For both subjects, electrodes were located in the contralateral and
ipsilateral upper trapezius (cUTr, iUTr) and ipsilateral posterior deltoid (iPDlt).
Additional implants in subject C were located on the ipsilateral side in latissimus dorsi
(iLat), long head of triceps (iTri), long head of biceps (iBic), and in contralateral middle
trapezius (cMTr). For subject D, additional implants were located in the ipsilateral
anterior deltoid (iADlt) and ipsilateral middle trapezius (iMTr). EMG data for ipsilateral
25 iBic and iTri were available from subject D for the six experiments conducted with
percutaneous EMG. Over the eight months of study most EMG implants remained
stable, except for iADlt in subject D, which failed in the last month.
EMG records were tested for the presence of crosstalk between muscles. For this
analysis, data were recorded for extended periods of time without stimulation. For these
data files, motor unit action potentials from each muscle were selected as triggers for compiling EMG triggered averages for the remaining muscles. Crosstalk was considered to be present if the EMG triggered averaged of any muscle exceeded 15% of the averaged amplitude of the triggering muscle (Cheney and Fetz, 1985;Fetz and Cheney, 1980).
Overall, one data file from each week of the study was examined for each subject.
Crosstalk was discovered from the outset between the chronic recordings of iPDlt and iUTr in subject D, and as a result, iPDlt data was removed for that subject. No other cases of crosstalk were observed.
Recording and Stimulation
Extracellular recording and stimulation were performed with Tungsten microelectrodes (0.2-mm stock) that were epoxy and polyimide insulated (Frederick Haer
& Co, Bowdoinham, ME). The electrode tips were conditioned with gold-plating to produce recording impedances in the range of 90-200 KΩ. Electrodes were inserted through a thin-walled 23 gauge stainless steel guide cannula positioned in the recording chamber by an X-Y grid, and lowered into the brainstem with a manual hydraulic microdrive.
26 All recording and stimulation sites were in the right mPMRF (Fig. 2.2). The
dorsal boundary of the recording/stimulating region was the abducens nucleus (defined
by cells with firing rates proportional to eye abduction) (Luschei and Fuchs 1972); the
ventral boundaries were the inferior olive and pyramidal tract. The facial nucleus defined
the lateral boundary, and the medial boundary was located 0.5 mm lateral to the midline.
Arm-related neurons were identified by their modulation of firing during arm movement
and by their responses to manipulation of the arm and shoulder. Neuronal activity, EMG
(sampled at 4.63 KHz), records of stimulation, hand position, and logic signals representing the state of the task were recorded with Spike2 software and a Power1401
acquisition unit (CED, Cambridge, U.K.). Neural data collected here was used as part of
a separate study of the mPMRF activity during reaching (Buford, 2000). After a
recording track was complete, the electrode was retracted and single pulse
microstimulation for StimulusTA was applied at or near sites where task related neurons
had been recorded.
To determine the appropriate stimulus current for each site, threshold testing was
conducted with repetitive microstimulation (12 biphasic pulses, negative, then positive,
200 µs/phase, 333 Hz) with a current ranging from 10 to 50 µA. Threshold was defined
as the current at which a muscle twitch or slight movement was consistently observed.
For StimulusTA, low frequency single pulse microstimulation (13.3 Hz, 75 ms interval,
2000 stimuli) were initially applied at a current intensity of about 1½ times the threshold.
If no response was observed during threshold testing, single pulse current for StimulusTA
was set at 30 µA. This current was chosen as a constant to permit testing for suppressive
effects that may not have been evident on observation. In some cases, the current used
27 for single pulses produced overt muscle twitches and current was decreased in 5 µA
increments until overt muscle twitches were abolished. Nevertheless, low intensity
stimuli (10 µA) sometimes produced overt (though slight) twitches at some sites, and
these records were included. The vast majority of the data reported here was associated
with current intensities that did not produce any overt contraction with a single pulse.
Averaging and Analysis
EMG records were adjusted to remove DC offsets, rectified, and averaged offline
using a custom script for Spike2. Averages were compiled over an 80 ms epoch that was
divided into a 20-ms pre-trigger period and a 60-ms post-trigger period. Triggers for periods when EMG exceeded the maximum range of the amplifier (4.5 V) were excluded
on a channel-by-channel basis. Consequently, the number of triggers in a single average
varied for each muscle. Approximately 1900 triggers were typically used for
StimulusTA analysis.
Criteria for a post-stimulus effect (PStE) were established relative to the mean and
standard deviation of a pre-stimulus baseline derived from the first 19 ms of each
average, ending 1 ms before the stimulus to prevent stimulus artifact from corrupting the
baseline period. Averaged EMG responses that differed from the mean of the pre-
stimulus EMG baseline by at least 2SD of the mean for at least 2 ms were considered
candidates for analysis (Mewes and Cheney, 1991). Onset and offset latencies were
determined by the points of intersection between the EMG averages and the ±2 SD
thresholds. The amplitudes of the PStEs were quantified by the standard deviation score
of the peak (SDPk), the Z-score (number of SD’s) of the peak displacement from
28 baseline, as well as by the mean percent change (MPC), the percent difference of mean
EMG activity of the PstE from the EMG baseline (Cheney and Fetz, 1985;Cheney et al.,
1991b;Kasser and Cheney, 1985).
By utilizing H-reflex testing in biceps and collision tests for reticulospinal
neurons from the cervical spinal cord in other subjects, we have estimated the earliest
onset latency for biceps from the mPMRF to be 4.5 ms (unpublished results, Buford).
Since more proximally located muscles (e.g., upper trapezius) were examined in the present study, 3.5 ms was chosen as the minimum acceptable onset latency for all muscles.
To examine the possibility that fluctuations in the EMG unrelated to the stimulus may have produced some of the PStEs, random triggered averages (RandomTA) were compiled for sites where post-stimulus effects were obtained. Using a custom script for
Spike2 (CED), 2000 fake stimulus events were created at random times within the period of stimulation and used as triggers for averaging. RandomTAs were computed and analyzed with procedures identical to those used for Stimulus TA.
There were 420 potentially significant events from StimulusTA, and 151 from
RandomTA. Examination of the distributions indicated that events beginning after 25-ms
were just as likely from RandomTA as they were from StimulusTA. The largest
difference was present for onset latencies between 3.5 ms and 12.5 ms, where events
from StimulusTA were eight times more frequent than those from RandomTA. This was
also a realistic time range for post-stimulus effects to begin. Therefore, we sought to develop additional criteria that would eliminate RandomTA events from the 3.5 – 12.5 ms window while preserving StimulusTA events. The parameters that discriminated
29 most effectively between StimulusTA and RandomTA events were duration and MPC.
As described in the RESULTS, by limiting the analysis to events with onset latencies from 3.5 – 12.5 ms (as detected with the 2SD threshold), duration of at least 2.5 ms, and a mean amplitude of at least 6% (MPC), we accepted 199 events from StimulusTA. Only
13 RandomTA events were not eliminated by these criteria.
A 2-way ANOVA by muscle and response type (facilitation or suppression) was used to compare effects on onset latency, duration, SDPk, and stimulus current level.
The frequencies of post-stimulus facilitation (PStF) and post-stimulus suppression (PStS) were compared across muscles with a chi-squared analysis. The significance level was set to P ≤ 0.05.
Anatomy and Histology
Electrolytic lesions were made with 20 µA for 20 s (DC anodal) in the final tracks at specific points of interest. After all lesions were made, subjects were deeply anesthetized with sodium pentabarbitol and perfused transcardially with phosphate buffered saline, followed by phosphate buffered formalin. The brain was removed and soaked in neutral buffered formalin with 30% sucrose for cryoprotection. Frontal sections were cut at 40 µm on a freezing microtome and every 4th section was mounted and stained with cresyl violet. The sections were scanned at 1200 dots per inch and the anatomical reconstructions were completed in software. Structures in the brainstem were identified according to a stereotaxic atlas (Szabo and Cowan, 1984) and a template atlas available online from the primate information center at the University of Washington
30 (Bowden et al., 2003). The locations of EMG recording electrodes were verified by post
mortem dissection.
2.4 Results
Task and EMG
A representative sample of EMG recorded from Monkey C during task performance is presented in Fig. 2.1. In this segment, the monkey began the trial by waiting in the start position, and then an instruction (target cue) was presented to direct
movement to one of the four targets. The monkey was then given the movement cue and
he subsequently moved to contact the correct target and held his hand on the target until
the reward was given. After the reward, a new trial began. For the purpose of
StimulusTA in the muscles studied, the task created moderate levels of EMG activity that
were sufficient to allow observation of post-stimulus facilitation or suppression. In iLat
and the upper and middle trapezius muscles, EMG activity was often highest as the
monkey held his hand in the start position. Modulation of iBic, iTri, and the deltoids was
most evident during reaching to the targets.
Selected analyses were performed to determine if the results of StimulusTA
varied depending on the direction of movement. Stimulation sites chosen for this analysis included sites from which single pulse stimulation produced substantial PStEs for several muscles, and sites near cells that demonstrated a directional preference for at least one of the four targets. These records were chosen for this analysis because the potential of observing movement or target related variation in the responses were
expected to be highest for these sites. In total, data from 10 sites (8%) were analyzed. In
31 no case was there a substantial difference in the PStEs when triggers were separated on
the basis of movement direction. There was also no substantial difference in the averages
constructed from within the movement periods and the averages constructed from the
overall period of stimulation. Therefore, only results from the overall period of
stimulation are presented here.
Locations of Stimulus Sites
The locations of stimulus sites were anatomically reconstructed and plotted by
muscle and type of response (PStF vs. PStS) to examine the organization of stimulus
sites. No somatotopic organization was evident for any muscle or response type.
Effective sites for each muscle were intermixed, and PStS and PStF were evoked from
sites throughout the region studied. The locations of stimulus sites in subject D that were
effective in producing post-stimulus effects in iUTr are presented in Fig. 2.2. Stimulus
sites for this muscle were chosen for this illustration because it was the most responsive
of all the muscles studied, and every site tested included a recording of this muscle. In
Fig. 2.2A, stimulus sites for a selected plane (AP -1) are superimposed on a coronal section; circles represent sites that evoked PStF, triangles represent PStS, and dashes
indicate sites from which no PStE was produced. For all locations, stimulus sites are
illustrated in the parasagittal view in Fig. 2.2B, and in a frontal view in Fig. 2.2C. As the
illustrations show, sites that produced PStF and PStS were intermixed.
Between the two subjects, stimulation was applied at 189 sites from 89 electrode
penetrations. After the anatomical reconstruction, 56 sites were found to be outside the
predetermined boundaries of the mPMRF (see METHODS) and were excluded. The
32 lateral boundary of the acceptable zone was the medial edge of the facial nucleus.
Stimulation at sites lateral to this resulted either in no movement whatsoever, or movement primarily of the face. Stimulation at sites that were too ventral produced no effect unless the electrode was so deep as to stimulate the corticospinal tract directly, resulting in brisk contralateral movements. Medial sties were excluded to prevent stimulation of the medial longitudinal fasciculis. Stimulation in sites that were too dorsal typically produced eye movements. This left 133 stimulation sites for the study. Of these, 109 (82%) were within 0.5 mm of a cell with task-related activity for the reaching task. The remaining 24 sites (18%) were within the same region of the mPMRF. Trains of stimuli (12 pulses at 333 Hz) within this zone produced muscle twitches in the arm, shoulder, and trunk, and occasionally resulted in movement of the face, ear, and neck.
The head was restrained, so it is not known if head movement would have resulted from these stimulus sites. It is presumed that activation of muscles such as the upper trapezius could have moved the head, if it had been free.
Selection of Post Stimulus Effects
Figure 2.3 illustrates the detection of PStEs using the ±2 SD criteria. As illustrated, sequential events with the same or opposite sign could be observed in the response of a single muscle. In this example, cUTr PStF was followed by two periods of
PStS. Because of the possibility that the second and third events may have resulted from segmental effects that were a consequence of the initial output, the present analyses were limited to the first PStE for each muscle. Accordingly, the PStEs analyzed for the
StimulusTA presented in Fig. 2.3 included only two events: PStF in cUTr and PStS in
33 iUTr. There were periods when the post-stimulus waveforms of iADlt and iMTR crossed
the 2SD threshold, but these responses and others like them did not meet the 2.5-ms
minimum duration or the 6% minimum value for MPC.
The distributions of onset latencies for RandomTA and StimulusTA (Fig. 2.4A)
were compared to a uniform distribution with a 1-sample Kolmogorov-Smirnov test. The
distribution for StimulusTA was significantly different from a uniform distribution (P <
0.001), but the RandomTA distribution was not (P = 0.399). Events with onsets at long latencies were suspect because they could have resulted from indirect routes, such as the gamma loop, through mechanisms like those proposed by Kasser and Cheney (Kasser and Cheney, 1985). To determine a cutoff latency beyond which StimulusTA data would not be analyzed, the mean and standard deviation of the number of events per 2-ms bin for the RandomTA data was calculated. On average, there were 2.5 ± 2.0 RandomTA events per 2-ms bin. Taking 2 SD over the mean as a cutoff (dashed horizontal line, Fig.
2.4A), it was evident that events with onset latencies of 26 – 28 ms (represented by the bar at 28 ms) or greater could not be accepted because they were most likely a result of random fluctuations in the levels of EMG. Further, most PStEs with onsets in the first 25 ms actually occurred within the first 12.5 ms. PStEs with onset latencies between 12.5 and 25 ms were 25% as common in the StimulusTA dataset as in the RandomTA dataset.
PStEs with onsets between 3.5 and 12.5 ms, however, were 15 times more likely for
StimulusTA than for RandomTA, so less than 7% of the earlier PStEs were likely to be due to chance. Consequently, all remaining analyses were limited to PStEs with onsets between 3.5 and 12.5 ms. This early window included 199 PStEs, of which 105 (53%) were PStS and 94 (47%) were PStF.
34 Stimulus Currents and Magnitudes of Post Stimulus Effects
Stimulus currents for StimulusTA ranged from 10 to 50 µA, and the average
current for all sites was 31 ± 7 µA. Because response amplitude was a primary criterion
for selection of significant PStEs and the stimulus level varied for single pulse
stimulation, it was important to determine whether differences in PStE amplitudes could
be explained by the use of different stimulation currents. For all events detected, neither
an ANOVA of response magnitude by current (F7,418 = 1.03, p = 0.41) nor a regression
of current against response magnitude ( r2 = 0.004, p = 0.10) showed any significant relationship between these two variables. Thus, variations in stimulus amplitude did not
explain variations in response amplitude.
Fig. 2.4B illustrates the distribution of responses amplitudes in terms of the
variable MPC. For PStEs accepted in the 3.5 – 12.5 ms window, MPC values ranged
from 6% to 31% (12.7% ±4.8) for PStS, and from 6% to 1296% (41.5% ± 142.7) for
PStF. The SDPk values ranged from 2.9 to 13.7 (mean 5.8 ± 2.1) for PStS and from 3.0
to 1176.9 (mean 47.6 ± 174.6) for PStF. Since there was a small number of PStF events
with very large amplitudes, the medians of the MPC (13.1% PStF, 12.2% PStS) and
SDPk (6.0 for PStF, 5.1 for PStS) values may be more reliable indicators of central
tendency for these measurements. For the medians, there was little difference between
the average amplitude of PStF and PStS.
35 Latency and Duration of Post Stimulus Events
Average onset latency was significantly earlier for PStF (6.99 ± 2.21 ms) than for
PStS (8.58 ms ± 2.04 ms), as indicated by the ANOVA (F1,182 = 5.736, p = 0.018). PStF
onset was sooner for six of the nine muscles, and for iMTr and iUTr this difference was
significant (Table 2.1). Although average onset latencies were earlier for PStS than for
PStF in iTri, iLat, and iADlt, these differences were not significant. There was no
significant difference found overall for the duration of PStS vs. PStF. iUTr responses
were significantly longer than those from all other muscles except iBic, iTri, and iLat.
The average duration was longer for PStF than for PStS in iUTr, which was the only
muscle to show a significant difference in duration by response type.
Effectiveness of Stimulation and Proportions of Facilitation and Suppression
The distributions of onset latencies for all PStF and PStS events with onsets
between 3.5 ms and 25 ms are presented in Fig. 2.5 for each muscle. As in Fig. 2.4, each
muscle was most likely to respond in the 3.5 – 12.5 ms interval; data after 12.5 ms are
shown for informational purposes, only. Of the 133 sites analyzed, single pulse stimulation at 97 (73%) produced a minimum of one PStE (PStF or PStS) for at least one
muscle. Since the number of muscles available for recording was not always the same
for each subject or electrode penetration, the percentage of muscles demonstrating a PStE
was calculated as a fraction of the total number of muscles recorded for each stimulus
site. On average, 38% of the muscles recorded demonstrated a PStE for each site tested.
