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Enhancement by ethanol of visually evoked responses in the goldfish optic tectum
ESI’EI
33, 329-342
NEYKOLOGY
Enhancement Responses
(1971)
by Ethanol of in the Goldfish
Visually Evoked Optic Tectum
Flash-evoked potentials and multiple unit discharge were recorded from the superficial layers of the goldfish optic tectum, and post-flash histograms were prepared. When goldfish were intoxicated by a level of ethyl alcohol just sufficient to depress maze-learning ability, the first of three components of these evoked responses was consistently enhanced. These results indicate that ethanol can unbalance neural processes near the periphery as well as central brain mechanisms. Furthermore, the data provide a basis for suggestion that the retina might be useful as a model system for further physiological and pharmacological analysis of the mechanisms by which ethanol disrupts normal brain functions. Introduction
Several behavioral studies within the last few years have suggested that the teleost fish provides a convenient experimental model for studying ethanol
intoxication.
The
ethanol-treated
fish
is deficient
in
fear-motivated
learning ( 11)) is over-aggressive (S) , and prone to amnesia (6). A particular advantage of the fish is the opportunity it affords to study effects of ethanol under steady-state conditions, since blood ethanol comes into equilibrium with that in the surrounding water within 2-3 hr (9). The present study represents a first attempt to study physiological effects of intoxication in the goldfish brain, using levels of alcohol found to retard, but not incapacitate, the fish in a maze-learning task. The strategy has been to explore the well-developed goldfish visual system, beginning with the optic projection to the tectum. The pathway from the retina to the tecum is easily accessible in the fish, and its structure and function are roughly comparable to that found among higher vertebrates. The recent success of other workers in showing clear effects of 1 This study was supported by an NIGH Research Grant (MH17729), by an NIH Grant (FR-05484) and by a grant from Licensed Beverages, Inc. During completion of this study the author was supported by NIMH Career Scientist Development Award (I<0213185 ). He extends thanks to Mr. David Butz for his valuable technical assistance in carrying out the present study. 329
330
INGLE
drugs on mammalian retina and tectum (1, 10, 11) provides some basis for hope that the pharmacological properties of behaviorally active drugs such as ethanol can be elucidated by systematic study of this well-defined neural system. Methods
Suvgevy. Large goldfish, 18-23 cm long, were deeply anesthetized in a solution of tricaine methanesulfonate (Finquel, Ayerst Lab.) and then fixed in a solid Plexiglas box, through which fresh water could be delivered via a mouth tube (Fig. 1). The cranium above forebrain and midbrain was opened quickly and the spinal cord severed just caudal to the
a
water inflow
FIG. 1. Apparatus for recording flash-evoked potentials (FEP) in the goldfish optic tectum. The fish is supported from below by a cut sponge and from above by screws tapped in to the cranium and cemented to an aluminum holding box. The microelectrode is inserted into the dorsolateral optic tectum contralateral to the stimulated eye, as the inset at the upper left illustrates. The lower inset further magnifies the view of the electrode tip, in order to schematically illustrate the terminations of optic tract axons within the region of dendritic arborizations ascending from deeper tectal neurons. The response thus recorded could be derived from either axonal or dendritic elements.
OPTIC
TECTCM
331
medulla. The optic tectum was visualized to the lateral margin by gentle aspiration under a dissecting microscope and small bleeding sources were blocked with bits of Gelfoam. Generally, the tectum remained clean for many hours, with clear fluid from the brain cavity bathing the surface. If the tectum was not soon covered with this fluid, mineral oil was added to protect the brain against drying. The fish’s mouth was fitted over a tube and the body supported in a wet sponge. For most experiments, the gills were held open and fixed tightly near the sides of the holding box by hard molding clay. For studies in which a microelectrode was to remain for some time fixed within the tecturn at a particular depth, fish were secured by cementing the cranium, into which tapered screws had been fixed, onto a special aluminum head holder. Water entered the fish’s mouth from a well-aerated tank outside the recording cage, ran over the gills, drained from the holding bo.x, and was continuously pumped back to the storage reservoir. Rccovding. During early experiments, evoked potentials were recorded from the tectal surface by means of silver-wire electrodes which were insulated (In&-X) escept on the bottom of the ball tips. These electrodes could rest lightly on the tectal surface for hours without causing visible damage to the tectum or changes in the essential form of the flash-evoked tectal potentials, For penetration into the tectum, either 60-p Teflon-insulated platinum-iridium wire (Medwire Corp.) or steel insect pins sharpened to a lo-15-1~ tip and coated with Insul-X were used. These electrodes were lowered via a micromanipulator, while tip distance from surface contact was monitored to the nearest 50 p. Although this distance measured on the manipulator scale did not represent the true depth of penetration, the values correlated well with the changes in the form of the evoked potential during successivepenetrations and among subjects. Evoked potentials were recorded between the electrode and a ground clip on the dorsal fin, led via a Grass P-14 or P-15 preamplifier (1 Hz-O.1 kHz) to a Tektronix 56.5 oscilloscope and to a Fabritek 1061 instrument computer. Flashes were delivered via a Grass PS-3 stroboscope, which triggered both the oscilloscope and computer. at a standard rate of 3 flash/set. Tectal unit discharge was filtered via a Grass preamplifier (100 Hz-10 kHz ) and again via a Tektronix 3A61 differential amplifier. TVhen optimal stimulus position was obtained, the internal triggering mechanism of the oscilloscope was used to convert the largest spikes to 0.05msec pulses, which were fed into the “pulse count” channel of the Fabritek. Both flashevoked potentials (FEP) or post-flash histograms (PFH) of unit activity were summed over 64 repeated sweeps and written out in illk by a Hewlett-Packard 7035’B S-Y recorder.
