Download Primary- and Secondary-Like Jaw-Muscle Spindle Afferents Have

Primary- and Secondary-Like Jaw-Muscle Spindle Afferents Have
Characteristic Topographic Distributions
DEAN DESSEM, REVERS DONGA, AND PIFU LUO
Department of Physiology, University of Maryland Dental School, Baltimore, Maryland 21201-1586
INTRODUCTION
Different types of afferent termination within the mammalian muscle spindle are known to be correlated with different
physiological responses (Boyd and Ward 1975). Much less
is known, however, about what correlations may exist between the types of peripheral afferent termination and the
axonal trajectory and terminations of these neurons within
the CNS. In the spinal cord, studies using intracellular staining (Brown and Fyffe 1978, 1981; Burke et al. 1979; Conradi
et al. 1983; Fyffe and Light 1984; Ishizuka et al. 1979;
Keirstead and Rose 1988a,b; Rose and Keirstead 1986) have
shed light on the central distribution of primary spindle afferents. Additional information about the distribution of primary afferents in the spinal cord comes from studies in
which the extracellular field potentials generated by single
Ia afferents have been mapped (Munson and Sypert 1979).
The relationship between primary spindle afferents and spinal motoneurons has been examined using both electrophysiological and intracellular staining techniques (Brown and
Fyffe 1981; Burke et al. 1979; Mendell and Henneman 1971;
Watt et al. 1976). Much less is known about the central
distribution of secondary muscle spindle afferents. Only one
small study (Fyffe 1979) subsequently discussed by Brown
(1981) has intracellularly labeled secondary muscle spindle
afferents. Additional information on the distribution of secondary spindle afferents in the spinal cord comes from extracellular electrophysiological studies (Edgley and Jankowska
1987) and studies in which the synaptic inputs from secondary spindle afferents to motoneurons were examined (Kirkwood and Sears 1982; Munson et al. 1982; Stauffer et al.
1976). In contrast to the spinal cord, there is no convincing
information demonstrating the central distribution of different jaw-muscle spindle afferent types. Studies in which retrograde neuroanatomic tracers have been injected into the muscles of mastication provide a macroscopic map of the distribution of jaw-muscle spindle afferents (Capra and Wax
1989; Gottlieb et al. 1984; Nomura and Mizuno 1985; Raappana and Arvidsson 1993; Rokx et al. 1985) but provide no
physiological information. Intracellular labeling studies in
the rat (Appenteng et al. 1985; Dessem and Taylor 1989;
Lingenhöhl and Friauf 1991; Luo and Dessem 1995; Luo et
al. 1991, 1995a) provide more detailed information about
the distribution of single jaw-muscle spindle afferents, but
none of these studies have characterized the responses of
these afferents in enough detail to distinguish between muscle spindle afferent types. Additional information concerning
the central distribution of jaw-elevator muscle spindle afferents can be gained from electrophysiological mapping studies in which jaw-elevator muscle spindles are characterized
and their central distribution is inferred from unitary spiketriggered averaging (Appenteng et al. 1978, 1989; Taylor
et al. 1993a). Although these studies may provide a gross
estimation of the afferent distribution, they cannot provide
0022-3077/97 $5.00 Copyright q 1997 The American Physiological Society
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Dessem, Dean, Revers Donga, and Pifu Luo. Primary- and secondary-like jaw-muscle spindle afferents have characteristic topographic distributions. J. Neurophysiol. 77: 2925–2944, 1997. Single jaw-muscle spindle afferent axons were characterized physiologically and intracellularly stained to determine whether particular
physiological types of spindle afferent show distinctive morphologies. Microelectrodes filled with either horseradish peroxidase
(HRP) or biotinamide (Neurobiotin) were advanced into the mesencephalic trigeminal nucleus (Vme) in anesthetized rats. Intracellular recordings then were characterized by their response: to palpation of the jaw muscles; when pressure was applied to the teeth
and during passive ramp and hold and sinusoidal jaw movement.
Seventy-one afferents were characterized physiologically and injected with HRP; an additional 61 afferents were typed and injected
with biotinamide. The response of 43 stained neurons was recorded
in the presence of suxamethonium. The major projection areas of
these afferents were the: trigeminal motor nucleus (Vmo); region
dorsal to Vmo; reticular formation, spinal trigeminal nucleus, superior cerebellar peduncle and Vme. One afferent type was modulated
strongly during stretching of the jaw-elevator muscles. Based on
their high sensitivity during stretching of the jaw muscles and/or
their silencing during the release phase of muscle stretch, these
afferents were classified as primary-like spindle afferents. These
afferents projected most strongly to Vmo. A second type of afferent
was modulated only modestly during stretching of the jaw-elevator
muscles. These tonic afferents were classified as secondary-like
spindle afferents because of their low dynamic sensitivity during
ramp muscle stretch and their continued discharge during the release phase of muscle stretch. Secondary-like afferents projected
most strongly to the region dorsal to Vmo. Boutons (n Å 3,834)
from 11 afferents were studied in detail. Secondary-like afferents
had statistically larger boutons within Vmo. In both secondaryand primary-like spindle afferents, only a small number of boutons
were associated closely with the somata and proximal dendrites of
trigeminal motoneurons. In these cases, however, two to five boutons appeared to contact individual motoneurons, implying multiple monosynaptic inputs to a selective subset of jaw-elevator motoneurons. Some ‘‘giant’’ boutons were present dorsal to Vmo and
in Vme. These results demonstrate that dynamically sensitive and
nondynamically sensitive jaw-elevator muscle spindle afferents
project preferentially to different regions. Primary-like spindle afferents are capable of providing feedback related to the dynamic
phases of muscle stretch and project most heavily to Vmo. Secondary-like spindle afferents can transmit a feedback signal associated
with muscle length and project most strongly to the supratrigeminal
region. Both types of afferent have projections caudal to Vmo that
may serve longer latency jaw-muscle stretch reflexes and/or the
projection of proprioceptive information to the thalamus and cerebellum.
2926
D. DESSEM, R. DONGA, AND P. LUO
METHODS
Male Wistar rats (300–350g) were anesthetized initially with
pentobarbital sodium (20 mg/kg ip) supplemented with additional
injections (8 mg/kg iv) every hour to maintain adequate anesthesia. To reduce secretions in the airways and trachea, atropine (1
mg/kg sc) was administered after the induction of anesthesia. The
femoral vein and artery then were cannulated, and systemic arterial
blood pressure was monitored for the duration of the experiment.
Body temperature was maintained at 377C by means of a thermostatically controlled heating pad. The animals then were placed in
a stereotaxic frame and ventilated (2 cm3 , rate: 100/min) for the
duration of the experiment with humidified air while maintaining
a positive end expiratory pressure of 1 cm H2O to prevent lung
collapse. Animals used for intracellular HRP labeling were paralyzed with gallamine triethiodide (20 mg/kg). Anesthesia in these
animals was maintained during paralysis by regular supplements
of anesthetic that were determined to be sufficient to prevent a
limb-withdrawal reflex before paralyzing the animal. The plane of
anesthesia also was checked periodically by allowing the paralysis
induced by gallamine to wear off. Rats used for intracellular biotinamide staining were not administered gallamine. All animal procedures were reviewed and approved by the Institutional Animal
Care and Use Committee of the University of Maryland before the
onset of experiments. To gain access to the mesencephalic trigeminal nucleus, the bone, dura, and pia mater overlying the dorsal and
posterior surfaces of the cerebellum were removed, and warmed
mineral oil was applied to the surface of the brain stem and cerebellar cortex. Before electrophysiological recording, chlorpromazine
(150 mg/kg) was administered to suppress background fusimotor
activity (Cody et al. 1972), and a pneumothorax was performed
to enhance intracellular recording stability. Microelectrodes then
were advanced via a stepping motor rostroventrally at an angle of
307 to the vertical through the posterior portion of the cerebellum
into the tract of the mesencephalic nucleus just dorsal and medial
to the trigeminal motor nucleus (P 0.0–1.0, L 1.1–2.0, depth 5–
6.5 mm). Jaw displacements of 2.5 mm then were produced via
an electromagnetic vibrator attached to the jaw at the diastema and
used as the search stimulus for stretch-sensitive afferents. Intraaxonal recordings made from afferents in this region of the brain
stem whose firing frequency increased during both jaw opening
(muscle stretch) and gentle probing of jaw muscles and that failed
to respond when pressure was applied to the teeth were tentatively
characterized as jaw-elevator muscle spindles. The intracellular
response of each of these stretch-sensitive afferents then was recorded on tape during 10 ramp and hold and 10 sinusoidal stretches
(0.6 Hz) for off-line analysis.
