Exp Brain Res (2005) 165: 328342
DOI 10.1007/s00221-005-2309-7
R ES E AR C H A RT I C L E
F. Hellstrom S. Roatta J. Thunberg M. Passatore
M. Djupsjobacka
Responses of muscle spindles in feline dorsal neck muscles
to electrical stimulation of the cervical sympathetic nerve
Received: 12 January 2005 / Accepted: 8 February 2005 / Published online: 10 May 2005

Springer-Verlag 2005
Abstract Previous studies performed in jaw muscles of
rabbits and rats have demonstrated that sympathetic
outow may aect the activity of muscle spindle aer-
ents (MSAs). The resulting impairment of MSA infor-
mation has been suggested to be involved in the genesis
and spread of chronic muscle pain. The present study
was designed to investigate sympathetic inuences on
muscle spindles in feline trapezius and splenius muscles
(TrSp), as these muscles are commonly aected by
chronic pain in humans. Experiments were carried out in
cats anesthetized with alpha-chloralose. The eect of
electrical stimulation (10 Hz for 90 s or 3 Hz for 5 min)
of the peripheral stump of the cervical sympathetic nerve
(CSN) was investigated on the discharge of TrSp MSAs
(units classied as Ia-like and II-like) and on their re-
sponses to sinusoidal stretching of these muscles. In
some of the experiments, the local microcirculation of
the muscles was monitored by laser Doppler owmetry.
In total, 46 MSAs were recorded. Stimulation of the
CSN at 10 Hz powerfully depressed the mean discharge
rate of the majority of the tested MSAs (73%) and also
aected the sensitivity of MSAs to sinusoidal changes of
muscle length, which were evaluated in terms of ampli-
tude and phase of the sinusoidal tting of unitary
activity. The amplitude was signicantly reduced in Ia-
like units and variably aected in II-like units, while in
general the phase was aected little and not changed
signicantly in either group. The discharge of a smaller
percentage of tested units was also modulated by 3-Hz
CSN stimulation. Blockade of the neuromuscular junc-
tions by pancuronium did not induce any changes in
MSA responses to CSN stimulation, showing that these
F. Hellstrom and S. Roatta equally contributed to this work.
F. Hellstrom J. Thunberg M. Djupsjobacka
Centre for Musculoskeletal Research, Gavle University,
Umea, Sweden
S. Roatta (&) M. Passatore
Department of Neuroscience-Physiology Division,
University of Torino Medical School, Torino, Italy
E-mail: silvestro.roatta@unito.it
responses were not secondary to changes in extrafusal or
fusimotor activity. Further data showed that the sym-
pathetically induced modulation of MSA discharge was
not secondary to the concomitant reduction of muscle
blood ow induced by the stimulation. Hence, changes
in sympathetic outow can modulate the aerent signals
from muscle spindles through an action exerted directly
on the spindles, independent of changes in blood ow. It
is suggested that such an action may be one of the
mechanisms mediating the onset of chronic muscle pain
in these muscles in humans.
Keywords Sympathetic nervous system
Muscle spindle Myalgia Neck muscles
Motor control
Introduction
Data reported on experimental animal models have
shown that proprioceptive information from some
muscle territories can be impaired by activation of the
sympathetic nervous system (SNS). In particular, acti-
vation of eerent sympathetic pathways was shown to
aect aerent activity of muscle spindle aerents
(MSAs) in jaw-elevator muscles of rabbits (Passatore
et al. 1996; Roatta et al. 2002) and rats (Matsuo et al.
1995). The interpretation of the few data reported on
hindlimb muscles is controversial in that the sympathetic
action was considered either relevant and directly
exerted on spindles (Francini et al. 1978; Hunt 1960),
small and therefore of scarce signicance in motor
function (Hunt et al. 1982), or secondary to the vaso-
constriction and consequent muscle hypoxia produced
by the stimulation (Eldred et al. 1960; Hunt et al. 1982).
Although generally characterized by depression of
stretch sensitivity, the responses of MSAs to sympathetic
stimulation showed some variability in the dierent
studies in terms of latency, time course, and magnitude
of the eects. Therefore, the need exists to study the

