ABSTRACT:
Extracellular recordings of Mauthner (M-) cell activity was carried out in the
Senorita wrasse, Ovyjulis californica Criteria for identifying the Mauthner
cell have been satisfied in our experimentation. We observed a C-start tail
flip behavior caused by both antidromic and orthodromic stimulation.
Electrophysiological recordings from the medullary area thought to contain
the M-cells reveal a short-latency, all-or-nothing spike of large amplitude.
Preliminary experiments on the effects of an as yet uncharacterized cone
snail toxin on M-cell activity were performed.
INTRODUCTION:
A dramatic and universal behavior pattern seen in teleost fish is the
characteristic "tail flip" escape response. This two phase startle response
consists of a fast body bend, where the fish assumes a"C"-like shape,
followed by a straightening of the tail which can propel the fish a full body
length (Eaton, 1976.) A single pair of cells in the medulla is responsible for
the coordination of spinal motor neurons to produce this behavior. These
cells, called the Mauthner (M-) cells, are the most conspicuous neurons in the
brain. In goldfish, the two main dendrites can measure up to half a
millimeter in length and the large myelinated axon extends down the length of
the spinal cord (Furshpan and Furukawa, 1962.) The M-soma receives input
from afferent neurons in the ear and lateral line. Thus a sudden vibrational
stimulus can activate one of the M-cells and cause the stereotypic body
contraction, resulting in rapid acceleration through the water.
Most behavioral research conducted on Mauthner cells has been directed at
a single behavior, the startle response. Although, the stereotypic escape
behavior has appealed to researchers because of its simplicity and
predictiblity, these responses are generally not all-or-none. Closer
examination of the startle response in fish reveal that the rapid escape
movements can be graded in amplitude (Aljure et al, 1980.) Thus it seems
that the execution of escape movements require some finer control. It is
conceivable that Mauthner cells may be involved in other fast movements
with a directed orientation, such as predatory and feeding behavior.
In hopes of understanding the M-cell in conjunction with activities other
than the tail flip, we have chosen the intertidal wrasse as the subject. The
Senorita, ovyjulis californica displays two separate "escape" reactions
when it senses danger, an extraordinary burrowing behavior as well a more
typical tail-flip response. It appears to use the same set of muscles for both
escape responses. Presumably, the tail-flip response is, as in other teleosts,
controlled by the M-cells. How then are the Mauthner cells involved in escape
burrowing and in the process of deciding between the two behaviors?
The following paper seeks to explore the above questions by first
developing a workable method to record electrophysiologic behavior from the
Senorita. We show by satisfying four criteria that we have indeed recorded
from the Mauthner cell. First, we demonstrated that antidromic and
orthodromic stimulations produce a c-start tail flip. Secondly, we have
measured large, single-unit spikes in an area of the medulla corresponding to
that where M-cells are located in goldfish. Third, the very short latency of
these responses are similar to those known to accompany antidromic
conduction along the large M-cell axons. Finally, we see that these short
latency responses are act ivated with the lowest threshold observed and bear
an all-or-nothing relationship to the strength of stimulus (Furshpan and
Furukawa, 1962.)
Our setup was applied to preliminary analysis of the effects of cone snail
toxins. The toxin used in this study is a peptide abstracted from Conus snails
of the Philippines that prey on fish. This as yet uncharacterized toxin is
thought to be the single component that allows snails to paralyze their prey
within seconds, but its mode of action has not been defined. (Olivera,
personal communication.) Given the speed of its effectiveness, it was
hypothesized that this "paralytic toxin" sends a train of rapidly firing action
potentials to the M-cells. The M-cells could then stimulate the contraction of
tail muscles on both sides thereby paralyzing the prey in a seizure of
uncoordinated activity. We show that our setup was adequate in measuring
M-cell activity as a response to toxin injection, though further study is
needed to clarify the effects.
HETHODS
Preparation of fish and operation procedure
Experiments were performed on adult Senorita (15.5-18)cm in length, Fish
were kept in fresh sea water at 14 C, and fed chopped squid. Fish were
anesthetized by a 5-10 minute imersion in sea water containing 0.0052
quinaldine and .028 isopropyl alcohol. Anesthetized fish were first wrapped
in gauze to reduce slipping from mucous and were then secured by means of
adjustable, plastic cable ties to a plexiglass platform fitted to a small tank.
