Department of Zoology, University of Oklahoma, Norman, Oklahoma 73019
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ABSTRACT |
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Berkowitz, Ari. Rhythmicity of Spinal Neurons Activated During Each Form of Fictive Scratching in Spinal Turtles. J. Neurophysiol. 86: 1026-1036, 2001. Are behaviors that rely on common muscles and motoneurons generated by separate or overlapping groups of pattern-generating neurons? This question was investigated for the three forms of scratching in immobilized, spinal turtles. Individual neurons were recorded extracellularly from the gray matter through most of the spinal cord hindlimb enlargement gray matter, but were avoided in the region of motoneuron cell bodies. Each form of fictive scratching was elicited by mechanical stimulation of the body surface. The rhythmic modulation of spinal neurons was assessed using phase histograms and circular statistics. The degree of rhythmic modulation and the phase preference of each rhythmically active neuron were measured with respect to the activity cycle of the ipsilateral hip flexor nerve. The action potentials of rhythmic neurons tended to be concentrated in a particular phase of the ipsilateral hip flexor activity cycle no matter which form of fictive scratching was elicited. This consistent phase preference suggests that some of these neurons may contribute to generation of the hip rhythm for all three forms of scratching, strengthening the case that vertebrate pattern-generating circuitry for distinct behaviors can be overlapping. The degree of rhythmic modulation of each unit during fictive scratching was consistently correlated with the dorsoventral location of the recording, but not with the mediolateral or rostrocaudal location; neurons located more ventrally tended to be more rhythmic. The phase preferences of units were related to the region of the body surface to which each neuron responded maximally (i.e., the region to which each unit was broadly tuned). Units tuned to the rostral scratch or pocket scratch region tended to have a phase preference during ipsilateral hip flexor activity, whereas units tuned to the caudal scratch region did not. This suggests the hypothesis that the hip flexes further during rostral and pocket scratching, and extends further during caudal scratching, due to the net effects of a population of spinal interneurons that are both broadly tuned and rhythmically active.
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INTRODUCTION |
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The same muscles and motoneurons
can be used to produce a wide variety of movements or behaviors. This
is possible because motoneurons are activated in a different pattern
during each behavior. Networks of neurons within the CNS are able to
generate the basic motor patterns underlying many rhythmic behaviors
(Marder and Calabrese 1996; Pearson
1993
). In some cases, there is a separate set of CNS neurons
dedicated to generating each of several distinct types of behaviors or
motor patterns (Heitler 1985
; Hennig
1990
; Ramirez and Pearson 1988
). In many other
cases, however, the same or overlapping sets of CNS neurons generate
several distinct types of motor patterns. Many known examples of such
multifunctional pattern-generating neurons are from invertebrates,
especially from the stomatogastric nervous system of crustaceans
(Dickinson and Moulins 1992
; Getting and Dekin
1985
; Heinzel et al. 1993
; Hooper and
Moulins 1989
; Lockery and Kristan 1990
;
Meyrand et al. 1991
; Weimann and Marder
1994
; Weimann et al. 1991
). There is now
substantial evidence for the existence of pattern-generating neurons
that are shared for multiple behaviors in vertebrates as well
(Berkowitz and Stein 1994b
; Grelot et al.
1993
; Larson et al. 1994
; Lieske et al.
2000
; Miller and Ezure 1992
; Nonaka and
Miller 1991
; Oku et al. 1994
; Perreault
et al. 1999
; Soffe 1993
; Soffe et al.
1984
; Svoboda and Fetcho 1996
; Westberg
et al. 1998
; Yajima and Larson 1993
), although
the evidence for vertebrate behaviors involving limb movements
(arguably the most complex movements) is more limited (Berkowitz
and Stein 1994b
; Perreault et al. 1999
).
One of the vertebrate behaviors for which this question can
conveniently be examined is turtle scratching. The turtle spinal cord
can generate three distinct forms of scratching, in response to
mechanical stimulation in three adjacent regions of its body surface
(Mortin et al. 1985). Each form of scratching is a
rhythmic behavior in which a distinct portion of the ipsilateral
hindlimb rubs against the stimulated site on the body surface and in
which the knee extends during a distinct phase of the hip movement
cycle (Mortin et al. 1985
). The turtle spinal cord can
generate three forms of fictive scratching when the animal is
spinalized and immobilized (Robertson et al. 1985
). The
three forms of fictive scratching occur in response to stimulation of
the same three regions of the body surface and display the same phase
relationships between knee extensor and hip flexor activities as
scratching movements (Robertson et al. 1985
).