eect of sympathetic action on other territories and
species while also checking whether such eects could be
related to the concomitant vascular action.
It is well known that proprioceptive information
from muscle spindles is important for control and
coordination of ongoing movements and posture (Abe-
lew et al. 2000; Bove et al. 2002; Cordo et al. 1994; Ghez
and Sainburg 1995) as well as for programming move-
ments (Bard et al. 1995; LaRue et al. 1995). In masti-
catory muscles, the proprioceptive impairment produced
by stimulation of sympathetic pathways was shown to
induce a consistent reduction of the myotatic reex
(Grassi et al. 1993a, 1993b) and also to aect more
complex motor patterns, such as the masticatory
movements evoked by stimulation of the cortical mas-
ticatory area in rabbits (Roatta et al. 2004). It is there-
fore reasonable to hypothesize that a strong sympathetic
activation and the ensuing depression of spindle aerent
activity may aect motor control and reduce eciency in
muscle use.
On this basis, the sympathetic modulation of motor
function has been recently taken into account among the
factors responsible for chronic myalgia (Roatta et al.
2002; Passatore and Roatta 2003; Johansson et al. 2003),
a complex and multifactorial syndrome whose patho-
physiology and etiology are scarcely known. This
hypothesis is motivated by the following: (1) Epidemi-
ological studies report the frequent association between
the development of chronic myalgias and stressful
working environments (reviewed by Punnett and Gold
2003), and (2) functional studies report, in human sub-
jects aected by chronic myalgia in the neck-shoulder
area, disturbances in the control and coordination of
ongoing movements that are attributed to deterioration
of proprioceptive information (Madeleine et al. 2004;
Michaelson et al. 2003). Among the possible causes of
the deterioration of proprioceptive information, sym-
pathetic hyperactivity due to stress and pain should
therefore be taken into account.
The aim of the present study was to investigate, in the
cat, whether stimulation of the sympathetic supply to
neck muscles aects the activity and sensitivity of MSAs
of splenius and trapezius muscles.
Methods
All procedures were performed in accordance with
Swedish law and were approved by the Ethical Com-
mittee on Animal Experiments at the University of
Umea (Sweden). The experiments were performed on 17
adult cats anesthetized with alpha-chloralose (initial
dose 60 mg/kg). To keep a sucient depth of anesthesia,
heart rate and arterial blood pressure (ABP) were
continuously monitored, and withdrawal reexes were
estimated every 15 min. Additional doses of alpha-
chloralose (25 mg/kg) were administered whenever
indications of a reduced level of anesthesia were pres-
entwhen heart rate and ABP increased or began to
329
spontaneously uctuate, when muscle tonus increased in
the right hindlimb, and/or when a withdrawal reex was
initiated as a response to nociceptive pinching of the
hindlimb paw. Tracheotomy was performed, and end-
expiratory CO2 was continuously monitored. The sys-
temic ABP, monitored via a catheter inserted into the
left femoral artery, was maintained above 80 mmHg,
and when necessary by infusion of dextran (Macrodex,
6% with sodium chloride, Pharmacia), glucose (55 mg/
ml) with sodium chloride, or sodium chloride acetate
into the left femoral vein. The left hindlimb was com-
pletely denervated to avoid aerent input from ischae-
mic necrotic tissues. The body temperature was
maintained between 37C and 38C through heating
lamps and a hot water cushion positioned under the
animals belly.
The dorsal neck muscles on the left side were exposed
by a midline longitudinal incision of the skin, which was
carefully denervated around the neck, and of the prox-
imal part of the ipsilateral forelimb. The maximum
physiological length of the ipsilateral trapezius and
splenius (TrSp) muscles was measured in situ, then both
muscles were freed from the surrounding tissues,
disconnected from their insertions to the skull, and
attached to an electromagnetic puller through a wire
(Fig. 1). The longissimus capitis, biventer cervicis, and
occipitoscapularis muscles were removed bilaterally. The
suboccipital muscles and the intervertebral muscles were
removed bilaterally to expose the dorsal aspect of the
cervical vertebral column. To gain access to the dorsal
roots, a laminectomy was performed between C2 and
C6. All exposed tissues were covered with paran oil,
which was held at a temperature of about 38C. The
skull and the cervical part of the vertebral column were
immobilized by clamps connected to a rigid metal frame.
In some experiments, the neuromuscular blocking
agent pancuronium bromide (1 mg/kg, administered
intravenously and repeated when needed) was given, and
the cat was articially ventilated to maintain the end-
tidal CO2 concentration at preparalysis levels.
Alpha-adrenergic blockade was performed in three
experiments by intravenous injection of phentolamine
methanesulfonate (Sigma, 2.53.5 mg/kg). In two cats,
the left subclavian artery, ipsilateral to the recorded MSA
units, was isolated, and a plastic snare was placed around
it so that it could be temporarily occluded to produce
hypoxia in the TrSp muscles. The location of the occlusion
was veried during the autopsy. At the end of the exper-
iments, all cats were sacriced by intravenous adminis-
tration of a lethal dose of pentobarbital sodium.
Preparation and stimulation of the cervical
sympathetic nerve
The cervical sympathetic nerve (CSN) ipsilateral to the
isolated TrSp muscles was dissected free from
surrounding tissues, and the depressor nerve, cut low in
the neck, and its peripheral stump were placed in a
330
Fig. 1 a Schematic picture of the registration set-up. Muscle
spindle aerents (MSAs) originating from the trapezius and
splenius muscle, isolated from dorsal roots, are placed on silver-
wire recording electrodes. The muscles are connected to an
electromagnetic puller. The head is xed to a metal frame. b
Schematic drawings of how muscle spindle aerent discharge was
quantied during sinusoidal stretches (1 Hz; peak ampli-
tude=1 mm). The stretch-evoked MSA activity was quantied by
tting a sine function to a spike density histogram, in which spikes
from four consecutive cycles were collected (the algorithm is
described in the appendix). The following were derived from the
quantication: mean discharge rate (MDR, number of spikes
divided by time duration of the interval considered, in pulses per
second [PPS]), peak amplitude (AMP, in PPS), and phase of the
tting sine function (PHASE, in deg). PHASE is equal to the phase
lead of the tting sine function with respect to the sinusoidal muscle
length signal
cylindrical polyethylene cu containing two platinum-
stimulating electrodes mounted 2 mm apart. The cu,
lled with warm mineral oil and covered with cotton
soaked in mineral oil for further insulation, was xed to
the surrounding tissue. Then the muscles and skin of the
neck were carefully stitched over the electrode cylinder
assembly. The CSN was routinely stimulated at 10 Hz
for 90 s and occasionally at 3 Hz for 5 min with 7 V
(0.4-ms pulse width). Successive stimulation trials were
separated by at least 15 min to allow for recovery. The
eectiveness of CSN stimulation was assessed through-
out the experiment by checking pupillary responses and
monitoring blood ow changes in muscle-cutaneous
tissues of the cheek through an inductive proximity
sensor (Selet Sensor Mod. 18/5 OC, Turin, Italy)
working as a surface plethysmograph (Roatta et al.
1996). The CSN isolation was performed before turning
the cat into the prone position and progressing with
dorsal root preparation.
Recording and analysis
Functionally single muscle aerents from the ipsilateral
TrSp muscles were isolated from cut laments of the
dorsal roots C3 and C4, and their activity was recorded
using silver-wire electrodes, amplied, and collected on
computers using an input-output interface device (CED
1401 power) with 16-bit AD-converter. The data
acquisition was performed by use of the program Spike
2 (CED). The activity of two to four units was recorded
at the same time.
All units were routinely tested with a sequence of
dierent stimuli in order to provide a basis for their
classication. After prestretching the muscle to
8 mm
(maximum physiological length=0 mm), the muscle was
further stretched with ramp-and-hold (R&H) stimuli
(amplitude=4 mm, rise time=2 s, plateau dura-
tion=6 s, passive tension developed <300 g). In addi-
tion, after prestretching the muscle to
2 mm, slow sine
waves (1 Hz, 1-mm peak amplitude) and 100-Hz and
150-Hz vibrations (amplitude 90140 lm and 3050 lm,
respectively) were applied. Aerent units were classied
as (1) Ia-like if they responded 1:1 to the 100-Hz
vibration and exhibited silencing of discharge during the
shortening phase of the slow sine wave and during the
release phase of the R&H, or (2) II-like if they did not
respond 1:1 to 100-Hz vibration and continued ring
during the release phase of sine waves and R&H. In
addition, the location of each receptor was assessed by
recording the units response to gentle pressure by a
probe on dierent areas of the muscle. Conduction
velocity measurements were not performed because of
the short conduction distance and the lack of bimodality
in the ber diameter spectrum (Price and Dutia 1989;

