Laboratorio de Neurociencia, Facultad de Biología, Universidad de Sevilla, 41012 Seville, Spain
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ABSTRACT |
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Trigo, José A.,
Agnès Gruart, and
José M. Delgado-García.
Discharge profiles of abducens, accessory abducens, and
orbicularis oculi motoneurons during reflex and conditioned blinks in
alert cats. The discharge profiles of identified abducens, accessory abducens, and orbicularis oculi motoneurons have been recorded extra- and intracellularly in alert behaving cats during spontaneous, reflexively evoked, and classically conditioned eyelid responses. The movement of the upper lid and the electromyographic activity of the orbicularis oculi muscle also were recorded. Animals were conditioned by short, weak air puffs or 350-ms tones as
conditioned stimuli (CS) and long, strong air puffs as unconditioned
stimulus (US) using both trace and delayed conditioning paradigms.
Motoneurons were identified by antidromic activation from their
respective cranial nerves. Orbicularis oculi and accessory abducens
motoneurons fired an early, double burst of action potentials (at 4-6
and 10-16 ms) in response to air puffs or to the electrical
stimulation of the supraorbital nerve. Orbicularis oculi, but not
accessory abducens, motoneurons fired in response to flash and tone
presentations. Only 10-15% of recorded abducens motoneurons fired a
late, weak burst after air puff, supraorbital nerve, and flash
stimulations. Spontaneous fasciculations of the orbicularis oculi
muscle and the activity of single orbicularis oculi motoneurons that
generated them also were recorded. The activation of orbicularis oculi
motoneurons during the acquisition of classically conditioned eyelid
responses happened in a gradual, sequential manner. Initially, some
putative excitatory synaptic potentials were observed in the time
window corresponding to the CS-US interval; by the second to the fourth conditioning session, some isolated action potentials appeared that
increased in number until some small movements were noticed in eyelid
position traces. No accessory abducens motoneuron fired and no abducens
motoneuron modified their discharge rate for conditioned eyelid
responses. The firing of orbicularis oculi motoneurons was related
linearly to lid velocity during reflex blinks but to lid position
during conditioned responses, a fact indicating the different neural
origin and coding of both types of motor commands. The power spectra of
both reflex and conditioned lid responses showed a dominant peak at
20 Hz. The wavy appearance of both reflex and conditioned eyelid
responses was clearly the result of the high phasic activity of
orbicularis oculi motor units. Orbicularis oculi motoneuron membrane
potentials oscillated at
20 Hz after supraorbital nerve stimulation
and during other reflex and conditioned eyelid movements. The
oscillation seemed to be the result of both intrinsic (spike
afterhyperpolarization lasting
50 ms, and late depolarizations) and
extrinsic properties of the motoneuronal pool and of the circuits
involved in eye blinks.
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INTRODUCTION |
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The motor system controlling eyelid responses is
an excellent experimental model to study how relatively simple,
although diverse, movements are generated by central neural circuits
(see references in Evinger 1995; Gruart et al.
1995
). The eyelid motor system is load free, has an almost
negligible mass, and, according to recent anatomic and functional data,
is free of proprioceptors (Porter et al. 1989
;
Trigo et al. 1997
, 1999
). Indeed, in spite of the
system's apparent simplicity, many different movements are
accomplished by the lids in diverse behavioral situations; in fact,
different motor systems are involved in blink responses. Essentially, a
blink is a reflex eyelid response to the mechanical activation of the
cornea and periorbital skin or to the electrical stimulation of the
supraorbital nerve (Baker et al. 1980
; Cruccu et
al. 1987
; Evinger et al. 1991
; Gordon
1951
; Gruart et al. 1995
; Hiraoka and
Shimamura 1977
; Kugelberg 1952
). However, blinks
also can be evoked by strong visual and acoustic stimuli
(Evinger and Manning 1993
; Gruart et al.
1995
; Manning and Evinger 1986
). The kinematics
of a reflex blink is the result of the brief, fast contraction of both
eyelids produced by the phasic activation of the orbicularis oculi
muscle, but it also involves the cocontraction of most of the
extraocular muscles, the relaxation of the levator palpebrae muscle,
and, in those species with a nictitating membrane, the activation of
the retractor bulbi muscle (Baker et al. 1980
; Evinger 1995
; Evinger and Manning 1993
).
Apart from reflexively evoked responses related to corneal wetting and
protection, lids move (mostly passively) accompanying rotational eye
movements in the orbit, in such a way that the pupil is not covered
during voluntary and reflex eye saccades and fixations (Becker
and Fuchs 1988
).
In addition to their participation in massive reflex blinks, lids play
an active role in complex and precise motor displays involved in
behavioral responses such as smiling and winking in humans
(Evinger 1995; Gordon 1951
) and during
grimacing and friendly displays in felines (Bateson and Turner
1988
; Darwin 1872
). Another example of graded
and precisely elaborated lid movement is the generation of classically
conditioned eyelid responses (CR). For the past 60 yr, the nictitating
membrane/eyelid response has been employed in the study of neural
mechanisms underlying motor learning (Gormezano et al.
1983
; Kim and Thompson 1997
; Marquis and
Porter 1939
; McCormick et al. 1982
; Welsh
and Harvey 1992
; Woody 1986
). The profile and
kinematics of CRs are different from those of reflex blinks, suggesting
a different origin and/or neural generation (Domingo et al.
1997
; Gruart et al. 1995
; Rescorla
1988
). The neural site where learning of eyelid response occurs
(Aou et al. 1992
; Bloedel 1992
;
Kim and Thompson 1997
; McEchron and Disterhoft 1997
), as well as the putative subcellular mechanisms involved (Bliss and Collingridge 1993
; Coulter et al.
1989
; Malenka 1995
; Woody et al.
1989
) are currently a subject of intensive research.
Studies about the functional involvement of neural centers and circuits
in reflex and conditioned eyelid responses have followed different
approaches. Thus while there are many studies on the firing profiles of
trigeminal, red nucleus, cerebellar cortex and nuclei, and cerebral
cortex neurons in relation to the acquisition and/or performance of new
motor skills (Aou et al. 1992; Bloedel 1992
; Kim and Thompson 1997
; McEchron and
Disterhoft 1997
; Richards et al. 1991
;
Woody 1986
), less attention has been paid to the functional properties of those brain stem motoneurons that represent the final common pathway for learned lid responses
namely abducens, accessory abducens, and orbicularis oculi motoneurons (Keifer et
al. 1995
; Matsumura and Woody 1986
). In
contrast, there are good descriptions of the electrophysiological
properties of these three types of blink-related motoneurons in both
acute and in vitro preparations (Aghajanian and Rasmusen
1989
; Baker et al. 1980
; Fanardjian et
al. 1983b
; Grantyn and Grantyn 1978
;
Gueritaud 1988
; McCall and Aghajanian
1979
; Nishimura 1985
; Nishimura et al.
1989
). The firing profiles of abducens
(Delgado-García et al. 1986
), accessory abducens
(Delgado-García et al. 1990
), and orbicularis
oculi (Hall and Hicks 1973
) motoneurons have been recorded in cats or rats during reflex blinks but not during the acquisition and performance of CRs.
It should be remembered that the eyelid CR is an adaptive response that
has to be accomplished by three different motor systems not equally
suited for this motor task. In two previous papers, we have described
the kinematics of spontaneous, reflexively induced, and classically
conditioned eyelid responses in alert behaving cats (Gruart et
al. 1995) and the frequency-domain and time-domain properties
of these facial movements (Domingo et al. 1997
). It was
found that both reflex and conditioned eyelid responses present movement discontinuities at a dominant frequency of
20 Hz. Thus lid
movements seem to be constructed from a basic 50-ms downward wave or
sag. Moreover reflex blinks seem to be the result of the massive,
synchronous activation of many orbicularis oculi motor units, whereas
CRs are elaborated in a succession of small downward sags in a
staircase-like manner. In this context, it seems necessary to study the
particular contribution of each blink-related motoneuronal pool to the
generation of these peculiar motor responses. The present experiments
were aimed at recording the firing activity of identified abducens,
accessory abducens, and orbicularis oculi motoneurons in the alert
behaving cat during the presentation of stimuli able to evoke reflex
blinks (puffs of air, flashes of light, tones, and electrical
stimulation of the supraorbital nerve). Recorded motoneurons were
identified by antidromic activation from their corresponding cranial
nerves. The discharge rate of these motoneurons was recorded during two
different (trace and delayed) conditioning paradigms. The unconditioned
stimulus (US) was always a long, strong air puff. As conditioned
stimulus (CS), either a short, weak air puff or a tone was used.
