Integration of visual signals in the crayfish brain: Multiunit recordings in eyestalk and brain

Comp. Biochem. Physiol. Vol. 114A, No. 3, pp. 211-217, Copyright 0 1996 Elsevier Science Inc.

1996

ISSN 0300-9629/96/$15.00 SSDI 0300-9629(95)02128-O

ELSEVIER

Integration of Visual Signals in the Crayfish Brain: Multiunit Recordings in Eyestalk and Brain Jeszis Serrate, * Oscar H. Hernhdez,

f and Fidel Rum6nS

*DEPTO. DE FISIOLOG~A,BIOF~SICAY NEUROCIENCIAS CINVESTAV DEL IPN APDO. POSTAL 14-740 MEXICO, D.F., 07000 MEXICO; tCENTRo DE INVESTIGACIONESEN ENFERMEDADESTROPICALES, UNIVERSIDAD AUT~NOMA DE CAMPECHE, CAMPECHE, CAMPECHE, MEXICO AND $DIVISI~N DE ESTUDIOS DE POSGRADOE INVESTIGACI~N, FACULTAD DE MEDICINA, UNIVERSIDAD NACIONAL AUT~NOMA DE MEXICO, CU Mfxrco, D.F., 04510 MEXICO

ABSTRACT.

We have used a preparation

the brain electrical electroretinogram

activity. (ERG)

of light of different

Besides,

in which crayfish are implanted

we placed electrodes

and central afferent activity.

wavelength

from a bright,

elicit trains of spikes that travel afferently

in the cornea

Stimulation

with chronic electrodes and the eyestalk

of eye photoreceptors

wavelength

small source 2 cm from the eye. Results

especially

is not the most effective

in the brain and the ERG-best

wavelength

brain, spikes elicited by different wavelengths

KEY

WORDS.

mechanism.

Invertebrate,

crustacea,

CNS,

visual system, integration

labeled-line; b) across-neuron response-pattern; c) matrixpattern; and d) across-region response-pattern (3 1,32). Of these, perhaps the concept of labeled-lines, introduced by Bullock and Horridge (6) is the most commonly

used (how-

ever, see Bullock, 1993, for a thoughtful description of possible coding systems at different neuronal levels) (5). results from stimulation

of gustatory sen-

sory systems of both invertebrates (8,9,10) and vertebrates (1,7,11,20,33) have been explained with the labeled-line or across-neuron pattern hypothesis. These hypotheses are not exclusive, and although occasionally it has been stated that the analyzing code can only be one of them (14), more frequently both have been used to explain coding of olfactory or gustatory CNS (8,20).

sensory modalities

at different

levels of the

Address reprint requests to: F. Ram& at: Divisibn de E-studios de Posgrado e Investigaci6n, Facultad de Medicina, Universidad National Autbnoma de M&co. CU M&co, D.F., 04510 Mexico. E-mail: fidelrr@servidor. unam.mx (Internet) Received 10 May 1995; revised 12 October 1995; accepted 2 November 1995.

a result compati-

1996. labeled-line

Although labeled-line

of peripheral sensory receptors elicits electrical

diverge,

in spikes per first 100 msec. At the

seem to be analyzed in different neuronal circuits,

activity that travels to the brain, where it can be analyzed in different ways. Four hypotheses have been proposed to describe possible analyzing mechanisms for food or odors: a)

Experimental

After the 13th spike, the intervals

is the most effective

COMP BIOCHEMPHYSIOL 114A;3:211-217,

INTRODUCTION Stimulation

irregular after that. Integration,

for the eyestalk or the brain in respect to first spike latency,

from the 1st to 13th are equal in spike interval.

