1Laboratorio de Neurociencia, Universidad de Sevilla, 41012 Seville, Spain; and 2Behavioral Neuroscience Unit, National Institute of Neurological Disorders and Stroke, National Insititutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Gruart, Agnès,
Bernard
G. Schreurs,
Eduardo Domínguez del Toro, and
José
María Delgado-García.
Kinetic and Frequency-Domain Properties of Reflex and Conditioned
Eyelid Responses in the Rabbit.
J. Neurophysiol. 83: 836-852, 2000.
Eyelid position and the
electromyographic activity of the orbicularis oculi muscle were
recorded unilaterally in rabbits during reflex and conditioned blinks.
Air-puff-evoked blinks consisted of a fast downward phase followed
sometimes by successive downward sags. The reopening phase had a much
longer duration and slower peak velocity. Onset latency, maximum
amplitude, peak velocity, and rise time of reflex blinks depended on
the intensity and duration of the air puff-evoking stimulus. A
flashlight focused on the eye also evoked reflex blinks, but not
flashes of light, or tones. Both delayed and trace classical
conditioning paradigms were used. For delayed conditioning, animals
were presented with a 350-ms, 90-dB, 600-Hz tone, as conditioned
stimulus (CS). For trace conditioning, animals were presented with a
10-ms, 1-k/cm2 air puff, as CS. The unconditioned stimulus
(US) consisted of a 100-ms, 3-k/cm2 air puff. The stimulus
interval between CS and US onsets was 250 ms. Conditioned responses
(CRs) to tones were composed of downward sags that increased in number
through the successive conditioning sessions. The onset latency of the
CR decreased across conditioning at the same time as its maximum
amplitude and its peak velocity increased, but the time-to-peak of the
CR remained unaltered. The topography of CRs evoked by short, weak air
puffs as the CS showed three different components: the alpha response to the CS, the CR, and the reflex response to the US. Through conditioning, CRs showed a decrease in onset latency, and an increase in maximum amplitude and peak velocity. The time-to-peak of the CR
remained unchanged. A power spectrum analysis of reflex and conditioned
blink acceleration profiles showed a significant 8-Hz oscillation
within a broadband of frequencies between 4 and 15 Hz. Nose and
mandible movements presented power spectrum profiles different from
those characterizing reflex and conditioned blinks. It is concluded
that eyelid reflex responses in the rabbit present significant
differences from CRs in their profiles and metric properties,
suggesting different neural origins, but that a common
8-Hz neural
oscillator underlies lid motor performance. According to available
data, the frequency of this putative oscillator seems to be related to
the species size.
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INTRODUCTION |
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The nictitating membrane/eyelid response has been
used for more than 60 yr as a primary experimental model for studying
the neuronal organization of reflex responses and, more importantly, for understanding the mechanisms underlying motor learning. Although early studies were carried out mostly in humans (Berstein
1934; Marquis and Porter 1939
), the rabbit has
been the animal of choice during the past four decades
(Gormezano et al. 1962
). Most of these studies were
carried out by recording the nictitating membrane response (that
passively follows eyeball retraction) as a measure of sensorimotor
changes taking place in blink-related circuits during learning of
different conditioning paradigms (Gormezano et al. 1983
;
Schreurs and Alkon 1990
; Thompson and Krupa
1994
; Welsh 1992
; Woody 1986
).
Although available data in rabbits have been extremely important for
understanding the basic processes related to the acquisition of new
motor skills, the passive character of nictitating membrane displacements makes certainly difficult its use in quantitative studies
of their kinetic and, mostly, frequency-domain properties. The search
coil in a magnetic field technique has been successfully used in
humans, cats, rabbits, and guinea pigs (Evinger et al. 1984, 1991
; Gruart et al. 1995
)
for the precise recording of upper eyelid movements. Because the
kinetics of conditioned eyelid responses have been recently described
in cats (Gruart et al. 1995
), the enormous amount of
available information on nictitating membrane conditioning in rabbits
suggests that a similar study of lid kinetics should be carried out in
the latter, for comparative and integrative purposes.
Moreover, a recent description of the frequency-domain properties of
reflex and conditioned eyelid reactions in cats has suggested the
existence of a 20-Hz oscillator underlying lid movements (Domingo et al. 1997
). Although not explicitly reported,
human eyelid learned responses present evident discontinuities in
downward lid displacements (Marquis and Porter 1939
). A
similar oscillation can be observed in nictitating membrane movements
(Welsh 1992
) or in the electromyographic (EMG) activity
of the orbicularis oculi muscle during classical conditioning of
blinking responses in rabbits (Berthier 1992
). The
reported oscillatory behavior (at
20 Hz) of pericruciate cortex
neurons in the cat during the conditioning of eyelid movements
(Aou et al. 1992
) suggests that these oscillations are
not the result of the inertial and viscoelastic properties of moving
lids, but a basic, neural background for the appropriate execution of
motor responses (Llinás 1991
). Also, cat
cerebellar interpositus neurons seem to fire in an oscillatory way
during reflex blinks, precisely following the successive eyelid excursions (Gruart and Delgado-García 1994
). A
similar oscillation has been reported underlying firing properties of
facial motoneurons innervating the orbicularis oculi muscle in alert
cats (Trigo et al. 1999a
) and in vitro studies in rats
(Magariños-Ascone 1999
). It seems interesting to
study the frequency-domain properties of rabbit eyelid responses as a
way to determine whether such oscillatory mechanisms are present in lid
motor performance, and to compare them with available data in other
species (Domingo et al. 1997
). Moreover, a complete
characterization of eyelid reflex and conditioned responses will help
to the identification of functional properties of underlying neural circuitry.
The present experiments were carried out in rabbits provided with an
upper lid search coil and with EMG recording electrodes implanted in
the ipsilateral orbicularis oculi muscle. Reflex blinks were induced by
puffs of air and by electrical stimulation of the ipsilateral
supraorbitary branch (SO) of the trigeminal nerve. Effects of other
blink-evoking stimuli (light flashes, tones) were also examined. The
profile and metric properties of evoked lid responses were analyzed as
a function of the intensity and duration of air puff stimulation.
Quantitative relationships between maximum lid velocity and duration of
the closing (downward) phase of the blink and lid displacement were
determined and discussed in relation with available data for cats
(Gruart et al. 1995) and humans (Evinger et al.