Animals were provided with eyelid search coils and with
electromyographic (EMG) recording electrodes. Results proved the
different involvement of each motoneuronal type in reflex and
conditioned eyelid responses and suggested some insights into the
peculiar way CRs are generated at the motoneuronal level.
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METHODS |
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Subjects
The present experiments were carried out on six adult cats weighing 2-2.6 kg obtained from an authorized supplier (Iffa-Credo). Animals were prepared for the chronic recording of upper eyelid movements, the EMG activity of the orbicularis oculi muscle, and the electrical activity of identified abducens, accessory abducens and orbicularis oculi motoneurons. All experimental procedures were carried out in accordance with the guidelines of the European Union Council Directive (86/609/EU) and following Spanish legislation (BOE 67/8509-12 1988) concerning the use of laboratory animals in chronic experiments.
Preexperimental surgical procedures
Each animal was anesthetized with pentobarbital sodium
(Nembutal, 35 mg/kg) following a protective injection of atropine
sulfate (0.5 mg/kg) to avoid unwanted vagal reflexes. A five-turn coil (3-mm diam) was implanted into the center of the upper left lid at ~2
mm from the lid margin. Coils were made from Teflon-coated multistranded stainless steel wire with an external diameter of 50 µm. Because of its low weight (12.5 mg, i.e., <1.5% of the cat's
lid weight), the implanted coil did not produce any noticeable lid
drooping or impairment of movement when compared with the nonimplanted
eyelid. Two hook electrodes, made of the same wire as the coils and
bared 1 mm at their tips, were implanted in the left orbicularis
oculi muscle. The two hook electrodes were aimed at the zygomatic
subdivision of the facial nerve, 1-2 mm posterior to the external
canthus, and at the tarsal portion of the upper lid, 1 mm medial to the
coil, respectively. Other two hook electrodes were implanted on the
ipsilateral supraorbitary branch of the trigeminal nerve. A silver
electrode (1-mm diam) was attached to the skull as ground. A silver
bipolar stimulating electrode was implanted on the left VIth nerve at
its exit from the brain stem (stereotaxic coordinates L = 3.5 and
P = 1). The position of the electrode was adjusted to produce an
abducting movement of the ipsilateral eye with a 50-µs single pulse
of <0.2 mA (Delgado-García et al. 1986
). To
allow transcerebellar access to selected brain stem motor nuclei during
recording sessions, a square (6 × 6 mm) window was drilled in the
occipital bone. The dura mater was removed, and an acrylic resin
chamber was constructed around the window. The cerebellar surface was
protected with a piece of silicone sheet, and the chamber was filled
with sterile gauze and capped with a plastic cover. A needle tip was
implanted stereotaxically in a corner of the chamber as reference point for the location of brain stem motor nuclei. Finally, a head-holding system was built in to give stability to unitary recordings and to have
a coil reference during the experimental sessions. Three bolts were
anchored to the skull in the vertical stereotaxic plane with the aid of
six small, self-tapping screws fastened to the bone. The bolts were
cemented to the skull with acrylic resin. Eyelid coil, EMG,
stimulating, and grounding wire terminals were soldered to a nine-pin
socket cemented to the holding system. Further details of this chronic
preparation have been described elsewhere
(Delgado-García et al. 1986
; Gruart et
al. 1995
).
General conditions of recording sessions
Recording sessions started 15 days after surgery and lasted 3
h/day. For each session, the animal was introduced into a fabric bag,
lightly wrapped with an elastic bandage, and placed in a foam-coated
Perspex box on the recording table. The animal's head was immobilized
by attaching the holding system to a bar affixed to the table. Data
illustrated in Figs. 1-11, corresponding to spontaneous and reflex
blink responses, were obtained during the first five recording
sessions. After these sessions, each animal was assigned randomly to
one of the two (A, B) conditioning paradigms described later
in METHODS. Conditioning sessions lasted
15 days, and
were preceded by two habituation sessions (Figs. 12-16). Unitary
recordings were carried out during all of the sessions in which reflex
or conditioned eyelid responses were evoked in the experimental
subjects. Special care was taken during recording sessions to avoid any discomfort to the animals. In fact, the continuous monitoring of heart
and respiratory rhythms with noninvasive procedures during the
recording sessions yielded values not significantly different from
those obtained while the cat was resting in the arms of one of us
(Gruart et al. 1995
). Figure
1 illustrates recording and stimulating
sites as well as other experimental procedures.
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Unitary recordings
The recording chamber was uncovered to allow electrode access to
blink-related brain stem motor nuclei. Unitary and field electrical
activities were recorded with glass electrodes filled with 1.5-2 M
NaCl. Intracellular recordings were carried out using pipettes filled
with 3 M potassium acetate. Field potentials were recorded with
low-impedance electrodes (1-3 M), while extra- and intracellular
unitary activity was recorded with smaller tips (3-8 and 8-12 M
,
respectively). Neuronal activity was recorded with the aid of a
high-impedance circuit located in a head-stage close to the animal's
head. Electrical signals were amplified and filtered with a bandwidth
of 1 Hz to 10 kHz for display and digitization purposes.
Micropipettes were advanced with a three-axis micromanipulator through
the intact cerebellum to reach the brain stem. The abducens and
accessory abducens nuclei were approached with the help of stereotaxic
coordinates and with the guidance of the patterns of the electrical
activity generated in the surrounding neuronal structures
(Delgado-García et al. 1986, 1990
). In the case
of orbicularis oculi motoneurons, the electrode was aimed at the dorsolateral subdivision of the facial nucleus, where those motoneurons are located (Shaw and Baker 1985
). The abducens and the
accessory abducens nuclei were located finally with the aid of the
antidromic field potential produced in their electrophysiological
limits by the electrical stimulation of the ipsilateral VIth nerve. The center of each nucleus was considered to be the point of maximum antidromic negativity. The VIth nerve was stimulated with 50-µs, 1- to 20-Hz cathodal square pulses with current intensities small enough
(<0.2 mA) to prevent its spreading to the Vth nerve. The accessory
abducens nucleus usually was located 2-2.5 mm lateral to the center of
the main abducens nucleus. The orbicularis oculi subdivision of the
facial nucleus was located by electrical stimulation of the zygomatic
nerve with electrical pulses similar to those applied to the VIth
nerve. The center of the dorsolateral subdivision of the facial nucleus
was found 3-4 mm lateral to the center of the main abducens nucleus
(Fig. 1, A-C) (see Delgado-García et al.
1986
, 1990
; Shaw and Baker 1985
for details).
Given the high density of blink-related units located in the
neighborhood of blink-related brain stem motor centers (Gruart et al. 1993), the collision test between orthodromic and
antidromic action potentials was used systematically to identify
recorded units. Only the recordings of properly identified motoneurons were stored and analyzed. Recorded units remained well isolated up to a
maximum of 2 h. Surprisingly, some accessory abducens and most
facial motoneurons could be recorded for a long time (
20 min) during
intracellular impalements. We decided to take advantage of this fact,
given the additional information offered by the intracellular recording
of neuronal electrical activity. In those cases, experimental
recordings started after membrane potential stabilization. Once stable
resting potentials (range:
67 to
45 mV) were reached, no
significant differences were found between the patterns of activity
recorded intra- or extracellularly (see Aou et al. 1992
;
Terzuolo and Araki 1961
). For technical reasons, no DC
recording was available during activation of the eyelid search coil
recording system. Averages and/or quantifications from intracellular
records (such as those illustrated in the insets of Figs. 11
and 15) were carried out only for spikes >45 mV (Kitai et al.