ble with a labeled-line

show that light pulses

(29)) seems to take place at both the eyestalk and the brain. The most effective

and Roach

for the ERG

but all wavelengths

the

was provided by pulses

and can be recorded at the eyestalk and at the brain. Spikes in single

units in these trains appear at regular intervals during the first 13 spikes, becoming as defined by Wiersma

for recording

for recording

and across-neuron

eses have been used predominantly

pattern hypoth-

to explain processing of

gustatory signals, other sensory signals, such as those evoked by cutaneous stimulation, have also been analyzed this way (22). However, it is noticeable that processing of visual signals by the vertebrate central nervous system has been explained differently, as a hierarchical array of feature-detecting cells (16) Rods and several kinds of cones are labelled lines but converge on overlapping sets of ganglion cells in the retina. Studies about the different neurons (fibers) of the crayfish visual system would seem to point towards a mechanism similar to that proposed to account for data from vertebrates. However, processing of electrical signals by the visual system of invertebrates could also be explained by either of those hypotheses. In support of the former possibility, crayfish visual units have been classified as sustaining, dimming, movement, space-constant, and multimodal, from the commissure and efferent responding to visual stimuli (26). Of these, the sustaining units have been more studied because they respond to light in their receptive fields with maintained discharges at frequencies that are proportional to the intensity of the light (23). In the studies referred to above, the behavior of visual units has been interpreted from recordings of fibers in the optic nerve, and we thought that it was important to obtain

J. Serrato et al.

212

information

about the behavior of visually evoked electrical

signals as they reach the brain. Furthermore,

this data would

be very useful for evaluating the appropriateness of a particular hypothesis about processing of visual signals by the invertebrate brain. Thus, in this study, we report results obtained by stimulating the visual system of crayfish with light pulses of different wavelengths and recording the electrical

ered at steps of 25 nm ascending and then descending in the interval 475 to 675 nm. Trains were repeated at 2-minute intervals and records for each train averaged. There

were

no false positives or negatives, but we did not search for omitted stimulus responses (5). The light pulses were obtained from a high-intensity monochromator (Bausch & Lomb) with a tungsten lamp of

signals elicited at three levels, the eye (electroretinogram, ERG), the visual path between the internal and external

150 W powered by a variable transformer, which was adjusted to deliver equal light intensity for each wavelength,

medulla, and the optic lobes of the brain (VEP). Our results

as measured with a calibrated photocell.

suggest that processing of visual signals occurs in different circuits within the crayfish brain, which agrees with the postulates of the labeled-line hypothesis. Preliminary accounts

tor was calibrated with a laser beam of He-Ne (Metrologic Inst., Blackwood, N.J., U.S.A.). Timing was provided by

of this data have been presented

electromechanical from a computer

(12,13).

(S88;

Grass Instruments

Co.)

controlling

an

shutter. Synchronization was obtained (Macintosh Quadra 610, Apple).

The electrical signals were recorded with AC-preamplifiers (P5 11; Grass Instruments Co.), filtered at 0.1 and 1

METHODS The experimental

a stimulator

The monochroma-

preparation was described in the compan-

ion paper, and it was developed for the chronic

recording

KHz for the ERG and 0.1 and 3 KHz for the other recordings. Signals were displayed on an oscilloscope (Gould, Model 1604) and in parallel digitized at 10 KHz (BIOPAC

of the electrical activity of the brain of unrestrained crayfish (15a). Briefly, experiments were performed on adult crayfish (Procambarus clarkii) of either sex. Animals were cold-anes-

System, Model MPlOO, WPI). Six-second segments of these records were stored in a computer (Macintosh Classic II,

thetized and an opening made in the dorsal carapace to introduce an electrode array, positioned in such a way that

Apple) for off-line analysis, such as power spectra (AcqKnowledge, Version 2.0, WPI) and multiunit spike histo-

the recording silver wires (50 pm) that it contains

grams in which all spikes above the obvious noise level were

were in

contact with the brain surface, on the visual lobe. In that paper, we describe control experiments performed to identify and, when necessary, eliminate nating from adjacent muscles.

electrical

counted U.S.A.).

