Center for Neurobiology and Behavior, Columbia University; and the New York State Psychiatric Institute, New York, New York 10032
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ABSTRACT |
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Chakrabarty, Samit and John H. Martin. Postnatal Development of the Motor Representation in Primary Motor Cortex. J. Neurophysiol. 84: 2582-2594, 2000. The purpose of this study was to examine when the muscles and joints of the forelimb become represented in primary motor cortex (M1) during postnatal life and how local representation patterns change. We examined these questions in cats that were anesthetized (45-90 days, n = 14; adults, n = 3) and awake (n = 4; 52-86 days). We used intracortical microstimulation (45 ms duration train, 330 Hz, 0.2-ms balanced biphasic pulses, with a leading cathodic pulse; up to 100 µA). In young animals (less than day 70), we also used stimulus trains and pulses that could produce greater temporal summation (up to 200-ms train duration, down to 143-Hz stimulus frequency, up to 0.8-ms pulse width). Anesthetized animals were areflexic, and muscle tone was similar to that of the awake cats (i.e., relaxed, not weight or load bearing, with minimal resistance to passive stretch). We monitored the kinematic effects of microstimulation and changes in electromyographic (EMG) activity in forelimb muscles. There was an age-dependent reduction in the number of sites where microstimulation did not produce a motor effect (i.e., ineffective sites), from 95% in animals younger than 60 days to 33% between 81 and 90 days. In adults, 24% of sites were ineffective. Median current thresholds for evoking movements dropped from 79 µA in animals younger than day 60 to 38 and 28 µA in day 81-90 animals and adults, respectively. There was a proximal-to-distal development of the somatotopic organization of the motor map. Stimulation at the majority of sites in animals younger than day 71 produced shoulder and elbow movement. Wrist sites were first present by day 71, and digit sites by day 81. Sites at which multiple responses were evoked, between 1.0 and 1.5 times threshold, were present after day 71, and increased with age. A higher percentage of distal joints were co-represented with other joints, rather than being represented alone. We found that effective sites initially were scattered and new sites representing proximal and distal joints filled in the gaps between effective sites. During most of the period examined, development of the caudal M1 subregion lagged that of the rostral subregion (percent of effective sites; threshold currents), although these differences were minimal or absent in adults. Our results show that the M1 motor representation is absent at day 45 and, during the subsequent month, the motor map is constructed by progressively representing more distal forelimb joints.
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INTRODUCTION |
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The primary motor cortex (M1) in
maturity represents contralateral limb muscles, through direct spinal
projections and indirect projections through the brain stem (for
review, see Porter and Lemon 1993). Classic stimulation
studies have shown that the limb motor representations have a basic
somatotopic organization, with distinctive arm and leg areas (for
example, Woolsey 1958
). Within these areas, there
appears to be a more complex organization because an individual muscle
can be represented at multiple sites and, at a single site, multiple
muscles can be represented (Armstrong and Drew 1984
;
Donoghue et al. 1992
; Sato and Tanji
1989
). These findings suggest that small regions of M1 could
represent motor synergies (Donoghue et al. 1992
;
Sanes et al. 1995
). Neurons within these representations
play important roles in organizing and controlling movement. For the
arm, inactivation, lesion, and recording studies point to important
roles for M1 in the control of the kinematic and dynamic parameters of
motor performance (for example, Cheney and Fetz 1980
;
Georgopoulos et al. 1982
; Kalaska et al.
1989
; for review, see Porter and Lemon 1993
) and
adaptive changes in limb trajectory (Drew et al. 1996
;
Martin and Ghez 1993
). M1 contains distinctive rostral
and caudal subregions (Pappas and Strick 1981a
,b
) that
have differential spinal projections (Martin 1996
).
These two subregions have been shown to contain limb representations that could serve different motor control functions (Geyer et al. 1996
; Martin and Ghez 1993
).
These features of the M1 motor representations have been described in
mature animals. However, it is not known whether the representation in
immature animals has a similar organization. This is an important
question to address because motor skills develop extensively during
early postnatal life (Hofsten 1982; Konczak and
Dichgans 1997
; Konczak et al. 1995
,
1997
). It is plausible that the motor map is modified
during this period as new motor skills are added to the behavioral
repertoire, similar to plasticity of the motor representation in
maturity (Donoghue and Sanes 1988
).
While there are no data on development of the characteristics of the M1
motor representation, there have been several studies examining the
postnatal development of the capacity to evoke motor responses with M1
stimulation. For example, in humans <2 yr of age, motor-evoked
potentials produced by transcranial magnetic stimulation (TMS) of the
motor strip occur only at maximal currents, and thereafter, stimulation
thresholds decrease until the 10th to 15th year (Eyre et al.
2000; Koh and Eyre 1988
; Müller et al. 1991
; Nezu et al. 1997
). Moreover, several
studies have reported the absence of motor effects in infants <1-2 yr
(Müller et al. 1991
; Nezu et al.
1997
). In the monkey, Lemon and colleagues have demonstrated
using TMS that the late and prolonged development of the motor effects
of M1 activation correlate with anatomical, physiological, and
behavioral maturation of the corticospinal (CS) projection
(Armand et al. 1997
; Flament et al. 1992
;
Olivier et al. 1997
). Delayed, but apparently sudden,
development of the capacity to evoke movements with intracortical
microstimulation (ICMS) of M1 has been suggested in the cat
(Bruce and Tatton 1980a
,b
).
The purpose of this study was to examine when the muscles and joints of
the forelimb become represented in M1 and how the local representation
patterns change during early postnatal life. We studied these questions
in the cat, using ICMS, between postnatal days 45 and
90 (PD 45 and 90) and in adults. In
the cat, CS axons grow into the cord around birth (Wise et al.