1972
). Intracellular records with spikes <20 mV were discarded (Aou et al. 1992
).
At the end of every recording session, the recording chamber was cleaned aseptically and instilled with antibiotics. The scar tissue was bathed with lidocaine and carefully removed. Finally, the chamber was sealed as previously described until the next recording session.
Recordings of eyelid movements and electromyographic activity of the orbicularis oculi muscle
Eyelid movements were recorded using the magnetic field
search-coil technique (Fuchs and Robinson 1966). The
coils were calibrated with the cat still under the effects of the
anesthetic. A transparent protractor was placed sagittally to the head
and centered at the external canthus of the lids (Gruart et al.
1995
). For the sake of homogeneity, the gain of the recording
system was always set at 1 V = 10°. Lid opening ranged 37-43°
for the six animals. The EMG activity of the orbicularis oculi muscle
was recorded with differential amplifiers with a bandwidth of 10 Hz to
10 kHz.
Stimuli evoking reflex eyelid responses
Reflex blinks were evoked with air puffs, light flashes, and
tones and by electrical stimulation of the supraorbital nerve. Air
puffs directed to the cornea and tarsal skin were applied through the
opening of a plastic pipette (2.5-mm diam) located 1 cm away from the
eye, at a lateral angle of 45° with respect to the central direction
of gaze. Air puff duration ranged from 20 to 100 ms, and air pressure
at the source was set at 1-3 kg/cm2. The onset of air puff
stimuli to skin mechanoreceptors was determined with a microphone
located at the same distance as the animal's cornea. The signal
recorded by the microphone was rectified, integrated, and fed into the
computer as 1-V square pulses for latency measurements. Visual blinks
were evoked with full-field flashes lasting 1 ms. Flashes were
produced by a xenon arc lamp located 1 m in front of the animal.
Tones of 600 or 6,000 Hz, 90 dB, applied for 10-100 ms were used as
acoustic stimuli. The loudspeaker was located 80 cm below the animal's
head. After the isolation of a motoneuron, each stimulus modality was
presented in blocks of 10 stimuli at variable intervals (5 ± 1 s), to
avoid interactions between subsequent blinks (Powers et al.
1997
). A minimum of 30 s was allowed between successive
presentations of stimuli of different modalities. The electrical
stimulation of the supraorbital nerve consisted of single or double (1- to 2-ms interval), 50-µs cathodal square pulses with current
intensities <0.2 mA.
No experimental procedure was used to evoke spontaneous eye blinks or
"eyelid friendly displays" (Gruart et al. 1995).
These eyelid motor responses were collected off-line from tape
recordings with the help of a window discriminator set to the detection
of the initial fast increase in velocity of these eyelid responses.
Classical conditioning paradigms
The classical conditioning of lid responses was achieved by the use of trace (A) or delayed (B) conditioning paradigms. For trace air puff-AIR PUFF (ap-AP) paradigm, the animal was presented with a short (20 ms), weak (1 kg/cm2) air puff, directed to the left cornea as CS. The CS was followed 250 ms later by a 100-ms, 3-kg/cm2 air puff directed to the ipsilateral cornea as US. For delayed TONE-AIR PUFF (T-AP) paradigm, the animal was presented with a 350-ms, 600-Hz, 90-dB tone as CS. The tone was followed 250 ms from its onset by a 100-ms, 3-kg/cm2 air puff stimulus directed to the left cornea as US. The tone and the air puff finished simultaneously.
Each of the two (A, B) conditioning paradigms was used to train three animals. An animal was considered to be conditioned when it could produce 95% of CRs per session to the CS-US paired presentation. Although this criterion was reached by the fifth conditioning session for the six animals, recordings were obtained up to the 15th session to obtain the maximum number of recorded motoneurons. Conditioning sessions were preceded by two habituation sessions.
Each conditioning session consisted of 12 blocks separated by a
variable time interval (range of 4-6 min). These intervals were used
to locate and identify recorded units. Each block consisted of 10 trials separated at random by intervals ranging from 20 to 40 s.
In a trial within each block, the CS was presented alone, that is, was
not followed by the US. A complete conditioning session lasted 2 h.
Only the stimulus selected as CS was presented during habituation
sessions with the same number of blocks per session and trials per
block and with a similar random distribution of interblock and
intertrial intervals (see details in Gruart et al.
1995
).
Data collection and analysis
Vertical and horizontal position of left upper eyelid, unrectified EMG activity of the ipsilateral orbicularis oculi muscle, neuronal activity, and 1-V rectangular pulses corresponding to blink-evoking stimuli or to CSs and USs presented during conditioning sessions were stored digitally on an eight-channel videotape recording system at a sampling frequency of 22 kHz for biopotentials and 11 kHz for the other signals. Data were transferred later through an analog digital converter (CED 1401 Plus) to a computer for off-line analysis. Most data were sampled at 1-4 kHz with an amplitude resolution of 12 bits. Selected unitary records were sampled at 22 kHz for representation purposes and precise analysis of spike profiles. Additionally, action potentials were fed into a window discriminator and the resulting Schmidt trigger pulses were stored on the computer, along with the analog signals, using the same A/D conversion card.
Commercial computer programs (Spike 2 and SIGAVG from CED and MATLAB)
were modified, and new programs were developed to display single,
overlapping, averaged, and raster representation of eyelid position,
velocity, and acceleration and of EMG and neuronal activities. Velocity
and acceleration traces were computed digitally as the first and second
derivative of lid position records after low-pass filtering of the data
(3 dB cutoff at 50 Hz and a 0 gain at
100 Hz). The single and
averaged histograms of instantaneous firing rate profiles of the
neuronal discharge were displayed in relation with the corresponding
EMG and eyelid activities. The instantaneous firing rate was calculated
as the inverse of the interspike intervals for segments of
1 s (see
Domingo et al. 1997
for details).
These computer programs also allowed the quantification, with the aid
of cursors, of lid position, EMG, and neuronal parameters, such as
onset latency, amplitude, duration, and peak and mean values of
recorded responses. Relationships between neuronal firing rate and lid
position and velocity were obtained by linear regression analysis to
calculate their respective slopes, i.e., the neuronal sensitivity to
lid position (in spikes per second per degree) and to lid velocity (in
spikes per second per degree per second). Because lid position was
sampled at 1 kHz, bin size for linear correlation was set at 1 ms.
The power of the spectral density function (i.e., the power spectrum)
of selected eye acceleration recordings was calculated using a fast
Fourier transform to define the relative strength of the different
frequencies present in lid responses. Analysis were carried out
according to procedures described in detail elsewhere (Bendat
and Piersol 1986
; Domingo et al. 1997
;
Wessberg and Vallbo 1995
). In short, acceleration recordings were divided in 1.024-s segments, starting 100 ms in advance
of the presentation of the reflex- or CR-evoking stimuli. Illustrated
power spectra (Figs. 11 and 15) correspond either to a single
acceleration segment (Fig. 11) or to the mean value of power spectra
computed from 10 different acceleration segments (Fig. 15). Data were
processed for statistical analysis with the SPSS/PC + package, for
two-tailed tests with a statistical significance level of
P = 0.05. Mean values are followed, when necessary, by the standard deviation (SD). Statistical differences of mean values were determined with the help of the Student's t-test for
variables of two categories or with the ANOVA for variables of more
than two categories. Rate-position and rate-velocity relationships for
spike activity and eyelid movements were calculated by linear regression analysis and nonlinear curve fits with the
SigmaStat/SigmaPlot software. For this, lid position or lid velocity
was plotted versus the instantaneous firing rate, in a point to point
procedure. Peaks of power spectra were tested with the
2-distributed test for spectral density functions (see
Delgado-García et al. 1986
; Domingo et
al. 1997
; Gruart et al. 1995
for details).