(Igor,

Stapleton

RESULTS Spontaneous

plete recovery. Then animals were cold-anesthetized, one of the eyestalks was fixed in an appropriate position using cyanoacrylate (“Krazy-glue”), and a square (2 X 3 mm) of

As described in the companion

the cuticle of the eyestalk was removed preventing

any loss

were returned to the tank for pe-

For the visual experiments,

Eugene,

Oregon

activity origi-

After the electrode array was implanted, we returned the animals to the aquarium for a period of 24 hr for their com-

of hemolymph. Animals riods of several hours.

Software,

besides the electrode

array

resting on the brain surface, we also placed other extracellular electrodes made of tungsten wires with 1 MR resistances (A-M Systems). These were located in the cornea for recording the photoreceptor activity (electroretinogram, ERG), few microns deep and perpendicular to the light beam to avoid shadows. We also placed two other ftne needle electrodes separated by 500 pm at a region between the internal and external medulla. Final positions were adjusted to maximize the response to light pulses. For the actual experiments (20-24”C), animals were held by the thorax with a clamp and maintained in a tank with well-aereated water, such that the level only reached the lower part of the eye peduncles. The other eye was covered to avoid stimulation. Light pulses were delivered by a dual fiber optic of 6 mm diameter. A branch was placed 2 cm away from the eye and the other one on a photocell. In all cases a train of 20 pulses each of 15 msec duration was deliv-

Activity paper (15a),

the spontane-

ous activity recorded by an electrode on the crayfish brain surface is comprised of numerous spikes of different amplitude, whose total number per unit time is slightly variable. The power spectrum of a 6-second segment of record shows maximum power at about 400 Hz, decreasing to noise values above 2 KHz.

Light-Triggered

Potentials

Eye Photoreceptors. The electrical activity produced by dark-adapting the crayfish for 30 min and then stimulating the eye photoreceptors with pulses of light of different wavelengths is an electroretinogram (ERG; Fig. 1A). All light pulses used for stimulation triggered ERGS whose morphologies depend on the wavelength. Pulses of short (475 nm) as well as long (675 nm) wavelength produce smallamplitude ERGS, with slow rates of rise and decay, and long latencies. On the contrary, pulses of intermediate wavelength (575 nm) produce the largest amplitude ERGS, which were also those with the fastest rates of rise and decay (Fig. 2). In these latter cases the latency was short. Subsequent to the activation of the eye photoreceptors (ERG) there are trains of spikes that travel along the visual

Visual Evoked Activity in Crayfish CNS

FIG. 1. Electrical activity from the visible spectrum. A. Records from and external medulla. C. Records records in Part C) were obtained for each column.

crayfish nervous system triggered by light pulses of different wavelengths in the mammalian the cornea (electroretinogram). B. Records from the eyestalk, at a level between the internal from the brain surface, on the optic lobe. Records for each wavelength (shown on top of simultaneously from the three regions and are the average of 20 sweeps. Calibrations are

pathway. These were recorded at two levels of the path, the eyestalk between the internal and external medulla, and the

optic lobes of the brain. Eyestalk. The trains of spikes recorded from the eyestalk appear after a delay that depends on the amplitude and/or rate of rise of the ERG. Thus, small ERGS with slow rates of rise (such as those produced with pulses of 475 and 675 nm) give origin to trains with longer latencies (38 ms), while large ERGS with a faster rate of rise (pulse of 575 nm) 60

40

20

1

000 450

trigger trains that appear with minimum delay (20 ms; Fig. 1B). Although somewhat similar, the trains of spikes are not equal when evoked with pulses of the same intensity but different wavelength and, besides their differences in latency, they have a different number of spikes. To determine 500

550

600

650

Wavelength 2. Amplitude of the corned electroretinogram expressed as percent of the maximum. Data for the plot is that shown in Fiie 1A. Note that the maximal amplitude (100%) corresponds to a wavelength of 575 nm.