Previous research has demonstrated that the anterior three segments of
the turtle spinal cord hindlimb enlargement are most important for
generation of each of the three forms of fictive scratching
(Mortin and Stein 1989). Moreover, individual spinal cord interneurons are rhythmically activated during both fictive rostral scratching and fictive pocket scratching (Berkowitz and Stein 1994b
). These findings suggest that some of the same
spinal cord interneurons may contribute to the generation of multiple forms of scratching. It was not known, however, whether individual spinal interneurons are rhythmically activated during all three forms
of fictive scratching; previous experiments were carried out in a
reduced preparation in which the receptive field for caudal scratching
was cut off from the remainder of the preparation (Berkowitz and
Stein 1994a
,b
). The hindlimb moves primarily forward during
rostral and pocket scratching but primarily backward during caudal
scratching (Mortin et al. 1985
). Thus the sets of
pattern-generating neurons for rostral and pocket scratching might
overlap more with each other than with the set of pattern-generating
neurons for caudal scratching. In the current study, therefore, spinal
neurons were recorded in a preparation that generates all three forms of fictive scratching, to address whether and how individual spinal neurons might contribute to generating each of the three forms of scratching.
Previous research also suggested that the descending axons of turtle
propriospinal neurons are not segregated within the spinal white matter
on the basis of their rhythmicities or their preferred phases of
activity within the hip activity cycle of fictive scratching (Berkowitz and Stein 1994a,b and unpublished
observations). That study, however, utilized axonal white matter
recordings and so could not address whether the cell bodies of such
neurons are organized within the gray matter according to their
rhythmic modulation. Therefore in the current study neurons were
recorded from the spinal gray matter, and these recording locations
were compared with the strength of rhythmic modulation and the
preferred phase of activity within the hip activity cycle for each neuron.
These findings have been previously described in an abstract
(Berkowitz 2000).
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METHODS |
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The neuronal data set used for this study is the same as that
used for the companion paper; complete descriptions of surgical, recording, stimulation, and histological methods can be found in that
paper (Berkowitz 2001). Briefly, the turtle's spinal
cord was transected between the 2nd dorsal (D2)
and D3 segments, and the spinal cord was also
exposed for recording in a segment or segments of the hindlimb
enlargement (D8, D9,
D10, S1, or
S2). Animals were immobilized with a chemical
neuromuscular blocker and were artificially respirated. Single-unit
recordings were made from the right hindlimb enlargement gray matter
while the right and left hip flexor muscle nerves and several right
knee extensor muscle nerves were simultaneously recorded. Recordings were obtained at depths of <1,200 µm in general and at <900 µm for penetrations of >60 laterality to avoid recording from motoneuron cell bodies. Each form of fictive scratching was elicited by mechanical stimulation of sites on the body surface. Neurons were studied only if
they were activated during some form of fictive scratching; stimulation
in the ipsilateral caudal scratch region was usually used as a search
stimulus. For each single-unit recording, the spinal segment, the
mediolateral location (visually estimated as a percentage of the
distance from the midline to Lissauer's tract), and the depth (read
out from the piezoelectric microdrive) of the recording were noted.
Dual-referent phase histograms (Berkowitz and Stein
1994b; see also Burns and Usherwood 1979
;
Drew and Doucet 1991
; Orlovsky 1972
;
Tresch and Kiehn 1999
; Westberg et al.
1998
) were used to measure the modulation of single-unit firing
rates within the ipsilateral hip flexor activity cycle. For each cycle
of fictive scratching, the hip flexor burst and the hip flexor
quiescent period were each divided into five equal-duration bins, and
the single-unit mean firing rate was calculated within each bin. For each of these 10 phases of the hip flexor activity cycle, the average
of the mean firing rates from all available cycles of fictive
scratching obtained with the same site of stimulation and the same
single-unit recording was then calculated, yielding a dual-referent
phase histogram. All scratch cycles that met certain standard criteria
(see Berkowitz 2001
) were used to produce these phase histograms.