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RESULTS |
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General properties of recorded motoneurons
The number of motoneurons recorded, identified, and analyzed in the present experiments was 317, subdivided as 61 abducens, 99 accessory abducens, and 157 orbicularis oculi motoneurons. Of those motoneurons, 163 units were recorded during conditioned eyelid responses (11 abducens, 22 accessory abducens, and 120 orbicularis oculi motoneurons). Motoneurons always were identified by their antidromic activation from the VIth nerve (abducens and accessory abducens) or from the zygomatic branch of the facial nerve (orbicularis oculi). Extracellular somatic spikes were identified by their characteristic positive-negative-positive or negative-positive shapes (see Fig. 1, A and B). Abducens motoneurons were activated antidromically at a mean latency of 0.66 ± 0.14 (SD) ms (n = 30; range, 0.4-0.8 ms), measured at the first negative peak of the spike. Accessory abducens motoneurons were activated antidromically at slightly shorter latencies: 0.51 ± 0.12 (n = 50; range, 0.4-0.7 ms). Considering the distance between the recording site and the position of the stimulating electrode, the mean conduction velocity of abducens motoneurons was 43.5 ± 15.3 m/s (n = 30; range, 20-60 m/s), whereas that of accessory abducens motoneurons was 47.5 ± 13.1 m/s (n = 50; range, 28-70 m/s). The mean latency for antidromic activation of orbicularis oculi motoneurons was 2.13 ± 0.23 ms (n = 100; range, 1.6-2.8 ms; Fig. 2C), with a mean conduction velocity of 46.9 ± 10.2 m/s (n = 100, range 27-75 m/s). These results indicate similar ranges of axonal conduction velocities for the three populations of blink-related brain stem motoneurons. The three motoneuron pools presented similar values for minimum intervals during double shock activation. Thus the mean minimum interval computed from well-isolated spikes (n > 15 for each group) was 1.2 ± 0.05 ms for abducens, 1.5 ± 0.9 ms for accessory abducens, and 1.7 ± 0.9 for orbicularis oculi motoneurons.
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All identified abducens motoneurons displayed a continuous, modulated
firing rate in relation to eye movements (Delgado-García et al. 1986). In contrast, accessory abducens motoneurons
recorded here seemed to lack a tonic firing rate. Instead they fired
almost exclusively during the early phase of reflexively evoked blinks. Additionally, they fired during large, voluntary, bilateral eye blinks.
Orbicularis oculi motoneurons fired during both early and late phases
of reflex blinks as well as during conditioned eyelid responses. About
10% of the recorded orbicularis oculi motoneurons fired tonically,
mainly when the animal voluntarily kept its eyes closed during alert
states. The other 90% of (phasic) orbicularis oculi motoneurons
sporadically showed some spontaneous, irregular, very-low-rate firing
not related to any overt motor behavior of the lids. On occasions, the
spontaneous, sustained, and isolated firing of identified orbicularis
oculi motoneurons was recorded and identified as corresponding to the
activity of a single orbicularis oculi motor unit. In such case, the
firing was not related to any behavior belonging to the animal's
repertoire but to fasciculations, observed in lid skin, and recorded
with the EMG electrodes implanted in the orbicularis oculi muscle. A
detailed account of all these behavioral peculiarities of blink-related motoneurons is presented in the following text.
Discharge profiles of abducens, accessory abducens, and orbicularis oculi motoneurons during reflexively evoked blinks
The three populations of blink-related brain stem neurons considered in this study showed quite different firing profiles during the presentation of air puffs, flashes of light, and tones and during single pulses applied to the ipsilateral supraorbital nerve (see Figs. 2 and 3).
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As illustrated in Fig. 2A, an air-puff-evoked blink
consisted of a fast downward displacement of the upper lid followed by a longer-lasting, wavy upward phase. Both the initial fast downward movement and the later small sags resulted from the phasic contraction of the orbicularis oculi muscle. For 100-ms, 3-kg/cm2 air
puffs (n = 60 presentations), the mean latency for
blink initiation was 11.8 ± 0.9 ms, the mean latency of the EMG
activity of the orbicularis oculi muscle was 7.2 ± 1.3 ms, and
the activation latency for orbicularis oculi motoneurons was 5.1 ± 0.7 ms. Thus the initial firing of orbicularis oculi motoneurons led
the EMG activity of its innervating muscle by 2 ms, and muscle
electrical activity preceded actual lid displacement by
4.5 ms. Long
(100 ms), strong (3 kg/cm2) air puffs evoked an initial
burst of action potentials in orbicularis oculi motoneurons followed by
a large-amplitude (range 0.5-7.2 mV) afterhyperpolarization (Fig.
2A). The amplitude of this afterhyperpolarization seemed to
be dependent on the number of spikes in the initial burst (Figs. 2,
8B, 10, and 11). This initial burst of activity usually was
followed, with a high degree of synchrony, by late single action
potentials, as suggested by the coincidence of each action potential
with phasic activities in the innervated orbicularis oculi muscle.
Single pulses applied to the ipsilateral supraorbital nerve evoked a
fast downward displacement of the lid followed by a slower upward
phase. Stimulation of the supraorbital nerve activated orbicularis
oculi motoneurons after a mean latency of 3.7 ± 0.9 ms
(n = 30; range, 3-6 ms). Usually, a single action
potential was evoked. A second action potential was seen at 9-12 ms
from stimulus presentation (see Figs. 1C and 2B).
This double activation of orbicularis oculi motoneurons by supraorbital
nerve stimulation corresponded quite well with the R1 and R2 responses
described in the EMG activity of the orbicularis oculi muscle as evoked by the same stimulus (Kugelberg 1952). Time intervals
between neural activity and muscle activation (
2 ms), and between
the latter and lid displacement (
4-5 ms), were similar to the
descriptions above for air-puff-evoked blinks.
Flash-evoked blinks showed a significantly (P < 0.01)
longer (n = 40; 49.1 ± 5.3 ms) latency than those
evoked by air puffs (Fig. 2C). Flash-evoked blinks were
usually of smaller amplitude and more-easily fatigable than
air-puff-evoked ones. Orbicularis oculi motoneurons fired a brief burst
of action potentials in response to flash presentations, sometimes
followed by a second burst (or just a single action potential) at an
interval of 50 ms. As illustrated in Fig. 2C (
), on
many occasions the depolarization corresponding to the late activation
of orbicularis oculi motoneurons during flash presentation did not
reach the threshold to produce an action potential.
Tones rarely evoked noticeable blinks in our experimental animals. In fact, two animals never blinked in response to tone presentation. When recorded, tone-evoked blinks presented a mean latency of 52.4 ± 10.2 ms (n = 25). Figure 2D illustrates a putative synaptic potential recorded intracellularly in an identified orbicularis oculi motoneuron during tone presentation, indicating that these cells probably are activated by acoustic inputs but only occasionally reach the threshold to evoke full spikes.
Figure 3 summarizes the more representative firing profiles of brain
stem, blink-related motoneurons during reflexively evoked blinks. Only
12% of the recorded abducens motoneurons (6 of 50) modified their
firing during reflex blinks, presenting a late and weak increase in
their discharge rate. For example, their latency of activation to
supraorbital nerve stimulation was >15 ms. Although eye movements were
not measured in the present experiments, the response of abducens
motoneurons was noticeably more related to eye rotational than to lid
downward blinking movements. This fact could explain the weakness and
variability in abducens motoneuron responses to reflexively evoked
blinks, depending probably on the adducted or abducted eye position in
the orbit before stimulus presentation (see
Delgado-García et al. 1986, 1990
). No response to tone presentation could be obtained from abducens motoneurons. Accessory abducens motoneurons presented a fast and brief increase in
their firing rates in response to air puff and supraorbital nerve
stimulations. However, the late components of air-puff-evoked lid
responses were not accompanied by any noticeable activity in accessory
abducens motoneurons (Fig. 3A). None of the recorded accessory abducens motoneurons (n = 40) fired in
response to flash or tone presentations even though synaptic potentials
were recorded both extra- and intracellularly in the accessory abducens
nucleus at the expected latencies (i.e.,
40-45 ms after stimulus
presentation, not illustrated). As shown in Fig.
