1Biological Computation Research Department, Bell Laboratories, Lucent Technologies, Murray Hill, New Jersey 07974; 2Department of Physiology and Neuroscience, NYU School of Medicine, New York, New York 10016; and 3Brain and Cognitive Sciences Department, MIT, Cambridge, Massachusetts 02139
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ABSTRACT |
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Aksay, E., R. Baker, H. S. Seung, and D. W. Tank. Anatomy and Discharge Properties of Pre-Motor Neurons in the Goldfish Medulla That Have Eye-Position Signals During Fixations. J. Neurophysiol. 84: 1035-1049, 2000. Previous work in goldfish has suggested that the oculomotor velocity-to-position neural integrator for horizontal eye movements may be confined bilaterally to a distinct group of medullary neurons that show an eye-position signal. To establish this localization, the anatomy and discharge properties of these position neurons were characterized with single-cell Neurobiotin labeling and extracellular recording in awake goldfish while monitoring eye movements with the scleral search-coil method. All labeled somata (n = 9) were identified within a region of a medially located column of the inferior reticular formation that was ~350 µm in length, ~250 µm in depth, and ~125 µm in width. The dendrites of position neurons arborized over a wide extent of the ventral half of the medulla with especially heavy ramification in the initial 500 µm rostral of cell somata (n = 9). The axons either followed a well-defined ventral pathway toward the ipsilateral abducens (n = 4) or crossed the midline (n = 2) and projected toward the contralateral group of position neurons and the contralateral abducens. A mapping of the somatic region using extracellular single unit recording revealed that position neurons (n > 120) were the dominant eye-movement-related cell type in this area. Position neurons did not discharge below a threshold value of horizontal fixation position of the ipsilateral eye. Above this threshold, firing rates increased linearly with increasing temporal position [mean position sensitivity = 2.8 (spikes/s)/°, n = 44]. For a given fixation position, average rates of firing were higher after a temporal saccade than a nasal one (n = 19/19); the magnitude of this hysteresis increased with increasing position sensitivity. Transitions in firing rate accompanying temporal saccades were overshooting (n = 43/44), beginning, on average, 17.2 ms before saccade onset (n = 17). Peak firing rate change accompanying temporal saccades was correlated with eye velocity (n = 36/41). The anatomical findings demonstrate that goldfish medullary position neurons have somata that are isolated from other parts of the oculomotor system, have dendritic fields overlapping with axonal terminations of neurons with velocity signals, and have axons that are capable of relaying commands to the abducens. The physiological findings demonstrate that the signals carried by position neurons could be used by motoneurons to set the fixation position of the eye. These results are consistent with a role for position neurons as elements of the velocity-to-position neural integrator for horizontal eye movements.
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INTRODUCTION |
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The
velocity-to-position neural integrator (VPNI) is a hypothesized part of
the oculomotor system that implements time integration in the
mathematical sense, transforming velocity-encoding neural inputs into
position-encoding outputs (Robinson 1975, 1989
;
Skavenski and Robinson 1973
). During saccades, VPNI
neurons should integrate a pulse-like velocity signal from saccadic
burst neurons into a sustained change in their own firing rate, thus
providing the position signal needed to hold the eye at a new fixation
point. The physiological processes by which transient inputs could be transformed into sustained changes in neural activity may include precisely tuned synaptic feedback in recurrent networks (Cannon and Robinson 1985
; Cannon et al. 1983
;
Seung 1996
), and cellular properties such as short-term
synaptic plasticity (Shen 1989
). To assess the potential
role played by these mechanisms, the neuronal elements of the VPNI must
first be identified.
Two medullary nuclei in mammals, the nucleus prepositus hypoglossi
(NPH) and medial vestibular nucleus (MVN), have been implicated as
sites of the VPNI for horizontal eye movements. In support of this
hypothesis, the NPH and MVN contain appropriate input and output
pathways to mediate integration of vestibular and saccadic commands
(Fukushima et al. 1992; McCrea 1988
;
Moschovakis 1997
). A subpopulation of neurons in the NPH
and MVN show sustained neural activity during fixations (Baker
et al. 1975
; Lisberger et al. 1994
;
Lopez-Barneo et al. 1982
; McConville et al.
1994
; McFarland and Fuchs 1992
). Below a
threshold eye position, these neurons are silent. Above this threshold,
firing rate is a linear function of horizontal eye position. Lesion and
pharmacological inactivation of regions including the MVN/NPH induced
profound centripetal drift during horizontal fixations and impairment
of the vestibuloocular reflex (VOR) and optokinetic reflex (OKR) that
is consistent with loss of integrator function (Cannon and
Robinson 1987
; Cheron et al. 1986
;
Moreno-Lopez et al. 1996
). Recently permanent lesions restricted to the rostral NPH in the primate led to deficits in horizontal fixations but largely spared horizontal sinusoidal VOR,
suggesting that integration of vestibular commands is primarily performed elsewhere (Kaneko 1997
, 1999
).
While mammalian systems have played a central role in efforts to
localize the VPNI, the goldfish oculomotor system may prove advantageous in elucidating the mechanisms by which the neural integrator operates. Both intracellular recording and optical imaging
can be performed in the awake goldfish to provide information about
synaptic currents and intrinsic electrophysiological properties in the
native neuromodulatory environment (Aksay et al. 1998; Graf et al. 1997
; Suwa et al. 1996
;
Svoboda et al. 1997
). Furthermore since goldfish eye
movements include a spontaneous scanning pattern of horizontal saccades
and fixations, the performance of the neural integrator can be assessed
without any behavioral training. In this system, two hindbrain regions,
termed area I and area II, have been implicated in oculomotor signal
generation and storage (Pastor et al. 1994
). Neurons in
area II have firing rates that modulate in phase with eye velocity
during VOR and OKR and do not change with fixation position, rendering
it unlikely that this region is part of the VPNI. Rather, lidocaine
inactivation studies suggest that it is a site for generation and
storage of eye-velocity signals. In contrast, within area I, the more
caudal of the two regions, neurons show eye-position signals. In
addition, lidocaine inactivation of regions including area I induced
profound centripetal drift during horizontal fixations and significant impairment of low-frequency (<0.25 Hz) VOR (Pastor et al.
1994
). Hence it is likely that area I composes at least part of
the goldfish VPNI for horizontal eye movements. The work presented here
is directed toward characterizing the anatomical and firing rate properties of neurons in area I that show an eye-position signal. These
neurons will be referred to as "position" neurons.
If area I is indeed an element of the VPNI, then it should have the appropriate input and output connectivity to mediate velocity-to-position integration. Neurons of the VPNI should have a soma/dendritic field that overlaps with the axonal termination fields of eye-velocity-signaling neurons. They should also have axonal projections terminating on the soma/dendritic fields of the abducens motoneurons and internuclear neurons. Therefore electrophysiologically identified position neurons in awake goldfish were labeled with Neurobiotin to visualize their somatic location, dendritic arborization, and axonal projection patterns.