1991
). Reflexively evoked blinks were also compared with the
activity induced in the EMG of the orbicularis oculi muscle. Animals
were trained with trace and delayed conditioning paradigms. In both
cases, the unconditioned stimulus (US) was a long, strong air puff. For
trace conditioning, a short, weak air puff (which finished 250 ms
before US onset) was used as a conditioned stimulus (CS), whereas
delayed conditioning was achieved with a long-lasting tone (which
finished simultaneously with the US). The topography and kinetics of
eyelid conditioned responses (CRs) were analyzed with the same
mathematical procedures used for reflex blinks. Finally,
frequency-domain properties of reflex and conditioned eyelid responses
were studied as well, and the results compared with available data for
the same motor system of other species (Domingo et al.
1997
). A preliminary report of this work has been presented in
abstract form (Gruart et al. 1997
).
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METHODS |
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Subjects
Experiments were carried out on 10 adult rabbits (New Zealand white albino) weighing 2.3-2.7 k on arrival from an authorized supplier (Iffa-Credo, France). Animals were prepared for the chronic recording of left upper eyelid displacements and of EMG activity of the ipsilateral orbicularis oculi muscle. In two of the rabbits, the SO nerve was stimulated. All experimental procedures were carried out in accordance with the guidelines of the European Union Council (86/609/EU) and following the Spanish regulations (BOE 67/8509-8512) for the use of laboratory animals in chronic experiments.
Surgical procedures
Animals were anesthetized with an intramuscular mixture of
ketamine (35-50 mg/kg), acepromazine (0.3 mg/kg), and xylazine (5 mg/kg) following a protective injection of atropine sulfate (0.4 mg/kg
im) to prevent unwanted vagal reflexes. Mepivacaine hydrochloride (2%)
was injected routinely into wound margins. A five-turn coil (3 mm diam)
was implanted into the center of the left upper eyelid as close as
possible to the lid margin. Coils were made of Teflon-coated stainless
steel wire (A-M Systems, Everett, WA) with an external diameter
of 50 µm. Coils weighed 10-15 mg and did not impair movement or
cause any lid drooping compared with the contralateral (right) upper
eyelid. Animals were also implanted with bipolar hook electrodes in the
left orbicularis oculi muscle. These electrodes were made of the same
wire as the coils and bared 1 mm at the tip. One of the orbicularis
oculi EMG electrodes was fixed 1-2 mm posterior to the external
canthus, close to the zygomatic subdivision of the facial nerve, and
the other was implanted
1-mm lateral to the eyelid coil. In two of the rabbits, a second bipolar hook electrode (made of the same wire)
was implanted. One of the electrode tips was fixed at the SO nerve, and
the other was anchored 1 mm laterally. A silver electrode (1 mm diam)
was attached to the skull as a ground. Finally, terminals of lid coil,
and of EMG, SO, and ground electrodes were soldered to a nine-pin
socket. All of these connectors were covered with acrylic resin, and
the whole system was attached to the skull with the aid of four small
screws fastened and cemented to the bone.
At the end of the recording sessions, animals were deeply anesthetized with pentobarbital sodium (50 mg/kg ip) and perfused transcardially with saline and 10% Formalin. The proper location of recording lid coil and EMG electrodes was then checked.
Recording sessions
Recording sessions began 2 wk after surgery. Sessions of ~80
min per day, to a total of 10-12, were carried out with each animal.
Each rabbit was placed in a Perspex restrainer specially designed for
limiting the animal's movements (Gormezano et al. 1983). The box was placed on the recording table and was
surrounded by a black cloth. The recording room was kept softly
illuminated and a 60-dB background white noise was switched on during
the experiments. For all the subjects, the first session consisted of
the adaptation of the rabbit to the restrainer and to the experimental conditions. Animals remained sitting for 1 h, and they were
presented, at random, with 6 air puffs of 50 ms of duration and 3 k/cm2 of pressure. Data illustrated in Figs.
1 and 2
were obtained during the second recording session, when a set of 48 air
puff stimuli of different pressures and duration were presented
randomly at time intervals of 20-40 s. Data shown in Figs. 4-10 were
obtained from classical conditioning of eyelid responses using two
different conditioning paradigms as described below. Four animals were
assigned at random to each of these paradigms. The remaining two
animals were submitted to seven recording sessions to collect
reflexively evoked blinks for parametric analyses. Data from these
sessions are presented in Figs. 3, 9, and
10.
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Recording of eyelid movements and EMG activity of the orbicularis oculi muscle
Eyelid movements were recorded with the magnetic field search
coil technique. As described in detail elsewhere (Gruart et al.
1995), eyelid coils were calibrated with a transparent
protractor placed sagittally to the animal's head and with its center
located at the external canthus of the lids. During eyelid calibration, rabbits were seated in the Perspex restrainer with the head
immobilized, and eyelid closures were evoked with light taps. Upper
eyelid maximum opening ranged from 32 to 48° for the 10 animals. For the sake of homogeneity, the gain of the recording system was adjusted
to yield 1 V per 10°. The EMG activity of the orbicularis oculi
muscle was recorded with the help of differential amplifiers (AM 502 Tektronix) at a bandwidth of 0.1 Hz to 10 kHz.
Stimuli evoking movements
Before classical conditioning sessions were started, the eight animals received a set of air puff stimuli of different pressures (1, 2, 3, and 4 k/cm2) and duration (10, 50, and 100 ms). To allow a complete return of the upper lid to its resting position during air puff presentations, stimuli were applied with a random time interval of 20-40 s. With respect to air puff duration and intensity, the order of presentation was also determined at random. Air puffs were applied through the opening of a plastic pipette (3 mm diam) attached to a metal holder fixed to the animal's nine-pin socket. This allowed the pipette to follow the spontaneous movements of the animal's head. The tip of the pipette was placed 1 cm away from the upper part of the cornea. The latency of the air puff in reaching corneal and lid mechanoreceptors was calculated at the beginning of the recording sessions with a microphone located at the same site as the eye. The recorded signal was rectified, integrated, and fed into the computer as 1-V square pulses for latency measurements.
Two of the rabbits were not subjected to the classical conditioning
procedure but were presented with different blink-evoking stimuli.
Auditory stimulation consisted of two different tones (600 and 6,000 Hz) applied for 10-100 ms at 90 dB. The loudspeaker was placed 1 m in front of the animal's head. Bright full-field flashes lasting for
1 ms were used as visual stimulation. These two animals also
received a set of air puff stimuli of different pressures and duration,
as indicated above. In addition, sets of air puffs were applied to the
whiskers, and superior and inferior eyelids. A set of single SO stimuli
(cathodal, square, 50 µs, <1-mA pulses) was also presented to these
two animals. To avoid habituation or response fatigue, stimuli of the
same modality, but of different values, were presented in blocks of
10-50 stimuli at varying intervals (20-40 s). A minimum of 4-6 min
was allowed between successive presentations of stimuli of different modalities.
