1Laboratoire de Physiologie de la Perception
et de l'Action, College de FranceCentre National de la Recherche
Scientifique, F-75231 Paris Cedex 05, France;
2Department of Anatomy, Medical College of
Virginia, Richmond, Virginia 23298; 3Department
of Neurology, Burke Rehabilitation Center, White Plains 10605; and
4Department of Physiology and Neuroscience, New
York University Medical Center, New York, New York 10016
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ABSTRACT |
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Graf, Werner,
Robert Spencer,
Harriet Baker, and
Robert Baker.
Vestibuloocular Reflex of the Adult Flatfish. III. A
Species-Specific Reciprocal Pattern of Excitation and Inhibition.
J. Neurophysiol. 86: 1376-1388, 2001.
In juvenile
flatfish the vestibuloocular reflex (VOR) circuitry that underlies
compensatory eye movements adapts to a 90° relative displacement of
vestibular and oculomotor reference frames during metamorphosis. VOR
pathways are rearranged to allow horizontal canal-activated
second-order vestibular neurons in adult flatfish to control
extraocular motoneurons innervating vertical eye muscles. This study
describes the anatomy and physiology of identified flatfish-specific
excitatory and inhibitory vestibular pathways. In antidromically
identified oculomotor and trochlear motoneurons, excitatory
postsynaptic potentials (EPSPs) were elicited after electrical
stimulation of the horizontal canal nerve expected to provide
excitatory input. Electrotonic depolarizations (0.8-0.9 ms) preceded
small amplitude (<0.5 mV) chemical EPSPs at 1.2-1.6 ms with much
larger EPSPs (>1 mV) recorded around 2.5 ms. Stimulation of the
opposite horizontal canal nerve produced inhibitory postsynaptic potentials (IPSPs) at a disynaptic latency of 1.6-1.8 ms that were
depolarizing at membrane resting potentials around 60 mV. Injection
of chloride ions increased IPSP amplitude, and current-clamp analysis
showed the IPSP equilibrium potential to be near the membrane resting
potential. Repeated electrical stimulation of either the excitatory or
inhibitory horizontal canal vestibular nerve greatly increased the
amplitude of the respective synaptic responses. These observations
suggest that the large terminal arborizations of each VOR neuron
imposes an electrotonic load requiring multiple action potentials to
maximize synaptic efficacy. GABA antibodies labeled axons in the medial
longitudinal fasciculus (MLF) some of which were hypothesized to
originate from horizontal canal-activated inhibitory vestibular
neurons. GABAergic terminal arborizations were distributed largely on
the somata and proximal dendrites of oculomotor and trochlear
motoneurons. These findings suggest that the species-specific
horizontal canal inhibitory pathway exhibits similar
electrophysiological and synaptic transmitter profiles as the anterior
and posterior canal inhibitory projections to oculomotor and trochlear
motoneurons. Electron microscopy showed axosomatic and axodendritic
synaptic endings containing spheroidal synaptic vesicles to establish
chemical excitatory synaptic contacts characterized by asymmetrical
pre/postsynaptic membrane specializations as well as gap junctional
contacts consistent with electrotonic coupling. Another type of
axosomatic synaptic ending contained pleiomorphic synaptic vesicles
forming chemical, presumed inhibitory, synaptic contacts on motoneurons
that never included gap junctions. Altogether these data provide
electrophysiological, immunohistochemical, and ultrastructural evidence
for reciprocal excitatory/inhibitory organization of the novel
vestibulooculomotor projections in adult flatfish. The appearance of
unique second-order vestibular neurons linking the horizontal canal to
vertical oculomotor neurons suggests that reciprocal excitation and
inhibition are a fundamental, developmentally linked trait of
compensatory eye movement circuits in vertebrates.
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INTRODUCTION |
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Flatfish are a natural paradigm to study a
developmental adaptation to a changing living situation. Larvae begin
their lives in an upright body posture with the eyes placed on the left
and right sides of the head (Fig.
1A). At this stage,
vestibuloocular reflex (VOR) eye movements elicited during swimming
involve detection of horizontal head rotation about the vertical axis
by the horizontal semicircular canals. Horizontal conjugate eye
movements are produced by the lateral and medial rectus eye muscles in
the earth-horizontal plane (Graf and Baker 1985a) (Fig.
1A). The neuronal circuitry mediating this reflex involves
the classical three-neuron arcs along with the specialized abducens
internuclear pathway (Fig. 1A).
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During metamorphosis, flatfish rotate 90° about the longitudinal axis
to become better adapted to an adult lifestyle on the bottom. The eye
ipsilateral to the side that now faces the sea bottom migrates across
the dorsal aspect of the head to the upper side of the flatfish (Fig.
1B). The eyes, including the extraocular muscle apparatus
retain their original orientation with respect to the environment;
however, since the labyrinths remain in their original position in the
head (Fig. 1B), the horizontal canals detect swimming
motions that now occur in the vertical plane. The appropriate
compensatory eye movements, by contrast, are parallel backward and
forward rotations of the eyes produced by contraction of vertical eye
muscles. This behavior requires a principal VOR circuitry connecting
the horizontal canals to vertical extraocular muscles as shown in Fig.