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Intracellular HRP labeling
Microelectrodes used for HRP labeling were fabricated from 1.0
mm OD borosilicate glass and initially filled with Tris HCl buffer
(pH 8.6). After the electrode tips had filled, 20–30% HRP (Sigma
VI) was placed into the electrode and allowed to diffuse into the
tips for 24 h. These electrodes were bevelled just before recording
to impedances of 80–120 MV.
In cases where stable intra-axonal penetrations with resting
membrane potentials more negative than 040 mV were maintained
from well-characterized spindle afferents, depolarizing current
pulses (40 ms, 100 Hz, 0.5–5 nA) were applied through the microelectrode for 6–18 min, resulting in total injection times of 14–
43 nA minutes.
Current injections were stopped every 30 s and discontinued if
the membrane potential became more positive than 030 mV. Two
to 3 h after the injection of HRP, heparin (500 units iv) was
administered, followed after 15 min by an overdose of pentobarbital sodium. The animals then were perfused through the ascending
aorta with 700 ml of 0.9% saline solution (387C) containing 500
units of heparin and 1 ml of 2% xylocaine. This was followed by
infusion of two liters of 1.25% glutaraldehyde and 1% paraformaldehyde in a phosphate buffer (pH 7.4) during 30 min. Finally, 1
l of 10% phosphate-buffered sucrose solution (pH 7.4) was infused
for 30 min. The brain then was removed and stored overnight at 47C
in a 10% phosphate-buffered sucrose solution (pH 7.4). Sagittal
sections (100 mm thickness) were cut on a vibratome and processed
for the demonstration of HRP according to the method of Metz et
al. (1989). These sections then were mounted on chrom-alum
slides, air-dried, and cover-slipped.
Intracellular biotinamide labeling
Electrodes for biotinamide labeling were made from 1.0 mm
OD borosilicate glass and filled with 3% biotinamide (Neurobiotin,
Vector Laboratories) dissolved in 0.25 M KCl and 0.5 M TrisHCl buffer (pH 7.6). Before use, these electrodes were beveled
to impedances of 60–80 MV. After physiological characterization
of a stable intracellular jaw-muscle spindle afferent impalement,
biotinamide was injected into the axon using DC currents ranging
from 1 to 14 nA for 0.75–15 min for a total of 5–105 nA minutes.
After a survival time of 165–270 min, the animals were killed
with sodium pentobarbital and perfused through the ascending
aorta with a solution composed of 0.8% NaCl, 0.025% KCl, 0.05%
NaHCO3 , 0.05%NaNO2 in phosphate-buffered saline (pH 7.4).
A fixative solution consisting of 4% paraformaldehyde and 0.5%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) then was circulated followed by a 5% sucrose solution in phosphate-buffered
saline (pH 7.4). The brain then was removed and placed in 30%
sucrose in phosphate-buffered saline (pH 7.4) at 47C for 12 h.
Sixty- to 70-mm serial sections of the brain stem were then cut on
a vibratome in either the sagittal or horizontal orientation. Sections
then were washed in 0.1 M phosphate-buffered saline (PBS; pH
7.4) and placed in 1% Triton X-100 in 0.1 M PBS at 257C for 30
min. The tissue was incubated then for 1–2 h in 1–2% normal
goat serum (Vector S-100) and 1% Triton X-100. The sections
were then placed in avidin biotin complex (1:50 Elite Vectastain;
Vector) mixed with 1% Triton X-100 and 1% normal goat serum
in 0.01 M PBS for 8–12 h at 47C. The tissue then was first rinsed
in 0.01 M PBS followed by 0.05 M tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.6) before being reacted using
nickel-diaminobenzidine (DAB)/H2O2 for 10–15 min. The reaction solution consisted of 0.05 M Tris-HCl buffer (pH 7.6),
0.025% nickel ammonium sulphate, 0.02% DAB, and 0.00018–
0.00024% H2O2 . After these procedures the sections were washed
in PBS, air-dried, dehydrated, defatted in xylene, mounted onto
slides coated with chrom-alum, and cover-slipped.
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detailed information about the branching of the axon, the
size and number of boutons, and the relationship between
the terminations of the afferents and the distribution of trigeminal motoneurons.
In the cat, Shigenaga and co-workers (1988, 1990), have
attempted to classify primary and secondary jaw-muscle
spindle afferents based on their response during a 1-s step
muscle stretch. Using this unconventional criterion, these
authors concluded that there was no correlation between the
afferent response and central trajectory of jaw-muscle spindle afferents. The experiments described in this paper were
carried out to examine the relationship between jaw-muscle
spindle afferents identified by classical physiological methods with their central distribution. Jaw-muscle spindle afferents were impaled intracellularly, physiologically classified
using controlled stretching of the jaw muscles, and intracellularly stained with either horseradish peroxidase (HRP) or
biotinamide to determine the central distribution of jaw-muscle spindle afferent types.
JAW-MUSCLE SPINDLE AFFERENTS
Physiological data analysis
The intracellular afferent responses of each well-stained neuron
and the jaw displacement signal were replayed from tape and digitized off-line (80 kHz; Cambridge Electronic Design, 1401plus).
Instantaneous and peak frequencies were computed with spike
analysis software (Spike2, Cambridge Electronic Design). Dynamic indices were calculated from the averaged response of 10
ramp stretches by taking the difference between the maximum
instantaneous frequency during the ramp stretch and 0.5 s later
(Crowe and Matthews 1964).
Morphological analysis
shapefactor Å
4pArea
Perimeter 2
Parametric and nonparametric statistical tests (SYSTAT) were
used to determine differences in the central tendency of bouton
area, perimeter, and shape within and outside the trigeminal motor
nucleus.
RESULTS
HRP staining
Seventy-one afferents were intracellularly impaled, physiologically characterized and injected with HRP in 37 rats.
Following histochemical procedures for the visualization of
HRP, 31 afferents in 16 rats were considered to be well
stained because their processes could be followed peripherally into the motor root of the trigeminal nerve, rostrally
into the tract of the mesencephalic trigeminal nerve and
caudally into the tract of Probst.
Biotinamide staining
Sixty-one afferents, which initially were identified as jawmuscle spindle afferents on the basis of their afferent firing
behavior, were impaled intracellularly, physiologically characterized, and injected with biotinamide in 23 rats. After
histochemical processing for the visualization of biotinamide, 41 afferents in 17 rats were judged to be well stained
because their processes could be visualized in the motor root
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of the trigeminal nerve, the tract of mesencephalic trigeminal
nucleus (Vme) and the tract of Probst.
Physiological characteristics
All of the afferent neurons labeled in this study responded
with an increased firing when the jaw-elevator muscles were
palpated but failed to respond when pressure was applied to
the teeth and gingiva. Two basic types of afferent response
could be distinguished. One type was modulated strongly
during stretching of the jaw-closing muscles. This kind of
response exhibited a high dynamic sensitivity and high peak
frequency during stretching of the jaw-elevator muscles and
either a large reduction or a silencing of the afferent response
during the release phase of muscle stretch (Fig. 1, A and
B). The second type of afferent response was modulated
only weakly during stretching of the jaw-elevator muscles.
This variety of response showed little dynamic sensitivity,
a low peak frequency during muscle stretch, and responded
continuously during all phases of ramp and hold and sinusoidal stretching (Fig. 1, C and D).