1977) and terminate bilaterally in the cervical gray matter by
PD 14-17 (Alisky et al. 1992
;
Theriault and Tatton 1989
). The aberrant ipsilateral terminations are largely eliminated during the subsequent 4-5 wk
(Alisky et al. 1992
; Theriault and Tatton
1989
), which depends on neural activity in sensory-motor cortex
(Martin and Lee 1999
; Martin et al.
1999
). We have recently shown that there also is extensive
postnatal refinement of contralateral terminations (Li and
Martin 2000
).
In this study, we determined the relationship between age-dependent
changes in the stimulation threshold to evoke movements and changes in
the topographic characteristics of the representation. We were
particularly interested in determining whether all forelimb joints were
represented from early in postnatal development or whether there were
age-dependent changes in the representation of particular joints. We
also were interested in determining whether the motor representations
in rostral and caudal subregions of M1 (Pappas and Strick
1981a,b
) have different characteristics from early in postnatal
development. Recently, we showed that the developmental program for
achieving corticospinal connectional specificity by the two subregions
is different (Li and Martin 2000
).
We will show that, while the capacity for evoking motor responses first
develops during the period immediately following anatomical refinement,
the M1 motor map initially is incomplete, sparsely representing only
proximal joints. The motor map is constructed during the subsequent
month: the number of sites at which ICMS produced movement increased,
the thresholds decreased, and M1 represented progressively more distal
forelimb joints. A preliminary report of some of these results were
published as an abstract (Chakrabarty and Martin
1999).
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METHODS |
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General
All cats were obtained from a supplier accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC, International; see Table 1 for listing of animals used in this study). All animals were delivered to the animal care facility after weaning. Kittens were housed in a group cage that complied with the standard established by the USDA, with up to four animals of similar age. Adults were housed individually. ICMS was performed acutely in anesthetized animals and chronically, in awake animals, with a stimulation/recording chamber implanted during a prior surgery. Procedures for the two preparations are described separately. All experiments were conducted with the approval of the New York State Psychiatric Institute animal care and use committee.
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ICMS experiments in anesthetized animals
For animals between PD 43 and 90, anesthesia was induced with ketamine hydrochloride (30 mg/kg im) and
maintained with ketamine alone (1.2-3.0 mg/kg im as needed). For
adults, we induced anesthesia using ketamine (30 mg/kg im) and xylazine
(0.6 mg/kg im) and maintained anesthesia using intravenous ketamine
infusion (10 mg · kg1 · h
1). Using this anesthesia protocol, animals
remained areflexive during surgery and testing. Electromyographic (EMG)
recordings from forelimb muscles revealed spontaneous activity (see
RESULTS; Fig. 8). The level of limb muscle tone during
stimulation was similar to that of the awake animals in this
study (i.e., relaxed, not weight or load bearing, with minimal
resistance to passive stretch).
Animals were placed in a conventional stereotaxic frame. A craniotomy was made over the lateral portion of the anterior parietal and frontal lobes to expose the forelimb areas of the sensory-motor cortex. A barrier was constructed with dental acrylic cement to create a saline well over the cortex. The dura over the lateral pericruciate cortex was incised and removed, enabling a radius of at least 3-5 mm of exposed cortex around the lateral margin of the cruciate sulcus. We photographed the exposed cortex using a charge-coupled device (CCD) camera attached to a dissecting microscope and referenced the coordinates of the electrode penetrations to locations on the cortical surface. Body temperature was maintained at 39° by a heating pad.
Electrode penetrations were made orthogonal to the cortical surface (see Fig. 1; range of penetration angles: 10-40°). In most of the younger animals (<PD 80-85) the motor cortex is relatively flat, which allowed us to use the same electrode angle for all penetrations. This resulted in one x-y coordinate axis for the experiment. For the older animals (>PD 85), when the motor cortex has a domed shape, we had to adjust the angle of penetrations rostral and caudal to the cruciate sulcus. This resulted in two x-y coordinate axes for the experiment. When this occurred, we calibrated the two axes according to the actual surface entry points of the electrodes. When a blood vessel was located at a stimulation site, we skipped to the next site.
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ICMS experiments in awake animals
To control for possible effects of anesthesia, we conducted experiments in awake animals chronically prepared for ICMS. Animals were administered atropine (0.04 mg/kg im) and a broad spectrum antibiotic (benzathene penicillin; 20,000 units/kg im). During surgery, body temperature was maintained at 39° by a heating pad. Anesthesia was induced using a mixture of ketamine hydrochloride (30 mg/kg im) and acepromazine (0.03 mg/kg im) and maintained with ketamine alone (1.2-3.0 mg/kg im as needed).
Animals were placed in a stereotaxic frame. A craniotomy was made over the lateral pericruciate cortex under aseptic conditions, and the bone at the margins of the craniotomy and immediately surrounding area was covered with dental acrylic cement. Several stainless steel screws were implanted into the frontal sinus and the frontal and parietal bones surrounding the craniotomy site. We cemented a cylindrical stainless steel stimulation/recording chamber (1 cm ID) over the craniotomy. Animals received buprenorphine (0.03 mg/kg im) following surgery for analgesia. Animals recovered from anesthesia within 6-12 h.
We began the stimulation experiments the day after recovery. During the stimulation experiments, the animals were gently restrained in a supine position on an assistant's lap. Animals were relaxed, and when the limbs were moved by the examiner, there was minimal resistance to passive limb stretch. We used a Kopf X-Y stage mounted onto the cylinder to position the electrodes. The weight of the assembly was 64 g, which was between 9 and 13% of the animals' body weight. Electrodes were carried through the dura while protected in a stainless steel hypodermic needle guide (26 ga). After penetrating the dura (which could be felt as a transient increase in resistance to advance the electrode-guide assembly), the electrode was advanced deeper into the cortex.