1B (Graf and Baker 1983,
1985b
). The previous neuroanatomical experiments
described the structural basis of the novel horizontal
canal-to-vertical eye muscle connections, and the current work
addresses the electrophysiology of the excitatory and inhibitory pathways.
In all vertebrates, VOR pathways exhibit a reciprocal excitatory and
inhibitory innervation of extraocular motoneurons in which a
stereotyped, constant relationship is maintained between each of the
three semicircular canals (anterior, posterior, and horizontal) and one
extraocular muscle pair (reviewed in Evinger 1988;
Graf 1988
; Graf and Ezure 1986
;
Graf et al. 1983
). In particular, with regard to the
vertical canals (anterior and posterior), second-order VOR neurons
exhibit a unilateral innervation pattern of the oculomotor neurons
(Graf and Ezure 1986
; Graf et al. 1983
,
1997
). Excitatory VOR neurons activated from the
anterior canal (AC) contact contralateral superior rectus (SR) and
inferior oblique (IO) motoneurons, whereas posterior canal (PC) VOR
neurons terminate on contralateral superior oblique (SO; i.e.,
trochlear) and inferior rectus (IR) motoneurons. Inhibitory pathways
originating from the same set of canals reach the motoneuron
populations of the antagonists of the above muscles via ipsilateral
projections. Thus each vertical oculomotor neuron receives disynaptic
excitatory and inhibitory input from the appropriate contralateral and
ipsilateral semicircular canals, respectively. This representative VOR
plan has been described in all vertebrates including flatfish
(Baker et al. 1973
; Graf et al.
1997
; Magherini et al. 1974
; Precht and
Baker 1972
).
By contrast to the above-described horizontal canal reflex circuitry,
adult flatfish develop a "novel" set of second-order vestibular
neuron connections with extraocular motoneurons hypothesized to perform
a VOR role appropriate for the 90°-lateral rotation postural
adaptation (Graf and Baker 1983, 1985b
).
These previous experiments showed that vestibular neurons activated
from the left horizontal semicircular canal (HC) nerve in the flatfish terminated bilaterally either in the oculomotor nuclei in motoneuron pools containing SR and IO motoneurons (Fig. 1B, yellow), or
in the trochlear and oculomotor nuclei with SO and IR motoneurons (Fig.
1B, blue). The first termination pattern was considered to
be excitatory and the second, inhibitory. The parent axons of both
these prospective excitatory and inhibitory vestibular neurons crossed
the midline in the hindbrain, and axon collaterals then recrossed the
midline within the midbrain to contact oculomotor motoneurons on both sides.
Experiments in the right-side winter flounder, Pseudopleuronectes
americanus, identified vestibular neurons by electrical stimulation of the downside (left) horizontal canal nerve branches (lHC) and injected horseradish peroxidase (HRP) for subsequent morphological reconstruction (Graf and Baker 1983,
1985b
). The pattern of oculomotor termination was
considered appropriate to mediate parallel compensatory eye movements
during head motion (Fig. 1B). In later experiments,
second-order vestibular neurons were labeled from the upside (right)
horizontal canal system of winter flounders (Graf and Baker
1986
; Graf et al. 1991
). Together, these
anatomical reconstructions unveiled a bilaterally symmetric innervation
scheme of flatfish horizontal canal VOR pathways that would be well
suited for the requirements of compensatory eye movements in
postmetamorphic animals within the conceptual framework of reciprocal
excitatory/inhibitory innervation of the VOR.
The present studies therefore were aimed at corroborating this
reciprocal excitatory/inhibitory projection onto oculomotor motoneurons
in adult flatfish with electrophysiology and anatomy. Evaluating the
pattern of excitatory and inhibitory postsynaptic potential (EPSP and
IPSP, respectively) allowed the operational mode of this
species-specific pathway to be tested as well as a basis for
structure/function comparison to be established with the reciprocal
excitatory/inhibitory organization of other vertebrate eye movement
circuits (Graf and Ezure 1986; Graf et al.
1997
). The physiology of the excitatory/inhibitory innervation
pattern of VOR pathways in flatfish was extended with
immunohistochemistry and electron microscopy for comparison to similar
integrated approaches undertaken in oculomotor and postural control in
goldfish (Aksay et al. 2000
; Baker et al.
1998
; Faber et al. 1989
; Korn et al. 1992
; Kumar and Faber 1999
; Straka and
Baker 1998
; Suwa et al. 1999
).
Preliminary results of this study have been previously summarized in
abstract form (Graf and Baker 1984; Graf
et al. 1985
; Spencer et al. 1986
).
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METHODS |
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All experiments were performed at the Marine Biological
Laboratory, Woods Hole, Massachusetts, in 26 adult (25-30 cm body length), right-sided winter flounders, Pseudopleuronectes
americanus. Neither left-sided examples of this species
(Policanski 1982) nor the indigenous left-sided summer
flounder, Paralichthys dentatus were examined in this study.
Experimental flounders were wild-caught by the trawler of the
institution in the Atlantic Ocean off Cape Cod and kept in large
indoor-outdoor tanks with cooled running seawater of 18°C.