General morphology of jaw-muscle spindle afferents
All of the axons labeled in this study entered the brain
stem ventrally in the motor root of the trigeminal nerve and
coursed dorsomedially toward the trigeminal motor nucleus
(Vmo). Either within or slightly dorsal to the trigeminal
motor nucleus these axons bifurcated with one branch coursing rostrally into the tract of the mesencephalic trigeminal
nucleus and the other turning caudally to enter the tract of
Probst. In most cases, the rostral process could be traced
to a HRP- or biotinamide-stained cell body located in the
mesencephalic trigeminal nucleus (Vme). All of these intracellularly stained somata exhibited a pseudounipolar morphology (Figs. 3, 5, 11, A and B, and 13) except one, which
possessed a small dendrite (Fig. 11C). In the caudal direction, each afferent possessed a prominent process that could
be followed in the tract of Probst (Probst 1899; Corbin 1942)
to the region between the facial motor nucleus and the inferior olivary nucleus (Fig. 13). All well-stained afferents
possessed axon collaterals that emanated from the main axon
into Vmo and the region dorsal to Vmo. These axons also
possessed collaterals that emerged from the tract of Probst
at the level of the facial motor nucleus and coursed ventrolaterally into the reticular formation. In addition to these major
projections, a few afferents had collaterals with en passant
and terminal swellings that coursed into the caudal part of
Vme. A few intracellularly stained afferents also had axon
collaterals that could be followed lateral and dorsolateral to
Vmo. At more caudal levels, some afferents possessed axon
collaterals that emerged from the tract of Probst at the level
of the facial motor nucleus and coursed laterally through the
reticular formation into the dorsomedial portion of the spinal
trigeminal nucleus.
Morphological characteristics of dynamically sensitive
jaw-muscle spindle afferents
The afferent response of a strongly modulated afferent
(R161) labeled with HRP is shown in Fig. 2. This afferent
was tested 15 min after the infusion of suxamethonium (22.8
mg/kg ip) and showed a high dynamic sensitivity (dynamic
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Photomicrographs and camera lucida drawings of the intracellularly stained neurons were made before counterstaining the unstained tissue. Axonal morphology was reconstructed using a software assisted three-dimensional computer reconstruction system
(Capowski and Sedivec 1981). The locations of swellings on fine
axon collaterals, comparable with structures demonstrated in previous studies to contain synapses (Luo et al. 1995a,b), were examined at 11,000 magnification. After reconstruction and photography, the coverslips were removed from the slides, and the tissue
was counterstained with either cresyl violet or neutral red to accurately determine the location of the intracellularly stained axons
and boutons in relation to cell bodies of various brain stem nuclei.
The morphology of boutons stained with HRP was analyzed using
a manual image analysis computer (Zeiss Videoplan). Boutons
were observed at 11,000 magnification, and their outlines traced
to determine bouton perimeter and area. Digital images of boutons
stained with biotinamide initially were produced and stored on
computer disk using a computer camera (Electrim, EDC 1000U)
attached to an Olympus (BH-2) microscope. Bouton perimeter and
area then were measured from these stored images using image
measurement software (Jandel, Sigmascan). A shape factor also
was calculated for these boutons using the formula
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D. DESSEM, R. DONGA, AND P. LUO
FIG . 1. Afferent responses of biotinamide-stained, jaw-muscle spindle
afferents. A: primary-like afferent (PAR8r) during ramp displacement of
jaw. B: afferent PAR8r during sinusoidal jaw movement. C: secondarylike afferent PAR21r during ramp and hold jaw movement. D: afferent
PAR21r during sinusoidal jaw displacement. Arrow, direction of jaw opening. Time bar is 1 s.
index Å 61 impulses/s) and high peak frequency (185 impulses/s) during jaw opening and a large reduction in firing
during the release phase of muscle stretch. This afferent
responded to palpation in the region of the posterior portion
of the masseter muscle and failed to respond when pressure
was applied to the teeth and surrounding gingiva. The pseudounipolar cell body of this afferent was located in the caudal portion of Vme between the locus coeruleus and the
medial parabrachial nucleus. The largest number of axon
collaterals, en passant and terminal boutons for R161 were
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Morphological characteristics of nondynamically sensitive
jaw-muscle spindle afferents
The behavior of a weakly modulated afferent is shown in
Fig. 4. The response of this neuron (R170) increased during
palpation of the region overlying the posterior masseter muscle but failed to respond to probing of the teeth and gingiva.
This afferent showed a low dynamic sensitivity (dynamic
index Å 10.0 impulses/s) and low peak frequency (62.9
impulses/s) during stretching of the jaw closing muscles
and showed only a modest reduction in firing during the
release phase of muscle stretch. The pseudounipolar cell
body of this afferent was located in the rostral part of the
mesencephalic trigeminal nucleus. Figures 5 and 14C show
the location of en passant and terminal boutons from this
afferent in relation to the Nissl-stained boundaries of the
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located within Vmo (Figs. 3 and 14A). Several of the axon
collaterals that coursed into Vmo traversed the nucleus and
could be followed into the region lateral to Vmo. This dynamically sensitive afferent also possessed collaterals that
coursed into the region dorsal to Vmo, the reticular formation
at the level of the facial motor nucleus, and the superior
cerebellar peduncle. The peak frequencies of dynamically
sensitive afferents labeled using the biotinamide protocol
ranged from 290 to 320 impulses/s and exhibited dynamic
indices between 152 and 178.5 impulses/s with a mean of
168.2 impulses/s (Table 1). The basic morphology of dynamically sensitive jaw-muscle spindle afferents labeled
with biotinamide was equivalent to that of the dynamically
sensitive spindle afferents stained with HRP. All dynamically sensitive, biotinamide-stained afferents possessed a
process in the motor root of the trigeminal nerve, a process
that extended rostrally into the tract of Vme and ended in
a pseudounipolar-shaped soma and a thinner process that
projected caudally in the tract of Probst. The greatest number
of boutons and axon collaterals of dynamically sensitive
afferents were located in the trigeminal motor nucleus (Table
2). More caudally, dynamically sensitive jaw-muscle spindle afferents possessed a process in the tract of Probst that
extended beyond the caudal extent of the facial motor nucleus. Additional axon collaterals emerged from this process
and coursed ventrolaterally through the reticular formation
to reach the dorsomedial portion of the spinal trigeminal
nucleus. No projections to the cerebellum were observed
emanating from dynamically sensitive biotinamide-stained
afferents.
The largest number of axon collaterals and boutons of
dynamically sensitive afferents stained in this study either
with HRP or biotinamide were located within the confines
of the trigeminal motor nucleus. These boutons, however,
only were found occasionally in close association to trigeminal motoneuron somata or proximal dendrites. All strongly
modulated afferents also possessed axon collaterals and boutons throughout the region dorsal to the trigeminal motor
nucleus and collaterals, which emerged from the tract of
Probst and coursed into the reticular formation. In a few
instances, dynamically sensitive afferents had axon collaterals with boutons that were overlying or adjacent to the somata of caudal Vme neurons. One dynamically sensitive
afferent labeled with HRP possessed a collateral which could
be traced into the superior cerebellar peduncle.
JAW-MUSCLE SPINDLE AFFERENTS
2929
trigeminal motor nucleus. As is apparent in these figures,
the axon collaterals and boutons of R170 were distributed
preferentially to the region overlying the trigeminal motor
nucleus. Additional but smaller projections to Vmo, Vme,
and the reticular formation were present.
A more restricted distribution of axon collaterals and boutons in the region dorsal to the trigeminal motor nucleus can
be seen in the afferent illustrated in Fig. 6. This neuron
(R186) responded to palpation of the ipsilateral temporalis
muscle and failed to respond to palpation of the teeth and
gingiva. This afferent showed little dynamic sensitivity (dynamic index Å 4.5 impulses/s), a low peak frequency (78.4
impulses/s), and failed to silence during the release phase
of muscle stretch (Fig. 7). The pseudounipolar cell body of
this weakly modulated afferent was located within a cluster
of Vme neurons in the rostral portion of the mesencephalic
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trigeminal nucleus. Note that the axon collaterals and boutons of this afferent, which are dorsal to the trigeminal motor
nucleus, are located primarily above the middle portion of
Vmo (Figs. 6 and 14D) in contrast to the more caudal distribution of boutons overlying Vmo in R170 (Figs. 5 and 14C).
