1División de Neurociencias, Laboratorio Andaluz de Biología, Universidad Pablo de Olavide, 41013 Sevilla; 2Departament de Psicobiologia i de Metodologia de les Ciències de la Salut, Facultat de Psicologia, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; and 3División de Neurociencias, Instituto Mexicano de Psiquiatría, San Lorenzo Huipulco, 14370 México, DF, Mexico
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ABSTRACT |
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Gruart, A., G. Guillazo-Blanch, R. Fernández-Mas, L. Jiménez-Díaz, and J. M. Delgado-García. Cerebellar Posterior Interpositus Nucleus as an Enhancer of Classically Conditioned Eyelid Responses in Alert Cats. J. Neurophysiol. 84: 2680-2690, 2000. Cerebellar posterior interpositus neurons were recorded in cats during delayed and trace conditioning of eyeblinks. Type A neurons increased their firing in the time interval between conditioned and unconditioned stimulus presentations for both paradigms, while type B neurons decreased it. The discharge of different type A neurons recorded across successive conditioning sessions increased, with slopes of 0.061-0.078 spikes/s/trial. Both types of neurons modified their firing several trials in advance of the appearance of eyelid conditioned responses, but for each conditioned stimulus presentation their response started after conditioned response onset. Interpositus microstimulation evoked eyelid responses similar in amplitude and profiles to conditioned responses, and microinjection of muscimol decreased conditioned response amplitude. It is proposed that the interpositus nucleus is an enhancer, but not the initiator, of eyelid conditioned responses.
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INTRODUCTION |
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The eyelid motor system is an
excellent experimental model for the study of how central neural
circuits generate motor responses (Evinger 1995;
Gormezano et al. 1983
). This motor system is peculiar, because eyelid movements are load free and have a minute mass. Also,
lid displacements depend on the exclusive action of a few extraocular
and facial muscles, apparently free of proprioceptors (Evinger
1995
; Evinger et al. 1991
; Gruart et al.
1995
; Trigo et al. 1999
). The kinetic,
frequency-domain, and time-domain properties of reflex and conditioned
eyelid blinks, as well as the functional properties of innervating
brain stem motoneurons, have been described recently for both cats and
rabbits (Domingo et al. 1997
; Evinger 1995
; Evinger et al. 1991
; Gruart et al.
1995
; Trigo et al. 1999
; Welsh
1992
).
Besides this apparent simplicity in their sensorimotor organization,
eyelid motor responses are involved in spontaneous, reflex, emotional,
and eye-related movements. Moreover, since its popularization in the
1960s, the nictitating membrane/eyelid response has been repeatedly
employed in the study of the neural mechanisms underlying motor
learning (Gormezano et al. 1983; Kim and Thompson
1997
; McCormick et al. 1982
; Welsh
1992
; Woody 1986
).
It is now well known that the profile and kinematics of eyelid
conditioned responses (CRs) are different from those of reflex blinks,
a fact suggesting a distinct origin and/or neural generation process
(Domingo et al. 1997; Evinger 1995
;
Gruart et al. 1995
; Trigo et al. 1999
;
Welsh 1992
). For example, in contrast to the fast
downward upper lid displacement characterizing reflex blinks, CRs are
usually ramp-like, reaching peak velocities that never surpass 1/6 of
those reached during air puff-evoked blinks (Gruart et al.
1995
). Moreover, CRs seem to be built up over an underlying oscillatory mechanism, already described in cats for reflex and conditioned eyeblinks. This underlying oscillatory mechanism has also
been determined in the electromyographic (EMG) activity of the
orbicularis oculi muscle, and in the activity of identified facial
motoneurons both in vivo and in vitro (Domingo et al.
1997
; Magariños-Ascone et al. 1999
;
Trigo et al. 1999
).
The neural site where learning of eyelid CRs occurs,
as well as the putative subcellular mechanisms involved, are currently a matter of intensive research (Bliss and Collingridge
1993; Kim and Thompson 1997
; Malenka
1995
; Mauk 1997
; Woody 1986
). One
of the suggested sites of motor learning is the deep cerebellar nuclei, particularly some not yet well-defined regions of the anterior and/or
posterior interpositus nuclei (Bracha et al. 1999
;
García and Mauk 1998
; Krupa et al.
1993
; Mauk 1997
; Schreurs et al.
1998
). The role of interpositus nuclei in eyeblink motor
learning has been extended to other classically conditioned withdrawal
reflexes (Bracha et al. 1999
). These proposals have been
supported mostly on lesion and pharmacological studies (Bracha
et al. 1999
; Krupa et al. 1993
;
García and Mauk 1998
; Mauk 1997
;
Yeo et al. 1985
). Nevertheless, specific areas of the
interpositus nucleus are indeed involved in eyelid responses, as
recently confirmed by retrograde transneuronal tracing with rabies
virus (Ugolini et al. 1999
).