Surgical procedures
Animals were mounted in a "vertical" position in a
fish-holder system (Graf and Baker 1985b), and cooled
(18°C), aerated seawater was continuously circulated over the gills
by an electric pump through a tube in the mouth. Animals were kept
immersed as deeply as possible in water to submerge the gills. All
surgery and physiological experiments were done under tricaine
methanesulfonate (1:20,000 wt/vol) anesthesia. In addition, incisions
were infiltrated with local anesthetics (2% lidocaine with
epinephrine). During recording, animals were immobilized with gallamine
triethiodide (Flaxedil). Access to the labyrinths was obtained
by removal of the skull bone overlying the fourth ventricle. Individual
labyrinthine nerve branches to the bilateral anterior, horizontal, and
posterior canal ampullae, as well as the utricular, saccular, and
lagenar endorgans were dissected and exposed. The bone overlying the
midbrain was removed, so the medial longitudinal fasciculus (MLF) could be reached through the intertectal cleft. Bipolar electrodes were positioned on the ampullary nerves for orthodromic activation of
vestibular pathways to extraocular motoneurons. The stimulation electrodes were held in place by Narishige microdrives. The horizontal canal nerves were selectively isolated and stimulated in 19 experiments, the vertical canals in 5 cases, and the entire vestibular
nerve in the 2 other experiments. In all experiments, two silver ball electrodes were placed into the orbits of both eyes to activate antidromically extraocular motoneurons.
Electrophysiology
Vestibular synaptic input to oculomotor motoneurons was recorded
with glass micropipettes containing 1 M KCl or 1 M KAcetate (approximately 20-30 M resistance, 0.5-1.0 µm tip size). The recording electrodes were positioned and advanced by a three-axis micromanipulator (Narishige, Canberra type). Motoneurons were penetrated in the oculomotor and the trochlear nucleus. These nuclei
were reached through the intertecal cleft that was widened by cutting
the commissural fibers for direct visualization of the third ventricle.
Earlier neuroanatomical studies had indicated the location of the
various motoneuron pools (Graf and Baker 1985a
), which
were identified by their antidromic potentials following electrical
activation (70 µA) via the stimulation electrodes located in the
orbits. Subsequently, synaptic potentials were elicited by electrical
stimulation from the labyrinthine electrodes (20 µA). Each penetrated
motoneuron could be identified by its location within the oculomotor
nuclei with respect to the eye from which it was antidromically
activated. The electrophysiological data were recorded either on
magnetic tape (Neurocorder: Data 6000), or on magnetic disks (Nicolet
4096), and later printed out on a laser printer for analysis and quantification.
Neurotransmitters
Protocols for GABA immunohistochemistry were described in detail
in Graf et al. (1997) and are briefly summarized here.
Animals were perfused with fixative solution containing 4.0%
paraformaldehyde in 0.1 M phosphate buffer with 0.002% calcium
chloride (pH 7.2). Vibratome sections were obtained through the
abducens, trochlear, and oculomotor nuclei (25-50 µm) and
subsequently processed for immunohistochemical localization of GABA
using an antibody generated in rabbit against GABA conjugated to bovine
serum albumin (Immunonuclear). Following primary and secondary antibody
incubation, the tissue was incubated in an avidin/biotin-HRP complex
(Vector). Addition of the chromogen 3,3'-diaminobenzidine and hydrogen
peroxide produced a brown diffuse reaction product. The material was
mounted for light microscopic examination using brightfield or
Normarski differential interference contrast optics.
Electron microscopy
Protocols were identical to those described in Graf et
al. (1997) and are summarized here briefly. Animals were
anesthetized and perfused with 1% paraformaldehyde and 1.25%
glutaraldehyde in 0.1 M phosphate buffer with 0.002 calcium chloride
(pH 7.4). Serial coronal sections (50-75 µm) through the oculomotor
nuclei were cut on a vibratome. Sections containing the oculomotor
nuclei were trimmed, postfixed in 1% osmium tetroxide, stained en bloc with 0.5% uranyl acetate, dehydrated, and embedded in resin between vinyl plastic microscope slides and cover slips.
Selected loci containing oculomotor neurons were analyzed by serial
ultrathin sectioning and electron microscopy. Serial sections (5 µm)
through the plastic-embedded vibratome sections were examined by light
microscopy and remounted on blank BEEM capsules from which ultrathin
sections were cut and collected on single-slot Formvar-coated
grids. Sections were then stained with uranyl acetate and lead citrate,
examined, and photographed with a Zeiss EM-10CA electron microscope.
The same procedure was followed in three animals who had received, 1 day earlier, an intracellular injections of HRP into second-order
vestibular neurons of the horizontal canal system. The brains were also
treated for HRP histochemistry before selected sections were prepared
for electron microscopy (Graf and Baker 1985a,b
).