The mounted fish was placed in the experimental tank and, the head of the
fish was immobilized by fixing the mouth over a disposable pipette tip.
Cooled (14 C) and oxygenated bath solution was perfused at rate of
10Oml/min through the pipette tip during the entire experiment. The body was
kept completely submerged at all times in a solution of 50% seawater and
50% deionized water which served as a simple and satisfactory saline
solution. The water pump circulating the bath solution was powered by a
12V DC battery to avoid 60 cycle noise (Fig. 1).
Äfter lowering the water level to expose the skull, a small amount of local
anesthetic (1.52 Dibucane) was applied to the area with cotton swab before
incision. A 1.Ox0.5 cm section of skin was removed from a central area
approximately 1 cm posterior to line drawn between the fish's pupils (Fig .2).
Muscle and connective tissues were scraped away to reveal the cranial ridge.
Using a scalpel, a small slot was made through the skull and forceps were
Used to peel away the bone. A Amm diameter hole was finally made through
which an electrode could be inserted.
Electrophysiological Observations
Conventional methods were used for electrophysiological recordings and for
electrode micromanipulation. M-cell activity was recorded with tungsten
microelectrodes insulated with 0-dope varnish except at the tip. The
recording electrode was first positioned over the center of the cerebellum
slightly off the midline (Eaton, 1981). The trajectory of the electrode was at
à 90 angle to the dorsal surface of the cerebellum. Data was recorded on a
digital oscilloscope and photographed with Polaroid film.
Since it was known that the M-cells in goldfish are situated approximately
1.5mm below the medulla oblongata (Zottoli,1978), and we measured the
Senorita cerebellar thickness to be 2.6mm, we predicted that Senorita
M-cells should be approximately 4.Omm below the surface of the cerebellum.
Thus in most experiments for recording M-cell activity, the M-cell was found
by lowering the electrode to a depth of 3-Amm below the surface of the brain
while monitoring responses to antidromic stimulation. A large negative going
extracellular action potential is generated at the M-axon in response to
antidromic stimulation of the spinal cord (Faber and Korn, 1978; Diamond
1971.) (See figure 2 for area of insertion.) Care was taken not to lower the
electrode beyond the region of M-spike focus. Survival of the animal appeared
to be greatly impaired after electrode penetration into these deeper regions
of the medulla.
Antidromic stimuli were delivered to the spinal cord by means of
stainless steel bipolar electrodes made from 25 gauge syringe needles
insulated with nailpolish except at the tip. The negative electrode was
inserted through the tail musclature down to the spinal cord at a location
6-8cm behind the cranium (Diamond 1971). The positive electrode was
positioned just outside the skin.
To ascertain the rate of repetitive stimulation useful for later
experiments, we tested the limits of habituation on a fish that had not
undergone surgery. By delivering a stimulus of 2 volts above threshhold value
at regular intervals, it was determined that the animal quickly habituated to
stimulus rates greater than 1shock per 5 seconds. Application of shocks was
therefore kept at a frequency less than O.1Hz.
RESULTS
Orthodromic Stimulation
Orthodromic depolarizations via a tungsten microelectrode in the medulla
produced the characteristic all-or-nothing tail flip at a sharp threshold.
Closing of both opercula and a slight bobbing of the head accompanied the tail
flip. Reversing the stimulus polarity elicited an obviously different behavior
best described as a twitch of the entire body of undef ined direction.
At an electrode tip depth of 3mm below the surface of the cerebellum, we
varied the stimulus duration and strength for depolarizing pulses to defined
strength-duration relation for threshold activation of what appeared to be the
all-or-none tail flip (Fig. 3). As expected, stronger stimull were necessary
to elicit the behavior as the duration grew shorter. Stimulus strength lower
than 2 volts did not produce a response even when the stimulus was very long.
In general, threshold values were distinct within one experiment at any time
but usually increased as the fish expired.