36 The effectiveness of stimulation for each muscle was quantified as the percentage
of sites where a PStE was observed for that muscle as a fraction of the number of
stimulation sites for which that muscle’s EMG was recorded (Fig. 2.5). The most
commonly affected muscles were iUTr, cUTr, iTri, and iADlt, which responded to
stimulation at 35 - 37% of the sites for which they were tested. The least commonly affected muscles were iBic (13%) and cMTr (11%). iPDlt and iLat responded for 27 –
28% of the sites for which they were tested.
As shown in Fig. 2.6, there were different proportions of PStF and PStS for most
muscles studied (χ2 = 64.1, p < 0.000). PStF was the most frequent response for the
elbow flexor, iBic (100%), and the shoulder flexor, iADlt (77%). PStS was most frequent for the elbow extensor, iTri (96%), and for the extensor/adductor of the humerus, iLat (81%). The frequencies of PStF and PStS were similar in the shoulder extensor iPDlt (59% PStS). Overall, for the arm and shoulder muscles, flexors demonstrated higher proportions of PStF and extensors demonstrated higher proportions of PStS. In the upper trapezius, an upward rotator and elevator of the scapula, 74% of ipsilateral events (iUTr) were PStS, while 78% of contralateral (cUTr) events were PStF.
For the middle trapezius muscle, a scapular retractor, PStF was more prevalent ispilaterally (iMTr, 64%), and PStS was more prevalent contralaterally (cMTr, 67%).
Representative Post-Stimulus Effects
An example of a typical StimulusTA is presented in Fig. 2.7. In this example, the majority of the muscles sampled (5/7) demonstrated a PStE, each of which was consistent with the prevalent response for that muscle. PStF was present in iBic and cUTr, and PStS
37 was present in iUTr, iTri, and iLat (see legend for response magnitudes). iPDlt and cMTr
did not respond for this stimulus site. In Fig. 2.8A, iTri, iPDlt, iLat, and iUTr responded
with PStS, their most prevalent sign of response. Fig. 2.8B, however, shows PStS in
cUTr and iMTr, which were usually facilitated, as well as the typical responses of PStF in
iBic and iADlt. Thus, outputs from the mPMRF demonstrated a variety of patterns.
Although PStS was the most prevalent response for iUTr, PStF events with very
large amplitudes were sometimes observed for this muscle. In eight cases, the magnitude
of PStF in iUTr exceeded 48 SDPk; five ranged from 430 to 1177, with onset latencies
between 4.4 ms and 8.3 ms. Stimulus currents for these 8 sites were consistent with those
for other sites and ranged from 10 to 30 µA. Even with a single pulse at 10 µA, EMG deflections (not artifact) were visible in the raw record for some of these low-threshold
sites. An example of such a response is illustrated in Fig. 2.9. At 30 µA, the SDPk of the
iUTr PStF event reached 85, compared to the relatively weak responses observed in other
muscles for this site. Although some strong, early latency responses did occur in other
muscles, they were consistent with their most prevalent response and did not exceed 40
SDPk (typically less than 20). Many of these occurred concurrently with a large
magnitude, short latency PStF event in iUTr. Sites that elicited these strong PStF
responses in iUTr were found throughout the area studied; no unique location was
apparent.
38 Responses among Antagonist and Bilateral Muscle Pairs
The arrangement of the EMG implants in subject C permitted analysis of
interactions between the anatomical antagonists iBic and iTri at 38 sites. In two cases,
both muscles responded to stimulation at a single site. Both of these were reciprocal,
with PStF in iBic concurrent with PStS in iTri (Fig. 2.7). The bilateral muscle pair, cUTr
and iUTr, was recorded concurrently for all 133 sites tested. There were 22 cases where
both muscles responded to stimulation at a given site, so that 37% of the iUTr responses
were paired with a cUTr response. These bilateral responses included a variety f
combinations. In ten of the 22 cases, the bilateral responses were concurrent. The most
frequent type of concurrent response, present in seven cases, was cUTr PStF concurrent with iUTr PStS, as illustrated in Fig. 2.10A. There were also two cases in which cUTr and iUTr were both facilitated, and all of these included strong PStF in iUTr (Fig. 2.9).
In one case, both muscles were suppressed. In addition to concurrent responses, there were ten serial responses, which were defined as cases in which the onset for one muscle came with ± 2-ms of the offset for the other (if a response fit the criteria for serial, it was
not classified as concurrent). Eight of these serial responses included cUTr PStF
followed by iUTr PStS, as illustrated in Fig. 2.10B. There were also two cases where
bilateral PStS responses were serial.
2.5 Discussion
In summary, StimulusTA of EMG was used in the context of a reaching task to
characterize the motor output of the mPMRF in the monkey. The results are consistent
with previous reports from the cat (Drew and Rossignol, 1990b;Drew and Rossignol,
39 1990a;Sprague and Chambers, 1953;Sprague and Chambers, 1954), and indicate that the
primate reticulospinal tract can facilitate or suppress muscles used for reaching. The
ipsilateral arm most frequently responded with extensor suppression and flexor
facilitation. In the trapezius, the most frequent ipsilateral responses were facilitation of middle trapezius and inhibition of upper trapezius, while the opposite pattern was observed contralaterally. Bilateral responses in the upper trapezius muscles were common, and most often this included PStS in iUTr and PStF in cUTr. The iUTr was unique in having a preponderance of responses that were PStS, but a small number of extremely large PStF responses. Onset latencies were shorter for PStF than for PStS.
Reliability of the Results
In a study that is based on responses to electrical stimulation, it is important to be
sure that stimulus intensities were appropriate to prevent excessive current spread. In the
present study, currents ranged from 10 µA to 50 µA, and the average current for all sites
was 31 µA, which was similar to current ranges employed in comparable studies. Cowie
and Robinson (Cowie and Robinson, 1994) used 10 µA to 100 µA stimulus trains (40 ms,
400 Hz) to study the output of the mPMRF for the control of head movements in Rhesus
monkeys, and effects were typically evoked with 40 µA. For StimulusTA of proximal
arm and shoulder muscles from the magnocellular region of the primate red nucleus,
Belhaj-Saif et al. (Belhaj-Saif et al., 1998) employed currents in the range of 20 µA. In a
StimulusTA study designed to map sites in the primary motor cortex with outputs to
shoulder, elbow, wrist, and hand muscles, Park et al. (2001) used 15 µA, increasing to 30
µA only when 15 µA was ineffective. In the reticular formation of the cat, Drew and
40 Rossignol (1990a) estimated the effective spread of a 35 µA stimulus train (33-ms train at
333 Hz) to be less than 0.5-mm. Since the stimuli for StimulusTA are single pulses, temporal summation should have been limited for the present study.
It is unlikely that the present results reflected the activation of structures outside of the mPMRF. Nevertheless, the potential for unintended activation of the CST was a
major concern for the present study. With single pulse and repetitive microstimulation,
movement of the hand or fingers for the ipsilateral or contralateral side was never
observed at any site studied, so it seems unlikely likely that the CST was directly
activated. CST collaterals, however, do terminate in the mPMRF of the cat (Kably and
Drew, 1998;Keizer and Kuypers, 1984) and monkey (Keizer and Kuypers, 1989).
Stimulation of CST collaterals could account for the present results if they excited root
axons that projected exclusively to motoneurons or interneurons of proximal arm and
shoulder muscles. A similar concern exists for the vestibular nuclei, which also project to
the mPMRF (Wilson and Peterson, 1981;Bolton et al., 1992;Matsuyama and Drew,
2000b;Matsuyama and Drew, 2000a). This projection raises the possibility of indirect
activation of the vestibular tracts as a consequence of mPMRF stimulation.
Shapovalov (1972) tested the hypothesis that CST collaterals were activated by
mPMRF stimulation by recording lumbar α motoneuron responses to stimulation of the
medial longitudinal fasciculus, NRGc, and Nucleus Reticularis Pontis caudalis in
pyramidotomized Rhesus monkeys. In 38 subjects, the CST was interrupted by
transection of the pyramids in the brainstem, by acute lesions of the spinal cord, or with
unilateral or bilateral lesions of the precentral gyrus (allowing sufficient time for CST
degeneration). Motoneuron responses to reticular formation stimulation for control
41 subjects were similar to those of the pyramidotomized and cortically lesioned monkeys,
and there was no evidence that the responses evoked from the reticular formation were a
result of CST collateral stimulation. In a separate study performed by Shapovalov and
colleagues (Shapovalov, 1973), the ipsilateral lateral vestibular nucleus and the
contralateral red nucleus were destroyed in the cat and lumbar α motoneurons responses
to reticular formation stimulation were examined. From these experiments, the authors
determined that the effects of mPMRF stimulation were not due to unintended activation
of the LVST or the rubrospinal tract.
Overall, Shapovalov’s findings suggest that the PStEs resulting from mPMRF
stimulation in the present study were mediated by the reticulospinal tract and were not
likely a consequence of unintended activation of the CST, the LVST, or their collaterals.
These findings, however, do not disprove the argument that these structures were
activated with single pulse stimulation in the present study. It would be necessary to
compare StimulusTA effects for arm and shoulder muscles in pyramidotomized or LVST lesioned monkeys to determine if activation of these structures contributes to the pattern
of PStEs reported here.
Comparison with Studies of the Motor Output of the Cat Reticular Formation
As mentioned in the Introduction, Sprague and Chambers (Sprague and
Chambers, 1954) and Drew and Rossignol (Drew and Rossignol, 1990b;Drew and
Rossignol, 1990a) reported that the most common effects of reticular formation
stimulation were ipsilateral elbow flexion, contralateral elbow extension, and turning of
the head to the stimulated side. In the monkey, the prevalence of flexor PStF with
42 extensor PStS in the proximal arm and shoulder would be consistent with a behavioral
response of flexion of the ipsilateral forelimb. Indeed, when PStEs were observed
concurrently in iBic and iTri, the predominant pattern was iBic PStF with iTri PStS. It is
important to note, however, that iBic was the least affected muscle while iTri was among
the most commonly affected muscles in the present results. It is uncertain whether the low effectiveness for iBic reflects the actual output of the reticulospinal tract, or merely reflects the low amplitude and short duration of biceps activity involved in the behavioral
task, which could have decreased the likelihood of observing PStEs in this muscle. EMG
responses were not recorded for the contralateral arm in this study, so it was not possible
to measure contralateral responses. On several occasions, however, contractions in the
contralateral triceps were visible with repetitive microstimulation. Recordings from the contralateral arm could reveal if extensor facilitation is prevalent contralaterally.
Head movement towards the ipsilateral limb was also noted in response to reticular formation stimulation in the cat (Drew and Rossignol, 1990a). In the rhesus
monkey, Cowie and Robinson (Cowie and Robinson, 1994) reported that ipsiversive head
rotation was elicited by repetitive microstimulation in and around the region of the
NRGc. In the present study, the head was restrained and it was impossible to observe
head movement. The most common effects in the upper trapezius muscle, however,
would have been consistent with turning of the head to the same side. Contraction of
sternocleidomastoid was also observed with stimulus trains at some sites, so it seems
likely that head movement would have resulted in these situations if the head had not
been restrained.
43 A difference between the study by Drew and Rossignol (Drew and Rossignol,
1990a) and the present study is that the cats were at rest when stimulation was applied.
Stimulus trains applied at rest are sufficient to allow observation of movement or EMG facilitation, but without activation of the muscle, suppression may be difficult to observe.
In fact, Drew and Rossignol rarely observed suppression, and the only cases of suppression noted were in the ipsilateral triceps, a muscle that also demonstrated a prevalence of PStS in the monkey. In the present study, most stimuli were applied during periods of activity. Presumably, higher baseline levels of EMG were present for subjects in the present study, so suppressive and facilitative effects of the EMG may both have been more readily revealed.
In the thalamic cat, responses observed in flexor and extensor muscles during locomotion depended on the phase of locomotion when mPMRF stimulation was applied
(Drew and Rossignol, 1984). Generally, responses for a muscle were most common when that muscle was most active (e.g. stance for extensors). In the intact, behaving cat, stimulus trains applied to the mPMRF during locomotion typically facilitated contralateral and ipsilateral limb flexors and inhibited ipsilateral limb extensors (Drew,
1991). As in the thalamic cat, stimulation was most effective for a muscle when applied in the phase of locomotion in which the muscle was most active. In the present study, no differences in PStEs were observed for different directions of reaching. During locomotion, spinal networks are governed by a central pattern generator, and motor responses to peripheral and supraspinal inputs depend heavily on whether the stimulus is applied during swing vs. stance (Forssberg, 1979;Gossard et al., 1996;Pearson et al.,
1998). Voluntary reaching in different directions within the workspace in front of the
44 animal and against no substantial external load might not involve such strong differences
in the state of spinal networks. To determine whether substantially different patterns of
output for StimulusTA in the monkey could be observed on the basis of the movement
underway, it would probably be necessary for the different reaching movements to be
forceful, mimicking the requirements of brachiation; at a minimum, it would probably be
necessary for shoulder girdle movements to differ substantially.
Bilateral Actions of the Reticulospinal Tract
Bilateral responses have been consistently reported between forelimb and
hindlimb muscles in both decerebrate and intact preparations at rest and during
locomotion (Jankowska et al., 2003;Peterson et al., 1975;Peterson et al., 1979;Drew and
Rossignol, 1984;Drew and Rossignol, 1990b;Drew and Rossignol, 1990a;Drew, 1991).
The serial and reciprocal responses observed between the ipsilateral and contralateral
upper trapezius in the present study are consistent with previous reports of the bilateral
actions of the reticulospinal tract in the cat. Most bilateral responses included
contralateral PStF with ipsilateral PStS for both serial and reciprocal responses. Bilateral
reciprocal and serial responses are unlike the analogous responses between anatomical
antagonists within a limb because bilateral responses could involve commissural
interneurons. Jankowska et al. (Jankowska et al., 2003) proposed several pathways, some
involving commissural interneurons, through which contralateral responses could be mediated by the reticular formation. Although these observations were made in the lower extremity of the cat, similar pathways could be responsible for bilateral actions in the upper trapezius muscles of the monkey. In one pathway, the reticulospinal tract would
45 concurrently activate an excitatory commissural interneuron (e.g., to cUTr) and an ipsilateral inhibitory interneuron (e.g., to iUTr), with both interneurons synapsing onto motoneurons. This would cause concurrent effects bilaterally, resulting in a reciprocal response. The occurrence of serial responses could be explained by monosynaptic excitation of motoneurons on one side in conjunction with oligosynaptic projections to inhibitory interneurons affecting motoneurons on the opposite side. The overall finding in the present results that PStF began sooner than PStS is consistent with an inhibitory interneuron being required to mediate PStS.
Whether or not these complex actions could represent the effects of a single mPMRF neuron is uncertain given that the present results were produced by stimulation.
Single reticulospinal axons can project to both sides of the spinal cord (Matsuyama et al.,
1997;Matsuyama et al., 1999), so the anatomical substrate for this is present.
Alternatively, bilateral responses could be mediated by several reticulospinal neurons synapsing at the segmental level on separate interneuron and motoneuron populations.
The use of spike triggered averaging (SpikeTA) could help reveal the circuitry of these pathways because this method can examine the motor output of a single neuron, if synchrony effects are excluded (Fetz and Cheney, 1980).
Ipsilateral Upper Trapezius
Compared to the other muscles included in the present study, the ipsilateral upper trapezius (iUTr) demonstrated a unique pattern of PStEs. Although suppression was the most prevalent response for iUTr, the strongest facilitation events observed for all muscles were in iUTr. Single pulse currents as low as 10 µA produced strong, short
46 latency PStF events, and in some cases, a muscle twitch could be visually observed for single pulses with these low stimulus currents. This suggests that, in the primate, ipsilateral upper trapezius motoneurons may receive monosynaptic projections from the mPMRF. This is consistent with Peterson et al.’s (Peterson et al., 1979) report of monosynaptic excitation of cat neck motoneurons and motoneurons projecting in the spinal accessory nerve. Peterson et al. (Peterson et al., 1979) reported that, in addition to the neck sites distributed throughout the mPMRF, a position caudal to the abducens nucleus was especially effective in producing monosynaptic responses in neck motoneurons.
Comparison with Primary Motor Cortex and Red Nucleus
Since reticulospinal neurons are caudal to the red nucleus and conduction velocities have been reported to be faster for reticulospinal axons (Shapovalov, 1972),
PStF onset latencies could be expected to be earlier for the mPMRF than for the red nucleus. In the red nucleus, average onset latencies for the proximal and distal arm combined were reported to be 7.9 ms for PStF and 12.3 ms for PStS (Belhaj-Saif et al.,
1998). The average PStF onset latency of 7.1 ms for the mPMRF was similar to that for the red nucleus, although PStS was considerably earlier for the mPMRF (8.4 ms). These figures, however, may not provide an accurate comparison for these two regions because different muscles were included in the latency measurements, and latencies for specific muscles should be considered instead. For the shoulder muscles, onset latencies were slightly later for the mPMRF (7.9 ms; iPDlt, iADlt, iLat) than for the red nucleus (7.3 ms). PStF onset latencies of 8.0 ms for iBic and iTri were almost equal to PStF latencies
47 for the elbow muscles from the red nucleus (8.2 ms). Thus, latencies were similar for
similar muscles.
