- Email: [email protected]
Evoked Potentials Elicited by Natural Stimuli in the Brain of Unanesthetized Crayfish
Physiology & Behavior, Vol. 66, No. 3, pp. 397–407, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99/$–see front matter
PII S0031-9384(98)00279-0
Evoked Potentials Elicited by Natural Stimuli in the Brain of Unanesthetized Crayfish JESÚS HERNÁNDEZ–FALCÓN,*1 JESÚS SERRATO,† AND FIDEL RAMÓN‡ *Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City, México, †Depto. de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, México, D.F. 07000, Mexico City, Mexico, and ‡División de Estudios de Posgrado e Investigación, Facultad de Medicina,Universidad Nacional Autónoma de México. Ciudad Universitaria México, Mexico City, Mexico Received 29 August 1997; Accepted 1 October 1998 HERNÁNDEZ–FALCÓN, J., J. SERRATO AND F. RAMÓN. Evoked potentials elicited by natural stimuli in the brain of unanesthetized crayfish. PHYSIOL BEHAV 66(3) 397–407, 1999.—Experiments were conducted to test some characteristics of vision by crayfish underwater and in air, and determine possible motion reactions elicited in response to naturalistic or quasi-ethological visual stimuli. Chronically implanted electrodes on the brain were used to record visually evoked potentials in response to moving bars at different speeds or to fish of different sizes. Electroretinograms were also recorded to detect when an object or a shadow appeared in the crayfish visual field. Ongoing brain activity is mild under basal conditions, but increases in RMS by z6% in response to bar passage and 12 to 53% in response to fish motionless or swimming in front of the crayfish. When crayfish are free to move, fish swimming in front of them elicit intense brain activity, together with displacement toward them and an attempt to grab them. Visual evoked potentials are elicited by moving objects as small as 18 at a distance of 30 cm in air as well as underwater. None of the stimuli used induced evident behavioral responses under our conditions. We conclude that vision-action activities can be divided into (a) vision of irrelevant objects with short lasting electrical activity and no motion in response to it; (b) vision of mildly interesting objects with long-lasting electrical effects, but no motion in response to it; and (c) vision of relevant objects with appropriate motion reaction. © 1999 Elsevier Science Inc. Invertebrate
Vision
Brain recording
Electroretinogram
shown by crayfish under laboratory conditions designed to stimulate characteristics of its underwater natural environment. Recording electrical activity from the crayfish brain in response to visual stimuli that seem relevant judging by behavioral responses, requires working with animals under nearly physiological conditions. To tackle this problem we developed a preparation of crayfish chronically implanted with electrodes in the brain for recording its electrical activity. We tested the visual system with light pulses of different wavelengths and determined the presence of evoked potentials at the brain. We found sites of integration [as defined in (24)] of visual signals at both eyestalk and brain. Furthermore, as pulses of light of different wavelength elicited electrical activity that seemed to follow different neural paths in the brain, we concluded that the integrating mechanism of these signals is compatible with postulates of the labeled line hypothesis (20).
STUDIES of crayfish optical apparatus as well as neural processing of visually evoked activity point to the presence of an elaborate visual sensory system; however, it is not known if this system is used to guide any specific behavior. In fact, studies on crayfish and lobsters point to olfactory and tactile sensory systems as most relevant for food searching, mating, and defense (3,16). Thus, our main interest in these studies is in the use of crayfish vision to guide some behaviors. To this end we have developed a preparation for chronic recording of brain multiunit activity from unanesthetized animals. In previous studies we described characteristics of spontaneous activity and signals elicited by light pulses of different wavelengths. In this article we deal with visual signals evoked by the presence of more natural stimuli than light flashes, such as bars, shadows, and fish, and motion responses elicited by them. The purpose of this work is to determine vision-action patterns
1To
Multiunit activity
whom requests for reprints should be addressed. E-mail: [email protected]
397
398
HERNÁNDEZ–FALCÓN, SERRATO AND RAMÓN
However, although these studies illuminate some neuronal circuits used to codify signals, they do not throw light into relationships between vision and resultant motion. To this end, in this article we describe experiments designed to gain insight into characteristics of crayfish vision in air and underwater, and stimuli that might elicit appropriate motion reactions. Stimuli used were objects, shadows, and animals moving at different speeds on clear backgrounds. Similar to results obtained by recordings from the optic nerve (1,23), spikes at the brain are elicited preferentially when an object or a shadow appears in the visual field or changes speed. However, some visual stimuli elicit behavioral responses that can be better described as of “attention,” and none elicit tail-flip escape responses. Unexpectedly, barely detectable visually evoked potentials are elicited by objects as small as 18 at a distance of 30 cm both in air or underwater, suggesting that the crayfish either has the same visual detecting threshold in both media, or is reacting to motion of an effectively point source. However, of stimuli used, only swimming fish elicit a locomotive response. MATERIALS AND METHODS
Animals We used crayfish Procambarus clarkii 8–10 cm in length and of either sex obtained from a local provider. Animals were maintained for 2–3 months in a large aquarium under natural light–dark cycles, with well-aerated water at room temperature and fed two to three times a week. Preparations Crayfish were implanted with a chronic electrode for recording multiunit brain activity as described in a previous work (20). Briefly, the procedure is as follows: animals are cold anesthetized, and a hole made in the dorsal carapace at the level of the junction between head and thorax. The brain becomes accessible after separating muscles and internal organs, mainly stomach and green gland, and electrodes (see below) are introduced and positioned on the dorsal surface of the brain. The electrode array is cemented to the carapace, and the animal returned to the tank and left undisturbed for 1–2 days. Experiments were performed in a specifically designed aquarium (Fig. 1). For recording with crayfish eyes in air animals were tethered in the larger aquarium facing the glass wall of the small aquarium, and water level was lowered. Additionally, just before experiments we placed wick electrodes on the corneal surface of one or both eyes without blocking their vision to record electroretinograms (ERG), which were used to indicate when the crayfish was exposed to the shadows or objects. For experiments recording brain activity underwater, crayfish were placed in the V-shaped chamber of the aquarium, which limited their movements when the glass partition was in place. When the partition was removed, crayfish could move into the rectangular chamber where fish were swimming. For these experiments the wick electrodes were modified as described below. In some experiments we used a manually controlled switch to time the bar entering the crayfish visual field. This signal was also sampled by a computer, making it easier to locate the wave on the ERG produced by objects tested. Stimuli To stimulate with flashes of light we used a Photic Stimulator (Model PS33-Plus; Grass Inst. Co., Quincy, MA) that trig-
FIG. 1. Diagram of the experimental glass aquaria. (A) Top view of the small aquarium with two compartments that can be separated by a removable glass partition (dotted line). The “V”-shaped compartment was used to place the crayfish and the rectangular one for fish. (B) Side view of the small aquarium placed inside a larger one (shown here as a separate compartment at the left). Water levels in both aquaria were controlled independently.
gers flashes of 10-ms duration and variable intensity. Unless stated otherwise, we used a medium setting and manually triggered single flashes. Other stimuli were bars of various widths (2–10 cm) made from black cardboard or rubber. Bars were hung from a cord pulled through a set of pulleys by a low-velocity motor, such that they passed in front of the crayfish every 1–2 min at a speed of 15 cm/s and a distance of 30 cm. Segments of the cord between pulleys moved horizontally or vertically, and to study the effects of bars moving in these directions the animal was placed in front of appropriate segments. Occasionally, the visual field was restricted with black paper to a window, horizontally about 1358 and vertically to 458. We visually stimulated animals underwater with black rubber vertical bars moving horizontally at 10 cm from crayfish in the rectangular compartment of the aquarium. In this case we did not search for responses to bars moving vertically. We also used live fish of different sizes and colors that, when standing still, were encouraged to move by lightly tapping them with a long thin glass pipette. They were visible to the crayfish at a distance of 8 cm on average. Background light was 0.3–1.0 lx, as measured with a Light Meter (Li-COR, Inc Model LI-1189; Lincoln, Nebraska; probe Model Photometric).