Circular statistics were then calculated from these phase histograms
(Batschelet 1981; Berkowitz and Stein
1994b
; Drew and Doucet 1991
; Mardia
1972
; Tresch and Kiehn 1999
; Westberg et
al. 1998
). The onset and offset of each ipsilateral hip flexor
burst were set at 0° (=360°) and 180°, respectively. Each
single-unit phase histogram was thus treated as a set of 10 vectors,
with each vector angle given by the position of the bin within the hip
flexor activity cycle and each vector length by the single-unit mean
firing rate for that bin. The normalized vector sum of these vectors
was then calculated, yielding the mean vector (see Batschelet 1981
; Berkowitz and Stein 1994b
; Mardia
1972
). The mean vector length measures the extent to which
single-unit action potentials were concentrated within one part of the
scratch cycle. A mean vector length of 1 would signify that all
single-unit action potentials occurred within 1 of the 10 phases of the
cycle; a mean vector length of 0 would signify that the unit's action
potentials were distributed evenly or randomly over the entire cycle.
The mean vector angle is a measure of the preferred phase of
single-unit firing within the ipsilateral hip flexor activity cycle
(Berkowitz and Stein 1994b
). The angular deviation is a
measure of the variability of the mean vector angle and is a function
of the mean vector length (Batschelet 1981
). If a unit
fired fewer than 10 action potentials for a particular site of
stimulation, it was not analyzed at that site using circular
statistics. When one mean vector length value was plotted for each
unit, this value was the average of the mean vector length values for
all ipsilateral sites stimulated.
The Rayleigh test was used to evaluate the null hypothesis that
single-unit action potentials were distributed evenly or randomly over
the entire hip flexor activity cycle (Batschelet 1981;
Berkowitz and Stein 1994b
; Drew and Doucet
1991
; Mardia 1972
; Tresch and Kiehn
1999
; Westberg et al. 1998
); for this
statistical test, n was the total number of single-unit
action potentials used to calculate the phase histogram. The mean
vector angle for a particular single unit and a particular stimulation
site was used for further analysis only if the corresponding phase
histogram passed the Rayleigh test with a probability of <0.01 that
the null hypothesis was correct. When the mean vector angle for each
form of fictive scratching was plotted (Figs. 3 and 4), the mean vector
angle selected was from whichever ipsilateral stimulation site in the scratch region provided the distribution of single-unit firing rates
that passed the Rayleigh test with the lowest probability of the null
hypothesis, unless multiple distributions had P < 0.001; in this case, the site among these with the highest mean vector
length was used. Sites in transition zones between scratch receptive
fields (see Berkowitz 2001
; Mortin et al.
1985
) were not used in this analysis. When a single mean vector
angle for each neuron was plotted (Figs. 7-9), the mean vector angle
selected was from whichever ipsilateral stimulation site (in any
scratch receptive field, including the transition zones) provided the distribution of single-unit firing rates that passed the Rayleigh test
with the lowest probability of the null hypothesis (or had the highest
mean vector length among those distributions with P < 0.001). When the mean or "summary" of a set of mean vector angles
was calculated (Figs. 8B and 9B), each mean
vector angle was treated as a single angular data point; the mean
vector of this distribution was then calculated in the usual way
(Batschelet 1981
; Mardia 1972
).
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RESULTS |
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Preferred phases of firing
Sixty-four spinal neurons were recorded during fictive scratching
in 16 animals. Fifty-six (88%) of these neurons displayed sufficient
rhythmic modulation during fictive scratching to pass the Rayleigh test
with P < 0.01 (indicating that the null
hypothesisthat single-unit action potentials were evenly or randomly
distributed across all phases of the hip flexor activity cycle
could
be rejected; see METHODS). Many of the spinal neurons
recorded were activated to some extent during stimulation in much or
all of the ipsilateral (and often contralateral) scratch receptive
fields (Berkowitz 2001
). For those neurons that passed
the Rayleigh test at each of several sites of stimulation, the
neuron's preferred phase of firing was examined by comparing the
neuron's mean vector angle across these stimulation sites (see
METHODS). For most neurons, the preferred phase of
single-unit firing within the ipsilateral hip flexor activity cycle was
approximately the same regardless of the form of ipsilateral fictive
scratching generated and regardless of the stimulation site within the
ipsilateral scratch receptive fields (Fig.
1). Different neurons typically had quite
different preferred phases, but each neuron maintained
approximately the same preferred phase no matter where fictive
scratching was elicited ipsilaterally (Fig. 1). The ipsilateral hip
flexor nerve typically is weakly but rhythmically activated during
contralateral fictive scratching (Berkowitz and Stein
1994a
,b
; Currie and Stein 1989
). In some cases,
the ipsilateral hip flexor activity during contralateral fictive
scratching was strong enough that a single units's mean vector angle
with respect to the ipsilateral hip flexor activity cycle could be
calculated for contralateral fictive scratching as well. In most such
cases, the neuron's preferred phase was approximately the same for
ipsilateral and contralateral fictive scratching (Fig. 1E).