Within Vmo itself, the distribution of axon collaterals was
sparser than in the region dorsal to Vmo. A few axon collaterals were also found in Vme, and one collateral projected
into the ipsilateral superior cerebellar peduncle. Axon collaterals emerging from the tract of Probst that coursed into the
reticular formation also were present at the level of the facial
motor nucleus.
The afferent illustrated in Fig. 8 provides an even more
extreme example of a preferential distribution of axon collaterals and boutons dorsal to Vmo. This neuron (R160) exhibited an increased firing during jaw opening (Fig. 9) and
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FIG . 2. Afferent response of a primary-like jaw-muscle spindle
afferent (R161). A: intra-axonal response during ramp and hold
displacement of jaw (top); bottom: jaw displacement signal. B:
instantaneous frequency of afferent during ramp and hold jaw
displacement. C: instantaneous frequency of afferent during sinusoidal displacement of jaw. Arrow, direction of jaw opening. Time
bar in A is 0.1 s, in B and C, it is 1 s.
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D. DESSEM, R. DONGA, AND P. LUO
when the posterior portion of the masseter muscle was palpated but failed to respond when the teeth and gingiva were
probed. The firing of this afferent did not silence during the
release phase of muscle stretch. Even though the response
of this afferent was recorded 20 min after the infusion of
suxamethonium (28.6 mg/kg ip), the afferent showed very
TABLE
1.
little dynamic sensitivity (dynamic index Å 17.8 impulses/
s) and followed all phases of sinusoidal jaw movement. The
HRP-stained, pseudounipolar cell body of R160 was located
in the rostral part of the mesencephalic trigeminal nucleus.
As seen in Fig. 14E, the majority of this afferent’s en passant
and terminal boutons were located in a region dorsal to the
Bouton measurements
Animal
Labeling
n
Dynamic
Index
R161
PAR13r
PAR14l
PAR8r
R170
R186
R160
PAR19r
PAR20r
PAR21r
R174
HRP
Biotin.
Biotin.
Biotin.
HRP
HRP
HRP
Biotin.
Biotin.
Biotin.
HRP
162
264
294
160
513
627
451
116
456
155
636
61.0
152.0
174.0
178.5
10.0
4.5
17.8
19.0
8.4
4.0
N/A
Perimeter, mm
11.5
9.0
8.5
9.7
12.0
12.0
11.7
6.2
9.5
9.6
11.8
{
{
{
{
{
{
{
{
{
{
{
16.2
2.4
2.2
2.2
4.0
4.5
4.1
2.4
2.9
2.6
4.3
(3.9–21.7)
(3.8–16.8)
(3.5–16.4)
(4.8–19.3)
(2.7–28.0)
(2.0–32.9)
(4.3–41.4)
(1.9–13.8)
(3.4–18.9)
(3.7–18.0)
(3.0–38.1)
Area, mm
8.3
5.1
4.6
5.6
9.3
6.0
8.2
2.6
5.6
5.5
7.9
{
{
{
{
{
{
{
{
{
{
{
4.7
2.4
2.2
2.3
6.1
2.2
5.5
2.2
3.0
2.6
5.7
(0.9–23.1)
(1.0–12.0)
(0.9–13.5)
(1.5–15.9)
(0.5–35.3)
(0.2–37.6)
(1.2–51.4)
(0.3–12.7)
(0.7–17.2)
(0.9–14.4)
(0.6–53.5)
Area and perimeter values are means { SD with range in parentheses. HRP, horseradish peroxidase; Biotin, biotinamide; N/A, not applicable.
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FIG . 3. Computer-assisted reconstruction of primary-like jaw elevator muscle spindle afferent R161 in
sagittal plane. Dotted line, outline of trigeminal motor
nucleus. Dorsal is toward page top, rostral toward right.
TrPb , tract of Probst; TrVme , tract of mesencephalic trigeminal nucleus; Cb, cerebellar collateral; PP, peripheral process; S, soma. Scale bar: 200 mm.
JAW-MUSCLE SPINDLE AFFERENTS
TABLE
2.
2931
Distribution of boutons in the region of the trigeminal motor nucleus
Number of Boutons
Animal
Dynamic afferent type
R161
PAR121
PAR13r
PAR14l
PAR8r
Labeling
HRP
Biotin.
Biotin.
Biotin.
Biotin.
Subtotal
HRP
HRP
HRP
Biotin.
Biotin.
Biotin.
Subtotal
Unclassified afferent type
R174
HRP
Outside Vmo
Total Boutons
123
405
198
326
269
39
225
160
129
131
162
630
358
455
400
1,321
684
2,005
197
123
83
93
257
141
316
504
368
203
302
241
513
627
451
296
559
382
894
1,934
2,828
306
330
636
Percentage
Inside Vmo
76
64
55
72
67
mean Å 67
38
20
18
31
46
37
mean Å 32
48
Vmo, trigeminal motor nucleus. For other abbreviations, see Table 1.
most rostral portion of Vmo. Some axon collaterals also
projected to Vmo, Vme, the dorsomedial portion of the principal sensory trigeminal nucleus (Vpdm), and the reticular
formation.
Nondynamically sensitive jaw-muscle spindle afferents labeled with biotinamide had peak frequencies between 52
and 105 impulses/s and dynamic indices that ranged from
4 to 19 impulses/s with a mean of 10.5 impulses/s (Table
1). The general morphology of nondynamically sensitive
afferents stained with biotinamide corresponded to the morphology of nondynamically sensitive afferents labeled with
HRP. All of the nondynamic biotinamide-stained afferents
had a single process that could be followed laterally to exit
the brain stem in the motor root of the trigeminal nerve.
These afferents also possessed single processes that coursed
rostrally in the tract of the mesencephalic nucleus of the
trigeminal nerve and terminated in a pseudounipolar cell
body. A thinner process bifurcated at the level of the trigeminal motor nucleus and traveled caudally in the tract of Probst.
Axon collaterals from this caudally directed process coursed
ventrolaterally through the reticular formation to reach the
dorsomedial portion of the spinal trigeminal nucleus. The
largest number of axon collaterals from these neurons were
located in the region dorsal to the trigeminal motor nucleus.
No axon collaterals of biotinamide-stained nondynamically
sensitive afferents were found in the cerebellar peduncle.
The most distinctive morphological feature of nondynamically sensitive afferents, stained either with HRP or biotinamide, was that the distribution of axon collaterals and
boutons were densest in the region dorsal to the trigeminal
motor nucleus. The local distribution and density of this
projection within the region dorsal to Vmo, however, varied
among nondynamically sensitive afferents. In some neurons
(Figs. 5 and 14C), axon collaterals and boutons tended to
be located dorsal to the middle and caudal portions of Vmo,
whereas in other instances, the distribution was located more
rostrally (Figs. 8 and 14E). Although swellings on axon
collaterals in the area dorsal to Vmo were generally not
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closely associated with Nissl-stained neurons, a case in
which they were is shown in Fig. 10A. Although the largest
number of collaterals and boutons of nondynamically sensitive afferents were located dorsal to Vmo, all of these afferents also possessed some axon collaterals and boutons within
Vmo. In most instances, these terminal and en passant swellings were not closely associated with trigeminal motoneuron
somata. Figure 10 (E and F), however, shows examples in
which boutons were adjacent to or overlying Nissl-stained
trigeminal motoneurons. In these instances of close apposition, multiple swellings often were found approximating the
somata and proximal dendrites of trigeminal motoneurons.
Several nondynamically sensitive afferents also possessed
axon collaterals with boutons closely opposed to or overlying
other Vme neurons (Fig. 11F). More caudally at the level
of the facial motor nucleus, axon collaterals emerged from
the tract of Probst of all nondynamically sensitive afferents
and coursed ventrolaterally into the reticular formation and
dorsomedial portion of the spinal trigeminal nucleus. An
additional projection into the ipsilateral cerebellar peduncle
was observed in two nondynamically sensitive jaw-muscle
spindle afferents.