332
INGLE
During early experiments the full strobe flash was directed at the eye from a lateral position 15-20 cm along the optic axis. This flash was diffused in some cases by inserting a piece of 20 X 20-cm white paper over the side of the holding box 2.5 cm distant from the eye. In later studies, the strobe was fitted with a black funnel with a 13-mm aperture that delivered a bright spot from a distance of 15-20 cm. The strobe was rigidly supported dorsal and lateral to the fish so that the funnel pointed downward at the eye, at a 30-45” angle. This location optimally activated units near the center of the dorsal tectum where electrode penetration was orthogonal to the tectal surface. Where visual field location was to be systematically altered, strobe position was marked either via a plumb-line from the strobe aperture to the table top, or on a 30-cm high Plexiglas hemisphere that was nearly centered on the fish’s eye. The optical conditions were abnormal for the fish, whose cornea has the refractive index of water. Therefore, the angular deviations here required to produce a given change in the visually evoked response were larger than would have been the case for a fish immersed in water. Experimental Protocol. Goldfish recovered from anesthesia within 30 min, as judged by growth and stabilization of evoked potential size. Control records were taken over periods of 30-120 min with electrodes on orjust below (100-200 ,k) the tectal surface. When the small strobe spot was used, it was placed in a position such that a standard “optimal” potential form (Results) was consistently obtained. Since the potential form changed noticeably during initial flashes, averaging was not begun until the strobe had flashed for about 5 sec. Normal room lighting was maintained throughout to avoid any modifications due to dark adaptation. Pairs of records were taken each 5-15 min after the water was switched by a valve to a solution of ethanol (600-700 mg/lOO ml). Since depressive effects of alcohol on fish behavior vary somewhat throughout the year, a given experiment used a concentration just high enough to retard fish in a mazelearning test at that particular time. Effects of intoxication were followed for l-2 hr until clear changes were produced. Then the valve was switched to the normal water supply and the fish was allowed a further hour to recover from effects of intoxication. Results
Full Strobe Stinzulation. During the initial series of experiments, diffused strobe flash produced a somewhat variable FEP form (type 1) that consisted of an initial sharp deflection followed by a series of three or four waves (Fig. 2). Late waves (150-300 msec) were observed consistently only when the strobe was placed in the peripheral visual field, a procedure
OPTIC
333
TECTLTIII
Control
*
30min Recovery
2 hr
Alcohol
3 hr
Alcohol
20min
Recovery
FIG. 2. Type I tectal responses to diffuse strobe flashes are given for two different fish. Records on the left are each traced from Polaroid photographs of five superimposed oscilloscope traces, while those on the right are copies of Fabritek write-outs after averaging 64 successive FEP. With type 1 responses, ethanol intoxication lasting l-2 hr resulted in enhancement of an early component contiguous with the first positive wave. In these records, positive polarity is up.
which much reduced the size of early wave complex As expected from the observations of Buser (3), these waveforms reversed polarity on penetration of the elctrode past a depth of 400-500 p (manipulator scale). When fish were intoxicated with a dose of 600 mg ethanol/100 ml water, one effect was consistently observed, i.e., after 45-90 min a conspicuous “hump” either appeared de noz’o or enlarged greatly just following the initial wave. This effect could be seen within superimposed oscilloscope traces. as well as with averaging (Fig. Zj. The effect appeared in each of six subjects and in each instance disappeared or was markedly reduced after only 15-30 min recovery from alcohol. The five subjects that received the direct strobe flash without a diffusing paper tended to show the potential (type 2) illustrated in Fig. 3. Two positive peaks with typical latencies of 30 and 50 msec, respectively, were observed instead of the somewhat more complex waveform produced by diffusion. Since this type of FEP closely resembled the FEP form obtained with the spot flash (below ), it seemslikely that the bright center of the stroke flash contributed to the difference between this waveform and that found with light diffusion. The effect of ethanol on this second type of FEP was confined to the first wave (Fig. 3). Because of the potential danger that ethanol effects could be confused with effects of anoxia, two fish were intentionally made anoxic by removing the airstone from the water supply. Figure 4 shows the progressive increase in responselatency that followed. No early wave enhancement, typical of ethanol intoxication, could be found. During a few later experiments,
331
INGLE
C. Left
Tectum
D. Right
Tectum
FIG. 3. Examples of the type 2 FEP record. In this illustration polarity is negative up in order to facilitate visual comparison with the type 3 waveform (Figs. 5 and 6) in which both waves are negative. A and B represent different fish, each showing enhancement of the first positive wave during ethanol intoxication, and subsequent reduction in first wave amplitude during the recovery phase. C and D show pairs of simultaneous records from opposite tecta of the same fish. The development of two distinct components within the second positive wave (B, C, and D) was often
seen in these
anoxia noted. ethanol
experiments.
was introduced inadvertently, and similar depressive effects were The results from these fish were excluded from the summary of effects. Spot Flash Procedure. The evoked potential waveform could be standardized among virtually all subjects by using the funneled strobe spot as stimulus, if care were given to the relative positions of the stimulus and the electrode on the tectum. Figure 5 shows the typical results of moving the strobe from the optimal position laterally along the horizontal axis, at each of depths of penetration (Medwire electrode). The standard waveform at the tectal surface (type 3) always consited of two sharp peaks with latencies of about 30 and 50 msec and usually followed by a third wave at 90-100 msec. With penetration, the third wave increased in amplitude relatively more than the initial waves. At a depth of 450-550 p in different experiments, the first two waves began to invert and, by 600-700 I”, they formed a mirror image of the surface potential. When the strobe was moved 20-30” laterally from this optimal location.