To interpret the role of position neurons in behavioral deficits
induced by lesion or pharmacological inactivation of area I
(Pastor et al. 1994), it is necessary to determine if
other eye-movement-related neurons are located within the same region. Therefore a careful mapping of cell types within the medulla was performed using extracellular single-unit recording. The spatial extent
of neurons with eye-position signals was determined, and the spatial
segregation of these neurons from other oculomotor neuronal types was established.
During fixations, the output of the horizontal VPNI is expected to provide a position signal to the abducens; hence, VPNI neuron firing rates should correlate strongly with horizontal eye position. Therefore the relationship between firing rate and eye position during fixations was quantified for position neurons in area I. Also, hysteresis in the rate-position relationship, latency of change in neuronal activity to the onset of saccades, and sensitivity to saccade velocity were quantified to investigate the contribution of position neuron signals to the activity of abducens neurons during saccades and fixations. All properties were studied in both light and dark conditions to assess the role of visual feedback on position neuron activity.
Portions of this work have been presented elsewhere in abstract form
(Aksay et al. 1997).
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METHODS |
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Eye-movement measurements
Eye position was measured using the scleral search-coil
technique (Robinson 1963). A cylindrical acrylic water
tank (12-in diam, 5-in depth) was mounted inside a Helmholtz coil
system (15-in diam, CNC Engineering, Seattle, WA), and all support
posts, subject restraints, and tank components within and around the
coils were made of nonmetallic materials. After preamplification, the
signal from each of the two eye coils (5.4-mm, 40-turn, Sokymat SA) was separated into the vertical and horizontal components by
phase-sensitive detectors (CNC Engineering). The resolution of the
eye-position detection system was between 0.05 and 0.1°. When dark
conditions were necessary, the coil system was shrouded in vinyl-backed
black cloth. Voltage signals induced by eye movement were digitized during acquisition of electrophysiological signals and subsequently downsampled to a final sampling rate of 200.00 Hz (166.67 Hz when two
electrodes were used). Signals were converted to degrees off-line in
Matlab software (MathWorks, Natick, MA) using calibration parameters determined by fitting appropriate trigonometric functions to a voltage
data set obtained by rotating protractor-mounted coils through a grid
of angles spanning 80 degrees in the horizontal plane and 60° in the
vertical plane.
Animal care and surgical preparation
All experiments were performed in strict compliance with the Guide for the Care and Use of Laboratory Animals (http://www.nap.edu/readingroom/books/labrats/). Specific protocols were approved by the local Institutional Animal Care and Use Committee. Goldfish (Carassius auratus, 4-5-in tip to peduncle, 35-50 g), purchased from a commercial supplier (Hunting Creek Fisheries, Thurmont, MD), were kept at 20-22°C in a 50-gallon aquarium with daily exposure to light. At least 24 h before electrophysiological experiments, goldfish were anesthetized with MS-222 (1:2000 wt/vol; 3-aminobenzoic acid ethyl ester, Sigma, St. Louis, MO), and a stabilizing lug was attached to the exposed skull with dental acrylic and self-tapping screws (R-TX00-2, Small Parts, Miami Lakes, FL). During experiments, goldfish were head-stabilized by attachment to a mount holding the microelectrode manipulators, and body-stabilized by clamping the torso between two sponge-covered curved acrylic plates, leaving the operculum free to move. Goldfish were submerged under room temperature aquarium water (20-22°C), completely covering the eyes and nostrils but leaving dorsal regions of the cranium above the waterline. To eliminate or reduce motion of the eyes associated with respiratory movements, recirculated aerated tank water was pumped through a close-fitting mouth tube at a flow rate between 400 and 600 ml/min (pump P/N 80864 and 82059, Cole Parmer Instruments, Vernon Hills, IL). Most goldfish stopped respiratory movements within 1 min, while the rest produced less vigorous movements at a lower frequency. No qualitative change in spontaneous eye movements was produced by this procedure.
Before continuing with cranial surgery, spontaneous saccades and fixations were monitored for a period of 10 min in the light and 10 min in the dark. These data served as a baseline with which postoperative eye motions could be compared to determine if any deficits in fixation ability were induced by surgery.
Surgical procedures were performed under local anesthesia with lidocaine (2% in saline). A window (5×5 mm) in the bone above the hindbrain was cut caudal to the semi-circular canals, and the cranial cavity was rinsed with ACSF (in mM, 140 NaCl, 2.55 KCl, 5 NaHCO3, 0.42 Na2HPO4, 5 HEPES, 1.16 CaCl2, 1 MgCl2, pH 7.2, 270 mOsm). The brain was exposed, revealing the vagal lobes, facial lobe, spinal cord to the level of C2, and the caudal regions of the cerebellum. In some cases, to prevent body motion, spinal nerve C1 and the spinal cord between C1 and C2 were compressed with forceps. The exposed brain was covered with a fluorinated hydrocarbon (Fluorinert, Sigma), which was effective in reducing vascular bleeding while providing good visibility.
Single-unit electrophysiology and spike detection
RECORDING METHODS.
Single-unit recordings were made with glass microelectrodes filled with
extracellular recording solution [2 M NaCl, 10 mM Fast Green (F-99,
Fisher Scientific), pH = 7.4] and beveled to a resistance between
2 and 5 M. This range in tip size was optimal for obtaining
well-isolated, high signal-to-noise ratio (>3:1) recordings.
Preamplified signals were band-pass filtered (300 Hz to 10 kHz) prior
to being digitized at 20.00 kHz (16.67 kHz for dual recordings).
Acquisitions were made either with a 12 bit A-D MacAdios board (GW
Instruments, Somerville, MA) and custom software or a Digidata 1200B
data-acquisition system and Clampex 7.0 software (Axon Instruments,
Foster City, CA). In both cases, eye movements and electrophysiological
recordings were digitized on the same system to ensure synchronization
of data over long recording periods.
SPIKE DETECTION.
Digitized voltage traces were processed off-line by custom
spike-detection software written in Matlab. Events exceeding a negative
voltage threshold (typically 50 to
150 µV) were detected to
initially select action potential waveforms. Two parameters of detected
spike shape, the peak-to-peak amplitude and the time separation between
peaks, were plotted against each other, and a bounded region defining a
single cluster of points was specified. The times of all spikes within
the boundary were used in subsequent data analyses. Comparison of
detected spike times with the original voltage trace indicated that the
number of inappropriately omitted or included spikes was <1% of the total.
IDENTIFICATION OF AREA I. Electrodes were advanced into the hindbrain along vertical tracks aligned with the dorsal-ventral axis. Spiking activity from position neurons was located with the guidance of three types of anatomical landmarks: morphological boundaries, vasculature, and fiber tracts (Fig. 4C). Activity was centered ~1 mm rostral of the obex, 0.8 mm caudal of the caudal face of the facial lobe, and 0.4 mm lateral of midline; the center of area I in any given preparation could deviate by as much as 20% from these values. The midline bisector of two prominent blood vessels, one just caudal of the facial lobe and the other identified by its midline division into three or more branches, was within 250 µm of the center of position neuron activity. The center of activity was consistently 150-250 µm from the lateral edge of the visible medial longitudinal fasciculus (MLF). These indicators place area I in the vicinity of the border of rhombomeric segments 7 and 8. Typically, these indicators, taken together, allowed the identification of the center of area I within 5-10 exploratory penetrations.