The effects of StimulusTA of proximal arm and shoulder muscles from the mPMRF, however, were generally opposite to those reported for the red nucleus.
StimulusTA studies in red nucleus of the monkey have consistently reported a clear extensor bias throughout the arm and shoulder (Belhaj-Saif et al., 1998;Cheney,
1980;Cheney et al., 1991b;Fetz et al., 1989). Belhaj-Saif et al. (1998) observed PStEs in
the muscles of the wrist, elbow, and shoulder joints, with PStEs most frequent in distal
arm muscles. PStF was most frequent in extensor muscles for the shoulder, elbow, and wrist, with the highest percentages of PStF shown distally. Conversely, PStF was uncommon in flexors, which showed higher proportions of PStS. Overall, PStS effects were less frequent than PStF effects. The authors concluded that the red nucleus preferentially controls extensor muscles in the distal and proximal arm, with a preference for distal arm muscles.
StimulusTA of proximal arm and shoulder muscles has also been performed from the primary motor cortex of the monkey (Park et al., 2001). Since this was a mapping study, only PStF events were analyzed and patterns of PStEs were not analyzed among muscles and onset latencies were not reported. Using SpikeTA in the primary motor cortex, McKiernan et al. (1998) demonstrated that post spike effects can be observed in proximal arm and shoulder muscles, although less frequently than in the distal arm.
Interestingly, the reported pattern of post spike effects was similar to the pattern of post stimulus effects observed from the mPMRF. This included a prevalence of flexor facilitation and extensor suppression in the muscles of the proximal arm and shoulder.
48 McKiernan et al. reported that, of the neurons that produced post spike effects in
proximal muscles, the majority also produced post spike effects in wrist and hand
muscles. One explanation for this similarity could be that the PStEs reported in the
present study represent the effects of CST collateral stimulation. As discussed above,
however, evidence suggests that the effects of mPMRF stimulation are not a result of
CST collateral stimulation. The tendency for post spike effects to be observed
concurrently in proximal and distal muscles suggests that CST collateral stimulation
would activate both proximal and distal limb muscles. The similarity in these results may indicate that, for proximal control, the primary motor cortex and the mPMRF could work synergistically to produce similar motor output patterns, with the primary cortex directing the reticulospinal system through CST collaterals and corticoreticular projections.
Clinical Relevance
In human, there are no direct data on the consequences of electrical stimulation in
the mPMRF, but inferences may be drawn from the motor patterns present in the paretic
limb of patients with hemiplegia after stroke. Dewald et al. (1995) demonstrated that
stroke patients display a predominance of certain muscle synergies after stroke and
argued that increased reliance on the reticulospinal systems for voluntary control might
explain this dominance. When the subjects studied by Dewald et al. (1995) elevated their
shoulder, they typically flexed their elbow, and had great difficulty recruiting the elbow
extensors along with the shoulder elevators. This is consistent with the finding in the
present study of a prevalence of concurrent iBic PStF and iTri PStS from the same
49 stimulation site and the strong PStF effects seen in iUTr, but not with the prevalence of
PStS in iUTr.
As demonstrated in these results and other studies (Matsuyama et al.,
1997;Wilson and Peterson, 1981), outputs from the mPMRF can have bilateral effects.
This bilateral control could be an important substrate for recovery of function after stroke
(Dewald et al., 1995;Freund and Hummelsheim, 1985), because either side of the cortex
could presumably access both sides of the body through reticulospinal outputs. Thus, the
present data could support the hypothesis that the motor patterns common in the upper extremity after stroke might represent increased reliance on outputs from the mPMRF for voluntary control. Further study is required to understand how reticulospinal outputs contribute to the control of reaching in the normal nervous system as well as after stroke.
50
Average Onset and Duration (Mean ± SD) Muscle n Facilitation n Suppression Onset 7.49 ± 2.06 — — iBic 8 0 Duration 8.88 ± 10.28 — — Onset 7.87 ± 2.84 7.07 ± 2.58 iADlt 10 3 Duration 6.63 ± 3.63 3.89 ± 1.35 Onset 6.75 ± 1.48 7.52 ± 1.68 iPDlt 7 10 Duration 4.57 ± 1.57 4.60 ± 2.30 Onset 5.06 ± — 7.48 ± 1.64 iTri 1 21 Duration 10.58 ± — 6.72 ± 3.27 Onset 10.10 ± 2.49 8.84 ± 1.10 iLat 3 13 Duration 7.34 ± 5.56 8.96 ± 7.65 Onset 7.28 ± 2.55 9.51 ± 2.02 iUTr 13 36 Duration 14.39 ± 9.20 7.79 ± 4.36 Onset 6.72 ± 1.92 8.04 ± 2.90 cUTr 38 8 Duration 5.16 ± 3.17 5.49 ± 3.02 Onset 5.91 ± 2.05 9.11 ± 1.32 iMTr 14 8 Duration 5.80 ± 3.89 5.56 ± 2.78 Onset 8.30 ± 0.00 9.21 ± 2.21 cMTr 2 4 Duration 2.70 ± 0.16 8.26 ± 4.54
Table 2.1. Average onset latency and duration of post-stimulus effects.
51 cUTr
iUTr
iLat
cMTr
iPDlt
iTri
iBic Move Target Hand Start Target Contacted Released Position
New Movement Reward Target Cue Trial Cue Task State
Stimulation
1s
Figure 2.1. Representative sample of EMG during task performance. EMG recorded from Monkey C is represented for a 5 s segment of the task. Although this record did not include stimulation, a trace labeled stimulation is provided to illustrate the frequency of stimulation in the context of the task. (iUTr), ipsilateral upper trapezius; (cUTr), contralateral upper trapezius; (iLat) ipsilateral latissimus dorsi; (iMTr), ipsilateral middle trapezius; (iPDlt), ipsilateral posterior deltoid; (iTri), ipsilateral trapezius; (iBic), ipsilateral biceps.
52 A B C
L A V SVe -6 -6 IV e p P MVe -7 -7 IVe VI VI -8 -8 B B B B A B B B BA B BA B A B A A S -9 B B -9 BA B A B BA B A A B A B A BA p A B A A 5 A A BA B AB BA B A B B A V A A B -10 -10 BA B BA D B B B B A B A B B BA B B A B B -11 -11 A A A -12 -12
-13 IO yr -13 IO IO P -14 -14 -3 -2 -1 0 1 2 3 0 1 2 3 4 AP ML Response Pyr A Fac B 1-mm iUTr Spr None
Figure 2.2. Anatomical location of effective stimulation sites for iUTr (subject D). The key indicates the type of response represented by each symbol. In A, stimulation sites for one plane (AP -1) are superimposed on a corresponding brainstem section. In B and C, all stimulation sites are illustrated for the anterior- posterior (AP, B) and medial-lateral (ML, C) planes. The arrow in B (labeled A) indicates the plane illustrated in A. The approximate location of the facial nucleus (VII), abducens nucleus (VI), inferior olive (IO), and the pyramidal tract (Pyr) are indicated in B and C. AP, ML, and dorsal-ventral (DV) scales are in Horsley- Clarke stereotaxic coordinates. (IV), fourth ventricle; (Pp), pedunculopontine nucleus; (MVe), (LVe), (SVe), and (IVe), medial, lateral, superior, and inferior vestibular nuclei, respectively; (Sp5), spinal trigeminal nucleus.
53 D572xStTA2
cUTr
iUTr
iADlt
iMTr
-20 0 20 40 60
Time (ms)
Figure 2.3. Detection of post-stimulus events. For each trace, the mean EMG level for the baseline period is illustrated by a solid horizontal line, and dotted horizontal lines above and below the mean indicate ± 2 SD. Filled bars represent periods of PStF; open bars represent PStS. Stimuli were applied at 20 µA. (iUTr), ipsilateral middle trapezius; (iADlt), ipsilateral anterior deltoid. SDPk for events: iUTr (17.6), cUTr (27.4).
54 A 80
70
60 RandomTA 50 StimulusTA
40
30
20
10
0 4 12 20 28 36 44 52
Onset Latency (ms)
B 40
30
20
10
0 6 10 20 30 40 50+
Mean Amplitude (% Change from Baseline)
Figure 2.4. A. Number of responses by onset latency for all acceptable events detected in response to actual stimulation (StimulusTA, filled bars) and to randomly timed fake stimuli from the same data files (RandomTA, open bars). The dashed horizontal line is drawn at a level 2 standard deviations above the mean number of RandomTA events per 2-ms bin. B. Distribution of response amplitudes for
55 i Biceps i Triceps i Latissimus Dorsi 63 Sites Tested, 13% Effective 63 Sites Tested, 35% Effective 57 Sites Tested, 28% Effective
i Anterior Deltoid i Posterior Deltoid i Middle Trapezius 38 Sites Tested, 35% Effective 63 Sites Tested, 27% Effective 70 Sites Tested, 31% Effective
i Upper Trapezius c Upper Trapezius c Middle Trapezius 133 Sites Tested, 37% Effective 133 Sites Tested, 35% Effective 57 Sites Tested, 11% Effective
4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24
Onset Time Relative to Stimulus (ms)
Figure 2.5. Histograms of PStEs by onset latency for each muscle; bin size is 2-ms, and there are 5 responses per vertical tick mark. Only events with onsets prior to 25 ms are represented. PStF is represented by upward, filled bars; PStS is represented by downward, empty bars. For each muscle, the number of sites stimulated for which a recording of that muscle's EMG was available is noted as the number of sites tested. The percentage of those sites for which a response occurred with an onset between 3.5 and 12.5 ms is indicated as the percent effective.
56 PStF PStS )
% 8/8 ( 100%
s 21/22
e 13/16 38/46
s 10/13
n 80% 36/49
o 14/22 p
s 60% 4/6 e 10/17 R
f
o 40%
n o i
t 20% r o p
o 0% i t r r t t r r r r l l c i a T T T T P T D D i L B U U i i M M i A P i c i i c
Figure 2.6. Proportions of PStF (solid line) and PStS (dashed line) events by muscle. Only events with onset latencies from 3.5 12.5 ms are represented. The number of times that response was observed is indicated as a fraction of the total number of responses observed for that muscle.
57 C775xStTA
iBic
iTri
iPDlt
cMTr
iLat
iUTr
cUTr
-20 0 20 40 60 Latency (ms)
Figure 2.7. A typical stimulus-triggered average. Filled bars represent PStF, open bars represent PStS. Stimuli were applied at 25 µA. SDPk of events: iBic (4.8), iTri (10.6), iLat (5.6), iUTr (7.8), cUTr (25.4).
58 A C673xStTA B D401DStTA_2
iBic
iBic
iTri
iADlt iPDlt iMTr cMTr
iLat iUTr
iUTr cUTr
cUTr
-20 0 20 40 60 -20 0 20 40 60
Latency (ms) Latency (ms)
Figure 2.8. Additional examples of stimulus-triggered averages. Filled bars represent facilitation and open bars suppression. A. Stimuli were applied at 30 µA. SDPk of events: iTri (5.8), iPDlt (4.6), iLat (8.2), iUTr (5.8). B. Stimuli were applied at 30 µA. SDPk of events: iBic (11.5), iADlt (6.5), iMTr (8.6), cUTr (11.5).
59 D601CStTA_1
iUTr
iADlt
iMTr
cUTr
-20 0 20 40 60 Latency (ms)
Figure 2.9. Large amplitude, short latency response in ipsilateral upper trapezius. Stimuli were applied at 30 µA. SDPk of events: iUTr (85.0), iMTr (4.4).
60 A C591BStTA
iUTr
cUTr
-20 0 20 40 60 Latency (ms)
B D571CStTA_1
iUTr
cUTr
-20 0 20 40 60 Latency (ms)
Figure 2.10. Bilateral responses in upper trapezius. A. Reciprocal response of iUTr PStS and cUTr PStF. Stimuli were applied at 40 µA. SDPk of events: iUTr (8.4), cUTr (8.4) B. Serial response of iUTr PStS and cUTr PStF. Stimuli were applied at 30 µA. SDPk of events: iUTr (15.7), cUTr (18.9).
61
CHAPTER 3
BILATERAL ACTIONS OF THE RETICULOSPINAL TRACT
ON ARM AND SHOULDER MUSCLES IN THE MONKEY:
SPIKE AND STIMULUS TRIGGERED AVERAGING
3.1 Abstract
The motor output of the pontomedullary reticular formation (PMRF) was
investigated using spike- and stimulus triggered averaging (SpikeTA, StimulusTA) of 12
bilateral muscle pairs in awake, behaving monkeys trained to perform a reaching task.
Muscles studied for the contralateral (c) and ipsilateral (i) side were extensor carpi ulnaris
(ECU), flexor carpi radialis (FCR), brachioradialis (Brac), biceps (Bic), triceps-long head
(TrLo), triceps-lateral head (TrLa), anterior deltoid (ADlt), posterior deltoid (PDlt), pectoralis major (PMj), latissimus dorsi (Lat), middle trapezius (MTr), and upper trapezius (UTr). For StimulusTA, a post-stimulus effect (PStE) was detected at 435
(81%) of the 535 sites tested. Overall, 1611 events were detected within 15 ms of the stimulus; 58% (941/1611) of responses were post-stimulus suppression (PStS) and 42%
(670/1611) were post-stimulus facilitation. Average onset latency was significantly
62 earlier for PStF than PStS, but average duration was significantly longer for PStS than
PStF. Average response magnitude was significantly larger for PStF. Overall, there was no difference in the number or characteristics of ipsilateral and contralateral PStEs. A bilateral pattern of PStEs was present for all muscles. For muscles of the arm and shoulder, ipsilateral flexors and contralateral extensors were typically facilitated and
contralateral flexors and ipsilateral extensors were typically suppressed. Similar patterns
were observed bilaterally for muscles acting on the scapula. Sites where PStEs were
observed in UTr demonstrated a gross rostrocaudal orientation, with PStF represented
caudally and PStS rostrally. Sites producing strong iUTr responses were concentrated in a
region ventral to the abducens nucleus. 398 cells were recorded for SpikeTA; post-spike
effects (PSpEs) were identified in only 9 (2%) of these cells. Of the 11 PSpEs detected, 8 were post-spike facilitation. 4 PSpEs matched the corresponding StimulusTA obtained from the same stimulus site. Overall, the pattern of responses observed with SpikeTA and
StimulusTA indicates that the PMRF is involved in the coordination of bilateral movements, with reciprocal actions both within a limb and typically between sides.
3.2 Introduction
Since most movements require some degree of bilateral coordination, it is important to understand the underlying mechanisms that mediate bilateral movements.
The corticospinal tract, rubrospinal tract, and vestibulospinal tract are primarily unilateral
(Kuypers, 1981), and therefore must rely on another motor system to coordinate bilateral movements. The most likely candidate for this is the pontomedullary reticular formation
(PMRF) and the reticulospinal tract (RST). Anatomical studies have shown that
63 reticulospinal axons have wide ranging patterns of projections and often terminate in the
ipsilateral and contralateral side of the spinal cord (Peterson et al., 1975;TORVIK and
Brodal, 1957;Petras, 1967;Nyberg-Hansen, 1965). Kably and Drew (Kably and Drew,
1998) have demonstrated that the PMRF also receives bilateral corticoreticular
projections. These authors also state that the pattern of corticoreticular projections
provide a substrate for the PMRF to control general postural movements. The reports of
Drew and Rossignol (1990 a,b) and Sprague and Chambers (1954) describe bilateral
movement patterns evoked with PMRF stimulation in the intact, awake cat. Sprague and
Chambers first emphasized the presence of bilateral responses in intact and decerebrate
cats, with opposite limbs generally demonstrating opposite responses at a single stimulus
site. The most prevalent response described in both reports consisted of flexion of the
ipsilateral limb, extension of the contralateral limb, and turning of the head towards the
flexed limb.
In chapter 2, the results of stimulus triggered averaging (StimulusTA) of
ipsilateral arm and shoulder muscles from the PMRF of the monkey are presented. In that
study, patterns of facilitation and suppression in flexors and extensors were sometimes
reciprocally organized around a single joint. The patterns of post stimulus facilitation and
suppression observed from the PMRF of the monkey resembled the responses described
by Drew and Rossignol and Sprague and Chambers; however, the bilateral analysis was
limited to the upper and middle trapezius. A bilateral examination of post stimulus effects
in the upper trapezius muscles did reveal a bilateral organization of responses, with these
muscles often responding with post stimulus effects of the opposite sign at a single stimulus site.