OPTIC
Development
335
TECTUM
of Anoxia
Control
35min
Recovery
after
aeration of water
4min 15min 3% FIG. 4. Typical type 2 record from a fish subjected to progressive anoxia by removing the aerator from the water supply tank. These examples, as well as intervening records not illustrated, show the gradual increase of latencies for each of the two primary waves. This depressive effect is always accompanied by reduction of amplitude of the first wave, unlike the typical enhancement found under ethanol. Note, however, that during recovery from anoxia, the second wave has divided into two components, similar to those illustrated in Fig. 3 during ethanol intoxication.
the first wave partially or wholly inverted (as with depth penetration) but the second wave remained negative and may actually have increased in amplitude. When the spot was displaced by 60” both waves were inverted, and the record resembled that of type 2, obtained from the full strobe flash. These results establish the principle that the receptive field for the first wave of a type 3 response is much smaller than that for the second wave. In all ethanol experiments, therefore, the standard type 3 waveform was obtained by placing the spot flash within the inner receptive field region. When this was done, each of eight fish treated with ethanol (650-700 mg/lOO ml) showed a distinct enhancement of the first, but not the second wave (Fig. 6). In some experiments a slight but consistent reduction of size in the second wave was observed. In all instances, recovery was obtained 30-40 min after returning to fresh water. Unit Responses Under Ethanol. When multiple unit records were taken, with electrodes penetrating to 100-200 p, the three peaks of the resulting
336
INGLE
FIG. 5. The effects of strobe position and electrode depth on the type 3 FEP waveform as obtained with a small flashing light spot. When the spot is near the center of the receptive field two sharp negative waves are always obtained. These two waves reverse at about the same depth, typically between 400 and 500 p as measured on the electrode positioner scale. When the spot is moved horizontally Z&30” off center, the first, but not the second, wave becomes largely or entire positive. With a further displacement to 50 or 60” both waves are found to be positive, and such a record does not change significantly during penetration of the electrode through the tectum.
PFH corresponded closely to the three FEP waves, except that unit activity peaked 5-10 msec before the FEP peaks. The first two tectal PFH peaks corresponded closely to PFH recorded from the optic tract in each of three subjects. When the electrode reached the depth at which both FEP waves began to invert, unit discharge became sparse or disappeared altogether. Deeper unit activity was often recorded, but these units never fired to flash stimulation with the same short latencies and in most instances would not give consistent PFH at all. Furthermore, with lateral strobe displacement, the first PFH peak disappeared as the first FEP wave began to invert. In fact, the relative change in amplitude of first and second PFH volleys was more dramatic (Fig. 7) during these position changesthan were the FEP changes. Therefore, in order to use the second peak as a base line against which to measure enhancement of the first peak, it provied useful to set the strobe slightly off center such that the first peak was measurably smaller than the second peak. In four experiments, the Medwire electrodes were left in place for the entire ethanol-recovery duration and FEP and PFH were intermittently recorded. In each case, the increase of the first wave of the FEP was paralleled by a proportionate increase in the first unit volley, with subsequent recovery toward the pattern before ethanol. In four further goldfish, steel
OPTIC
337
TECTUM
Cont
rot
I hr Alcohol FIG. 6. Example of ethanol effect on FEP and PFH records recorded at the same time intervals during intoxication. At the same time that the typical enhancement of the FEP first wave is well developed (B) the corresponding unit histogram (D) shows a corresponding enhancement of the first peak, but not the second. Note that the PFH peaks coincide with the falling phase of the corresponding negative FEP wave.
electrodes were moved up and down at 50-1~distances at each sampling and at each depth two PFH were taken using two voltage levels for the internal trigger, one at which no spontaneous activity was admitted to the Fabritek and a lower selection level at which a few spontaneous spikes per second were admitted, and so that these latter PFH contained at least twice as many flash-evoked spikes. By using four different population samples at each interval after ethanol, the generality of the enhancement effect was determined. In these four experiments the ratio of spike counts in first and second peaks was determined via the integration mode of the Fabritek which provided a cumulative total of spike counts. Figure S shows a typical record of successive PFH and their corresponding integrations. An increase in the ratio of counts in the first to the second peak thus provided a measure of the expected ethanol enhancement. Although the ratios varied from 0.2 to 0.7 among different sampled populations, a l-hr ethanol treatment (700 mg/100 ml) resulted in ratios exceeding 1.0 in all casesfor each of four subjects. That is to say, the rank order of amplitudes between first and second peaks always reversed during ethanol intoxication, even though dif-
338
INGLE
FIG. 7. Illustration of relative amplitude reversal of PFH peaksby moving the strobe spot only 10” laterally. At the right of each PFH is a tracing of the “integra-
tion” computationby the Fabritek: a cumulativerecord of spike countsduring the 1%msec epoch, as summed over 64 sweeps. Peak reversal with spot displacement is seen more clearly using PFH records than with FEP records, as illustrated in Fig. 5.
ferent populations of units were monitored at successiveintervals. Figure 9 shows a particularly clear, but not exceptional, result, i.e. a reversal in relative amplitude of first and second peaks during intoxication, followed by a recovery to the ratios obtained before ethanol. Increase in FEP Field in the Vertical Dimension. One might further ask whether the enhancement following ethanol treatment is confined to units that already fire regularly to a light flash, or whether new units are added to the responsive population under the influence of ethanol. As one approach to this question, we measured the depth of the reversal point of FEP during successiveelectrode penetrations. Since unit discharge rate diminishes quickly at this level, the firing of previously silent units at the lower boundary might cause a deepening of the reversal point of the FEP. In each of four subjects. the reversal point was twice determined before ethanol treatment by advancing 50 ,LIat a time and averaging the FEP for the usual 64 sweeps, As Fig. 9 illustrates, the “isoelectric point” was obtained at the same level during a second descent, as measured from the
OPTIC
I ntsgration
Histogram
339
TECTLYM
ratio
FIG. 8. Example of PFH changes using a multipopulation sampling procedure. Since the electrodes were moved systematically up and down and different triggering levels used at each tip position throughout the experiment, the ratio of unit counts in each peak provides the essential criterion of ethanol-induced enhancement. In the experiment here illustrated, the second PFH record was written out at one-half amplitude, since the total spike count in the first peak was much larger than that in control or recovery records. However, spike counts in the second peaks were about the same, when the same triggering levels were used. The copy of the integration write-out was measured, as illustrated, to provide data for calculation of the integration ratio.