FIG.

this we counted the number of spikes in the first lOO-msec interval following the onset of the ERG, and found that spikes are more numerous with pulses of 550 nm (about 18), while pulses of 475 and 675 produce no more than about 15 spikes (Fig. 3A). We chose to measure the spikes occurring in an interval of 100 msec because it includes most spikes evoked by the light pulses (i.e. Fig. 1B and 1C). Al-

214

J. Serrato

et al.

and so on, up until the last spike of the arbitrary 100 msec interval. As Figure 4A shows, data points follow a linear relationship fcr the first 13-14 spikes, after which there is a change in slope, which becomes irregular. The total number of spikes in those 100 msec is 22-24. A similar behavior to that described above for the intervals between spikes elicited with the three wavelengths used as example was seen for all wavelengths used. In a11those cases, after the linear relationship

seen for the first 13-14

there is a change in slope and a variable relationship.

spikes In all

cases it is clearly seen that the data points follow different trajectories for each wavelength used in these experiments. 0 450

500

550

600

650

Brain. The electrical activity elicited at the eye by the light pulses evokes a train of spikes that was recorded at

700

the eyestalk, and which also reaches the visual lobes. At the brain the train, seen in favorable single or few-unit recordings, is comprised of about 15-30 spikes of 5- 10 ,uV amplitude and less than 1 msec in duration. The train is briefer

n

for short and long wavelengths (475 and 675 nm) and longer for those around 575 nm. This is shown with bars in Figure 3B, where it is clear that the maximum number of spikes is reached, with pulses of 575 nm. The graph of latencies has a linear relationship

for the

first 8 spikes recorded at the brain during the first 100 msec after the onset of the ERG, becoming irregular after that. Furthermore, the intervals between these spikes also show different patterns for each wavelength used, as it can be

0 4ki0

5b0

550

6ii0

Wauelength

6b

7i0

seen with the spikes triggered with 475-, 575- and 675-nm wavelengths shown in Figure 4B. As the same Figure 4B shows, the length of the train of

Inm)

spikes recorded at the brain is different for different wavelengths. Pulses of 575 nm trigger more action potentials,

FIG. 3. Number of spikes elicited in a few unit preparation by equal intensity pulses of different wavelengths and recorded at the eyestalk and the brain during the first 100 msec after the onset of the corresponding ERG. A. Number of spikes recorded at the eyestalk. Note that spikes reach a maximum number with pulses of 550 nm. B. Number of spikes recorded at the brain. Note that the number is maximal with pulses of 575 nm. In both cases the number of spikes is smaller for shorter and longer wavelengths than for the optimal ones.

DISCUSSION

though

100

As initially shown by Bullock (2), spontaneous electrical activity from invertebrate brains (apparently with the only

msec, they appear at irregular intervals, do not seem to be time-locked with the stimulus, and are smeared out in averaged records such as those of Figure 1. Thus, we are co&dent that we have included in the count at least most of the appropriate spikes. Another characteristic of the trains of spikes provides a basis for proposing a mechanism responsible for analyzing

with a small component of slow-frequency signals. This pattern is different from the electrical activity of the vertebrate brain, although some attempts have been made to relate specific frequency components of the invertebrate records to known vertebrate EEG waves (18). Similar slow waves were seen in insect optic lobes long ago (17,30).

there are still some spikes after those initial

these signals in the central nervous system. In Figure 4A we have plotted the intervals between spikes elicited by pulses of 475, 575 and 675 nm, versus the order in which they arrive to the area of the recording electrode. The tirst spike of all trains arrives about 40 msec after the beginning of the corresponding ERG, the second one slightly later,

approximately 35 in the arbitrary first 100 msec period after the onset of the corresponding ERG, while longer or shorter wavelengths trigger fewer spikes, 26 at 675 nm and 20 at 475 nm. Other experiments performed under similar conditions lead to similar results, although slopes varied somewhat.

exception of cephalopods)

Visual

Evoked Potentials

(4) IScomprised mainly of spikes,

(VIPs)

The electrical events recorded from the crayfish brain in response to stimulation of eye photoreceptors with pulses of light have their counterpart in the event-related poten-