Morphological characteristics of an unclassified jawmuscle spindle afferent
The response of an HRP-stained afferent that was modulated phasically in a different manner is shown in Fig. 12.
This afferent (R174) responded with an increase in firing
during the initial portion of the jaw opening phase followed
by a silencing when the jaw was held open. During jaw
closing, the afferent responded and continued to discharge
when the jaw was held closed. Manual manipulation of the
mandible revealed that this afferent also could be activated
by anteroposterior movement of the mandible and palpation
in the region of the posterior temporalis muscle. This afferent
failed to respond, however, to palpation of the teeth or gingiva. Afferent R174 exhibited a well-stained central axon
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Nondynamic afferent type
R170
R186
R160
PAR19r
PAR20r
PAR21r
Inside Vmo
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D. DESSEM, R. DONGA, AND P. LUO
located in the tract of Vme that could be traced to a pseudounipolar cell body located in the caudal portion of the mesencephalic trigeminal nucleus underlying the anterior portion
of the cerebellum. This cell body was juxtaposed to a cluster
of several other unlabeled Vme somata. The largest number
of axon collaterals of afferent R174 were located within
Vmo (Fig. 13). A smaller number of axon collaterals emanated from the tract of Vme into the region dorsal to Vmo
with a few collaterals reaching the dorsomedial portion of
the trigeminal principal sensory nucleus. More caudally, additional collaterals emanated from the tract of Probst at the
level of the facial motor nucleus and coursed ventrolaterally
into the reticular formation with a small number of collaterals reaching the dorsomedial portion of the spinal trigeminal
nucleus.
Morphology and distribution of boutons
The morphology of 2,389 HRP-stained en passant and
terminal swellings on axon collaterals presumed to be synap-
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tic boutons were examined in detail for five afferents in five
animals (Table 1). The total number of boutons located on
these HRP-stained afferents ranged from 162 to 636 (Table
2). One hundred sixty-two boutons were found on the HRPstained, dynamically sensitive afferent. The average number
of boutons on nondynamically sensitive afferents stained
with HRP was 530. The perimeter of these boutons ranged
in size from 2.0 to 41.4 mm. The mean perimeter of boutons
on the dynamically sensitive afferent was 11.5 mm, whereas
that for the nondynamically sensitive afferents was 11.9 mm.
The average area of the boutons on dynamically sensitive
afferents was 8.3 mm2 , whereas that for the boutons on nondynamically sensitive afferents was 8.7 mm2 . In three neurons, the presence of a few very large boutons, which ranged
from 16 1 12 to 30 1 10 mm, were observed. These ‘‘giant’’
boutons were located in the region dorsal to Vmo and within
the caudal portion of Vme.
The morphology of 1,445 biotinamide-stained en passant
and terminal boutons was measured from six afferents in an
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FIG . 4. Afferent response of secondary-like jaw-muscle spindle
afferent . A: intra-axonal response generated during ramp displacement of jaw. B: instantaneous frequency of afferent during ramp
and hold displacement of jaw. C: instantaneous frequency of afferent during sinusoidal displacement of jaw. Arrow, direction of jaw
opening. Time bar in A is 0.1 s, in B and C, time bars are 1 s.
JAW-MUSCLE SPINDLE AFFERENTS
2933
FIG . 5. Sagittal view of a computer-assisted reconstruction of masseter secondary-like muscle spindle afferent. Dotted
line, location of trigeminal motor nucleus.
Dorsal is toward page’s top, rostral is toward page right. S, soma. Scale bar: 200
mm.
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To examine the distribution of boutons in more detail, the
location of afferent boutons in relation to the trigeminal
motor nucleus was plotted for five HRP-stained afferents
(Fig. 14). Note that for both dynamically sensitive and nondynamically sensitive afferents, the majority of those boutons that are located outside Vmo are located dorsal to Vmo.
Within the trigeminal motor nucleus, boutons were distributed to relatively restricted regions for both dynamically
sensitive and nondynamically sensitive afferents.
The size of boutons located inside the trigeminal motor
nucleus was compared with boutons located in regions surrounding Vmo for individual HRP- or biotinamide-stained
afferents (Table 3). The boutons of all six of the nondynamically sensitive afferents examined were significantly larger
within Vmo than outside it. In only one of four cases
(PAR8r) was a significant difference in bouton size inside
and outside of Vmo found for dynamically sensitive afferents.
DISCUSSION
Two morphological features demonstrate that the intracellularly labeled cells presented in this study are first-order
neurons. First of all, their peripheral processes were observed
exiting the brain stem in the motor root of the trigeminal
nerve. Second, the intracellularly labeled somata of these
afferents exhibited the pseudounipolar morphology, which,
within the brain stem, is unique to Vme neurons and corresponds to that reported in studies where retrograde neuroanatomic tracers have been injected into the muscles of mastication (Capra and Wax 1989; Gottlieb et al. 1984; Nomura
and Mizuno 1985; Rokx et al. 1985).
Physiological response of the afferents
Several features evident in the physiological responses of
the neurons stained in this study indicate that they are jaw-
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additional six animals. Every axonal swelling in the trigeminal motor nucleus and surrounding regions was examined
and its position recorded. Due to the thickness of the tissue
sections, however, the perimeters of some boutons could not
be resolved exactly enough to measure accurately. The total
number of boutons that could be measured precisely ranged
from 116 to 456. The average number of boutons measured
on dynamically sensitive, biotinamide-stained afferents was
239, whereas the average number of boutons measured on
nondynamically sensitive afferents stained with biotinamide
was 242. The perimeter measurements of the total population
of biotinamide-stained boutons ranged from 1.9 to 19.3 mm.
The mean perimeter of the dynamically sensitive afferents
was 9.1 mm, whereas that of the nondynamically sensitive
afferents was 8.4 mm. The average area of the dynamically
sensitive afferents was 5.1 mm2 in contrast to 4.6 mm2 for
the nondynamically sensitive afferents. The shape factor for
biotinamide-stained boutons ranged from 0.35 to 0.91. The
mean shape factor for dynamically sensitive afferents was
0.75, whereas that for nondynamically sensitive afferents
also was 0.75. No statistical difference was found between
the shape factors of dynamically sensitive versus nondynamically sensitive afferent boutons (t-test; P Å 0.920; MannWhitney U-test, P Å 1.0).
The distribution of 5,469 boutons located in the region of
the trigeminal motor nucleus was examined in five HRPstained and seven biotinamide-stained afferents (Table 2).
For dynamically sensitive afferents, an average of 66.8% of
boutons located in the region of Vmo were distributed within
the confines of the trigeminal motor nucleus. In contrast to
this, an average of only 31.7% of boutons on nondynamically
sensitive afferents were located within Vmo. When examined as a group, jaw-muscle spindle afferents with high dynamic sensitivity, as indicated by a large dynamic index,
consistently distribute a greater percentage of their boutons
within the Vmo as compared with surrounding regions.
2934
D. DESSEM, R. DONGA, AND P. LUO
muscle spindle afferents. One of these distinguishing features is that the afferents responded with increased firing
during passive stretching of the jaw-elevator muscles. Additional support for this identification is given by the fact that
all of the afferents had single receptive fields restricted to
the region of the jaw-elevator muscles. These characteristics
are comparable with those previously attributed to jaw-muscle spindle afferents (Appenteng et al. 1978; Inoue et al.
1981; Miyazaki and Luschei 1987). The possibility that the
afferents labeled in this study are Vme periodontal afferents
was excluded because they failed to respond to probing of
the teeth and gingiva and were modulated when the jawelevator muscles were stretched. It is also important to consider the level of fusimotor activity during these experiments
because fusimotor drive has the capability to alter the afferent response of muscle spindles. Because trigeminal fusimotor axons exit the brain stem in the motor root of the trigeminal nerve, it is impractical to deefferent jaw-elevator muscle
spindles. Therefore fusimotor drive in the HRP-labeling experiments was reduced by deeply anesthetizing the animals
with barbiturate anesthesia and administering chlorpromazine (Cody et al. 1972). The constant level of fusimotor
drive obtained under these conditions is evident in the repro-
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ducible patterns of afferent discharge observed during repeated muscle stretches.