ICMS protocol
Microstimulation was applied through paralene-insulated tungsten
microelectrodes (Microprobe; 0.5 M nominal impedance). For all ages, stimuli (45 ms duration train, 330-Hz, 0.2-ms biphasic balanced pulses; with a leading cathodic pulse) were delivered once
every 3 s using a commercial constant current stimulator (AM
Systems). These parameters are routinely used in adults (Asanuma et al. 1976
) and have been reported to be effective in cats
>PD 49 (Bruce and Tatton 1980a
,b
).
In the initial experiments we also determined whether the immature CS
system could be activated by different stimulation patterns. Since it
was likely that the electrical stimulus produced motor effects
primarily by trans-synaptic activation in cortex
(Jankowska et al. 1975
), we prolonged the duration of the burst, from 45 ms up to 200 ms, reduced stimulus frequency (from
333 down to 143 Hz), and increased the pulse width (from 0.2 up to 0.8 ms) to produce greater temporal summation. These changes could also
lead to more effective summation of spinal responses (if present).
For experiments in anesthetized animals, we sampled cortical sites at 500-µm intervals over as much of the lateral pericruciate cortex as possible before we felt that the preparation was becoming compromised (i.e., prior to edema and significant fibrin production, which could prevent us from penetrating the pial surface close to blood vessels). For the experiments in awake animals, we sampled at 1-mm intervals. For experiments in both anesthetized and awake animals, motor effects produced by ICMS occurred at lowest stimulus currents at depths where we recorded multiunit activity with the largest amplitude spikes. The cell layer depth was typically between 1 and 1.5 mm from the pial surface (measured directly in acute experiments and estimated in the chronic experiments). This cell layer corresponded to layer 5, since we typically lost multiunit activity after advancing the electrode an additional several hundred micrometers. We also stimulated at other depths, within 500 µm of the cell layer. Except where noted (see RESULTS), we only included data from within the first 2 mm of cortex. This minimized the likelihood of biasing our sample with data from penetrations not orthogonal to the cortical surface.
In determining the threshold and topography of ICMS effects, we kept the limb in a posture in which the shoulder was slightly retracted, the elbow was approximately half-way between flexion and extension, and the wrist was plantarflexed. Sometimes it was necessary to stabilize a proximal joint during stimulation to verify that distal joint movement was not due to an inertial inter-joint interaction. When this was done, or whenever the limb was moved, we waited several seconds before microstimulating to minimize the effect of mechanical limb stimulation on current threshold. We characterized movements at the shoulder (retraction, protraction, abduction, adduction), elbow (flexion, extension), wrist (dorsiflexion, plantarflexion, supination, pronation), and at digit joints (dorsiflexion/digit closure, plantarflexion).
When ICMS produced a motor effect, we determined the current threshold, defined as the lowest current that consistently produced a motor effect. At first, we quickly raised the current to suprathreshold values, then reduced the current to below threshold, noting the lowest current at which the effect was present. Next we increased the current and noted when the effect reappeared. If necessary, we repeated this procedure until we were confident of the threshold value. Each threshold value represents testing over at least one descending and one ascending stimulation run.
We used a maximal current of 100 µA, which is higher than what is
commonly used in mature animals, to minimize the likelihood of missing
effective sites. We did not systematically exceed this value because
using higher currents (e.g., up to 150 µA for 1 or 2 trains) did not
typically produce an effect when one was not present at 100 µA.
Moreover, 100 µA produces current spread of approximately 350 µm
(Asanuma and Sakata 1967), which was an acceptable
distance considering the sizes of joint representation zones in cat M1
(Keller 1993
). However, it should be noted that ICMS
could spread farther due to the branching patterns of stimulated axons:
spikes produced by the stimulus in axons at the stimulation site can be
antidromically conducted to other sites. In practice, however, 100-µA
currents were only used in the younger animals, when the thresholds
were high and the number of effective sites was low (see Fig. 3). Such
currents did not appear to produce lesions. In the younger animals, in
which we routinely used higher currents, the histology was not
noticeably different from that of adults, where high currents were used
only rarely. Immediately following stimulation, we were also able to
record multiunit activity, further suggesting that the stimulus did not
damage the cortex. Moreover, in PD 70-90 animals, where
there was a mix of high- and low-threshold sites (see Fig. 3), the
presence of an ineffective site tested at 100 µA did not preclude
identifying adjacent effective sites at much lower thresholds.
We determined the type of motor effect primarily on the basis of the evoked phasic kinematic change. Concurrent EMG recordings generally validated this approach (see Fig. 8). There was a close correspondence between the occurrence of a short-latency burst in a particular muscle and the presence of a phasic kinematic change that was appropriate for the activated muscle. Occasionally, late EMG activity occurred without a frank kinematic change (e.g., see Fig. 8).
In addition to stimulating at each site, we recorded single- and multiunit activity and determined the presence and location of peripheral receptive fields. We used mechanical stimuli: gentle tapping of the skin, joint rotation, and brushing of the hairs. We examined the contralateral and ipsilateral forelimbs and adjoining neck and trunk regions, the face, and the hind limbs. At completion of the experiments, marking lesions were made to help reconstruct the location of the penetrations.
For experiments in anesthetized and awake animals, we compiled data for each stimulation site, on the presence or absence of 1) multiunit activity and 2) a motor effect produced by ICMS. We created a database of compiled values, for individual cats and pooled data (in 10-day blocks) across the entire population. While we explored the cortex within the walls of the lateral portion of the cruciate sulcus to verify that we had not missed effective sites in young animals (see RESULTS), our database only included stimulation and recording sites from within the surface layers of cortex (i.e., within 2 mm of the pial surface). We examined a total of 863 sites in anesthetized animals and 97 sites in awake animals. We determined the significance of age-related and site-related differences using paired and unpaired t-tests, ANOVAs, and linear regressions using the program Statview, and we computed an exponential fit using KaleidaGraph.