at the surface where electrode noise suddenly diminished. A\fter 60-90 min of ethanol treatment the reversal point was found to have deepened by at least 100 IL, as measured on the manipulator scale. Reversal points of two fish were remeasured after recovery from ethanol and each showed a restitution to about the former control level. These recovery data eliminate the possibility of a measurement error due to progressively easier passageof the electrode through the sametrack. In the reversal point experiments, the PFH records were not taken so that the desired correlation between slow-wave and Lmit changes at this
point
critical boundary are not available. However, in one experiment a new technique was employed to obtain relevant data. Instead of evoking unit
activity by flash stimulation, the vertical bar of the X-Y recorder was covered with black tape and was moved through the multim~it receptive field driven by the output of the Fabritek itself. In this way motion-evoked Lmit histograms were obtained at progressively deeper electrode positions until the electrode passed below the active retinotectal axon terminals. The electrode remained at this position through 45 min of ethanol intoxication. A progressive increment of unit activity was seen from 25 to 45 min, and then during the recovery period the response quickly waned (Fig. 10). The
result
of this single
esperiment
strengthens
the assumption
that dur-
340
INGLE
A. 40min
Alcohol
B. 90min
C. 90 min
Alcohol
Recovery
FIG. 9. Determination of the reversal point, i. e., the isoelectric boundary of the negative wave source, during and after the peak effect of ethanol. Both first and second waves recorded at 500 p were found to be reversed (i. e., positive) before the peak ethanol effect (A) and likewise following recovery (C). However, the 90-min ethanol record (C) shows strong negative polarity at this depth, and even at 600 p. Although the second wave appears equal in amplitude to the first at 500 p in (B) the surface records showed the typical first wave enhancement.
ing ethanol intoxication some deeper units can be released from a normally unresponsive state. Discussion
The present series of experiments indicates that moderate levels of ethanol intoxication in goldfish can selectively enhance the initial component of the flash-evoked tectal-evoked potential, as well as the corresponding component of tectal unit discharge. The ethanol-sensitive units are presumed to be presynaptic optic tract terminals since the two peaks noted in the PFH are found within multiple unit responsesrecorded directly from the optic tract, and each peak slightly leads the corresponding wave of slow activity in the FEP. Furthermore, Jacobson and Gaze (7) also concluded that tectal units within the upper half of the tectum were axon terminals of retinal ganglion cells. While it seems likely that the ethanol-induced enhancement we have documented reflects an imbalance among retinal components, I have not excluded the possibility that these effects are secondary to changes in the modulation functions of efferent fibers to the retina. Although popular belief supposesalcohol to induce its characteristic behavioral effects through a depression of “higher” brain centers, it is in-
OPTIC
341
TECTUM
Control
25min Alcohol
45min Alcohol
--------,\ I,-II i I ‘. - ----
I f f , __-- -w-
30min Recovery
5 SQC >I Stimulus sweep
FIG. 10. Appearance of deep unit activity evoked by a moving black bar during ethanol treatment. During the condition before ethanol, the electrode tip had been moved just beyond the boundary of the active zone, so that no unit activity was evoked (A). However, after 25 min of ethanol intoxication (B) the stimulus began to activate units and a peak effect was obtained by 45 min (C). The response again disappeared after 30 min recovery (D).
structive to find in the fish a clear effect of ethanol in the periphery. AS with the vestibular system, the net effect is not one of depression but enhancement (2, 7). Behavioral “disinhibition” during alcohol intoxication is also found with innate behaviors such as aggressive display in fish (8). Thus, further analysis of ethanol effects on the retina may provide a useful model for analysis of the cellular mechanism by which ethanol can unbalance a variety of central neural systems. References 1. AMES, A. III, and D. A. POLLEN. 1969. Neurotransmission tissue: a study of the isolated rabbit retina. J. Nc~vophysiol. 2.
G., M. BERGSTEDT, nystagmus in man during 17 : 381-105.
.%SCHAN,
3. BUSER, P. stimulation 4.
central nervous 32: 424-442.
L. GOLDBERG, and L. LACRELL. 1956. Positional and after alcohol intoxication. Qrrnvt. J. Str~!. Air.
1955. I. Description et analyses du nerf optique. J. Phj~iol Paris
topographiques 47 : 737-768.
M., and R. IX?. GAZF. 1961. Types of visual in the optic tectum and optic nerve of the goldfish. (No. 2) : 199-209.
JACOBSON,
in
des
responses
response from @rrt. J. Exp.
a la
single units Pt’zysiol. 49
342
INGLE
5. RAYNES, A. E., RYBACK, R., and D. INGLE. 1968. The effects of ethanol on aggress sion in the Siamese fighting fish. Betta splendcrts. Conwvw~ Brlzav. Biol. Par1 A 2 : 141-146. 6. RYBACK, R. S. 1969. The use of goldfish as a model for alcohol amnesia in man
Quart. J. Stud. Ale. 30 : 877-882. 7. RYBACK, R. S., and P. J. DOWD. 1970. Aftereffects on positional 8. RYBACK, R. and shock 9. RYBACK, R., ethanol in 10. STRASCHILL,
of various nystagmus and coriolis acceleration. Aerosp. S., and D. INGLE. 1970. Effect of ethanol and avoidance in the goldfish. Quart. J. Stud. Ale. PERCAKPIO, B., and J. VITALE. 1969. Equilibration the goldfish. Nature Londoft 222 : 106%1070. M. 1968. Action of drugs on single neurons
alcoholic
beverages
Med. 41: 429-434. bourbon
on Y-maze
Suppl. 5: 136-140. and metabolism in
the
cat’s
of retina.
Vision. Res. 8 : 35-47. 11.