Visual Evoked Activity

in Crayfish

CNS

215

A

FIG. 4. Plot of the latency of the spikes recorded from a certain few unit preparation during the fu-st 100 msec after the onset of the ERG corresponding to the appropriate wavelength. A. Latency of spikes recorded at the eyestalk. The relationship is approximately linear for the first 13 spikes, with a clear change in slope after that. B. Latency of spikes recorded at the brain. The relationship has a nearly linear slope for the first 16 spikes elicited with pulses of 675 nm, but it is linear for only the first 8 spikes elicited with pulses of 475 and 575 nm. After these 8 spikes, the changes in slope are in different directions for spikes elicited with 475 and 575 nm. Note also that the total number of spikes is about the same (23) in the first 100 msec for records from the visual path (Part A), but different in the records from the brain (20 for pulses of 475 nm, 26 for 675 nm, and 36 for 575 nm; Part

-

120

B

lco!

575

2

Bo-

E ICI (P

60.

nm

40:

0

5

10

20

15

Spike

475

30

25

number

PI

loo-

675

IIP

.

I. 30

575

Em

SO80706050-

B).

4030-

,

201 0

.,.,.I., 5

. 10

15

Spike tials of vertebrates (5). However, although these recordings are not surprising, the VEPs from the crayfish brain are mainly comprised of relatively high-frequency signals with some components

of slow frequencies.

On the other hand,

20

, 25

I 35

1 40

number

per, as we have not stimulated the eye with pulses of a similar wavelength at different intensities. What we have demonstrated here is that pulses of different wavelengths are able to stimulate different photoreceptor combinations and

the vertebrate event-related potentials have no obvious spikes but only slow waves. In crayfish, these VEPs are a consequence of the arrival of the train of impulses initiated

that these generate trains of spikes. We have used these trains to locate the region along the visual path where inte-

at the eye photoreceptors, as we demonstrated with recordings from the eye, the visual path and the brain (Fig. lA-1C).

the eyestalk, at the level between the internal and external medulla (see below). Trevino and Larimer (23) stimulated the crayfish eye with pulses of monochromatic light and recorded the firing of sustaining neurons in response to input from blue-sensitive and yellow-sensitive photoreceptors. The authors suggested that crayfish have the capacity todiscriminate between colors, although they indicated that behavioral evidence is lacking.

Color Vision The question of whether or not crayfish has color vision has not been addressed by the experiments reported in this pa-

gration might take place, and have found that it starts in

J. Serrato et al.

216

This suggestion was supported by the finding that the crayfish eye contains two color pigments (2 1).

and the recording point at the eyestalk, the original activity has encountered some internal activity; therefore, and ac-

Our results show that light pulses of all wavelengths in the visible spectrum stimulate the crayfish eye photorecep-

cording to the definition used, this would be a region at which integration is taking place. A similar conclusion in

tors and generate electrical signals that travel to the brain. Thus, we can reach a conclusion similar to that of Trevino and Larimer (23).

relay station, but also as an integrational reached by other authors (24,25,27).

Integration

in the trains of spikes. Figure 4B shows that spikes elicited with pulses of 675 nm have similar intervals through the

relation to the capacity of the eyestalk to act not only as a site, has been

As the spikes reach the brain, there are further changes by Neural Structures

It is tempting to speculate that the initial 13 spikes seen at the visual path after the light pulse are due to firing of the sustaining

neurons described by Wiersma

and Yamaguchi

13th-16th

spike, and can be fitted with a straight line. On

the contrary, in the cases of the spikes elicited with pulses of 475 and 575 nm, only the first eight spikes can be fitted

(28). These neurons receive input from first-order visual interneurons and their rate of firing is a linear function of the logarithm of the light intensity (15). At the intensities used

with a straight line. After that the spike interval changes, resulting in an irregular slope.

for these studies the logarithm should be at least 2, a value

is a group of about 6-8 spikes that travels unaltered from the eye to the brain, while others are the result of, or encounter,

at which the sustaining

neurons should fire continuously

The data shown in Figures 4A and B suggest that there

during the 15 msec that the light pulse lasts. Therefore,

it

internal activity. Therefore,

is very likely that these spikes are due to the activation sustaining neurons.

of

taking place both at the visual path and at the brain.