Two basic kinds of jaw-muscle spindle afferent response
were observed in this study. One type was modulated phasically during stretching of the jaw-closing muscles. This modulation consisted of a marked dynamic sensitivity during
muscle stretch and a cessation or substantial reduction when
the stretch was released. Afferent response behavior like this
is known to be produced by primary muscle spindle afferents
(Boyd and Ward 1975; Cooper 1961). Examples of this
type of afferent response that show a dramatic sensitivity
during stretching of the jaw-closing muscles are illustrated
in Figs. 1, A and B, and 2. Because these afferents were
recorded after the infusion of suxamethonium, which contracts intrafusal bag fibers (Boyd 1985; Gladden 1976; Smith
1966; Taylor et al. 1993b), the large dynamic response of
these afferents is most likely due to bag fiber contraction
and is indicative of a primary spindle afferent ending (Taylor
et al. 1995). Intracellular recordings during the infusion of
suxamethonium were difficult to maintain, and therefore no
attempt was made to further subdivide the spindle afferent
response types using depolarizing drugs as some extracellular electrophysiological studies have done (Donga and Tay-
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FIG . 6. Computer-assisted reconstruction in sagittal view
of a temporalis secondary-like jaw muscle spindle afferent
(R186). Dotted line, location of trigeminal motor nucleus.
Dorsal is toward page top, rostral is toward right. Scale bar:
200 mm.
JAW-MUSCLE SPINDLE AFFERENTS
2935
lor 1995; Price and Dutia 1987; Taylor et al. 1992). Larger
peak frequencies and dynamic indices were recorded from
dynamically sensitive afferents encountered during the biotinamide staining experiments than during HRP labeling. A
strong contributor to these differences was the presence of
suxamethonium, which increases the dynamic sensitivity of
primary muscle spindle afferents (Rack and Westbury 1966)
and which was administered to all animals used for biotinamide-labeling but only a few employed for HRP labeling.
When the afferent responses of dynamically sensitive, biotinamide-stained afferents were compared with dynamically
sensitive, HRP-stained afferents, which also were exposed
to suxamethonium, biotinamide-stained afferents exhibited
larger peak frequencies and dynamic indices. A likely contributor to these differences is the presence of gallamine,
which more readily blocks neurotransmission from dynamic
fusimotor axons onto intrafusal bag fibers (Proske and Carr
1995; Yamamoto et al. 1994) and was not used in the biotinamide protocol. An additional consideration is that chlorpromazine, which was administered in the HRP-staining experiments, has been shown to reduce fusimotor activity and
produce a more passive spindle (Cody et al. 1972; Henatsch
and Ingvar 1956).
Figure 12 shows an afferent with a more modest dynamic
response during the initial phase of jaw opening followed
by a strong silencing. This type of response implies that the
spindle was only stretched during the initial jaw-opening
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movement and then was unloaded. Afferents like this characteristically were activated more effectively by anteroposterior movement of the mandible. These afferents were interpreted as primary-like jaw-muscle spindle afferents because
of the silencing of their afferent response. It is likely that
the spindle unloading that creates this silencing is due to the
peripheral muscle receptor being at an oblique angle to the
direction of jaw opening and closing. Spindle afferents like
this indicate that masticatory muscle spindle afferent feedback can exist during mandibular movements of various directions.
The second type of afferent response seen in this study
was only slightly modulated during stretching of the jawclosing muscles and exhibited tonic activity. This response
type showed little dynamic sensitivity during muscle stretch
as evidenced by a dynamic index, which ranged from 4.5 to
17.8 impulses/s in the reconstructed afferents illustrated
here. Characteristically these afferents also discharged continuously during all phases of stretching of the jaw-closing
muscles and essentially mimicked the displacement of the
jaw. This type of afferent response previously has been demonstrated to originate from secondary muscle spindle afferents contacting nuclear chain fibers (Boyd and Ward 1975;
Cooper 1959). We were also able to examine this afferent
response type after the infusion of large doses of suxamethonium and even under these circumstances, the afferent response showed little dynamic sensitivity during stretching
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FIG . 7. Afferent response of spindle afferent R186.
A: instantaneous frequency of afferent during ramp and
hold displacement of jaw. B: instantaneous frequency
of afferent during sinusoidal displacement of jaw.
Arrow, jaw opening, time bars are 1 s.
2936
D. DESSEM, R. DONGA, AND P. LUO
FIG . 8. Computer-assisted reconstruction in sagittal view of a masseter secondary-like jaw-elevator muscle spindle afferent (R160). Dotted line, location of trigeminal motor nucleus. Dorsal is toward page’s
top, rostral is toward page right. Scale bar:
500 mm.
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FIG . 9. Afferent response of jaw-muscle spindle
afferent R160. A: instantaneous frequency of afferent
during ramp and hold displacement of jaw. B: instantaneous frequency of afferent during sinusoidal displacement of jaw. Time bars are 1 s; Arrow, jaw
opening.
JAW-MUSCLE SPINDLE AFFERENTS
2937
of the jaw muscles and no evidence of silencing during the
release of muscle stretch. The afferent responses of nondynamically sensitive afferents stained with biotinamide were
comparable with those stained with HRP.
Basic axonal morphology and projections
The basic axonal trajectory of the afferents stained in
this study is comparable with that described previously for
unclassified jaw-muscle spindle afferents stained with HRP
(Dessem and Taylor 1989; Lingenhöhl and Friauf 1991; Luo
et al. 1991; Shigenaga et al. 1988, 1990) and biotinamide
(Luo and Dessem 1995; Luo et al. 1995a) but differs from
that reported by Appenteng and co-workers (1985) using
Lucifer yellow. Appenteng et al. (1985) contended that the
jaw-muscle spindle afferent impulses first reach Vmo and
then invade the spinal trigeminal subnucleus oralis followed
by supratrigeminal region (Vsup) and finally Vme. In this
study, we found no jaw-muscle spindle afferent axonal trajectories consistent with the projection of spindle afferent
impulses from Vmo through the spinal trigeminal nuclei to
the supratrigeminal region.
Projection areas of primary-like jaw muscle spindle
afferents
The most distinguishing feature of primary-like jaw-muscle spindle afferents is that their strongest projection, based
on the number of axon collaterals and axonal swellings, is
to the trigeminal motor nucleus. Consistent with this finding
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is the previous report by Taylor et al. (1993a) that the largest
extracellular field potential generated by primary jaw-muscle
spindle afferents in cats is also within the trigeminal motor
nucleus. Even though a large number of intracellularly
stained primary-like muscle spindle afferent axon collaterals
and boutons are located in Vmo, most of the en passant
and terminal boutons of these afferents were not closely
associated with Nissl-stained trigeminal motoneurons. There
was typically, however, a small subpopulation of trigeminal
motoneurons, whose somata and proximal dendrites were
apposed closely by up to five labeled, primary-like jawmuscle spindle afferent boutons (Fig. 10, C and D). Although some previous electrophysiological studies have
implied that masticatory muscle spindle afferents contact
trigeminal motoneuron distal dendrites (Appenteng et al.
1978; Chandler et al. 1980), others (Grimwood et al. 1992;
Nozaki et al. 1985) report larger EPSPs with sharp rising
phases that may be indicative of the proximal dendritic and
somatic contacts found in this study.
In addition to a strong projection to Vmo, all primarylike jaw muscle spindle afferents possessed axon collaterals
with en passant and terminal boutons, which were distributed
throughout the region dorsal to the trigeminal motor nucleus
including an area more medial than the supratrigeminal nucleus described by Lorente de Nó (1922). Because no contacts between primary-like jaw-muscle spindle afferents and
Vsup neurons were observed in this study, it remains to be
determined how many spindle afferent boutons contact the
distal dendrites of trigeminal motoneurons that extend into
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FIG . 10. Photomicrographs
of horseradish peroxidase
(HRP)-stained axons. A: HRP-stained boutons (arrows) from a
secondary-like jaw-muscle spindle afferent in close association
with a Nissl-stained neuron in supratrigeminal region. B: HRPstained axon collaterals from an unclassified jaw-muscle spindle
afferent (R174) possessing boutons that closely approximate
Nissl-stained neurons (arrows) in lateral reticular formation. C
and D: HRP-stained boutons (arrows) from primary-like jawmuscle spindle afferents overlying or in close association with
cell bodies of Nissl-stained trigeminal motoneurons. E and F:
HRP-stained boutons (arrows) from secondary-like jaw-muscle
spindle afferents in close association or overlying trigeminal motoneurons. Scale bars: A, 25 mm; B, 50 mm; C–F, 10 mm.