Richmond and Abrahams 1979). All units classied as
nonmuscle spindles were discarded. In particular, units
exhibiting high discharge thresholds to stretch stimulus
and failing to maintain a tonic ring during the hold
phase of the R&H were excluded, as this pattern is more
typical of Golgi tendon organs (GTOs) (Jami 1992;
Richmond and Abrahams 1979). However, the non-
parallel orientation of the muscle bers in TrSp muscles
causes the applied stretches to be detected dierently by
receptors positioned in dierent muscle areas. For the
same reason, the classical GTO identication technique
based on the response to muscle twitch, attempted in a
few experiments, proved to be ineective, also due to the
incomplete muscle contraction elicited by direct muscle
stimulation (Richmond and Abrahams 1979). Therefore,
we cannot exclude that some GTOs may have been
erroneously identied as muscle spindles. However, the
low ratio of GTO/MSAs reported for cat neck muscles,
8/129 (Richmond and Abrahams 1979), makes the
inclusion of GTOs in the muscle spindle sample rather
unlikely.
While testing the eect of CSN stimulation, the
TrSp muscles were subjected to sinusoidal stretches
(1 Hz; peak-to-peak amplitude=2 mm), which were
superimposed on the plateau of large R&H stretches
(from
10 mm to the plateau at
2 mm; plateau
duration=60 s). The R&H stretches were separated by
short intervals of 13 s. The stretch-evoked MSA
activity was quantied by means of a least square
tting, with a 1-Hz sine function, of the spike density
histogram generated from a single MSA. This analysis
was performed over intervals including four consecu-
tive cycles, each interval being moved one cycle for-
ward with respect to the previous interval. In this way,
a time resolution of 1 s was achieved in the estimate
of the tting parameters. The tting algorithm adop-
ted is a modied version of the classical spike-density
cycle histogram tting (Matthews and Stein 1969;
Hulliger et al. 1977) and is described in the appendix.
The following parameters were then extracted from
the t: (1) the mean rate of discharge (MDR, number
of spikes divided by time duration of the interval
considered, in pulses per second [PPS]); (2) the
amplitude of the tted sine function (AMP, peak
amplitude of the tted sine function, in PPS; (3) the
phase advance of the tted sine function with respect
to the input (muscle length) signal (PHASE, in
degrees), a zero value corresponding to perfect align-
ment between the two traces (Fig. 1).
The eects of CSN stimulation on MSA discharge
were evaluated by assessing the changes exhibited by
the above parameters during and after CSN stimula-
tion. The parameters were time-averaged over 2040-s
intervals positioned in the following sequence: (1) 40 s
before the start of CSN stimulation (control); (2)
around the point of maximum eect on the MDR
(early phase); (3) 100300 s after the end of CSN
331
stimulation, during the recovery period, depending on
the time, when the MDR started to return toward
control value (late phase).
Possible changes in extrafusal muscle ber activity of
the TrSp muscles were indirectly assessed by monitoring
the force output from the muscles through a force
transducer connected to the electromagnetic puller
(sensitivity=0.01 N). In seven cats, trials were per-
formed to investigate whether the actions induced by
sympathetic stimulation on the MSA discharge could be
secondary to the hypoxic condition induced by the
concomitant vasoconstriction. In these experiments,
microvascular blood ow was measured by a laser
Doppler owmeter (PeriFlux PF 2b, Perimed, Sweden)
via a needle probe (PF 302) that estimates microcircu-
lation within a tissue hemisphere having a radius of
11.5 mm. The probe was inserted into the trapezius or
splenius muscle, close to the location (see above) of at
least one of the muscle spindles currently recorded. In
this group of trials, a particular muscle spindle aerent
was discarded whenever this maneuver drastically
changed either its discharge frequency or its pattern of
discharge, which was interpreted as a sign that the probe
had produced a lesion. During the trial in which the
probe was inserted, the muscle was kept in a xed po-
sition; that is, sinusoidal stretches were not delivered in
order to avoid movement artifacts and damages in
microcirculation by the probe. In two of these experi-
ments, the subclavian artery was occluded to induce a
large sudden reduction or abolition of blood supply to
the TrSp muscles.
Statistical analysis was performed using two non-
parametric tests, the Wilcoxon signed rank test and the
Mann-Whitney U-test (signicance level at 0.05 in all
tests). The Wilcoxon signed rank test was used to
investigate the eect of CSN stimulation on MDR,
AMP, and PHASE in the early and late periods com-
pared with control within either Ia-like or II-like units,
as well as to investigate the eects of neuromuscular
junction blockade on the changes induced by CSN
stimulation. The Mann-Whitney U-test was used for
investigating dierences in MDR, AMP, and Phase
during control, early, and late periods in relation to unit
type (Ia-like or II-like). All statistics were conducted
using the SPSS statistical package.
Results
According to our identication procedure, 46 spindle
units were classied as Ia-like (n=33) or II-like
(n=13). In the control condition (before CSN stimu-
lation), Ia-like units diered from II-like units in
having a signicantly lower MDR (26.511.5 PPS vs.
37.77.0 PPS, P<0.01), a higher stretch sensitivity
(AMP: 22.67.5 PPS vs. 10.25.3 PPS, P<0.01),
and a slightly higher dynamic sensitivity as evidenced
332

by the larger phase lead (PHASE: 64.413.4 vs.
51.110.3, P<0.01).
Eects of CSN stimulation at 10 Hz
Stimulation of the CSN was tested on the discharge of
all 46 MSAs (33 Ia-like and 13 II-like) innervating
spindles located in TrSp muscles. Two to four dierent
units were often recorded at the same time so that their
response to the same CSN stimulation trial could be
compared. CSN stimulation at 10 Hz lasting 90 s
changed the MDR in 34 of the 46 MSAs tested (73%).
The remaining 12 units were unresponsive to the stim-
ulation. Both responsive and unresponsive units were
recorded during the same experiments, in some cases
simultaneously. An example of the most common re-
sponse pattern is shown in Fig. 2a (thick line), with the
stimulation period indicated by the solid black line be-
neath the time axis. In general, the eect consisted of a
strong depression of the discharge rate, hereafter termed
early eect, followed by a fast partial recovery, which
often began with a transient excitatory rebound of
varying size. Thereafter, the discharge exhibited a long-
lasting aftereect (late eect) in which it remained
slightly depressed before returning to the control value,
within a time between 30 s and 4 min after the end of
stimulation.
Another example of rapid depression is shown in
Fig. 3 (upper panel). The least common pattern of
response to CSN stimulation, occurring in only three
units, consisted of a slight increase in MDR coupled
with either a decrease (two units) or an increase (one
unit) in amplitude modulation (AMP) in response to
sinusoidal muscle stretch (see below).
To quantify possible changes in stretch sensitivity,
sinusoidal stretches were continuously delivered to the
muscle in 38 (27 Ia-like and 11 II-like) of the 46 tested
units, and the unit responses to CSN stimulation were
evaluated whenever possible in terms of AMP and
PHASE of the sine-tted discharge rate (see Methods).
Early eect
As mentioned above, the most common pattern of
response, occurring in 30 of the 46 tested units, consisted
of a powerful depression in MDR that usually outlasted
the stimulation time. In 21 units, the depression was
strong enough to completely silence the unit for a period
ranging from 5 s to 160 s, during which the aerent
discharge was not even transiently activated by the
ongoing sinusoidal stretch. The latency of the eect was
rather long and variable (range 2580 s, mean 50.7 s).
This variability could not be attributed to dierent
animal conditions because widely dierent latencies also
occurred between dierent units recorded simulta-
neously. In the remaining 16 tested units, the average
decrease in MDR was 21.4% (
7.012.4 PPS,
P<0.05), with no signicant dierence between Ia-like
and II-like units. Figure 4a shows the distribution of
these eects for Ia-like (lled circles) and II-like (lled