Occasionally, an external 10-turn coil was located on the nose or under the mandible for recording movements during the smelling or eating of food. This coil was made of 1-mm diam, enamel-coated copper wire.
Classical conditioning paradigms
Classical conditioning of eyelid movements was achieved by the use of either delayed or trace conditioning paradigms. For delayed tone-air puff (T-AP) conditioning, a 350-ms, 600-Hz, 90-dB tone was presented to the animal as CS. The tone was followed 250 ms from its onset by a 100-ms, 3-k/cm2 air puff directed to the left cornea as US. The tone and the air puff co-terminated. For trace weak air puff-strong air puff (ap-AP) conditioning paradigm, animals were presented with a short (10 ms), weak (1 k/cm2) air puff as CS, followed 250 ms later by the same US as that presented during the delayed conditioning paradigm. Both CS and US were presented to the left side. Each of the classical conditioning paradigms was used to train four animals.
For both conditioning paradigms, the conditioning session consisted of
66 trials separated at random by 50- to 70-s intervals. Six of the 66 trials were test trials, and the CS was presented alone. The daily
conditioning session lasted for 80 min, and each animal was trained
for seven successive days. An animal was considered to be conditioned
when it was able to produce 95% of CRs per session to the CS-US paired presentation.
Ancillary observations
To complete data illustrated in Fig. 11, additional experiments were carried out in four Wistar rats (230-260 g) and three pigmented guinea pigs (420-580 g) obtained from local official suppliers. Those animals were prepared for the chronic recording of eyelid position as described here for rabbits. During recordings sessions (n = 2 per animal) they were only presented with air puffs as blink-evoking stimulus. No attempt was made to obtain classically conditioned blinks from them. The holding, stimulation, and recording systems were adapted to animals' size.
Data collection and analysis
The horizontal and vertical position of the upper eyelid, the unrectified EMG activity of the orbicularis oculi muscle, 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. Data were transferred later through an analogue digital converter (CED 1401-plus, CED, Cambridge, UK) to a computer for quantitative off-line analysis. Data were sampled at 1,000 Hz, with an amplitude resolution of 12 bits.
Commercial computer programs (Spike2 and SIGAVG from CED; MATLAB, The Mathworks; and Corel Draw, Corel Corporation, Ontario, Canada) were used to display single, overlapping, averaged, and raster representations of eyelid position, velocity, and acceleration, and of EMG activity of the orbicularis oculi muscle. These programs also allowed the quantification, with the aid of cursors, of the onset latency, latency to the peak, amplitude and peak velocity of the eyelid displacement, and the onset latency, peak amplitude and area of the rectified EMG activity of the orbicularis oculi muscle.
Some analyses required the development of new programs. Velocity and
acceleration traces were computed digitally as the first and second
derivative of lid position records, following low-pass filtering of the
data (3 dB cutoff at 50 Hz and a zero gain at
100 Hz). As
explained in detail elsewhere (Domingo et al. 1997
), the
power of the spectral density function (i.e., the power spectrum) of
selected data were calculated using a fast Fourier transform to
determine the relative strength of the different frequencies present in
lid displacements and EMG recordings. The power spectra of lid
movements were calculated exclusively from the corresponding acceleration (Domingo et al. 1997
; Wessberg and
Vallbo 1995
). Concisely, acceleration recordings were divided
into 1.024-s segments, starting 100 ms in advance to any blink-evoking
stimulus. Segments containing CRs were selected exclusively from those
obtained during the presentation of the CS alone. This design allowed
the complete evoked response (from eyelid, nose, and mandible) to be
contained in the segment, with a spectral resolution of 0.97 Hz. The
spectral power of EMG records was calculated in the same way.
Autocorrelation of acceleration recordings as illustrated in Fig.
8E, and cross-spectral values between acceleration and EMG
recordings as illustrated in Fig. 8D, were calculated
according to available statistical tools (Bendat and Piersol
1986
; Domingo et al. 1997
). The coherence spectrum was normalized to a 0 to 1 scale.
Statistical analyses were carried out using the SPSS package (SPSS,
ILL), for a statistical significance level of P = 0.05. Mean values are followed when necessary by their standard deviations (SD). Unless otherwise indicated, mean values were calculated from 16
measurements collected from a minimum of 2 animals. Latency measurements were carried out with the help of a special graphic program determining the intersection point between resting and deflected eyelid positions. The rise time of downward phase of reflex
responses (Fig. 2F) was calculated with the help of the same
program. Statistical differences of mean values were determined by
ANOVA. Regression analyses were carried out using
100 measurements collected from at least two animals. Peaks of power spectra were tested
with the
2-distributed test for spectral
density functions.
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RESULTS |
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General characteristics of experimentally induced eyelid movements
Before presentation of any kind of blink-evoking stimulus, animals rarely made spontaneous blinks (1-2 per hour). Once air puff stimulation sessions were started, 60% of rabbits made spontaneous eye blinks of large amplitude, mostly within a few tenths of a second after evoked reflex responses.
Air-puff-induced blinks consisted of a fast downward phase followed by
a much slower upward movement until the lid's initial position was
reached (Fig. 1A). For air puffs of 50 ms duration, a
double slope was noticed during the initial fast downward phase. This
double slope was more easily detected in the lid velocity and
acceleration profiles (Fig. 1A). Mean latency of blinks
elicited by 50-ms, 3-k/cm2 air puffs was
19.8 ± 4.4 ms (mean ± SD; n = 16). For this
stimulus, the downward phase of the elicited blink presented a mean
amplitude of 14.2 ± 7.9° (n = 16), which was
not enough to produce the complete closure of the eye, even with the
addition of the upward movement of the lower lid (not measured). Mean
peak velocity reached by the lid during its downward trajectory was
626.3 ± 386.5°/s (n = 16). The time elapsed
from stimulus presentation until maximum lid displacement was 94.3 ±19.3 ms (n = 16). When an air puff lasting for 100 ms
was applied, the initial, fast downward lid displacement was followed
by successive small sags also in the direction of closure (Fig.
2B).
Although in some sessions the number of blink-evoking stimuli was considerable, animals always reopened the eye after stimulation. The duration of complete reopening of the lid to the initial position could last >1 s. For this reason, eyelid upward movements were measured until 90% of the total eyelid displacement was reached. The mean duration of upward phases of eye blinks induced by 50-ms, 3-k/cm2 air puffs was 463 ± 92.1 ms (n = 16), and the mean peak velocity reached during upward displacements was 243.3 ± 19.3°/s (n = 16). The time constant, calculated as the time needed by the lid to return two-thirds of the distance to its initial position showed a mean value of 73.2 ± 21.2 ms (n = 16).