64 Several studies have attempted to define a somatotopic organization of the motor output of the PMRF. In a series of experiments, Peterson and colleagues (Peterson, 1979;
Peterson et al., 1979) have defined a rough organization of monosynaptic projections to limb and axial motoneurons from the cat PMRF. In these experiments, the authors applied stimulation in the PMRF of the decerebellate cat and recorded intracellular motoneuron potentials in the ipsilateral spinal cord. Sites evoking monosynaptic responses in neck and back motoneurons were distributed over the entire region of the
PMRF. Excitatory monosynaptic projections to limb motoneurons were less common than those for neck or back motoneurons, and were confined to an area rostral to the abducens nucleus to a dorsal region at the level of the rostral half of the inferior olive.
Polysynaptic projections to limb and axial motoneurons, were found throughout the
PMRF. The authors failed to find a difference in the prevalence of hindlimb and forelimb projections, which has been confirmed by Takakusaki et al. (2001).
Drew and Rossignol (1990 a,b) examined the somatotopic organization of movements evoked with stimulus trains in the awake, intact cat. In a pair of companion reports, the authors presented a rostrocaudal/dorsoventral organization of movements.
Neck movements were more often evoked from caudal regions, hindlimb movements were confined to the rostroventral medulla, and forelimb movements were more commonly evoked from regions in between. The authors emphasized that this organization was not specific, with some overlap between these regions; head and forelimb movements were evoked over the entire medullary reticular formation. In the dorsoventral plane, forelimbs were more often represented dorsally, while hindlimbs were typically represented ventrally (Drew and Rossignol, 1984). In a bilateral
65 comparison, contralateral limb movements were evoked from more ventral areas of the
PMRF, while ipsilateral limb movement was evoked from dorsal areas of this region.
Similar to Sprague and Chambers (1954), Drew and Rossignol (1990a,b) found that
excitation of flexors was more common medially, compared to extensor excitation that
was typical in lateral regions, though the prevalence of extensor excitation may be
attributed to the presence of descending fibers from the lateral vestibular nucleus.
The PMRF in the rhesus monkey has been previously explored with stimulus
trains, with the intent to study head movement (Quessy and Freedman 2004; Cowie and
Robinson 1994; Cowie et al.1994). In addition to head movement, Cowie and Robinson
were able observe to neck, back, shoulder, and arm movements with threshold level
currents (~40 µA). These responses, however, were never quantified with EMG. Sites
producing movement were found to have a dorsoventral organization, with head and neck
movements found more ventral, arm movements more dorsal, and shoulder back movements in between.
Several studies have employed the method of StimulusTA in the monkey to map
the somatotopic organization of the motor cortex (Park et al., 2001) and red nucleus
(Belhaj-Saif and Cheney, 2000;Belhaj-Saif et al., 1998). In the present study, single pulse
microstimulation for StimulusTA, in addition to spike-triggered averaging (SpikeTA),
were used to determine if there was a bilateral organization of the motor output of the
PMRF in the awake, behaving monkey as has been described for the cat. The muscles
studied were ipsilateral and contralateral proximal arm and shoulder muscles. Forearm
muscles were also included to determine if patterns of facilitation or suppression could be
observed in wrist flexors and extensors.
66 3.3 Methods
Subjects and Task
Subjects were two male Macaca fasicularis monkeys trained to perform an
instructed delay, bilateral reaching task administered by Tempo software for Windows.
For the task, subjects were required to sit in a primate chair with their head restrained and
reach to targets presented on a touch screen. A schematic of the task is presented in Fig.
3.1. The task began with the presentation of two dim, white circular cues (start) that instructed the monkey to depress two start buttons located in front of each arm on a
horizontal work surface. Once the start buttons were closed, the start cues were
illuminated and a blue circular cue (ready) was presented in the center of the screen.
Following a 1 to 2.5 ms delay, a square target was shown to the left or right of the ready
cue. A red target instructed the monkey to reach with the right hand; a green target
instructed a reach with the left. The monkey held both hands on the start buttons for a
randomized delay (1 to 2.5 s), after which time the ready cue turned purple (movement
cue), instructing the monkey to touch the appropriate target with the appropriate hand.
The monkey was rewarded for correctly performing a trial with a banana flavored pellet
delivered to a food well located immediately below the touch screen. Failure to correctly
perform the trial for any reason resulted in termination of the trial, and an extra delay was
added before the start of the next trial.
The shading in Fig. 3.1 shows the task was divided into two periods. The grey
shaded region, or the instruction period, consisted of the time between depression of the
start switches and the presentation of the go cue. The duration of this period was
randomized, depending on the individual times between presentation of the target and the
67 movement cue. The movement period, represented by the yellow shaded region, was the
time between the presentation of the go cue and the delivery of the reward. In total, four
different reaching movements were possible in the movement period: right hand-right
target, right hand-left target, left hand-left target, left hand-right target. The neural
activity associated with the performance of the task will be the subject of a separate
report.
Surgical procedures for implanting the recording chamber and EMG are also
detailed in the previous chapter. Briefly, a stainless steel recording chamber was mounted
to the skull over a craniotomy of the left parietal bone and secured with dental acrylic.
The recording chamber was aimed to stereotaxic coordinates AP 0, ML 0, DV -12, and tilted 10 degrees lateral to allow bilateral access to the PMRF. For collection of EMG data, pairs of stainless steel wires were implanted in twelve bilateral muscle pairs, for a total of 24 muscles. The muscles implanted are presented in Table 3.1.
Subjects were cared for by laboratory personnel and the veterinary staff of the
Ohio State University. Animals were housed and cared for under conditions that comply with NIH standards as stated in the Guide for the Care and Use of Laboratory Animals,
OSU-ULAR recommendations, and AAALAC accreditation standards for animals of this species. Surgeries were performed under veterinary supervision in aseptic conditions and in compliance with NIH guidelines. Animals were pretreated with antibiotics and were pre-anesthetized with ketamine HCl (13 mg/kg im) followed by isoflurane gas (1 – 2%) through endotracheal intubation. While under anesthesia, subject’s respirations, heart rate, and body temperature were monitored. Antibiotics and non-steroidal anti-
68 inflammatory analgesics (e.g., Buprenorphine, Ibuprofen, Acetaminophen) were
administered postoperatively to prevent infection and discomfort.
Recording and Stimulating Procedures
Extracellular recordings were made from PMRF neurons. The recording region of the PMRF was defined by the ventral border of the abducens nucleus dorsally, the dorsal
border of the inferior olive and/or corticospinal tract ventrally, the medial border of the
facial nucleus laterally (~ 4 mm lateral to the midline), and 0.5 mm lateral to the midline
medially. Specifically, the pontis caudalis, gigantocellularis, and magnocellularis reticular nuclei were studied. Polyimide insulated tungsten microelectrodes that were bared at the tip (Frederick Haer & Co.) were positioned in the recording chamber with an
X-Y grid and lowered into the brainstem with a manual hydraulic microdrive. The
electrode was inserted through a stainless steel cannula to guide it through the cerebrum
and cerebellum. Both the left and right side of the brainstem were explored for
recordings.
Extracellular potentials were amplified with a differential amplifier (AM-
Systems) and isolated online with a window discriminator (BAK Electronics). Once a
neuron was identified in the PMRF, all neural, EMG, and task related data were recorded with Spike2 software (CED). EMG data was sampled at 4.0 KHz and amplified at 1K, with a 20 -2000 Hz cutoff range. Neural data was fed directly to Spike2 from the differential amplifier, and re-examined offline to sort neurons on the basis of isolated
action potentials. Typically, one to two neurons could be recorded at a single site. To
compare the results of StimulusTA and SpikeTA, single pulse microstimulation for
69 StimulusTA was applied immediately following a recording, without changing the
electrode position. Once the bottom of the recording track was reached, the electrode was
withdrawn and single pulse microstimulation was applied in 0.5 mm intervals.
The equipment configuration and procedures for SpikeTA and StimulusTA are
presented in Fig. 3.2. For StimulusTA, averaging stimuli consisted of 4000 biphasic
pulses (first negative then positive) with a 0.2 ms phase duration and each pulse separated
by 100 ms. Stimuli were controlled with a Master 8 stimulus controller (AMPI) and delivered at 30 µA with an analog stimulus isolator (AM Systems). In the previous study, the typical stimulus threshold for a PMRF site was found to be approximately 30 µA, and the average current for all sites was 31 µA. This stimulus level was adequate to evoke post-stimulus effects at a majority of the sites studied, and as a result, was chosen as the stimulus intensity for all sites in this study. Since the purpose of StimulusTA is to
examine small changes in motor unit potentials over time, stimulus current was reduced
when overt muscle twitches were observed with the initial current. In 18 cases, current
was reduced in 5 µA increments until the twitches were abolished. Overall, the range of
stimulus current used was 10 to 30 µA.
Averaging and Analysis
Peri-event averages of EMG were compiled offline with a custom script for
Spike2 (CED) utilizing isolated action potentials (SpikeTA) or single pulse stimuli
(StimulusTA) as averaging triggers (Fig. 3.2). EMG data were corrected for DC offsets,
rectified, and averaged over an 80 ms epoch that was divided into a 20 ms pre-trigger and
60 ms post-trigger period. Previous investigation with SpikeTA has shown that
70 suppressive effects are more readily revealed from averages compiled with higher EMG
levels (McKiernan et al., 1998). In the present study, the peak of the baseline noise
associated with periods of inactivity was typically .045 V. To increase the likelihood of observing suppressive effects, the minimum EMG level was increased by approximately
10% to 0.05 V. Triggers were selected for averaging if the mean EMG of the 80 ms
window was greater than 0.05 v. Triggers with EMG that was greater than or equal to the maximum output of the amplifier (4.5 V) were discarded to prevent excessive, unrelated
EMG from corrupting the averages. Since triggers were accepted on a channel by channel basis, the number of triggers for each muscle in a single SpikeTA or StimulusTA varied.
A minimum of 1,000 triggers were required for SpikeTA, and a minimum of 500 triggers were required for StimulusTA (McKiernan et al., 1998).
All offline data analysis was conducted with DataPac2002 software (Run
Technologies). Criteria for selecting a post-stimulus (PStE) or post-spike effect (PSpE) were established relative to a pre-trigger EMG baseline. The EMG baseline was the mean activity of the first 15 ms of the average, ending 5 ms prior to the trigger (-20 to -5 ms).
Periods where the averaged EMG equaled or exceeded 2 standard deviations of the EMG baseline for at least 2.5 ms were considered for analysis. Events that demonstrated peak
EMG levels that equaled or exceeded +2 standard deviations were classified as post-spike or post-stimulus facilitation (PSpF, PStF). Events with peak EMG levels less than or equal to -2 standard deviations were classified as post-spike or post-stimulus suppression
(PSpS, PStS). The onset and offset latencies of PSpEs and PStEs were taken as the times were the averaged EMG crossed the ±2 SD threshold. In the previous chapter, post- stimulus effects occurring between 3.5 and 25 ms were much more frequent than random-
71 triggered events occurring within the same window. Events with onsets less than or equal
to 12.5 ms were selected for analysis in that study because these events were determined
to most likely represent the fast, direct outputs of the reticulospinal system. For the
present analysis, the same event period was analyzed, except 2.5 ms was added to account for the contralateral and distal muscles that are included here (Fig. 3.4).
Therefore, events with onset latencies between 3.5 and 15 ms were included in this
analysis.
The magnitudes of PSpEs and PStEs were quantified by two measures: mean
percent change (MPC) (Cheney and Fetz, 1985) and the standard deviation peak (SDPk).
MPC was the average displacement of an event from the EMG baseline expressed as a
percentage; SDPk was the peak displacement of an event from the EMG baseline
expressed as a Z score. Statistical analyses for onset latency, duration, SDPk, and MPC
consisted of 2-way ANOVAs with muscle and response type (PStS or PStF). For all
statistical analyses, the significance level was set to p ≤ 0.05.
Following the initial detection with the 2 SD, 2.5 ms criteria, post-stimulus and
post-spike effects were filtered using more stringent criteria to increase the likelihood that
the most reliable responses were analyzed. These criteria included a minimum 3.5 ms
duration, a minimum MPC of 3%, and a minimum SDPk of 4. The MPC threshold was
reduced from 6% in the previous chapter to 3% to account for PSpEs, which are typically
smaller in magnitude compared to PStEs (Lemon et al., 1986). Previous reports of
SpikeTA and StimulusTA have varied the minimum SDPk required to determine
significant effects. Cheney et al. (Belhaj-Saif et al., 1998) eliminated weak PStEs and
PSpEs by excluding responses with a SDPk less than 3. In our analysis, we found that a
72 minimum SDPk of 4, in addition to the MPC criteria, most effectively eliminated weak
PStEs and PSpEs. To further prevent random fluctuations from corrupting the results, the minimum duration in which a response was required to exceed the 2 standard deviation was increased to 3.5 ms. It was not uncommon for a muscle to demonstrate more than one response in a single average, and only the first response in a muscle that met the above criteria was accepted for analysis.
Random triggered averages (RandomTA) were compiled from SpikeTA data files.
Data that included stimulation were not used for RandomTA to prevent the effects of stimulation from corrupting the RandomTAs. For these RandomTAs, times for triggers
(isolated action potentials) were randomly reassigned and used as averaging triggers.
Random triggers were realigned within the period in which the original action potentials were recorded. Methods for computing and analyzing RandomTAs were identical to those used for SpikeTA and StimulusTA. The frequencies of detected effects in the
RandomTA data were compared with those for StimulusTA and SpikeTA to ensure that the criteria were strict enough to avoid most RandomTA effects. Overall, there were
1611 StimulusTA responses that met the criteria, compared to 96 RandomTA responses.
This was 6% of all StimulusTA responses; a smaller proportion than what was found in a similar analysis in the preceding chapter.
A cross-talk analysis was performed for all muscles from data files randomly selected from each week of the study (Fetz and Cheney, 1980). The purpose of this analysis was to eliminate the possibility that an adjacent pair of EMG electrodes recorded the same motor units. For this analysis, a motor unit potential from each muscle was used as a trigger for averaging the remaining muscles. Cross-talk was considered to be
73 present between muscles if the average EMG of a muscle exceeded 15% of the amplitude of the triggering motor unit potential. Cross talk was revealed between RFCR – RECU and TrLa –TrLo bilaterally in Monkey I, and between ADlt – PMj bilaterally in Monkey
G. These muscle pairs were examined and the data from the more reliable implant was retained for analysis. As a result, RECU, RTrLa, and LTrLo from Monkey I and R/L
ADlt from Monkey G were removed from the analysis. The integrity of the EMG implants was checked for the duration of a subject’s time in service by testing impedances and stimulating through the EMG wires. It was discovered that some implants became unreliable during the study, and these data were also removed the analysis. This included R/LBrac, RADlt, RBic, LPMj, and LPDlt in Monkey I and RPDlt,
RLat, LPDlt, LPMj, LMTr, LUTr, and LBic in Monkey G.
Anatomy and Histology
After all recording and stimulation was complete, electrolytic lesions were made with 20 µA for 20 s (DC anodal). Subjects were deeply anesthetized with sodium pentabarbitol and perfused transcardially with phosphate buffered saline, followed by phosphate buffered para-formaldehyde. This was followed by a perfusion with 10% sucrose in phosphate buffer, followed by 30% sucrose in phosphate buffer. The brain was removed and soaked in neutral buffered sucrose (30%) for cryoprotection. Frontal sections (Monkey G) and transverse sections (Monkey I) were cut at 50 µm on a freezing microtome and every 4th section was mounted and stained with cresyl violet. The sections were scanned at 1200 dots per inch and the anatomical reconstructions were completed in software. Structures in the brainstem were identified according to a
74 stereotaxic atlas (Szabo and Cowan, 1984) and a template atlas available online from the
primate center at the University of Washington (Bowden et al., 2003). The locations of
EMG recording electrodes were verified by post mortem dissection.
3.3 Results
A sample of EMG recorded while Monkey I performed the task is presented in
Fig. 3.3. As can be seen in the figure, activity was present in all muscles while the
monkey performed the task. EMG levels were highest for arm and shoulder muscles with
reaching, while moderate levels of EMG were present for most shoulder muscles when
reaches were made with the opposite arm. The majority of muscles showed moderate levels of activity during the instruction period, but EMG activity was greatest for the
movement period. During the movement period, the EMG appeared to be phasic for
reaches with the right and left arm. Monkey I adopted a strategy of touching the target
with a repetitive motion, like knocking on a door with an open hand. Attempts to eliminate this behavior with training were not successful and the monkey continued to perform in this manner for the duration of the study. Fortunately, this behavior was not detrimental to the study and may have been effective in increasing muscular activity for the wrist during reaching. Overall, levels of EMG were adequate to allow the observation of suppressive as well as facilitative effects.