Although very valuable evidences have been accumulated until now
regarding the firing activities and functional properties of
interpositus neurons during the acquisition and performance of eyelid
CRs (Berthier and Moore 1990; Freeman and
Nicholson 1999
; Gruart et al. 1997
), more
specific information is still needed. For example, to accomplish their
putative role in motor learning, interpositus neurons should fire a few
milliseconds in advance of the initiation of the CR, and, perhaps,
their firing should be time-locked to CR profiles. Moreover,
interpositus neurons should start increasing their firing monotonically
in the time window between conditioning (CS) and unconditioning (US)
stimuli some trials in advance of the appearance of the CR. Finally,
microstimulation of the learning-related zone of the interpositus
nuclei should evoke an eyelid response resembling the profile and
kinetic properties of CRs, while the chemical inactivation of
interpositus neurons should remove (or at least modify) the normal
performance of CRs.
To assess those fundamental questions in a suitable experimental model,
cats were prepared for the chronic recording of eyelid movements and of
the EMG activity of the ipsilateral orbicularis oculi muscle. The
unitary activity of antidromically identified posterior interpositus
neurons was recorded during controls and during classic conditioning of
eyeblinks. Both trace and delayed conditioning paradigms were used. The
US was always a long, strong air puff. The CS was either a short, weak
air puff or a tone. Electrical microstimulation and microinjection of
muscimol [a -aminobutyric acid-A (GABAA)
receptor agonist; see Krupa et al. 1993
] were carried
out at selected recording sites. Present results suggest that although
posterior interpositus neurons are probably not involved in the
initiation of eyelid CRs, they do contribute to the reinforcement of
motor commands of a yet unknown origin.
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METHODS |
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Subjects
The present experiments were carried out on eight adult cats weighing 2.5-3 kg obtained from an authorized supplier (Iffa-Credo). Experimental procedures were performed in accordance with the guidelines of the European Union Council regulations (86/609/EU) and following Spanish legislation (B.O.E., 67: 8509-8512, 1988) for the use of laboratory animals in chronic experiments.
Preexperimental surgical procedures
Animals were anesthetized with pentobarbital sodium (Nembutal,
35 mg/kg, ip) following a protective injection of atropine sulfate (0.5 mg/kg) to prevent vagal reflexes. As illustrated in Fig.
1A, a five-turn coil (3 mm in
diameter) 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. Two hook
electrodes, made of the same wire and bared 1 mm at their tips, were
implanted in the ipsilateral orbicularis oculi muscle. A bipolar
stimulating electrode (200 µm in diameter, enamel-coated silver wire)
was implanted in the vicinity of the magnocellular division of the red
nucleus following stereotaxic coordinates (Berman 1968)
to activate axons located in the ascending limb of the superior
peduncle. A bare silver electrode (1 mm in diameter) was attached to
the skull as a ground. To allow a transcortical access to deep
cerebellar nuclei, a recording window of 5 × 5 mm was drilled in
the occipital bone. The dura mater was removed, and an acrylic
recording 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 chamber corner for reference purposes during unitary recordings. Finally, a head-holding system consisting of three bolts cemented to the skull perpendicular to the
stereotaxic plane was implanted. Wire terminals from recording and
stimulating electrodes were soldered to a socket cemented to the
holding system. Further details of this chronic preparation have been
described elsewhere (Gruart and Delgado-García
1994
; Gruart et al. 1995
; Trigo et al.
1999
)
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General conditions of recording sessions
Recording sessions started 2 wk after surgery and lasted for
<0.3 h per day for a maximum of 25 days. For experiments, animals were
lightly restrained with elastic bandages and placed on the recording
table with their head immobilized in the head-holding system. Recording
sessions to find the selected area lasted 4-9 days. The recording
chamber was opened and a microelectrode advanced toward the
interpositus nucleus. The correct location of the micropipette was
confirmed by the antidromic identification of recorded units and by the
characteristic firing of interpositus neurons to blink-evoking stimuli
(see Gruart and Delgado-García 1994). After
these sessions, each animal was assigned randomly to one of the two
(trace: 3, delayed: 5) conditioning paradigms. Conditioning sessions
lasted for
10 days, were preceded by two habituation sessions, and
followed by
4 extinction sessions. Muscimol injection was carried out in two animals conditioned with a delayed paradigm. A microstimulation session was carried out in all the animals, but in two of them (1 trace, 1 delayed) the session took place before extinction sessions.
Unitary recordings and neuron identification
Neuronal electrical activity was recorded with glass
micropipettes filled with 2 M NaCl. Field potentials were recorded with low-resistance electrodes (1-3 M), while the unitary activity was
recorded with microelectrodes of 3-6 M
of resistance. Neuronal activity was filtered in a bandwidth of 10 to 10 kHz. The recording electrode was tilted anteriorly by 30 deg and moved in the sagittal and
coronal planes in 0.2-mm steps. The recording area was approached with
the help of stereotaxic coordinates (Berman 1968
) and
the field potentials were induced by electrical stimulation of the red
nucleus. Electrical stimuli consisted of single- or double- (1- to 2-ms
interval) cathodal 50-µs square pulses with current intensities <0.2
mA. The recording of single-unit activity was restricted to those
neurons identified by their antidromic activation. The collision test
was used to determine whether the recorded and the activated neuron
were the same (Fig. 1B). Criteria for the discrimination of
somatic versus axonic recording were systematically followed (see
Gruart and Delgado-García 1994
). Data
corresponding to identified orbicularis oculi facial motoneurons
illustrated in Fig. 6 were collected from unpublished records from a
recent work from our group (Trigo et al. 1999
).