Air puffs presented to the cornea evoked a double EMG activation of the
orbicularis oculi muscle at 12.5 ± 1.8 and 28.7 ± 3.5 ms
(n = 16), as computed for stimuli of 3
k/cm2 of pressure. Thus muscle activity preceded
the initial downward phase of the blink by 7 ± 1.2 ms
(n = 16). For air puffs of 50 ms and 3 k/cm2, the peak amplitude of the rectified EMG
showed a mean value of 1.3 ± 1.2 mV (n = 16), and
its total area had a mean value of 21.82 ± 18.9 µV · s
(n = 16). The area of the rectified EMG was calculated
for the 300 ms from stimulus presentation (see Gruart et al.
1995
).
Power spectrum analyses were carried out to determine the frequency
components of the successive downward waves of the air-puff-evoked blinks. The mean power spectra of 10 acceleration records from air-puff-evoked blinks showed a dominant (P = 0.01)
peak at 8 Hz over a broadband of frequencies between 4 and 15 Hz
(Fig. 1B).
No eyelid movement could be reflexively induced by flash or tone
stimulation in any of the animals. As already described (Manning and Evinger 1986), only when a flashlight was located very
close to the cornea did the animal close its eyelids. The mean power spectra of flashlight-evoked blinks showed a dominant peak
(P < 0.001) at a lower (6 Hz) frequency.
Relationships of amplitude with duration and peak velocity for air-puff-evoked blinks
As illustrated in Fig. 1, mean values presented by the down- and upward phases of reflex blinks showed a significant variation when air puff parameters were modified. All the animals tested (n = 6) showed a statistically significant (P < 0.0001) linear relationship between the peak velocity achieved during the downward phase of the blink and the maximum amplitude of the evoked movement (Fig. 1C, bottom). The same significant (P < 0.0001) linear relationship was found for the upward phase of reflex blinks, but with a slope of one-third the value for the downward phase. These results indicate that for a 10° movement of the upper eyelid, if the movement is in the downward direction its maximum velocity will be threefold that when the movement corresponds to the upward phase of air-puff-induced blinks.
Although the relationship between the maximum amplitude of eyelid
reflex responses and their duration was also statistically significant
(P < 0.0001), it showed very low coefficients of
correlation (r < 0.4) for both upward and downward
phases of the blink (Fig. 1C, top). The low slope recorded
for this relationship (0.4 ms/deg for the downward phase and 6.1 ms/deg
for the upward phase) is in agreement with the constancy in rise time
of the downward phase during the presentation of air puffs of different
intensity and/or duration (see Fig. 2F). According to these
amplitude-peak velocity and amplitude-duration relationships, the
duration of a 10° lid opening movement induced by a puff of air is
15 times longer than the duration of lid displacement in the closing
direction during the same blink.
Dependence of air-puff-evoked blinks on stimulus parameters
Results of the quantitative study of lid movements during the presentation of air puff stimuli of different pressures and duration are shown in Fig. 2. The latency to blink onset decreased with the increase in air puff pressure, and it was unrelated to stimulus duration (Fig. 2C), except for 10-ms, 1-k/cm2 air puffs, which rarely evoked lid responses, and when they did, the responses showed a large onset latency (52.6 ± 7.4 ms). Latency values decreased in a statistically significant (P < 0.01), exponential-like manner, from 52.6-41.6 ms for responses evoked by air puffs of 1 k/cm2 to 18.3-17.7 ms those ones evoked by air puffs of 4 k/cm2. Maximum amplitude of reflexively evoked downward lid movements increased linearly (P < 0.01) with increased air puff pressure for stimuli lasting 50 and 100 ms (Fig. 2D). Mean maximum amplitude values increased from 6.8-7.3° for air puffs of 1 k/cm2 to 20.4-22.7° for air puffs of 4 k/cm2. No significant trend was observed in the mean maximum amplitude for reflex responses evoked by stimuli of 10 ms during increasing air puff pressures. In this latter case, blink amplitude remained between 3.8 and 6.1° for the different air puff pressures.
Peak velocity of the downward phase of air-puff-evoked blinks increased with a statistically significant (P < 0.01) linear trend with air puff pressure for stimuli of 50 and 100 ms (Fig. 2E). Mean peak velocity values increased from 278.7-284.1°/s for 1 k/cm2 air puffs to 1,210-1,352°/s for 4 k/cm2 air puffs. Although mean peak velocity for 10-ms air puffs increased slightly with the increase in air pressure, no significant trend was observed. The mean peak velocity values for 10-ms air puffs ranged from 175.5 to 274.2°/s.
The rise time of the downward phase of air-puff-evoked blinks was calculated as the time spent by the lid in producing 80% of its displacement, as measured from the initial 10% to the final 90% of lid displacement. Mean values for the rise time were similar (26.4-21.7 ms) for pressures of 2, 3, and 4 k/cm2 regardless of the duration of the stimulus (Fig. 2F). However, rise time mean values (28.6-30.1 ms) of reflex blinks evoked by 1-k/cm2 air puffs were significantly (P < 0.01) longer than those obtained for air puff presentations of 2-4 k/cm2. In fact, responses to air puffs of 1 k/cm2 (and especially of 10 ms duration) were both small and variable, as observed in the four parameters measured.
Figure 3 illustrates the EMG activity of the orbicularis oculi muscle
and lid movements of different responses evoked by the same 100-ms,
3-k/cm2 air puff when applied ipsilaterally to
different parts of the animal's face: cornea, whiskers pad, and
superior and inferior eyelids. Mean onset latencies of all these
responses had similar values around 16.5 ms, except for lid responses
during inferior eyelid stimulation, which was 32.5 ms. The amplitude
of the air-puff-evoked eyelid responses as well as the profile were
modified depending on the stimulus application site. The maximum eyelid
displacement and the largest EMG-rectified area were recorded for
stimuli applied to the cornea. Stimuli applied to whiskers and superior
and inferior eyelids evoked lid movements of about one-third those
evoked by corneal stimulation. Frequently, an oscillatory activity of
~8 Hz was noticed in lid position traces in correspondence with
vigorous movements of the animal's nose after receiving an air puff to the whiskers. The oscillation of the lid in the absence of any noticeable EMG activity helped to determine the foreign origin of the
oscillation recorded with the eyelid coil (see Comparison of
power spectra from eyelid, nose, and mandible movements).