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RESULTS |
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A total of 102 extraocular motoneurons was recorded in the trochlear, oculomotor, and abducens nuclei. The latency for antidromic invasion averaged 0.7 ms. Disynaptic depolarizing synaptic potentials were observed in all motoneurons following whole vestibular nerve branch stimulation with latencies between 1.2 and 1.8 ms. As expected, selective activation of individual canal nerves also evoked disynaptic depolarizations that were shown to be EPSPs in the appropriate populations of motoneurons (Figs. 2-4). However, stimulation of the presumed antagonistic canal nerve supposed to provide inhibitory input, typically produced membrane depolarizations as well (Figs. 2, C and D, and 4C). Hyperpolarizing IPSPs were recorded in only nine motoneurons (i.e., 9% of all cases; Fig. 3, B and D), and these IPSPs were about equally distributed between the vertical oculomotor subgroups (2 each in IR, IO, and SR motoneurons, and 3 in SO motoneurons). The existence of inhibition was supported by finding GABAergic contacts on extraocular motoneurons (Fig. 5) and by ultrastructural anatomical features associated with inhibitory synapses (Figs. 7 and 8).
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Neurophysiology
Synaptic potentials recorded in extraocular motoneurons could be
compared with those recorded in goldfish (Graf et al.
1997) as well as interpreted with respect to the
vestibulooculomotor pathways shown earlier in flatfish (Graf and
Baker 1985b
) (see also Figs. 1B and 9). To do so, it
was necessary to recognize the recorded motoneuron pools (Graf
and Baker 1985a
). Hence, in each experiment the trochlear
nuclei (SO motoneurons) were located first by the antidromic field
potential profile initiated from the contralateral orbits (Fig.
4A)
(Baker et al. 1973
; Graf et al. 1997
). SR
motoneurons were identified in the caudal oculomotor complex following
stimulation of the contralateral orbits. IR and IO motoneurons were
recognized by antidromic activation from the ipsilateral orbits, while
abducens (LR) motoneurons were recorded in the posterior brain stem.
Motoneuron somata were located adjacent to the midline (Fig.
5A), and antidromic
intracellular records were characterized by initial segment-soma
dendritic (IS-SD) invasion followed by a large afterdepolarization
typical of an intrasomatic penetration (Figs. 2A and 3,
A and C).
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Since synaptic potentials recorded in all vertical oculomotor
motoneurons exhibited similar profiles, the electrophysiology is thus
illustrated for four key somatic recording sites (2 left-side trochlear
motoneurons, Fig. 2, and 1 left-side and 1 right-side SR motoneuron,
Fig. 3) and one intradendritic recording from a right-side trochlear
motoneuron (Fig. 4). The cases selected were stable penetrations with
resting potentials between 50 and
60 mV. Membrane depolarization
was elicited from stimulation of the right horizontal canal
labyrinthine nerve branch (rHCe) predicted to provide excitatory input
to trochlear motoneurons (Figs. 1B and 9). Stimulation of
the prospective left horizontal canal inhibitory input (lHCi; Fig.
1B, blue pathway) did not produce significant membrane
hyperpolarization at this level of resting potential. In fact, in
93/102 cases, the stimulus that should have produced a hyperpolarizing
IPSP induced membrane depolarization (Fig. 2, C and
D). In only nine cells could hyperpolarizing IPSPs be
demonstrated without the application of extrinsic current following
stimulation of the appropriate canal nerves (Fig. 3, B and
D). Depolarizing current injection between +5 and +15 nA, in
the absence of action potentials, showed that stimulation of the
vestibular nerve branch expected to provide inhibitory input, produced
IPSPs associated with a membrane conductance. This observation
suggested that the IPSP equilibrium potential might be close to this
motoneuron's resting potential. In these cases, the synaptic
potentials could also be re-reversed to depolarization by injection of
hyperpolarizing current ranging from
4 to
11 nA. Injection of
chloride ions also significantly displaced the IPSP equilibrium
potential in some motoneurons to a level equal to that of the
contralateral EPSP (not illustrated). However, in contrast to goldfish
motoneurons, use of depolarizing current and chloride injection to
change the membrane resting potential and/or IPSP equilibrium potential
did not reveal either robust hyperpolarizing IPSPs or modified EPSP amplitudes (Graf et al. 1997
).
Chemically elicited EPSPs were always preceded by a short-latency electrotonic potential at a latency of 0.8-0.9 ms (Figs. 2, B-D, 3B, and 4C). The ubiquitous presence of electrotonic coupling indicates distributed gap junction connections throughout the extraocular motoneuron pools that was corroborated by electron microscopy (Figs. 6-8). Electrotonic coupling could be demonstrated by collision block of the action potential elicited from direct activation with that from antidromic invasion (Fig. 3C). Following electrical activation of the horizontal canal nerve, small-amplitude chemical depolarizations were recorded with latencies of 1.2-1.6 ms (Figs. 2, B-D, and 3B). Larger excitatory depolarizations, presumed to be EPSPs, occurred from 2.2 to 2.6 ms (Fig. 2, B-D). Stimulation of a vestibular nerve branch expected to produce IPSPs, in general, also produced small membrane depolarizations beginning at a latency of 1.6-1.8 ms that gradually increased in amplitude resulting in larger depolarizations at 2.6 ms (Figs. 2, C and D, and 3B).