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For 12 neurons, the preferred phase within the ipsilateral hip flexor activity cycle was found to differ by >90° among different forms of ipsilateral fictive scratching. The neuron that exhibited the most systematic change in preferred phase with ipsilateral stimulation site is shown in Fig. 2. During fictive rostral scratching, this neuron fired mainly during ipsilateral hip flexor bursts; during fictive caudal scratching, however, it fired mainly between hip flexor bursts; its preferred phase changed within the pocket scratch receptive field (Fig. 2).
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For all recorded neurons that were rhythmically modulated during multiple forms of ipsilateral fictive scratching, the neuron's preferred phase of firing (i.e., its mean vector angle) as a function of the form of ipsilateral fictive scratching was assessed quantitatively (Figs. 3 and 4). In pairwise comparisons of the three forms of fictive scratching, most points fell within a 90°-wide y = x band, indicating that the vast majority of neurons had a similar mean vector angle for any two of the three forms of fictive scratching (Fig. 3). The correlation between mean vector angles in different forms of fictive scratching was highly significant (P < 0.001) in each pairwise comparison (Fig. 3). This was especially evident in the comparison of mean vector angles in pocket and rostral fictive scratching (Fig. 3C; r = 0.91); mean vector angles in caudal fictive scratching were more likely to differ (Fig. 3, A and B). Some neurons were sufficiently rhythmically active during each of the three forms of fictive scratching to do a three-way comparison of mean vector angles (Fig. 4). In these cases, neurons tended to have similar mean vector angles for all three forms of ipsilateral fictive scratching (Fig. 4).
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Rhythmic modulation and recording locations
The strength of rhythmic modulation, as assessed by the mean
vector length of the distribution of single-unit action potentials within the ipsilateral hip flexor activity cycle, tended to be higher
for neurons that were recorded further ventrally in the spinal gray
matter. Figure 5 illustrates this
tendency for four representative neurons in a single spinal segment of
a single animal. Figure 6 shows that
there was a clear correlation between the mean vector length and the
depth of the recording across the entire sample of spinal neurons
studied (Fig. 6A; r = 0.48;
P < 0.001). This tendency was also observed within
individual electrode penetrations in which three or more units were
recorded (Fig. 6A, ,
,
, and
; different symbol for each penetration).
This tendency was also observed in an additional set of recordings in
which three or more units were recorded on each electrode penetration, and an electrolytic lesion was made to mark the site of the deepest unit recorded on the penetration (Fig. 6B; different symbol
for each penetration). In contrast, there was no apparent relationship between the mean vector length and either the mediolateral location (Fig. 6C) or the spinal segment (Fig. 6D) of the
single-unit recording.
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Preferred phases of firing and recording locations
To assess whether there was a relationship between the preferred phase of firing of a neuron and its recording location, the hip flexor activity cycle was divided into 90° phase quadrants: 1) mid-hip flexion, 2) late hip flexion/early hip extension, 3) mid-hip extension, and 4) late hip extension/early hip flexion (Fig. 7). Each neuron was placed into one of these phase quadrants based on its mean vector angle (at whichever stimulation site gave the most rhythmic response; see METHODS). The recording locations of all rhythmic neurons are displayed in Fig. 7A, with a different symbol for each phase quadrant; the average of the distribution of recording locations for each quadrant is shown in Fig. 7B. Neurons in each phase quadrant were scattered both dorsoventrally and mediolaterally (Fig. 7A). The average locations are clustered together, with widely overlapping error bars (Fig. 7B). Nonetheless, there was a slight tendency for neurons with a phase preference in mid-hip flexion to be located more laterally than neurons with a phase preference in mid-hip extension, while neurons with phase preferences near the flexion-extension and extension-flexion transitions tended to be in intermediate locations (Fig. 7B).