Recordings of eyelid movements and electromyograms
Eyelid movements were recorded using the search coil in a
magnetic field technique (Gruart et al. 1995). Eyelid
movements were calibrated with the help of a transparent protractor
placed sagittally to the head and with its center located at the lid external canthus. Lid opening ranged 36-42 deg in the eight animals. The EMG activity of the orbicularis oculi muscle was recorded with
differential amplifiers at a bandwidth of 10 Hz to 10 kHz.
Stimuli-evoking reflex blinks
Air puffs directed to the cornea and periorbital region were
applied through the opening of a plastic pipette (3 mm in diameter) located 2 cm from the eye. Air pressure was regulated at the source from 1-3 kg/cm2 and lasted 20-100 ms. Bright
full-field xenon flashes were used as visual stimulus. Tones (600 or
6,000 Hz) were presented for 10-100 ms at 90 dB. During control
sessions, stimuli were presented at random, with intervals >5 s (Fig.
1A).
Electrical stimulation of cerebellar nuclear sites
Selected sites of the interpositus nucleus were stimulated for
1 s with trains of cathodal 50 µs square pulses, at 5, 10, 20, 30, 40, and 60 Hz. Stimuli were delivered with tungsten electrodes of
1-5 M. Unitary recordings in the aimed area were always carried out
before the start of electrical microstimulation sessions.
Injection experiments
A guide tube consisting of a 26-gauge stainless steel needle was implanted up to 1.5 mm over the desired injection sites in two of the animals 1 day before classical conditioning. The guide tube was maintained sterile and protected by a removable 33-gauge stainless steel rod. A total of 2.5 µg of muscimol (Sigma) dissolved in 1 µl artificial cerebrospinal fluid (pH = 7.4) was injected 20 min before selected conditioning sessions. Injection was carried out at a rate of 0.25 µl/min with the help of a 1-µl Hamilton syringe connected by a calibrated plastic tubing to a 33-gauge stainless steel needle inserted inside the guide tube. The internal needle projected 1.2 mm beyond the guide tube tip.
Classical conditioning paradigms
The classical conditioning of eyelid responses was achieved by the use of delayed and trace conditioning paradigms (Fig. 1C). For delayed (T-AP) conditioning, a 370-ms, 600-Hz, 90-dB tone (T) was used as CS. The tone was followed 270 ms from its onset by a 100-ms, 3-kg/cm2 air puff (AP) directed at the left cornea as US. Thus the tone and the air puff terminated simultaneously. For trace (ap-AP) conditioning, the animal was presented with a short (20 ms), weak (1 kg/cm2) air puff (ap) directed at the left cornea as CS. The CS was followed 250 ms later by a 100-ms, 3-kg/cm2 AP directed at the same eye as US.
Each conditioning session consisted of 12 blocks separated by a
variable (4-6 min) interval. 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. The CS was presented
alone in the first trial of each block. A complete conditioning session
lasted for 2 h. The CS was presented during habituation and
extinction sessions for the same number of blocks/session and
trials/block and with similar random interblock and intertrial
distribution (see Gruart et al. 1995
). Although conditioned criterion (95% of CRs/session) was reached in the six
unit-recording animals by the fourth to sixth conditioning session,
conditioning was maintained for 10 sessions to obtain the maximum
number of recorded neurons.
Histology
At the end of recording sessions, animals were deeply anesthetized (pentobarbital sodium, 50 mg/kg, ip). Electrolytic marks were placed at selected recording sites with a tungsten microelectrode and at stimulating sites with the help of the implanted electrode (1 mA for 10 s). Animals were then perfused transcardially with saline and phosphate-buffered formalin. Serial 50-µm sections of the cerebellum and brain stem were mounted on glass slides and stained with toluidine blue or neutral red.
Data collection and analysis
Eyelid position, EMG and neuronal activity, and 1-V rectangular pulses corresponding to blink-evoking stimuli or to CS and US presentations were stored digitally on a videotape recording system at a sampling frequency of 22 kHz for biopotentials and 11 kHz for the other signals. Data were transferred 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, but selected unitary records were sampled at 22 kHz for representation purposes. In addition, action potentials were fed into a window discriminator and the resulting Schmidt trigger pulses were stored on the computer 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 representations of eyelid position,
velocity and acceleration, and EMG and neuronal activities. The color
raster shown in Fig. 5 was made with the help of a representation program written in Java language by one of us (R.F.-M.). Velocity and
acceleration profiles 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 instantaneous
firing rate was calculated as the inverse of the interspike intervals
(see Trigo et al. 1999
for details).