Antidromic Stimulation
Stimulation at the spinal cord with a bipolar electrode produced
contraction of tail muscles ipsilateral to the stimulus. In addition, the
stimulus also caused simultaneous closing of both opercula. Both behaviors
appeared to be similar to those produced by orthodromic stimulation.
Stimulus strength necessary to produce antidromic responses varied from as
low as 1.5V at a duration of O.06ms to as much as 40 volts depending on the
condition of the fish and the location of the electrode. Reversing the
stimulus polarity (i.e. making the internal electrode positive) gave rise to a
tail bob in the vertical direction and, at a higher voltages, caused the tail to
curve in the opposite direction. Depending on the extent that experimentation
worsened the health of the animal, the tail flip varied from strong, sweeping
motions to only slight flicks. Although the behavior were not as well defined
as those following orthodromic stimulation, both probably involve M-cells as
supported by results below.
Electrophysiological recordings made in a region dorsal to the M-cells
typically reveal two fast negative spikes and one broader wave, 1.5ms in
duration (Fig 44). As measured from the start of the shock artifact to the
beginning of the initial positivity, the latencies were very short,
approximately 1.2, 1.6 and 3.Oms (Fig. 4B). The two early components begin to
appear at a depth of 1.5-2.Omm below the surface of the brain and their
amplitudes increased with depth. From the last trace in figure 4C, we see
that the one of the early components focuses to a large spike of 0.6V at a
depth of 4.8 where the electrode is now in the medulla. The late potential
change occuring at 3.Oms can be measured from the within the cranial cavity
above the brain and increases as the electrode enters the cerebellum.
However, as the electrode is lowered to the medulla, this late component
disappears. In sum, the two early spikes seem to originate from the medulla
while the slowest spike appears to originate from the cerebellum.
The two early spikes and later single component vary differently as a
function of voltage. Two separate experiments, figure 5A and 5B, confirm
that both sets of spikes appear to have the same threshhold value (4.0 and
4.2V respectively). Once breaching that value, it is clear that the amplitude of
the two early spikes do not increase with stimulus strength while the late
one does. Thus it can be said that the two early spikes bear an all-or-nothing
relationship with strength of the stimulus, and the single later component
can be graded with voltage.
To assess the level of background "cranial noise," the recording electrode
was inserted in a location believed to be distant from the Mauthner location.
In this experiment, the M-spike was first located by methods described above.
Then the electrode was placed 1.3mm anterior and approximately 0.75mm to
the right of the location of the M-spike, and lowered to a depth of 3.Smm.
Medullary recordings in this region show no spikes corresponding to any of
the three responses measured in the M-cell region nor any brief latency
responses. On a slower time base, it is apparent that electrodes in the
non-M-cell region recorded spontaneous and stimulated brain activity not
seen in the M-cell regions (Figs 6B1,B2,B3.)
In addition to spinal cord electrical stimulation, a strobe light flash was
used to trigger a startle response and action potentials in the M-cell were
measured from the same two regions. The trace from the M-cell region (fig
7A) shows only one spike at a latency of 1.Oms. The same stimulus failed to
produced any short latency mauthner-like spikes in the non M-cell region but
elicited a broad peak at 33ms after the shock artifact (fig 7B).
Cnus Pentides and M-cell activity
Preliminary studies were also performed on M-cell involvement with
paralysis of Senorita with a toxin from cone snails. The peptide was injected
within Icm radius of stimulating electrode insertion while delivering
superthreshhold shock to tail at a rate of 6 shocks/minute. Amplitudes of
both the M-cell spikes and cerebellar components decreased until
disappearing. The trunk of the fish became very stiff as if muscles both sides
were completely contracted. Its caudal and dorsal fins were fanned out like
that of a the tail in mid-flip response. It was confirmed that the fish was
partially paralyzed when the released fish could not move its tail but could
flap its pectoral fins. In another experiment where less than the required
dosage to paralyzed was injected, the fish was seen to twitch convulsively
in frregular sessions while electrophysiological data is presently being
analysed.
DISCUSSION
Observations of behavior induced by anti- and orthodromic stimulations,
and electrophysiological data have shown we located and recorded from the
Mauthner cell. Data from cranial recordings support the hypothesis of direct
influence by M-cells on neurons in the cerebellum.