As shown in Fig. 3, B and C, lid movements evoked by corneal, whiskers pad, and eyelid (superior and inferior) stimulation shared similar, statistically significant (P < 0.05) relationships for EMG amplitude versus lid peak velocity, as well as for integrated EMG area versus lid amplitude plots. All the coefficients of correlation for the plotted data were r > 0.7. Regression lines for data obtained after superior eyelid stimulation with air puffs showed higher slope values than those obtained by stimulating the cornea, the whiskers or the inferior eyelid for the same set of air puffs.
Selected animals (n = 2) were presented with
electrical stimulation of the ipsilateral SO nerve. The relatively weak
SO nerve stimulation (<1 mA) used here was able to evoke early and
stable EMG responses at a mean latency of 9.1 ± 1.8 ms, and late
and more variable responses at >20 ms. Both early and late EMG evoked responses were named R1 and R2 following Kugelberg
(1952). The EMG activity of the orbicularis oculi muscle
preceded lid movement by 4-5 ms (not illustrated).
Kinetic characteristics of eyelid conditioned responses
Raster displays shown in Fig. 4, A and B, illustrate the evolution through seven successive classical conditioning sessions of the blink reflex of one of the animals trained with the T-AP paradigm. The CS in this paradigm was a 350-ms, 90-dB, and 600-Hz tone followed at 250 ms from tone onset by a US consisting of a 100-ms, 3-k/cm2 air puff. In three rabbits trained with the T-AP paradigm, clear conditioned responses appeared during the third conditioning session, and in the fourth animal appeared during the second conditioning session. The CRs seemed to move forward from the UR as small, downward waves or sags toward CS onset. Once animals were consistently conditioned, they showed an eyelid response in which the CR was integrated with the UR. Rabbits presented only a few responses in a ramplike manner even after seven sessions of training. The evolution of the CR could be seen more clearly during test trials when the US (Fig. 4B) did not follow the CS. Two of the animals sometimes showed late downward lid displacements just after the end of unconditioned responses during paired trials, or following CRs during CS-alone trials. As clearly illustrated in Fig. 6C, onset latency of CRs decreased through the successive conditioning sessions from a mean value of 215.7 ± 23.2 ms for the first conditioning session to 121.4 ± 20.5 ms for the seventh. Mean and standard deviation values were calculated for the four animals trained with the T-AP paradigm.
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In the ap-AP paradigm, the CS consisted of a 20-ms, 1-k/cm2 air puff applied to the ipsilateral cornea followed 250 ms later by a 100-ms, 3-k/cm2 air puff as US. The raster displays shown in Fig. 5, A and B, illustrate the evolution through seven consecutive sessions of blink conditioning in one of the animals trained with the ap-AP paradigm. During the first two conditioning sessions, the reflex response to the CS increased in amplitude and peak velocity in a clear process of sensitization. This alpha response started to decrease during the fourth session and almost completely disappeared during the seventh session (see Figs. 5B and 6, C-F). For all four animals, the CR evoked with the ap-AP paradigm appeared during the second conditioning session. After the second session, the CR was a succession of downward, small sags that produced an almost complete lid closure (Fig. 5B). Because the reflex response to the short air puff (CS) appeared in almost all the traces throughout conditioning, the complete response topography consisted of an alpha response to the CS, the CR, and the reflex response to the US. Only late in conditioning (i.e., 5th-7th session, see Fig. 5B) were ramplike CRs obtained with onset in coincidence with CS reflex response onset, and maximum amplitude at the time of the unconditioned response to US. In those cases, the CS alpha response seemed integrated with the CR.
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As illustrated in Fig. 6C, mean latency for CR onset across the conditioning sessions decreased from 228 ± 18.6 ms to 191.4 ± 9.6 ms (n = 4 animals) for the ap-AP paradigm. Mean latency values for CRs for the ap-AP paradigm were computed without the inclusion of the alpha response evoked by the short, weak air puff used as CS (see Fig. 6B). This decrease in onset latency values through the seven conditioning sessions was even more evident with the T-AP paradigm (from 215.7 ± 23.2 ms for session 1 to 121.4 ± 20.5 ms for session 7). From session 2 to session 7, all the CRs obtained with the ap-AP paradigm showed significantly (P < 0.05) longer onset latency values than that shown by CRs obtained with the T-AP paradigm.
The time-to-peak amplitude of the CR measured from CS onset (Fig. 6D) did not show any significant variation across the seven conditioning sessions for either paradigm, as computed for data collected from CS-alone trials. Obviously, because the latency for CS onset decreased through the conditioning sessions (Fig. 7C), the duration of CRs increased with training. Thus mean time-to-peak CR values measured from CR onset increased from 60.2 ± 17.8 ms (session 1) to 134.6 ± 6.8 ms (session 7) for those CRs evoked by the T-AP paradigm, and from 47.2 ± 15.5 ms (session 1) to 91.3 ± 3.9 ms (session 7) for those obtained with the ap-AP paradigm. In both cases, data were collected from test trials (not illustrated).
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Mean values of the maximum amplitude of CRs showed a statistically significant (P < 0.001) linear trend through different conditioning sessions, as illustrated in Fig. 6E, during T-AP and ap-AP conditioning paradigms. From session 3 to session 7, differences observed between CRs obtained with both paradigms were statistically significant (P < 0.05). During the seventh session, the maximum amplitude of the CR was large enough to produce a complete eyelid closure in one of the animals (trained with the T-AP paradigm). The other animals had an upper eyelid displacement around two-thirds of the complete closure, for animals submitted to T-AP conditioning, and around one-half for those submitted to ap-AP conditioning. Individual differences could depend on the animal's strategies for motor learning and, perhaps, on the precise location of the coil in the upper eyelid.
With regard to maximum CR amplitude, peak velocity had a statistically significant linear trend (P < 0.01) across the successive training sessions (Fig. 6F) for both conditioning paradigms. Although mean peak velocity values from session 2 to session 7 were smaller for the ap-AP than for the T-AP paradigm, no significant differences were recorded, probably due to the wide variability of these values, as noticed in the high standard deviation values.
The kinetic characteristics of the CRs in CS alone trials were also studied. As illustrated in Fig. 7, D and F, the peak velocity of CRs obtained during T-AP or ap-AP conditioning paradigms increased linearly as a function of their maximum amplitude, with coefficients of correlation, r, between 0.75 and 0.96 (P < 0.01), for both downward (Fig. 7D) and upward (Fig. 7F) CR phases. Moreover, slopes of regression lines computed from data of CR downward phases were twofold those for the return movement. These results indicate that for a 10° CR, the peak velocity during the downward phase was twice the peak velocity during the upward phase. Maximum CR amplitude values during ap-AP conditioning paradigms were significantly (P < 0.01) smaller than those recorded for reflex blinks, whereas maximum CR amplitude values during T-AP conditioning paradigms were significantly (P < 0.01) larger than those recorded for reflex responses to 50-ms, 3-k/cm2 air puffs (Figs. 1 and 7). However, CR peak velocity values during both paradigms reached one-sixth the peak amplitude values for reflex blinks, for downward (Fig. 7D) and upward (Fig. 7F) lid displacements. As in the case of air-puff-evoked lid movements, very low coefficients of correlation were obtained between duration and maximum amplitude of CRs for downward (Fig. 7C) and upward (Fig. 7E) eyelid movements for both conditioning paradigms.