Dye-labeling and histology
Neurons were filled with Neurobiotin (Vector Laboratories,
Burlingame, CA) by iontophoresis with sharp intracellular electrodes or
by use of the juxtacellular technique (Pinault 1996).
Sharp electrodes (1.0 mm OD, 0.58 mm ID, borosilicate omega dot, AM Systems, Seattle, WA) used for intracellular dye injection were pulled
on a horizontal laser puller (P-2000, Sutter Instruments, Novato, CA).
Electrodes were first back-filled with dye solution (1-2%
Neurobiotin, 2 M KCl, 10 mM Fast Green, pH = 7.4), then with
normal intracellular recording solution (2 M KCl, 10 mM Fast Green,
pH = 7.4), achieving a final resistance of 60-100 M
. Position neurons were penetrated with sinusoidal current bursts (0.5-2 ms, 2-5
kHz, ± 10 nA) or brief high-voltage pulses (0.1-1 ms, 2-7.5 V).
Iontophoretic labeling (50 ms, 50% duty cycle, 0.2- to 5-nA positive
current, 1-10 min) was initiated after monitoring spiking activity for
a period of 20-60 s to determine correlation with motions of the
ipsilateral eye. Electrodes used for juxtacellular dye injection were
pulled to a tip diameter of ~1 µm, first back-filled with dye
solution (5-10% Neurobiotin, 2 M NaCl, 10 mM Fast Green, pH = 7.4), then with normal extracellular recording solution. After
maximizing (to 1-2 mV) the size of the recorded action potential from
a position neuron (with
10 µm of lateral repositioning of the
electrode when near the cell), units were dye injected with 2-50 nA of
positive current (50 ms, 50% duty cycle) for 5-10 min. The amplitude
of the injection current was attenuated if the width of the action
potential increased.
Following a minimum 1-h period after Neurobiotin injection, goldfish
were anesthetized with MS-222 and transcardially perfused with ACSF
(with 1,000 U of heparin) followed by fixation solution (4%
paraformaldehyde, 0.5% glutaraldehyde, 20 mM NaOH, in ACSF). Perfused
brains were fixed overnight (4% paraformaldehyde), gelatin-embedded, and frozen. Sections (100 µm thick) were processed with the use of
the avidin-biotin-peroxidase complex (Horikawa and Armstrong 1988). Neurobiotin label was revealed with diaminobenzidine and NiCo histochemistry (Mesulam 1982
). Mounted sections
were counter-stained with cresyl violet. Tissue typically exhibited
10-20% shrinkage.
Experimental protocol
Twelve goldfish were used for the labeling studies. After extracellular localization of area I, penetrations aimed at sites of the highest density of position neurons were made with injection electrodes. To qualify for labeling, neural firing rate had to exhibit sharp transitions during saccades and a steady rate of activity during fixations, consistent with patterns of firing recorded extracellularly from position neurons (Fig. 1A). Only one position neuron in each half of the brain was injected. In some cases, 40 µg gallamine triethiodide (Flaxedil, American Cyanamid, Wayne, NJ) or 1 µg doxacurium chloride (Nuromax, Glaxo Wellcome, Research Triangle Park, NC) was intra-muscularly injected into the trunk after localization of area I to afford increased stability of recording. In these situations, attenuated eye motions could still be recorded. All Neurobiotin injections were performed in the light.
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Six goldfish were used to develop a functional map of cell types in the medulla. After surgical exposure of the floor of fourth ventricle, recording penetrations spanning the depth of the brain stem were made in a region bounded rostrocaudally by the facial lobe and the obex, and mediolaterally by the MLF and the vagal lobes; penetrations were made at 100-µm intervals on a grid. In locations where position neurons were detected, penetration intervals were reduced to 25 µm. All of these experiments were performed in the light.
Fifteen other goldfish were used for the studies relating firing rates of position neurons to eye movements. Following area I localization, up to four recording sessions of different position units were performed in each fish, with each session lasting from 5 min to 2 h (total of 44 cells). All dark data were taken during the 10 min of dark immediately following a light period that was minimally 5 min long. In some cases, goldfish spent up to an hour in the dark during a session.
Analysis of eye motions and firing rates
SACCADE DETECTION. Time blocks containing saccades were determined by grouping together those time points for which the average eye velocity in the preceding 30-ms window matched or exceeded a threshold value of 10 °/s. The leading edge of each time block defined the saccade onset time. During some saccades, a brief dip in eye velocity below the threshold level caused the algorithm to assign two time blocks to one saccade. Therefore onset times that were <250 ms after a preceding onset time were omitted from the detected set.
FIXATION PARAMETERS.
Average firing rate and eye position was calculated over a 1-s window
in each fixation beginning 0.5 s after saccade onset. Fixations of
<1.6 s between saccades were excluded from study. To eliminate
undetected saccades or significant respiration artifact, fixations were
also excluded if the mean sum-squared deviation from the best-fit line
to the calculation segment (all best-fit lines in this study are
calculated in the least-squares sense) was >0.2
deg2. Fixations were also excluded if the
preceding saccade onset time was within 1 s of a stretch (brief,
large amplitude deviations directed downward and laterally) (see
Easter 1971). Position data were normalized for
comparison between cells by a linear transformation which set the new
extreme values to
1 and 1.
FIRING RATE CALCULATIONS. Firing rate functions were calculated in one of three ways. In the first method, average firing rate was calculated by dividing the number of spikes occurring within a given window by the duration of the window. In the second method, average firing rate was calculated by taking the reciprocal of the average inter-spike intervals (ISIs) between the spikes occurring within a given window. In the third method, calculation began by determining the instantaneous ISI function for the spike train (in binwidths of at most 1.2 ms). This function was then convolved with a box-window, and the reciprocal of each element of the smoothed function was taken to produce an average firing rate function. The first method was the default, and the second one was employed when the expected spike count in a chosen calculation window was low. The third method was used when the rate function accompanying a sequence of saccades and fixations was of interest. The rate functions produced had sharper transitions associated with saccades than functions calculated by either of the other methods. The method used with a particular analysis is noted in the text.
LATENCIES. Separate algorithms, one for temporal and another for nasal saccades, were employed for the analysis of the latency between the onset of neural transitions and the onset of saccades. Transitions in firing rate associated with temporal saccades were analyzed within a window beginning 150 ms before and ending 50 ms after the moment of saccade onset. To minimize noise, transition onset times were only assigned if there was no spiking activity in the first half of the analysis window, the length of the first two ISIs in the second half of the analysis window was <20 ms, and the firing rate during the subsequent fixation was >10 spikes/s. For these cases, the time of the first spike within the second half of the analysis window was taken as the time of transition onset.