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Phase preferences were not strongly related to the hindlimb enlargement spinal segment from which the neuron was recorded (Fig. 8). Neurons from each spinal segment except D10 had mean vector angles spread over much of the hip activity cycle (Fig. 8A). Nonetheless, there was a slight tendency for neurons recorded in the D8-D10 segments to have a phase preference within ipsilateral hip flexion and those in the S2 segment to have a phase preference within ipsilateral hip extension. Nineteen of 30 D8 neurons (63%), 7 of 10 D9 neurons (70%), and 4 of 4 D10 neurons (100%) had mean vector angles between 0 and 180° (i.e., during the ipsilateral hip flexor burst); in contrast, 6 of 8 S2 neurons (75%) had mean vector angles between 180 and 360° (i.e., during the hip flexor quiescent period); S1 neuron mean vector angles were approximately evenly divided between ipsilateral hip flexion and extension (4 of 7 between 0 and 180°). These distributions were summarized by calculating the mean vector of the entire distribution of mean vector angles for each spinal segment (see METHODS). These summary mean vector angles for D8, D9, D10, and S1 all fell within 0-180° (i.e., during the ipsilateral hip flexor burst), while the summary mean vector angle for S2 was near 270° (i.e., near the midpoint of hip flexor quiescence). However, all but the D10 mean vector length failed to pass the Rayleigh test as a group (P > 0.2 for each), indicating that the preferred phase of firing was not very consistent across the sample of neurons recorded in each segment; the D10 mean vector length was 0.96, but only four rhythmic neurons were recorded in D10 (the smallest segmental sample size).
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Preferred phases of firing and tuning to regions of the body surface
Each neuron's preferred phase of firing within the ipsilateral
hip flexor activity cycle was also examined as a function of the region
of the ipsilateral body surface to which the neuron was tuned, for the
set of neurons that were both rhythmically modulated and broadly tuned
(Fig. 9) (see Berkowitz
2001 for methods used to assess tuning to regions of the body
surface). Neurons tuned to more anterior regions tended to have a phase
preference within the ipsilateral hip flexor burst, while neurons tuned
to more posterior regions did not. Seven of 10 rostral-tuned neurons (70%), 3 of 3 pocket/rostral-tuned neurons (100%), and 7 of 8 pocket-tuned neurons (88%) had mean vector angles between 0 and 180°
(i.e., during the ipsilateral hip flexor burst); in contrast, 3 of 7 caudal/pocket-tuned neurons (43%) and 8 of 14 caudal-tuned neurons
(57%) had mean vector angles between 180 and 360° (i.e., during the
hip flexor quiescent period). The summary mean vector angles for
rostral-tuned, pocket/rostral-tuned, and pocket-tuned neurons were all
between 56 and 82°, with summary mean vector lengths between 0.36 and
1.0, indicating a concentration of phase preferences during ipsilateral
hip flexion (Fig. 9B). In contrast, the summary mean vector
length for caudal/pocket-tuned neurons was near zero (0.08), and the
summary mean vector angle for caudal-tuned neurons was 216°, with a
summary mean vector length of 0.26 (Fig. 9B). These summary
mean vectors indicate that neurons tuned to a region of the body
surface anterior to the hindlimb tend to be active in fictive
scratching mainly during ipsilateral hip flexor bursts, while neurons
tuned to a region posterior to the hindlimb do not show this tendency.
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DISCUSSION |
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The firing rates of the vast majority (88%) of spinal neurons
activated during fictive scratching were rhythmically modulated with
the scratching rhythms. This is similar to the percentage of
rhythmically active neurons found during fictive scratching in previous
studies of turtles (Berkowitz and Stein 1994a) and cats
(Berkinblit et al. 1978
) and during fictive locomotion
in neonatal rats (Tresch and Kiehn 1999
). Together with
the companion paper describing broad tuning of these same neurons
(Berkowitz 2001
), this study shows that the firing rates
of many turtle spinal neurons carry both directional information (the
location of a stimulus and/or the direction of the ensuing scratch) and
phasic information simultaneously.
Preferred phases and forms of fictive scratching
Spinal neurons that were rhythmically activated during fictive
scratching tended to be active in a particular phase of the ipsilateral
hip flexor activity cycle during all three forms of ipsilateral fictive
scratching (Figs. 1, 3, and 4). Previous research showed that many
descending, propriospinal neurons are rhythmically active in a
particular phase of the ipsilateral hip flexor activity cycle during
both ipsilateral rostral and pocket fictive scratching (Berkowitz and Stein 1994b). The current study extends
this result to all three forms of ipsilateral fictive scratching.
Rhythmically active spinal interneurons typically are active in a
consistent phase of the hip rhythm during fictive scratching in cats as
well (Berkinblit et al. 1978
).