EMG recordings
We recorded EMG activity using Ni-chrome wires inserted percutaneously into muscle. We routinely recorded from shoulder extensors, triceps, biceps, and either wrist dorsiflexors or wrist plantarflexors because these muscles are the agonists for the most common kinematic changes produced by ICMS. The contralateral forelimb was shaved to facilitate muscle identification and to ensure accurate electrode placement. We used conventional amplification and filtration. For this study, we inspected the recorded EMG during the experiment for concordance between the kinematic and EMG changes and present representative data below (see RESULTS).
Histology
After completing experiments in the awake animals, they were
deeply anesthetized and perfused transcardially with warm saline, followed by 10% formal-saline. The brain was removed and placed in
fixative. After each acute experiment, the animal was killed with an
overdose of Nembutal, and the brain was removed and transferred to
fixative solution for at least 1 mo. Sections were cut (in the
parasagittal plane) through the region of the penetrations and
Nissl-stained. Only sites located within M1 (area 4) are included in
this study. These sites were located rostral to the area 4-3a border,
identified histologically by the presence of an attenuated layer 4 (granular layer) (Avendaño and Verdu 1992).
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RESULTS |
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Animal database and histology
Acute experiments were conducted on 17 cats (PD 43 to adult) to determine age-dependent changes in the motor effects produced by ICMS of the forelimb areas of M1. Chronic experiments were conducted in an additional four cats to determine the effects of anesthesia on ICMS. Table 1 lists the animals, the ages examined, and the number of sites stimulated.
Figure 1 shows representative parasagittal sections through the lateral
portions of the anterior and posterior sigmoid gyri, which correspond
to the rostral and caudal M1 sectors, respectively in a PD
52 (A) animal and an adult (B). Stimulation
sites reported in this study were localized to M1 on the basis of
surface landmarks and histological criteria postmortem
(Avendaño and Verdu 1992). The arrows in
C and D point to layer 5 at the junction of areas 3a and 4. The general appearance of cortical lamination and cell density for the immature animals and the adults was the same. Note
that, as in adults, M1 in the younger animal shown in Fig. 1A had a prominent layer 5 (see arrow, Fig. 1A).
Figure 1B shows evidence of an electrode tract in rostral M1
(see arrow). Camera lucida drawings (Fig. 1, C and
D) show the locations of electrode tracts identified over
several nearby sections.
Age-dependent decreases in ineffective sites and ICMS thresholds
Figure 2 plots the percent of
sites from which ICMS at 100 µA did not produce a motor
response. Data from all acute experiments in animals
PD 90 are shown. When data were fit to a linear regression, we found that
there was a significant age-dependent reduction in the number of
ineffective sites (R = 0.853; n = 14;
P < 0.01). The inset plots data from both
young animals and adults. We extrapolated 100% ineffective sites back
to PD 5, based on data in the PD 43-45 animals
and the data trend. Fitting these data to a linear regression, the
correlation coefficient was significant (R = 0.641;
P < 0.01) but did not adequately explain the
variability (R2 = 0.411). By contrast,
there was an excellent fit to a sigmoidal function (see
inset Fig. 2), which resulted in a significant correlation coefficient for the exponential fit (R = 0.903;
P < 0.01) that better explained the variance
(R2 = 0.816). Without the back
extrapolation, the correlation coefficient dropped to 0.82 (R2 = 0.67) for the sigmoid function
(Y-intercept at 74% rather than 95%).
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Similar to the age-dependent decrease in ineffective sites, there was a decrease in the thresholds for evoking motor responses with ICMS. Figure 3 presents histograms of threshold currents for five age groups. (The PD 45 animals are not shown because ICMS did not produce any effects at that age.) The filled columns to the right re-plot the percent of ineffective sites from Fig. 2, but for each age group. In the PD 51-61 group (Fig. 3A), where ICMS evoked responses at only a small minority of sites, thresholds were distributed above 40 µA. In older animals, there was a progressive increase in the numbers of low-threshold sites and a concomitant decrease in high-threshold sites. These changes parallel the percent decrease in ineffective sites (see Fig. 3, right). While there was a clear shift in the threshold distributions toward lower values with increasing age, a substantial percentage of higher threshold sites prevailed. There also was a clear age-dependent decrease in median (and mean) thresholds (see Fig. 10).
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To verify that the absence of effects in the younger animals was not due to the effect of anesthesia, we studied the age-dependent changes in ineffective sites and ICMS thresholds in four awake animals prepared for chronic stimulation. We focused on PD 51-70, when there are the fewest effective sites and the highest thresholds. For these experiments, we were able to examine several sites each day, thereby obtaining sufficient daily threshold samples to determine trends. Table 1 summarizes the dates for the chronic experiments and the total number of sites examined.
Data from two animals were compiled for the PD 51-60 block and from three animals, for the PD 61-70 block. The percentage of ineffective sites was slightly higher for the anesthetized animals in both age groups (difference between anesthetized and awake: PD 51-60 = 2%; PD 61-70: 9%). Average ICMS thresholds were higher for the anesthetized animals in the two age groups, by 16 and 17 µA, respectively. An additional animal, which was examined over a later 2-wk period (PD 69-86), showed a significant reduction in threshold from an average of 36 µA for the first week to 28 µA, for the second week (t = 1.82; P = 0.09). Comparison of the threshold distributions in Fig. 3 with the average increase in threshold for the anesthetized animals (16-17 µA) shows that the effect of ketamine was not sufficient to mask ICMS effects in the younger animals.
We determined the presence and location of receptive fields on the body surface at the stimulation sites in the young animals and adults. During the same period in development when there were systematic reductions in stimulus threshold and the percentage of effective sites, there were no differences in the percent of sites where multiunit activity could be consistently driven with peripheral mechanical stimulation (PD 41-90; adults: F = 1.943; P = 0.39).
We conducted three control procedures to verify that the absence
of effects in the younger animals reflected the particular developmental plan of the M1 motor representation rather than artifacts
of the ICMS technique or the sampling of cortical stimulation sites.