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Intra-axonally recorded action potentials in the MLF (Fig. 2B) from horizontal canal vestibular neurons were recorded at a latency of 0.6-0.8 ms after stimulation (Fig. 2B). The first vestibular action potential was coincident with the electrotonic synaptic potential at 0.8 ms (Fig. 2, B and E), whereas the second vestibular neuron action potential overlapped with both an electrotonic and chemical depolarization at 1.6 ms (Fig. 2, B and C). In these experiments, multiple HC stimuli were required to produce a large enough EPSP to initiate oculomotor motoneuronal action potentials (Fig. 2B, lTro Mn).
In all 102 motoneurons, the presence of excitatory and putative
inhibitory synaptic potentials correlated well with the proposed innervation scheme of horizontal canal second-order input to oculomotor neurons in flatfish. Activation of the left horizontal canal nerve produced EPSPs in superior rectus and inferior oblique motoneurons on
both sides, whereas IPSPs were elicited from the right horizontal canal
(cf. Figs. 1B; 3, B and D; and 9).
Similarly, the bilateral trochlear (superior oblique) and inferior
rectus motoneurons received excitatory input from the right horizontal
canal nerve and inhibitory input from the left horizontal canal nerve
(Figs. 1B; 2, B-D; 4, B and
C; and 9). Synaptic responses from the anterior and
posterior canals (not illustrated) were qualitatively similar to those
observed in mammals and goldfish (Graf et al. 1997).
In all animals, averaging of synaptic potentials following single
shock stimuli was sufficient to determine latency and polarity (Figs.
2, B-D; 3B; and 4C); however,
multiple shock stimulation was required to elicit synaptic potentials
large enough to activate spikes in the penetrated motoneurons (Figs.
2B and 4B). This phenomenon, in part, may have
been due to either anesthetic (MS222 and Flaxedil) and/or surgical
effects on central vestibular excitability. Alternatively, the small
synaptic potentials, especially IPSPs, after single electrical stimuli
could be due to a larger than normal electrotonic load provided by the
dense, bilateral terminal arborization (Figs. 1B and 9; see
also DISCUSSION) (Graf and Baker 1985b).
GABA immunohistochemistry
Corroborative evidence for vestibular inhibition of oculomotor
neurons was provided by the immunohistochemical localization of GABA,
which is the inhibitory transmitter in the vertical VOR of higher
vertebrate species (Precht et al. 1973; Spencer
et al. 1989
, 1992
). GABA immunohistochemistry
was used in goldfish to demonstrate the axonal trajectories of
inhibitory vestibular neurons in the MLF, as well as terminations on
extraocular motoneurons in the trochlear and oculomotor nucleus
(Graf et al. 1997
). In flatfish, axons of HC inhibitory
neurons were physiologically identified to course in the MLF
(Graf and Baker 1985b
), and the GABA reaction product
was also found in the transversely cut axonal profiles in the MLF (Fig.
5, C and D). Unlike in goldfish, these HC
inhibitory axons did not travel together in bundles, but were dispersed
throughout the coronal sections of this pathway (compare Fig.
5D with Fig. 5C of Graf et al.
1997
). In the flatfish oculomotor nucleus, GABA-positive
boutons were observed on the motoneurons in all subdivisions (Fig. 5,
A and B). Terminal arborizations were distributed
largely on the somata and proximal dendritic trees (Fig. 6,
B and E).
Electron microscopy
To provide evidence for the presence of electrotonic and
chemical excitation and chemical inhibition, the pattern and mode of
terminal arborizations were studied in the oculomotor nuclei of four
flatfish after intracellular HRP labeling of physiologically identified
HC activated neurons (Graf and Baker 1985b) (Figs. 6 and
7). Axons of horizontal canal VOR neurons
were injected in the MLF for 3-5 min to minimize masking the
cytoplasmic details of the contacts onto oculomotor neurons. The somata
of neurons in individual subnuclei were identified first in thick
plastic sections (e.g., Fig. 7A) that subsequently were thin
sectioned for electron microscopy (Spencer and Baker
1983
). Cytoplasmic organelles typical of other vertebrates,
e.g., Golgi apparatus and mitochondria, were observed in the motoneuron
somata (Figs. 6A and 7B). In addition, cisternal
arrays of granular endoplasmic reticulum typical of mammalian
oculomotor motoneurons were well-developed (Spencer and Baker
1983
).
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A moderate to high density of axosomatic synaptic endings was found in all oculomotor subdivisions and on trochlear motoneurons (e.g., arrow in Fig. 6B). Electron microscopic reconstruction of synaptic contacts on oculomotor neurons showed axosomatic and axodendritic synaptic endings that contained spheroidal synaptic vesicles and established chemical (presumed excitatory) synaptic contacts characterized by asymmetrical pre/postsynaptic membrane specializations (Fig. 8, B and D). Many HRP-labeled HC axosomatic synaptic endings also exhibited gap junction contacts consistent with electrotonic coupling (Figs. 7F and 8, C and D). Chemical synaptic contacts exhibited an intersynaptic space of approximately 20 nm, while gap junction contacts displayed a separation of approximately 2 nm between the pre- and postsynaptic membranes (Fig. 8D). Mixed chemical and electrotonic axosomatic synaptic endings contained fewer spheroidal vesicles than axosomatic endings establishing only chemical synaptic contacts (Fig. 8B). By contrast, axodendritic endings, including those on spines, established only chemical contact zones (Fig. 7E). When individual synaptic contacts were serially reconstructed, gap junctions were associated with other unlabeled axodendritic synaptic endings (Fig. 7F). Both types of contacts could be observed in relation to different (Fig. 7E) or the same (Fig. 8, C and D) postsynaptic profiles.
|
Other axodendritic and axosomatic synaptic endings contained pleiomorphic vesicles and established single chemical synaptic contacts (Figs. 7, C and D, and 8A). Synaptic contact zones exhibited symmetrical pre-/postsynaptic membrane specializations (Fig. 8A). These presumed inhibitory synaptic endings were never observed to establish gap junctions and were located on either somata or proximal dendrites (Fig. 7, B and D).