2938
D. DESSEM, R. DONGA, AND P. LUO
this region (Mong et al. 1988) versus interneurons and therefore whether afferent information from primary-like jawmuscle spindle afferents is relayed through Vsup.
Caudal to the trigeminal motor nucleus, primary-like jawmuscle spindle afferents typically possessed five to eight
axon collaterals that emerged from the tract of Probst and
coursed ventrolaterally into the parvicellular reticular formation. In some instances, collaterals with en passant and terminal boutons were located in the dorsolateral part of the parvicellular reticular formation and the adjacent dorsomedial
portion of the spinal trigeminal subnucleus oralis (Vo). Several large, multipolar neurons, in the region of Vo were
closely apposed by intracellularly stained primary-like jaw
muscle spindle afferent boutons. Falls et al. (1985) have
described similar large, multipolar neurons in this region
that project to orofacial regions of the cerebellum, suggesting
that the contacts observed in this study may represent a
disynaptic mossy fiber pathway for the transmission of trigeminal proprioceptive feedback to the cerebellum. Because
neurons in Vo and the adjacent reticular formation also are
known to project to the thalamus (Travers and Norgren
1983), the trigeminal motor nucleus (Luo and Dessem
1996b; Mogoseanu et al. 1993; Yoshida et al. 1994) and the
cervical spinal cord (Dessem and Luo 1996), the possibility
also exists that this projection conveys polysynaptic stretch
reflexes or proprioceptive information to the thalamus, trigeminal motor nucleus and spinal cord.
Convincing evidence for a projection of primary-like jaw
muscle spindle afferents to the mesencephalic trigeminal nucleus and the cerebellum also was found in the present study.
The close association between primary-like spindle afferent
boutons and caudal Vme neurons that was observed implies
that neuronal communication can occur directly between the
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rostral and caudal portions of the mesencephalic trigeminal
nucleus. The occurrence of a primary-like jaw-muscle afferent axon collateral in the ipsilateral superior cerebellar peduncle suggests that muscle spindle feedback can reach the
cerebellum without relay and may allow the gain of the jawstretch reflex to be regulated (Donga and Dessem 1993).
Projection areas of secondary-like jaw-muscle spindle
afferents
The strongest projection of secondary-like jaw-muscle
spindle afferents, as indicated by both axon collaterals and
boutons, was in the region dorsal to the trigeminal motor
nucleus. As was the case with primary-like spindle afferent
boutons, most of the secondary-like spindle afferent boutons
in this region do not appear to contact Nissl-stained neurons.
In a few instances, however, intracellularly labeled, secondary-like boutons were observed in close association with
neurons dorsal to Vmo. Previous studies have reported that
some neurons in this region project to the contralateral trigeminal motor nucleus (Kamogawa et al. 1988; Mizuno et
al. 1978; Rokx et al. 1986; Travers and Norgren 1983).
Secondary jaw-muscle spindle afferent projections to this
area therefore may be involved in polysynaptic jaw muscle
stretch reflexes. Neurons in this region also project to the
cerebellum (Somana et al. 1980), making this region a potential relay for secondary jaw-muscle spindle proprioceptive feedback to the cerebellum. Although it remains uncertain what percentage of secondary-like jaw-muscle spindle
afferent boutons in this region contact interneurons and how
many contact the distal dendrites of trigeminal motoneurons,
at least some information transmitted via secondary-like jawmuscle spindle afferents is relayed through interneurons dorsal to Vmo.
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FIG . 11. Photomicrographs of HRP-stained jaw-muscle spindle afferents. A: soma of secondary-like afferent . B: cell body
of unclassified spindle afferent R174. C: secondary-like afferent
possessing a dendritic process (arrow). D: HRP-stained jawmuscle spindle afferent soma juxtapositioned with soma of an
unlabeled mesencephalic trigeminal neuron. E: HRP-stained
boutons (arrows) from a primary-like jaw-muscle spindle afferent
closely approximating cell body of another mesencephalic trigeminal neuron. F: HRP-stained ‘‘giant’’ bouton (arrow) from
a secondary-like jaw-muscle spindle afferent overlying soma of
a Nissl-stained mesencephalic trigeminal neuron. Scale bars: A,
100 mm; B and C, 50 mm; D–F, 25 mm.
JAW-MUSCLE SPINDLE AFFERENTS
2939
A smaller number of secondary-like jaw-muscle spindle
afferent boutons were located within the trigeminal motor
nucleus than in the region dorsal to Vmo. In a manner similar
to the primary-like spindle afferent boutons, only a few secondary-like jaw-muscle spindle afferent boutons were associated closely with the cell bodies and proximal dendrites
of trigeminal motoneurons. Even though the number of motoneurons receiving this input is small, multiple boutons
were found overlying single motoneurons in some cases,
implying that these inputs may be powerful. These inputs
from secondary-like jaw-muscle spindle afferents to trigeminal motoneurons provide morphological corroboration of
previous electrophysiological data (Appenteng et al. 1978;
Taylor et al. 1993a).
In addition to projections at the level of the trigeminal
motor nucleus, all secondary-like jaw-muscle spindle afferents had collaterals that emerged from the tract of Probst
caudal to Vmo and coursed into the reticular formation and
adjacent dorsomedial portion of the spinal trigeminal nucleus. As with primary-like afferents, boutons were observed
in the reticular formation and spinal trigeminal nucleus.
Evidence also was found that some secondary-like jawmuscle spindle afferents project directly to the cerebellum
via processes in the ipsilateral superior cerebellar peduncle.
Both primary and secondary jaw-muscle spindle afferent
feedback, therefore, can be transmitted to the cerebellum
without relay.
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Secondary-like jaw-muscle spindle afferents whose somata were located rostrally in Vme also were seen to possess
axon collaterals with boutons overlying more caudally located Vme neurons. Luo and Dessem (1996a) recently have
reported that some Vme, jaw-muscle spindle afferent boutons contact the somata of other Vme, jaw-muscle spindle
afferents, although these experiments did not differentiate
between muscle spindle afferent types. In this study, we
have been able to characterize this circuitry further by demonstrating the involvement of both primary and secondary
jaw-muscle spindle afferents in the interaction of Vme neurons. An additional observation within the mesencephalic
trigeminal nucleus is that one labeled secondary-like Vme
neuron possessed a small dendrite (Fig. 11C). Some previous studies (Capra et al. 1984; Gottlieb et al. 1984; Nomura
and Mizuno 1985; Shigenaga et al. 1988; Walberg 1984)
have reported a subpopulation of Vme neurons with small
dendrites after the injection of neuroanatomic tracers into
the jaw-elevator muscles. This study demonstrates that in
the rat, some of these multipolar Vme neurons are secondary
jaw-elevator muscle spindle afferents.
Comparison with previous attempts to correlate the
physiology and morphology of jaw-muscle spindle
afferents
Previous attempts to correlate the physiology and morphology of jaw-muscle spindle afferents are limited. Tay-
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FIG . 12. Afferent response of an unclassified jawmuscle spindle afferent R174. A: instantaneous frequency of afferent during ramp and hold displacement
of jaw. B: instantaneous frequency of afferent during
sinusoidal displacement of jaw. Arrow, direction of jaw
opening; time bars are 1 s.