These computer programs also allowed the quantification of lid
position, EMG, and neuronal parameters. Data were processed for
statistical analysis with the SPSS for Windows package, for two-tailed
tests with a statistical significance level of P = 0.05. Unless otherwise indicated, mean values are followed by the
standard deviation (SD). The possible relationship between neuronal
firing rate and lid position and velocity was checked by linear
regression analysis. For this, eyelid position or velocity was plotted
versus the instantaneous firing rate, in 1-ms point-to-point process.
The power of the spectral density function (i.e., the power spectrum)
of selected 1.024-s segments from eye acceleration recordings was
calculated using a fast Fourier transform to define the relative
strength of the different frequencies present in eyelid responses.
Significance of power spectra peaks was tested with the
2-distributed test for spectral density
functions. Statistical differences of mean values were determined with
the help of Student's t-test for variables of two
categories, or with the analysis of variance (ANOVA) for variables of
more than two categories, followed by a contrast analysis when needed
(see Domingo et al. 1997
, and Trigo et al.
1999
for details).
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RESULTS |
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Neuronal response types
The recording area was selected according to available
morphological and neurophysiological reports indicating its putative involvement in the neuronal control of facial muscles for both cats
(Gruart et al. 1995, 1997
) and monkeys (van Kan
et al. 1993
). Thus the posterior interpositus nucleus was
explored during the random presentation of blink-evoking stimuli (Fig.
1A). This procedure took 4-9 recording sessions. For all
the animals (n = 8), this area was found to contain
neurons related to eyeblinks. Recorded neurons could be classified as
type A and B (Gruart et al. 1994
). Type A neurons were
activated antidromically from the red nucleus at latencies of 0.76 ± 0.11 ms (measured from the stimulus artifact to the first negative
peak, n = 50). Type B were activated at significantly
longer latencies (0.98 ± 0.08, n = 50, P < 0.01, Fig. 1B). The firing rate for
type A neurons was rather irregular, with mean values ranging from
10-60 spikes/s (n = 50) and peak firing reaching 400 spikes/s. The spontaneous firing of type B neurons was more regular,
ranging between 30 and 80 spikes/s (n = 50) and
reaching peak values of 300 spikes/s. The main functional difference
between type A and type B neurons was that type A cells increased their
firing during air puff, flash, and (on occasions) tone presentations,
while type B cells paused or even stopped firing during the
presentation of similar blink-evoking stimuli. No attempt was made here
to quantify the response of type A and B neurons to blink-evoking
stimuli, as these functional properties have been described elsewhere
(Gruart et al. 1994
). Once the selected recording area
was found, six of the animals were assigned at random to a trace
(n = 3) or a delayed (n = 3) classical
conditioning paradigm (Fig. 1C). In the other two animals, a
guide tube was implanted over the recording site and no further
recordings of neuronal activity was carried out.
Neuronal activities during classical conditioning of eyelid responses
The precise location area where neurons used in this study were recorded is shown in Fig. 2. Recorded neurons (n = 320) were concentrated in the dorsal part of the anterior pole of the posterior interpositus nucleus. Of this, 192 neurons were classified as type A and 128 as type B. Since a total of 96 conditioning sessions were carried out (2 habituation, 10 conditioning, and 4 extinction sessions in six animals), on average, 3.5 neurons per session were recorded. In fact, many recorded neurons were held for the whole conditioning session.
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Recordings carried out outside the shaded area illustrated in Fig. 2
(but within the stereotaxic coordinates of both anterior and posterior
interpositus nucleus, Berman 1968) did not show unitary
activity related to lid responses. Neurons with firing profiles
qualitatively related to ear and/or mouth movements were found in the
immediate vicinity of the area selected for this study. Those neurons
were not further considered in this study.
As illustrated in Figs. 3,
A-D and 4, A-C,
type A neurons discharged during CRs similarly to the way already
described for blink-evoking stimuli (Gruart et al.
1994). They fired a burst of action potentials following
initiation of the eyelid CR. This burst of activity could be
individualized for the successive CS and US presentations (Figs.
3A and 4A) or could be present through the whole
CR (Fig. 4C). Some intermediate responses of type A neurons
could also be observed (Fig. 4B). The increase in firing rate of type A neurons during the CS-US interval over control values
ranged from 2-4.5 times when calculated from averaged records (n
20) for n = 50 neurons. In
contrast, type B neurons decreased their firing slightly after
initiation of CRs. The decrease in firing rate of type B neurons
remained until US presentation (Fig. 4D).
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As already reported for cats (Gruart et al. 1995), the
kinematics of eyelid CRs was very different during delayed (Figs.