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DISCUSSION |
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Summary
In our previous morphological study, second-order VOR neurons were
identified by electrical stimulation of the left-side (down-side) HC
nerve, injected with HRP and their axonal trajectories and termination
patterns reconstructed in the oculomotor complex (Graf and Baker
1983, 1985b
). These observations suggested the
presence of reciprocal HC species-specific excitatory and inhibitory
vestibular projections to vertical extraocular motoneurons as
illustrated in Fig. 9 (red and blue
pathways, respectively). The electrophysiology (Figs. 2-4) and anatomy
(Figs. 5-8) presented here strongly support this interpretation and,
in particular, also confirm the presence of a second-order excitatory
and inhibitory pathways originating from the right-side (up-side) HC
nerve (Fig. 9, green and orange pathways, respectively). As a result,
either right-side or left-side horizontal semicircular canal
stimulation initiates a pattern of electrotonic/chemical synaptic
depolarization and inhibition. This is consistent with the idea of
equally weighted, bilaterally symmetric, reciprocal
excitatory/inhibitory pathways contacting the four vertical oculomotor
motoneuron pools. Since the ultrastructural and immunochemical synaptic
profiles in flatfish were similar to some aspects of those described in
the vertical VOR pathways of goldfish (Graf et al.
1997
), we suggest that the development, hindbrain location and
function of VOR neurons may be an unchanging trait in not only teleost
fish, but also possibly throughout vertebrate phylogeny.
|
Electrophysiology
In antidromically identified oculomotor motoneurons innervating
either the bilateral SO/IR or IO/SR muscle pairs, electrical stimulation of either the rHC or lHC vestibular nerve produced short-latency disynaptic electrotonic EPSPs (0.8-0.9 ms) followed by
disynaptic chemical depolarizations with latencies of 1.2-1.8 ms,
respectively. In each motoneuronal population, and for all experiments,
the amplitude of the single shock EPSPs were small (<1 mV), whereas
the depolarization was significantly enhanced in response to multiple
stimuli. While the surgical and anesthetic conditions likely
contributed to this reduced synaptic efficacy, an alternative, more
physiological explanation might be related to the observation that
unitary action potentials do not completely invade large terminal
arborizations as seen, for example, in tectobulbospinal neurons
(Grantyn and Grantyn 1982) and other neuronal models
(e.g., Mackenzie and Murphy 1998
; Toth and
Crunelli 1998
). Intra-axonal records from HC excitatory and
inhibitory vestibular neurons ascending in the MLF always responded
with a single or double action potential of 0.6-0.8 ms latency after
single shock stimulation of the appropriate HC nerve (Fig.
2B) (Graf and Baker 1985b
). Multiple HC
stimuli were required, however, to produce additional action potentials that clearly were more effective in producing transmitter release as
evidenced from the intracellular records in oculomotor neurons. The
latter interpretation suggests that orthodromic invasion of the
multiple branch points requires a short presynaptic interspike interval
and/or a background of spontaneous activity likely present under
natural conditions (Graf and Baker 1990
).
In the appropriate motoneurons, stimulation of the vestibular nerve
branch expected to be inhibitory produced small-amplitude depolarizations beginning at a latency of 1.6-1.8 ms that gradually increased in amplitude to peak at 3.0 ms. Direct evidence for disynaptic inhibitory input to extraocular motoneurons in the form of
hyperpolarizing IPSPs was only seen in 9/102 motoneurons (i.e., 9%).
Postsynaptic inhibition in teleost fish largely appears as a shunting
membrane conductance at the soma and proximal dendrites to reduce the
input resistance of the cell and thereby short-circuiting excitation
arriving at, and from, the more distal dendrites (Faber and Korn
1987; Graf et al. 1997
). The location of
inhibitory (GABAergic) terminals overlying the somata and proximal
dendrites supports a similar interpretation in flatfish (Figs. 5, 7,
and 8A). Manipulation of membrane potential by current
injection through the microelectrode demonstrated the short-latency
polarization to be associated with a membrane conductance exhibiting an
equilibrium potential slightly more negative than resting potential
(Fig. 3, C and F). However, IPSP reversal by
injected current appeared to be independent of either intrasomatic or
intradendritic recording sites and, surprisingly, in neither case did
the injection of chloride ions significantly displace the equilibrium potential.