Fig. 2 Response of a Ia-like muscle spindle aerent (MSA) to

cervical sympathetic nerve (CSN) stimulation while trapezius and
splenius muscles were subjected to sinusoidal stretches. a CSN
stimulation at 10 Hz for 90 s produces a powerful depression in the
mean discharge rate (MDR), the unit falling silent and slowly
recovering in the late period, after a rebound eect. Similar long-
lasting depression is exhibited by the amplitude of modulation
(AMP). b CSN stimulation at 3 Hz for 300 s elicits in the same unit
a similar depressant eect; however, in this case the latency is
increased, the duration of the silencing is decreased, and the return
to the control condition is faster and occurs before the end of the
stimulation. Sinusoidal stretches: 1 Hz, peak-to-peak ampli-
tude=2 mm. CSN stimulations (7 V) are signaled by horizontal
bars and vertical dashed lines
Fig. 3 Response of a Ia-like muscle spindle aerent (MSA) to
stimulation of the cervical sympathetic nerve (CSN) during
sinusoidal stretches of trapezius and splenius muscles. The eect
during the early period consists of a complete suppression of the
discharge (MDR trace); the response to sinusoidal stretches (AMP
trace) is absent. Notice, however, the dierent time course of the
recovery of these two signals; MDR shows a long-lasting late eect
whereas AMP quickly returns to control. PHASE cannot be
evaluated during the early period, and increases during the long-
lasting depression of the MDR that follows CSN stimulation.
Parameters of CSN stimulation and sinusoidal stretches are as in
Fig. 2
 333

squares) units. A high number of units silenced by the
CSN stimulation are visible; this eect occurred preva-
lently in Ia-like units and is independent of initial values
of MDR.
In the many units that rapidly fell silent in response to
CSN stimulation (n=16, out of 38 tested with sine
stimulus), changes in AMP and PHASE could not be
measured. In the remaining 22 units (13 Ia-like, 9 II-
like), a signicant AMP reduction (
19.8%, P<0.05)
occurred only in Ia-like units, which, however, showed
no signicant change in PHASE (+1.44.7). II-like
units exhibited both small increases and decreases in
AMP without signicant average changes (+1.33.7
PPS), while exhibiting a small decrease in PHASE
(
3.74.3, P<0.05). Scatter plots describing the
eects of CSN stimulation on AMP and PHASE are
illustrated in Fig. 4b, c, respectively (lled symbols).
In some units, the depressant eect was preceded by a
small and transient increase in MDR (see, for instance,
Fig. 3, upper panel).
Late eect
The early depressant eect described above terminated
with a fairly rapid return of the MDR toward control
values. However, in many cases (31/46), a slight
depression of ring rate persisted for several minutes.
Analysis of this late phase (which could now be per-
formed over all the 38 units tested with the sinusoidal

Fig. 4 Mean discharge rate

(MDR; a), amplitude (AMP; b),
and phase (PHASE; c) of the
tted sine function after cervical
sympathetic nerve (CSN)
stimulation compared with
control (ctrl). Early eect (lled
symbols, on left) and late eect
(open symbols, on right) for all
units divided into Ia-like
(circles) and II-like (squares)
plotted against their own
controls. Diagonal lines
represent no change between
control and early or late eects.
a Ia-like early (n=33), II-like
early (n= 13), Ia-like late
(n=33), II-like late (n=13).
b Ia-like early (n=13), II-like
early (n=9), Ia-like late
(n=26), II-like late (n=11).
c Ia-like early (n=12), II-like
early (n=9), Ia-like late
(n=27), II-like late (n=11)
334
stimulus) showed that, on average, MDR remained
signicantly decreased in the Ia-like group (
4.37.1
PPS,
14%, P<0.01) and was not aected in the II-
like group (
2.2% compared with control). The gen-
eral picture is shown in Fig. 4a, right, for Ia-like (open
circle) and II-like (open squares) aerents. On average,
neither Ia-like nor II-like units exhibited signicant
changes in AMP and PHASE. In single trials, a few
net increases or decreases occurred in AMP (equally
represented), while four units showed a clear phase
advance (for example, Fig. 3, bottom panel) ranging
between 6 and 17 (see Fig. 4b, c, right, open sym-
bols).
Correlation among parameters
Scatter plots are shown in Fig. 5 in which sympa-
thetically-induced changes in AMP (Fig. 5a) and in
PHASE (Fig. 5b) are related to relative changes in
MDR for early (lled symbols, left) and late (open
symbols, right) eects in Ia-like (circles) and II-like
(squares) units. In the early eect, some correlation
appears to exist in Ia-like units between the early ef-
fect on MDR and the variation of both AMP
(r=0.86, P<0.01) and PHASE (r=
0.89, P<0.01),
although estimated from only nine units (those that
were not silenced by CSN stimulation). In the late
eect, only the correlation between PHASE and MDR
remains signicant (r=
0.77, P<0.01). Conversely,
II-like units never exhibit signicant correlations
between these parameters.
Fig. 5 Relative changes in
amplitude of modulation
(AMP, panel a) and phase
(PHASE, panel b) are plotted
versus the relative change in
mean discharge rate (MDR).
Early eect (lled symbols) is
displayed on the left side, and
late eect (open symbols) on the
right. In all cases, changes are
evaluated with respect to the
control condition. Circles refer
to Ia-like and squares to II-like
units
Eects of CSN stimulation at 3 Hz (5 min)
CSN stimulation at 3 Hz was tested on 12 of the units,
which were modulated by 10-Hz stimulation. Six of
them displayed signicant alterations in stretch re-
sponse, even though the eects were generally smaller
and exhibited latencies from stimulus onset, which were
longer than the ones observed for the routinely applied
10-Hz stimulations. The 3-Hz stimulation did not gen-
erally succeed in producing a moderate and stable
inhibition of the ring pattern, rather, in some cases
(n=3), it still silenced the receptor (Fig. 2b). The other
six units were unaected by the stimulation. MSAs not
responsive to 10-Hz CSN stimulation did not respond to
the 3-Hz stimulation either.
Absence of changes in fusimotor activity
and extrafusal activity
Although electromyographic activity was not recorded
from the studied muscles, activation of extrafusal bers
in both anesthetized and anesthetized-curarized prepa-
rations can be excluded because no change in muscle
force was detected during the trials.
In addition, to ensure that the observed eects were
not due to changes in extrafusal and fusimotor activity,
some of the animals were administered a neuromuscular
blocking agent (pancuronium) either throughout the
experiment or during part of it. The majority of trials
were performed under neuromuscular blockade (n=31),
and the others in nonblocked preparations (n=15). The