3A and 4, A, C, and D) versus trace
(Fig. 4B) conditioning paradigms. Nevertheless, for present
experiments, no significant difference was noticed between type A
firing rates during eyelid CRs evoked by delayed versus trace
conditioning paradigms (see Figs. 3A and 4, A and
C, for delayed and 4B for trace conditioning).
Single (Figs. 3A and
5A) and averaged (Figs.
3D and 4, A-C) firing profiles of type A neurons
sometimes seemed to reproduce eyelid position records, i.e., type A
neuron firing was apparently related to eyelid position and/or velocity
profiles. Linear regression analysis of firing rate versus eyelid
position and velocity yielded slopes of 2-9 spikes/s/deg and
0.02-0.13 spikes/s/deg/s (quantified from n = 100 records per neuron; n = 30 neurons). However, the low
coefficients of correlation obtained for these linear relationships (r 0.42, P
0.05 for firing
rate/eyelid position and r
0.37, P
0.01 for firing rate/eyelid velocity, n = 30 neurons) precluded us from proposing that type A neurons were encoding
eyelid position and/or velocity signals. Similar negative results were
obtained for type B neurons when analyzed for eyelid position and/or
velocity signals (n = 30, r
0.31, P
0.05 for firing rate/eyelid position relationships). Finally, cross-correlation analyses of neuronal firing
versus the EMG activity of the orbicularis oculi muscle or lid position
profiles did not show any significant relationship (not illustrated).
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It was evident from the simple observation of firing records obtained
across successive conditioning and neuronal recording sessions that the
discharge rate profiles were increasing in their averaged values.
Figure 5 illustrates the time-course in both firing rate and in
acceleration profiles of CRs across conditioning in a representative
animal. As shown, the firing rate of different posterior interpositus
neurons recorded in successive sessions across conditioning increased
when analyzed at a fixed time in relation to CS presentation, mainly
when compared with a fixed time before CS (Fig. 5D). Eyelid
acceleration similarly increased across conditioning. It is important
to note that the increase in firing rate for different neurons across
conditioning was evident trials in advance of the appearance of a
noticeable CR (Fig. 5, B-D). Linear regression analyses of
data illustrated in Fig. 5D indicated that recorded
interpositus neurons increased their firing at a rate of 0.078 spikes/s
per trial (r = 0.71, P < 0.001, Fig. 5B), while eyelid acceleration during the first downward
movement of the CR (Fig. 5C) increased at 6.99 degrees/s2 per trial (r = 0.8, P < 0.01). The same analysis carried out in two other
animals conditioned with a delayed paradigm yielded values for the
increase in the firing rate of identified posterior interpositus
neurons of 0.06 and 0.072 spikes/s per trial (r 0.65; P < 0.01). As illustrated in Fig. 5D,
the neuronal activity in the CS-US interval decreased more slowly than
CRs across extinction sessions, i.e., extinction of CRs slightly
preceded that of neural activity.
It has been reported (Domingo et al. 1997) that eyelid
CRs present a wavy profile, easily noticeable in eyelid acceleration traces (Fig. 4). The Fourier transform analysis of eyelid acceleration profiles such as the ones illustrated in Fig. 5, A and
C, showed a significant peak (P < 0.01) in
the power spectra at 20 Hz. Surprisingly, none of the posterior
interpositus neurons recorded here presented any evidence of a bursting
firing. Thus, the interspike interval distributions for type A and B
neurons (n = 20 for each) were always leptokurtic,
skewed to the right and unimodal (i.e., with a single discrete peak),
both during spontaneous firing and when quantified during the CS-US
interval (Fig. 3B). Mean interspike intervals were 22.2 ± 17.7 ms for type A and 14.4 ± 9.1 ms for type B neurons. No
significant peak was observed in the distribution at 50 ms, a fact
indicating that these neurons did not modulate their firing at 20 Hz.
Similarly, the autocorrelation profiles of the same neurons
(n = 40) showed no statistically significant sinusoidal
tendency (that is, no evidence of autorhythmicity) either during
spontaneous firing or when measured in the CS-US interval (Fig.
3C).
As illustrated in Figs. 3 and 4, the firing rate increase evoked in
posterior interpositus neurons by CS presentation started after the
initiation of the eyelid CR. An attempt was made to analyze the
temporal relationships between the neuronal groups involved in blinks.