Frequency- and time-domain analyses of conditioned eyelid and orbicularis oculi EMG responses
Figure 8 illustrates the mean power
spectra of selected segments of lid acceleration records during the
performance of CRs evoked by the tone CS. These power spectra showed a
significant (P < 0.001) peak at 8 Hz accompanied by
other peaks at lower and, particularly, higher frequencies (Fig.
8B). The mean power spectra of selected EMG segments during
the same evoked CRs are shown in Fig. 8C. In this case, EMG
records were not rectified but were filtered to avoid their
high-frequency components' masking the signal. The EMG mean power
spectra showed a broad, dominant peak at 6-10 Hz, although other
significant peaks were recorded, especially at higher frequencies. The
coherence between acceleration (Fig.
9B) and EMG (Fig.
9C) mean power spectra from reflex responses is shown in
Fig. 9D and was significant (99% confidence limits) for
frequencies <18 Hz. The coherence between acceleration and EMG power
spectra from CRs evoked during ap-AP and T-AP conditioning paradigms
(n = 2 animals each) was always significant (99%
confidence limit) for frequencies between 2 and 20 Hz.
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The autocorrelation function of acceleration records was also
calculated to check the rhythmicity of acceleration profiles for CRs
obtained during ap-AP and T-AP conditioning paradigms (Fig.
8E). This autocorrelation function showed repeated peaks at
125-ms intervals. Nevertheless, the autocorrelation time of the
signal was rather short, fading away in two to four waves, possibly due
to the shortness (<0.5 s) of the evoked response (see Domingo
et al. 1997
).
As illustrated in Figs. 4 and 5, eyelid displacement during CRs did not
occur in all-or-nothing fashion, but seemed to increase by the
successive inclusion of waves, or quanta of lid displacement, which
added to the downward movement produced by the preceding wave. The
increase in the number of waves present in CRs during 7 consecutive
conditioning sessions with the T-AP paradigm (Fig. 4B)
produced a progressive increase in the height of a significantly (P < 0.01) dominant frequency of 8 Hz, measured in
acceleration profiles of CRs. In the same way, CRs recorded during the
ap-AP conditioning paradigm also seemed to be elaborated by the
addition of successive downward waves or sags. The increase in the
number of waves present in CRs during consecutive conditioning sessions (Fig. 5B) produced a parallel increase in the height of the
dominant
8 Hz component (P < 0.001), also computed
from the acceleration records of CRs (Fig. 5C).
Comparison of power spectra from eyelid, nose, and mandible movements
It could be argued that frequency-domain properties ascribed here
to eyelid reflex and learned responses could be motor artifacts propagated from other facial muscles, mostly those controlling nose or
mandible movements. To test this possibility, nose movements were
recorded while the animal was smelling a piece of apple placed 1 cm
from its nose, and mandible movements were recorded when the animal was
eating pieces of carrot. For the sake of comparison, nose and mandible
position during these movements and eyelid position during reflex and
learned lid responses are shown in Fig. 9, A-D. As already
illustrated (Figs. 1, 8, and 9E) both reflex and classically conditioned eyelid responses present a dominant peak at 8 Hz in
their corresponding power spectra within a broadband of lower and
higher frequencies (Fig. 9E, arrows A and B). The power
spectra of acceleration profiles corresponding to nose movements showed a significant (P < 0.001) dominant peak of
8 Hz
(Fig. 9E, arrow C). In this case, it was a very well-defined
frequency domain with a narrow range of other accompanying peaks, from
7 to 10 Hz. As illustrated in Fig. 9C, nose movement proved
to be a small-amplitude, but stereotyped movement. The amplitude of
nose movement was only
to
the amplitude of reflex and conditioned eyelid responses, a fact that may explain the
smaller peak in the spectral power of nose movement when compared with
corresponding power spectra for lid movements. The power spectra of
acceleration records corresponding to mandible movements showed a
significant (P < 0.001) peak at
4 Hz (Fig.
9E, arrow D), accompanied by two other peaks of lower
spectral power values at 6 and 9 Hz.
Spectral power of acceleration profiles corresponding to reflex blinks
evoked by air puffs of increasing pressure also increased in their
relative strength around the dominant frequency of 8 Hz (Fig.
10A). The substantial
increase of blink response when the air puff was increased from 3 k/cm2 to 4 k/cm2 was
accompanied by a corresponding increase in the power spectra. Similarly, the increase in the duration of the reflex-evoking air puffs
also increased the height of the corresponding dominant frequency (
8
Hz) in the power spectrum. As shown in Fig. 4C for T-AP, and
in Fig. 5C for ap-AP conditioning paradigms, the spectral power of acceleration profiles of CRs from successive conditioning sessions showed an increase in the height of the corresponding dominant
frequency (
8 Hz) in parallel with the appearance of new waves in
eyelid position traces during the progressive build-up of the CR (Fig.
10C).
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DISCUSSION |
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General overview
The present experiments show the profiles and metric properties of
reflex and conditioned eyelid responses in conscious rabbits using the
search coil in a magnetic field technique as a precise way of recording
lid movements. Previous reports of this technique in different species
(Evinger et al. 1984, 1991
; Gruart
et al. 1995
) prompted us to apply it to the study of eyelid
blinks in rabbits, because this is the species of choice for studying
classical conditioning of the nictitating membrane/eyelid response.
Because eyelid coils have negligible mass, they do not interfere with lid displacements, allowing a power spectrum analysis to be applied to
selected lid responses (Bendat and Piersol 1986
;
Domingo et al. 1997
; Wessberg and Vallbo
1995
). We show here that the kinetics of lid CRs is different
from that of reflexively evoked blinks. Moreover, we also show that an
8-Hz oscillator characterizes both reflex and conditioned eyelid
responses, and probably its generator circuit underlies the genesis and
control of those lid responses. According to available data, the
dominant frequency of this neural oscillator is species-specific, being
adapted to the inertial and viscoelastic needs of each species (Fig.
11).