In the best preparations of Senorita which were studied, behaviors
produced by anti- and orthodromic stimulations of the medulla are in
agreement with those expected to be produced by excitation of the Mauthner
cell, based on published work. The trunk, gill and eye movements observed in
our studies are all characteristics of M-initated responses in goldfish
(Diamond, 1971.)
The optically activated Mauthner activity produced the same behavior as
that evoked by electrical stimulus. Detwiler (1927) has also demonstrated
that visually-evoked startle responses are mediated by the M-cell in
Amblystoma. This fact was sufficient to show that a "naturally" occuring
stimulus would produce not only the same mechanical behavior but also the
same electrical discharge characteristic of what we identify as M-cell
related.
From observations of antidromic stimulated behavior, it appeared that the
tail flick response was sometimes graded with stimulus strength. The
weaker tail flicks did not correspond in form to that of the naturally occuring
startle response though they were accompanied by an M-spike. Eaton and
Bombardieri (1978) recognized that the Mauthner response is not simply an
explosive and random reflex contraction of the animal's body musclature.
Rather, it is a highly organized motor pattern involving the M-cell and
probably many other neurons as well. Studies conducted by Aljure (1980)
indicate that the gradations result from inhibition onto motorneurons.
Clearly, the ambiquity and speed of behavior renders unaided visual
observation inadequate for adequately document ing M-cell related behavior.
The above behaviors were observed to have a well-def ined threshold
consistent with that of M-cell stimulated behavior in other studies. The
strength-duration curve for tail-flick threshold obtained from orthodromic
stimulation (fig3) indicates that the behavior observed is probably due to
stimulation of a small number of neurons. The sharpness of the curve would
be consistent with selective stimulation of the large M-cell in the medulla
and supports the proposal that we are in fact stimulating M-cells to produce
the tail flip.
It has been shown that recordings from the brain concurring with
Mauthner tail flip arise from two different depths. We believe the two early
components measured in the medulla are the result of the asymmetrical
activation of the two M-cells. This is confirmed by their all-or-nothing
character and very short latencies of 1.2 and 1.5ms. The mean spike latency
measured in a similar manner in goldfish is 0.78ms (Eaton 1981.) The
apparent conduction velocity in our experiments was calculated by dividing
the negative spike latency into the distance from the stimulation cathode to
recording electrode. The result, a conduction velocity of 80m/s, agrees well
with published values for the goldfish M-, ranging from 55 (Dorsett, 1980)to
10Oms (Eaton, 1981; Furshpan 1962.) Although the diameter of the M-axon in
Senorita is not yet known, it would appear to be very close to that in goldfish.
Further data which support that these are M-cell initiated spikes comes
from inspection of the relative amplitudes of the two spikes. In Figure 48,
the first of the two early spikes is larger, wheras in Fig 4C the second spike
is very much larger. Since the M-cell axons are known to cross in the medulla
(Diamond, 1971), one would expect stimulation on the one side of the fish to
excite the contralateral M-cell first. In this case of Fig. 4B the recording
electrode in the medulla was positioned to the left of the midline, and
stimulation was on the right-hand side of the fish. Thus, we expect the first
early spike to be the larger of the two, because the electrode is closer to the
left M-cell. Exactly the opposite result is seen( l.e., the second early spike is
larger) in Fig. 4C, when the recording and stimulating electrodes were plac
on the same side of the fish.
Since the slower component can be measured from outside the brain and
essentially disappears once the electrode enters the medulla, we believe that
neurons excited in the cerebellum caused this signal. The slow, i11-defined
time course of the signal suggests the massive recruitment of many
cerebellar neurons firing at slightly different times. In addition, we did not
find any changes in the tail-flip behavior when the cerebellum was
completely removed, but the slow component was no longer recordable. This
lends further evidence to our hypothesis that the third component does
originate from the cerebellum and does not play a necessary role in the M-cell
activated tail flip.