Figure 6, A-C, illustrates
the EMG activity of the orbicularis oculi muscle and eyelid
displacement evoked by electrical stimulation of the posterior
interpositus nucleus (A), the red nucleus (B), and the dorsomedial
division of the facial nucleus (C). The electrical stimulation of the
interpositus nucleus evoked lid movements at a mean latency of
10.6 ± 0.9 ms (n = 10). The latency of lid
response to red nucleus stimulation was 8.2 ± 0.6 ms
(n = 10). Finally, lid displacement triggered by
orbicularis oculi motoneurons (n = 5 neurons) was
5.9 ± 0.6 ms. Accordingly, an interpositus neuron should start
firing 4-5 ms in advance of facial motoneurons to be accepted as
directly premotor to them. On the contrary, as illustrated in Fig. 6,
D-F, recorded interpositus neurons fired following the
burst of activity characterizing the orbicularis oculi motoneurons
(Trigo et al. 1999
). Thus antidromically identified
orbicularis oculi motoneurons fired at 53.1 ± 9.1 ms (range
46-64, n = 100) with respect to CS presentation during
the delayed conditioning paradigm. The mean latency for the beginning
of EMG activity in the orbicularis oculi muscle was 55.2 ± 7 ms
(range 48-69; n = 100). Eyelid CRs started at 59 ± 9 ms (range 51-70, n = 100) following CS
presentation. Finally, mean latency with respect to CS presentation for
type A posterior interpositus neurons was significantly larger
(P < 0.001) than values for eyelid CRs: 71.5 ± 13.9 ms (range 54-144, n = 100).
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Since CR latency with respect to CS presentation has some variability, perimovement histograms were also carried out by triggering spike data after CR onset. In this case, the mean delay between CR initiation and type A neuron firing during delayed conditioning was 22.5 ± 9.1 ms (range 3-75, n = 100), with the neuron lagging CR initiation. Similar significant values (P < 0.01 at least) were obtained for type A neurons during trace conditioning (35 ms, n = 100), and for type B neurons during delayed (29.2 ms, n = 100) and trace (31 ms, n = 100) conditioning paradigms. Accordingly, these data confirmed latency analyses of neuronal activity carried out with respect to CS presentation, as illustrated in Fig. 6F.
Eyelid movements evoked by microstimulation of the interpositus nucleus
A further attempt was made to determine the exact contribution of
posterior interpositus neurons to eyelid CRs. For this, microstimulation sessions at selected recording sites were carried out
following or preceding extinction sessions in four and two animals,
respectively. The two animals subjected to muscimol injections were
stimulated at the end of the eighth conditioning session. Several
stimulation patterns were tried: 5, 10, 20, 30, 40, and 60 Hz trains
lasting for 1 s. The 20-Hz train proved to be the most efficient
to evoke a ramp-like downward lid movement with the minimum of current
applied (45 µA). As illustrated in Fig. 7B, microstimulation of the
posterior interpositus nucleus evoked an eyelid response more similar
in its kinetics to a CR than to a reflex blink (see Gruart et
al. 1995
). Moreover, microstimulation of this area did not
produce any observable motor response (in mouth, nose, whiskers, or
ears) in addition to eyelid movement. When compared with CR responses
evoked in the same animal by CS-alone presentations in a delayed
conditioning (Fig. 7A), the eyelid response evoked by
microstimulation was significantly (P < 0.001) smaller in amplitude than the natural CR (it was less than one half). However, the simultaneous presentation of the CS and the train
of electrical impulses significantly (P < 0.001)
increased the size of the evoked eyelid response (Fig. 7C).
It should be noted that this pairing of stimuli increased the amplitude
of the evoked eyelid response, but did not significantly modify its latency or duration, even considering that the train of electrical stimuli started
50 ms before and finished
200 ms after the CR.
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Effects of muscimol injections in the posterior interpositus nucleus
Two of the animals were conditioned following the implantation of a guide tube to allow the injection of muscimol in a site of the posterior interpositus nucleus selected after recording blink-related neuronal activity in it (Fig. 8). In control trials, it was checked that injections of 1-µl artificial cerebrospinal fluid or of 2.5 µg muscimol dissolved in the same volume of fluid did not produce any significant modification of air puff- (100 ms, 3 kg/cm2) and flash-evoked blinks. Following controls, both animals were conditioned using a delayed paradigm. Mean values of CR maximum amplitude increased exponentially through the first four conditioning sessions. However, CR maximum amplitude decreased significantly (P < 0.05 at least) with respect to the fourth session for the two conditioning sessions (fifth and sixth) during which the muscimol was injected 20 min in advance of session start. The mean decrease was 35.1% for the fifth and 18.2% for the sixth conditioning sessions. The next two conditioning sessions (seventh and eighth) showed mean values in peak amplitude of the evoked CRs corresponding to the expected evolution of the learning acquisition curve (Fig. 8B). A significant decrease in CR peak velocity (30.3% for the fifth and 21.1% for the sixth conditioning sessions, P < 0.05 at least) during the two muscimol-injection sessions was also observed. Nevertheless, no change was observed in CR latency during the two muscimol sessions.
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DISCUSSION |
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Posterior interpositus as a site for motor learning
Different sorts of irreversible (Welsh 1992;
Yeo et al. 1985
) and reversible (Bracha et al.
1999
; Garcia and Mauk 1998
; Krupa et al.
