NA SA-CR-202 154

FINAL TECHNICAL REPORT

BASIC GRAVITATIONAL REFLEXES IN THE LARVAL FROG

NASA SPACE BIOLOGY PROGRAM--GRANT NUMBER

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NAG 2-780

DURATION OF GRANT: 1/1/92-8/31/96

PRINCIPAL INVESTIGATOR: Stephen L. Cochran, Ph.D.

Assistant Professor

INSTITUTION: University of Texas Medical Branch

Department of Otolaryngology, Rt. J63

Galveston, TX 77555-1063

Description of Research

Little is known about how vertebrates are able to sense gravity and how they process

this information to generate appropriate motor responses. This investigation was designed to

determine how a primitive vertebrate, the bullfrog tadpole, is able to sense and process

gravitational stimuli. Because of the phylogenetic similarities of the vestibular systems in all

vertebrates, the understanding of the gravitational reflexes in this relatively simple vertebrate

should elucidate a skeletal framework on a elementary level, upon which the more elaborate

reflexes of higher vertebrates may be constructed.

The tadpole manifests a powerful counter-rolling response of the eyes to static tilt of

the head. In addition, the fact that amphibians are cold blooded means that their cellular

metabolism is much lower than that of mammals. Consequently, the entire head can be

maintained in vitro with the brain, sensory structures, and eye muscles exposed. The strong

gravitational reflexes are still evident under these conditions and persist for several days.

Such conditions permit detailed and reproducible electrophysiological and anatomical

investigations of this reflexive behavior.

The purpose of this study was to understand how the nervous system of the larval

amphibian processes gravitational information. This study involved predominantly

electrophysiological investigations of the isolated, alert (forebrain removed) bullfrog tadpole

head. The focus of these experiments is threefold: 1) to understand from whole extraocular

nerve recordings the signals sent to the eye following static gravitational tilt of the head; 2) to

localize neuronal centers responsible for generating these signals through reversible

pharmacological ablation of these centers; 3) to record intracellularly from neurons within

these centers in order to determine the single neuron's role in the overall processing of the

center. This study has provided information on the mechanisms by which a primitive

vertebrate processes gravitational reflexes.

Accomplishments

Quantification of the behavior. A software event detection technique, which has been

developed over the last fifteen years, has been finalized and published. This technique
allows for analysis of spontaneously-occurring voltage deflections over time.

Electrophysiological recordings digitized (at 50 KHz) over many minutes can be rapidly and

automatically analyzed. The publication illustrates this technique for spontaneously occurring

synaptic potentials recorded intracellularly from VIIIth nerve afferents that are consequent

from convergent hair cell innervation of the afferents. This technique allows for tens of

thousands of EPSPs to be analyzed during this period. With respect to the experiments in

this study that involve whole nerve recordings from the trochlear and medial rectus nerves,

this same technique can be applied to detect and quantify the extracellularly recorded nerve

action potentials. Since these whole nerve recordings are more stable than the intracellular

recordings of EPSPs, digitized periods are often over an hour in duration, allowing for

continuous quantitation of hundreds of thousands of spike potentials that occur during the

recording period. Since the amplitude (as well as other parameters) of each spike potential is

quantified, whole nerve recorded spike potentials can be parceled according to size, and their

size can then be related to the time of occurrence during a tilt stimulus.

Responses to static tilt of the head. Trochlear motoneurons increase their frequency of firing

with nose up tilts and decrease it with nose down tilts. Motoneurons innervating the medial

rectus muscle respond reciprocally--i.e, they increase their activity with nose down tilts and

decrease their activity with nose up tilts. For both the trochlear and medial rectus

motoneurons, the larger sized units are phasic and adapt more rapidly than the smaller sized

units, which appear to have a larger tonic component and which adapt less rapidly. Typically

larger units fire only during a movement (relating them more to velocity), while smaller units

appear to be modulated both by the table movement as well as by the absolute position of the

table (relating them both to table velocity and position). During the period of this grant, the

principal investigator constructed a servo-controlled tilting system for his electrophysiological

setup. This system allowed for precisely controlled movements of the table of a range of + 10

degrees and at velocities between 0.023 and 2 degrees per second. Clearly compensatory

trochlear responses were seen even at the lowest velocities and involved principally the

smallest amplitude units, suggested a tilt position signal is communicated from the inner ear

(probably the utricle) through the central nervous system to the trochlear motoneurons.

Pharmacological studies. Iontophoresis of the kynurenic acid, an antagonist of the excitatory

transmitter glutamate, into the extraocular motor nuclei reversibly abolishes spike activity in

the extraocular motor nerves. Intracellular recordings from oculomotor neurons show complex

mixtures of EPSPs and IPSPs following electrical stimulation of the vestibular nerves and

afferent brainstem pathways to this nucleus. Bath application of kynurenic acid reversibly

blocks over 95 % of the EPSPs. Both these findings are consistent with the hypothesis that

the principal excitatory transmitter to the oculomotor nucleus is glutamate or a related

compound.