STRASCHILL, M., and K. P. HOFFMANN. 1969. Effect of D-amphetamine ity of single neurons of the cats’ optic tectum. Experetztia 25: 373.
on activ-
33, 329-342
NEYKOLOGY
Enhancement Responses
(1971)
by Ethanol of in the Goldfish
Visually Evoked Optic Tectum
Flash-evoked potentials and multiple unit discharge were recorded from the superficial layers of the goldfish optic tectum, and post-flash histograms were prepared. When goldfish were intoxicated by a level of ethyl alcohol just sufficient to depress maze-learning ability, the first of three components of these evoked responses was consistently enhanced. These results indicate that ethanol can unbalance neural processes near the periphery as well as central brain mechanisms. Furthermore, the data provide a basis for suggestion that the retina might be useful as a model system for further physiological and pharmacological analysis of the mechanisms by which ethanol disrupts normal brain functions. Introduction
Several behavioral studies within the last few years have suggested that the teleost fish provides a convenient experimental model for studying ethanol
intoxication.
The
ethanol-treated
fish
is deficient
in
fear-motivated
learning ( 11)) is over-aggressive (S) , and prone to amnesia (6). A particular advantage of the fish is the opportunity it affords to study effects of ethanol under steady-state conditions, since blood ethanol comes into equilibrium with that in the surrounding water within 2-3 hr (9). The present study represents a first attempt to study physiological effects of intoxication in the goldfish brain, using levels of alcohol found to retard, but not incapacitate, the fish in a maze-learning task. The strategy has been to explore the well-developed goldfish visual system, beginning with the optic projection to the tectum. The pathway from the retina to the tecum is easily accessible in the fish, and its structure and function are roughly comparable to that found among higher vertebrates. The recent success of other workers in showing clear effects of 1 This study was supported by an NIGH Research Grant (MH17729), by an NIH Grant (FR-05484) and by a grant from Licensed Beverages, Inc. During completion of this study the author was supported by NIMH Career Scientist Development Award (I<0213185 ). He extends thanks to Mr. David Butz for his valuable technical assistance in carrying out the present study. 329
330
INGLE
drugs on mammalian retina and tectum (1, 10, 11) provides some basis for hope that the pharmacological properties of behaviorally active drugs such as ethanol can be elucidated by systematic study of this well-defined neural system. Methods
Suvgevy. Large goldfish, 18-23 cm long, were deeply anesthetized in a solution of tricaine methanesulfonate (Finquel, Ayerst Lab.) and then fixed in a solid Plexiglas box, through which fresh water could be delivered via a mouth tube (Fig. 1). The cranium above forebrain and midbrain was opened quickly and the spinal cord severed just caudal to the
a
water inflow
FIG. 1. Apparatus for recording flash-evoked potentials (FEP) in the goldfish optic tectum. The fish is supported from below by a cut sponge and from above by screws tapped in to the cranium and cemented to an aluminum holding box. The microelectrode is inserted into the dorsolateral optic tectum contralateral to the stimulated eye, as the inset at the upper left illustrates. The lower inset further magnifies the view of the electrode tip, in order to schematically illustrate the terminations of optic tract axons within the region of dendritic arborizations ascending from deeper tectal neurons. The response thus recorded could be derived from either axonal or dendritic elements.
OPTIC
TECTCM
331
medulla. The optic tectum was visualized to the lateral margin by gentle aspiration under a dissecting microscope and small bleeding sources were blocked with bits of Gelfoam. Generally, the tectum remained clean for many hours, with clear fluid from the brain cavity bathing the surface. If the tectum was not soon covered with this fluid, mineral oil was added to protect the brain against drying. The fish’s mouth was fitted over a tube and the body supported in a wet sponge. For most experiments, the gills were held open and fixed tightly near the sides of the holding box by hard molding clay. For studies in which a microelectrode was to remain for some time fixed within the tecturn at a particular depth, fish were secured by cementing the cranium, into which tapered screws had been fixed, onto a special aluminum head holder. Water entered the fish’s mouth from a well-aerated tank outside the recording cage, ran over the gills, drained from the holding bo.x, and was continuously pumped back to the storage reservoir. Rccovding. During early experiments, evoked potentials were recorded from the tectal surface by means of silver-wire electrodes which were insulated (In&-X) escept on the bottom of the ball tips. These electrodes could rest lightly on the tectal surface for hours without causing visible damage to the tectum or changes in the essential form of the flash-evoked tectal potentials, For penetration into the tectum, either 60-p Teflon-insulated platinum-iridium wire (Medwire Corp.) or steel insect pins sharpened to a lo-15-1~ tip and coated with Insul-X were used. These electrodes were lowered via a micromanipulator, while tip distance from surface contact was monitored to the nearest 50 p. Although this distance measured on the manipulator scale did not represent the true depth of penetration, the values correlated well with the changes in the form of the evoked potential during successivepenetrations and among subjects. Evoked potentials were recorded between the electrode and a ground clip on the dorsal fin, led via a Grass P-14 or P-15 preamplifier (1 Hz-O.1 kHz) to a Tektronix 56.5 oscilloscope and to a Fabritek 1061 instrument computer. Flashes were delivered via a Grass PS-3 stroboscope, which triggered both the oscilloscope and computer. at a standard rate of 3 flash/set. Tectal unit discharge was filtered via a Grass preamplifier (100 Hz-10 kHz ) and again via a Tektronix 3A61 differential amplifier. TVhen optimal stimulus position was obtained, the internal triggering mechanism of the oscilloscope was used to convert the largest spikes to 0.05msec pulses, which were fed into the “pulse count” channel of the Fabritek. Both flashevoked potentials (FEP) or post-flash histograms (PFH) of unit activity were summed over 64 repeated sweeps and written out in illk by a Hewlett-Packard 7035’B S-Y recorder.