4, accessory abducens motoneurons restricted their firing in the alert behaving cat to highly demanding eyelid (and, supposedly, nictitating membrane) responses. Thus air
puffs presented to the same animal on different occasions evoked
smaller or larger lid downward displacements depending on the
participation of accessory abducens motor units. In fact, a
displacement of the nictitating membrane in the cat was observed only
during eye retractions accompanying large eyelid blinks.
|
Quantitative relationships between the firing rate of abducens, accessory abducens and orbicularis oculi motoneurons and reflexively evoked and spontaneous eye blinks
The possible linear relationships between the firing rate of
selected abducens (n = 10), accessory abducens
(n = 10), and orbicularis oculi (n = 10) motoneurons and eyelid position and/or velocity during
air-puff-evoked blinks were analyzed (see Figs. 5 and 6).
Separate rate-position and rate-velocity plots were made for the early
(I) and late (II) components of eye blinks (Fig. 5A). The
firing rate of orbicularis oculi motoneurons was not linearly related
to either early (I, Fig. 5B) or late (II, not illustrated)
phases of eye blinks. Also the firing rate of abducens and accessory
abducens motoneurons did not seem be linearly related to lid position
(not illustrated). The coefficients of correlation (r) for
rate-position plots during both early and late phases of the blink
calculated for a total of 30 motoneurons (10 of each type) yielded
values of r 0.41 (P
0.05).
Conversely, orbicularis oculi motoneurons firing was related linearly
to lid velocity during both early and late phases of eye blinks (see I
and II in Fig. 5A). Figure 5, C and D,
illustrates the linear relationships between the firing rate of an
orbicularis oculi motoneuron and lid velocity during the early (Fig.
5C) and late (Fig. 5D) phases of air-puff-evoked
blinks. It should be pointed out that the slope of these linear
relationships was larger for the same motoneuron during the late (2.34 spikes/s per °/s) phase of the blink than for the early one (0.39 spikes/s per °/s). As illustrated in Fig. 6, C and
D, the mean slope of the linear relationship between the
firing rate of 10 orbicularis oculi motoneurons and the late component
of air-puff-evoked blinks (1.99 ± 0.32 spikes/s per °/s; range,
1.57-2.46) was about four times larger (P < 0.01) than the corresponding value for the early component of the same set of
blinks (n = 10; 0.41 ± 0.09 spikes/s per °/s;
range 0.27-0.57).
|
|
The firing rate of accessory abducens motoneurons was linearly related with lid velocity during the early downward phase of the blink (Fig. 6B). The mean slope of the linear relationship was 0.16 ± 0.03 spikes/s per °/s (n = 10; range 0.12-0.22). Because accessory abducens motoneurons were rarely active during the late phase of eye blinks, no numerical relationship could be established between the two variables. The firing rate of abducens motoneurons (n = 10) was not significantly related to lid velocity during either early (I) or late (II) phases of the blink (see Fig. 6A).
For purposes of comparison, Fig. 7
illustrates an air-puff-evoked eye blink and an spontaneous downward
lid movement with the kinetic characteristics of an "eyelid friendly
display" (Gruart et al. 1995). As opposed to reflex
blinks, eyelid friendly displays have a symmetric bilateral
presentation and consist of a slow, long-lasting downward lid
displacement followed by a slow upward phase. These spontaneous lid
movements were made by the animal from time to time (0.5-2/min),
usually when viewing the experimenters moving around in the recording
room. The profile of eyelid friendly responses was similar to that of
the late component of air-puff-evoked blinks (Figs. 5A and
7). Indeed, the firing rate of orbicularis oculi motoneurons during
eyelid friendly displays was related linearly to lid velocity but not
to lid position. The mean slope of the relationship between neuronal
firing rate and eyelid velocity during "eyelid friendly displays"
was 2.31 ± 0.43 spikes/s per °/s (n = 10;
range, 1.57-2.46; r
0.73; P
0.01), that is, similar to the high values (1.99 spikes/s per °/s)
obtained during the late phase (II in Fig. 5A) of
air-puff-evoked blinks, and five times larger than those (0.41 spikes/s
per °/s) obtained for the same set of orbicularis oculi motoneurons
during the early phase (I in Fig. 5A) of reflex blinks. The
long-lasting record shown in Fig. 7 illustrates the lack of tonic
activity in most of the identified orbicularis oculi motoneurons
recorded in the present study. Only when the lids were maintained
actively closed for a few seconds was a tonic activity in
10% of
recorded neurons observed.
|
Probable origin of spontaneous lid fasciculations
The spontaneous firing activity of isolated (i.e., single)
orbicularis oculi motoneurons was observed on a few occasions
(n = 9 motoneurons) during which the animal did not
make any overt spontaneous, reflex, or conditioned eyelid movements,
and only minute eyelid displacements could be observed on the
ipsilateral side. Sometimes, a small-amplitude, although evident,
fasciculation of the upper or lower subdivision of the orbicularis
oculi muscle was observed under the orbital skin. No stimulus was being
applied during those recordings, although all of the observations were made during the late recording sessions in the two animals showing this
spontaneous activity. The low amplitude of the evoked lid response
(0.1-1°), the absence of any EMG activity not accompanied by the
corresponding spike in the neuronal recording trace (see Fig.
8A), and the lack of any
noticeable lid movement on the contralateral side further suggest that
we were recording the spontaneous activity of a single motor unit.
Accordingly, we concluded that fasciculations recorded in lid position
traces and in the EMG activity of the orbicularis oculi muscle were the
result of the sole action of the recorded motoneuron (see Fig. 8,
A and B). Interestingly, when firing single
action potentials, these motoneurons presented a spike
afterhyperpolarization lasting for 40-60 ms (Fig. 8A, inset). Moreover, the units tended to fire in doublets, the
second spike evoking a larger displacement than the preceding one, as a
function of the interspike interval (Fig. 8C). Available
data indicated that the optimum interval for this potentiating effect was
10 ms, i.e., close to the time-interval values between R1 and R2
components of blinks evoked by the electrical stimulation of the
supraorbital nerve (Gruart et al. 1996
; Kugelberg
1952
). This optimum interval was one-fifth and one-eighth the
values described for fast (49.4 ms) and slow (86.9 ms) gastrocnemius muscle units also in cats (Burke et al. 1976
).
|
Spike-triggered averaging of lid movement and of the electromyographic activity of the orbicularis oculi muscle from abducens, accessory abducens, and orbicularis oculi motoneurons
The degree of synchrony between spikes produced by identified
blink-related motoneurons, the EMG activity of the orbicularis oculi
muscle, and the actual lid displacement was checked in a few
(n = 5) selected motoneurons from each of the three
motor groups considered in the present study. As shown in Fig.