It is known that the output of sustaining neurons feeds the command neurons to elicit the crayfish withdrawal response (19); therefore, integration of spikes coming from these neurons at different levels, the visual path and the brain, would be expected.

Other visual neurons that could

be stimulated by light are fewer; for instance, only 4 or 5 types of neurons detect movement (28). A further argument to support the idea that the trains of spikes are due to firing of sustaining neurons is that the spectral sensitivity curve of neuron O-38 in the dark-adapted state peaks at 575 nm, and neuron O-l at 570 nm (23,28), which is also where we obtained the maximum effects. Thus, it is likely that the first few spikes traveling along the visual path and arriving at the brain after a pulse of light are due to firing of sustaining neurons. In this work we use the term integration as used by Bullock (3) to describe neurons that do not act as one-to-one relays, and defined explicitly by Wiersma and Roach (29): “the interaction of sensory stimuli and internal processes.” However, Bullock (5) provides a description of several other

“integration”

would seem to be

Perhaps the most significant finding from these experiments is that the curves relating the spike interval to their number

are not equal for light pulses of different

wave-

length. That is, spikes generated by stimulating the eye photoreceptors are recorded at the brain at intervals that are different for each wavelength used (Fig. 4B). Unless the eye is monochromatic,

with a different function of effective in-

tensity-to-wavelength arities with intensity, ated by different

than our photocell, and has nonlinethis result suggests that spikes gener-

wavelengths

travel

different

neuronal

circuits in the brain, and that these are part of the integrating mechanisms used to analyze them. Another possibility could be that the obviously greater effectiveness of some wavelength than others only begins to influence intervals after an initial burst. Central processing of gustatory signals has been explained by two hypotheses. The “labeled-line,” which assumes that identifiable groups of neurons code the sensory quality with their activity (a temporal pattern), while an alternative hyassumes that sensory pothesis, L‘across-neuron-pattem,”

displayed by neurons and

quality is coded by the differential magnitudes of response across many neurons (a spatial pattern) (3 1). In our experi-

From this point of view, the train of spikes generated by

ments, we did not identify the discharge of individual neurons and therefore cannot obtain across-neuron correla-

stimulation of the eye photoreceptors travels along the visual pathway towards the brain, and the constancy in the number of spikes (about 23) in the fu-st 100 msec for each wavelength suggests that they are generated by equivalent combinations of photoreceptors. However, it is worth noticing that it is only the first 13 spikes that produce a straight line in the plot of Figure 4A, with a change in slope from then onwards. Again, this is similar for all light wavelengths.

tions; however, our results seem compatible with interpretation by the labeled-line hypothesis. The possibility that crustacean nervous system analyses sensory signals with a mechanism such as labeled-lines could have as an anatomical basis the small number of cells dedicated to these tasks. The crayfish has only about 70,000 neurons in the brain (25); thus, it may be that labeled-lines are simpler to handle than, for example, hierarchical levels, and it would not be a surprise to find that in invertebrates

A possible reason for the change in slope at about the 13th spike is that between the generating point at the eye

all sensory signals are analyzed by such a mechanism. In summary, the activity generated at the crayfish eye

possible integrating neural networks.

mechanisms

Visual Evoked Activity

photoreceptors

in Crayfish

CNS

by pulses of different

trains of spikes that travel afferently

217

wavelengths

elicits

and can be recorded

at the visual path and the brain. “Integration” seems to take place in the path as well as in the brain. At the brain, trains of spikes behave differently depending upon the pulse wavelength, and therefore upon the photoreceptor combination stimulated. These results suggest that different neuronal circuits analyze the activity coming from different photoreceptor combinations, and support an interpretation based on the labeled-line hypothesis. The authors would like to exbress their abbreciation to Prof. T. H. Bullock for continuous encourigement thro;ghout this work, k well as for his helpful comments on the original version of this manuscript. S. Eknes provided expert help with the experiments during the initial stages of this project.

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