2940
D. DESSEM, R. DONGA, AND P. LUO
lor and co-workers ( 1993a ) used spike-triggered averaging to determine the location of unitary field potentials
generated from single jaw-muscle spindle afferents in the
cat. Due to volume conduction of the recorded potential
and the large size of the recording electrode, however,
this technique cannot distinguish the detailed morphology
of individual afferents including axon collaterals and synaptic boutons. Taylor et al. ( 1993a ) , for instance, were
unable to detect jaw-muscle spindle afferent projections
within Vme or into the cerebellar peduncle nor were they
able to discern the relationship between afferent terminations and individual trigeminal motoneurons. In a general
sense, however, the results of Taylor et al. ( 1993a ) are
similar to those reported here in that Taylor and co-workers reported a large extracellular field potential generated
by single secondary jaw-muscle spindle afferents in the
region dorsal to the trigeminal motor nucleus and a large
field within the trigeminal motor nucleus by primary jawmuscle spindle afferents.
Shigenaga and co-workers (1988, 1990) also attempted to
compare the physiological responses of jaw-muscle spindle
afferents with their axonal morphology using intracellular
HRP staining in the cat. These authors were able to differentiate two morphological types of afferent. One type, which
they designated as type I, showed its strongest projection to
Vmo. The second type (type II) had the majority of its axon
collaterals and boutons in the supratrigeminal region with
much sparser projections to Vmo. The morphology of the
type I afferents described by Shigenaga et al. (1988) is
similar to the morphology of the primary-like jaw-muscle
spindle afferents that we describe here. The type II axonal
morphology reported by Shigenaga et al. (1988) resembles
that of the secondary-like jaw-muscle spindle afferents re-
/ 9k13$$ju29
J793-5
ported here. Shigenaga et al. (1990), however, reported no
relationship between the response of the spindle afferent and
these morphological types and therefore concluded that there
was no correlation between the type of spindle afferent response and its central morphology. This is completely the
opposite from what we report in this study in which it was
found that distinct morphological types of jaw-muscle spindle afferent corresponded to distinctly different afferent responses. The most likely explanation for this discrepancy is
that Shigenaga et al. (1990) recorded the response of afferents during a step displacement of the jaw, which is inadequate to physiologically characterize muscle spindle afferents. In addition, Shigenaga et al. (1990) did not try to
control the fusimotor drive to the spindles or to test any
afferents in the presence of suxamethonium to see if the bag
fibers could be activated. In a more recent study (Yabuta et
al. 1996), a few afferents were recorded during ramp and
hold displacement of the jaw before intracellular staining.
These authors, however, provide no quantitative measure of
the dynamic sensitivity of the afferents making their classification into traditional muscle spindle afferent types problematic.
Comparison with muscle spindle projections in the spinal
cord
The central projection of jaw-muscle spindle afferents
resembles the projection pattern of limb muscle spindle
afferents in a number of ways. In the lumbosacral spinal
cord, HRP-stained primary muscle spindle afferents show
their strongest projection to the motor nuclei ( Brown and
Fyffe 1978; Burke et al. 1979; Honga et al. 1987; Ishizuka
et al. 1979 ) . Similarly, the primary-like jaw-muscle spin-
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FIG . 13. Computer-assisted reconstruction of posterior temporalis, unclassified spindle afferent R174 in sagittal perspective. Dotted line, outline of trigeminal motor nucleus. Dorsal is toward page’s top, rostral toward page right. Scale bar:
1,000 mm.
JAW-MUSCLE SPINDLE AFFERENTS
2941
dle afferents reported here have their strongest projection
to the trigeminal motor nucleus. Primary jaw-muscle spindle afferents, however, appear to directly contact a smaller
percentage of the motoneuron pool than hindlimb spindle
afferents based on the number and distribution of boutons
located within the motor nucleus ( Honga et al. 1987; Ishizuka et al. 1979 ) . An additional consideration, however,
is that some jaw-muscle spindle afferent boutons dorsal
to the anatomically defined trigeminal motor nucleus may
be contacting the distal dendrites of trigeminal motoneurons. Presently, however, the relationship between masticatory muscle spindle afferents and motoneurons appears
to resemble more closely the distribution of primary muscle spindle afferents in the cervical spinal cord; these afferents make monosynaptic contacts with only Ç10% of
neck motoneurons ( Keirstead and Rose 1988a,b ) .
Hindlimb secondary muscle spindle afferents project
mainly to interneurons located in laminae V, VI, VII
( Fyffe 1979 ) . The region dorsal to the trigeminal motor
nucleus may be homologous because secondary-like jawmuscle spindle afferents project predominately to this region. Some spinal secondary spindle afferents make
monosynaptic contacts with motoneurons ( Kirkwood and
Sears 1974; Stauffer et al. 1976 ) ; the close association of
secondary-like jaw-muscle spindle afferents reported here
provides evidence for comparable monosynaptic connections in the trigeminal system.
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Bouton distribution and morphology
Previous studies (Bae et al. 1996; Conradi et al. 1983;
Luo and Li 1991; Luo et al. 1995a,b) provide ultrastructural
evidence that swellings on HRP- and biotinamide-filled axon
collaterals are synaptic boutons. In these studies, a few additional synapses usually are revealed during electron microscopic analysis, implying that the bouton counts presented
in this study are likely to be an underestimate of the total
number of jaw-muscle spindle afferent synapses.
The total number of boutons present on the spindle afferents stained with HRP in this study ranged from 162 to 636
with a mean of 478, whereas those labeled with biotinamide
ranged from 296 to 559 with a mean of 408. By comparison,
Shigenaga and co-workers (1990) reported a range of 245
to 2,182 HRP-stained boutons with a mean of 1,219 boutons
on intra-axonally stained cat jaw-muscle spindle afferents.
Although the sample sizes in both of these studies are small,
these data imply that rat jaw-muscle spindle afferents possess
fewer synaptic boutons than those of cats.
A small number of the swellings on primary- and secondary-like axon collaterals in Vme and the supratrigeminal
region are very large (16 1 12 to 30 1 10 mm). In the cat,
Shigenaga and co-workers (1990) also have reported a few
jaw-muscle spindle afferent boutons that were ú7 mm. These
putative giant boutons appear to be comparable with those
found on Ia fibers in Clarke’s column; those on Clarke’s
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FIG . 14. Location of boutons associated with trigeminal motor nucleus on reconstructed jaw-muscle spindle afferents.
A: primary-like afferent R161. B: unclassified spindle afferent R174. C: secondarylike afferent R170. D: secondary-like afferent R186. E: secondary-like afferent R160.
Dashed line, location of trigeminal motor
nucleus. Open circle, location of en passant
boutons; Open triangle, location of terminal
boutons. Dorsal is toward page top, rostral
is toward right. Scale bar: 500 mm.
2942
D. DESSEM, R. DONGA, AND P. LUO
3. Comparison of bouton perimeters inside and outside
of the trigeminal motor nucleus
TABLE
Animal
Dynamic afferent type
R161
PAR13r
PAR14l
PAR8r
Nondynamic afferent type
R170
R186
R160
PAR19r
PAR20r
PAR21r
Unclassified afferent type
R174
imply a partitioning of monosynaptic sensory feedback from
jaw-muscle spindle afferents to small groups of trigeminal
motoneurons similar to that described in the spinal cord
(Stuart et al. 1988). Future studies will be needed to determine how this sensory partitioning relates to the compartmentalization of the jaw muscles and their differential contraction.
t-Test
Mann-Whitney U Test
0.791
0.436
0.901
0.039*
0.602
0.267
0.819
0.201
0.049*
0.000*
0.015*
0.000*
0.000*
0.001*
0.048*
0.000*
0.049*
0.000*
0.000*
0.002*
We thank R. Druzinsky, C. J. Heckman, R. Meszler, and A. Taylor for
reading versions of this manuscript and N. Capra for use of the Zeiss image
analysis system.
This work was supported by National Institute of Dental Research Grant
DE-10132.
Address for reprint requests: D. Dessem, Dept. OCBS, Room 4G-15,
University of Maryland Dental School, 666 W. Baltimore St., Baltimore,
MD 21201-1586.
0.097
0.056
Received 28 November 1995; accepted in final form 12 February 1997.
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The distribution of boutons in the region surrounding the
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