Transitions in firing rate associated with nasal saccades were analyzed within a window beginning 270 ms before and ending 100 ms after the moment of saccade onset. To reduce noise, a bias against small transitions was introduced by requiring that the average rate of firing in the initial 200 ms of the analysis window exceed 30 spikes/s (rate method 2). For the selected cases, the spikes in the latter 200 ms of the analysis window were used to determine the time of transition onset. This time was identified either by the location of the first spike followed by an ISI ofVELOCITY SENSITIVITY DURING SACCADES. The velocity sensitivity of position neurons during temporal saccades was assessed by analyzing the relationship between eye velocity and burst amplitude. Saccade velocity was measured by calculating the peak eye velocity in the initial 200 ms following saccade onset. Burst amplitude was assessed two ways: first by subtracting the presaccadic firing rate from the peak rate during a burst, and second by subtracting the postsaccadic firing rate. The peak firing rate during a burst was calculated by finding the maximum rate within a 150-ms window centered on the moment of saccade initiation (rate method 3, 50-ms convolution window). Only those saccades where the presaccadic firing rate was >5 spikes/s were included. Saccade velocity was only calculated for those cells for which more than eight data points were obtained.
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RESULTS |
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The spontaneous eye movements of head-stabilized goldfish
typically consist of a scanning pattern of horizontal saccades and fixations in which both eyes are directed toward one extreme, then span
a range of 20-40° in two to four saccades to arrive at the other
extreme (Easter 1971; Hermann and Constantine
1971
). A segment of such a scanning pattern is shown in the
eye-position records in Fig. 1A, accompanied by a
single-unit recording from a representative position neuron from area
I. The neuron progressed through a sequential set of nearly tonic
levels of firing, one for each eye fixation. The onset of a temporal
saccade (Fig. 1B) of the ipsilateral eye was preceded by the
onset of an overshooting transition in firing rate that began with a
burst of action potentials. Nasal saccades (Fig. 1C) were
accompanied by undershooting transitions in firing rate that began with
a pause in action potential discharge. Following bursts, the rate of
firing decayed to a new tonic level within 500 ms, and this new level
of activity could be maintained with little decay or deviation. During
the slide in firing rate, a slide in eye position to a new fixation
value was frequently observed. This burst-tonic pattern was
qualitatively similar to that observed during the discharge of abducens
motoneurons (Pastor et al. 1991
).
In the following, the results of labeling experiments will be presented that focused on outlining the dendritic tree and axonal projection patterns of position neurons. Next, results will be presented of a systematic exploration of the medulla in which neurons were sampled for several minutes to develop a functional map of cell types in the vicinity of area I. Following this, a quantitative analysis will be presented of position neuron discharge properties associated with changes in fixation position.
Anatomy of position neurons
Fifteen physiologically identified position neurons were injected
with Neurobiotin using sharp intracellular microelectrode recording
techniques or the juxtacellular labeling technique (Pinault 1996); nine were labeled and recovered for analysis (intra:
n = 4, juxta: n = 5). A representative
example of changes in action potential firing associated with changes
in fixation during an intracellular sharp microelectrode recording is
shown in the inset of Fig.
2A. In all cases, discharge
frequency could be modulated by passing current through the electrode
(<1.0 nA for intracellular, <20 nA for juxtacellular, data not
shown), indicating proximity to the action potential initiation site.
In every preparation, only one labeled neuron was found within each
area I. Camera lucida reconstructions in the coronal plane of two
neurons are shown in Fig. 2, A and B. Photomicrographs illustrating morphological characteristics of position
neuron soma, dendrites, and axonal projections are shown in the coronal
sections of Fig. 3. In
Fig. 2C, a schematic diagram in the horizontal plane of the
brain stem is shown that depicts the axonal projections and primary
dendritic field of position neurons determined from camera lucida
reconstructions.
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The somata of electrophysiologically-identified position neurons were fusiform in shape and ~20 µm in length (Fig. 3, A-D; n = 9/9). All cell bodies were found 0.7-1.2 mm rostral of the obex, 280-420 µm from the midline, and 360-800 µm from the surface of the fourth ventricle. All somata were located within a column of the inferior reticular formation that was 100-150 µm wide and 200-300 µm thick (Fig. 3, A, B, and D, insets).
Two to three dendritic branches emanated from the somata of position neurons (n = 9/9). As evident in Fig. 2, A and B, these dendrites branched repeatedly while ramifying over a wide extent of the medulla, primarily in the ventral region. The dendritic arbor extended from the midline to the entrance of the Xth nerve and from the level of the facial lobe (1.2 mm rostral from the center of area I) to ~300 µm rostral from the obex. Arborization was especially heavy in the first 500 µm rostral of cell somata (Fig. 2C).
In heavily labeled cells (n = 4/9), spiny protrusions were visible on dendritic branchlets (Fig. 3, G and H, photomicrographs of the dendritic segments labeled G and H in Fig. 2B). These protrusions typically had shafts that were 2-4 µm in length, ending in ~1-µm-sized swellings. At their highest density, spiny protrusions were spaced at intervals of ~5 µm (Fig. 3H).
Position neuron somata gave rise to a single axon hillock (Fig. 3, A-D). In six of nine cases, it was possible to follow the axon of the cell and determine its projection pattern. The hillock led to a sub-micron diameter axonal initial segment that was ~30 µm in length (Fig. 3C). Axons exhibited two types of projection patterns, one ipsilaterally directed and the other contralaterally directed.
In four ipsilateral cases, axons turned ventrally and then coursed rostrally, 300-500 µm from the midline, along a ventrally located fiber bundle (vfb) above the inferior olive (Fig. 2B). Axons could be followed at least up to the level of the facial lobe (1.2 mm from the soma), where they began to overlap with the dendritic field of the caudal internuclear group of the abducens (Fig. 2C). In two cases, the cell was labeled well enough to visualize axonal collaterals, with presynaptic boutons (Fig. 3, E and F), emanating from the parent axon at multiple levels through the abducens complex (Fig. 2C). In these cases, axons ventured as far as 500 µm beyond the rostral group of abducens neurons before either becoming too weakly labeled to follow or ending.
In two contralateral cases, axons projected toward the contralateral area I, crossing the midline beneath the MLF (Fig. 2A). In one case, the cell was labeled well enough to see that the crossing axon gave rise to collaterals with presynaptic boutons near the somata and proximal dendrites of contralateral position neurons (Fig. 2, A and C). Following this initial collateralization, the axon coursed rostrally along the ventral pathway described above, before collateralizing and terminating within the dendritic field of contralateral internuclear neurons.
Axon collaterals and terminal arborizations were only visible in three of six cells with filled parent axons. Therefore the full extent of the axonal termination pattern of position neurons may have been underestimated.