9C, orbicularis oculi
motoneurons preceded by 2 ms the activation of the innervated muscle, and the latter also preceded by
4 ms lid downward
displacement (see Woody and Brozek 1969
). Although
accessory abducens motoneurons do not innervate the orbicularis oculi
muscle and, accordingly, do not act directly on lid displacement, their
action potentials also were synchronized with muscle activity and lid
reflex responses (Fig. 9B). These results suggested a
high degree of synchrony for the activation of both populations of
motoneurons in response to strong (3 kg/cm2) air puffs
presented to the cornea and tarsal skin. Because accessory abducens
motoneurons almost exclusively fired slightly preceding (
5-6 ms)
the initial, fast downward component of the reflex blink, their
apparent effect on lid movement was larger than that evoked by
orbicularis oculi motoneurons (compare the 2 set of records in Fig. 9,
B and C). On the other hand, we averaged
orbicularis oculi motoneuron spikes produced through both the early and
late phases of the blink, which probably contributed to a decrease in
the amplitude of the averaged lid displacement. Finally the firing of
abducens motoneuron action potentials was related poorly to lid
movement and, in fact, lagged behind orbicularis oculi muscle
activation by
5-7 ms (Fig. 9A).
|
Oscillatory properties of orbicularis oculi motoneurons
As illustrated in Figs. 2, 8, and 9C, the firing activities of orbicularis oculi motoneurons were correlated highly with even minute changes in the EMG activity of the orbicularis oculi muscle and with the smaller changes in eyelid position. This fact is further illustrated in Fig. 10 during a reflex lid response evoked by the electrical stimulation of the supraorbital nerve (Fig. 10B) and during spontaneous blinks (Fig. 10, A and C). In all of these intracellular records, it could be observed that a noticeable afterhyperpolarization followed each orbicularis oculi spike, with a mean duration of 47.3 ± 7.4 ms (n = 30; range 37-56 ms). As already indicated, the spike afterhyperpolarization depended in amplitude on the number of spikes present in the preceding burst (Figs. 2 and 10, A and B). Moreover, the afterhyperpolarization usually was followed by a late depolarization or rebound potential (Figs. 2C and 10, A-C). In Fig. 10B three intracellular record traces corresponding to the same orbicularis oculi motoneuron are overlapped, each evoked by a single electrical stimulus applied to the ipsilateral supraorbital nerve. As shown in Fig. 10, every single stimulation of the supraorbital nerve evoked the synaptic burst activation of the motoneuron as well as an evident oscillation of its membrane potential. Obviously subsequent spikes appeared with a higher probability riding on the top of each wave, as if it were a time window when the neuron was depolarized more easily to reach threshold for a full action potential (see Fig. 10, B and D).
|
A further attempt was made to correlate the oscillatory properties of
orbicularis oculi motoneurons and the wavy profile of air-puff-evoked
blinks. As already described (Domingo et al. 1997; Gruart et al. 1995
), and as illustrated in Fig.
11, the succession of downward sags
after the initial down phase of air-puff-evoked blinks outlasted the
duration of the stimulus, and when measured by hand from peaks in
velocity profiles, those sags yielded a mean duration of 40-60 ms. The
power spectra of the acceleration profile of the eye blink illustrated
in Fig. 11 presented a significant peak (P < 0.01) at
20 Hz, over a broadband of frequencies between 15-25 Hz. The figure
also illustrates the high degree of coincidence between the firing
profile of the orbicularis oculi motoneuron and the wavy appearance of
the lid position record. Interestingly, the average (n = 200) of single spikes corresponding to the same neuron showed a spike
afterhyperpolarization lasting
50 ms (see inset in Fig.
11). In consequence, the oscillation observed in lid acceleration
profiles could be the result of the intrinsic membrane properties of
innervating orbicularis oculi motoneurons. However, the participation
of some premotor circuits cannot be ruled out as depolarizing rebound
potentials also could be of synaptic origin (Fig. 10B).
|
Discharge profiles of abducens, accessory abducens, and orbicularis oculi motoneurons during conditioned eyelid responses
Representative profiles of eyelid CRs obtained through different
conditioning sessions are illustrated in Figs. 12-15. Latency, maximum
amplitude, peak velocity, and profile of these CRs were very different
depending on the animal and on the modality of the sensory cue used as
CS. Because this paper is devoted mostly to the study of the firing
properties of blink-related brain stem neurons, no further mention will
be made of the analysis of latency and profiles of eyelid learned
movements, which has been reported in two recent publications
(Domingo et al. 1997; Gruart et al. 1995
).
Abducens, accessory abducens, and orbicularis oculi motoneurons were
recorded systematically during the successive conditioning sessions in
the six animals. As illustrated in Fig.
12, the firing rate of identified
orbicularis oculi motoneurons changed dramatically from stages
preceding the appearance of a CR (Fig. 12A) to those during
which the CR was being formed (Fig. 12B). Being highly
phasic cells, orbicularis oculi motoneurons only rarely fired
spontaneously, unless excited experimentally by blink-evoking stimuli,
or during spontaneous eyelid movements (see preceding text). In this
situation, and during the first (1-3) conditioning sessions, the ap-AP
trace conditioning paradigm activated the firing of these motoneurons only during air puff presentations but not during the CS-US time interval. However, before a CR started to be formed, some synaptic potentials were observed during the CS-US interval (n = 2-5 per CS-US interval; see Figs. 12A and
13), some of them large enough to evoke
a single action potential (Fig. 12A). Moreover, the membrane potential background activity increased immediately after CS
presentation. By the fourth conditioning session (Fig. 12B),
the CS-US interval appeared full of action potentials (12-15 per
CS-US interval) distributed at more-or-less fixed intervals (see
averages in Fig. 14, B and
D). The progressive increase
in the number of synaptic potentials as well as in the number of full
spikes is better illustrated in Fig. 13, together with the subsequent
appearance and consolidation of the CR. Obviously, the continuous
increase in the number of action potentials during the time window
represented by the CS-US interval evoked an increase in the EMG
activity of the orbicularis oculi muscle and a ramp-like displacement
of the upper lid in the downward direction (Figs. 12 and 13). This
ramp-like profile of lid movement radiated from the CS until producing
the almost complete closing of the lids at the time of the US
presentation (see later set of intracellular records in Fig. 13). The
latency between CS presentation and the initiation of action potentials from identified orbicularis oculi motoneurons recorded in the CS-US
interval during trace ap-AP conditioning decreased from a mean value of
180 ± 17 ms (n = 10, range 158-205 ms), during the 4th conditioning session, to 52 ± 6 ms (n = 10, range 45-61 ms), during the 15th one (if the early
alpha response, clearly separated from the true CR by a
noticeable spike afterhyperpolarization is not taken into account; see
Fig. 13, left). The ramp movement of the lid was thus the
result of the low-rate, repetitive, and tonic-like firing of
orbicularis oculi motoneurons, a fact also noticed in the smaller
amplitude of muscle complex action potentials, mostly when compared
with the EMG activity of the muscle during reflex blinks (Figs.
2A and 12). It was concluded that the ramp-like profile of
CRs, as well as its wavy aspect, were the result of the low-rate and
sustained activity of orbicularis oculi motoneurons (at a dominant
oscillatory frequency of
20 Hz). Similar results were obtained when
recording the activity of identified orbicularis oculi motoneurons
during the T-AP delayed conditioning paradigm; that is, synaptic
potentials were observed during the first conditioning sessions, well
in advance of the appearance of action potentials and, obviously, of
any slight sign of a CR (not illustrated). In the case of the T-AP
conditioning paradigm, the latency between CS presentation and the
initiation of action potentials from identified orbicularis oculi
motoneurons recorded in the CS-US interval decreased from 137 ± 16 ms (n = 10, range 118-151 ms) during the 4th
conditioning session to 55 ± 8 ms (n = 10, range
45-64 ms) during the 15th one.
|
|
|
Figure 14 summarizes the activity of identified abducens, accessory
abducens, and orbicularis oculi motoneurons during the classical
conditioning of eyelid responses. To our surprise, none of the units
recorded and identified as an accessory abducens motoneuron
(n = 22) fired during the CS-US interval, i.e., during the time of CR presentation (see Fig. 14, middle set of
records). However, all of these motoneurons fired during US
presentations, indicating that their activity still was recorded by the
micropipette when firing. From the 11 abducens motoneurons recorded
during the classical conditioning of lid responses, only 1 (Fig.
14A) seemed to fire in relation to US presentation, but not
even 1 of them fired in response to the presentation of the two
different CSs (ap and T) used in the present study. It should be
pointed out that the 20-Hz, repetitive response of orbicularis oculi motoneurons triggered by CS presentation was still noticeable in the
averages (n = 10) illustrated in Fig. 14, B
and D (arrows), corresponding to the sole CS presentation.
The frequency-domain properties of CRs were analyzed quantitatively and
compared with the firing properties of recorded orbicularis oculi
motoneurons. As illustrated in Fig. 15,
CRs after CS presentation in well-conditioned animals had a power
spectrum in their acceleration profiles with a significant peak
(P < 0.01) at 20 Hz over a broadband of frequencies
between 10-30 Hz. There was also a high degree of coincidence between
the power spectra of acceleration profiles, those corresponding to the
EMG activity of the orbicularis oculi muscle, and those to the firing
rate profiles of simultaneously recorded neurons (not illustrated).