Efficacy of the inhibitory synaptic conductance could not be assessed
in flatfish as easily as in either goldfish extraocular motoneurons
(Graf et al. 1997) or Mauthner cells (Faber and
Korn 1975
, 1987
). The steep voltage dependence
of chloride channels envisioned to increase the magnitude and duration
of the inhibitory response in goldfish motoneurons (Graf et al.
1997
) was not seen in flatfish motoneurons. Hyperpolarizing
IPSPs also were not found in the VOR pathways of the puffer fish, a
marine teleost (Korn and Bennett 1972
,
1975
). Perhaps sharp electrode intracellular recordings
from hindbrain neurons in teleosts may not reflect sufficiently well
the extent of inhibitory conductance because reconstructed cells
exhibit an extensive cable-like appearance arguing against a uniform
change in potential throughout all compartments (Graf and Baker
1985a
; Highstein et al. 1992
; Pastor et
al. 1991
; Straka and Baker 1998
; Zottoli
and Faber 1980
). Nonetheless, in view of the voltage dependence
of glycine-activated Cl
channels in which the
kinetics of the synaptic response are enhanced in the face of
excitation (Faber and Korn 1988
), activation of the
inhibitory HC canal nerve would be expected to reduce the firing
frequency of oculomotor neurons. Indeed, in some isolated cases, when
spontaneous activity was present in the recorded motoneuron, stimulation of the putative inhibitory vestibular nerve markedly reduced the firing rate.
The reciprocal semicircular canal inhibitory and excitatory pathways
must play an important role in coordinated eye movements. If such
reciprocity were not present, vestibular input would result in a
counterproductive functional input to the oculomotor system (Graf and Baker 1990). Our current interpretation
therefore is that the special nature of excitable membrane properties
in fish permits an IPSP equilibrium potential close to membrane resting potential to be as effective as the much more negative equilibrium potential in mammalian oculomotor neurons (Llinás and
Baker 1972
).
GABA immunohistochemistry and electron microscopy
GABAergic terminals were found clustered around cell somata and
proximal dendrites in a similar pattern as described for goldfish oculomotor and abducens nuclei (Graf et al. 1997). GABA
antibodies localized inhibitory neurons in the vestibular nuclei (not
illustrated) and, as in goldfish, axons headed toward and into the
contralateral MLF. Terminal arborizations were observed to distribute
primarily over the somata and proximal dendritic trees of trochlear
(SO) and oculomotor motoneurons. Thus the presence, spatial
distribution, and magnitude of GABAergic inhibition from presumed
GABAergic neurons of the HC vestibular system in flatfish appears to be similar to the vertical VOR system in goldfish and mammals (Graf et al. 1997
; Spencer and Baker 1992
).
In the vestibulooculomotor system of mammals, the inhibitory
transmitter related to vertical canal reflexes is GABA; that related to
the horizontal canal reflex is glycine (Spencer et al.
1989). Thus an apparent paradox exists in the adapted
vestibuloocular reflex circuitry of adult flatfish: our
immunohistochemical data indicate the existence of a robust GABA
termination in the oculomotor and trochlear nucleus, while the
inhibitory input to these vertical eye muscle motoneurons is largely
derived from HC second-order vestibular neurons. The solution to this
apparent paradox will bear directly on the embryonic origin of the
adapted VOR circuits, and several scenarios may be envisioned. If the
novel VOR circuitry originates from extant classical and functional
horizontal canal VOR neurons with terminals originally in the abducens
nucleus where glycine exists as an inhibitory transmitter
(Spencer et al. 1989
), the neurotransmitter content must
switch from glycine to GABA during metamorphosis when new contacts
would be made with vertical extraocular motoneurons. Alternatively,
these inhibitory neurons might arise from neurons formerly related to
the vertical VOR, and secondarily became recruited by the horizontal
canals. A more parsimonious hypothesis, however, would be that these
second-order inhibitory neurons and their excitatory counterparts
originate from entirely different embryonic progenitors. Preliminary
results support the latter conjecture because newly born vestibular
neurons appear at a time when the postmetamorphic connectivity formed (Graf et al. 1991
). Establishing the embryonic origin of
these novel VOR pathways will reveal the relevant hindbrain rhombomeres and genes responsible for flatfish-specific neuronal adaptation (Baker 1998
).
Ultrastructure
The electron microscopic visualization of flatfish oculomotor and
trochlear nuclei revealed axosomatic and axodendritic synaptic endings
containing spheroidal synaptic vesicles that established chemical,
presumed excitatory, synaptic contacts characterized by asymmetrical
pre/postsynaptic membrane specializations. In addition, many axosomatic
synapses also showed gap junction contacts consistent with electrotonic
coupling. The latter were similar in morphological profile to the
"mixed" synapses described in the goldfish abducens nucleus
(Graf et al. 1997; Sterling 1977
). Other
axosomatic synaptic endings contained pleiomorphic synaptic vesicles
establishing chemical synaptic contacts on motoneurons. These presumed
inhibitory synapses were never observed to establish gap junctions.