First, while numerous sites were effective in the younger animals (as
low as 40 µA in Fig. 3A and 20 µA in Fig.
3B), we varied the pulse widths, frequencies, and train
durations (see METHODS) to rule out the possibility that
other ICMS stimulation parameters were more effective in activating the
immature CS system. The range of stimulation parameters were intended
to permit greater temporal summation (i.e., increasing train duration
to 200 ms; increasing pulse width to 0.8 ms). However, when the
standard parameters were ineffective, so too were other parameters.
Second, we also explored the cortex within the rostral and caudal banks of the cruciate sulcus in the younger animals when ICMS of the cortex
on the surface did not evoke movements. When the surface sites were
ineffective, so too were the deep sites within the sulcus. Third, if no
effect was seen at 100 µA, we attempted to reduce the threshold by
applying nonspecific somatic sensory stimulation (rubbing skin and
massaging muscles) just before ICMS. We had found that such stimulation
could sometimes reduce the threshold by a small amount, as others have
reported (e.g., Donoghue and Sanes 1988). This was only
modestly successful, resulting in a slightly higher incidence of sites
producing effects at 100 µA for the PD 51-80 animals. To
estimate the effect of this stimulation on the percent of ineffective
sites, we eliminated data from the 100-µA bin and recomputed the
averages for the different age groups. While there were small increases
in the percent of ineffective sites for all groups (PD 51:
4%; PD 61: 6%; PD 71: 6%; PD 81: 2.4%; Adult: 0.7%), the strong developmental trend remained.
Development of the forelimb joint representation
In addition to age-dependent decreases in the numbers of
ineffective sites and current thresholds, we also found that there were
consistent changes in the numbers of sites at which ICMS produced
movements of proximal and distal joints. Figure
4 plots the percent occurrence of
movement at each of the forelimb joints for the various age groups.
ICMS in animals PD 71 only produced movement of the
shoulder or elbow joints. In the older animals and adults, ICMS
produced effects at both proximal (shoulder and elbow) and distal
(wrist and digit) joints. Similar results were obtained in the awake
animals. Prior to PD 71, stimulation of 1 of 29 sites (3 animals) produced a distal response, while after PD 71, stimulation of 4 of 15 sites (1 animal) produced distal responses.
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There also was an age-dependent reduction in threshold for producing movement at each joint (Fig. 5), similar to the changes for the population (i.e., Fig. 3). While between PD 61 and 70, the thresholds for evoking elbow and shoulder were significantly different (P = 0.09), at older ages, the thresholds were not. These changes for individual joints were due to an increase in the number of sites with lower thresholds and a reduction in the number of high-threshold sites. These results show that as the M1 motor representation became established postnatally, there was a proximal-to-distal elaboration in map somatotopy.
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Topographic distribution of ICMS effects
Color-coded M1 microstimulation maps for five animals are shown in Fig. 6. Sites that were not tested because of the presence of blood vessels or bone around the craniotomy margins are white. The evoked joint movement is indicated by the overlying letter (see legend for abbreviations). Red squares without a letter indicate ineffective sites, tested at 100 µA (the maximal current). The most lateral penetrations were located close to the lateral margin of the frontal lobe, where the lateral sigmoid gyrus curves inferiorly into the coronal sulcus. All sites shown were several millimeters from the midline.
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These maps confirm, in individual animals, the age-dependent changes
reported above (i.e., decreasing numbers of ineffective sites;
decreasing thresholds; and increasing somatotopic diversity). In
addition, several new features of M1 motor representation development became evident. First, effective sites were more prevalent rostral to
the cruciate sulcus than caudal during early development. These two
regions correspond to the rostral and caudal M1 subregions (Martin 1996; Pappas and Strick 1981a
,b
).
Second, within both the rostral and caudal M1 subdivisions, effective
sites tended to be scattered early in development. Third, in the older
animals (>PD 71), there were sites at which ICMS produced
movement of multiple joints (and EMG activity in multiple muscles;
labeled M; see next section). These effects were produced using
currents between 1.0 and 1.5 times threshold (see below). Multijoint
sites shown in Fig. 6 are for threshold currents. Fourth, these
individual maps show that there was intersubject variability (also
evident in Fig. 2). For example, despite only a 3-day difference, the PD 75 (C) animal had a denser representation
within rostral M1 than the PD 72 animal (B).
Variability in motor map topographic features, current threshold, and
the density of effective sites (see Fig. 2) did not correlate with any
known differences in the animals' experiences, such as the length of
time spent in the animal care facility or the number (and activity
level) of cage mates. This could reflect biological variability.
Multijoint responses evoked at individual sites
While ICMS at most sites evoked a response at a single joint, we
noticed a minority of sites at which ICMS produced movement of multiple
joints (and contraction of multiple muscles). These multijoint sites
were only present after PD 71, as the representation of more
distal joints emerged and the incidence of effective sites increased.
Single joint sites typically continued to produce a single joint
response with increasing current strength. In contrast, multijoint
effects were present at threshold or emerged with small current
increments above threshold. The maps in Fig. 6, B-E, show sites from which multijoint movement were produced (labeled M). We
quantified the incidence of multijoint sites. We determined the
percentage of sites across the database where currents up to 1.5 times
threshold evoked multijoint movements. For this analysis, we included
only the effects produced at the threshold stimulation site (i.e.,
lowest current site in tract within 2 mm of pial surface; typically in
the large-cell layer) and at sites up to 500 µm superficial or deeper
to the threshold site (along the same penetration). The mean current
value for evoking multijoint effects was 30 µA, and 80% of the
effects were evoked by currents 40 µA. We chose 1.5 times threshold
within 500 µm of the threshold site, rather than only the threshold
site, to increase the sample size and increase the likelihood of
detecting emerging multijoint patterns. We reasoned that this was
acceptable because 1) the threshold is not absolute but
somewhat dependent on excitability changes (viz. anesthesia level, and
other uncontrolled factors), and 2) there is local current
spread (Asanuma and Sakata 1967
). Moreover, as we
discuss at the end of this section, similar effects were obtained at
threshold (i.e., with no threshold or distance margin), but with a
reduced number of sites.