Thus the oscillation observed in lid acceleration profiles during CRs
could be the result of the tendency of orbicularis oculi motoneurons to
fire at a dominant frequency of
20 Hz.
|
Quantitative relationships between conditioned eyelid responses and the firing rate of abducens, accessory abducens, and orbicularis oculi motoneurons
The firing rate of orbicularis oculi motoneurons was plotted
against eyelid position and velocity during CRs obtained after the sole
presentation of the CS (Fig. 16). As
shown, motoneuronal activity seemed to be linearly related to eyelid
position, but not to eyelid velocity, during CRs. These results were
opposite to those mentioned in the preceding text above and illustrated in Figs. 5 and 6, suggesting that orbicularis oculi motoneurons encode
eyelid velocity during reflexively evoked blinks. In fact, no
orbicularis oculi motoneuron (n = 10) presented a
firing rate linearly related to lid velocity during CRs. The mean slope
of rate-position plots was 7.18 spikes · s1
· deg
1 (n = 10; range 4.11-10.5).
Accordingly, the firing rate of orbicularis oculi motoneurons seemed
highly synchronized during reflex blinks (Fig. 2), a finding related to
the fact that single neurons used to fired in doublets (Figs. 2, 4, 7,
and 8). This latter circumstance favors a fast lid downward
displacement for optimal interspike intervals of
10 ms. Conversely,
orbicularis oculi motoneurons changed to a low-rate, tonic firing
during CRs, a fact that explains the ramp-like profile of these lid
displacements (Figs. 12-15). This tonic firing also explains the low
peak and mean velocity of CRs (about
that of reflex
blinks).
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DISCUSSION |
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Motor roles of blink-related brain stem motoneuronal pools during spontaneous and reflexively evoked blinks
The present work is an attempt to compare the firing
characteristics of identified abducens, accessory abducens, and
orbicularis oculi motoneurons during the performance of spontaneous,
reflexively induced and classically conditioned eyelid responses in the
alert behaving cat. As shown here, each motoneuronal pool contributed in a specific manner to the generation of the different types of eyelid
response. Air-puff-evoked blinks seemed to be produced by the
cooperative participation of the three motoneuronal pools. Abducens
motoneurons presented a weak and variable relationship with upper lid
blinking responses. As already reported (Delgado-García et al. 1986, 1990
), abducens motoneurons mostly are related to rotational eye movements. Only 10-15% of abducens motoneurons seemed
to have some trigeminal input signals, but even those motoneurons fired
during eyelid blinks, depending on the position of the eye in the
orbit, making their firing not linearly related to either eyelid
position or velocity. Their contribution to eye retraction and, as a
consequence, to the passive downward displacement of the upper lid
during blink responses had necessarily to depend on the synchronous
cocontraction of the other three recti muscles, a fact not confirmed in
the present experiments, but already described in the goldfish
(Pastor et al. 1991
) and in the rabbit (Evinger and Manning 1993
).
It has been described in cats that accessory abducens motoneurons fired
a fast burst of action potentials in response to long, strong air puffs
presented to the cornea and periorbital skin (Delgado-García et al. 1990). Their firing in
response to electrical stimulation of the supraorbital nerve consisted
of a double activation, usually reduced to a couple of spikes separated
by a 10-ms interval. This double activation corresponds to the R1 and
R2 responses recorded in the EMG activity of the orbicularis oculi
muscle in humans (Kugelberg 1952
) and also was observed
directly in eye retractional movements during air-puff-evoked blinks
(Delgado-García et al. 1990
). The firing rate of
accessory abducens motoneurons was found to be linearly related to lid
velocity, but not to lid position, during reflex blinks, which further
indicates the contribution of this motoneuron pool to a fast closing of
the eye during protective eyelid responses. Accessory abducens
motoneurons recorded here did not respond to flash or tone presentations.
The firing rate of identified orbicularis oculi motoneurons followed
the profile of lid displacements during blinks evoked by air puffs,
flashes of light, and tones, and during the electrical stimulation of
the supraorbital nerve. The EMG activity of the orbicularis oculi
muscle showed an extremely high degree of synchrony with the presence
of spikes in the neural recording, suggesting a phasic, synchronous
firing of orbicularis oculi motoneurons activated during a given eyelid
reflex response. The firing of identified orbicularis oculi motoneurons
was organized in an early double burst of spikes that evoked the R1 and
R2 responses in the innervated muscle (Cruccu et al.
1987; Hiraoka and Shimamura 1977
;
Kugelberg 1952
), and a fast downward displacement of the upper lid. This burst usually was followed by a late response involving
a sustained activation of the orbicularis oculi muscle and a slow
displacement of the lid further in the downward direction. The firing
of orbicularis oculi motoneurons could not be related linearly to
eyelid position, but it was related significantly to eyelid velocity
during both the early and late phases of air-puff-evoked blinks. This
finding confirms that orbicularis oculi motoneurons are exclusively
involved in the fast downward displacement of the lid. Thus the
maintenance of lid position during the intervals between successive
blinks is achieved by tonic position signals present in the levator
palpebrae muscle (Evinger 1995
; Fuchs et al.
1992
; Gruart et al. 1995
; Trigo et al.
1999
).
Involvement of abducens, accessory abducens, and orbicularis oculi motoneurons in eyelid classically conditioned responses
A CR consists of a ramp-like downward lid displacement that seems
to be generated in a quantal manner, from a basic 50-ms downward wave
or sag (Domingo et al. 1997). As shown here, even a
single motor unit can move the lid. Although it is more probable that
"quantal" lid movements observed during the acquisition of CRs
are the result of the synchronous activation of more than one motor
unit, the perfect time coordination between different motoneurons
inside the orbicularis oculi subdivision of the facial nucleus suggests
a repetitive mechanism intrinsic to the motoneuronal membrane, i.e., an
oscillation triggered by CS presentation. The present results further
confirm that the wavy aspect of CRs is the result of the peculiar
firing pattern of orbicularis oculi motoneurons during the learning
process. The finding that motoneuronal firing was correlated with
eyelid position during CRs, but with eyelid velocity during reflex
responses, suggests a putative mechanism involving the functional
peculiarities of the motoneuronal pool. For example, it could be
expected that tonic signals typical of CRs were generated at a
motoneuronal level by inputs arriving at distal dendrites, whereas
phasic firing characterizing the activity of the motoneuron during
reflex responses was produced by a strong input impinging on the soma
and proximal dendrites.
The electrical activity of identified orbicularis oculi motoneurons was modified by the classical conditioning process well in advance (i.e., days) of the appearance of the first single action potential, and even more in advance if correlated to the appearance of a noticeable downward wave in the lid position records during the CS-US interval. This finding indicates a slow building up of the CR that remains unnoticed unless recorded directly from motoneuronal membrane activity. In this sense, further experiments in a more stable, and better-controlled paradigm are necessary to unravel the early stages of the appearance of the CR at the motoneuronal level.
The lack of involvement of accessory abducens motoneurons in CRs was
rather a surprise. Nevertheless, given the high-threshold of this
motoneuronal pool in cats, as reported in both acute (Baker et
al. 1980) and chronic (Delgado-García et al.
1990
; present work) experiments, their behavior during
conditioned eyelid responses is understandable. In cats, CRs have a
peak velocity about one order of magnitude lower than that observed
during air-puff-evoked blinks (Domingo et al. 1997
;
Gruart et al. 1995
), a fact further confirmed here. In
this sense, CRs are generated, at least in cats, from a neural site
unable to provide enough synaptic excitation to generate full spikes in
accessory abducens motoneurons. The peculiar morphology and passive and
active membrane properties of accessory abducens motoneurons make them
suitable for burst firing during fast and huge depolarizing potentials,
mostly from corneal origin (Baker et al. 1980
;
Grant and Horcholle-Bossavit 1983
), but apparently
unable to fire in a low, stable rate during CRs.