ELECTRICAL ACTIVITY FROM THE CRAYFISH BRAIN Recording and Electrodes During recording sessions an implanted animal was tethered in a water tank. Electrodes for recording multiunit brain activity were made as previously described (12). Briefly, electrodes were a grounded stainless steel cannula containing four Teflon-coated silver wires (100-mm diameter) secured with dental cement; spacing between wires was 100 mm. Electrode position on the brain optic lobe was determined in postmortem observations as small depressions on the brain surface, and did not produce macroscopic damage. Because experiments were performed on chronic animals, we did not want to damage their cornea by superficially introducing small stainless steel needless to record ERGs. Therefore, when crayfish were in air we used small wick electrodes, made with a silver/silver-chloride wire and a narrow cotton thread in contact with the dorsal part of the eye. This electrode was frequently moistened with saline solution. It did not obstruct the field of view. For experiments with animals underwater, wick electrodes were modified as follows. A glass capillary tube was filled with electrode cream (EC2; Grass Inst. Co.) and a small drop applied to the dorsal surface of the eye. A silver wire was immersed in the electrode cream in the tube. This electrode was useful for about 30 min, after which the cream dissolved and we had to make a new electrode. Data Acquisition and Analysis Recordings from electrodes were amplified with AC amplifiers (P511, Grass Inst. Co.) and filtered to pass between 1 Hz and 3 KHz; a 60 Hz band-reject filter was occasionally used. The output of amplifiers was displayed on an oscilloscope (Gould, Model 1604) and 5-s samples were digitized, usually at 8 KHz, with A/D converters (BIOPAC Systems, WPI) and stored in a computer (Power Macintosh 711000/ 666AV) for off-line analysis. Digitized traces were analyzed with Igor (Stapleton Software, Eugene, OR). RESULTS
Crayfish Eyes in Air Experimental procedure. For experiments with crayfish eyes in air we proceeded through the following steps: 1) claws were immobilized with a rubber band and animals tethered at the border of the experimental tank, with eyes slightly above water level; 2) with a micromanipulator we placed wick electrodes on the upper surface of the cornea of both eyes; 3) implanted electrodes were tested for the level of spontaneous brain activity they recorded, and the one showing the largest spikes and the most per unit time was selected; 4) a light pulse from a flash lamp about 20 cm over the animal was triggered and recordings displayed on the oscilloscope. If necessary, wick electrodes were adjusted and/or recording electrode changed until flashes obtained the largest response in both electrodes; 5) the animal was left undisturbed in the tank for 15–30 min in a room with dim illumination. After the crayfish is tethered in the tank there is a period of 10–15 min during which it rhythmically moves pleopods and pereiopods. After this period animal remains quite most of the time, with occasional bursts of activity that last 15–30 s. Records were obtained when the animal was motionless. Spontaneous multiunit activity. As previously described (12), spontaneous multiunit activity recorded by a macroelectrode on the crayfish brain is mainly comprised of numerous spikes
399
of different amplitude and morphology. Spikes appear continuously, but their frequency per unit time is slightly variable. Power spectra of 6-s segments of records show maximum power at about 400 Hz, decreasing to noise level above 2 KHz. Spike histograms show that higher frequency spikes are small (5–10 mV), while spikes of large amplitude (40–50 mV) are seldom seen. Electroretinogram (ERG). As some visual stimuli elicited brain responses that were barely discernible above the spontaneous activity, we needed to determine when objects entering the crayfish visual field were “seen.” Thus, we used as monitor a wave produced in the ERG when objects enter the visual field. Figure 2C shows the characteristics of the ERG in response to a flash of light of medium intensity (225 lx). After a stimulus artifact there is a brief positive wave followed by a slower negative one, which in some records overshoots the zero line after recovery. These are typical H-I and H-II components described from recordings with needle electrodes (18). Figure 2A plots the absolute amplitude of those two components of the ERG during flashes of light of variable intensity. Both components increase approximately linearly in amplitude as flash intensity increases. Similarity in morphology between ERGs recorded with wick electrodes (Fig. 2C) and those recorded with needle electrodes (18) suggests that wick electrodes faithfully record photoreceptor activity elicited by flashes of light. Thus, we use them to record signals due to introduction of objects in the crayfish visual field. Passage of a black bar (22.5 cm wide, 10 cm long) in both horizontal directions in front of a crayfish elicits reproducible waves in the ERG. Figure 3A shows four wave complexes (vertical lines) elicited in response to a black bar passing four times in front of a crayfish. ERGs are similar to those described previously; a brief positive-going wave (H-II) followed by a larger negative-going (H-I). During each H-I wave an electrode placed on the brain (Fig. 3B) recorded few large spikes, more noticeable during the first and fourth passage. Integrated brain activity (Fig. 3C) more clearly shows the increase in electrical brain activity associated with the H-I ERG wave. Evoked activity. We recorded multiunit brain activity elicited in response to black bars of different widths moving in air across a white background in front of crayfish. Bars did not cast shadows on the animal, which was maintained under constant illumination. Figure 4 shows records obtained from an experiment in which blacks bars of different widths passed at the same speed (15 cm/s) at a distance of 30 cm in front of tethered crayfish. A black bar 1.2 cm wide (Fig. 4B) passing in front of the animal elicits a discharge comprised of spikes of different amplitude lasting about 35 ms. Similar discharges are seen during passage of black bars of 2.5, 5, 7.5, and 10 cm width (Fig. 4C, D, E, and F), although discharge duration is longer for wide bars. Discharges seem to have intermediate periods of decreased activity, more noticeable in records 4B and C. Record 4A is a control, obtained while moving cord and pulleys without the bar. In general, records in Fig. 4 show that in response to a black bar entering a crayfish visual field there is an initial train of spikes, followed by an increased activity period roughly related to bar width. A precise correlation between spikes and bar borders cannot be done with these records; however, increased brain activity related to bar width can be demonstrated by comparing Vrms values of records shown in Fig. 4 (Table 1). Vrms was calculated with Igor for 6-s segments starting at stimulus onset.
400
HERNÁNDEZ–FALCÓN, SERRATO AND RAMÓN
FIG. 2. Electroretinogram of crayfish. (A) Stimulus response relationships for ERG components H-I and H-II (absolute values) in air. Stimuli of increasing intensity (from 122 to 1800 lx) were applied at 2-min intervals. (B) Stimulus response relationships for ERG components H-I and H-II (absolute values) underwater from the same animal as in A. (C) Typical ERG recorded in response to a light flash (z2225 lx, 10 ms); H-I and H-II components are labeled.
White bars of similar widths passing in front of an animal at the same speed as black ones described above did not elicit spike discharges at the brain (not shown). With our illumination they were about the same brightness as the background. In other experiments we moved square pieces of black cardboard of various sizes in front of crayfish at 15 cm/s. Figure 5A shows that a 2-cm black square elicits a bimodal discharge of spikes (circles), similar to those described above. The time between peaks corresponds to the time for the two edges of the card to pass by. A 1-cm square elicits only a single obvious spike of large amplitude (Fig. 5B). Similar squares made with white cardboard and moving at the same speed as black ones did not elicit any response. In other experiments (Fig. 6A) we worked with shadows covering entire crayfish. Black bars (20 3 28 cm) were moved in front of a light source such that they cast shadows on entire crayfish. When a shadow moved rapidly (25 cm/s) we recorded two prominent discharges lasting about 40 and 50 ms, separated by a period of about 140 ms during which there was less activity. In contrast, slow movement (5 cm/s) of the same shadow elicited two spike discharges (125 and 135 ms, respectively) separated by about 1.4 s. In both cases spike discharges have similar characteristics to those seen in response to moving black bars; that is, two groups of spikes separated by a silent period, which is seen better when movement was slow. Shadows moving in either direction produced similar spike discharges. In a few experiments (Fig. 6B) fast shadow movement elicited a discharge (170 ms) followed by several seconds of in-
creased activity with occasional large spikes. This activity was not seen after a slowly moving shadow, nor was it correlated with movement of the animal. Although these results clearly show that crayfish are able to detect movement of black objects as small as 18 at a speed of about 208/s, experiments were done with animals tethered with eyes in air slightly above the water level. Thus, it was of interest to determine also the crayfish visual sensitivity in water, its natural habitat. Crayfish Underwater Spontaneous multiunit activity. Records of spontaneous multiunit brain activity show numerous spikes both isolated and in short bursts, with a small component of slow frequency waves. These characteristics are similar to those seen in similar records obtained while crayfish was in air (compare Figs. 4A and 7A). Electroretinogram. To record ERG underwater we tethered crayfish and slightly reduced eye movements with a small piece of cotton placed through a glass partition in front of the crayfish from a lamp in air at a distance of 15 cm. Recorded ERG H-I and H-II components are similar to those seen in air, but their amplitude is substantially reduced. Similarly to results obtained in air, stimulus–response relationships are linear (Fig. 2B). Evoked activity. We performed experiments placing bars or fish of different sizes in the rectangular compartment of the aquarium described in the Methods section. Initially we com-
ELECTRICAL ACTIVITY FROM THE CRAYFISH BRAIN
401
FIG. 3. ERG, multiunit brain activity, and integral of this activity during two horizontal passages of a black bar in front of crayfish in air. Left and right electroretinograms (A) were recorded with wick electrodes simultaneously with multiunit brain activity (B). Vertical lines are drawn at the peaks of the integral of recorded brain activity (C) synchronous with the H-I component of the ERG; this helps to localize the resulting spike discharge.
pared electrical activity recorded by four electrodes on the brain in response to passage of a black bar in front of crayfish tethered underwater. Passage of a black bar in front of a crayfish elicits spike discharges in all four channels, with characteristics similar to those seen when crayfish eyes were in air. That is, discharges are comprised of two groups of spikes, separated by a period of less activity. Number of spikes and discharge duration are rather similar in all four channels (not shown), and also to those described above. Next, we used as stimulus fish swimming in front of crayfish. Initially, we recorded spontaneous electrical activity from the brain of crayfish facing the tank without fish. However, as records from all four implanted electrodes are similar, here we show only channel 4. Thus, Fig. 7A shows that spontaneous activity recorded on the brain has characteristics similar to those seen when crayfish eyes were in air, and shown above
and elsewhere (12). Then we introduced fish of different sizes in the tank in front of crayfish. Figure 7C contains a record obtained when a gold fish (about 6 3 2 cm, side view) moved in front of a crayfish at a distance of about 8 cm. Record shows a large activity mostly comprised of small spikes, but numerous large spikes isolated and in short bursts can be also seen. This large activity is due to fish moving in front of the crayfish, as it can be ascertained by noticing that when fish remain motionless (Fig. 7B), the number of spikes decreases; nevertheless, even in this case there is a large number of small amplitude spikes. We also introduced in the tank fish of slightly larger size (about 7.5 3 2.5 cm, side view). In this case (Fig. 7D) spikes recorded from the crayfish brain are even more numerous and larger than those recorded with the previous smaller fish. Largest spikes seem to occur only when the fish is changing
402
HERNÁNDEZ–FALCÓN, SERRATO AND RAMÓN
FIG. 4. Multiunit brain activity in response to horizontal passage of black bars of different widths. Control records moving cord and pulleys without bar (A) were followed by the passage of bars 1.2 (B), 2.5 (C), 5.0 (D), 7.5 (E), and 10 (F) cm wide. A spike discharge is associated with entrance of the leading edge into the crayfish visual field.