75 Selection of Post Stimulus Events
Single pulse stimulation for StimulusTA was applied at 790 sites. Following an
anatomical reconstruction of stimulus sites (a detailed discussion of the location of
stimulus sites is presented below), 255 sites were determined to be outside the defined
region of the PMRF (see METHODS) and were removed from analysis. The remaining 535
sites (244 on the left, 291 on the right) were examined for the presence of PStEs. As shown in the histogram in Fig. 3.4, most PStEs occurred within 25 ms of the stimulus, and most of these responses were confined to the first half of the 25 ms period. 2170
PStEs were observed in the 25 ms window, of which, 939 (43%) were PStF and 1231
(57%) were PStS. Selecting for PStEs that occurred in the first 15 ms reduced the total
number of PStEs analyzed to 1611, which was 74% of the 2170 events in the 25 ms window. A PStE was counted at 435 sites, 81% of all sites tested in the PMRF.
Differences in Post-Stimulus Facilitation and Suppression
Differences between the overall characteristics of PStS and PStF responses were evident. For all muscles combined, PStS responses occurred more often than PStF responses; of the 1611 PStEs, 941 (58%) were PStS and 670 (42%) were PStF. PStS was also evoked from a larger number of sites (367/535, 69%) than PStF (314/535, 59%). The
range of onset latencies was identical (3.5 to 15 ms) for both PStS and PStF, although this
was simply a consequence of the analysis criteria. Overall, average onset latency was
significantly earlier for PStF (7.2 ± 2.8 ms) than for PStS (8.4 ± 2.8 ms) (p<0.001).
Response duration ranged from 3.5 to 27.5 ms for PStS and 3.5 to 26.3 ms for PStF. On
76 average, the duration of PStS (8.0 ± 3.4 ms) was longer than the duration of PStF (7.4 ±
3.4 ms) (p= 0.001).
In general, the magnitudes of PStF responses were larger than PStS responses.
The average SDPk of PStF was significantly larger than PStS SDPk (PStF: 11.4 ±21.7,
PStS: 7.2 ±3.1; p<0.001). This was also true for MPC: PStF (17.0 ±14%) was significantly larger than PStS MPC (13.2 ±5%; p<0.001). The magnitudes of some PStF events were considerably large and had a clear effect on the average magnitude of all
PStF events. A non-parametric Mann-Whitney test for these measures revealed that, despite the differences in the distributions of response magnitudes, PStF was significantly larger for PStF (p<0.001). Since the distribution of PStF magnitudes was much larger, the median of response magnitudes should also be considered. In this comparison, the median of PStF magnitude (SDPk: 7.0, MPC: 14.0%) was only slightly larger than PStS magnitude (SDPk: 6.2, MPC: 13%).
Ipsilateral vs. Contralateral Responses
The number of PStEs occurring in ipsilateral and contralateral muscles was examined for all effective stimulus sites. Of the 1611 PStEs observed, 783 (49%) were observed in contralateral muscles, while the remaining 828 (51%) PStEs were observed in ipsilateral muscles. Bilateral responses were evoked at 62% (269/435) of all effective sites. The remaining 166 sites were evenly divided for sites that were exclusive for ipsilateral or contralateral PStEs (83/435, 19%). Since the numbers of left and right stimulus sites were not equal and the number of muscles studied on each side varied by monkey, it was important to consider the number of responses observed relative to the
77 number of responses possible for a side. On the ipsilateral side, the 828 PStEs observed were 21% of the 3,997 possible ipsilateral responses. This ratio was similar for the contralateral side: 783 contralateral PStEs were 19% of the 4,028 possible contralateral responses. These numbers indicate there was little difference in the number of ipsilateral and contralateral PStEs, with contralateral responses occurring as often as ipsilateral responses.
Differences in the characteristics of PStS and PStF were also compared for ipsilateral and contralateral PStEs. Overall, PStS responses were more common and accounted for 60% (496/828) of all ipsilateral PStEs and 57% (445/783) of all contralateral PStEs. The most considerable difference between ipsilateral and contralateral PStEs was in response onset latency. Interestingly, average onset latency of
PStF was earlier for contralateral responses (6.7 ±2.5 ms) than for ipsilateral responses
(7.7 ±2.9 ms) (p<0.001). The opposite was true for the average onset latency of PStS; contralateral PStS (8.8 ±2.9 ms) was later than ipsilateral PStS (8.0 ±2.7 ms) (p<0.001).
There was little difference for contralateral and ipsilateral muscles for measures of duration and magnitude. The average duration of contralateral (7.4 ±3.5 ms) and ipsilateral (7.3 ±3.4 ms) PStF were similar, though contralateral PStS (8.2 ±3.8 ms) duration was slightly longer than ipsilateral PStS (7.7 ±3.0 ms) duration (p<0.05). Among the measures of magnitude, the only comparison of contralateral (contr.) and ipsilateral
(ips.) responses that differed significantly was the average MPC of PStS (contr.:13 ±5%, ips.: 14 ±5 %; p= 0.001). The average magnitude of ipsilateral PStF responses was similar to that for contralateral PStF responses for both SDPk (contr.: 9.9 ± 6.4, ips.: 12.9
78 ± 30.2) and MPC (contr.: 16.0 ± 8%, ips.: 18.0 ± 17%). The average SDPks of ipsilateral
and contralateral PStS responses was almost equal (contr.: 7.2 ±3.2; ips.: 7.2 ±3.0).
Divergence of Motor Output for PMRF Stimulus Sites
The percentage of muscles responding at a stimulus site was used as a measure of the divergence of motor output, the number of muscles that were affected from a single stimulus site. This number was determined for each stimulus site where a PStE was evoked by dividing the number of muscles responding by the number of muscles available for recording for that site. In total, 15 muscles were available for study for each stimulus site in both monkeys. For Monkey I, 7 muscles were located on the right and 8 were on the left. In monkey G, 9 were located on the right and 6 were on the left. On average, 24.7 ± 18.4% of muscles responded per effective stimulus site, which corresponds to approximately 3 to 4 muscles. For all effective sites, the average number of muscles affected ranged from 6.7% to 93.3%. Thus, some sites evoked a wide range of responses, while stimulation at others produced little change in most muscles studied. On average, a higher percentage of muscles demonstrated PStS (14.4 ±12%) than PStF (10.3
±9%) for a single stimulus site. PStS without PStF was observed at 121 sites, while PStF alone was observed at 68 sites. The finding that PStS was more common for individual
stimulus sites is consistent with responses being more prevalent for PStS overall.
There was little difference in the percentage of muscles facilitated or suppressed
based on the laterality of the muscle. On average, 10.0 (±11) % of ipsilateral muscles and
10.5 (±12) % of contralateral muscles were facilitated for effective stimulus sites. By the
same analysis, 15.2 (±16) % of ipsilateral muscles and 13.6 (±13) % of contralateral
79 muscles demonstrated PStS. The number of ipsilateral and contralateral muscles
facilitated or suppressed relative to the number of possible responses was also examined.
These numbers were similar to those observed on a site by site comparison in that there was little difference between ipsilateral and contralateral responses: 18.0% of all ipsilateral and 19.8% of all contralateral muscles were facilitated and 25.4% of all ipsilateral and 24.6% of contralateral muscles were suppressed by stimulation at any given site.
Distribution of Post-Stimulus Effects by Muscle
The percent effectiveness of stimulation was determined for each muscle by counting the number of sites at which a PStE was observed for a muscle and dividing this number by the total number of sites where EMG was recorded for that muscle. These values are presented in Fig. 3.5.A. This analysis revealed that stimulation was most effective for cPMj (56%) and cUTr (40%), followed by the iUTr (38%) and iPMj (31%), the major muscles of the shoulder girdle. For arm and shoulder muscles, extensors tended to be more often affected from a stimulus site; the arm flexors were the least responsive muscles of all muscles studied. There appeared to be little difference between the ipsilateral and contralateral muscle for bilateral comparisons within the same muscle.
Percentages of PStF responses are presented for each muscle in Fig. 3.5.B. PStS was more prevalent in the ipsilateral elbow extensors, iTrLo (80/94, 85%) and iTrLa
(82/92, 89%), and in the shoulder extensor, iPDlt (25/33, 76%). PStS was also the prevalent response for the ipsilateral wrist extensor, iECU (61/72, 85%). The opposite response was observed in contralateral extensors: PStF was the prevalent response for all
80 contralateral wrist (cECU 28/37, 76%), elbow (cTrLo 67/99, 68%; cTrLa 59/75, 79%)
and shoulder (PDlt 13/16, 81%) extensors.
For the ipsilateral shoulder flexor, ADlt, PStF was the prevalent response (7/11,
67%), though there were few responses in ADlt when compared to other muscles. The
ipsilateral elbow flexors demonstrated a prevalence of PStF (iBic 24/27, 89%; iBrac 7/7,
100%), while the ipsilateral wrist flexor showed only a slightly higher proportion of PStF
(35/62, 57%). On the contralateral side, PStS was the prevalent response for the wrist
flexor, cFCR (39/54, 72%), the elbow flexor, cBic (12/20, 60%), and the shoulder flexor,
cADlt (9/11, 82%). No significant PStEs were observed in the contralateral elbow flexor,
cBrac.
Differences in the proportions of PStS and PStF were also present for the more
proximally located muscles (PMj, Lat, UTr, MTr). In PMj, a shoulder flexor and
protractor, PStF was more prevalent for the ipsilateral muscle (80/91, 88%) compared to
a prevalence of PStS in the contralateral muscle (132/136, 97%). In the upper trapezius muscles, PStF was more prevalent for cUTr (120/169, 71%) while PStS was most prevalent for iUTr (117/163, 72%). The opposite was true for the middle trapezius muscles; PStF was more common for iMTr (51/60, 85%) and PStS was more common for cMTr (55/63, 87%). Of all the muscles studied, the latissimus dorsi was the only muscle to demonstrate a prevalence of PStS bilaterally, with a higher proportion of PStS observed contralaterally (cLat 89/103, 86%; iLat 77/116, 66%).
A StimulusTA demonstrating the prevalent response for the majority of muscles is presented in Fig. 3.6. In this average, every response present is consistent with the most
common response for each muscle; however, 5 of the 15 muscles did not respond at this
81 site. In fact, there were no sites where a PStE was detected in every muscle recorded. For
the 5 muscles that did not respond, 3 were ipsilateral arm flexors. This is consistent with the observation that flexors were least often affected by stimulation. The 2 strongest responses were observed in iUTr and cUTr.
Large magnitude, short latency responses were observed in the iUTr. An example of this type of response, and all other muscles responding at the same site, is presented in
Fig. 3.7. The SDPk of the strong iUTr responses always exceeded 40, and these responses were the largest observed among all responses. The iUTr response illustrated in Fig. 3.7
was one of the largest responses counted in the present study. The SDPk of this event
reached 246, while the SDPk values for the other muscles studied at the same site were
between 4.9 and 13. In total, 13 responses with a magnitude greater than 40 SDPk were
counted in the iUTr, and only 2 were found in any other muscle. These responses were in
cUTr and iADlt and were 2 of the 3 weakest responses with an SDPk over 40. Current
was reduced from 30 µA (20 – 25 µA) at 4 of the 13 sites where the strong iUTr responses were observed.
Comparison of Onset Latency, Duration, and Magnitude of Post Stimulus Effects among
Muscles
Values for onset latency are presented for each muscle in Table 3.2. For most
muscles, average PStF onset was sooner than average PStS onset. Interestingly, PStS was
the prevalent response for the 3 muscles that demonstrated earlier latencies for PStS. The latissimus dorsi was the only muscle to demonstrate significantly earlier onset latencies
for PStS for both the ipsilateral (p<0.05) and contralateral muscles (p<0.001). iUTr was
82 the only other case where average PStS onset latency was significantly earlier than PStF
onset (p<0.001). Among muscles, PStS was significantly earlier for iUTr than for all
other muscles except i/cPMj, i/cLat, cPDlt, cTrLo, iTrLa, and iBic. There were fewer
significant differences among muscles for PStF latencies. The earliest average PStF onset
latencies were observed in the contralateral elbow and wrist flexors cADlt, cBic, and cFCR, but the average PStF onset latency was significantly earlier for cUTr than for all
other muscles except iUTr, iADlt, cLat, and iLat (p<0.05). Average PStS duration was
longer than average PStF for most muscles (Table 3.3). Even though cUTr demonstrated
higher proportions of PStF, PStS duration was longer in this muscle than any other
muscle except for cPDlt and iBic (p<.05). The average duration of PStF was longer for
iPMj than for cECU, cFCR, cPDlt, cTrLa, iECU, iFCR, iMTr, and iTrLa (p<.05).
Within muscles, average magnitudes tended to be larger for PStF than for PStS.
iLat was the only muscle where the average PStS SDPk was greater than the average
PStF SDPk (p<.05) (Table 3.5). The average PStF MPC was greater than average PStS
MPC for iUTr, cUTr, iTrLo, cTrLa, and iPDlt (p<.05) (Table 3.4). Among muscles, the
average SDPk and MPC for PStF was largest for iUTr, which was significantly greater
than all muscles except iADlt, cADlt, and cPMj (p<0.05). Average SDPk of PStS was
largest in cTrLo (significant with 9 muscles, p<0.05), and average MPC of PStS was
largest in iTrLa (significant only with cTrLo and cPMj, p=0.001).
83 Reciprocal and Bilateral Response Patterns among Muscles
Since there was an overall organization of PStF and PStS for ipsilateral vs. contralateral muscles, it was important to examine the response patterns between individual muscles pairs at individual stimulus sites. Muscles pairs for antagonists around the wrist, elbow, and shoulder/scapula were included in this analysis. Comparisons between other muscle pairs are also presented below. The PStEs for these muscles were examined for the presence of reciprocal, cofacilitation, or cosuppression responses.
Reciprocal responses consisted of PStEs between the paired muscles that were opposite in sign (i.e. PStF with PStS). Cosuppression and cofacilitation responses occurred when both muscles in a pair were either facilitated or suppressed. These paired response patterns were further divided into concurrent, serial, and staggered patterns. In a concurrent pattern, one muscle’s response overlapped with the response in the paired muscle for at least half of the duration of the shortest response between the pair. Two examples of concurrent reciprocal responses are shown in Fig. 3.8. In this figure, a concurrent reciprocal response between the ipsilateral and contralateral UTr (A) and iBic with cTrLa (B) are illustrated. Serial responses were similar to concurrent patterns, except the overlap between responses was less than half the duration of either response, or the onset of the second response began within 2 ms of the offset of the first response.
An example of a serial reciprocal between response between cTrLa and cBic is presented in Fig. 3.9.A. In total there were two possible reciprocal patterns: concurrent reciprocal and serial reciprocal responses. The remaining possible responses were concurrent and serial cosuppression or cofacilitation. Although a range of responses were possible, only the most common response between the muscle pairs is reported for simplicity.
84 Comparisons in the ipsilateral arm were available for iBic/iTrLa, iBrac/iTrLA,
and iFCR/iECU; cTrLa/cBic and cFCR/cECU were available in the contralateral arm. In
most cases, reciprocal responses were the typical response between anatomical
antagonists, and concurrent reciprocal responses were more frequent than serial
reciprocal responses. Within a pair, the responses were consistent with the prevalent responses for each muscle within the pair. Cofacilitation and cosuppression were not uncommon, but were much less frequent than reciprocal responses. For iBic/iTrLa, most
responses were concurrent reciprocal (8/10) with iBic facilitated. Slightly more than half
of the cBic/cTrLa responses were serial reciprocal (6/10) with cBic suppressed. A similar
trend was seen between iBrac/iTrLa: all of these responses were concurrent reciprocal (5)
with the ipsilateral flexor facilitated. Interestingly, the timing of reciprocal responses for
FCR/ECU was different from that observed for Bic and TrLa. Reciprocal responses were
still the most common responses between iFCR/iECU, but most were serial (8/14) instead
of concurrent. All of these responses consisted of iFCR PStF with iECU PStS. There
were few responses counted between cFCR/cECU and most of these were reciprocal (2
serial, 1 concurrent). cFCR was suppressed in 2 of the 3 reciprocal responses.
Response patterns for antagonist muscles around the shoulder were also
examined. These included comparisons of the shoulder flexor and protractor, PMj, with
the shoulder extensor, PDlt, and PMj with the humerous extensor, Lat. Since there were
no cases where PDlt and ADlt were recorded together, this comparison was not available.