Origin of the 20-Hz oscillation underlying reflex and conditioned eyelid responses
Brain stem and cortical projections to the facial nucleus have
been described in detail based on histological (Courville
1966; Holstege et al. 1986a
,b
; May et al.
1996
; Mizuno and Nakamura 1971
; Pozo and
Cerveró 1993
; van Ham and Yeo 1996
) and
electrophysiological experiments (Fanardjian and Manvelyan
1984
, 1987a
,b
; Fanardjian et al. 1983a
,b
;
Hiraoka and Shimamura 1977
; Vidal et al.
1988
) carried out in rats, rabbits, cats, and monkeys. Briefly,
the principal and trigeminal nuclei, the dorsal red nucleus, the
superior colliculus, specific regions of the mesencephalic, pontine and medullary reticular formation, and different cortical areas were described as projecting directly (i.e., monosynaptically) or indirectly (di- or polysynaptically) to blink-related brain stem motoneuronal pools, mainly to the accessory abducens nucleus and to the orbicularis oculi subdivision of the facial complex. Most of those descriptions indicated that trigeminal signals arriving at the main abducens nucleus
follow a polysynaptic pathway. Taken together, these hodological and
electrophysiological descriptions are in agreement with data reported
here on the firing peculiarities of abducens, accessory abducens, and
orbicularis oculi motoneurons. However, some comments should be made
about discrepancies between the described pathways and the functional
findings. For example, a similar pathway, originating at the olivary
pretectal nucleus and at the nucleus of the optic tract, has been
reported to arrive at both the accessory abducens and orbicularis oculi
motoneuronal pools carrying signals related to light-evoked blinks
(Holstege et al. 1986a
,b
). Because, as shown here,
accessory abducens motoneurons are not activated by light flashes, we
must accept that the actual function of a given pool of neurons is not
determined exclusively by the presence of a given neural pathway or
circuit and that other factors, such as intrinsic neuronal properties
and particular behavioral situations, also can play an important role.
A similar comment could be made regarding the lack of involvement of
accessory abducens motoneurons in CRs, when this motoneuronal pool has
been reported to be one of the two final common pathways involved in
CRs (Holstege et al. 1986a
,b
; Kim and Thompson
1997
). A parsimonious explanation could be that, perhaps, the
weakness of the stimulus used here as CS rendered unnecessary any
nictitating membrane displacement during the CR. In
electrophysiological terms, synaptic inputs to accessory abducens
motoneurons produced less depolarization than that needed to evoke full
action potentials in those motoneurons.
Another important point is the proposal of preferred neural pathways of
functional relevance, such as the projection of the cerebellar
interpositus nucleus that reaches facial and accessory abducens
motoneurons via the dorsal red nucleus (Bloedel 1992; Fanardjian et al. 1983a
; Kim and Thompson
1997
) or the cerebral pathway that reaches the facial nucleus
via the spinal trigeminal nucleus (Fanardjian et al. 1983a
,
1987a
,b
). These indirect, polysynaptic pathways are of major
importance because they could be involved in slowly building, tonic,
late motor responses similar to those described here as CRs, in
opposition to fast, precise and strong motor responses supported on
somatic projections, such as the disynaptic arc involved in the blink
reflex (Baker et al. 1980
; Cruccu et al.
1987
; Evinger 1995
; Evinger et al.
1991
; Gruart et al. 1995
; Hiraoka and
Shimamura 1977
; Kugelberg 1952
). In fact, some
of these slow-building, polysynaptic inputs are mediated preferentially
by axon terminals endings on distal dendrites, such as those arriving
at orbicularis oculi motoneurons from the red nucleus (Mizuno
and Nakamura 1971
) or from the spinal trigeminal nucleus
(Fanardjian et al. 1983a
). The presence of neurons
firing at dominant frequencies ~20-30 Hz and highly synchronized
with the EMG activity of the orbicularis oculi muscle or with actual lid displacements has been reported in both the cerebral cortex (Aou et al. 1992
) and the interpositus nucleus
(Gruart and Delgado-García 1994
). The
contributions of both neural pathways should not be ruled out as
priority functional elements for generating eyelid learned responses.
The present findings further confirm previous suggestions
(Domingo et al. 1997) that orbicularis oculi motoneurons
by themselves play an important role in the kinematics, time-domain,
and frequency-domain properties of eyelid motor responses. For example,
the presence of postspike rebound potentials (Llinás
1984
; Steriade et al. 1990
) in orbicularis oculi
motoneurons could endow them with the possibility of generating
repetitive spikes, at a low tonic rate, similar to those discharges
observed in the EMG activity of the orbicularis oculi muscle and in the
power spectra of acceleration records corresponding to lid reflex and
conditioned responses (Domingo et al. 1997
;
Gruart et al. 1995
).
The oscillations observed in lid responses are not produced by a delay
in stretch-reflex feedback signals because proprioceptors have not been
found in the orbicularis oculi muscle from either structural
(Porter et al. 1989) or functional (Trigo et al.
1997
, 1998
) evidence. Facial motoneurons seem to have some
intrinsic membrane properties that allow them to fire repetitively at
20 Hz as a basic cellular functional substrate for actual reflex and
learned eyelid responses. First, as suggested by reported here and
described in acute experiments in cats for both accessory abducens
(Baker et al. 1980
) and facial (Fanardjian et al.
1983b
) motoneurons, these blink-related motoneurons present a
spike afterhyperpolarization lasting
50 ms. Moreover, the membrane
potential of orbicularis oculi motoneurons has a tendency to oscillate
at
20 Hz in response to a membrane depolarization as reported here
and as recently described in rats in vitro
(Magariños-Ascone et al. 1997
). These membrane
potential oscillations were blocked by TTX, suggesting the involvement
of a Na2+ conductance in their generation. Moreover, late
afterdepolarizations in the form of small humps at the end of the
afterhyperpolarization have been observed here and described in more
precise recordings in extraocular motoneurons (Gueritaud
1988
). Recently, the presence of specific membrane ionic
conductances able to produce rebound responses after their
hyperpolarization has been described in rat facial motoneurons
(Magariños-Ascone et al. 1997
). These rat facial
motoneurons are susceptible to change to a stable, tonic, repetitive
firing after the application of carbachol
(Magariños-Ascone et al. 1997
). Nevertheless, it
cannot be ruled out completely that these late-depolarizing rebound
responses could have a synaptic origin because, as reported in acute
experiments (Baker et al. 1980
; Grant and
Horcholle-Bossavit 1983
), accessory abducens motoneurons present a 10- to 140-ms depression during conditioning-test
stimulations, suggesting the presence of local (trigemino-facial)
circuits able to facilitate repetitive firing at selected time
intervals. Indeed, the high degree of synchronization between different
muscles (extraocular recti, retractor bulbi and orbicularis oculi)
during reflex and conditioned eyelid responses (Berthier
1992
; McCormick et al. 1982
) also suggests the
presence of higher-level centers triggering the activity of the set of
motoneurons involved. Some cortical areas and the cerebellar
interpositus nucleus could be involved in this synchronizing mechanism
because both structures have been reported to fire at dominant
frequencies of
20 Hz in alert behaving cats during reflex and
conditioned eyelid responses (Aou et al. 1992
;
Gruart and Delgado-García 1994
).
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ACKNOWLEDGMENTS |
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We thank R. Churchill for help in editing the manuscript.
This experimental work was supported by grants from the Spanish Comisión Interministerial de Ciencia y Tecnología (SAF 96-0160) and Dirección General de Investigación Científica y Técnica (PB93-1175), and the Junta de Andalucía (PAI, CVI-122).
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FOOTNOTES |
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Address for reprint requests: J. M. Delgado-García, Laboratorio de Neurociencia, Facultad de Biología, Avda. Reina Mercedes, 6, 41012-Sevilla, Spain.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 June 1998; accepted in final form 14 December 1998.
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REFERENCES |
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