position, although continuous movement of dorsal and lateral fins elicits many spikes of different amplitudes. During all these recordings the only visible sign that the crayfish had seen a fish was an “attentive” position that it maintained during the whole time it faced the tank with fish. As multiunit activity evoked in response to crayfish facing fish appears as single large spikes or in short bursts, to compare records shown in Fig. 7 we calculated their Vrms, and Table 2 shows those values. Vrms values are smallest during spontaneous activity and increase during the passage of black
bars or facing fish. Values of records obtained when crayfish face a motionless small fish (Fig. 7B) are slightly larger than those due to a bar passing (Fig. 6), but both are smaller than those obtained when the same fish (Fig. 7C) or a slightly larger one (Fig. 7D) swims. Responses to black bars moving across the crayfish visual field were elicited almost independently of the background level of illumination, within the range 11–2000 lx (24). DISCUSSION
Electroretinogram (ERG) TABLE 1 CRAYFISH BRAIN ELECTRICAL ACTIVITY FIG. 4 Record
A B C D E F
Bar Width (cm)
Vrms (MV)
Response
control 1.2 2.5 5.0 7.5 10.0
9.2389 9.5995 9.7917 9.8109 10.2677 10.7426
— 0.3606 0.5528 0.5720 1.0288 1.5037
Bar widths and Vrms values for 6-s segments of records shown in Fig. 4. Values in response column were obtained by subtracting control Vrms from each experimental value.
In response to flashes of light, electroretinograms recorded with needle electrodes in the corneal surface of dark adapted crayfish eyes showed two components, H-I and H-II (18). After penetrating the cornea, while in the crystalline cone layer, the ERG shows a negative peak (H-I) followed by a slow plateau (H-II). The H-I component amplitude depends on light intensity, while the amplitude and duration of H-II depend on the duration of the light stimulus. In response to a flash of light, a wick electrode records ERG components (H-I and H-II) similar to those seen after penetrating the cornea and similar, inverted, components can be also recorded during the passage of a black bar in front of crayfish. This indicates that photoreceptors detect both big changes in light intensity produced by a flash of light and small changes produced by the bar passage, activating in both
ELECTRICAL ACTIVITY FROM THE CRAYFISH BRAIN
403
FIG. 5. Multiunit brain activity elicited in response to the horizontal passage of 2-cm square (A) or 1-cm square (B) black cardboard at 30 cm distance. Two brief discharges are elicited by the large square and only one obvious extra spike in response to the small one (circles).
cases afferent fibers. The small contrast produced by the bar in some experiments suggests a great visual sensitivity. Brain Recordings Recordings of electrical activity form the brain of invertebrates have been mainly acute (4,7,17), but there are a few from chronically implanted animals, such as crayfish (1), octopus and cuttlefish (2,5,6,8), and horseshoe crab (9). An aim of this work is to find out if whether the technique of implanting electrodes on the brain and recording in the intact, behaving animal still permits responses to naturalistic or quasi-ethological visual stimuli to be observed and replicated over useful length of time. A second aim is to ask how small a physiolocigal stimulus can evoke a visible response, what features such as movement, are more effective and how such tests are comparable for vision under water vs. that in air. We show that in the absence of any overt behavioral response, one can record objective, central neuronal responses that at the least manifest detection or perception. Activity evoked at the brain by visual stimuli is mainly comprised of fast spikes with a few low-amplitude, low-frequency signals. Although some spikes coming from muscles located under the brain are picked up by our electrodes, they can be easily identified by their slow frequency components and filtered out [see also (12)]. Spikes shown in our records are only of neural origin. In most experiments several types of units might be excited simultaneously and contribute to the overall activity recorded
at the brain. Furthermore, as we placed animals facing a white screen over which black bars were moving, we were stimulating homo-, hetero-, and bilateral visual interneurons, and no effort was made to separate their contribution. Thus, increased brain activity in our records seems mainly due to visual fibers. Visual Fibers Characteristics of purely visual fibers to the brain were reported by Wiersma and Yamaguchi (22), resulting in a classification based on the type of stimulus to which they responded best, into sustaining, dimming, and movement units. Only a small number of each type of fibers was isolated, 14 sustaining, 17 dimming, and 13 movement units. A usual response of sustaining fibers (SuFs) to illumination is a tonic discharge with a latency of 20–40 ms and, although it starts with a burst of about 300 spikes per second, it stops as soon as light is turned off. Illumination on the retina outside the SuFs excitatory field inhibits a discharge. However, SuFs have also been shown to burst after shadows at a frequency of 4–10 spikes per second, without signs of adaptation (24). Nonetheless, SuFs characteristic of constant discharge in the presence of light makes us suspect that most spikes recorded at the brain by our electrodes do not come from them. As opposed to SuFs, movement fibers, in particular of jittery type (JMFs), have firing characteristics that make them likely to be contributing largely to spikes recorded at the
404
HERNÁNDEZ–FALCÓN, SERRATO AND RAMÓN
FIG. 6. Multiunit brain activity elicited in response to horizontal passage of a black bar (20 3 28 cm), which cast shadows covering the entire crayfish, at two speeds—fast and slow. (A) In both cases there are two spike discharges, but time interval is greater during slow than fast passage. (B) In some experiments fast shadow passage elicited a long-lasting discharge. Two white horizontal lines are drawn to facilitate comparison of spike activity level before and after stimulus.
ELECTRICAL ACTIVITY FROM THE CRAYFISH BRAIN
405
FIG. 7. Multiunit brain activity elicited in response to several stimuli. Only one of four recorded channels is shown. (A) Spontaneous activity. (B) A goldfish (z6 3 2 cm, side view; 8 cm from the crayfish eyes) motionless stimulates isolated but frequent spikes or short bursts. (C) Same fish swimming movements elicit bursts of spikes. (D) A burst of large spikes was seen whenever a fish (7. 5 3 2.5 cm side view) moved in a separate compartment visible to the crayfish.
brain. For example, JMFs react strongly to dark edges entering their field with a mean response frequency that is a linear function of edge velocity, from 18/s to 358/s (10). Furthermore, in response to dark edges moving across their visual field there is an inhibitory wave that seems to precede the moving edge, and such inhibition is apparent in some of our records (i.e., Fig. 6A). In particular, they react with a strong discharge when an object moves in a jittery fashion within their visual field, and this is how one would describe movement of a fish in water. Fish movements, in particular those of fins and tail, stop and start at every stroke, so that we would expect no adaptation of JMFs.
ment across the whole field will not be seen by these fibers. A response at the end of the bar passage could be due to sustaining fibers, as these respond to this type of stimulus. Although crayfish do not seem to have fibers (medium and fast movement) that react to approaching dark objects, occasionally we recorded brief spike discharges in response to this type of stimulus. It is possible that in these cases we were stimulating both jittery and sustained fibers, or higher order
TABLE 2 CRAYFISH BRAIN ELECTRICAL ACTIVITY UNDERWATER
Responses to Movement of Black Bars and Shadows As jittery movement fibers show a pronounced refractory period, stimuli were presented with a frequency of less than one every 2 min. These presentations were kept usually at a total of less than 10, as more produce a profound and long lasting habituation (10,11). In our experiments black bars moved across a crayfish visual field at a uniform velocity, and the inhibitory response that travels in front of the response of jittery movement fibers was not seen in our records, most likely because of large background activity. We expect that jittery movement fibers react with a single adapting burst as a bar enters the field and move-
Ch1 Ch2 Ch3 Ch4
Spont. Act
Black Bar
Small Fish (Motionless)
Small Fish (Moving)
Big Fish (Moving)
0 0 0 0
0.7385 0.5952 0.5612 0.6704
1.2155 0.9866 0.9936 1.4217
2.3760 1.8157 1.8729 1.9861
5.1010 4.9046 5.0664 5.5580
Change in Vrms values for 6-s segments of records obtained during experiments underwater. Values for spontaneous activity were subtracted from each experimental Vrms value.