Functional map of area I
A systematic exploration of the medulla was made in six goldfish, spanning the area bounded by the vagal lobes, facial lobe, and obex (Fig. 4). In each fish, >20 extracellular single-unit recordings were obtained from position neurons. Spiking activity from position neurons was separable into two distinct classes according to spike waveform: one class had triphasic action potentials (initially positive going, Fig. 4A), and the second had biphasic ones (initially negative going, Fig. 4B). Biphasic action potential waveforms were very stable; a recording from a single unit could be monitored over an electrode displacement of ~100 µm along the dorsoventral axis and maintained for several hours. Biphasic recordings were only seen in a restricted region that, as outlined in the following text, coincided with the locations of labeled position neuron somata. Recordings of triphasic waveforms were unstable; recordings could only be monitored over distances of ~20 µm and were frequently associated with sudden wave-form amplitude changes (for example, a decrease in peak-to-peak amplitude from 0.8 to 0.3 mV). Triphasic waveforms were recorded over a wide range of the medulla that, as outlined in the following text, coincided with the range of the dendritic arbor of labeled position neurons. The stability and localization of biphasic waveforms indicate that they were recorded in proximity to somata, while the instability and widespread occurrence of triphasic waveforms indicate that they were recorded in proximity to processes.
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The ellipse in Fig. 4C is centered at a location that corresponds to the approximate mean position of area I across the fish studied (anatomy is drawn to scale). As mentioned in METHODS, the center of position neuron (somatic) activity could deviate by as much as 20% from the average distances indicated. During vertical penetrations, the probability of encountering a position neuron soma was >80% in the caudal half of area I, ~50% in the rostral half, and <20% near the boundaries; this encounter scale is represented by the sizes of the filled circles in Fig. 4C. The encounter probability outside the bounding ellipse was <5%. The width of the ellipse along the mediolateral axes was ~125 µm, in close agreement with the width of the inferior reticular formation column in which labeled somata were identified. The length of the ellipse along the rostrocaudal axes was ~350 µm, consistent with the range over which labeled somata were found. Somatic recordings of position neurons in any given fish were encountered over a ~250 µm vertical range during penetrations, in close agreement with the thickness of the column in which labeled somata were identified. Across the population, this range was always encountered between the depths of 400-1,000 µm below the surface of the medulla. Therefore this functionally defined ellipsoidal region, ~350 µm in length, 125 µm in width, 250 µm in depth and coinciding closely with the location of position neuron somata, will be used to define the extent of area I.
On average, any given penetration in area I would yield approximately one encounter with a position neuron soma; at locations indicated by the largest circles, encounter rates could be as high as two position units every 100 µm. The lateral extent over which a somatic recording could be monitored was typically only 40 µm. This was established by obtaining a stable biphasic recording, retracting the electrode, moving laterally, advancing to record the same neuron (as determined by the position sensitivity and onset threshold), and repeating these steps until spikes from the cell of interest were undetectable.
The likelihood of obtaining a triphasic recording from a position neuron process during a single penetration within the boundary of area I was ~20%. Encounter likelihood beyond this boundary was not the same in all directions: recording probability was <20% during rostral penetrations and <5% during caudal penetrations. Also as penetrations further rostral were made, the mediolateral and dorsoventral range over which processes were encountered widened. A dramatic decrease in probability of encounter was observed beyond ~0.5 mm past the rostral border of area I.
The majority (>95%) of position neurons recorded had ipsilateral on directions, increasing firing rate with temporal (on) saccades and decreasing firing rate with nasal (off) saccades of the ipsilateral eye. Recordings of units with contralateral on directions were always of triphasic waveforms. This suggests that such recordings were from axons of position neurons with somata in the contralateral area I.
Approximately two-thirds of all single-unit recordings within the ellipsoidal boundary of area I were from position neurons. Four other cell types, with biphasic waveform recordings indicative of proximity to somata, were also encountered. One cell type was active in correlation with respiratory rhythms. This cell type was not correlated with eye movements when respiratory movements were not present. A second type was tonically active, not exceeding 1-2 spikes/s in discharge rate. A third type was clearly related to motions of the body and tail, discharging in bursts when the tail was flicked toward the ipsilateral side. The fourth type, encountered very infrequently (<2% of recordings) and not in every goldfish, exhibited a build-up in firing rate during a fixation, and, in contrast to position neurons, paused during temporally directed saccades. The first three cell types also composed the great majority (>90%) of single-unit recordings obtained in the few hundred micrometers rostral or caudal of area I. In the regions within a few hundred micrometers medial or lateral of area I, very few recordings were indicative of proximity to a soma, consistent with area I residing along a column of the inferior reticular formation.
Discharge properties
To quantify saccade and fixation related discharge properties of position neurons, long-duration extracellular recordings were obtained from 44 neurons. Of these, 24 were recorded both in the light and dark, 5 were recorded only in the dark, and another 15 were recorded only in the light. Data were grouped into dark (n = 29), light (n = 39), and composite sets (n = 44; 29 dark and 15 light). Data, in both light and dark conditions, on position sensitivity, velocity sensitivity, latency to saccades, and the relation of position sensitivity to threshold of firing and hysteresis are summarized in Table 1.
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The difference in means of the dark and light set population averages for position sensitivity, threshold, hysteresis, velocity sensitivity, and latency was not significant at the P < 0.05 level (t-test; used for all tests of difference in population means). Therefore summary information for the composite set is also provided in Table 1.
POSITION SENSITIVITY. As shown in Fig. 1A, each new eye position after the occurrence of a saccade was accompanied by a different mean firing rate. For all 29 cells in the dark, fixation-averaged firing rate (rate method 2) and fixation-averaged horizontal position of the ipsilateral eye were well correlated above an eye-position threshold (mean r = 0.84, all r > 0.6, P < 0.005; 2-tailed Student's t-test, used for all tests of correlation; fixations following on- and off-direction saccades over a period of 5-10 min were pooled). Representative data from two position cells in the dark are shown in Fig. 5, A and B. Light data for the cell of Fig. 5A is shown in Fig. 5C; note the qualitative similarity in slope and threshold. The best-fit lines determined for the population of neurons recorded in the dark are shown in Fig. 5D. For all cells in the dark, the slope (k) of the relationship between firing rate and eye position, a measure of the position sensitivity given in units of (spikes/s)/°, ranged from 0.5 to 8.0, with a mean of 2.5. The thresholds and slopes of cells were not topographically organized. All units in the light and dark had ipsilateral on direction, increasing firing rate for temporal saccades and decreasing firing rate for nasal saccades of the ipsilateral eye.
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FIRING RATE HYSTERESIS.
The firing rate following an on saccade to a given eye position
was typically less than that following an off saccade to the same
position. An example of this hysteresis in the relationship between
firing rate and eye position is shown in Fig.
6A. In this figure for data
from a single position neuron in the dark, are from fixations
following on saccades, and
from fixations following off saccades.