Earlier propriospinal axonal recordings (Berkowitz and Stein
1994b) and the current gray matter recordings have revealed a preponderance of neurons whose rhythmic activity is correlated with the
hip activity cycle during each form of fictive scratching, despite the
fact that the knee-hip synergy differs for each form of scratching. One
possibility is that both studies missed recording a large number of
interneurons specialized for knee control due to the location, size, or
morphology of knee-related neurons. This seems unlikely, however,
because the earlier study (Berkowitz and Stein 1994a
,b
)
sampled large cross-sectional regions of the spinal white matter and
the current study sampled large regions of the spinal hindlimb
enlargement gray matter. Another possibility is that the turtle spinal
cord is able to generate the appropriate knee-hip synergy for each form
of scratching without using interneurons that are specialized for knee
control [see Preferred phases and broad tuning and Fig.
10, as well as the discussion in
Berkowitz and Stein (1994b)
].
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One cannot determine whether any one of the recorded neurons actively
contributes to the generation of scratching rhythms, as opposed to
merely receiving feedback from pattern-generating neurons. However,
given that such a large proportion of spinal neurons that are activated
during fictive scratching are rhythmically activated during multiple
forms of fictive scratching, it seems likely that at least some of
these neurons actively contribute to the generation of multiple forms
of fictive scratching. If so, this adds to growing evidence that
individual CNS neurons contribute to the generation of multiple types
of behaviors in vertebrates. Such evidence is now substantial for the
control of vertebrate axial body movements (Soffe 1993;
Soffe et al. 1984
; Svoboda and Fetcho
1996
) and oral or respiratory movements (Grelot et al.
1993
; Larson et al. 1994
; Lieske et al.
2000
; Miller and Ezure 1992
; Nonaka and
Miller 1991
; Oku et al. 1994
; Westberg et
al. 1998
; Yajima and Larson 1993
); evidence for
the control of vertebrate limb movements is currently more limited
(Berkowitz and Stein 1994b
; Perreault et al.
1999
) or circumstantial (Arshavsky et al. 1972
,
1978
; Deliagina and Feldman 1981
;
Deliagina and Orlovsky 1980
; Feldman and Orlovsky
1975
; Pratt and Jordan 1987
). In invertebrates, it has been conclusively demonstrated that individual CNS neurons can
contribute to the generation of multiple types of behaviors or motor
patterns (Dickinson and Moulins 1992
; Getting and
Dekin 1985
; Heinzel et al. 1993
; Hooper
and Moulins 1989
; Lockery and Kristan 1990
;
Meyrand et al. 1991
; Weimann and Marder
1994
; Weimann et al. 1991
).
A few neurons had substantially different phase preferences when
fictive scratching was elicited via different stimulation sites (Figs.
2 and 3). In principle, a small number of phase-shifting neurons could
be responsible for generating knee extension, which occurs in a
different phase of the hip activity cycle during each form of
scratching (Mortin et al. 1985; Robertson et al.
1985
). This seems unlikely, however, given that shifts in phase
preference with respect to the hip flexor activity cycle were both
uncommon and inconsistent in this data set. The clearest and most
consistent example of a phase-shifting neuron is shown in Fig. 2. This
neuron, however, changed its phase preference in the middle of the
pocket scratch receptive field; neurons that would excite knee
extensors in the appropriate phase of the hip cycle would be expected
to change their phase preference in the transition zone between forms of scratching instead (Mortin et al. 1985
).
Rhythmicities and locations of spinal neurons
The degree of a neuron's rhythmic modulation was correlated with
the depth of the recording in the spinal gray matter (Figs. 5 and 6,
A and B). The method used to measure depth of the
electrode appears to be sufficiently precise to detect such overall
trends (see Fig. 5 in Berkowitz 2001). Moreover, this
trend was also apparent in most individual electrode penetrations in
which three or more units were recorded (Fig. 6, A and
B). This trend was apparent even though the recordings
avoided motoneuron cell bodies (see METHODS). This suggests
that there may be a dorsoventral gradient of rhythmicity among spinal
interneurons. An important caveat is that some recordings could have
been from dendrites or axons, rather than from cell bodies. If this
occurred, however, it would be expected to weaken, rather than
strengthen, any relationship between recording location and functional
properties. Thus the observed correlation between recording depth and
rhythmicity is likely to reflect a real organizational principle of the
turtle spinal cord; deeper neurons may be more likely to be
pattern-generating neurons or to receive stronger feedback from
pattern-generating neurons.