The pattern of responses observed between the shoulder antagonists was similar to that
observed for the elbow. For iPMj/iPDlt, most responses were concurrent reciprocal
(14/20) and iPMj was facilitated in all of these. Almost all of the cPMj/cPDlt responses
85 were reciprocal (12/14), with the number of serial and concurrent responses evenly
divided (6). cPMj PStF never occurred with a cPDlt response, so all of these responses
consisted of cPMj PStS and cPDlt PStF. Most of the responses counted between iPMj/iLat were reciprocal (13/18), and again, iPMJ was facilitated in all of these. cPMj
PStF was never paired with a cLat response and most of the cPMj/cLat responses were
cosuppression (11).
Other comparisons were also made for muscles that produce opposite movements
about the scapula. Most of these pairs included the UTr, which was compared to MTr,
PMj, and Lat. Comparisons were also made for MTr between Lat and PMj; Lat was
compared with PMj. The most common response pattern between each of these muscle
pairs, as well as bilateral homologues (discussed below), are indicated in Fig. 3.11.
Overall, the pattern observed between these muscles were similar to responses observed between arm and shoulder antagonists: reciprocal responses were more common than any other type of response. Concurrent reciprocal responses were most common for iPMj/iLat
(13/18), iUTr/iMTr (17/43), cUTr/cLat (27/58), and cUTr/cPMj (18/41), while serial reciprocal responses were most common for cUTr/cMTr (16/42) and iUTr/iPMj (13/38).
Compared to the arm and shoulder, cofacilitation and cosuppression responses were more often observed among the scapular pairs. Cosuppression was most frequent for cPMj/cMTr (18/26), cPMj/cLat (11/17), and iUTr/iLat (30/59); cofacilitation was the most frequent response for iPMj/iMTr (10/16).
Another question of interest was if there were similar patterns observed between the antagonist pairs compared above when the muscles were contralateral to one another
(i.e. iPMj/cLat). For some of these pairs, reciprocal effects were most common. For
86 iPMj/cMTr, 8 of 16 responses were serial reciprocal and contained iPMj PStF. For
cPMj/iMTr, 16 of 26 responses were serial reciprocal, and cPMj was suppressed in all of
these. Concurrent reciprocal responses were most frequent for iPMj/cLat (29/44) and
iPMj was facilitated in all of these, however, cosuppression was much more frequent for
cPMj/iLat (39/67). The remainder of the muscle pairs included in this particular analysis
typically showed responses of the same sign. Cosuppression was common for iUTr/cPMj
(52/61), iUTr/cMTr (31/38), and iUTr/cLat (29/41). Cofacilitation was common for cUTr/iPMj (32/43) and cUTr/iMTr (36/48).
Muscles recorded from the ipsilateral and contralateral side at a single stimulus
site, or bilateral homologues, were also compared. Concurrent reciprocal responses most
often occured for iUTr/cUTr (40/74) and 39 of these included cUTr PStF. Bilateral
reciprocal responses were also common between iMTr/cMTr; 14 of 29 responses for this
pair were serial reciprocal and iMTr showed PStF in all of these. Cosuppression
accounted for slightly more than half of the bilateral Lat responses (16/27). For elbow
and wrist muscles, bilateral comparisons were available for the TrLa, TrLo, and FCR. In
all three cases, reciprocal patterns were the most common response between these muscle
pairs and were typically consistent with the prevalent response for each muscle.
Concurrent reciprocal responses were common for iTrLa/cTrLa (13/17), and iPStS was
observed in all of these bilateral responses. For TrLo, 6 of 9 bilateral responses contained
ipsilateral PStS. 6 serial reciprocal responses were counted in this pair, with only one
containing iTrLo PStF. For the wrist, 7/17 FCR bilateral responses were serial reciprocal
(6 iPStF), while there were only two bilateral responses observed in ECU.
87 Spike Triggered Averaging
SpikeTA allowed the opportunity to examine the output of individual PMRF
neurons. Overall, 398 cells were discriminated in the offline examination of data
collected at recording sites. SpikeTA were compiled in a manner identical to
StimulusTA: events were first identified with the initial criteria and filtered with the
criteria derived from the RandomTA analysis. SpikeTA were subjected to a third criteria
in which events were judged in a visual inspection by two investigators. Only PSpEs that demonstrated characteristics similar to a PStE were accepted. These characteristics included a well defined peak within the event and an appropriate shape without sloping or ramping EMG that began prior to the time of the trigger. An example of a PSpE that fit these criteria is presented in Fig. 3.10. In total, 42 PSpEs were selected independently by two investigators. The majority of these PSpEs (28) were agreed upon; however, there were 14 discrepancies where a response was selected by only one of the two investigators. Following a second review with both investigators in conference, 3 of the
14 discrepancies were retained for analysis. The resulting 31 PSpEs were subjected to a comparison with RandomTAs complied from data collected from the same sites, and no
RandomTA event appeared similar to a detected SpikeTA event. In the final test, the data files for these 31 cells were split into halves and averaged separately to ensure that PSpEs were not merely a result of large, random deflections in the EMG that occurred in a small segment of the data file. For a PSpE to be accepted, the event needed to be detected with the 2 standard deviation, 2.5 ms criteria in at least one half of the file, and at minimum, the shape of the EMG needed to be similar to the detected PSpE in the remaining half.
88 After all these criteria were applied, only 11 PSpEs with onsets between 3.5 and 15 ms
were accepted for analysis.
Overall, PMRF cells producing acceptable PSpEs were rare. Only 9 cells were found to produce a PSpE, which was 2% of all cells recorded in the PMRF. 8 of the 11 responses were PStF, and the number of ipsilateral (6) and contralateral PSpEs (5) was almost equal. Onset latencies ranged from 4 to 9.3 ms for PSpF and 5.8 to 9.3 for PSpS and the range of duration was much more distributed for PSpF (3.8 to 28.3 ms) than PSpS
(4.3 to 5.5 ms). For PSpF, the range of magnitude measures was 4.4 to 7.6 for SDPk and
4% to 11% for MPC. For PSpS the range was 4.3 to 4.6 for SDPk and 3% to 8% for
MPC. With such a small number of PSpEs detected, statistical analyses of PSpEs were inappropriate.
Of the 11 PSpEs observed, 6 were in the more proximal muscles studied (UTr: 3,
PMj: 2, Lat: 1). The other, 5 PSpEs were observed in elbow flexors (Bic: 1) and extensors (TrLa: 3, TrLo: 1). For 2 cells, PSpEs were observed in 2 muscles. In one of these averages, two ipsilateral muscles were facilitated (iTrLa, iUTr). In the second, iBic and cTrLa both showed PSpEs, with PStF observed ipsilaterally and contralaterally (Fig.
3.10.B).
The 9 SpikeTAs containing acceptable PSpEs were compared to StimulusTAs obtained from the corresponding stimulus site. This comparison revealed 4 of 11 PSpEs that corresponded to a PStE in the same muscle. One of the two SpikeTAs with two muscles demonstrating a PSpE is illustrated with its corresponding StimulusTA in Fig.
3.10.B. As illustrated, the two PSpEs matched the sign and approximate onset latencies
for the PStEs resulting from stimulation at the same site. It should be noted that these
89 were not the only PStEs observed at this site, StimulusTAs showed more responses than
corresponding SpikeTAs. An opposite response for SpikeTA and StimulusTA was
observed in only one comparison. At this site, cTrLo responded with PStF and PSpS. In
the remaining cases, there was no corresponding PStE for a PSpE.
Location and Organization of Stimulus Sites
In Fig. 3.11, sites producing PStF and PStS were plotted for the ipsilateral and contralateral upper trapezius. Sites where single pulse stimulation resulted in no PStEs are also indicated in the figure. Upper trapezuius was selected for this demonstration because it was recorded at all stimulus sites and, compared to the other muscles studied, this muscle was one of the most responsive to PMRF stimulation. Stimulus sites that were excluded from analysis are not indicated in the figure. Excluded sites were located
on the midline, or in or near the vestibular nuclei, the facial nucleus, inferior olive, and
the pyramidal tract. As illustrated, stimulus sites that were selected for analysis were
confined to our predetermined boundaries.
An examination of the stimulus sites plotted for the upper trapezius revealed that
PStS sites were located more rostrally and PStF sites were more caudal. In addition to
this, a region ventral to the abducens nucleus was found to have a higher proportion of
the large magnitude (>40 SDPk) iUTr responses described above. Sites where stimulus
current was reduced due to evoked movements were typically located in this region as
well. The results of the WGA-HRP injections indicated that large cells projecting to the
spinal cord are located in the same region ventral to the abducens nucleus, as well as
regions within the NRGc and NRPc.
90 Overall, there was no apparent organization for cells that produced PSpEs or sites that produced PStEs. There also were no clear boundaries for stimulus sites or cells producing facilitation or suppression; both responses were evoked from regions located throughout the PMRF. There was no obvious organization for sites or cells representing contralateral or ipsilateral muscles. This was also true for individual muscles. Excluding
UTr, there were no obvious regions producing facilitation or suppression.
3.4 Discussion
In summary, SpikeTA and StimulusTA were used to study the bilateral motor outputs of the primate PMRF. Similar to previous studies in the cat PMRF (Peterson et al., 1979;Peterson, 1979;Drew and Rossignol, 1990b;Drew and Rossignol,
1990a;Sprague and Chambers, 1954), StimulusTA revealed a wide range of bilateral outputs to arm and shoulder muscles from the primate PMRF. Bilateral effects were common, and there was almost no difference in the number of ipsilateral and contralateral
PStEs. Overall, PStS was more common than PStF, which was also true when ipsilateral and contralateral PStEs were examined separately. For individual muscles, PStF was more common for ipsilateral flexors and contralateral extensors, while PStS was more common for ipsilateral extensors and contralateral flexors. A similar organization was observed between the ipsilateral and contralateral muscles for more proximally located muscles. At individual stimulus sites, reticulospinal output was often reciprocal for bilateral muscle pairs, as well as for antagonists situated around a single joint. SpikeTA in the primate PMRF was found to produce few PSpEs, which may indicate that monosynaptic projections in the primate RST are limited. PSpF accounted for 8 of the 11
91 PSpEs, but there were too few responses to determine if there were differences in the proportions of PStF and PStS for individual muscles. Of the averages where PSpEs were observed, most showed a PSpE in only one muscle. In 2 cases, PSpEs were observed in 2 muscles that were either separated by at least one joint, or were in muscles that were on opposite sides of the body.
Spike-Triggered Averaging
Of the 11 detected PSpEs, most were found in the upper trapezius and triceps.
Because there were 1600 more PStEs than PSpEs, there are few comparisons that can be made between the results of SpikeTA and StimulusTA. Similar to the findings of
StimulusTA, there was no difference in the number of ipsilateral and contralateral PSpEs.
Both PSpF and PStS suppression were observed, although PSpF was the most common response. This was in contrast to the overall pattern of PStEs, where PStS was more common.
It has been reported from several studies of rubromotoneuronal and corticomotoneuronal (CM) cells that the method of SpikeTA is typically more effective when monosynaptic connections are present (Fetz and Cheney, 1980;Lemon et al., 1987).
The lack of PSpEs in the present study is somewhat surprising, considering that monosynaptic projections from the PMRF to neck, back, and limb motoneurons have been well documented in the cat (Peterson, 1979;Shapovalov et al., 1967).
Reticulomotoneuronal (RM) connections have also been identified in the monkey with intracellular recordings; however, these projections were to lumbar motoneurons
(Shapovalov, 1972;Shapovalov, 1973). Other than Shapovalov’s investigations, we are
92 unaware of any other electrophysiological study that has examined RM projections in the monkey. In the present study, neurons were recorded from over 200 sites located over a large extent of the PMRF, and most, if not all regions within the boundaries of the recording region (see Methods) were represented by a recording site. Our inability to find a large number of cells producing PSpEs suggests that there are a limited number of monosynaptic projections to the upper extremity in the monkey. Another possibility may be that RST terminals may only contact the distal regions of motoneuron dendrites, resulting in weaker effects (Matsuyama et al. 2004).
Lloyd (1941) first described the motor effects observed with reticular formation stimulation and attributed a majority of the resulting effects to activation of interneuronal populations. Drew and Rossignol (1990a) also attributed most of the effects they observed with PMRF stimulation to interneuronal networks. In the decerebrate cat,
Takakusaki et al. (2001) found that inhibitory reticulospinal pathways influencing motoneurons and interneurons acted in parallel. Our results obtained from SpikeTA of arm and shoulder muscles appear to support previous investigator’s claims that reticulospinal control of movement is mediated primarily by interneurons, which may be especially true for the upper extremity in the monkey. The possible mechanisms of these pathways are presented below in the discussion of the StimulusTA results.
The output of single PMRF neurons, as measured by SpikeTA, was limited. The muscle fields of PMRF neurons were small, typically affecting only one muscle. Only 2 of the 11 cells produced PSpEs in more than one muscle, and in both cases, PSpEs were detected in only two muscles. In one case, output was directed to a contralateral elbow extensor and an ipsilateral elbow flexor, with both muscles responding with PSpF. In the
93 other case, output was solely ipsilateral, producing PSpF in iTrLa and iUTr. The muscle
fields demonstrated by PMRF cells are smaller than those reported for rubrospinal and
corticospinal cells in similar SpikeTA studies in the monkey (Fetz and Cheney,
1980;Cheney and Fetz, 1985;Kasser and Cheney, 1985). The disparity in the size of
muscle fields, compared to CM and rubromotoneuronal cells, is likely a result of the
difference in the number of monosynaptic projections among these motor systems. As
discussed above, the present results indicate that there may not be a significant number of
RM cells for the upper extremity in the primate, whereas CM and rubromotoneuronal
connections are much more common and are more specific for distal arm muscles (Fetz
and Cheney, 1980;McKiernan et al., 1998;Mewes and Cheney, 1991). The tendency to
find PSpEs in more proximally located muscles (UTr, PMj) suggests that RM projections
are more prevalent for these motoneurons, and to a lesser extent, motoneurons for the
triceps. Hence, there appears to be little overlap for targets of rubromotoneuronal and CM cells with RM cells.
For a single neuron to produce PSpEs in ipsilateral and contralateral elbow muscles, or in muscles spanning more than one joint, it would be necessary for a RST axon to branch and send terminals to more than one level of the spinal cord. This has been reported with anatomical examinations of the RST in the cat, where single axons were described as branching over several levels of the spinal cord and contacting both sides of the cord (Peterson et al., 1975;Matsuyama et al., 1997;Nyberg-Hansen,
1965;Petras, 1967;Matsuyama et al., 1999). We are unaware of any similar anatomical study that has been performed in the primate, and it is not known if the branching patterns of primate RST axons are indeed similar to those in the cat. With the PSpEs
94 observed in the present study, it is likely that the branching patterns of primate RST
neurons are indeed similar to those of the cat.
The task employed in the present study may have limited the ability to observe
PSpEs. Compared to the task employed in the previous study, the present task was chosen to increase the range of movement and to increase the recruitment of reaching muscles.
This task was successful in meeting the criteria it was designed for, except that to observe
a larger number of PSpEs, it may be necessary to utilize a task that generates more
forceful movements. While a wide range of movements were necessary to perform the
present task, there was little need for forceful movements. As a result, fewer muscles may
have been recruited during task performance. Additionally, RST recruitment of muscles
may increase when larger forces are generated. A study that would employ a mechanism
such as a manipulandum to regulate force may determine if SpikeTAs are more
commonly observed in the PMRF when forceful movements are made.
Stimulus-Triggered Averaging
A discussion on the effectiveness of the stimulation methods used for this study is presented in the previous chapter. In the present study, current for the majority of stimulus sites was set to 30 µA and varied only when muscle twitches were observed. For both studies, 30 µA was sufficient for the purposes of StimulusTA in the PMRF of the monkey. Additionally, the stimulus intensities chosen were lower than those used in previous investigations of the PMRF of the cat and monkey (Cowie and Robinson 1984;
Drew and Rossignol 1990a). As discussed in the previous chapter, single pulse stimulation was likely successful in stimulating only PMRF neurons and their axons. The
95 possibility still remains, however, that CST collaterals in the PMRF are activated
indirectly with stimulation. To determine if the PStEs are a result of stimulating PMRF
neurons and axons, rather than CST collateral activation, it would be necessary to
compare the effects of pyramidal tract stimulation in the caudal medulla with PMRF
stimulation. If the EMG responses of pyramidal tract and CST stimulation were to
summate, then it is likely that the two pathways are independent and the PStEs observed
with PMRF stimulation are the actual output of this region.