The ordinate is the deviation in firing rate from the best-fit line of
the rate-position relationship. As a measure of hysteresis, the
difference in the means of on and off groups was calculated for data
from the central 40% of fixations, where there was the highest degree
of overlap between the groups. The hysteresis values in the dark for 13 analyzed cells (those units with 3 fixations in each group) ranged
from 1.3 to 54 spikes/s (P < 0.05), with a mean of
12.3; as a fraction of maximum firing, values ranged from 5.1 to 25%,
with a mean and standard deviation of 15.5 ± 6.6%. When
hysteresis values are plotted against position sensitivity, a
significant trend emerges (Fig. 6B). Hysteresis scaled
linearly with position sensitivity, with a slope of the regression line
of 6.0° (r = 0.93, P < 0.001).
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VELOCITY SENSITIVITY.
Position neurons exhibited burst-tonic behavior similar to that shown
in Fig. 1. Typically, a burst of action potentials lasting no longer
than 100 ms accompanied on-direction saccades, with the onset of bursts
preceding saccadic motion of the eye (Fig. 1B). Burst
amplitude was assessed two ways: first, by subtracting the presaccadic
firing rate from the peak rate during a burst, and second, by
subtracting the postsaccadic firing rate. The first method assumes that
all of the burst is related to saccadic motion, while the second
assumes that only the portion of the burst that is overshooting is
related to saccadic motion. Forty-three units exhibited an overshoot in
firing rate during on-saccades (mean value ranging from 2.7 to 54.6 spikes/s, population average = 26.0 spikes/s). One unit did not
exhibit an overshoot (mean = 8.1 spikes/s).
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LATENCY. The mean delay between the onset of a burst and the onset of a saccade for the population of units in the dark was 18.4 ± 8.6 ms (SD) (n = 82 bursts, n = 9 cells). The ranges of the mean and standard deviation for those units (n = 9) with at least three analyzed bursts were 6.2-24.1 and 1.3-11.2 ms, respectively.
Off-direction saccades were typically (>80% of the time) accompanied by undershoots in firing rate that began before saccade onset (Fig. 1C). The mean delay time from the last action potential before the beginning of this undershoot to the onset time of the saccade for the dark set was 22.0 ± 21.2 ms (n = 165 transitions, n = 22 cells). The ranges of the mean and standard deviation for those units (n = 16) with at least three analyzed off transitions were 11.3-45.7 and 8.7-34.6 ms, respectively. Relaxation to new tonic levels of firing occurred rapidly, within 500 ms. One position neuron showed no burst activity associated with on-direction saccades. The first spike associated with a transition in firing rate accompanying an on-direction step in fixation position began after the initiation of the saccade, with a mean delay of 9.6 ± 5.6 ms (n = 17 bursts). However, this unit paused before onsets of off-direction saccades in the same manner as the rest of the quantified population (22.1 ± 7.1-ms delay, n = 25 transitions). ![]() |
DISCUSSION |
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The results presented here extend knowledge of the properties of
premotor neurons in the goldfish inferior reticular formation that have
an eye-position signal, building on an initial description of neurons
recorded in a coarsely defined region termed area I (Pastor et
al. 1994). Lidocaine injections into this general area disrupted fixation ability, providing evidence that area I was part of
the hypothesized VPNI for horizontal eye movements. Here, a description
of the anatomical characteristics of position neurons has been provided
to determine whether or not they possess the appropriate connectivity
to be elements of the VPNI. The spatial segregation of position neurons
was assessed to determine what the results of area I inactivation
experiments imply about the function of position neurons. Finally, a
quantitative description of the discharge characteristics of position
neurons during saccades and fixations has been provided to determine
whether or not they carry the signals appropriate for the VPNI during
this behavior.
In the following, these results will be first discussed in the context of the role of area I position neurons in oculomotor control. Next, comparisons will be made between characteristics of goldfish and mammalian position neurons. Following this will be a discussion of properties of the area I to abducens projection, then of issues raised by these results that are relevant to the mechanism of integration.
Role of area I position neurons in oculomotor control
Expected characteristics for elements of the VPNI include
dendritic fields consistent with the axonal terminations of neurons carrying premotor eye-velocity commands (inputs to the integrator), axonal projections to the extraocular motor nuclei for horizontal eye
movements, and appropriate eye-position signals during oculomotor behavior. All three of these expectations were met, at least in part,
by position neurons in area I of the goldfish. Position neurons had
extensive dendritic arborization ventral and rostral to the cluster of
somata. Second-order vestibular neurons and horizontal canal afferents
are known to terminate extensively along the ventral half of the
medulla rostral of area I (Baker et al. 1998). Thus
position neuron dendrites are in the correct location to receive head
velocity signals, consistent with the modulation of position neuron
discharge observed during sinusoidal VOR stimuli (Pastor et al.
1994
). Position neurons either had axons that followed a
well-defined ventral pathway toward the ipsilateral abducens complex,
or had axons that crossed the midline, projecting toward the
contralateral group of position neurons and the contralateral abducens
complex. Finally, the firing rate of position neurons was well
correlated with horizontal position of the ipsilateral eye. These
results provide evidence that area I position neurons are a central
component of the horizontal VPNI.
One manner in which the role of area I in oculomotor behavior can be
tested is through pharmacological inactivation experiments. Previous
work has demonstrated that injection of lidocaine, a sodium-channel
blocker, in a region including area I induced centripetally directed
drift of the eye during fixations (Pastor et al. 1994). This result, by itself, shows that this region of the medulla may be
part of the VPNI but does not distinguish between the role played by
position neurons and other eye movement related neurons that may also
have been inactivated. Evidence presented here indicates that the
somata of position neurons in the medulla of the goldfish form a
spatially localized cluster. Recordings from position neurons composed
~2/3 of those obtained from area I. No other cell type encountered in
this region appears to be relevant to the issue of defining the role
that this locus plays in oculomotor behavior: neurons with activity
correlated to respiratory rhythms are likely to be involved in
respiratory function, those related to tail motion are likely involved
in axial movement, and those with low tonic firing rate do not
characterize any known vestibular-related (Green et al.
1997
) or optokinetic-related (Pastor et al.
1994
) neurons. Neurons in which activity built-up during
fixations were encountered too infrequently to play a significant part
in defining the function of this region during normal saccades and
fixations. Therefore in respect to the oculomotor system, area I can be
viewed as a spatially segregated locus that is composed almost
exclusively of neurons carrying horizontal eye-position signals. This
result, when coupled with results of inactivation experiments, provides further evidence that area I position neurons are elements of the
goldfish VPNI for horizontal eye movements. A quantitative assessment
of the precise contribution of these units to oculomotor function will
require experiments in which the spatial extent of inactivation of area
I and other medullary regions are monitored. Given the localization
results presented here, such experiments can now be performed with a
high degree of accuracy (Aksay et al. 1998
).
There were no significant differences in the position sensitivities
(k) or thresholds (Eth) of
position neuron firing rates in the light versus in the dark (Table 1).
Even though goldfish are afoveate, stabilization of the eye in the
light is improved over the dark condition (Mensh et al.