332
INGLE
During early experiments the full strobe flash was directed at the eye from a lateral position 15-20 cm along the optic axis. This flash was diffused in some cases by inserting a piece of 20 X 20-cm white paper over the side of the holding box 2.5 cm distant from the eye. In later studies, the strobe was fitted with a black funnel with a 13-mm aperture that delivered a bright spot from a distance of 15-20 cm. The strobe was rigidly supported dorsal and lateral to the fish so that the funnel pointed downward at the eye, at a 30-45” angle. This location optimally activated units near the center of the dorsal tectum where electrode penetration was orthogonal to the tectal surface. Where visual field location was to be systematically altered, strobe position was marked either via a plumb-line from the strobe aperture to the table top, or on a 30-cm high Plexiglas hemisphere that was nearly centered on the fish’s eye. The optical conditions were abnormal for the fish, whose cornea has the refractive index of water. Therefore, the angular deviations here required to produce a given change in the visually evoked response were larger than would have been the case for a fish immersed in water. Experimental Protocol. Goldfish recovered from anesthesia within 30 min, as judged by growth and stabilization of evoked potential size. Control records were taken over periods of 30-120 min with electrodes on orjust below (100-200 ,k) the tectal surface. When the small strobe spot was used, it was placed in a position such that a standard “optimal” potential form (Results) was consistently obtained. Since the potential form changed noticeably during initial flashes, averaging was not begun until the strobe had flashed for about 5 sec. Normal room lighting was maintained throughout to avoid any modifications due to dark adaptation. Pairs of records were taken each 5-15 min after the water was switched by a valve to a solution of ethanol (600-700 mg/lOO ml). Since depressive effects of alcohol on fish behavior vary somewhat throughout the year, a given experiment used a concentration just high enough to retard fish in a mazelearning test at that particular time. Effects of intoxication were followed for l-2 hr until clear changes were produced. Then the valve was switched to the normal water supply and the fish was allowed a further hour to recover from effects of intoxication. Results
Full Strobe Stinzulation. During the initial series of experiments, diffused strobe flash produced a somewhat variable FEP form (type 1) that consisted of an initial sharp deflection followed by a series of three or four waves (Fig. 2). Late waves (150-300 msec) were observed consistently only when the strobe was placed in the peripheral visual field, a procedure
OPTIC
333
TECTLTIII
Control
*
30min Recovery
2 hr
Alcohol
3 hr
Alcohol
20min
Recovery
FIG. 2. Type I tectal responses to diffuse strobe flashes are given for two different fish. Records on the left are each traced from Polaroid photographs of five superimposed oscilloscope traces, while those on the right are copies of Fabritek write-outs after averaging 64 successive FEP. With type 1 responses, ethanol intoxication lasting l-2 hr resulted in enhancement of an early component contiguous with the first positive wave. In these records, positive polarity is up.
which much reduced the size of early wave complex As expected from the observations of Buser (3), these waveforms reversed polarity on penetration of the elctrode past a depth of 400-500 p (manipulator scale). When fish were intoxicated with a dose of 600 mg ethanol/100 ml water, one effect was consistently observed, i.e., after 45-90 min a conspicuous “hump” either appeared de noz’o or enlarged greatly just following the initial wave. This effect could be seen within superimposed oscilloscope traces. as well as with averaging (Fig. Zj. The effect appeared in each of six subjects and in each instance disappeared or was markedly reduced after only 15-30 min recovery from alcohol. The five subjects that received the direct strobe flash without a diffusing paper tended to show the potential (type 2) illustrated in Fig. 3. Two positive peaks with typical latencies of 30 and 50 msec, respectively, were observed instead of the somewhat more complex waveform produced by diffusion. Since this type of FEP closely resembled the FEP form obtained with the spot flash (below ), it seemslikely that the bright center of the stroke flash contributed to the difference between this waveform and that found with light diffusion. The effect of ethanol on this second type of FEP was confined to the first wave (Fig. 3). Because of the potential danger that ethanol effects could be confused with effects of anoxia, two fish were intentionally made anoxic by removing the airstone from the water supply. Figure 4 shows the progressive increase in responselatency that followed. No early wave enhancement, typical of ethanol intoxication, could be found. During a few later experiments,
331
INGLE
C. Left
Tectum
D. Right
Tectum
FIG. 3. Examples of the type 2 FEP record. In this illustration polarity is negative up in order to facilitate visual comparison with the type 3 waveform (Figs. 5 and 6) in which both waves are negative. A and B represent different fish, each showing enhancement of the first positive wave during ethanol intoxication, and subsequent reduction in first wave amplitude during the recovery phase. C and D show pairs of simultaneous records from opposite tecta of the same fish. The development of two distinct components within the second positive wave (B, C, and D) was often
seen in these
anoxia noted. ethanol
experiments.
was introduced inadvertently, and similar depressive effects were The results from these fish were excluded from the summary of effects. Spot Flash Procedure. The evoked potential waveform could be standardized among virtually all subjects by using the funneled strobe spot as stimulus, if care were given to the relative positions of the stimulus and the electrode on the tectum. Figure 5 shows the typical results of moving the strobe from the optimal position laterally along the horizontal axis, at each of depths of penetration (Medwire electrode). The standard waveform at the tectal surface (type 3) always consited of two sharp peaks with latencies of about 30 and 50 msec and usually followed by a third wave at 90-100 msec. With penetration, the third wave increased in amplitude relatively more than the initial waves. At a depth of 450-550 p in different experiments, the first two waves began to invert and, by 600-700 I”, they formed a mirror image of the surface potential. When the strobe was moved 20-30” laterally from this optimal location.