406 neurons not isolated previously (23). Other fibers studied in lobsters and crabs, such as “light,” undirectional,” and “seeing” types, have not been found or have been little studied in crayfish (24); thus, we cannot invoke them to explain our findings. In all records shown here it is clear that there is a spike discharge as the leading edge of a bar enters a crayfish visual field, and occasionally again in response to the trailing edge; however, activity during passage of the bar itself is very small. This suggests that jittery movement units rather than sustained fibers are being activated, as with the latter one expects a prolonged response. This conclusion is supported by large responses elicited during passage of a swimming fish in front of crayfish, a condition that would produce maximal activation of jittery fibers due to apparently very irregular movements of fish fins. The fact that shadows are more effective than bars in eliciting brain spike responses supports this conclusion. Thus, in general, data shown here support the viewpoint that crustaceans see mainly movement as opposed to objects (13). Vision Depth of water in the fish tank was about 9 cm, and because fish measured 2 to 6 cm in a vertical axis, displacement of fish as stimuli is mostly in the horizontal direction. Thus, most spikes evoked by fish movement correspond to activation of units sensitive to horizontal movement. It is also apparent that an equal number of spikes is evoked by fish moving in either direction; however, our data do not allow us to say if the same movement units are being activated or if there are different fibers sensing each direction. Controls taken when fish were not displacing but only moving their fins show that this is not a very effective stimulus, as few spikes were produced by this movement. However, when fish change position, the stimulus was large and numerous spikes were elicited. Many stimuli produce photoreceptor activation and input signals to the brain, where some of them might be integrated while some others continue through the circumesophageal connectives. If electrical activity evoked at the brain is taken as vision, crayfish is able to see moving objects placed in its visual field, as well as all wavelengths in the mammalian visual spectrum (14,20). However, as shown above, many objects in the crayfish visual field evoke activity at the brain without a motion reaction. Tethered crayfish react with evoked potentials to fish swimming in their visual field, with no attempt to free themselves or to move. Thus, it seems that fish, as used in our experiments, are not adequate stimuli to trigger specific behaviors in the situation and motivational state tested. It is possible that tethering or some other aspect of our setup alters crayfish behavior. On the contrary, when crayfish are free, they slowly walk towards fish, and if these are cornered, attempt to grab them. Although we used fish of different colors, we did not observe differences in associated brain recordings. This could indicate that movement detectors are not sensitive to colors, and that it is only “borders” that stimulate them. However, this is not strong evidence, because measured responses could easily miss color sensitivity, if present. Comparison of crayfish brain activity in air and underwater suggests that visual responses are equivalent in both environments, and raise a question about its adaptive utility in an almost exclusively aquatic animal. The crayfish visual system seem designed to gain sensitivity more than visual acuity. A flat cornea and radial plane mirrors orthogonally arranged (in
HERNÁNDEZ–FALCÓN, SERRATO AND RAMÓN the squared shaped crystalline cone) of the superposition eye (19,21) facilitate capture of impinging photons, a design where sensitivity is high and independent of the environment. In air, light dispersion is small due to a thin layer of water apposed on the corneal surface, and a small f number characteristic of superposition eyes (19) must produce a bright and blurred image, such that only dark edges passing through different fields of the eye might be detected and turn significant. Underwater light dispersion is large, but the system detects small changes in light intensity produced by moving dark objects (fish fins and tails), which give rise to random light fluctuations (9). These are integrated, inducing a behavioral response when the crayfish is free (an attempt to grab fish). We speculate that in air brain discharge is mostly due to whole activation of both retinas; in contrast, activity recorded underwater might reveal detection and/or integration of visual significant stimuli. Motion Responses Despite the variety of fish shapes and sizes that we have used, we have not seen crayfish try to escape or have sudden movements. This can be partly due to our tethering of animals, which has been shown to inhibit escape reflex (15), and which could also inhibit other types of movement. However, because in other experiments with animals free in water their reactions were always similar, that is, no motion or slow displacements towards fish, we can conclude that none of those visual stimuli were considered threatening or aroused obvious curiosity. When crayfish are free in the tank and a fish is cornered, crayfish try to grab them in a sudden movement with the chelas; however, crayfish unmyelinated nerve fibers are no match for myelinated ones, and fish always escape in our tanks. Occasionally our records show in all recording channels slow signals associated with crayfish movements and/or displacement. Similar slow potentials can appear in only one channel, and in this case their origin is not clear. Although a possibility exists of movement artifact in only one electrode, it is more likely that they are due to muscle movements near the electrode. In most of our records we could not detect slow waves such as those described in gastropods (7), possibly because we did not filter out the spikes. As described in the Results section, the motion response of crayfish to different visual stimuli applied was different. Thus, black bars did not elicit any visible movement from crayfish, even when it produced a brief but clear discharge response to both leading and trailing edge (Fig. 6) or lasted longer than the time it took the bar to pass (Fig. 6B). A similar lack of movement response was seen when crayfish faced small motionless or swimming fish (Fig. 7A and B); in this case, however, crayfish seemed to maintain a very “attentive” attitude and the spike discharge lasted longer than the time it took the fish to pass by. However, a clear motion response was obtained when a larger fish passed swimming in front of crayfish, as in this case there was a slow displacement toward the fish and, in the few occasions in which crayfish cornered the fish, it attempted to grab it. These different motion responses to visual stimuli suggest the presence of an “integrator circuit” with a high threshold that has to be overcome to trigger a motion response. It would seem as if a brief stimulus (i.e., the passage of a bar), even though intense (i.e., a light flash), is not able to raise brain activity level above this putative threshold. Furthermore, as even the presence of a small motionless or slowly swimming fish that elicits more spikes that a black bar does not trigger a
ELECTRICAL ACTIVITY FROM THE CRAYFISH BRAIN motion response, this suggests the need of not only high but also sustained brain activity. This threshold, however, is finally reached when the visual stimulus is as intense and sustained as that produced by a large fish swimming in front of crayfish. It should be interesting to localize a brain neuronal circuit with such characteristics.
407 ACKNOWLEDGEMENTS
The authors would like to express their appreciation to Prof. T. H. Bullock for continuous encouragement throughout this work, as well as for his helpful comments on the original version of this manuscript. The help of J. C. Gómez and C. Ramírez with illustrations is also acknowledged.
REFERENCES 1. Aréchiga, H.; Wiersma, C. A. G.: The effects of motor activity on the reactivity of single visual units in the crayfish. J. Neurobiol. 1:53–69; 1969. 2. Bleckmann, H.; Budelmann, B. U.; Bullock, T. H.: Peripheral and central nervous responses evoked by small water movements in a cephalopod. J. Comp. Physiol. 168:247–257; 1991. 3. Bovbjerg, R. V.: Dominance order in the crayfish Orconectes virilis (Hagen). Physiol. Zool. 26:173–178; 1956. 4. Bullock, T. H.: Problems in the comparative study of brain waves. Yale J. Biol. Med. 17:657–679; 1945. 5. Bullock, T. H.: Electrical signs of activity in assemblies of neurons: Compound field potentials as objects of study in their own right. Acta Morphol. Acad. Sci. Hung 31:39–62; 1983. 6. Bullock T. H.: Ongoing compound field potentials from octopus brain are labile and vertebrate-like. Electroencephalogr. Clin. Neurophysiol. 57:473–483; 1984. 7. Bullock, T. H.; Basar, E.: Comparison of ongoing compound field potentials in the brains of invertebrates and vertebrates. Brain Res. Rev. 13:57–75; 1988. 8. Bullock, T. H.; Budelman, B. U.: Sensory evoked potentials in unanesthetized unrestrained cuttlefish: A new preparation for brain physiology in cephalopods. J. Comp. Physiol. A 168:141– 150; 1991. 9. Dodge, F. A.; Porcello, D. M.; Dodge, S. A.; Kaplan, E.; Barlow, R. E.: Noise components in Limulus vision. Biol. Bull. 187:261– 262; 1994. 10. Glantz, R. M.: Habituation of the motion detectors of the crayfish optic nerve. J. Neurobiol. 5:489–510; 1974. 11. Glantz, R. M.: Defense reflex and motion detection responsiveness to approaching targets: The motion detector triggers to the defense reflex pathway. J. Comp. Physiol. 95:297–314; 1974. 12. Hernández, O. H.; Serrato, J.; Ramón, F.: Chronic recording of electrical activity from the brain of unrestrained crayfish: The basal, unstimulated activity. Comp. Biochem. Physiol. 114A:219– 226; 1996.
13. Horridge, G. A.: The evolution of visual processing and the construction of seeing systems. Proc. R. Soc. Lond. B 230:279–292; 1987. 14. Kennedy, D.; Bruno, M. S.: The spectral sensitivity of crayfish and lobster vision. J. Gen. Physiol. 44:1089–1101; 1961. 15. Krasne, F. B.; Wine, J. J.: Extrinsic modulation of crayfish escape behavior. J. Exp. Biol. 63:433–456; 1975. 16. Kravitz, E. A.; Beltz, B.; Glusman, S.; Goy, M.; Harris-Warrick, R.; Johnston, M.; Livingstone, M.; Schwartz, T.; Siwicki, K. K.: The well-modulated lobster: The roles of serotonin, octopamine, and proctolin in the lobster nervous system. In: Selverston, A., ed. Model neural networks and behavior. New York: Plenum Press; 1985: 339–360. 17. Maynard, D. M.: Electrical activity in the cockroach cerebrum. Nature 177:529–530; 1956. 18. Naka, K.; Kuwabara, M: Two components from the compound eye of the crayfish. J. Exp. Biol. 36:51–61; 1959. 19. Nilsson, D. E.: From cornea to retinal image in invertebrate eyes. Trends Neurosci. 13:55–64; 1990. 20. Serrato, J.; Hernández, O. H.; Ramón, F.: Integration of visual signals in the crayfish brain: Multiunit recordings in eyestalk and brain. Comp. Biochem. Physiol. 114A:211–217; 1996. 21. Vogt, K.: Die Spiegeloptik des Flusskrebsauges. J. Comp. Physiol. 135:1–19; 1980. 22. Wiersma, C. A. G.; Yamaguchi, T.: Integration of visually stimuli by the crayfish central nervous system. J. Exp. Biol. 47:409–431; 1967. 23. Wiersma, C. A. G.; Roach, J. L. M.: Principles of organization of invertebrate sensory systems. In: Brookhart, J. M.; Mountcastle, V. B., ed. Handbook of physiology. Section I: The nervous system, vol. 1, part 2. Bethesda, MD: American Physiological Society; 1977:1089–1135. 24. Wiersma, C. A. G.; Roach, J. L. M.; Glantz, R. M.: Neural integration in the optic system. In: Bliss, D., ed. The biology of crustacea, vol. 4. New York: Academic Press; 1982:1–31.