1993
) inactivation procedures of the cerebellar interpositus nucleus in both rabbits and cats have addressed its possible
participation in the acquisition of motor abilities, mainly that of the
classical conditioning of the nictitating membrane/eyelid motor
response. Although there is general agreement regarding the involvement of the cerebellar interpositus nucleus in the generation of eyelid CRs
(Bracha et al. 1999
; Garcia and Mauk
1998
; Kim and Thompson 1997
; Mauk
1997
), some controversy remains regarding its preferred role in
the acquisition of new eyeblink responses (Garcia and Mauk
1998
; Krupa et al. 1993
; Mauk
1997
) or in their performance (Welsh 1992
;
Welsh and Harvey 1992
). Available information about unitary recordings of putative deep cerebellar nuclei neurons also
indicates their involvement in eyelid CRs (Berthier and Moore 1990
; Freeman and Nicholson 1999
, 2000
;
Gruart et al. 1997
).
Present results indicate that a very definite area of the posterior
interpositus nucleus is involved in the genesis and control of newly
acquired eyelid CRs in cats. Indeed, the activity of these neurons
fulfilled some of the conditions required for this role. First, they
increased their firing in the CS-US time interval across conditioning
several trials in advance of the appearance of the CR. In this regard,
it has been shown that the amplitude of field potentials recorded in
dorsal posterior interpositus by contralateral inferior olive
stimulation increases across conditioning sessions in advance of the
appearance of eyelid CRs (Gruart et al. 1997). This fact
indicates that some facilitation for the firing of posterior
interpositus neurons takes place in this area in the CS-US time
interval during successive conditioning trials. Second, the
microstimulation of the same recording area evoked ramp-like eyelid
movement profiles similar in their kinetic characteristics to cat
eyelid CRs (Gruart et al. 1995
). Third, the
microinjection of muscimol in the recording area decreased the
amplitude of eyelid CRs across conditioning sessions, although did not
completely abolish it (see Krupa et al. 1993
). However,
although the discharge rate profiles of these posterior interpositus
neurons were apparently related to the profiles of eyelid CRs, their
firing could not be proved to encode eyelid position and/or velocity
signals. Moreover, the rapid decrease in neuronal activity in parallel
with CR extinction suggests a tight relationship with the learned motor
response, since the extinction process should be considered a new form
of learning during which the meaning of the CS is reinterpreted. For
all of those reasons, and because firing at the CS-US interval was
initiated after CR onset, it cannot be proposed that this region is the
place were eyelid CRs are initiated. Nevertheless, the interpositus
nucleus certainly seems to be a site were CRs are facilitated and/or
enhanced for an appropriate motor expression. The present results fit
well with some proposed models regarding cerebellar functions on motor
control and learning (see Houk et al. 1996
).
This study has also shown that posterior interpositus neurons are
equally involved in both delayed and trace conditioning paradigms and
that the two functional types of neuron (A and B) described here could
be related to the antagonist roles of the orbicularis oculi muscle (for
closing the lids) and of the levator palpebrae muscle (for opening
them). The same types of interpositus neuron were described previously
in a study restricted to reflexively evoked eyelid blinks
(Gruart and Delgado-García 1994).
A damping role for posterior interpositus neurons
As confirmed here with the Fourier analysis of eyelid acceleration
profiles, a significant oscillation at 20 Hz was observed during CRs
(Domingo et al. 1997
). A surprising fact regarding the
activity of these posterior interpositus neurons was the absence of
oscillation, i.e., of a repetitive bursting firing in their discharge
rate. Thus, their interspike time intervals were unimodal, and their
autocorrelation functions showed no sinusoidal profiles. The absence of
any noticeable oscillation in the firing rate of eyelid-related
posterior interpositus neurons is in evident contrast with reports
regarding the wavy form of eyelid CRs, formed in the cat by a
succession of small waves at a dominant frequency of
20 Hz
(Domingo et al. 1997
). This oscillatory behavior is by
no means an exclusive property of cat eyelid CRs, since, according to
recent data, oscillation underlying reflex and/or conditioned responses
has been recorded in up to five different species (Gruart et al.
2000
). Thus we have to assume that this population of
posterior interpositus neurons might play a role more related to the
damping of the oscillation already observed in eyelid motor responses than to its generation. A role in this direction, i.e., the predominant involvement of interpositus neurons in the control of terminal tremor,
has already been proposed from monkey studies (Thach et al.
1992
) and apparently confirmed recently in dystonic rats
(LeDoux et al. 1998
). In fact, a neuronal oscillation at
the same (
20 Hz) dominant frequency has been recorded in the
facial nucleus (Magariños-Ascone et al. 1999
;
Trigo et al. 1999
), motor cortex (Aou et al.
1992
), and dentate nucleus (Gruart et al. 1993
)
of alert cats. Accordingly, it could be necessary to have a neural site
taking charge of canceling an overt oscillation of the lids, mostly
during the acquisition of a new motor ability. In this sense, the
irregular firing and the wide range of latencies at which these neurons
are recruited could contribute to their role as a neuronal damping
device (Thach et al. 1992
). According to the present
results, muscimol injections did not produce an increase in eyelid
oscillations, a fact that contradicts the putative role of interpositus
nucleus as a damping device (Thach et al. 1992
). Perhaps
the low dose of muscimol used here did not allow us to evoke a
significant eyelid oscillation during CRs (see Mason et al.