Comparison of flatfish oculomotor and trochlear nuclear complex to that
in goldfish showed that the relative placement of chemical versus
electrotonic contacts of excitatory second-order vestibular neurons was
different (Spencer et al. 1986
). In goldfish, chemical
contacts were located at some distance from gap junctions (Graf
et al. 1997
), whereas in the flatfish, gap and chemical junctions were adjacent to each other (Fig. 8, C and
D). In flatfish, the spatial relationship of chemical and
electrotonic contacts was closer, and thus more like the arrangement in
the goldfish abducens nucleus (Graf et al. 1997
). The
functional significance of this difference in chemical versus
electrical junction placement is undetermined in both species as is the
significance of electrotonic coupling per se in VOR behavior. A prior
interpretation argued that electrotonic transmission played a more
important role in goldfish VOR (Graf et al. 1997
) than
in synchronizing motoneuronal discharge during either fast phases or
saccades (Korn and Bennett 1971
). Nonetheless,
electronic coupling apparently is not essential for normal VOR behavior
in all vertebrates, because gap junctions are not found in mammals.
Reciprocal excitatory/inhibitory innervation
The pattern of excitation and inhibition in the
vestibulooculomotor system of flatfish is in agreement with previously
obtained morphological results and also supports the structural
requirements to produce compensatory eye movements during swimming in
the adult animals (Graf and Baker 1985b,
1986
, 1990
). In particular, HC innervation of the SR, SO, IR, and IO extraocular motoneurons is quite
unique, but its blueprint fits well into the classical reciprocal
excitatory/inhibitory innervation scheme of semicircular canal-related
VOR function. In eye movement plants of upright symmetrical
vertebrates, e.g., mammals and goldfish, excitation arrives from the
contralateral, and inhibition from the ipsilateral labyrinth. The
flatfish innervation pattern of the right-sided winter flounder can
only be interpreted in a context-specific manner. In such case, the
bilateral SR and IO motoneurons receive excitatory input from the
downside (left) horizontal canal and reciprocal inhibitory input from
the upside (right) horizontal canal. The motoneurons innervating the
antagonists of these muscles, the bilateral SOs and IRs, would be
innervated with opposite-directed signals, i.e., excitation from the
(upside) rHC, and inhibition from the (downside) lHC. Unlike VOR
organization in mammals and goldfish, wherein excitatory and inhibitory
VOR neurons ascend on the contralateral and ipsilateral side,
respectively, all ascending excitatory and inhibitory
horizontal canal VOR neurons in flatfish ascend on the contralateral
side (Figs. 1 and 9).
Spatial coordination of eye movements
VOR connections are quite remarkable, if not coincident, when
classical co-contraction patterns of yoke eye muscles are compared between flatfish and other vertebrates (Szentágothai
1943, 1950
). The SR muscle of one eye and the IO
muscle of the other eye are defined as yoke muscles as in similar
fashion are the SO and contralateral IR counterpart. Flatfish
co-contract either the bilateral SR and IO for backward rotation (i.e.,
extorsion) or the bilateral SO and IR for forward rotation (i.e.,
intorsion) to produce compensatory eye movements during downward or
upward head movements, respectively (Graf and Baker
1983
, 1985b
). In light of extraocular muscle
kinematics (Graf and Baker 1985a
), this arrangement is
biologically meaningful and not different from either other upright
fish or lateral-eyed vertebrates (Graf and Brunken 1984
;
Graf and McGurk 1985
; Simpson and Graf
1985
). However, the similar VOR behavior in flatfish is brought
about by a VOR innervation scheme that is different from all other
vertebrates. In the latter case both anterior (vertical) canals provide
excitatory signals to the bilateral SR and IO motoneurons during
downward head movements. In flatfish, comparable excitation is
transmitted from the downside horizontal canal to the bilateral SR and
IO motoneurons. A similar scenario holds for posterior (vertical) canal
reflexes in upright vertebrates as well as that elicited by the upside
horizontal canal in flatfish. Inhibitory input from the same canals in
the above-mentioned cases reaches the antagonists of the respective
muscles. Thus to implement the evolutionarily adapted state, a
previously established innervation scheme based on retained spatial
geometry and orientation of the eye muscles is employed in the
postmetamorphic flatfish VOR circuits. In upright vertebrates, the
sensory information signaling upward and downward head movement arises
from the four vertically oriented canals, whereas in flatfish, the
two, now vertically oriented, horizontal canals fulfill this role (Fig.
1B).
Conclusion
The conceptual view of reciprocal excitatory-inhibitory organization of VOR circuits as necessary and essential for compensatory eye movement production has been upheld by electrophysiological, immunohistochemical, and ultrastructural evidence obtained from the species-specific expression found in flatfish. These observations also suggest that second-order vestibular neurons can use a novel developmental plasticity to achieve new structural and physiological requirements. Thus the unique VOR organization in flatfish can be used as a model to study the embryonic, endocrine, environmental, and genetic mechanisms underlying this pre- to postmetamorphic transformation.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health Grants DC-00239, EY-04613, EY-02007, and NS-13742.
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FOOTNOTES |
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Address for reprint requests: R. Baker, Dept. of Physiology and Neuroscience, New York University School of Medicine, 550 First Ave., New York, NY 10016 (E-mail: bakerr01{at}popmail.med.nyu.edu).
Received 10 December 1999; accepted in final form 13 April 2001.
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REFERENCES |
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