Figure 7A plots the percentage of multijoint sites (produced by currents up to 1.5 times threshold) for the various age groups. It was more common for adjacent joints to be co-represented and for two rather than three joints. ICMS at one site never produced motion of all four forelimb joints. In the adult, where it was common for all joints to be represented, all multijoint combinations were present, although shoulder-elbow and elbow-wrist were most common. In the adult, we noted that there was a proximal-to-distal gradient in the percentage of multijoint effects. Figure 7B plots the percent of occurrence of effects at each joint when produced together with an effect at another joint (up to 1.5 times threshold). We found that a higher percentage of distal joints were co-represented with other joints, rather than being represented alone. For example, 81% of digit effects occurred together with an effect at another joint compared with only 22% of shoulder effects. There was a tendency for an increased percentage of multijoint effects in the older animals, although this also could have been due to the increased likelihood of such sites as the M1 map represents more joints later in development (viz. Fig. 4).
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As expected, the kinematic changes at multiple joints reflected
activation of multiple muscles rather than inertial interactions between one joint and another (i.e., interjoint interaction torques) (Hollerbach and Flash 1982). Figure
8 shows phasic responses evoked by
ICMS in triceps, biceps, and a wrist dorsiflexor. The stimulus consistently evoked responses in the biceps and wrist dorsiflexor at
short latency. The triceps was weakly activated after the end of the
biceps burst. This could have been due to reciprocal organization in
the spinal cord (Baldissera et al. 1981
). The kinematic
changes reported at the site were forearm flexion and wrist
dorsiflexion (both at 40 µA).
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We also examined the numbers and patterns of multijoint responses at
1.0 times threshold to verify that the effects we are reporting were
not simply due to use of a suprathreshold current intensity. While the
number of multijoint effects were reduced overall (1.5 times threshold;
500 µm distance = 47; 1.0 times threshold = 22), the late
emergence was clearly present (PD 80: 0%; PD
81-90: 11.5%; Adult: 12.2%).
Differential development of representational parameters in rostral and caudal motor cortex
As briefly described for the maps shown in Fig. 6, we noted more ineffective sites and higher current thresholds for caudal than rostral M1. Figure 9 compares the percentage of ineffective sites (A) and mean threshold (B) for caudal (dark gray bars) and rostral (light gray bars) M1. Beginning at PD 51, the percent of ineffective sites was consistently higher for caudal M1 than rostral. This difference was significant (paired t-test; t = 3.798; P < 0.002). Between PD 61 and 90, the thresholds to evoke motor responses also were consistently higher in caudal than rostral M1. The difference in the adults was not significant.
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DISCUSSION |
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Maturation of the motor representation in cat M1 occurred during a prolonged postnatal period. ICMS became progressively more effective at evoking forelimb motor responses and the somatotopic characteristics of the forelimb M1 representation developed in a proximal to distal sequence. In parallel with these somatotopic changes, there was a progressive increase in the number sites at which ICMS produced effects at multiple forelimb joints. These results are summarized in Fig. 10. Our findings are consistent with two stages in the development of the M1 motor representation. Between PD 49 and 70, there is an increase in ICMS efficacy, without a substantial change in the type of joints represented. After PD 70, the major changes were increases in the representation of different forelimb joints and sites producing multijoint effects.
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We were surprised with the paucity of effective sites and high currents
needed to evoke responses in the younger animals, considering the
findings of Bruce and Tatton (1980a,b
). Those authors
reported the sudden emergence of forelimb EMG responses at about
PD 41-45. In those studies, neither the number of sites from which effects were evoked nor the specific current thresholds were
reported making comparison with our study difficult. Moreover, it was
not indicated whether there was an attempt to sample a large portion of
the forelimb area or whether they extensively explored areas
surrounding sites where ICMS produced effects. We too encountered
effective sites in younger animals (PD 51-60; Fig.
3A), but we did not make additional penetrations close to those sites.
An important concern is the impact that general anesthesia could have
on assessment of the motor map using ICMS, at any age. While our data
show that ketamine anesthesia elevated current thresholds (see
RESULTS), we kept experimental conditions constant across
ages. We reason therefore that the changes in the motor representation
reflected particular developmental processes not an effect of the
anesthesia. Similar age-dependent reductions in TMS threshold occur in
awake humans (Koh and Eyre 1988; Müller et
al. 1991
; Nezu et al. 1997
), also suggesting the
importance of developmental changes and not anesthesia.
Sequential development of anatomical specificity of CS terminations and maturation of M1 motor representation
A period of activity-dependent refinement of CS terminations
immediately precedes the emergence of the M1 representation
(Martin et al. 1999). During this early refinement
period, there is elimination of transient CS axon terminations
(Alisky et al. 1992
; Theriault and Tatton
1989
) and axon terminal growth (Li et al. 1999
),
resulting in a topographic distribution of CS terminations similar to
that of mature animals. It appears that the synaptic contacts made by
these developing CS terminations with spinal neurons, while capable of
mediating early activity-dependent maturation, have insufficient
strength to activate motoneurons. In the monkey, responses to TMS are
first evoked by 4-6 mo, which follows most of the development of CS
projections to the lateral motor nuclei in the cervical cord
(Armand et al. 1997
). These findings in cat and monkey
suggest that the CS system waits until the specificity of connections
with spinal target regions is largely achieved before the M1 motor map
becomes established. The sequence of anatomical then physiological
maturation of the motor representation is different from development of
the visual responses in primary visual cortex. While motor responses
produced by ICMS occur after development of the CS
projection, visual responses in primary visual cortex occur
during visual pathway development, although the immature responses are less robust and selective than in maturity (e.g., Crair et al. 1998
). Recently, Eyre and colleagues
(2000)
found that TMS in humans produced motor effects
beginning at 26 wk (in premature infants), albeit at higher intensity
and longer latency than in older children and adults. They suggested
that there are some functional corticomotoneuronal connections at these
young ages. While this study shows that human CS development begins at
earlier ages than either monkey or cat, more research on the time
course of maturation of CS terminations is needed to determine whether
functional contacts with spinal target neurons and cortical-evoked motor effects develop concurrently or sequentially.