Preliminary investigations in the turtle. The advantage of investigating static gravitation

responses in the tadpole is that the head can be maintained viably in vitro for a number of

hours after decapitation. The disadvantage is that tadpoles and frogs do not possess both

types of hair cells found in mammals, but only have type II hair cells. Consequently,

interpretations of results from tadpoles cannot easily be extended to mammals and birds,

because of this increased complexity. Homeothermic organisms such as birds and mammals

have higher metabolic demands than poikilothermic vertebrates such as frogs, and

consequently isolation of relatively intact portions of the nervous system or inner ear do not
survive in vitro. Turtles, however, have both types of hair cells as is found in mammals and

birds and also are poikilothermic, so portions of the inner ear and nervous system (and

possibly the whole head) can be maintained viably in vitro. It is thus of interest to compare

the tilt sensitivity of vestibular and extraocular motoneurons in the turtle to that in the tadpole

to attempt to discover the additional role(s) type I hair cells have in producing compensatory

behavior to static tilt. Preliminary investigations at the end of this grant period have focused

upon investigations of the inner ear of the turtle and have attempted to compare the basic

aspects of inner ear organization in the turtle with that known in the frog and tadpole. These

investigations indicate that the turtle inner ear can be maintained in vitro with a similar viability

as is found in the frog, and that the hair cell transmitter is likely to be the same in both these

animals. In addition, ultrastructural investigation of the types of synaptic contact found

between hair cells, afferents, and efferents is quite similar to that found in the other amniotes

(i.e. birds and mammals). This research has promising directions with respect to

understanding the comparative aspects of the static vestibular reflexes.

Significance of the Accomplishments

In general, the above accomplishments provide a step toward the understanding of how these

primitive vertebrates sense gravitational stimuli and how they process these stimuli and

integrate them into reflex control.

The development of the quantification technique is an important step in the ability to

investigate how gravity can influence eye movements. Moreover, this technique is

generalizable to any spontaneously occurring events that can be represented as voltage

deflections over time, indicating that it has a potential use other than its strict application in

this study.

The similarity of the responses from the trochlear versus medial rectus motoneurons (although

their receptive field properties are different) suggests that different inner ear endorgan signals

converge upon these motoneurons, but that the quality of the processing of these signals is

similar. The contribution of individual endorgans and specific vestibular nuclear complex

neurons in channeling the static and dynamic signals remains to be determined.

The finding that glutamate is the main excitatory transmitter to the oculomotor nucleus

indicates that glutamate is the principal excitatory transmitter of the entire vestibulo-ocular

reflex. Glutamate (or a related compound) is the hair cell transmitter, it is the transmitter of

VIIIth nerve afferents, it is the transmitter of excitatory commissural vestibular neurons, and it

is the transmitter of vestibulo-oculomotor neurons.

These findings provide basic information as to the organization of gravitationo-ocular reflexes

in a relatively simple lower vertebrate. These studies can provide a basic framework for

understanding how these reflexes are organized and elaborated upon in the more complex

vestibulo-ocular behaviors exhibited by higher vertebrates.

The preliminary investigations in the turtle are a new promising route towards understanding

the different contributions of type I versus type II hair cells in generating compensatory reflex

behavior, and should they continue, would suggest how the neuronal organization responsible
for mediating this behavior is tailored to suit the individual animals and their repertoire of

behaviors.

Publications

MANUSCRIPTS

Cochran, S.L. (1992): Peripheral Vestibular Transmission. In Sensing and Controlling

Motion (B. Cohen, D.L. Tomko, and F. Guedry, eds.), Ann. N.Y. Acad. Sci. 656:

580-593.

Cochran, S.L. (1993): Algorithms for detection and measurement of spontaneous

events. J. Neurosci. Meth. 50: 105-121.

Cochran, S.L (1994): Evidence against a hypothesis of vestibular efferent function.

Brain Res. 642: 344-347.

Cochran, S.L. and M.J. Correia (1995): Functional support of glutamate as a vestibular

hair cell transmitter in an amniote. Brain Res. 670: 321-325.

Cochran, S.L. (1995) Cationic influences upon synaptic transmission at the hair cellafferent fiber synapse of the frog. Neurosci. 68:1147-1165.

Cochran, S.L. (in press) Vestibular hair cell-afferent transmission. In Neurochemistry of

the vestibular system (A. Beitz and J. Anderson, eds.). CRC Press.

ABSTRACTS

Cochran, S.L. (1992): Responses of medial rectus motoneurons to static tilt of the isolated

tadpole head. Amer. Soc. Gravitat. Space Biol., 8th Ann. Meeting; ASGSB Bull. 6: 93.

Cochran, S.L. (1992): Synaptic input to oculomotor neurons of the frog. Soc. Neurosci. Abstr.

18: 28.

Cochran, S.L. and M.J. Correia (1994): Electrophysiological evidence that 'glutamate' is a

hair cell transmitter in the turtle. Soc. Neurosci. Abstr., 969.

Cochran, S.L. (1994): Responses of trochlear motoneurons to constant velocity head tilt.

ASGSB Bull., 7.

Cochran, S.L. (1995): Synaptic ultrastructure in the semicircular canals and utricle of the

turtle. ASGSB Bull. 9: 34.

Cochran, S.L. (1995): U ltrastructural and electrophysiological correlates of synaptic

transmission in the turtle inner ear. Soc. Neurosci. Abstr. 21: 399.