1997). This improvement is likely due to the use of a retinal
slip signal in a closed-loop control system that minimizes drift
(Robinson 1981
). If the feedback were to bypass area I,
then the relationship between firing rate and eye position might be
different in the light and the dark. Our finding of no difference
between light and dark conditions is consistent with the idea that area
I is part of the neural system that integrates these feedback signals
to produce an improved signal for stabilization.
Comparisons with mammalian position neurons
The anatomy of position neurons in the goldfish most closely
matches that of the "principal" cells in the NPH of the cat
(McCrea and Baker 1985a,b
). These cells, recorded in the
anesthetized preparation, projected unilaterally and distributed
terminal fields within the abducens complex, leading to the suggestion
that this cell type generates the position signal recorded in the NPH
in the awake preparation. The anatomy of position neurons in the goldfish did not match that of the "small" cells of the
dorsolateral NPH in the cat, which had bilateral projections. Nor did
it match that of the "multidendritic" cells of the caudal part of
the cat NPH, which exhibited projections to the cerebellum. As
described in previous work (Pastor et al. 1994
),
pressure injections of biocytin into the vestibulo-cerebellum of
goldfish selectively labeled the inferior olive and brainstem area II,
not cells in area I.
Field potentials recorded in the abducens nucleus of the cat following
discharge of position neurons in the NPH are consistent with excitatory
ipsilateral and inhibitory contralateral projections (Escudero
and Delgado-Garcia 1988; Escudero et al. 1992
).
In support of the inhibitory nature of the contralateral projections,
injection of tritiated glycine, a putative inhibitory transmitter in
this system, into the abducens complex of cats resulted in label of the
somata of neurons in the contralateral NPH (Spencer et al. 1989
). The possibility that ipsilaterally and contralaterally projecting position neurons of goldfish have different physiological properties remains unexplored. Since all goldfish position neurons have
ipsilateral on directions, the contralateral projection to the abducens
would only make functional sense if it were inhibitory. A similar
argument has been made for contralaterally projecting neurons in the
primate NPH/MVN (McFarland and Fuchs 1992
). Similarly, the ipsilateral projection from goldfish position neurons would only
make functional sense if it were excitatory.
In this study, the term "position neuron" was used for any
cell in area I whose tonic discharge during fixations correlated with
eye position. In primate, two types of position neurons have been
identified: those that were sensitive to eye-position, eye-velocity, and head-velocity, and those that were sensitive primarily to eye-position and -velocity only (Fukushima et al. 1992;
McFarland and Fuchs 1992
). The first type has been
termed "eye/head-velocity" and the second "burst-tonic." In
cat, only position neurons that can be broadly termed "burst-tonic"
have been described (reviewed in Fukushima et al. 1992
).
The contribution of head-velocity to the discharge of position neurons
in cat and goldfish has not been determined.
The relationship between eye movements and neural activity for position
neurons in area I was very similar to that for burst-tonic neurons in
the MVN/NPH of cat (Delgado-Garcia et al. 1989;
Lopez-Barneo et al. 1982
) and monkey (Fukushima
et al. 1992
; McFarland and Fuchs 1992
) under
similar behavioral conditions (saccades and fixations). Units exhibited
bursts of activity preceding the onset of on-direction saccades,
relaxed to steady-state values of firing rate within 500 ms, and
exhibited undershoots in firing rate accompanying saccades in the off
direction. Cells were capable of maintaining steady firing during a
fixation at values between 3 and 200 spikes/s. The position
sensitivities of goldfish position neurons ranged from 0.5 to 8.4 (spikes/s)/°, comparable to those in the cat and monkey. Onset
thresholds were distributed in the nasal half of the range of eye
motion, and the relationship between position sensitivity and threshold
indicated a recruitment order, again, in agreement with the trend in
the cat and monkey. Such functional homology suggests that a common
mechanism of integration may be used and that understanding the
cellular and circuit mechanisms of the goldfish VPNI may generalize
across vertebrate species.
Based on qualitative consideration of the saccade-related burst, the
burst-tonic neurons of mammals have been separated into two or more
groups: in primate, "burst-position" and "pure-position" (McFarland and Fuchs 1992), and in cat,
"velocity-position," "position-velocity," and pure-position
(Delgado-Garcia et al. 1989
). Is it possible to assign
area I burst-tonic neurons to similar subgroups? In primate, analysis
of the latencies to on-direction saccades indicated that those neurons
classified as pure-position generally began firing after the initiation
of saccades, while those classified as burst-position generally began
firing before. In goldfish, only one neuron was identified that
consistently did not burst during saccades and began transitions in
rate after saccades were initiated. Based on the low frequency of
occurrence of this type of response in goldfish (n = 1/44), it is difficult to determine if pure-position neurons represent
a distinct class or the tail end of a distribution. In the cat, it was
noted that units were "distributed as a continuum in which a
progressive decrease of eye-velocity sensitivity was accompanied by a
proportional increase in eye-position sensitivity"
(Delgado-Garcia et al. 1989
). In the goldfish, however,
there was a weak correlation between velocity and position
sensitivities with a slope indicating direct proportionality. Thus it
is inappropriate to attempt to divide these data into the same classes
used in analysis of cat NPH/MVN neurons.
Area I projection to the abducens
Based on differences in the latencies of field potentials recorded
in the abducens complex following position-velocity and pure-position
neuron discharge in cat, it was hypothesized that pure-position neurons
are the sole means by which position signals are supplied to the
motoneurons (Escudero et al. 1992). For this hypothesis
to be consistent across species, the activity of the injected area I
neurons that projected to the abducens complex should have been
characterized by a lack of burst activity during on-saccades. But this
was not the case, as seen for a representative recording in Fig.
2A, where clear burst activity accompanied on-direction saccades. Furthermore it has been noted that in the monkey, the major
output to the abducens is from the marginal zone of the NPH and that
the majority of position neurons in the marginal zone are of the
burst-tonic variety (Belknap and McCrea 1988
; Langer et al. 1986
). Therefore this hypotheses is either
invalid or applies only to certain species.
Goldfish position neurons exhibited a significant rate-position
relationship hysteresis associated with the direction of the preceding
saccade; furthermore this hysteresis was correlated with the position
sensitivity of the cell. The presence of hysteresis in motoneurons has
been noted in previous work in the goldfish (Pastor et al.
1991), cat (Delgado-Garcia et al. 1986
), and
monkey (Goldstein and Robinson 1986
). The origin of this
hysteresis is unknown, but a significant muscle hysteresis may be a
contributing factor (Collins et al. 1975
;
Goldberg et al. 1998
). Abducens motoneurons of the
goldfish were reported to exhibit a hysteresis corresponding to a
constant difference of ~10 spikes/s over most of the oculomotor range
in the rate-position plots obtained after on- and off-directed saccades
(Pastor et al. 1991
). In the primate, abducens neurons (motoneurons and internuclear neurons) were reported to exhibit a
significant hysteresis in their rate-position curves, corresponding to
an average difference of 5.4 spikes/s near the middle of the range of
eye motion (Goldstein and Robinson 1986
). In those
studies, no correlation was found between the static position
sensitivity of an abducens neuron and its degree of hysteresis, in
contrast with results presented here for position neurons. If the
projection from the position neurons to the motoneurons was organized
according to position sensitivity (high to high, low to low), then one
would expect that the motoneurons would also have shown a correlation between hysteresis and sensitivity. Thus this difference is consistent with the idea that all abducens motoneurons receive the same position signal, a "common drive" derived from an average of the outputs of
many position neurons. This would imply that the recruitment order seen
at the level of the motoneurons (Pastor et al. 1991
) is
established independently of the recruitment order seen for position neurons.