1998
).
Functional properties of type A and B neurons
This study has supplied evidence indicating that the activity of
posterior interpositus neurons is related to some aspects of the
acquisition and/or performance of eyelid CRs, but not to the modality
and/or properties of the sensory stimuli. Thus two different sensory
modalities were used as a CS, but no functional difference was observed
in the response of recorded neurons, other than their different
activation time, probably related to the different latencies presented
by air puff- and tone-evoked eyelid CRs (Gruart et al.
1995). Furthermore, Berthier and Moore (1990)
have reported that deep cerebellar nuclear neurons do not contain specific frequency information regarding auditory stimuli, a fact confirmed for tone-evoked blinks in cats (Gruart and
Delgado-García 1994
). Moreover, the origin of signals
present in posterior interpositus neurons cannot be a feedback from
external cues, as it has been demonstrated recently that muscles
involved in eyelid responses are devoid of typical proprioceptors and
of a stretch reflex (see Trigo et al. 1999
for
references). In addition, the response of these neurons was not
strictly time-locked to CS presentation or to CR performance,
suggesting a more diffuse generation system for the motor-learning
signals present in their firing. In this sense, their activation could
be related to attentive activation processes mainly present in advance
of well-defined eyelid CRs (Allen et al. 1997
).
Type A neurons increased their activity during the CR, but not when the
cat kept its eyes open. This could be related to the facilitating role
of interpositus neurons in flexor activities during movement involving
inhibition of antigravitatory muscles (Armstrong and Edgley
1988; Bracha et al. 1999
; Orlovsky
1972
). Flexor activity is accomplished for the upper lid by the
orbicularis oculi muscle, and interpositus nucleus neural signals could
reach facial motoneurons by the red nucleus (Gonzalo-Ruiz and
Leichnetz 1990
). In the same line of thought, type B neurons
decreased their firing during eyelid CRs, as already described for this
type of neuron during reflexively evoked blinks (Gruart and
Delgado-García 1994
). Their putative role could be to
disfacilitate the continuous antigravitatory action of the levator
palpebrae muscle. It has been described that interpositus neurons
project by the ascending limb of the posterior peduncle not only to the
red nucleus, but also, among other structures, to the perioculomotor
area (Gonzalo-Ruiz and Leichnetz 1990
). Accordingly, the
role of type A neurons could be to facilitate the acquisition of an
eyelid-closing response, and that of type B cells to disfacilitate the
antagonic action of the neural mechanism active during alertness to
maintain the eyes open. This opposing activity of type A and B
posterior interpositus neurons raises an interesting question about the
possible antagonistic organization of overlying Purkinje cell pools, a
problem addressed recently using in vitro procedures (Schreurs
et al. 1998
).
Microstimulation and chemical inactivation of the posterior interpositus nucleus
The (opposite) effects of microstimulation and drug depression of
the posterior interpositus nucleus reported here are further complementary proofs of the involvement of the posterior interpositus nucleus in eyelid motor learning. It is accepted generally that the
chemical inactivation of the interpositus neurons with muscimol prevents the acquisition of eyelid CRs (see Kim and Thompson
1997 and Mauk 1997
for details). In present
experiments, the CRs were already acquired when muscimol was applied.
In this situation, a significant decrease (but not a complete
abolition) of the CR was observed. A different susceptibility between
rabbits (Garcia and Mauk 1998
; Krupa et al.
1993
) and cats (Bracha et al. 1999
) to muscimol
injections in cerebellar structures has recently been proposed to
explain the different intensity in the evoked responses observed in the
two species (Bracha et al. 1999
). On the other hand,
microstimulation of this deep cerebellar area after conditioning evoked
an eyelid response resembling the profile of a CR, but which did not
reach the amplitude of a normal CR. Nevertheless, the combined
presentation of CS and microstimulation produced a significant increase
in the amplitude of the evoked eyelid CR.
In summary, results obtained with microstimulation and muscimol infusions in the dorsal posterior interpositus nucleus further support data collected with unitary recordings in the same area, demonstrating a reinforcing role of interpositus neurons in the acquisition and performance of eyelid learned movements, but probably not in its initiation.
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ACKNOWLEDGMENTS |
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We thank R. Churchill for help in editing the manuscript. G. Guillazo-Blanch and R. Fernández-Mas were visiting fellows to the Laboratory.
Experiments were supported by BI04-CT98-0546, SAF 96-0160, PM98-0011, and CVI-122 grants.
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FOOTNOTES |
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Address for reprint requests: J. M. Delgado-García, División de Neurociencias, Laboratorio Andaluz de Biología, Universidad Pablo de Olavide, Ctra. de Utrera, Km. 1, 41013 Sevilla, Spain (E-mail: jmdelgar{at}dex.upo.es).
Received 13 April 2000; accepted in final form 5 July 2000.
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REFERENCES |
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