OPTIC
Development
335
TECTUM
of Anoxia
Control
35min
Recovery
after
aeration of water
4min 15min 3% FIG. 4. Typical type 2 record from a fish subjected to progressive anoxia by removing the aerator from the water supply tank. These examples, as well as intervening records not illustrated, show the gradual increase of latencies for each of the two primary waves. This depressive effect is always accompanied by reduction of amplitude of the first wave, unlike the typical enhancement found under ethanol. Note, however, that during recovery from anoxia, the second wave has divided into two components, similar to those illustrated in Fig. 3 during ethanol intoxication.
the first wave partially or wholly inverted (as with depth penetration) but the second wave remained negative and may actually have increased in amplitude. When the spot was displaced by 60” both waves were inverted, and the record resembled that of type 2, obtained from the full strobe flash. These results establish the principle that the receptive field for the first wave of a type 3 response is much smaller than that for the second wave. In all ethanol experiments, therefore, the standard type 3 waveform was obtained by placing the spot flash within the inner receptive field region. When this was done, each of eight fish treated with ethanol (650-700 mg/lOO ml) showed a distinct enhancement of the first, but not the second wave (Fig. 6). In some experiments a slight but consistent reduction of size in the second wave was observed. In all instances, recovery was obtained 30-40 min after returning to fresh water. Unit Responses Under Ethanol. When multiple unit records were taken, with electrodes penetrating to 100-200 p, the three peaks of the resulting
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INGLE
FIG. 5. The effects of strobe position and electrode depth on the type 3 FEP waveform as obtained with a small flashing light spot. When the spot is near the center of the receptive field two sharp negative waves are always obtained. These two waves reverse at about the same depth, typically between 400 and 500 p as measured on the electrode positioner scale. When the spot is moved horizontally Z&30” off center, the first, but not the second, wave becomes largely or entire positive. With a further displacement to 50 or 60” both waves are found to be positive, and such a record does not change significantly during penetration of the electrode through the tectum.
PFH corresponded closely to the three FEP waves, except that unit activity peaked 5-10 msec before the FEP peaks. The first two tectal PFH peaks corresponded closely to PFH recorded from the optic tract in each of three subjects. When the electrode reached the depth at which both FEP waves began to invert, unit discharge became sparse or disappeared altogether. Deeper unit activity was often recorded, but these units never fired to flash stimulation with the same short latencies and in most instances would not give consistent PFH at all. Furthermore, with lateral strobe displacement, the first PFH peak disappeared as the first FEP wave began to invert. In fact, the relative change in amplitude of first and second PFH volleys was more dramatic (Fig. 7) during these position changesthan were the FEP changes. Therefore, in order to use the second peak as a base line against which to measure enhancement of the first peak, it provied useful to set the strobe slightly off center such that the first peak was measurably smaller than the second peak. In four experiments, the Medwire electrodes were left in place for the entire ethanol-recovery duration and FEP and PFH were intermittently recorded. In each case, the increase of the first wave of the FEP was paralleled by a proportionate increase in the first unit volley, with subsequent recovery toward the pattern before ethanol. In four further goldfish, steel
OPTIC
337
TECTUM
Cont
rot
I hr Alcohol FIG. 6. Example of ethanol effect on FEP and PFH records recorded at the same time intervals during intoxication. At the same time that the typical enhancement of the FEP first wave is well developed (B) the corresponding unit histogram (D) shows a corresponding enhancement of the first peak, but not the second. Note that the PFH peaks coincide with the falling phase of the corresponding negative FEP wave.
electrodes were moved up and down at 50-1~distances at each sampling and at each depth two PFH were taken using two voltage levels for the internal trigger, one at which no spontaneous activity was admitted to the Fabritek and a lower selection level at which a few spontaneous spikes per second were admitted, and so that these latter PFH contained at least twice as many flash-evoked spikes. By using four different population samples at each interval after ethanol, the generality of the enhancement effect was determined. In these four experiments the ratio of spike counts in first and second peaks was determined via the integration mode of the Fabritek which provided a cumulative total of spike counts. Figure S shows a typical record of successive PFH and their corresponding integrations. An increase in the ratio of counts in the first to the second peak thus provided a measure of the expected ethanol enhancement. Although the ratios varied from 0.2 to 0.7 among different sampled populations, a l-hr ethanol treatment (700 mg/100 ml) resulted in ratios exceeding 1.0 in all casesfor each of four subjects. That is to say, the rank order of amplitudes between first and second peaks always reversed during ethanol intoxication, even though dif-
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INGLE
FIG. 7. Illustration of relative amplitude reversal of PFH peaksby moving the strobe spot only 10” laterally. At the right of each PFH is a tracing of the “integra-
tion” computationby the Fabritek: a cumulativerecord of spike countsduring the 1%msec epoch, as summed over 64 sweeps. Peak reversal with spot displacement is seen more clearly using PFH records than with FEP records, as illustrated in Fig. 5.
ferent populations of units were monitored at successiveintervals. Figure 9 shows a particularly clear, but not exceptional, result, i.e. a reversal in relative amplitude of first and second peaks during intoxication, followed by a recovery to the ratios obtained before ethanol. Increase in FEP Field in the Vertical Dimension. One might further ask whether the enhancement following ethanol treatment is confined to units that already fire regularly to a light flash, or whether new units are added to the responsive population under the influence of ethanol. As one approach to this question, we measured the depth of the reversal point of FEP during successiveelectrode penetrations. Since unit discharge rate diminishes quickly at this level, the firing of previously silent units at the lower boundary might cause a deepening of the reversal point of the FEP. In each of four subjects. the reversal point was twice determined before ethanol treatment by advancing 50 ,LIat a time and averaging the FEP for the usual 64 sweeps, As Fig. 9 illustrates, the “isoelectric point” was obtained at the same level during a second descent, as measured from the
OPTIC
I ntsgration
Histogram
339
TECTLYM
ratio
FIG. 8. Example of PFH changes using a multipopulation sampling procedure. Since the electrodes were moved systematically up and down and different triggering levels used at each tip position throughout the experiment, the ratio of unit counts in each peak provides the essential criterion of ethanol-induced enhancement. In the experiment here illustrated, the second PFH record was written out at one-half amplitude, since the total spike count in the first peak was much larger than that in control or recovery records. However, spike counts in the second peaks were about the same, when the same triggering levels were used. The copy of the integration write-out was measured, as illustrated, to provide data for calculation of the integration ratio.