data obtained from the two groups did not show sig-
nicant dierences in terms of time course and size of
response to sympathetic stimulation. In addition, in ve
of these units (one II-like and four II-like), the response
to CSN stimulation was tested both before and after
blockade. The blockade could produce a slight decrease
in MDR in the control phase but did not aect the ex-
tent and time course of the response. Therefore, all units
were pooled into one single group for analysis.
Action of alpha-adrenergic blockade
on the sympathetically induced response
The eect of CSN stimulation was tested before and
after alpha-adrenergic blockade by phentolamine on ve
MSAs collected from three curarized animals. After the
blockade, the muscle-cutaneous vasomotor response to
CSN stimulation was almost completely abolished in all
animals. Alpha-adrenergic blockade completely abol-
ished the response to sympathetic stimulation in all
MSAs tested.
Tests for vasomotor eects
Tests were performed to assess whether sympathetically
induced vasoconstriction, and the potentially resulting
hypoxic condition, could be responsible for the observed
eects on MSA discharge.
During eight CSN stimulation trials, performed in
ve experiments, microvascular blood ow was mea-
sured from the muscle area containing the MSAs being
recorded. In some of these trials, two dierent MSAs
located close to each other (approximate distance 1 cm)
were simultaneously recorded while the blood ow was
measured. One of these trials is shown in Fig. 6. Sym-
pathetic stimulation induced a powerful depression of
the discharge of the unit labeled unit 1 and did not
substantially aect unit 2, while producing a large de-
crease in blood ow. After the end of stimulation, the
discharge of unit 1 steeply increased again, while the
blood ow remained reduced.
In two experiments, the following trials were per-
formed while recording from MSAs located in trapezius
muscle. One of these trials is shown in Fig. 7. Standard
CSN stimulation was performed (Fig. 7a) and then re-
peated under condition of muscle hypoxia (Fig. 7b), that
is, 3 min after starting the occlusion of the ipsilateral
subclavian artery, which was maintained for 78 min
altogether. This occlusion practically eliminated the ow
in the area of trapezius muscle, where both tested spin-
dles were located. The comparison between these two
trials shows that the latency and the onset of the
depressant response (early eect) to CSN stimulation
were identical for both MSAs. If the muscle hypoxia per
se were responsible for the eect induced by CSN
stimulation, occlusion (Fig. 7b) should modify the MSA
discharge during the rst 3 min, and the latency of the
335
Fig. 6 Eect of cervical sympathetic nerve (CSN) stimulation on
the discharge of two muscle spindle aerents in trapezius muscle
and on the microvascular blood ow recorded from an area of the
muscle close to the location of both the recorded spindles. Unit 1 is
Ia-like, and unit 2 is II-like. Note that the small increase in arterial
blood pressure (ABP) at the start of stimulation, as well as all
rhythmical changes along the trial, are parallel to the changes in
muscle microcirculation, which indicates the high sensitivity of the
intramuscular laser Doppler measurement. CSN stimulation at
10 Hz (7 V, 90 s) is signaled with a horizontal bar and vertical
dashed lines. Microvascular blood ow is calibrated in arbitrary
units (a.u.)
early eect of CSN stimulation should be much shorter.
This was never the case. In addition, the recovery of the
discharge of both units took place while the blood ow
in trapezius was still close to zero, due to the maintained
arterial occlusion. The only dierence between the two
conditions is that the eect of CSN stimulation lasted
longer in the trial performed during occlusion, which is
explained by the fact that the washout of the released
catecholamines was slower than in the control condition.
The above data indicate that the sympathetically induced
modulation of MSA discharge was not secondary to
the concomitant reduction of muscle blood ow.
Discussion
The present study has shown, for the rst time, that the
SNS can strongly aect the proprioceptive aerent
information from MSAs in cat neck muscles. The pat-
terns of response to stimulation of the sympathetic
nerves were fairly homogeneous and mainly consisted of
a marked depression of MSA discharge, which was more
prominent in Ia-like than in II-like units and often
strong enough to completely suppress the activity of the
MSAs. Whenever such depression silenced the MSA, the
stretch sensitivity could not be assessed. Such an
assessment was thus restricted to a small number of units
that were not silenced during the early phase and ex-
tended to all units during the recovery period (late ef-
fect). These assessments revealed a signicant decrease
in the amplitude of the sine-tted discharge rate in Ia-
like units in the early eect, while the phase was on
average aected but little.
The following aspects of the present ndings are in
agreement with other recent studies (Matsuo et al.
336

Fig. 7 Comparison of the

responses of the same two Ia-
like MSAs from trapezius
muscle to cervical sympathetic
nerve (CSN) stimulation
performed in control (a) and in
hypoxic conditions induced by
occlusion of the ipsilateral
subclavian artery (b). The laser
Doppler needle probe
measuring microvascular blood
ow was inserted into the
trapezius muscle, in an area
close to both spindles whose
aerent activity was recorded.
Note that the response of the
two units is very similar in the
two conditions in terms of
latency and magnitude of the
eect; the longer duration of the
eect under hypoxic condition
is motivated by the slower
washout in the latter condition
due to the persistent drastic
reduction-zeroing in blood
ow. The displacement of the
cheek surface (surf. pleth.),
routinely used as a surface
plethysmography record
(Roatta et al. 1996; see
Methods) shows that CSN
stimulation was eective in
reducing blood volume in
masseter muscle in both trials;
the small decrease in the
amplitude of this response, as
well as the artifacts visible on
both this and on the ow
record, are due to some
stretching of the common
carotid artery during the
subclavian artery occlusion
maneuver. CSN stimulations
(10 Hz, 7 V, 90 s) are signaled
by horizontal bars and vertical
dashed lines

1995; Roatta et al. 2002). We found that the dis-
charge of 73% of the tested MSAs was modulated by
sympathetic stimulation, which is comparable to the
values of 85% reported for rabbit jaw muscles (Ro-
atta et al. 2002) and 68% for rat jaw muscles (Ma-
tsuo et al. 1995). All studies describe a prevalently
depressant eect on MSAs, although with dierent
latencies and time courses. This depression may be
responsible for the decrease in the reex muscle re-
sponse to stretch or vibration that was observed
during sympathetic stimulation and intraarterial cate-
cholamine injection (Francini et al. 1978; Grassi et al.
1993a, b; Matsuo et al. 1995). In addition, as in
rabbit jaw muscles, the eect of sympathetic stimu-
lation was independent of gamma eerent activity and
appeared to be largely mediated by alpha-adrenore-
ceptors.
However, at partial variance with results reported on
jaw muscles, the present data did not show a consistent
sympathetic eect on MSA stretch sensitivity as inves-
tigated with continuously supplied sinusoidal muscle
stretch. In the few Ia aerents that did not fall silent
after sympathetic stimulation, the stretch response
amplitude (AMP) was decreased proportionally to the
relative change in the MDR, suggesting that both eects
could be produced by the same depressant mechanism.
However, the observation that a few II-like units
exhibited clearcut changes in stretch sensitivity, both in
terms of amplitude and phase, unrelated to changes in
MDR, suggests that these eects may be mediated by
dierent mechanisms. Such eects as well as other
smaller eects, such as the initial increase in MDR often
observed, might have been masked in many instances by
the main depressant eect.