PII S0031-9384(98)00279-0
Evoked Potentials Elicited by Natural Stimuli in the Brain of Unanesthetized Crayfish JESÚS HERNÁNDEZ–FALCÓN,*1 JESÚS SERRATO,† AND FIDEL RAMÓN‡ *Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City, México, †Depto. de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, México, D.F. 07000, Mexico City, Mexico, and ‡División de Estudios de Posgrado e Investigación, Facultad de Medicina,Universidad Nacional Autónoma de México. Ciudad Universitaria México, Mexico City, Mexico Received 29 August 1997; Accepted 1 October 1998 HERNÁNDEZ–FALCÓN, J., J. SERRATO AND F. RAMÓN. Evoked potentials elicited by natural stimuli in the brain of unanesthetized crayfish. PHYSIOL BEHAV 66(3) 397–407, 1999.—Experiments were conducted to test some characteristics of vision by crayfish underwater and in air, and determine possible motion reactions elicited in response to naturalistic or quasi-ethological visual stimuli. Chronically implanted electrodes on the brain were used to record visually evoked potentials in response to moving bars at different speeds or to fish of different sizes. Electroretinograms were also recorded to detect when an object or a shadow appeared in the crayfish visual field. Ongoing brain activity is mild under basal conditions, but increases in RMS by z6% in response to bar passage and 12 to 53% in response to fish motionless or swimming in front of the crayfish. When crayfish are free to move, fish swimming in front of them elicit intense brain activity, together with displacement toward them and an attempt to grab them. Visual evoked potentials are elicited by moving objects as small as 18 at a distance of 30 cm in air as well as underwater. None of the stimuli used induced evident behavioral responses under our conditions. We conclude that vision-action activities can be divided into (a) vision of irrelevant objects with short lasting electrical activity and no motion in response to it; (b) vision of mildly interesting objects with long-lasting electrical effects, but no motion in response to it; and (c) vision of relevant objects with appropriate motion reaction. © 1999 Elsevier Science Inc. Invertebrate
Vision
Brain recording
Electroretinogram
shown by crayfish under laboratory conditions designed to stimulate characteristics of its underwater natural environment. Recording electrical activity from the crayfish brain in response to visual stimuli that seem relevant judging by behavioral responses, requires working with animals under nearly physiological conditions. To tackle this problem we developed a preparation of crayfish chronically implanted with electrodes in the brain for recording its electrical activity. We tested the visual system with light pulses of different wavelengths and determined the presence of evoked potentials at the brain. We found sites of integration [as defined in (24)] of visual signals at both eyestalk and brain. Furthermore, as pulses of light of different wavelength elicited electrical activity that seemed to follow different neural paths in the brain, we concluded that the integrating mechanism of these signals is compatible with postulates of the labeled line hypothesis (20).
STUDIES of crayfish optical apparatus as well as neural processing of visually evoked activity point to the presence of an elaborate visual sensory system; however, it is not known if this system is used to guide any specific behavior. In fact, studies on crayfish and lobsters point to olfactory and tactile sensory systems as most relevant for food searching, mating, and defense (3,16). Thus, our main interest in these studies is in the use of crayfish vision to guide some behaviors. To this end we have developed a preparation for chronic recording of brain multiunit activity from unanesthetized animals. In previous studies we described characteristics of spontaneous activity and signals elicited by light pulses of different wavelengths. In this article we deal with visual signals evoked by the presence of more natural stimuli than light flashes, such as bars, shadows, and fish, and motion responses elicited by them. The purpose of this work is to determine vision-action patterns
1To
Multiunit activity
whom requests for reprints should be addressed. E-mail: [email protected]
397
398
HERNÁNDEZ–FALCÓN, SERRATO AND RAMÓN
However, although these studies illuminate some neuronal circuits used to codify signals, they do not throw light into relationships between vision and resultant motion. To this end, in this article we describe experiments designed to gain insight into characteristics of crayfish vision in air and underwater, and stimuli that might elicit appropriate motion reactions. Stimuli used were objects, shadows, and animals moving at different speeds on clear backgrounds. Similar to results obtained by recordings from the optic nerve (1,23), spikes at the brain are elicited preferentially when an object or a shadow appears in the visual field or changes speed. However, some visual stimuli elicit behavioral responses that can be better described as of “attention,” and none elicit tail-flip escape responses. Unexpectedly, barely detectable visually evoked potentials are elicited by objects as small as 18 at a distance of 30 cm both in air or underwater, suggesting that the crayfish either has the same visual detecting threshold in both media, or is reacting to motion of an effectively point source. However, of stimuli used, only swimming fish elicit a locomotive response. MATERIALS AND METHODS
Animals We used crayfish Procambarus clarkii 8–10 cm in length and of either sex obtained from a local provider. Animals were maintained for 2–3 months in a large aquarium under natural light–dark cycles, with well-aerated water at room temperature and fed two to three times a week. Preparations Crayfish were implanted with a chronic electrode for recording multiunit brain activity as described in a previous work (20). Briefly, the procedure is as follows: animals are cold anesthetized, and a hole made in the dorsal carapace at the level of the junction between head and thorax. The brain becomes accessible after separating muscles and internal organs, mainly stomach and green gland, and electrodes (see below) are introduced and positioned on the dorsal surface of the brain. The electrode array is cemented to the carapace, and the animal returned to the tank and left undisturbed for 1–2 days. Experiments were performed in a specifically designed aquarium (Fig. 1). For recording with crayfish eyes in air animals were tethered in the larger aquarium facing the glass wall of the small aquarium, and water level was lowered. Additionally, just before experiments we placed wick electrodes on the corneal surface of one or both eyes without blocking their vision to record electroretinograms (ERG), which were used to indicate when the crayfish was exposed to the shadows or objects. For experiments recording brain activity underwater, crayfish were placed in the V-shaped chamber of the aquarium, which limited their movements when the glass partition was in place. When the partition was removed, crayfish could move into the rectangular chamber where fish were swimming. For these experiments the wick electrodes were modified as described below. In some experiments we used a manually controlled switch to time the bar entering the crayfish visual field. This signal was also sampled by a computer, making it easier to locate the wave on the ERG produced by objects tested. Stimuli To stimulate with flashes of light we used a Photic Stimulator (Model PS33-Plus; Grass Inst. Co., Quincy, MA) that trig-
FIG. 1. Diagram of the experimental glass aquaria. (A) Top view of the small aquarium with two compartments that can be separated by a removable glass partition (dotted line). The “V”-shaped compartment was used to place the crayfish and the rectangular one for fish. (B) Side view of the small aquarium placed inside a larger one (shown here as a separate compartment at the left). Water levels in both aquaria were controlled independently.
gers flashes of 10-ms duration and variable intensity. Unless stated otherwise, we used a medium setting and manually triggered single flashes. Other stimuli were bars of various widths (2–10 cm) made from black cardboard or rubber. Bars were hung from a cord pulled through a set of pulleys by a low-velocity motor, such that they passed in front of the crayfish every 1–2 min at a speed of 15 cm/s and a distance of 30 cm. Segments of the cord between pulleys moved horizontally or vertically, and to study the effects of bars moving in these directions the animal was placed in front of appropriate segments. Occasionally, the visual field was restricted with black paper to a window, horizontally about 1358 and vertically to 458. We visually stimulated animals underwater with black rubber vertical bars moving horizontally at 10 cm from crayfish in the rectangular compartment of the aquarium. In this case we did not search for responses to bars moving vertically. We also used live fish of different sizes and colors that, when standing still, were encouraged to move by lightly tapping them with a long thin glass pipette. They were visible to the crayfish at a distance of 8 cm on average. Background light was 0.3–1.0 lx, as measured with a Light Meter (Li-COR, Inc Model LI-1189; Lincoln, Nebraska; probe Model Photometric).