The distribution of spinal neurons that were rhythmically active in
this study was generally consistent with the distributions of spinal
neurons that have been found to be rhythmically active during fictive
scratching or fictive locomotion in other limbed vertebrates (see
Kiehn and Kjaerulff 1998 for review). It has been found
repeatedly that neurons that are rhythmically active or are most
important for rhythmogenesis are located primarily in the intermediate
zone and ventral horn rather than the dorsal horn (Barajon et
al. 1992
; Berkinblit et al. 1978
; Huang
et al. 2000
; Kjaerulff et al. 1994
; Noga
et al. 1995
; O'Donovan et al. 1994
;
Orlovsky and Feldman 1972
; Viala et al.
1991
).
In contrast, in this study there were no consistent correlations
between the degree of rhythmic modulation of neurons and the spinal
segment or the mediolateral location of the recording within the turtle
hindlimb enlargement gray matter. Localization to a single spinal
segment is unambiguous, so the lack of a correlation between
rhythmicity and spinal segment probably indicates that the cell bodies
of spinal neurons that are strongly rhythmically modulated during
fictive scratching are widely distributed through the five segments of
the turtle hindlimb enlargement gray matter. Previous studies have
found that the capacity to generate rhythmic motor activity is
distributed throughout the hindlimb enlargement, but predominantly in
the rostral and middle segments of the hindlimb enlargement, in turtles
(Mortin and Stein 1989), chicks (Ho and O'Donovan 1993
), rats (Kjaerulff and Kiehn
1996
), and cats (Deliagina et al. 1983
;
Marcoux and Rossignol 2000
). Rostral
(L2) hindlimb enlargement neurons tend to be more
rhythmic than more caudal (L5) neurons during
pharmacologically evoked locomotor activity in neonatal rats
(Tresch and Kiehn 1999
), but spinal neurons activated when locomotor activity is evoked by electrical stimulation of the
mesencephalic locomotor region in cats tend to be most concentrated in
the L4-L6 segments
(Noga et al. 1995
) or the L6
segment (Huang et al. 2000
).
Preferred phases and cell locations
There was only a weak relationship between the preferred phase of
a neuron and its recording location (Figs. 7 and 8). Neurons with a
preferred phase within the ipsilateral hip flexor burst showed a slight
tendency to be located more medially (Fig. 7) and in the anterior
segments of the hindlimb enlargement (Fig. 8). However, neurons with
any particular phase preference were widely scattered (Figs. 7 and 8).
A single recording often (although not always) included rhythmically
active neurons with distinctly different phase preferences (not shown).
This same observation has been made for recordings of rhythmically
active spinal neurons during cat fictive scratching (Berkinblit
et al. 1978).
Hip motoneurons show some rostrocaudal localization within the turtle
spinal cord hindlimb enlargement: hip flexor motoneurons are found in
segments D8-D9 and hip
extensor motoneurons in segments D9-S2 (Ruigrok and
Crowe 1984). The rostrocaudal organization of putative
interneurons that are rhythmically active in the current study
parallels this motoneuron organization but suggests that turtle spinal
interneurons with a particular phase preference within the hip flexor
activity cycle show an even greater rostrocaudal spread than individual
turtle hip motoneuron pools. Neonatal rat spinal interneurons that are
rhythmically active during in vitro drug-induced locomotor activity
also show a wide distribution of phase preferences in each spinal cord
segment, but are somewhat more likely to have a phase preference that
matches the modulation of the ventral root activity of the same cord
spinal segment (Tresch and Kiehn 1999
).
Preferred phases and broad tuning
How might these spinal neurons contribute to generating the
distinct motor synergy underlying each form of turtle scratching? During each form of scratching, hip flexor activity alternates with hip
extensor activity. However, rostral scratching occurs for stimuli
anterior to the hindlimb, whereas caudal scratching occurs for stimuli
posterior to the hindlimb. Thus to bring the hindlimb to the correct
location during each cycle of scratching, hip flexor activity needs to
be greater during rostral scratching and hip extensor activity greater
during caudal scratching; pocket scratching is intermediate. Indeed,
hip flexor bursts are typically longest and strongest during rostral
scratching and shortest and weakest during caudal scratching; hip
extensor bursts typically vary in the opposite way (Robertson et
al. 1985). Thus one might expect that the population of
rhythmically active spinal interneurons as a whole would be weighted
toward hip flexion during fictive rostral scratching but toward hip
extension during fictive caudal scratching.