Potential mechanisms
An increasing body of evidence shows that the SNS is
involved not only in the traditionally recognized veg-
etative functions but also in modulating the somatic
sensitivity and thus sensorimotor integration. In par-
ticular, the SNS has been reported to aect the gen-
eration and transmission of sensory information from
a number of mechanical and chemical receptors
through an action exerted at the receptor level (re-
viewed by Akoev 1981; Koltzenburg 1997). In some
cases, the ndings have been supported by morpho-
logical data. Ballard (1978) and Barker and Saito
(1981) described adrenergic terminals in the spindle
capsule that were not associated with intracapsular
blood vessels but were either adjacent to sensory
endings or in neuroeective association with intrafusal
bers. Similar evidence has recently been obtained on
human muscle spindles, in which the noradrenaline
cotransmitter NPY has been identied in intrafusal
bers of lumbrical muscles (L.-E. Thornell, personal
communication).
The present results on cat neck muscles add to the
few previous studies reporting a modulatory action of
sympathetic stimulation and catecholamine injection
on MSAs from cat hindlimb muscles (Eldred et al.
1960; Francini et al. 1978; Hunt 1960; Hunt et al.
1982) and from jaw muscles in rabbit and rat (Matsuo
et al. 1995; Passatore et al. 1996; Roatta et al. 2002).
However, there is still no general agreement concern-
ing the mechanism of such an action regarding whe-
ther the observed eects are due to a direct action on
the receptor (Francini et al. 1978; Matsuo et al. 1995;
Passatore et al. 1996; Roatta et al. 2002) or are sec-
ondary to the concomitant reduction of blood ow to
the muscle tissue (Eldred et al. 1960; Hunt et al.
1982). To our knowledge, no study has been able to
convincingly show that the eects on MSAs are due
to hypoxia consequent to a reduction in blood supply
to the muscle. This point deserves consideration be-
cause, in the present work, the latency of the eect
was on average longer (50.7 s) than reported by other
studies, these latencies ranging from 10 s to 45 s in
rabbit jaw muscles (Roatta et al. 2002) and lying be-
low 1 s in rat jaw muscles (Matsuo et al. 1995). We
tested the possible eect of hypoxia by performing a
series of trials with the subclavian artery clamped. The
virtually complete interruption of blood ow to the
test muscles did not aect MSA discharge per se, nor
did it reduce the latency of the sympathetically in-
duced eect, nor did it prevent the return of spindle
discharge to control while the artery was still occluded
and the blood ow was still close to zero. These
observations rule out muscle ischemia as a major
cause of the observed sympathetic eects on spindle
discharge while supporting the hypothesis of a direct
sympathetic action on the spindle.
Consequently, other mechanisms should be con-
sidered for explaining the dierence in latencies ob-
337
served in the dierent preparations. For instance, the
density as well as the distance between the terminals
releasing catecholamines and the eectors could dier
for the spindles of dierent muscles or animal spe-
cies, in analogy with what has been reported in
other sympathetically innervated structures. Thus, the
density and nerve varicosity-smooth muscle cell sep-
aration of cerebral and ear arterial beds in the rabbit
are reported by Dodge et al. (1994) to vary by a
factor of up to ve. In these studies, they attribute
to the lower density of sympathetic terminals and to
the higher terminal-eector distances found in the
middle cerebral artery territory the weaker action
exerted by the sympathetic innervation on cerebral
blood vessels, compared with the ear territory. At
present, no morphological data are available on
terminal-eector distances in muscle spindles, how-
ever.
Sympathetic modulation of muscle-spindle activity
might take place at dierent receptor sites, such as the
gamma neuromuscular junction, the intrafusal muscle
ber, the aerent receptive terminal, or the aerent
encoding site. Because the eects were not altered by
blockade of the neuromuscular junction, a modulating
action at the gamma endplate can be excluded. In a
recent paper on rabbit jaw muscles (Roatta et al.
2002), an action at the level of the aerent ber was
hypothesized because the sympathetically induced
depression of the MSA activity overpowered the
excitation induced by the succinylcholine-mediated
contraction of intrafusal bers. This possibility de-
serves additional considerations. Several studies have
shown that calcium channels, in addition to sodium
channels, are involved in generating the receptor po-
tential in MSA terminals. In particular, calcium
channel blockers were shown to exert an inhibitory
action on the amplitude of the receptor potential and
on the ring frequency in these receptors (Fischer and
Schafer 2002; Ito et al. 1990; Kruse and Poppele
1991). For instance, Fischer and Schafer (2002)
recently found that in isolated muscle spindles, the
calcium antagonist nifedipine (at a concentration of
25 lM) could strongly depress the activity in Ia and II
aerents for several minutes. Although comparisons
between recordings from MSAs in isolated and in-vivo
preparations must be made with caution, the eects of
nifedipine are impressively similar to the eects of
CSN stimulation described here (compare their Fig. 2
with our Fig. 6a, unit 1). The possibility for nor-
adrenaline to inhibit Ca2+ inux is well established
and has been demonstrated specically for sensory
neurons in dierent animal species (Cox and Dunlap
1992; Dolphin 1995; Marchetti et al. 1986). Therefore,
it may be hypothesized that a noradrenaline-mediated
modulation of calcium channels underlies the eects
produced by CSN stimulation on MSA discharge.
However, specic investigations on the adrenergic
receptors involved and their intracellular pathways are
needed to conrm this hypothesis.
338
Methodological considerations and physiological
signicance
A stimulation frequency of 10 Hz is often considered to
be beyond the physiological range of operation of the
eerent sympathetic system, at least in humans (Janig
and Habler 2000a; Maceeld et al. 2003). However,
studies performed in animal models, in which reductions
in muscle blood ow produced by reex sympathetic
activation were compared with eects produced by
sympathetic stimulation at dierent frequencies, showed
that the eects of strong sympathetic activation were
reproduced by electrical stimulations in the range of 8
10 Hz, and estimates of maximum physiological fre-
quencies reached 1520 Hz (Folkow 1952; Kendrick
et al. 1972; Mellander and Johansson 1968). Moreover,
in the anesthetized cat, a condition known to depress
sympathetic outow, unitary sympathetic bers of the
cervical sympathetic trunk showed spontaneous activity
at frequencies between 0.4 and 7.5 PPS, with an average
around 2 PPS (Janig and Schmidt 1970; Mannard and
Polosa 1973; see also Janig 1985 for review), while in the
anesthetized rabbit, the same types of ber responded to
hypoxia by increasing their average discharge rate from
2 to 12 PPS (Dorward et al. 1987). These data suggest
that the physiological values of sympathetic frequen- pathetic bers supplying muscle spindles is presently
cies are not yet clearly identiable and most likely de-
pend on the animal species, organ, or body district and
on whether pre- or postganglionic activity is considered.
Of course, the electrical stimulation of a sympathetic
nerve diers from physiological sympathetic activation
in another respect. With electrical stimulation, all sym-
pathetic bers are excited synchronously at a constant
frequency; while physiologically the sympathetic bers
may be recruited partially and variably, re asynchro-
nously and preferentially in bursts. Moreover, sympa-
thetic bers are now known to be dierentially controlled
in dierent territories, organs, and tissues, depending on
the context and type of stressful stimulus (for review and
references, see Janig and Habler 2000b; Morrison 2001),
and their action is in general potentiated by circulating
catecholamines. For all the above reasons, CSN stimu-
lation is a poor approximation of physiological sympa-
thetic activation, and the pattern of response observed
may not faithfully predict the MSA behavior occurring
under stressful conditions. Nevertheless, having ruled
out circulatory mechanisms and having observed similar
eects at 3-Hz stimulation frequency, we believe that
increased sympathetic outow under physiological con-
ditions may entail a depression of MSA activity.
Several research groups have recently investigated
possible alterations in proprioception or motor control in
healthy humans in whom increases in sympathetic
activity were provoked by stimuli of dierent types
(Christou et al. 2004; Maceeld et al. 2003; Matre and
Knardahl 2003). Inspiratory capacity apnea and maximal
expiratory apnea, maneuvers increasing muscle sympa-
thetic nerve activity, did not aect MSA activity as de-
tected by microneurography (Maceeld et al. 2003).
Along the same lines, Matre and Knardahl (2003) showed
that proprioceptive acuity at the ankle joint was not re-
duced by sympathetic activation induced either by a cold-
pressor test (foot in 7 water for 3.5 min) or glucose
ingestion. In contrast, Christou et al. (2004) did observe
an increase in the uctuations of the pinch-grip force
(which had to be maintained at 2% of maximal voluntary
contraction) when stressing the subject with electrical
painful stimuli to the contralateral hand. Eectiveness of
the stressor was evidenced by both the results of cognitive
assessment and by the signicant increase in the plasma
concentrations of stress hormones (epinephrine, norepi-
nephrine, ACTH). This result clearly indicates a decrease
in the precision in the control of muscle force in stress
conditions. In other studies, stressors of various qualities
and intensities have been shown to impair motor per-
formances in humans, with the same stressor eliciting
dierent eects depending on the intensity as well as basal
conditions of the subjects anxiety (see, for instance,
Noteboom et al. 2001; Sade et al. 1990).
The variability of results is likely related to the dif-
ferent tests of sympathetic activation used. As pointed
out above, dierent stressful stimuli likely produce
dierent actions on the sympathetic outow to dierent
territories. Moreover, the adequate stimulus for sym-
unknown. Therefore, if a given test fails to evoke
changes in MSA discharge and/or proprioception and
motor performance, the possibility should be considered
that the resulting change in sympathetic outow is
inadequate in terms of type, intensity, or duration.
Concluding remarks
The data collected in trapezius and splenius muscles, as
well as similar data reported previously on jaw and some
hindlimb muscles (see above), suggest a direct depressant
action exerted by the sympathetic system on MSAs.
These data also suggest that such sympathetic action is
not conned to certain muscles or animal species but
likely is a general feature of the control of MSA dis-
charge under conditions producing an increase of sym-
pathetic outow, such as stress. This action would
potentially impair the dierent body functions in which
muscle spindles play a relevant role, including proprio-
ception and motor control. In particular, muscle spindles
are known to mediate moment-to-moment control of
muscle length through the fast spinal negative feedback
loops that also mediate the stretch reex. Activation of
the sympathetic system would, in this respect, reduce the
gain of this control, a condition considered useful for
achieving stability in rapidly executed movements
(Prochazka 1989), such as a ght-or-ight reaction. In
dierent motor contexts, however, including ne move-
ments requiring stability and precision, the reduced
control of muscle length may be detrimental. This
condition of worsened motor control may call for
alternative and suboptimal motor strategies, such as the
 339