ELECTRICAL ACTIVITY FROM THE CRAYFISH BRAIN Recording and Electrodes During recording sessions an implanted animal was tethered in a water tank. Electrodes for recording multiunit brain activity were made as previously described (12). Briefly, electrodes were a grounded stainless steel cannula containing four Teflon-coated silver wires (100-mm diameter) secured with dental cement; spacing between wires was 100 mm. Electrode position on the brain optic lobe was determined in postmortem observations as small depressions on the brain surface, and did not produce macroscopic damage. Because experiments were performed on chronic animals, we did not want to damage their cornea by superficially introducing small stainless steel needless to record ERGs. Therefore, when crayfish were in air we used small wick electrodes, made with a silver/silver-chloride wire and a narrow cotton thread in contact with the dorsal part of the eye. This electrode was frequently moistened with saline solution. It did not obstruct the field of view. For experiments with animals underwater, wick electrodes were modified as follows. A glass capillary tube was filled with electrode cream (EC2; Grass Inst. Co.) and a small drop applied to the dorsal surface of the eye. A silver wire was immersed in the electrode cream in the tube. This electrode was useful for about 30 min, after which the cream dissolved and we had to make a new electrode. Data Acquisition and Analysis Recordings from electrodes were amplified with AC amplifiers (P511, Grass Inst. Co.) and filtered to pass between 1 Hz and 3 KHz; a 60 Hz band-reject filter was occasionally used. The output of amplifiers was displayed on an oscilloscope (Gould, Model 1604) and 5-s samples were digitized, usually at 8 KHz, with A/D converters (BIOPAC Systems, WPI) and stored in a computer (Power Macintosh 711000/ 666AV) for off-line analysis. Digitized traces were analyzed with Igor (Stapleton Software, Eugene, OR). RESULTS
Crayfish Eyes in Air Experimental procedure. For experiments with crayfish eyes in air we proceeded through the following steps: 1) claws were immobilized with a rubber band and animals tethered at the border of the experimental tank, with eyes slightly above water level; 2) with a micromanipulator we placed wick electrodes on the upper surface of the cornea of both eyes; 3) implanted electrodes were tested for the level of spontaneous brain activity they recorded, and the one showing the largest spikes and the most per unit time was selected; 4) a light pulse from a flash lamp about 20 cm over the animal was triggered and recordings displayed on the oscilloscope. If necessary, wick electrodes were adjusted and/or recording electrode changed until flashes obtained the largest response in both electrodes; 5) the animal was left undisturbed in the tank for 15–30 min in a room with dim illumination. After the crayfish is tethered in the tank there is a period of 10–15 min during which it rhythmically moves pleopods and pereiopods. After this period animal remains quite most of the time, with occasional bursts of activity that last 15–30 s. Records were obtained when the animal was motionless. Spontaneous multiunit activity. As previously described (12), spontaneous multiunit activity recorded by a macroelectrode on the crayfish brain is mainly comprised of numerous spikes
399
of different amplitude and morphology. Spikes appear continuously, but their frequency per unit time is slightly variable. Power spectra of 6-s segments of records show maximum power at about 400 Hz, decreasing to noise level above 2 KHz. Spike histograms show that higher frequency spikes are small (5–10 mV), while spikes of large amplitude (40–50 mV) are seldom seen. Electroretinogram (ERG). As some visual stimuli elicited brain responses that were barely discernible above the spontaneous activity, we needed to determine when objects entering the crayfish visual field were “seen.” Thus, we used as monitor a wave produced in the ERG when objects enter the visual field. Figure 2C shows the characteristics of the ERG in response to a flash of light of medium intensity (225 lx). After a stimulus artifact there is a brief positive wave followed by a slower negative one, which in some records overshoots the zero line after recovery. These are typical H-I and H-II components described from recordings with needle electrodes (18). Figure 2A plots the absolute amplitude of those two components of the ERG during flashes of light of variable intensity. Both components increase approximately linearly in amplitude as flash intensity increases. Similarity in morphology between ERGs recorded with wick electrodes (Fig. 2C) and those recorded with needle electrodes (18) suggests that wick electrodes faithfully record photoreceptor activity elicited by flashes of light. Thus, we use them to record signals due to introduction of objects in the crayfish visual field. Passage of a black bar (22.5 cm wide, 10 cm long) in both horizontal directions in front of a crayfish elicits reproducible waves in the ERG. Figure 3A shows four wave complexes (vertical lines) elicited in response to a black bar passing four times in front of a crayfish. ERGs are similar to those described previously; a brief positive-going wave (H-II) followed by a larger negative-going (H-I). During each H-I wave an electrode placed on the brain (Fig. 3B) recorded few large spikes, more noticeable during the first and fourth passage. Integrated brain activity (Fig. 3C) more clearly shows the increase in electrical brain activity associated with the H-I ERG wave. Evoked activity. We recorded multiunit brain activity elicited in response to black bars of different widths moving in air across a white background in front of crayfish. Bars did not cast shadows on the animal, which was maintained under constant illumination. Figure 4 shows records obtained from an experiment in which blacks bars of different widths passed at the same speed (15 cm/s) at a distance of 30 cm in front of tethered crayfish. A black bar 1.2 cm wide (Fig. 4B) passing in front of the animal elicits a discharge comprised of spikes of different amplitude lasting about 35 ms. Similar discharges are seen during passage of black bars of 2.5, 5, 7.5, and 10 cm width (Fig. 4C, D, E, and F), although discharge duration is longer for wide bars. Discharges seem to have intermediate periods of decreased activity, more noticeable in records 4B and C. Record 4A is a control, obtained while moving cord and pulleys without the bar. In general, records in Fig. 4 show that in response to a black bar entering a crayfish visual field there is an initial train of spikes, followed by an increased activity period roughly related to bar width. A precise correlation between spikes and bar borders cannot be done with these records; however, increased brain activity related to bar width can be demonstrated by comparing Vrms values of records shown in Fig. 4 (Table 1). Vrms was calculated with Igor for 6-s segments starting at stimulus onset.
400
HERNÁNDEZ–FALCÓN, SERRATO AND RAMÓN
FIG. 2. Electroretinogram of crayfish. (A) Stimulus response relationships for ERG components H-I and H-II (absolute values) in air. Stimuli of increasing intensity (from 122 to 1800 lx) were applied at 2-min intervals. (B) Stimulus response relationships for ERG components H-I and H-II (absolute values) underwater from the same animal as in A. (C) Typical ERG recorded in response to a light flash (z2225 lx, 10 ms); H-I and H-II components are labeled.
White bars of similar widths passing in front of an animal at the same speed as black ones described above did not elicit spike discharges at the brain (not shown). With our illumination they were about the same brightness as the background. In other experiments we moved square pieces of black cardboard of various sizes in front of crayfish at 15 cm/s. Figure 5A shows that a 2-cm black square elicits a bimodal discharge of spikes (circles), similar to those described above. The time between peaks corresponds to the time for the two edges of the card to pass by. A 1-cm square elicits only a single obvious spike of large amplitude (Fig. 5B). Similar squares made with white cardboard and moving at the same speed as black ones did not elicit any response. In other experiments (Fig. 6A) we worked with shadows covering entire crayfish. Black bars (20 3 28 cm) were moved in front of a light source such that they cast shadows on entire crayfish. When a shadow moved rapidly (25 cm/s) we recorded two prominent discharges lasting about 40 and 50 ms, separated by a period of about 140 ms during which there was less activity. In contrast, slow movement (5 cm/s) of the same shadow elicited two spike discharges (125 and 135 ms, respectively) separated by about 1.4 s. In both cases spike discharges have similar characteristics to those seen in response to moving black bars; that is, two groups of spikes separated by a silent period, which is seen better when movement was slow. Shadows moving in either direction produced similar spike discharges. In a few experiments (Fig. 6B) fast shadow movement elicited a discharge (170 ms) followed by several seconds of in-
creased activity with occasional large spikes. This activity was not seen after a slowly moving shadow, nor was it correlated with movement of the animal. Although these results clearly show that crayfish are able to detect movement of black objects as small as 18 at a speed of about 208/s, experiments were done with animals tethered with eyes in air slightly above the water level. Thus, it was of interest to determine also the crayfish visual sensitivity in water, its natural habitat. Crayfish Underwater Spontaneous multiunit activity. Records of spontaneous multiunit brain activity show numerous spikes both isolated and in short bursts, with a small component of slow frequency waves. These characteristics are similar to those seen in similar records obtained while crayfish was in air (compare Figs. 4A and 7A). Electroretinogram. To record ERG underwater we tethered crayfish and slightly reduced eye movements with a small piece of cotton placed through a glass partition in front of the crayfish from a lamp in air at a distance of 15 cm. Recorded ERG H-I and H-II components are similar to those seen in air, but their amplitude is substantially reduced. Similarly to results obtained in air, stimulus–response relationships are linear (Fig. 2B). Evoked activity. We performed experiments placing bars or fish of different sizes in the rectangular compartment of the aquarium described in the Methods section. Initially we com-
ELECTRICAL ACTIVITY FROM THE CRAYFISH BRAIN
401
FIG. 3. ERG, multiunit brain activity, and integral of this activity during two horizontal passages of a black bar in front of crayfish in air. Left and right electroretinograms (A) were recorded with wick electrodes simultaneously with multiunit brain activity (B). Vertical lines are drawn at the peaks of the integral of recorded brain activity (C) synchronous with the H-I component of the ERG; this helps to localize the resulting spike discharge.
pared electrical activity recorded by four electrodes on the brain in response to passage of a black bar in front of crayfish tethered underwater. Passage of a black bar in front of a crayfish elicits spike discharges in all four channels, with characteristics similar to those seen when crayfish eyes were in air. That is, discharges are comprised of two groups of spikes, separated by a period of less activity. Number of spikes and discharge duration are rather similar in all four channels (not shown), and also to those described above. Next, we used as stimulus fish swimming in front of crayfish. Initially, we recorded spontaneous electrical activity from the brain of crayfish facing the tank without fish. However, as records from all four implanted electrodes are similar, here we show only channel 4. Thus, Fig. 7A shows that spontaneous activity recorded on the brain has characteristics similar to those seen when crayfish eyes were in air, and shown above
and elsewhere (12). Then we introduced fish of different sizes in the tank in front of crayfish. Figure 7C contains a record obtained when a gold fish (about 6 3 2 cm, side view) moved in front of a crayfish at a distance of about 8 cm. Record shows a large activity mostly comprised of small spikes, but numerous large spikes isolated and in short bursts can be also seen. This large activity is due to fish moving in front of the crayfish, as it can be ascertained by noticing that when fish remain motionless (Fig. 7B), the number of spikes decreases; nevertheless, even in this case there is a large number of small amplitude spikes. We also introduced in the tank fish of slightly larger size (about 7.5 3 2.5 cm, side view). In this case (Fig. 7D) spikes recorded from the crayfish brain are even more numerous and larger than those recorded with the previous smaller fish. Largest spikes seem to occur only when the fish is changing
402
HERNÁNDEZ–FALCÓN, SERRATO AND RAMÓN
FIG. 4. Multiunit brain activity in response to horizontal passage of black bars of different widths. Control records moving cord and pulleys without bar (A) were followed by the passage of bars 1.2 (B), 2.5 (C), 5.0 (D), 7.5 (E), and 10 (F) cm wide. A spike discharge is associated with entrance of the leading edge into the crayfish visual field.