This was assessed in the current study by comparing the phase
preference of each neuron to the region of the body surface to which it
responded maximally, for the population of spinal neurons that were
both rhythmically modulated and broadly tuned (Fig. 9) (see
Berkowitz 2001 for methods used to assess tuning). Indeed, neurons that were broadly tuned to the rostral, pocket/rostral, or pocket scratch regions tended to have a preferred phase during the
ipsilateral hip flexor burst, while neurons that were broadly tuned to
the caudal scratch region did not. Thus if many of the recorded neurons
project directly or indirectly to hip muscle motoneurons, then the
summed or averaged activity of this sample of spinal neurons would tend
to move the hip appropriately for each form of scratching, despite the
fact that each neuron was active during multiple forms of fictive
scratching (see Berkowitz 2001
). This suggests the
possibility that the strength of hip flexion and the strength of hip
extension within each half-cycle of scratching may result from the
summed effects of a large population of rhythmically active premotor
interneurons having diverse effects. Evidence for this type of
organization, in which individual CNS neurons have diverse and even
competing effects, and yet the population of neurons acts to create an
appropriate motor output, has been found in a variety of other systems
as well (Georgopoulos et al. 1986
; Groh et al.
1997
; Heiligenberg 1987
; Kristan and Shaw
1997
; Lee et al. 1988
; Levi and Camhi
2000
; Lewis and Kristan 1998
; Liebenthal
et al. 1994
; Lockery and Kristan 1990
;
Shaw and Kristan 1997
; Sparks et al.
1976
, 1997
).
The most obvious distinction among the three forms of fictive
scratching, however, is the phase of the hip activity cycle in which
knee extensor activity occurs (Robertson et al. 1985). Knee extensor motoneurons, particularly those that innervate the monoarticular knee extensor muscle (FT-KE) (see Robertson et al. 1985
) are active in a burst that occurs during the latter
portion of hip flexor activity in rostral scratching, during hip
extensor activity in pocket scratching, and just after hip extensor
activity ends (but just before hip flexor activity begins) in caudal
scratching (Robertson et al. 1985
). How might the
rhythmically active spinal neurons recorded in this study contribute to
generating the distinct knee-hip synergy for each form of scratching?
It has been hypothesized that populations of spinal interneurons active
in a particular phase of the hip activity cycle could generate the
appropriate knee-hip synergies for rostral scratching and pocket
scratching, provided that each such neuron projects to both a knee
motor pool and a hip motor pool; neurons broadly tuned to the rostral
region would project both to knee extensors and hip flexors, whereas
neurons broadly tuned to the pocket region would project both to knee
extensors and hip extensors (Fig. 10) (Berkowitz and Stein
1994b). Thus the appropriate knee-hip synergy for each of these
two forms of scratching would result from the summed effects of a
population of broadly tuned synergy-generating interneurons, with most
"votes" cast for the appropriate synergy and a smaller number of
votes cast for the inappropriate synergy. But what about caudal
scratching? An extension of the previous hypothesis would be that
rhythmically active neurons that are broadly tuned to the caudal
scratch region would project to knee extensors but would not project to
any hip motoneuron pool (Fig. 10), because knee extension occurs
primarily in between the hip extensor and hip flexor burst in caudal
scratching (Robertson et al. 1985
). Such neurons would
be expected to have a preferred phase near the end of hip flexor
quiescence, to generate knee extension in the appropriate phase of the
hip movement cycle. The results of the current study provide partial
support to this hypothesis. Ten of the 12 broadly tuned neurons with a
preferred phase between 200 and 350° were tuned to either the caudal
region (n = 7) or the caudal/pocket region
(n = 3). However, this is a relatively small sample,
and the preferred phases of these neurons were spread throughout the
period of hip flexor quiescence rather than being concentrated in the
last quarter of hip flexor quiescence (270-360°) as would be
expected for the caudal scratch synergy. Clearly, this issue will
require additional investigation.
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ACKNOWLEDGMENTS |
---|
The author thanks H. Sabapathy for technical assistance, Drs. Joseph Bastian, Gammon M. Earhart, Paul S. G. Stein, Donald A. Wilson, and two anonymous reviewers for comments on a previous version of the manuscript, and Dr. Joseph Bastian for many useful discussions.
This research was supported by National Science Foundation Award 9807991.
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FOOTNOTES |
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Author E-mail: ari{at}ou.edu.
Received 15 August 2000; accepted in final form 2 April 2001.
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REFERENCES |
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