Distribution of Post-Stimulus Effects
The most striking finding with StimulusTA in the present study was the
organization of bilateral motor output. Bilateral movement patterns resulting from PMRF
stimulation were first described in the cat by Sprague and Chambers (1954) and more
recently by Drew and Rossignol (1990 a,b). Movements described by these investigators
included flexion of the spine, head movement, and combinations of independent or
inclusive movements with all four limbs. Sprague and Chambers emphasized the
occurrence of bilateral reciprocal-like responses, with opposite responses or movements
observed for opposite sides of the body. Reciprocal responses within a limb were also
reported by Drew and colleagues. The most common response described in both reports was ipsilateral arm flexion, contralateral arm extension, and head turning towards the ipsilateral limb. The results of StimulusTA in the monkey PMRF were comparable to the reports of previous investigations with stimulation in the cat PMRF. In the present study, the prevalence of flexor PStF and extensor PStS in the ipsilateral arm, in combination with extensor PStF and flexor PStS in the contralateral arm, is consistent with ipsilateral
96 arm flexion and contralateral arm extension. Additionally, the pattern of PStEs observed between the bilateral upper trapezius muscles is consistent with head turning to the ipsilateral side. We also found that the PStEs observed in the present study were consistent with our previous findings with StimulusTA in ipsilateral arm and shoulder muscles in the monkey. It should be emphasized, however, PStEs in either study were not limited to this type of response and a wide range of PStF and PStS was observed. In fact
PStF and PStS were observed in every muscle except for Brac.
StimulusTA of arm and shoulder muscles in the primate PMRF revealed that stimulation at individual stimulus sites could evoke reciprocal responses between anatomical antagonists and bilateral homologues that were typically consistent with the prevalent response for both muscles involved. Almost all of the reciprocal responses observed were concurrent or serial, but were more often concurrent. Interestingly, for arm and shoulder muscles, concurrent reciprocal responses were more common on the ipsilateral side, whereas serial reciprocal responses were more common for the contralateral side. The occurrence of two types of reciprocal response patterns in either limb suggests these responses may be mediated by separate pathways. Whether or not these pathways are the same for the ipsilateral and contralateral arm is not certain, although, given the differences in the prevalence of reciprocal responses observed here and the findings of other investigators, it is likely some of the pathways mediating contralateral RST responses are indeed different than those for the ipsilateral side
(Jankowska et al. 2003). In the spinal cord of the cat, RST terminals are present in both sides of the spinal cord and are capable of synapsing directly on motoneurons (Petras,
1967;Matsuyama et al., 1999;Matsuyama et al., 1997), and these monosynaptic pathways
97 could mediate similar responses regardless of side. Pathways utilizing interneurons, however, may be different depending on the side. It can be hypothesized that pathways for the ipsilateral side may have fewer synapses between RST terminals and motoneurons since input for contralateral responses would have to originate from the opposite side. It should be noted that, in the present study, contralateral PStF was significantly earlier than ipsilateral PStF. Whether or not this reflects an actual physiological difference in the two pathways, or was merely a consequence of the combination of muscles studied or the number of ipsilateral or contralateral stimulus sites is not certain. Another possibility may be these different responses reflect the differences in medial RST and lateral RST projections that control ipsilateral and contralateral movements. The medial RST is descends ipsilaterally, whereas the lateral RST descends bilaterally; however, the pattern of terminations in the spinal cord for these two pathways is not known (TORVIK and
Brodal, 1957;Peterson et al., 1975). Despite that, one pathway is primarily ipsilateral and the other is bilateral, they may or may not have access to the same interneuron populations in the spinal cord and can influence motoneurons in a similar manner.
Further investigation into the differences in the patterns of terminations for these two pathways is necessary.
The exact pathways that may be involved in reticulospinal responses in the ipsilateral forelimb are not certain. In a pathway that could account for concurrent reciprocal responses, flexor and extensor motoneurons would be independently targeted by interneurons that concurrently excite flexor motoneurons and inhibit extensors. For a serial reciprocal pathway, an excitatory interneuron could excite the flexor motoneuron directly while also exciting an inhibitory interneuron for the extensor motoneuron.
98 Another serial reciprocal could contain flexor motoneurons that receive monosynaptic
contacts form RST terminals that also excite inhibitory interneurons for extensor motoneurons. In each of these pathways, the activity of the primary interneuron would be driven by reticulospinal projections. In hindlimb motoneurons of the cat, Ib and FRA pathways have been reported to mediate reticulospinal inhibition (Takakusaki et al.,
2001;Engberg et al., 1968a;Engberg et al., 1968b). Specifically, Engberg et al. reported that PMRF stimulation attenuates EPSPs and IPSPs in FRA interneurons and reduces Ib responses in motoneurons via the dorsal reticulospinal system. Takakusaki et al. reported
the same effects on FRA and Ib interneurons from the ventral reticulospinal system,
except some Ib interneurons were also facilitated. Even though most of these
investigations have been conducted in cat hindlimb, it seems likely that these same
pathways are also involved in reticulospinal control in the forelimb (Jankowska, 1992).
The fact that reciprocal responses for the contralateral arm and shoulder were
usually serial, rather than concurrent, suggests that different pathways are involved in
reticulospinal control of contralateral muscles. The FRA pathways are capable of
mediating such a response (Jankowska, 1992) and are likely involved in some
contralateral responses. The focus of a recent series of reports by Jankowska and
colleagues (Bannatyne et al., 2003;Jankowska et al., 2003;Krutki et al., 2003) has been
the crossed reticulospinal response in cat hindlimb motoneurons. In these experiments,
contralateral motoneuron responses to reticular formation (MLF) stimulation were
examined. In accordance with our results with SpikeTA, responses in the contralateral
muscles likely involve monosynaptic connections from the contralateral and ipsilateral
reticular formation. Monosynaptic connections, however, are not the only pathways for
99 contralateral responses; Jankowska and colleagues reported that responses in contralateral
motoneurons were still observed after the contralateral half of the spinal cord was
lesioned rostral to the lumbar cord. These authors present several pathways that can
account for contralateral responses. Some of these pathways include ipsilateral or
contralateral interneurons receiving terminals from the ipsilateral RST, that in turn
synapse directly contralateral motoneurons. In another possible pathway, commissural
interneurons that receive projections from ipsilateral interneurons synapse on
contralateral motoneurons. In a separate report, it was discovered that the commissural
interneurons mediating contralateral RST responses may also mediate contralateral
vestibulospinal responses (Krutki et al., 2003). Thus, commissural interneurons are also
candidate for mediating contralateral responses.
Wrist flexors and Extensors
There are no known reports that have described movement below the elbow with
stimulation in the primate PMRF. PMRF projections to distal cat forelimb motoneurons
have been described in some reports; however, Drew and Rossignol (1990a) reported that
movement below the elbow was never observed with stimulus trains in the cat PMRF. In the present study, we observed PStEs in the ipsilateral and contralateral wrist flexors and
extensors, as well as in the distal elbow flexor brachioradialis. Responses were not as
common in these muscles as they were for others, and PSpEs were not detected in these
muscles. It may be possible that there are no RM projections to wrist muscles in the
monkey, which would explain the lack of PSpEs in these muscles. In spite of this, the
overall pattern of responses for distal arm muscles matched those for other arm muscles
100 studied (i.e. ipsilateral flexor PStF). Although all stimulus sites were located in the
PMRF, it is possible that these responses resulted from stimulating corticospinal tract
collaterals. Since responses were observed in the contralateral and ipsilateral arm from
some stimulus sites, this would require collaterals from both sides of the cortex to be present in one side of the PMRF. It would be necessary to perform the experiment with
pyramidal tract stimulation described above to determine origin of the wrist PStEs.
Reticulospinal control of movement
Studies in our lab have shown that PMRF neurons are active in the control and
preparation of movement (Buford and Davidson 2004). A question that remains, however, is how PMRF neurons are recruited for the control of movement.
Corticoreticular projections and corticospinal tract collaterals are bilateral (Kuypers
1981), which leaves the possibility that neurons in both sides of the PMRF can be recruited together by either side of the cortex. Another possibility may be that cortical recruitment is more selective and the output of the two halves of the PMRF can be selected independently, depending on the requirements of the movement. Since there is a strong bilateral component to PMRF motor output, either possibility could account for the coordination of bilateral movements between these two motor systems. Furthermore, studies have shown the superior colliculus (Werner et al., 1997a;Werner et al.,
1997b;Kawamura et al., 1974), and the cerebellum (Sprague and Chambers, 1953;Brodal,
1956) also send efferents to the PMRF. With such a variety of input to the PMRF, it is
difficult to determine which combinations are active in controlling movement.
101 Even though stimulation in the PMRF may more often evoke ipsilateral limb flexion, contralateral limb extension, and head turning to one side, the results of stimulation studies may merely reflect the sum of the outputs of the PMRF. If the prevalent response of the ipsilateral and contralateral PMRF were evoked concurrently, the net output would likely result in co-contraction of most limb muscles, as well as proximal shoulder and back muscles, given the wide ranging bilateral PMRF outputs that have been revealed. Indeed, stimulation in the PMRF may indicate a preference for flexor facilitation and extensor suppression ipsilaterally and the opposite responses for the contralateral side; the results of StimulusTA in both the red nucleus and motor cortex are similar and have reported a preference for extensor facilitation (red nucleus) (Cheney et al., 1991b) or flexor and extensor facilitation (motor cortex) (McKiernan et al., 1998).
Nevertheless, these responses are not the only response observed with PMRF stimulation and a wide range of movements or effects on motoneurons have consistently been reported. In some reports, these responses were found to be dependent on the state of the nervous system at the time stimulation was applied (i.e. swing vs. stance phase of locomotion) (Drew and Rossignol, 1984;Drew et al., 1986;Drew, 1991). In other words,
RST output is not an all or none phenomenon. An independent selection of RST outputs would almost be necessary for the brain to produce coordinated movements with the
PMRF involved, and these outputs are likely selected differently by the various regions that send projections to the PMRF. As pointed out by Canedo (1997) in a review of descending motor control, stimulation may not mimic the convergence of these inputs on
PMRF neurons.
102 Ito et al. (1970b) suggested the presence of inhibitory PMRF neurons that do not
send their axons to the spinal cord. As these authors suggest, these inhibitory neurons are
likely involved in local inhibitory networks within the PMRF. The presence of these local
inhibitory interneurons would further complicate the PMRF circuits involved in the
control of movement. They may also help to explain why the results of SpikeTA and
StimulusTA are not always consistent. In this situation, stimulation at a particular site
would also activate inhibitory neurons that may have an inhibitory effect on some RST neurons and not others. This would provide one mechanism in which specific RS outputs can be independently selected and is reminiscent to the inhibitory interneuronal networks present in the spinal cord. Given this organization, the components of the PMRF may be much more complicated, yet more organized than originally thought.
Organization of Stimulus Sites
If the cortex were capable of independently selecting PMRF output, then it could be hypothesized that PMRF neurons demonstrate some gross organization based on their output. UTr, one of the two most affected muscles demonstrated the most noticeable distribution of sites where PStS and PStF were evoked, with PStS found more rostral and
PStF found more caudal. This organization of stimulus sites was not specific, especially when compared to previous descriptions of the organization of RST output. It is possible that this organization was most noticeable in UTr because this muscle had one of the highest number of responses. An examination of organization of stimulus sites in other muscles with similar numbers of responses may reveal a similar organization of output.
103 Drew and Rossignol (1990a,b) reported that ipsilateral limbs were represented
more dorsally and contralateral limbs were represented more ventrally. We found no
evidence for the organization of flexor or extensor responses in the medial-lateral or
dorsal-ventral orientations. The region studied here included areas that were more rostral
than those examined by Drew and Rossignol and may explain our inability to find a
difference in sites representing flexion or extension, and ipsilateral or contralateral limbs.
Drew and Rossignol also reported head movement was evoked from all regions of the
PMRF, but were concentrated in the caudal medulla. Because the head was restrained for this study, we could not compare stimulus sites on the basis of head movement.
Nevertheless, sites where UTr responses were evoked were located over the extent of the region studied. The sign of response observed depended on the location of the stimulus sites, which were oriented in a rostrocaudal organization. PStF responses were more common caudally, and may suggest head movements may be more often evoked from caudal regions. This is consistent with Drew and Rossignol’s observations that head movements were more often evoked from caudal regions in the cat..
Our results were consistent with Peterson and colleagues description of a region caudal to the abducens nucleus that contained a large number of monosynaptic connections to neck motoneurons. Although we can not confirm the presence of monosynaptic connections with StimulusTA, we were able to identify responses in the iUTr that are consistent with monosynaptic responses. Similar to what was described in the previous chapter, some of the responses in the iUTr were considerably larger than responses in all other muscles. These responses were typically evoked with the lowest stimulus currents; in some cases, these responses were observed with currents as low as
104 10 µA. Additionally, every case where stimulation current was reduced was a result of
movement in the ipsilateral shoulder. Thus, consistent with Peterson and colleagues, we
have identified a region in the primate PMRF located caudal to the abducens nucleus that
appears to be specific for strong, fast latency facilitative responses in the iUTr.
Clinical Relevance
The coordination of bilateral movements is necessary for the performance of daily activities. Many of the activities that are required day after day depend on bilateral coordination of posture or stabilization to perform specific movements (Prentice and
Drew, 2001;Schepens and Drew, 2003b;Schepens and Drew, 2003a). The results from previous studies in the cat reticulospinal system have shown that this motor system plays a major role in coordinating organized bilateral movements. The results of the present study indicate that this is also true in the primate. The corticospinal system has access to
the reticulospinal system through corticoreticular projections, as well as pyramidal tract
collaterals, and may normally direct some bilateral movements through these connections. In cases where unilateral corticospinal damage has occurred, it may be possible to recover function through alternate pathways by emphasizing recovery with the remaining, intact motor systems. With its potential for bilateral control, the reticulospinal pathway is a likely substrate for such recovery.
105
Location of EMG Implants (12 / side, 24 tot.) Function (s) Muscle 1 Wrist flexion Flexor carpi radialis FCR 2 Wrist Extension Extensor carpi ulnaris ECU
3 Elbow flexion Brachioradialis Brac
4 Elbow Flexion Biceps – long head Bic
5 Elbow Extension Triceps – long head TrLo 6 Elbow Extension Triceps – lateral head TrLa
7 Humerus Flexion & Deltoids – anterior head ADlt Med Rotation
8 Humerus Extension & Deltoids – posterior head PDlt Lat Rotation 9 Humerus Flexion & Pectoralis Major PMj Adduction
10 Humerus Extension, Latissimus dorsi Lat Adduction, & Rotation 11 Scapula Retraction Middle Trapezius MTr 12 Scapula Elevation Upper Trapezius UTr
Table 3.1. Location of EMG implants. Implants were located in left and right muscles, 12 muscles per side, for a total of 24 muscles.
106
Average Onset of PStEs (Mean ± SD) Muscle nIpsilateral n Contralateral ADlt Fac 7 11.8 ± 2.2 2 4.6 ±0.5
Spr 4 10.9 ± 2.2 9 10.4 ±1.4 Bic Fac 24 7.4 ± 2.6 8 5.6 ±1.2
Spr 3 9.2 ± 3.8 12 10.5 ±1.9 Brac Fac 7 7.8 ± 2.1 - - - ECU Fac 11 7.7 ±2.128 7.7 ±2.1
Spr 61 11.0 ±2.39 12.3 ±1.6 FCR Fac 35 7.8 ±2.115 6.3 ±2.1 Spr 27 10.1 ±2.53910.2 ±1.9
Lat Fac 39 8.6 ±3.214 9.2 ±3.9 Spr 77 7.0 ±3.089 6.7 ±1.9 MTr Fac 51 6.8 ± 2.8 8 7.8 ± 3.6 Spr 9 11.1 ± 2.7 55 9.9 ± 2.6
PDlt Fac 8 6.3 ±2.313 6.8 ±2.4 Spr 25 9.0 ±2.13 10.7 ±1.1 PMj Fac 80 7.7 ± 2.7 4 6.4 ± 5.6
Spr 11 8.5 ± 2.8 132 7.5 ± 2.0 TrLa Fac 10 7.6 ± 2.0 59 6.6 ±2.2
Spr 82 7.2 ± 2.3 16 11.7 ±3.4 TrLo Fac 14 6.7 ± 4.0 67 6.8 ± 3.4
Spr 80 8.1 ± 2.0 32 8.1 ± 3.0 UTr Fac 46 8.3 ± 3.1 120 6.3 ±1.7 Spr 117 6.6 ±1.64911.7 ±2.3
p<0.05
Table 3.2. Average onset latency of post-stimulus effects.