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Metric properties of reflex blinks
According to the present study, reflex eye blinks in rabbits have
a shorter latency than reported values for nictitating membrane responses following air puff presentations or the electrical
stimulation of the SO nerve. The mean latency of reflex eyelid blinks
to air puff stimuli was 20 ms, which is 25-70% shorter than the
corresponding values for air puff-evoked nictitating membrane
displacements (Marshall-Goodell et al. 1992
;
Thompson and Krupa 1994
). The latency of reflex eyelid
blinks in response to SO nerve stimulation was also 35-45% shorter
than nictitating membrane displacements evoked by similar stimuli
(Marshall-Goodell et al. 1992
). In general, reflex
eyelid blinks presented a more wavy appearance and a more consistent
profile than reflexively evoked nictitating membrane responses. When
compared with the same motor response in cats (i.e., an animal of
similar body weight), reflex blinks in rabbits presented a longer
latency and rise time and a lower peak velocity (Gruart et al.
1995
). Moreover, the time constant of lid upward phases for
rabbits (
73 ms) presented almost a value double that reported for
cats (
37 ms) (Gruart et al. 1995
). Apart from
quantitative differences in the behavioral characterization of reflex
blinks between different species, the present data show that blink
topography (i.e., latency, amplitude, duration, and peak velocity) in
the rabbit is highly dependent on the intensity and duration of the applied stimulus (Manning and Evinger 1986
;
Schreurs and Alkon 1990
). Moreover, rabbits did not seem
to respond to light flashes or tones of similar intensity to those able
to evoke reflex blinks in cats.
The electrical activity of the orbicularis oculi muscle preceded the
onset of lid movement by 7 ms, a value larger than the one reported for
cats (4 ms) (Gruart et al. 1995
), but shorter than in
humans (10-12 ms) (Evinger et al. 1991
). As already
reported for humans (Kugelberg 1952
) and cats
(Gruart et al. 1995
), the initial downward phase of
reflex blinks in rabbits was the result of a double activation of the
orbicularis oculi muscle, evoked either by puffs of air
(Rap1 and Rap2) or by
electrical stimulation of the SO nerve (R1 and R2). As already
described (Lindquist and Mårtensson 1970
), the weak SO
stimulus used here (<1 mA) evoked a stable R1 response and a late,
more variable R2 response in the EMG activity of the orbicularis oculi muscle.
In agreement with previous descriptions for humans (Evinger et
al. 1991) and cats (Gruart et al. 1995
), the
peak velocity of the initial downward phase of reflex blinks in rabbits
was a linear function of total lid displacement; that is, larger lid responses were achieved by increasing the velocity of the movement. Accordingly, the rise time of the eyelid closing response remained unchanged for the whole range of lid responses, and, obviously, lid
movements were not linearly related to the duration of the movement.
Thus the metric properties of eyelid blinks made them similar to
skeletal ballistic movements, but different from eye saccades, because
the latter depend on both the velocity and the duration of eye
movements (Evinger et al. 1984
). The upward phases of
reflex blinks reported here were much slower than the downward phases
and presented lower gains in peak velocity for increasing lid
displacement, a result that has also been reported previously (Evinger et al. 1991
; Gruart et al.
1995
).
It seems very important to characterize the effects of stimulus
parameters rabbit eyelid responses to provide much-needed background
information for plasticity studies of conditioned blinks. For
comparative purposes (Gruart et al. 1995;
Marshall-Goodell et al. 1992
), air puffs of different
duration and/or pressure were selected here for a systematic analysis
of stimulus parameters on reflex eyelid blinks (Schreurs et al.
1995
). Latency of evoked blinks to stimulus onset presented an
inverse exponential-like relationship with air puff pressure. Indeed,
the eyelid response to short (10 ms) and weak (1 k/cm2) air puffs was both weak and variable,
being very much dependent on the attentive state of the animal. Both
amplitude and peak velocity of the downward phase of the evoked blinks
increased with air puff pressure for stimuli >50 ms in duration. Here
again, stimuli
10 ms were apparently unable to activate corneal
receptors in a constant way. Another interesting finding was that the
set of activated sensory receptors is a determinant of the metric and
profile of the evoked response, because small displacements of the
pipette used for air puff presentations significantly modified the
evoked reflex (see Fig. 3). Compared with those in cats,
air-puff-evoked reflex blinks in rabbits seem to be more dependent on
direct application of the stimulus to corneal receptors.
The duration of air puff stimulation did not affect the latency,
amplitude, or peak velocity of the initial downward phase of reflex
blinks. More importantly, the rise time of evoked blinks did not change
for stimuli >2 k/cm2. As a consequence, further
increases in the duration of eye blinks can be accomplished only by
increasing the number of downward waves or sags, as already
demonstrated for cats (Gruart et al. 1995).
In agreement with previous reports (Evinger et al. 1991;
Gruart et al. 1995
), peak EMG amplitude was linearly
related with peak downward lid velocity, suggesting that lid velocity
depends on the number of motor units simultaneously active. On the
other hand, the rectified EMG area was related to lid maximum
displacement, that is, an indication that the final lid position is
dependent on the total number of activated motor units. A recent study
on the firing activities of cat facial motoneurons innervating the orbicularis oculi muscle further supports these contentions
(Trigo et al. 1999a
).
Metric properties of conditioned eyelid responses
Animals used here for classical conditioning of blink responses
reached criterion in a similar number of trails to those previously described (Bartha and Thompson 1992; Gormezano et
al. 1962
, 1983
; Mauk and Ruiz
1992
; Smith 1968
). The accepted criterion for
rabbit nictitating membrane CR is
0.5 mm (Marshall-Goodell et
al. 1992
). The 0.5-mm criterion represents 2-3° of eyelid
displacement, which is within the range of the small waves or downward
sags shown here as the constitutive (or quantum) elements of CRs. The
resolution of the eye coil system used here was
15 min of arc, which
allows an excellent signal/noise ratio, even for the smallest eyelid responses.
The profile and metric properties of rabbit CRs were different from
those of reflex blinks. The CR showed a slow buildup throughout the
successive conditioning sessions and had a wavy shape and smaller
amplitude than maximum reflexively evoked blinks. Moreover, CRs have
always a slower building-up than reflex responses. The peak velocity of
the CR was only one-fifth of that reached by reflex blinks. The peak
velocity of the CR (considered as a single motor process) was shown to
be a linear function of CR amplitude, but the gain of this relationship
was that of air-puff-evoked blinks. The gain of the CR
peak velocity-amplitude relationship was also related to the
conditioning paradigm, with ap > T. As observed for reflex
blinks, CR duration was not significantly related to CR amplitude,
mostly because the CR tended to reach its maximum amplitude at the
(already fixed) CS-US interval. All these metric properties are similar
(with slight differences) to a previous report on the topography and
kinetics of eyelid CRs in the cat (Gruart et al. 1995
).