cocontraction of agonists and antagonists muscles, Consider the cycle divided into n bins, the center of
aimed at increasing joint stiness and limb stability the ith bin being located at the angle hi (1 i n) and
(Gribble et al. 2003). In turn, these altered motor strat- the bin width being D h=2p/ n radians. A cycle histo-
egies and postures may in the long run lead to muscu- gram is generated from the spike activity of a single
loskeletal problems and diseases. In fact, cocontraction MSA collected over four consecutive cycles. The jth
as well as low-intensity long-duration static contractions spike occurs at a time tj, which corresponds to a specic
are recognized among the main risk factors for the angle hj =2pf tj within the cycle, 1 j m, with m
development of myalgias (Hagg 1991; Rissen et al. 2000, being the total number of spikes collected from the
see Sjogaard et al. 2000 and Van Dieen et al. 2003 for current sample. The number of spikes collected into the
review). ith bin, Yi, divided by the number of cycles (nc=4) and
Numerous experimental and clinical data suggest a the bin width in seconds (Dt=1/(nf)), yields bin contents
correlation between chronic muscle pain of various ori- in terms of spike densities.
gins and disturbances in motor coordination and pro- The histogram thus obtained is tted with a sine wave
prioception, particularly in the cervical region (e.g., whose amplitude, phase, and oset are to be determined
Eriksson et al. 2004; Karlberg et al. 1995; Michaelson by the tting algorithm. For mathematical convenience,
et al. 2003; Revel et al. 1991, 1994; Thunberg et al. 2001; we dened this sine wave by the sum of three terms: a
Wenngren et al. 1998; for review and references, see sine (in phase with the length stimulus), a cosine (90 in
Djupsiobacka 2003; Blair 2003; Johansson et al. 2003). advance of the length stimulus), and a constant term, C,
The data presented in this study may be interpreted to corresponding to the mean ring rate (bias):
suggest that the sympathetically-induced derangement
of proprioceptive information may be one of the F h A sinh B cosh C 2
mechanisms underlying chronic muscle pain, whichas The three unknown variables (A, B, and C) are deter-
pointed out in the Introductionis frequently associ- mined by solving the system of n equations, dened in
ated with stress conditions. matrix terms as
Acknowledgements We thank Monica Edstrom, Stina Langendoen, Mx Y; 3
and Margareta Marklund for valuable assistance in the experi-
ments; Lars Backstrom and Goran Sandstrom for implementing where M is a numerical matrix, Y is the array of spike
the data processing routines and for technical support; and Pro- densities from the above histogram, and x contains the
fessor Uwe Windhorst for enlightening and fruitful comments on variables to be estimated:
the manuscript. This study was supported by grants from The
Swedish Council for Work Life Research, IngaBritt and Arne 2 3
sinh1 cosh1 1
Lundbergs Forskningsstiftelse, and the Italian Ministry of Scien- 2 3
6 sinh2 cosh2 1 7
tic Research (MIUR). 6 7 A
6 7
M 6 sinh3 cosh3 1 7 x 4 B 5
6 .. .. .. 7
4 . . .5 C 4
sinh