The latencies reported here between saccade onset and firing rate
transition onset for burst-tonic neurons were slightly greater than
those reported for goldfish abducens motoneurons (Pastor et al.
1991) (17.2 ± 8.1 vs. 14.1 ± 4.8 ms for
on-saccades, 21.8 ± 19.9 vs. 20.2 ± 5.6 ms for
off-saccades). Assuming no offset was introduced by differing latency
measurement methodologies, these results are consistent with
bursts/pauses in position neurons contributing to the development of
bursts/pauses in motoneurons. Furthermore firing rate bursts during on
saccades were correlated with eye velocity, again, consistent with a
role in the generation of saccades. However, during bilateral lidocaine
inactivation of regions including area I (Pastor et al.
1994
), large amplitude temporal and nasal saccades were still
produced. This suggests that the burst activity of area I position
neurons may not be a significant source of saccadic drive and that the
parallel pathway of burst input to the motoneurons may be sufficient
for the generation of saccadic motion.
What might be the primary function of position neuron bursts? One
possibility is that they are necessary to produce the postburst slide
in firing rate that was observed in goldfish area I position neurons.
Similar postburst slides have been observed on the position neurons of
the cat (Lopez-Barneo et al. 1982) and primate
(McFarland and Fuchs 1992
). Since burst neurons do not
exhibit a slide, the slide in position neuron firing rate may serve as
the primary determinant of the slide of motoneuron firing rates, a
common feature in the abducens of goldfish (Pastor et al.
1991
), cat (Delgado-Garcia et al. 1986
), and
monkey (Fuchs et al. 1988
). This slide in motoneuron
firing is thought to be important in overcoming the viscoelastic
properties of the oculomotor plant during rapid eye movements
(Goldstein and Robinson 1984
; Optican and Miles
1985
).
Implications for mechanisms of integration
The identification of position neuron axons projecting to the
contralateral area I suggests a pattern of reciprocal connectivity, a
form of recurrent synaptic drive. Since the on directions of area I
cells in each half of the hindbrain are opposite to each other, it
seems likely that this projection is inhibitory as was also suggested
for the continuing projection of the main axon of these cells to the
contralateral abducens. It is interesting to note that reciprocal
inhibition can produce net positive feedback, a mechanism employed in
network models of the VPNI (Cannon et al. 1983;
Galiana and Outerbridge 1984
). Such a substrate for feedback activity has been observed in the cat (McCrea and Baker 1985b
; Spencer et al. 1989
) and monkey
(McCrea 1988
). Preliminary efforts exploring the role of
a reciprocal feedback pathway through midline-sections and unilateral
inactivation led to the conclusion that a single intact area I could
process position signals capable of driving both eyes (Pastor et
al. 1994
), although some eye movement deficits were observed
after unilateral inactivation. However, the effects of these
experiments were not studied in a quantitative manner, and therefore
should be repeated in light of the results presented here.
The principal cells of cat NPH have axon collaterals within the nucleus
(McCrea and Baker 1985a,b
). Likewise, many cells in the
NPH of the monkey also give rise to local collaterals (McCrea 1988
). These local collaterals may serve as a means by which
recurrent synaptic drive is produced among position neurons.
Unfortunately, the results presented here do not contribute to an
understanding of the presence or absence of local collaterals from area
I position neurons in the goldfish because of incomplete labeling of
the axonal process. It is likely that the Neurobiotin injections were at the soma or the large proximal dendrites. Given the small diameter of the axon initial segment and the injection location, it is possible
that the flux of Neurobiotin to the axon was too small to significantly
fill its collaterals and terminations. Also, very light labeling of
spines on dendrites suggests the presence of diffusion barriers in
these neurons. Given the present identification of a ventral pathway by
which the larger diameter distal axons course rostrally, an alternative
future strategy to determine position neuron morphology could be to
label through axonal microinjections, a strategy that has been
successfully employed in the primate (Moschovakis 1997
).
The region of heavy dendritic arborization rostral of cell somata
closely coincided with a region in which extracellular recordings of
triphasic action potentials were common. Since the extent of this
region of triphasic signals was much greater than the narrow pathway
that axons were seen to follow rostral of area I, it is likely that
most of the processes from which the recordings were obtained were
dendritic. Thus these results suggest that action potentials propagate
along the dendrites of position neurons. Dendritic action potentials
could activate dendro-dendritic chemical synapses mediating recurrent
feedback among position neurons (Mori et al. 1982) or be
involved with the gating of synaptic plasticity (Yuste and Tank
1996
).
Neurons in the abducens complex of a teleost fish are electrically
coupled through gap-junctions (Korn and Bennett 1975). In this study, intracellular microinjection of Neurobiotin produced at
most only one labeled cell in each attempt. The absence of dye-coupling
suggests that gap junctions are not present between position neurons in
area I. Therefore it is likely any feedback between position neurons is
not mediated through electrical synapses.
The threshold distribution pattern found in this study may have
important consequences for the ability of the VPNI to hold the eyes at
temporal positions. In a conductance-based recurrent-feedback model of
the VPNI (Seung et al. 2000), it was observed that
recruitment of new position neurons was necessary to compensate for the
shortfall in synaptic feedback caused by synaptic saturation. In this
model, a restriction of the onset thresholds to the nasal half of the range of eye motion resulted in a supra-linear increase in the magnitude of nasally directed drift with increasing temporal eye position. This pattern of drift closely matches what has been experimentally observed during spontaneous behavior in goldfish (Mensh et al. 1997
).
The lateral range over which a position unit could be monitored was
~40 µm, and, on average, the number of units encountered on a
single vertical penetration into area I was close to one. This,
together with the overall size of area I shown in Fig. 4C, implies that there are ~30 position units contained within each area
I. This number is comparable to the ~60 motoneurons in the abducens
(Pastor et al. 1991) and the ~40 neurons in the medial rectus motor pool (Pastor et al. 1991
). If area I
provided all of the position signal driving fixations, then, on
average, each position neuron would account for a few percent of the
total. This raises the interesting possibility that lesion or
stimulation of a single position neuron might have an experimentally
detectable effect on oculomotor behavior.
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ACKNOWLEDGMENTS |
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We thank Drs. Brett Mensh and Daniel Lee for contributing to software development and Dr. Hiroshi Suwa for assistance with intracellular labeling.
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FOOTNOTES |
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Address for reprint requests: R. Baker or D. W. Tank.
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 21 January 2000; accepted in final form 24 April 2000.
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REFERENCES |
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