We found that once motor effects could be evoked by ICMS, the threshold
decreased with age. This is similar to results in both monkey and
humans, using TMS, showing that thresholds decrease over a prolonged
postnatal period. For monkeys, there is an age-dependent reduction in
threshold until 8 mo (Flament et al. 1992), and, in
humans, thresholds decrease over a 10- to 15-yr period (Eyre et
al. 1991
; Nezu et al. 1997
). Both the delayed
onset of motor effects produced by M1 activation (ICMS and TMS) and the
subsequent prolonged reduction in activation thresholds could reflect
delayed and prolonged maturation of 1) local connections
within M1 itself that lead to the generation of descending CS control
signals and 2) CS transmission in the spinal cord. While our
method does not distinguish between these two mechanisms, several
published studies, as well as our preliminary data, provide insight
into their relative contributions.
M1 activation in immature cats may not generate sufficient CS activity
to drive movement, irrespective of maturation of the CS axons and their
terminations. This is an important concern because ICMS using trains of
pulses produce motor effects primarily by trans-synaptic
activation in cortex (Jankowska et al. 1975). If ICMS
were inefficient in generating CS activity, it is not simply due to
generalized immaturity of intracortical circuits because several
studies have shown maturation of synaptic transmission from subcortical
inputs at ages prior to emergence of ICMS. Electrical stimulation of
the cerebellar nuclei evokes synaptic responses in M1 at birth, and the
latency and rise time of the potentials decrease rapidly by PD
30-40, at which time they are not different from adults
(Kawaguchi et al. 1983
). By PD 28, stimulation of the pyramid evokes excitatory-inhibitory postsynaptic
potential sequences in M1 pyramidal neurons, similar to that seen in
adults (Oka et al. 1985
). Moreover, intracellular
staining of pyramidal neurons revealed extensive basal and apical
dendritic arrays by PD 28, which were not remarkably
different from adults (Oka et al. 1985
). We have noted
that during the period when the M1 motor map is maturing, the presence
of responses of M1 neurons to peripheral mechanical stimulation
delivered to the contralateral forelimb does not change. This shows
that the immature M1 can organize somatic sensory information into
receptive fields. These findings strongly suggest that M1 is
electrophysiologically quite mature before the emergence of ICMS
effects and that the absence of ICMS effects in the younger animals is
not due to electrical inexcitability of M1. Nevertheless, it is
plausible that ICMS (or TMS) is inefficient in generating CS signals
that can evoke movement. This could be due to the need for further
maturation of local M1 circuits. Failure to generate motor responses
also could be due to inefficient transmission of descending signals in
the CS tract. There is a correspondence between the emergence of
consistent effects produced by TMS and the development of myelination
(Weidenheim et al. 1996
) along with associated increases
in conduction velocity (Eyre et al. 1991
; Olivier
et al. 1997
). Thicker myelination and faster conduction velocity would favor synchronization of descending volleys and, as a
consequence, more effective postsynaptic temporal summation in the cord.
The emergence of motor effects produced by M1 stimulation (ICMS, TMS)
and the subsequent reduction in activation thresholds also could
reflect development of more efficient synaptic transmission of control
signals in the spinal cord. For production of motor effects, especially
in the cat where CS effects on motoneurons are mediated through di- and
oligosynaptic connections, there must be a sufficient number of CS axon
terminal branches and presynaptic sites to adequately drive
postsynaptic target neurons. Consistent with this, there is a
progressive increase in the number of CS axon terminal branches during
the period of M1 representation development (Li et al.
1999). An alternative to the lack of axon terminal maturation
is that M1 stimulation-evoked responses could have been prevented
during early postnatal life by a process that limits CS synaptic
efficacy, such as inhibition (Asanuma and Sakata 1967
).
Further studies are needed to determine whether maturation of the
cortical motor map reflects predominantly one or several of these mechanisms.
Late development of M1 somatotopy
Before PD 70 only the shoulder and elbow were
represented in M1. These sites tended to be scattered, with fewer sites
in caudal than rostral M1. Our data are consistent with the idea that
the uncommitted sites were used for the expansion of previously
represented joints or for the new representation of more distal joints.
We found a clear proximal-to-distal joint somatotopic maturation. This
is reminiscent of the late development of fine distal control (Forssberg et al. 1991; Porter and Lemon
1993
).
We found that wrist and digit movements evoked by ICMS were often
associated with movement about a more proximal joint. By contrast,
proximal joint motion evoked by ICMS was more commonly produced in
isolation. Several studies have reported that ICMS at individual sites
in mature monkey, cat, and rat M1 evokes multiple responses
(Armstrong and Drew 1984; Donoghue and Sanes
1988
; Donoghue et al. 1992
; Martin and
Ghez 1993
). Using up to twice threshold currents, ICMS at most
sites in M1 of the Ketamine-anesthetized squirrel monkey evoked
contraction of multiple muscles (Donoghue et al. 1992
),
and in the cat, 40% of sites from which forelimb movements could be
evoked produced multijoint effects (Armstrong and Drew
1984
). These results in awake and Ketamine-anesthetized animals
differ from those reported by Asanuma and colleagues (for review, see
Asanuma 1981
) for animals anesthetized with a
barbiturate. Since GABAergic blockade promotes the expression of
multijoint coding in M1 (Matsumura et al. 1991
), using a
barbiturate [which is a GABA agonist (Simmonds and Turner
1987
)] as an anesthetic agent could have blocked the
multijoint effects.