However, it should also be stated that CRs were similar to reflex lid
responses regarding the dominant frequency of the power spectrum and to
the presence of downward sags.
The latency of CR decreased with successive sessions, and by the
seventh session was longer than the corresponding value for air-puff-evoked reflex blinks (Berstein 1934;
Gormezano et al. 1983
; Mauk and Ruiz
1992
; Smith 1968
). Latency values for T-AP conditioning for lid CRs (
120 ms) were smaller than those reported for a similar conditioning of nictitating membrane CRs (
180 ms) (McCormick et al. 1982
). On the other hand, the latency
of CRs during ap-AP conditioning was longer than for the T-AP
conditioning paradigm. This fact could be explained by the inhibitory
postsynaptic potential that follows the early reflex (i.e., alpha)
response evoked by CS (short, weak air puff in this case) presentation (Grant and Horcholle-Bossavit 1983
; Trigo et al.
1999a
).
As reported here, rabbits did not show detectable alpha responses to
tones, but did respond to short, weak air puffs used as CS. Alpha
responses reported here had latency (40 ms), magnitude (
1/8 of
the CR by the 7th conditioning session), and duration (
35 ms) values
similar to those reported previously in the same species
(Gormezano et al. 1983
). Alpha conditioning is easily produced in humans to light CS (Grant and Adams 1944
),
and in cats to short, weak air puffs and, sometimes, to tones
(Gruart et al. 1995
; Woody 1986
). The
main difference between cats and rabbits is that the former are able to
integrate the alpha response with the CR, because no temporal gap
exists in CR profiles (Gruart et al. 1995
), a result not
observed in rabbits (Schreurs and Alkon 1990
, and the
present experiments). In fact, different CR profiles can be induced by
different CSs, and different species may solve the same motor learning
problem in different ways (Gruart et al. 1995
;
Rescorla 1988
). The different functional organization of neural circuits controlling nictitating membrane/eyelid motor learning
in cats and rabbits should not be a surprise if one considers the
noticeable differences between species for the related oculomotor system (e.g., Graf and Simpson 1981
).
Oscillatory mechanisms underlying reflex and conditioned eyelid responses
The fact that reflex and conditioned blinks have a wavy profile
has been noticed even for nictitating membrane recordings (Bartha and Thompson 1992; Berthier 1992
;
Welsh 1992
) and was observed in early human experiments
(Marquis and Porter 1939
). Illustrations of reflex and
conditioned nictitating membrane responses in previous reports
(Berthier 1992
; Welsh 1992
) also present
noticeable oscillations at 8-10.5 Hz. The dominant frequency of eyelid
responses in the rabbit is about one-third of its resonant frequency
(
30-35 Hz) (see Evinger et al. 1984
). A similar
finding has been reported for finger movements in humans
(Halliday and Redfearn 1956
; Wessberg and Vallbo
1995
). In agreement with recent reports in cats (Domingo et al. 1997
), increases in the duration of reflex blinks were accomplished by the addition of successive downward waves of
125 ms.
Moreover, and as reported for finger movements in humans (Vallbo and Wessberg 1993
), larger lid CRs in rabbits were achieved by increasing the amplitude and number of waves composing them, but not by
modifying the dominant frequency of the movement. If we follow this
line of thought, CRs could be envisioned as the process of reaching a
target in a given time with the help of a fixed-frequency neuronal
oscillating machinery (Domingo et al. 1997
).
The fact that light-evoked blinks presented a dominant oscillation
frequency lower than air-puff-evoked ones has also been reported
recently in cats (Domingo et al. 1997) and suggests that functional properties of the circuits involved may contribute to
determine the dominant frequency of each reflex response. In this
sense, the number of synapses involved in each of the circuits and the
possibility of recurrent loops could contribute to the differences
reported here.
As illustrated in Fig. 11, when the available data on lid dominant
oscillation frequencies for different species are plotted against
species body weight, an inverse logarithmic relationship is obtained.
Indeed, these data suggest that lid biomechanics could be tuned to the
lid's weight and to its viscoelastic properties. A similar inverse
logarithmic relationship (with exactly the same slope) has been
demonstrated to exist between heart rate and body mass for mammals
(Stahl 1967). The slope of this relationship (
0.25)
indicates that lid oscillation frequency (as shown for heart rate)
could be related to oxygen consumption per unit body mass (in
l/kg/h), because the latter is also related to body mass (in
kg) by a linear relationship of the same slope (i.e.,
0.25) (see
Figs. 4.9 and 6.10 in Schmidt-Nielsen 1979
).
Because the eyelid is load free, and facial motoneurons receive
no feedback proprioceptive signals from the orbicularis muscle (Porter et al. 1989; Trigo et al. 1999b
),
it could be suggested that the oscillatory behavior of the eyelid is
the result of the activity of the neuronal mechanisms controlling it.
In fact, it has been shown recently in cats and rats that tremor of the
lids is an inherent rhythmical property of facial motoneurons
innervating the orbicularis oculi muscle (Magariños-Ascone
et al. 1999
; Trigo et al. 1999a
). Moreover, a
noticeable oscillatory behavior has been observed in cat pericruciate
cortex neurons during the classical conditioning of blink responses
(Aou et al. 1992
) and in cat cerebellar interpositus
neurons during reflexively evoked blinks (Gruart and
Delgado-García 1994
). Coherent 25- to 35-Hz
oscillations have also been reported in the sensorimotor cortex of
awake monkeys during exploratory and manipulative movements
(Murthy and Fetz 1992
). Taken together, these data
suggest that motor systems could be controlled by central neural
oscillators tuned to the inertial and viscoelastic needs of moving appendages.
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ACKNOWLEDGMENTS |
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We thank R. Churchill for help in editing the manuscript.
This study was supported by Grant PB93-1175 from the Spanish Direción General de Investigación Científica y Técnica, Grant SAF96-0160 from the Comisión Interministerial de Ciencia y Tecnologia, Junta de Andalucía Grant CVI122, and North Atlantic Treaty Organization Programme Grant 951197 to J. M. Delgado-García and D. L. Alkon.
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FOOTNOTES |
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Address for reprint requests: A. Gruart, Laboratorio de Neurociencia, Facultad de Biología, Avda. Reina Mercedes 6, 41012 Seville, 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 23 April 1999; accepted in final form 10 September 1999.
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REFERENCES |
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