at the surface where electrode noise suddenly diminished. A\fter 60-90 min of ethanol treatment the reversal point was found to have deepened by at least 100 IL, as measured on the manipulator scale. Reversal points of two fish were remeasured after recovery from ethanol and each showed a restitution to about the former control level. These recovery data eliminate the possibility of a measurement error due to progressively easier passageof the electrode through the sametrack. In the reversal point experiments, the PFH records were not taken so that the desired correlation between slow-wave and Lmit changes at this
point
critical boundary are not available. However, in one experiment a new technique was employed to obtain relevant data. Instead of evoking unit
activity by flash stimulation, the vertical bar of the X-Y recorder was covered with black tape and was moved through the multim~it receptive field driven by the output of the Fabritek itself. In this way motion-evoked Lmit histograms were obtained at progressively deeper electrode positions until the electrode passed below the active retinotectal axon terminals. The electrode remained at this position through 45 min of ethanol intoxication. A progressive increment of unit activity was seen from 25 to 45 min, and then during the recovery period the response quickly waned (Fig. 10). The
result
of this single
esperiment
strengthens
the assumption
that dur-
340
INGLE
A. 40min
Alcohol
B. 90min
C. 90 min
Alcohol
Recovery
FIG. 9. Determination of the reversal point, i. e., the isoelectric boundary of the negative wave source, during and after the peak effect of ethanol. Both first and second waves recorded at 500 p were found to be reversed (i. e., positive) before the peak ethanol effect (A) and likewise following recovery (C). However, the 90-min ethanol record (C) shows strong negative polarity at this depth, and even at 600 p. Although the second wave appears equal in amplitude to the first at 500 p in (B) the surface records showed the typical first wave enhancement.
ing ethanol intoxication some deeper units can be released from a normally unresponsive state. Discussion
The present series of experiments indicates that moderate levels of ethanol intoxication in goldfish can selectively enhance the initial component of the flash-evoked tectal-evoked potential, as well as the corresponding component of tectal unit discharge. The ethanol-sensitive units are presumed to be presynaptic optic tract terminals since the two peaks noted in the PFH are found within multiple unit responsesrecorded directly from the optic tract, and each peak slightly leads the corresponding wave of slow activity in the FEP. Furthermore, Jacobson and Gaze (7) also concluded that tectal units within the upper half of the tectum were axon terminals of retinal ganglion cells. While it seems likely that the ethanol-induced enhancement we have documented reflects an imbalance among retinal components, I have not excluded the possibility that these effects are secondary to changes in the modulation functions of efferent fibers to the retina. Although popular belief supposesalcohol to induce its characteristic behavioral effects through a depression of “higher” brain centers, it is in-
OPTIC
341
TECTUM
Control
25min Alcohol
45min Alcohol
--------,\ I,-II i I ‘. - ----
I f f , __-- -w-
30min Recovery
5 SQC >I Stimulus sweep
FIG. 10. Appearance of deep unit activity evoked by a moving black bar during ethanol treatment. During the condition before ethanol, the electrode tip had been moved just beyond the boundary of the active zone, so that no unit activity was evoked (A). However, after 25 min of ethanol intoxication (B) the stimulus began to activate units and a peak effect was obtained by 45 min (C). The response again disappeared after 30 min recovery (D).
structive to find in the fish a clear effect of ethanol in the periphery. AS with the vestibular system, the net effect is not one of depression but enhancement (2, 7). Behavioral “disinhibition” during alcohol intoxication is also found with innate behaviors such as aggressive display in fish (8). Thus, further analysis of ethanol effects on the retina may provide a useful model for analysis of the cellular mechanism by which ethanol can unbalance a variety of central neural systems. References 1. AMES, A. III, and D. A. POLLEN. 1969. Neurotransmission tissue: a study of the isolated rabbit retina. J. Nc~vophysiol. 2.
G., M. BERGSTEDT, nystagmus in man during 17 : 381-105.
.%SCHAN,
3. BUSER, P. stimulation 4.
central nervous 32: 424-442.
L. GOLDBERG, and L. LACRELL. 1956. Positional and after alcohol intoxication. Qrrnvt. J. Str~!. Air.
1955. I. Description et analyses du nerf optique. J. Phj~iol Paris
topographiques 47 : 737-768.
M., and R. IX?. GAZF. 1961. Types of visual in the optic tectum and optic nerve of the goldfish. (No. 2) : 199-209.
JACOBSON,
in
des
responses
response from @rrt. J. Exp.
a la
single units Pt’zysiol. 49
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INGLE
5. RAYNES, A. E., RYBACK, R., and D. INGLE. 1968. The effects of ethanol on aggress sion in the Siamese fighting fish. Betta splendcrts. Conwvw~ Brlzav. Biol. Par1 A 2 : 141-146. 6. RYBACK, R. S. 1969. The use of goldfish as a model for alcohol amnesia in man
Quart. J. Stud. Ale. 30 : 877-882. 7. RYBACK, R. S., and P. J. DOWD. 1970. Aftereffects on positional 8. RYBACK, R. and shock 9. RYBACK, R., ethanol in 10. STRASCHILL,
of various nystagmus and coriolis acceleration. Aerosp. S., and D. INGLE. 1970. Effect of ethanol and avoidance in the goldfish. Quart. J. Stud. Ale. PERCAKPIO, B., and J. VITALE. 1969. Equilibration the goldfish. Nature Londoft 222 : 106%1070. M. 1968. Action of drugs on single neurons
alcoholic
beverages
Med. 41: 429-434. bourbon
on Y-maze
Suppl. 5: 136-140. and metabolism in
the
cat’s
of retina.
Vision. Res. 8 : 35-47. 11.
STRASCHILL, M., and K. P. HOFFMANN. 1969. Effect of D-amphetamine ity of single neurons of the cats’ optic tectum. Experetztia 25: 373.
on activ-