Our present findings show a late developmental origin to such
multijoint sites. The particular pattern of evoked movement at distal
and proximal joints in this study suggests that the production of
multiple effects was not due to current spread from one, to an
adjoining, single-joint site but rather reflects the cortical control
of particular motor synergies. The lack of current spread to diverse
single joint sites is also consistent with the general dimensions of
single joint representation zones in the cat (Keller
1993) and that our mean current (i.e., 30 µA) would not have
been expected to spread more than about 200 µm (Asanuma and
Sakata 1967
).
Sites in M1 where ICMS can evoke multijoint effects could play a role
in coordinating the control of distal and proximal joints for producing
straight hand/paw paths during reaching (Martin et al.
1995). In humans, hand paths become straighter during the first
few postnatal months (Konczak et al. 1995
).
Proximal-distal joint synergies could also serve to stabilize distal
joint motion, which can be perturbed by inertial interactions produced
by proximal joint motion (Ghez et al. 1996
;
Hollerbach and Flash 1982
). Control signals from a
multijoint site could concurrently steer proximal joint movement and
distal joint stabilization for ensuring accurate movement endpoints.
Development of functional specificity
During normal human and animal development, there appears to be
safeguards against the production of errant movements when errant
(i.e., transient) CS connections are present. Thresholds for evoking
movements by TMS (Flament et al. 1992) or ICMS
(Bruce and Tatton 1980a
,b
; our present study)
are substantially higher early in development before CS connectional
specificity is established (Alisky et al. 1992
;
Li and Martin 2000
; Theriault and Tatton 1989
). This could be a developmental adaptation to prevent
activation of inappropriate CS connections leading to maladaptive
behaviors. Weak CS connections, or local connections in M1, that are
appropriate to steer the emerging movements could become strengthened
by activity-dependent mechanisms. In the spinal cord, plasticity of CS
connections could be an extension of the earlier activity-dependent
refinement of terminations (Martin et al. 1999
).
Normal children up to 10 yr of age sometimes produce mirror and other
unintentional movements together with production of an intended
movement (Connolly and Stratton 1968;
Müller et al. 1997
; Reitz and Müller
1998
), implicating a lack of somatotopic specificity of CS
control of voluntary action. Our data, showing initial single-joint
effects and later development of multijoint effects, suggest
that unintentional movements during normal motor development reflect a
lack of specificity in accessing the motor map by structures
presynaptic to M1 rather than the lack of specificity in its output to
the spinal cord.
Relationship between postnatal maturation of the M1 motor map and representational plasticity
There are similarities between postnatal maturation of the M1
representation and M1 representational plasticity in mature animals. In
maturity, the area of the forelimb representation becomes increased
with experience performing forelimb motor tasks (Kleim et al.
1998; Nudo et al. 1996
). Similarly, the areas of the shoulder and vibrissae representations are increased after forelimb
amputation (Donoghue and Sanes 1988
). Along with
increases in the area of representations, there also are decreases in
the current thresholds for eliciting movements (Donoghue and
Sanes 1988
; Sanes et al. 1990
). We found that
during development there were consistent increases in the area of M1
devoted to a particular joint and decreases in threshold.
We noted two consistent differences in the way the M1 motor map changes
during development and what has been reported for mature animals.
First, during early postnatal life the motor map was more like a tabula
rasa on which sites representing forelimb joints were added. Although
the connections with the spinal cord are established and
topographically appropriate by PD 45-50 (Alisky et
al. 1992; Theriault and Tatton 1989
), M1 does
not represent contralateral limb muscles and joints, other than at a
few scattered sites. When the density of effective sites is low, new
representational zones for one joint could be added without competing
with the addition of zones for other joints. In contrast, stimulation
of most of M1 in maturity produces motor effects because the density of
effective sites is high (e.g., 76%; see Fig. 10). Expansion of one
joint's representation in maturity could result in the contraction of
the representation of other joints (Kleim et al. 1998
;
Nudo et al. 1996
). The competing claims of the
representation zones of the different joints in maturity could thus
provide an effective regulation for representational plasticity, which
is not present in immaturity.
Second, in maturity representational changes (e.g., area, threshold)
can occur rapidly, as short as after 1.5 h following peripheral
motor nerve section (Donoghue et al. 1990) or up to 3 h after local M1 electrical stimulation (Nudo et al.
1990
). The changes we report during development occurred over a
month. This difference probably reflects the contributions of different biological mechanisms, which could rely more on axon terminal growth or
branching in development than maturity. The slower time course of
developmental changes is consistent with a greater dependence on
long-term (and possibly more consistent) behavioral trends for shaping
the representational changes than short-term (and possibly
inconsistent) behavioral events.
During early postnatal life, both innate and experiential factors could play important roles in establishing the M1 circuitry for the adaptive control of movements in maturity. While innate mechanisms could have a limited capacity to anticipate future motor control requirements, experience-dependent development could overcome this limitation. By leaving development of the specific pattern of representation of forelimb joints and muscles to a late postnatal period, when new control demands are placed on the emerging skilled limb movements, the CS system can match development of the adaptive control circuits to the animal's actual motor behaviors.
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ACKNOWLEDGMENTS |
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We thank P. Okonkwo for technical assistance and G. Johnson for construction of apparatus. We also thank Dr. Qun Li for helping with the histology and Dr. Mo Osman for veterinary care and developing the anesthesia protocols.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-36835 and a grant from the March of Dimes Foundation.
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FOOTNOTES |
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Address for reprint requests: J. H. Martin, Center for Neurobiology and Behavior, Columbia University, 1051 Riverside Dr., New York, NY 10032 (E-mail: jm17{at}columbia.edu).
Received 27 March 2000; accepted in final form 23 June 2000.
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REFERENCES |
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