The facts that 1) both M-spikes and the cerebellar discharge appear to
have the same threshold value and 2) there exists a consistant delay between
the two early and third components, may indicate that M-cell activity can
quite directly cause or influence the firing of the cerebellar neurons. We
observed the appearance of the first two spikes without the cerebellar
components only once in our entire study. This recording was made precisely
at threshold in the first two spikes at a depth where both early and late
components were measured in all other instances. The M-cell coupling to
cerebellum discharge would thus appear to be powerful, but still susceptible
to inhibition from other sources.
The exact relationship between the cerebellum and the M-cells is still
unclear. The most recent research, conducted by Bartelmez in 1915, suggested
the cerebellum as a possible source of afferent input to M-cells. Experiments
relating the maintainance equilibrium and M-cells were conducted in
Ambystoma by Detwiler (1947) who showed that animals missing M-cells
could hold normal posture at rest and during swimming. Thus M-cellsare not
necessary for functions of the cerebellum pertaining to these behaviors.
The Senorita proved to be an excellent subject for study of Mauthner
initiated behaviors. Senorita are surprisingly hearty animals; most fish were
able to live 1- 3 hours after the operation. Their brains, large for their body
mass, are easily accessible through their thin skull. Electrophysiological
recordings from freely mobile Senorita using techniques pioneered by Zottoli
(1977) might help to elucidate the role of M-cells in the decision-making
process such as that between the two escape responses, the C-start and the
burrowing.
Acknowlegement:
would like to sincerely thank Dr. William Gilly for all his
guidance and pat ience throughout the project.
BIBLIOGRAPHY
Aljure E, Day JW, Bennett MVL(1980) Postsynaptic depression fo
Mauthner cell-mediated startle reflex, a possible contributor
to habituation, JPhysiol, 45:533-564.
Detwiler SR (1947) Quantitative Studies on the Locomotor
Capacity of Larval Amblystoma (A. jetfersoniamun) Lacking
Mauthner's Neuron or the Ear, J Exp Zool, 104.343-351.
Diamond J(1971) The Mauthner cell. In: Hoar WS, Randall Do(eds)
Fish Physiology vol 5. Academic Press, New York, pp
265-346.
Dorsett DA(1980) Design and Function of Giant Fiber Systems,
Trends in Neurosciences, 3:205-208.
Eaton RC, Bombardieri RA, and Meyer DL (1976) The Mauthner¬
Initiated Startle Response in Teleost Fish, J Exp. Biol. 66:65¬
81.
Eaton RC, Hackett JT (1984) The Role of the Mauthner Cell in Fast
Starts Involving Escape in Teleost Fishes. In: Neura/
Mechanisms of Startle Behavior, Eaton RC (ed) Neural
Mechanisms of Startle Behavior, Plenum, New York,pp213-260.
Eaton RC, Lavender WA, and Wieland CM(198 1)Identif ication of
Mauthner-Initiated Response Patterns in Goldf ish: Evidence
from Simultaneous Cinematography and Electrophysiology,
Comp. Physiol. 144.521-531.
Faber DS, Korn H (1978) Electrophysiology of the Mauthner cell:
Basic Properties, Synaptic Mechanisms, and Associated
Network. In: Faber DS, Korn H(eds) Neurobiology of the
Mauthner cell Raven Press, New York, pp 47-131.
Furshpan EJ, Furukawa T(1962) Intracellular and Extracellular
Responses of the Several Regions of the Mauthner cell of the
Goldfish, JNeurophysiology 25:732-771.
Zottoli SJ (1978) Comparative Morphology of the Mauthner Cell in
Fish and Amphibians. In: Faber DS, Korn H (eds) Neurobiology
of the Mauthner Cell Raven Press, New York, pp 13-45.
FIGURE LEGENDS
Fig 1. Schematic of Experimental Setup.
Fig 2. A. Drawing of Senorita brain and electrode insertion at
place marked X. B. Drawing of location of M-cells as seen
from above from goldfish. Cerebellum retracted to expose
medulla (Furshpan and Furukawa, 1962)
Fig 3. Strength-Duration Relationship of Medullary Shock to
Produce Escape Response.
Fig 4. Three components of brain activity measured at varying
depths.
Fig 5. Medullary components as a function of voltage.
Fig 6. Comparisons of brain activity in two regions of the medulla.
Fig 7. Response to strobe light stimulus measured in two regions
of the medulla.
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