position, although continuous movement of dorsal and lateral fins elicits many spikes of different amplitudes. During all these recordings the only visible sign that the crayfish had seen a fish was an “attentive” position that it maintained during the whole time it faced the tank with fish. As multiunit activity evoked in response to crayfish facing fish appears as single large spikes or in short bursts, to compare records shown in Fig. 7 we calculated their Vrms, and Table 2 shows those values. Vrms values are smallest during spontaneous activity and increase during the passage of black
bars or facing fish. Values of records obtained when crayfish face a motionless small fish (Fig. 7B) are slightly larger than those due to a bar passing (Fig. 6), but both are smaller than those obtained when the same fish (Fig. 7C) or a slightly larger one (Fig. 7D) swims. Responses to black bars moving across the crayfish visual field were elicited almost independently of the background level of illumination, within the range 11–2000 lx (24). DISCUSSION
Electroretinogram (ERG) TABLE 1 CRAYFISH BRAIN ELECTRICAL ACTIVITY FIG. 4 Record
A B C D E F
Bar Width (cm)
Vrms (MV)
Response
control 1.2 2.5 5.0 7.5 10.0
9.2389 9.5995 9.7917 9.8109 10.2677 10.7426
— 0.3606 0.5528 0.5720 1.0288 1.5037
Bar widths and Vrms values for 6-s segments of records shown in Fig. 4. Values in response column were obtained by subtracting control Vrms from each experimental value.
In response to flashes of light, electroretinograms recorded with needle electrodes in the corneal surface of dark adapted crayfish eyes showed two components, H-I and H-II (18). After penetrating the cornea, while in the crystalline cone layer, the ERG shows a negative peak (H-I) followed by a slow plateau (H-II). The H-I component amplitude depends on light intensity, while the amplitude and duration of H-II depend on the duration of the light stimulus. In response to a flash of light, a wick electrode records ERG components (H-I and H-II) similar to those seen after penetrating the cornea and similar, inverted, components can be also recorded during the passage of a black bar in front of crayfish. This indicates that photoreceptors detect both big changes in light intensity produced by a flash of light and small changes produced by the bar passage, activating in both
ELECTRICAL ACTIVITY FROM THE CRAYFISH BRAIN
403
FIG. 5. Multiunit brain activity elicited in response to the horizontal passage of 2-cm square (A) or 1-cm square (B) black cardboard at 30 cm distance. Two brief discharges are elicited by the large square and only one obvious extra spike in response to the small one (circles).
cases afferent fibers. The small contrast produced by the bar in some experiments suggests a great visual sensitivity. Brain Recordings Recordings of electrical activity form the brain of invertebrates have been mainly acute (4,7,17), but there are a few from chronically implanted animals, such as crayfish (1), octopus and cuttlefish (2,5,6,8), and horseshoe crab (9). An aim of this work is to find out if whether the technique of implanting electrodes on the brain and recording in the intact, behaving animal still permits responses to naturalistic or quasi-ethological visual stimuli to be observed and replicated over useful length of time. A second aim is to ask how small a physiolocigal stimulus can evoke a visible response, what features such as movement, are more effective and how such tests are comparable for vision under water vs. that in air. We show that in the absence of any overt behavioral response, one can record objective, central neuronal responses that at the least manifest detection or perception. Activity evoked at the brain by visual stimuli is mainly comprised of fast spikes with a few low-amplitude, low-frequency signals. Although some spikes coming from muscles located under the brain are picked up by our electrodes, they can be easily identified by their slow frequency components and filtered out [see also (12)]. Spikes shown in our records are only of neural origin. In most experiments several types of units might be excited simultaneously and contribute to the overall activity recorded
at the brain. Furthermore, as we placed animals facing a white screen over which black bars were moving, we were stimulating homo-, hetero-, and bilateral visual interneurons, and no effort was made to separate their contribution. Thus, increased brain activity in our records seems mainly due to visual fibers. Visual Fibers Characteristics of purely visual fibers to the brain were reported by Wiersma and Yamaguchi (22), resulting in a classification based on the type of stimulus to which they responded best, into sustaining, dimming, and movement units. Only a small number of each type of fibers was isolated, 14 sustaining, 17 dimming, and 13 movement units. A usual response of sustaining fibers (SuFs) to illumination is a tonic discharge with a latency of 20–40 ms and, although it starts with a burst of about 300 spikes per second, it stops as soon as light is turned off. Illumination on the retina outside the SuFs excitatory field inhibits a discharge. However, SuFs have also been shown to burst after shadows at a frequency of 4–10 spikes per second, without signs of adaptation (24). Nonetheless, SuFs characteristic of constant discharge in the presence of light makes us suspect that most spikes recorded at the brain by our electrodes do not come from them. As opposed to SuFs, movement fibers, in particular of jittery type (JMFs), have firing characteristics that make them likely to be contributing largely to spikes recorded at the
404
HERNÁNDEZ–FALCÓN, SERRATO AND RAMÓN
FIG. 6. Multiunit brain activity elicited in response to horizontal passage of a black bar (20 3 28 cm), which cast shadows covering the entire crayfish, at two speeds—fast and slow. (A) In both cases there are two spike discharges, but time interval is greater during slow than fast passage. (B) In some experiments fast shadow passage elicited a long-lasting discharge. Two white horizontal lines are drawn to facilitate comparison of spike activity level before and after stimulus.
ELECTRICAL ACTIVITY FROM THE CRAYFISH BRAIN
405
FIG. 7. Multiunit brain activity elicited in response to several stimuli. Only one of four recorded channels is shown. (A) Spontaneous activity. (B) A goldfish (z6 3 2 cm, side view; 8 cm from the crayfish eyes) motionless stimulates isolated but frequent spikes or short bursts. (C) Same fish swimming movements elicit bursts of spikes. (D) A burst of large spikes was seen whenever a fish (7. 5 3 2.5 cm side view) moved in a separate compartment visible to the crayfish.
brain. For example, JMFs react strongly to dark edges entering their field with a mean response frequency that is a linear function of edge velocity, from 18/s to 358/s (10). Furthermore, in response to dark edges moving across their visual field there is an inhibitory wave that seems to precede the moving edge, and such inhibition is apparent in some of our records (i.e., Fig. 6A). In particular, they react with a strong discharge when an object moves in a jittery fashion within their visual field, and this is how one would describe movement of a fish in water. Fish movements, in particular those of fins and tail, stop and start at every stroke, so that we would expect no adaptation of JMFs.
ment across the whole field will not be seen by these fibers. A response at the end of the bar passage could be due to sustaining fibers, as these respond to this type of stimulus. Although crayfish do not seem to have fibers (medium and fast movement) that react to approaching dark objects, occasionally we recorded brief spike discharges in response to this type of stimulus. It is possible that in these cases we were stimulating both jittery and sustained fibers, or higher order
TABLE 2 CRAYFISH BRAIN ELECTRICAL ACTIVITY UNDERWATER
Responses to Movement of Black Bars and Shadows As jittery movement fibers show a pronounced refractory period, stimuli were presented with a frequency of less than one every 2 min. These presentations were kept usually at a total of less than 10, as more produce a profound and long lasting habituation (10,11). In our experiments black bars moved across a crayfish visual field at a uniform velocity, and the inhibitory response that travels in front of the response of jittery movement fibers was not seen in our records, most likely because of large background activity. We expect that jittery movement fibers react with a single adapting burst as a bar enters the field and move-
Ch1 Ch2 Ch3 Ch4
Spont. Act
Black Bar
Small Fish (Motionless)
Small Fish (Moving)
Big Fish (Moving)
0 0 0 0
0.7385 0.5952 0.5612 0.6704
1.2155 0.9866 0.9936 1.4217
2.3760 1.8157 1.8729 1.9861
5.1010 4.9046 5.0664 5.5580
Change in Vrms values for 6-s segments of records obtained during experiments underwater. Values for spontaneous activity were subtracted from each experimental Vrms value.