n coshn 1 
Appendix
nf .
Y array20cY1 Y2 Y3 ..Yn
According to a widely used procedure, the stretch- nc
evoked MSA activity may be quantitatively character-
This system is solved by means of the least-square
ized by constructing a cycle histogram of spike density to
algorithm (LSA), which involves multiplying both sides
which a 1-Hz sine function is tted by means of a least-
with the transpose of M:
square algorithm (e.g., Matthews and Stein, 1969; Hul-
liger et al. 1977). A modied version of this procedure is MT Mx MT Y; 5
described here that grants improved performance in case T
the number of spikes in the histogram is low, such as where M is the transpose of the matrix M, and then
when the discharge rate is low and/or the spike sequence solve for x:
is collected from only a few consecutive cycles. Although
this algorithm does not require the construction of the x MT M
1 MT Y 6
histogram, this approach is nevertheless described be- The following matrices are generated in Eq. 5:
cause it helps explain how the algorithm works. 2 P 3
The sinusoidal length stimulus applied to the (neck) n P
n P
n
sin2 hi sinhi coshi sinhi
muscles was the following: 6 7
6 n 1 1 1 7
6 P Pn P
n 7
Stimulus L sinh; 1 T
M M6 6 sinh cosh cos 2
h cosh i 7

i i i 7
6 1 n 1 1 7
where h is the radian angle: h=x t=2p ft, with x being 4 P Pn 5
sinhi coshi n
the angular velocity, f being the frequency of the sine 1 1
wave (f=1 Hz in our case), and t the time. The range
7
0<h<2p (radians) corresponds to a single cycle.
340
2P
n 3 where hj is the angular occurrence of each spike.
Yi sinhi Expressions in Eqs.10 and 11 are then inserted in
6 1 7
6 n 7 Eq. 5, and the nonnite term n is eliminated from both
6
nf 6 P 7
MT Y 6 Yi coshi 7
7 8 sides. The resulting numerical equation is the one that is
nc 6 1 7 used by the algorithm and solved for x according to
4 Pn 5
Yi Eq. 6. Estimates A, B, and C as dened in Eq. 2 are
1 nally obtained.
Now, by letting the width of the histogram bins ap-
proach zero (Dt 0), i.e., n , the elements in the
MTM matrix (Eq. 7) become denite integrals computed Empty bins
over one cycle (0 h 2p); for instance, for the ele-
ment in row 1, column 1 we get the following: This second part deals with the method applied to ex-
X Z 2p clude empty bins in the silent period, which, if not
n
1 Xn
n!1 n
excluded, would tend to lower the amplitude of the tted
sin2 hi sin2 hi Dh
! sin2 hi dh;
1
Dh 1
2p 0 sine (Hulliger et al. 1977).
9 After the sine had been tted, the code checked
whether the amplitude was greater than the baseline
and expression Eq. 7 becomes (=negative swing). The part of the sine with negative
2 R 2 R R 3 values was then excluded in the integral of the sine, and
sin hdh sinhcoshdh sinhdh spikes occurring in the silent period were also ex-
T n 6R R 2
R 7 cluded. So, MTM and MTY were recalculated and solved
M M 4 sinhcoshdh cos hdh coshdh 5
2p R R as before.
sinhdh coshdh 2p
Finally, MDR, AMP, and PHASE (see Methods)
10 were computed from A, B, and C:
Therefore, excluding the common factor n (n ), the
nine elements in the matrix can be exactly solved for h, MDR C;  
resulting in simple constant (numeric) values. p A 12
2 2
AMP A B ; PHASE Atan
As for the MTY vector (Eq. 8), its elements now B
contain an innite number of terms, corresponding to
the innite number of bins, but only m terms will be
dierent from zero, m being the total number of spikes
collected into the histogram. In fact, because the bin Validation
width approaches zero (Dt 0), any bin will contain
either one or zero spike. It is therefore convenient to Comparisons of the classical 24-bin method and the
maintain the discrete form of the MTY matrix and only present algorithm were performed by tting a simulated
sum the nonzero terms: (articially generated) spike train with the following
2P m 3 characteristics: average discharge rate=30 Hz, ampli-
sinhj tude of modulation=10 Hz, frequency of modula-
6 1 7
6 m 7 tion=1 Hz, Phase=0. The better performance of the
nf 6 P 7
T
M Y 6 coshj 7 11 present algorithm is apparent from Table 1, which
6
nc 6 1 7;
7 shows the results of the tting in two examples with
4 P m 5
1 dierent sample sizes.
1

Table 1 Comparisons of the estimates produced by the tting of an articially generated spike train with sinusoidal modulation of the
ring frequency (average discharge rate: 30Hz, amplitude of the sinusoidal modulation: 10 Hz, frequency of the sinusoidal modulation:
1 Hz)

Data sample One cycle (31 spikes) Ten cycles (310 spikes)

Present algorithm Classical 24 bins Present algorithm Classical 24 bins

Mean discharge rate (Hz) 31,000 30,000 31,000 30,900

Amplitude of modulation (Hz) 11,000 12,460 11,000 12,114
Phase (Deg) 0,005
7,943 0,006
2,173

The present algorithm is compared with the classical technique of spikes (2nd and 3rd columns) and 310 spikes (4th and 5th columns).
tting a 24-bin spike density histogram (see text). Two cases are The phase parameter is the phase lead of the tting sine wave with
presented in which data samples of dierent sizes have been used: 31 respect to the modulating sine wave in the simulated data set.

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