107
Average Duration of PStEs (Mean ± SD) Muscle nnIpsilateral Contralateral ADlt Fac 7 6.9 ± 1.9 2 4.8 ± 1.1
Spr 4 5.6 ± 1.0 9 5.9 ± 2.9 Bic Fac 24 7.2 ± 2.3 8 6.0 ± 2.0 Spr 3 5.8 ± 3.0 12 7.8 ± 6.4 Brac Fac 7 5.8 ± 2.4 - - - ECU Fac 11 5.1 ± 0.7 28 6.3 ± 2.2
Spr 61 9.4 ±3.49 7.9±5.6 FCR Fac 35 5.4 ± 1.6 15 4.8 ± 1.2 Spr 27 6.4 ± 2.7 39 5.4 ± 2.1
Lat Fac 39 7.0 ± 3.5 14 7.5 ± 3.2 Spr 77 7.3 ± 3.0 89 7.0 ± 2.9
MTr Fac 51 6.2 ± 2.1 8 7.7 ± 5.0 Spr 9 5.1 ± 2.2 55 8.1 ± 2.9
PDlt Fac 8 5.3 ± 1.9 13 5.3 ±1.5 Spr 25 7.2 ± 2.6 3 8.2 ±3.2 PMj Fac 80 9.2 ±3.34 7.8±4.8
Spr 11 6.8 ± 4.0 132 8.9 ± 3.1 TrLa Fac 10 5.2 ±1.159 6.1 ±1.6 Spr 82 8.4 ±2.616 7.5 ±2.3
TrLo Fac 14 6.6 ±2.167 8.7 ±4.5 Spr 80 8.2 ±2.732 6.9 ±2.5 UTr Fac 46 8.9 ± 4.8 120 8.2 ±3.5 Spr 117 7.2 ±3.04912.3 ±5.3 p<0.05
Table 3.3. Average duration of post-stimulus effects.
108
Average MPC of PStEs (Mean ± SD) Muscle nnIpsilateral Contralateral ADlt Fac 7 0.35 ± 0.33 2 0.22 ±0.04
Spr 4 0.10 ± 0.04 9 0.15 ±0.03 Bic Fac 24 0.18 ± 0.05 8 0.17 ± 0.03
Spr 3 0.13 ± 0.05 12 0.16 ± 0.03 Brac Fac 7 0.19 ± 0.04 - - - ECU Fac 11 0.11 ± 0.03 28 0.14 ± 0.06
Spr 61 0.12 ± 0.03 9 0.10 ± 0.04 FCR Fac 35 0.15 ± 0.04 15 0.13 ± 0.04 Spr 27 0.13 ± 0.04 39 0.12 ± 0.04
Lat Fac 39 0.13 ± 0.06 14 0.13 ± 0.04 Spr 77 0.14 ± 0.05 89 0.14 ± 0.05 MTr Fac 51 0.15 ± 0.04 8 0.11 ± 0.07 Spr 9 0.14 ± 0.04 55 0.15 ± 0.05
PDlt Fac 8 0.29 ± 0.19 13 0.26 ± 0.17 Spr 25 0.16 ± 0.04 3 0.18 ± 0.04 PMj Fac 80 0.13 ± 0.07 4 0.16 ± 0.06
Spr 11 0.11 ± 0.03 132 0.12 ± 0.05 TrLa Fac 10 0.16 ± 0.07 59 0.19 ±0.07 Spr 82 0.19 ± 0.06 16 0.14 ±0.05 TrLo Fac 14 0.14 ± 0.10 67 0.14 ± 0.08
Spr 80 0.11 ± 0.03 32 0.11 ± 0.05 UTr Fac 46 0.34 ±0.371200.16 ±0.07 Spr 117 0.13 ±0.0649 0.10 ±0.04 p<0.05
Table 3.4. Average MPC of post-stimulus effects.
109
Average SDPk of PStEs (Mean ± SD) Muscle nnIpsilateral Contralateral ADlt Fac 7 16.8 ± 16.2 2 5.8 ± 0.1
Spr 4 5.1 ± 0.8 9 5.4 ± 1.1 Bic Fac 24 7.1 ± 3.3 8 6.5 ± 1.6 Spr 3 5.2 ± 1.9 12 5.5 ± 1.6 Brac Fac 7 6.7 ± 1.6 - - - ECU Fac 11 7.2 ± 2.1 28 10.3 ±6.9
Spr 61 6.4 ± 2.1 9 5.5 ±1.7 FCR Fac 35 6.3 ± 1.6 15 7.9 ±3.2 Spr 27 5.8 ± 2.0 39 5.3 ±1.1
Lat Fac 39 6.2 ± 1.5 14 6.8 ± 2.1 Spr 77 7.3 ± 3.1 89 6.9 ± 2.7
MTr Fac 51 6.6 ± 2.0 8 6.6 ± 2.0 Spr 9 5.2 ± 1.8 55 6.0 ± 1.7
PDlt Fac 8 15.0 ± 10.5 13 12.2 ± 11.8 Spr 25 5.9 ±1.33 6.1±2.1 PMj Fac 80 9.4 ± 6.1 4 11.8 ± 4.3
Spr 11 6.9 ± 2.7 132 8.3 ± 3.5 TrLa Fac 10 5.3 ± 1.3 59 7.3 ± 3.3 Spr 82 6.4 ± 1.9 16 5.7 ± 1.0
TrLo Fac 14 8.5 ± 5.8 67 9.9 ± 5.4 Spr 80 7.2 ± 2.3 32 9.3 ± 5.9 UTr Fac 46 44.4 ± 73.1 120 12.0 ±7.5 Spr 117 9.0 ±4.349 7.4 ±2.7 p<0.05
Table 3.5. Average SDPk of post-stimulus effects.
110 Figure 3.1. Diagram of the behavioral task. After the start switches were contacted, one of four trial types began (left hand left target = LHLT, etc.). After a variable waiting period, the instruction was given. The target's color indicated which hand to use (red = right, green = left). After another waiting period, the Go Cue instructed the subject to reach to the target with the appropriate hand while keeping the other hand on its start switch. The grey box surrounds the instruction period, and yellow surrounds the movement period. The inset (green box) at the top shows the apparatus and the approximate size of a subject compared to the touch screen.
111 Touch Screen
Display
Start Switches
Food Well
Start Switches Contacted
1 - 2.5 ms LHLT RHLT RHLT LHRT delay
Instruction
1 - 2.5 ms delay
Go Cue
Contact with Target
112 A Stimulator B Record H.S. + Extracellular
e d Potentials or EMG o - r t Stimulus Current c e Spikes e b l u RSN E T Recording e d Chamber i -20 0 20 40 60 u G Time Relative to Spike (ms)
+ Record - EMG C aMN EMG Stimuli
-20 0 20 40 60 Muscle Time Relative to Spike (ms)
Figure 3.2. Spike and Stimulus Triggered Averaging. A) Methodological setup for SpikeTA and StimulusTA. Electrode is inserted through a guide tube that is also used as (-) input to the head stage amplifier (H.S.). Electrode and guide tube can be attached to stimulus isolator by remote controlled relays to allow stimulation through the electrode. Stainless steel recording chamber served as a ground. B) Extracellular action potentials are shown relative to the recorded EMG for an 80 ms sweep. C) Single pulse stimulation relative to EMG for an 80 ms sweep. Procedures for averaging and analyzing SpikeTA and StimulusTA are explained in the text.
113 Left Arm, Left Target Right Arm, Left Target RUTr RMTr RLat RPMj RPDlt RTrLo RFCR LUTr 1
1 LMTr 4 LLat LADlt
LTrLa LBic LECU
LFCR 4.5 mV Unit
Stimulus 5 s
Figure 3.3. EMG records for reaches made with left and right arm to the left target in two consecutive trials. Grey shading indicates waiting periods; yellow shading indicates the movement periods. Stimuli are imposed from a separate StimulusTA file for demonstration purposes. The total length of this segment is 20 s. 200 Fac
s Spr
E 100 t S P
f o 0 r e b m u 100 N
200
0 20 40 60 Onset Latency (ms)
Figure 3.4. Histogram of onset latencies for PStF(Fac) and PStS (Spr). All events detected in the post-stimulus window are included. PStF are represented by the upward grey bars, PStS by the downward black bars.
115 A Effectiveness of Stimulation by Muscle d e k
60 6 o 5 v E s
a 50 w E 9 t 3 7 S
40 3 P 1 a 3 8 h 7 2 6 c
30 2 i 2 4 4 h 2 2 1 w 9 2 1 8 m 20 1 5 o 4 1 r 1 2 f 1 0 1 1 s 1 9 e 8 8 t 10 7 i 6 5 S f o 0 % j j t t t t t t r r c c c a a r r o o l l l l R R i i a a U U a T T L L T T L L r M M L L D C D C B D D B r C r C r r i i U U c c P P B P F P F i T E T M M E i T T A i A c i i c i i i i c c i c c c c c
B Proportion of Post-Stimulus Facilitation by Muscle Ipsilateral 100 Contralateral
d 80 e v r e s
b 60 O s E t
S 40 P f o
% 20
0 t j t t c c r a r l o l R i U a a T L T r L M C B D D L C r r U B P F P E T M A T
Figure 3.5. A) Percent effectiveness of stimulation by muscle. Percentages are indicated above each bar. B) Percentages of PStF responses for all responses observed in a muscle for the ipsilateral (ã) and contralateral (g) side.
116 cUTr cMTr
cLat cPMj
cPDlt
cTrLo cFCR iUTr
iMTr
iLat
iADlt iTrLa iBic iECU
iFCR 0.1 mV -20 0 20 40 60 Latency (ms)
Figure 3.6. Representative StimulusTA with the majority of muscles responding with their prevalent response. PStF is indicated by filled bars, PStS by open bars. Stimuli were applied at 30 µA. SDPk of events: iECU -7.7, iTrLa -4.7, iLat -7.8, iMTr 9.1, iUTr -23.7, cFCR -5.9, cTrLo 13.9, cPMj -10.3, cMTr -4.3, cUTr 22.4.
117 iUTr
iLat
iADlt
cPMj 0.1 mV
-20 0 20 40 60 Latency (ms)
Figure 3.7. Strong PStF in iUTr. PStF is indicated by filled bars, PStS by open bars. Stimuli were applied at 20 µA. SDPk of events: cPMj -4.9, iADlt 13.4, iLat -9.8, iUTr 246.6.
118 A
cBic
cTrLa
-20 0 20 40 60 Latency (ms)
B
cBic
cTrLa 0.1 mV
-20 0 20 40 60 Latency (ms)
Figure 3.9. Serial reciprocal response (A) and serial cofacilitation response (B) between cBic and cTrLa. PStF is indicated by filled bars, PStS by open bars. Stimuli were applied at 30 µA. SDPk of events: A) cTrLa 5.1 cBic -5.3 B) cTrLa 6.3, cBic 5.9.
120 A 1,807 iUTr
-20 0 20 40 60 Latency (ms)
B 766 A T
s iBic u l
u 658
m cTrLa i t S
3,268 A
T iBic e
k 2,754 i 0.1 mV p cTrLa S
-20 0 20 40 60 Latency (ms)
Figure 3.10. (A) Example of a PSpE that met all acceptance criteria. (B) Comparison between a SpikeTAand StimulusTA complied from the same site. PStF is indicated by filled bars. The number of triggers used for averaging are indicated above each muscle. Stimuli were applied at 30 µA. SDPk of events: A) iUTr 4.5. B) StimulusTA: cTrLa 4.0, cBic 6.1; SpikeTA: cTrLa 6.3, cBic 5.9.
121
iPMj
iPDltcPMj Reciprocal, Concurrent cPDlt Reciprocal, Serial
iMTriMTr Cosuppresion cMTr Cofacilitation r
iLatiLat Not available cLat cLat cMT
iUTr iUTr cUTr
Figure 3.11. Representation of frequent responses between antagonists and bilateral homologues for scapular muscle pairs.
122
Fig 3.12. Sites from which UTr responses were evoked by StimulusTA. Brainstem sections adapted from Szabo and Cowan (REF), with nomenclature from Martin et al. (REF). Symbols explained in legend. Abbreviations: n4, 4th cranial nerve; n5, 5th cranial nerve; 6, 6th cranial nerve nucleus; n7, 7th cranial nerve; 7, 7th cranial nerve nucleus; n8, 8th cranial nerve; 10, 10th cranial nerve nucleus; 12, 12th cranial nerve nucleus; icp, inferior cerebellar peduncle; mcp, middle cerebellar peduncle; scp, superior cerebellar peduncle; Py, pyramidal tract; tz, trapezoid body; IO, inferior olivary nucleus; SO , superior olivary nucleus; IVe, inferior vestibular nucleus; SVe, superior vestibular nucleus; LVe, lateral vestibular nucleus; MVe, medial vestibular nucleus; PrP, nucleus prepositus; ACu, anterior cuneate nucleus; Sol, nucleus solitarius; Pr5, principal nucleus of 5th cranial nerve; Sp5, spinal nucleus of 5th cranial nerve; LC, locus ceoruleus; IC , inferior colliculus; SC , superior colliculus; 4V, 4th ventricle; Aq, cerebral aqueduct.
123 p IVe AP -3 sc AP -1 MVe 10 p 4V LVe c u i C A 12 Sol A 4V B A A IVe MVe B A AB A A B Sp5 B B A B A Sol AB A A A AB AB B AB BA A AP -2 A AB AB A IO B BA AB AB A AB A Sp5 BA A AB A AB A AB IVe MVe 4V AB B A Py AB AB AB AB Sol PrP B A AB AB B A IO AB A Sp5 AB A AB A AB B A AB AB AB Py AB AB A AB B AB AB AB B AB B B A AB B IO scp e AP 1 Py SV cs p 4V LVe SVe AP 0 6 n7 A LVe 4V B AB 5 AB AB MVe p B B A n8 S B B B A AB LVe PrP AB B A AB B A AB AB B B A A Sp5 7 AB A n8 A AB B A A AB A A A B A AB AB A AB A A B A AB A AB IO AB A IO Py
Py AP 3
SC p AP 2 sc 4V mcp LC
LVe B IC B A B B B Sp5 AB A Pr5 B A BA A A B A n7 B BA BA AB A A 4n Aq B A p B B A A sc B A n8 B 4 A 7 B A B A AB mcp B A AB IO BA A B A B A Py A B B n5 A 1-mm BA DV -12, ML 0 SO LATERAL Response tz A CONTRA Fac B IPSI Spr Py
No Response SDPk > 40
124
CHAPTER 4
CONCLUSION
Compared to the corticospinal, rubrospinal, and vestibulospinal tracts, the least is
known about the reticulospinal tract. The reticulospinal system has been the focus of
investigation in several studies in the cat; however, there have been few studies that
examined this motor system in the monkey. The data presented in this dissertation have
helped to fill this void of information by providing maybe one of the most extensive
studies of the primate reticulospinal system to date. These reports are the first that have
described the use of spike and stimulus triggered averaging in the primate reticulospinal
system.
In some aspects, the results obtained from these experiments are not unexpected.
A variety of bilateral and reciprocal pattern of movements have been described for the
reticulospinal system for over 50 years, and have been corroborated by several investigators within this period of time (Drew and Rossignol, 1984;Ashe et al.,
1993;Matsuyama et al., 2004;Takakusaki et al., 2001;Peterson et al., 1974;Peterson et al.,
1979;Drew et al., 1986;Drew and Rossignol, 1990b;Drew and Rossignol, 1990a;Drew,
125 1991). The results presented in this dissertation also corroborate the reports of earlier
investigators that first described the reciprocal and bilateral outputs of the reticulospinal
tract, and extend these findings to include the data from the primate reticulospinal tract.
We found the motor outputs of the primate PMRF to be highly specific on the basis of movement, and produced consistent results with stimulation. This potential for specific
output appears to be a plausible mechanism that the nervous system may use as the basis
of coordinated bilateral movements such as locomotion, voluntary reaching, or other forceful movements that would require postural stabilization.
Some of these results were surprising in that few post-spike effects were observed
form the primate pontomedullary reticular formation (PMRF). According to the literature, there appears to be a considerable number of monosynaptic projections from the cat
PMRF to motoneurons. This, however, does not appear to be the case in the primate.
Other studies with spike triggered averaging of EMG in the primate have focused on the corticospinal and rubrospinal tracts, and have constantly reported post-spike effects from these regions (Fetz and Cheney, 1980;Poliakov and Schieber, 1998).This leads us to believe that the lack of PSpEs in the primate indicates that there fewer reticulomotneuronal projections in the primate reticulospinal system. Shapovalaov (1973) compared the characteristics of corticomotoneuronal and rubromotoneuronal projections to the lumber cord, and found that there were fewer projections for reticulomotoneuronal cells than for corticomotoneuronal cells. Additonaly, reticulomotoneuronal projections were found to be weaker than corticomotoneuronal projections. This result has been supported by anatomical studies (Matsuyama et al., 2004)
126 Therefore, it appears that the primate reticulospinal tract is capable of careful
coordination of bilateral and reciprocal movements through interneurons, and less
commonly, directly through monosynaptic projections. This information may be useful
for the clinician working with individuals suffering from corticospinal impairments
resulting from injury or stroke. The knowledge that the reticulospinal system, which was once thought of as a primitive system, is capable of coordinated movements may help the clinician develop new strategies of recovery for these patients.
127
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