406 neurons not isolated previously (23). Other fibers studied in lobsters and crabs, such as “light,” undirectional,” and “seeing” types, have not been found or have been little studied in crayfish (24); thus, we cannot invoke them to explain our findings. In all records shown here it is clear that there is a spike discharge as the leading edge of a bar enters a crayfish visual field, and occasionally again in response to the trailing edge; however, activity during passage of the bar itself is very small. This suggests that jittery movement units rather than sustained fibers are being activated, as with the latter one expects a prolonged response. This conclusion is supported by large responses elicited during passage of a swimming fish in front of crayfish, a condition that would produce maximal activation of jittery fibers due to apparently very irregular movements of fish fins. The fact that shadows are more effective than bars in eliciting brain spike responses supports this conclusion. Thus, in general, data shown here support the viewpoint that crustaceans see mainly movement as opposed to objects (13). Vision Depth of water in the fish tank was about 9 cm, and because fish measured 2 to 6 cm in a vertical axis, displacement of fish as stimuli is mostly in the horizontal direction. Thus, most spikes evoked by fish movement correspond to activation of units sensitive to horizontal movement. It is also apparent that an equal number of spikes is evoked by fish moving in either direction; however, our data do not allow us to say if the same movement units are being activated or if there are different fibers sensing each direction. Controls taken when fish were not displacing but only moving their fins show that this is not a very effective stimulus, as few spikes were produced by this movement. However, when fish change position, the stimulus was large and numerous spikes were elicited. Many stimuli produce photoreceptor activation and input signals to the brain, where some of them might be integrated while some others continue through the circumesophageal connectives. If electrical activity evoked at the brain is taken as vision, crayfish is able to see moving objects placed in its visual field, as well as all wavelengths in the mammalian visual spectrum (14,20). However, as shown above, many objects in the crayfish visual field evoke activity at the brain without a motion reaction. Tethered crayfish react with evoked potentials to fish swimming in their visual field, with no attempt to free themselves or to move. Thus, it seems that fish, as used in our experiments, are not adequate stimuli to trigger specific behaviors in the situation and motivational state tested. It is possible that tethering or some other aspect of our setup alters crayfish behavior. On the contrary, when crayfish are free, they slowly walk towards fish, and if these are cornered, attempt to grab them. Although we used fish of different colors, we did not observe differences in associated brain recordings. This could indicate that movement detectors are not sensitive to colors, and that it is only “borders” that stimulate them. However, this is not strong evidence, because measured responses could easily miss color sensitivity, if present. Comparison of crayfish brain activity in air and underwater suggests that visual responses are equivalent in both environments, and raise a question about its adaptive utility in an almost exclusively aquatic animal. The crayfish visual system seem designed to gain sensitivity more than visual acuity. A flat cornea and radial plane mirrors orthogonally arranged (in
HERNÁNDEZ–FALCÓN, SERRATO AND RAMÓN the squared shaped crystalline cone) of the superposition eye (19,21) facilitate capture of impinging photons, a design where sensitivity is high and independent of the environment. In air, light dispersion is small due to a thin layer of water apposed on the corneal surface, and a small f number characteristic of superposition eyes (19) must produce a bright and blurred image, such that only dark edges passing through different fields of the eye might be detected and turn significant. Underwater light dispersion is large, but the system detects small changes in light intensity produced by moving dark objects (fish fins and tails), which give rise to random light fluctuations (9). These are integrated, inducing a behavioral response when the crayfish is free (an attempt to grab fish). We speculate that in air brain discharge is mostly due to whole activation of both retinas; in contrast, activity recorded underwater might reveal detection and/or integration of visual significant stimuli. Motion Responses Despite the variety of fish shapes and sizes that we have used, we have not seen crayfish try to escape or have sudden movements. This can be partly due to our tethering of animals, which has been shown to inhibit escape reflex (15), and which could also inhibit other types of movement. However, because in other experiments with animals free in water their reactions were always similar, that is, no motion or slow displacements towards fish, we can conclude that none of those visual stimuli were considered threatening or aroused obvious curiosity. When crayfish are free in the tank and a fish is cornered, crayfish try to grab them in a sudden movement with the chelas; however, crayfish unmyelinated nerve fibers are no match for myelinated ones, and fish always escape in our tanks. Occasionally our records show in all recording channels slow signals associated with crayfish movements and/or displacement. Similar slow potentials can appear in only one channel, and in this case their origin is not clear. Although a possibility exists of movement artifact in only one electrode, it is more likely that they are due to muscle movements near the electrode. In most of our records we could not detect slow waves such as those described in gastropods (7), possibly because we did not filter out the spikes. As described in the Results section, the motion response of crayfish to different visual stimuli applied was different. Thus, black bars did not elicit any visible movement from crayfish, even when it produced a brief but clear discharge response to both leading and trailing edge (Fig. 6) or lasted longer than the time it took the bar to pass (Fig. 6B). A similar lack of movement response was seen when crayfish faced small motionless or swimming fish (Fig. 7A and B); in this case, however, crayfish seemed to maintain a very “attentive” attitude and the spike discharge lasted longer than the time it took the fish to pass by. However, a clear motion response was obtained when a larger fish passed swimming in front of crayfish, as in this case there was a slow displacement toward the fish and, in the few occasions in which crayfish cornered the fish, it attempted to grab it. These different motion responses to visual stimuli suggest the presence of an “integrator circuit” with a high threshold that has to be overcome to trigger a motion response. It would seem as if a brief stimulus (i.e., the passage of a bar), even though intense (i.e., a light flash), is not able to raise brain activity level above this putative threshold. Furthermore, as even the presence of a small motionless or slowly swimming fish that elicits more spikes that a black bar does not trigger a
ELECTRICAL ACTIVITY FROM THE CRAYFISH BRAIN motion response, this suggests the need of not only high but also sustained brain activity. This threshold, however, is finally reached when the visual stimulus is as intense and sustained as that produced by a large fish swimming in front of crayfish. It should be interesting to localize a brain neuronal circuit with such characteristics.
407 ACKNOWLEDGEMENTS
The authors would like to express their appreciation to Prof. T. H. Bullock for continuous encouragement throughout this work, as well as for his helpful comments on the original version of this manuscript. The help of J. C. Gómez and C. Ramírez with illustrations is also acknowledged.
REFERENCES 1. Aréchiga, H.; Wiersma, C. A. G.: The effects of motor activity on the reactivity of single visual units in the crayfish. J. Neurobiol. 1:53–69; 1969. 2. Bleckmann, H.; Budelmann, B. U.; Bullock, T. H.: Peripheral and central nervous responses evoked by small water movements in a cephalopod. J. Comp. Physiol. 168:247–257; 1991. 3. Bovbjerg, R. V.: Dominance order in the crayfish Orconectes virilis (Hagen). Physiol. Zool. 26:173–178; 1956. 4. Bullock, T. H.: Problems in the comparative study of brain waves. Yale J. Biol. Med. 17:657–679; 1945. 5. Bullock, T. H.: Electrical signs of activity in assemblies of neurons: Compound field potentials as objects of study in their own right. Acta Morphol. Acad. Sci. Hung 31:39–62; 1983. 6. Bullock T. H.: Ongoing compound field potentials from octopus brain are labile and vertebrate-like. Electroencephalogr. Clin. Neurophysiol. 57:473–483; 1984. 7. Bullock, T. H.; Basar, E.: Comparison of ongoing compound field potentials in the brains of invertebrates and vertebrates. Brain Res. Rev. 13:57–75; 1988. 8. Bullock, T. H.; Budelman, B. U.: Sensory evoked potentials in unanesthetized unrestrained cuttlefish: A new preparation for brain physiology in cephalopods. J. Comp. Physiol. A 168:141– 150; 1991. 9. Dodge, F. A.; Porcello, D. M.; Dodge, S. A.; Kaplan, E.; Barlow, R. E.: Noise components in Limulus vision. Biol. Bull. 187:261– 262; 1994. 10. Glantz, R. M.: Habituation of the motion detectors of the crayfish optic nerve. J. Neurobiol. 5:489–510; 1974. 11. Glantz, R. M.: Defense reflex and motion detection responsiveness to approaching targets: The motion detector triggers to the defense reflex pathway. J. Comp. Physiol. 95:297–314; 1974. 12. Hernández, O. H.; Serrato, J.; Ramón, F.: Chronic recording of electrical activity from the brain of unrestrained crayfish: The basal, unstimulated activity. Comp. Biochem. Physiol. 114A:219– 226; 1996.
13. Horridge, G. A.: The evolution of visual processing and the construction of seeing systems. Proc. R. Soc. Lond. B 230:279–292; 1987. 14. Kennedy, D.; Bruno, M. S.: The spectral sensitivity of crayfish and lobster vision. J. Gen. Physiol. 44:1089–1101; 1961. 15. Krasne, F. B.; Wine, J. J.: Extrinsic modulation of crayfish escape behavior. J. Exp. Biol. 63:433–456; 1975. 16. Kravitz, E. A.; Beltz, B.; Glusman, S.; Goy, M.; Harris-Warrick, R.; Johnston, M.; Livingstone, M.; Schwartz, T.; Siwicki, K. K.: The well-modulated lobster: The roles of serotonin, octopamine, and proctolin in the lobster nervous system. In: Selverston, A., ed. Model neural networks and behavior. New York: Plenum Press; 1985: 339–360. 17. Maynard, D. M.: Electrical activity in the cockroach cerebrum. Nature 177:529–530; 1956. 18. Naka, K.; Kuwabara, M: Two components from the compound eye of the crayfish. J. Exp. Biol. 36:51–61; 1959. 19. Nilsson, D. E.: From cornea to retinal image in invertebrate eyes. Trends Neurosci. 13:55–64; 1990. 20. Serrato, J.; Hernández, O. H.; Ramón, F.: Integration of visual signals in the crayfish brain: Multiunit recordings in eyestalk and brain. Comp. Biochem. Physiol. 114A:211–217; 1996. 21. Vogt, K.: Die Spiegeloptik des Flusskrebsauges. J. Comp. Physiol. 135:1–19; 1980. 22. Wiersma, C. A. G.; Yamaguchi, T.: Integration of visually stimuli by the crayfish central nervous system. J. Exp. Biol. 47:409–431; 1967. 23. Wiersma, C. A. G.; Roach, J. L. M.: Principles of organization of invertebrate sensory systems. In: Brookhart, J. M.; Mountcastle, V. B., ed. Handbook of physiology. Section I: The nervous system, vol. 1, part 2. Bethesda, MD: American Physiological Society; 1977:1089–1135. 24. Wiersma, C. A. G.; Roach, J. L. M.; Glantz, R. M.: Neural integration in the optic system. In: Bliss, D., ed. The biology of crustacea, vol. 4. New York: Academic Press; 1982:1–31.