Electrophysiological evidences of mutual modulatory influences on the retinal activity of the crayfish Procambarus bouvieri (O)

Brain Research Bulletin, Vol. 3, pp. 101-106.

Printed in the U.S.A.

Electrophysiological Evidences of Mutual Modulatory Influences on the Retinal Activity of the Crayfish Procambarus bouvieri (0)’ BALTAZAR BARRERA-MERA AND ESPERANZA M. ABASTA Department0

de Fisiologia,

Facultad de Medicinia,

UNAM, Apdo. Postal 70250, Mixico,

D. F. Mkxico

(Received 11 February 1977) BARRERA-MERA, B. AND E. M. ABASTA. Electrophysiological evidencesof mutual modulatory influenceson the retinal activity of the crayfish P. bouvieri. BRAIN RES. BULL. 3(2) 101-106, 1978. - Electroretinographic evoked potentials (ERGS) were recorded in dark adapted crayfish by the application of pulses of light (0.09 Cd/ft’) presented every 2.5 min. Heterolateral illumination (HI) for sixty min (0.06-0.3 Cd/ft2) induced up to 50% decrease in ERG after a latency of 12-25 min. ERG depression was proportional to the intensity of HI and also showed a circadian rhythm. During the a phase the ERG recovery started 3-10 min after HI was turned off. In contrast it started only after lo-20 min during the p phase. The time course of the ERG depression, which was abolished in splitbrain animals, strongly suggests that a mutual modulatory influence, probably of neuroendocrine nature, is present in the crayfish visual system.

ERG modulation

Circadian rhythms

Split ganglia

Crayfish

THE COMPLEX mechanism of light adaptation apparently involves different events at several levels of the vertebrate as well as in invertebrate eye. Recent studies on light adaptation in insects, mollusks and crustacea indicate that, aside from the major effect of light on the photolabile pigment of the receptor cell, changes in retinal sensitivity could be also due to other phenomena. In these animals synaptic interactions [ 14, 15, 18, 25, 271 and the position of the retinal shielding pigments (RSP) [3, 10, 11, 13, 191 probably have an important role in the development of light-dark adaptation. The electroretinographic (ERG) voltage in the crayfish, taken as an index of the position of the RSP [4,6] confirm the important role of these pigments, whose position is apparently under the control of humoral influences [ 16, 20, 21, 22, 231 ; and such as ERG [2], that position follows a circadian rhythm [9, 17, 321.

and right eyestalks through the heterolateral sustained response neurons, have been previously given in Podophthalamus [ 3 1,351 and in Procambarus [ 361. It is proposed that these specific neural pathways connect the contralateral retina to the neurohemal complex of the sinus gland, which releases the humoral factors involved in the light adapted position of the RSP. In the present paper we report that the depression in the ERG voltage, heterolaterally induced, shows a circadian course in intact but not in splitbrain animals. METHOD Crayfish P. bouvieri of either sex and lo-14 g body weight were used. Many of the technical aspects of stimulation and recording from the eye photoreceptors have been previously described [ 71. Each animal was kept in a small cage, half full of water and placed into a sound proof chamber under continuous darkness interrupted by brief test light stimuli, 0.09 Cd/ft*, applied to one eye. ERG voltage was recorded every 2.5 min during 24-48 hr periods by the application of such test light stimuli by means of a small lamp placed 1 mm in front of the cornea1 surface. Each stimulus also activated a photocell, which permitted to measure the intensity of the stimulus and the time of its delivery. The photocell signals were amplified by a Tektronix 122 preamplifier and recorded in one channel of a physiograph (Narco Bio Systems). In another channel the ERG was recorded. Both signals were also displayed on a dual beam Tektronix oscilloscope model 502 and photographed by a kymographic camera (Grass).

In a study of the genesis of the ERG rhythm in the crayfish Procambarus clarkii, a tendency to maintain the

same free running frequency and phase in both left and right ERG oscillations has been recently reported by Page and Larimer [29] . This phenomenon suggests that a coupling mechanism responsible for bringing into phase the ERG oscillations could be present in these animals. The clear heterolateral ERG voltage depression induced by surface illumination of one single eye in the intact crayfish [ 8 ] supports the existence of the above mentioned bilateral integrative mechanism. It was postulated [ 1 ] that liberation of specific humoral substances from the crayfish sinus gland, after the heterolateral eye surface illumination, could explain such ERG depression. Since electrophysiological evidences of mutual neural connections between both left

’ The authors are indebted to Drs. Albert0 Guevara-Rojas and Hector Brust-Carmona for their suggestions and review of this paper. 101

0361-9230/78/0302-0101$00.75

Copyright 0 1978 ANKHO

International Inc.

BARRERA-MERA

102 About one hour after the ERG voltage reached a complete dark adaptation, light (0.06-0.20 Cd/f?) was applied for 60 min to the heterolateral eye. In this case, the illumination was restricted to the cornea1 surface to reduce to a nlinj~~unl the possible light cor~tamination to the recorded eye. For the same reason the light intensity was always kept under 0.30 Cd/ft*, A similar experimental procedure was employed in long-term experiments in which the same heterolaternl illuJllination was applied every 3..-4 hr during two days. In these experiments the motor activity was simultaneously recorded to determine the general condition of the preparation. After a control period, partial or complete surgical bisection of the cerebral ganglion or the transection of the circumoesophageal connectives were carried out by the same technique described elsewhere [ 61. At the end of the experiments the cerebral ganglia were fixed in 5% aqueous Formalin, and frozen frontal sections 50 Hnr thick were stained by the Nissl method (Fig. 1).

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FIG. 2. ERG depression induced by ~ontr~latera1 eye ilIumination. At 0 time it shows the maximum response obtained after 80 min of dark adaptation. Notice the depression of the response during the illumination period (60 min) which persists 20 min after the stimulus was suppressed (signaled by 80). Lower traces photocell signals, shows that the test light stimuli 10.09 Cd/f@) was uniform in all cases. Lower numbers: minutes. Cal. 500 .uV, 200 msec.

induced is illustrated in Fig. 3, which shows the effects of three different light intensities. The ERG data were obtained from five animals under similar experimental conditions. In these animals microelectrodes had been implanted in both eyes. in order to test their mutual modulatory influence. To this end both eyes were alternatively tested allowing a thirty minute period after complete recovery of the ERG response on one side, before testing the opposite eye. With a light intensity of 0.3 Cd/f?. the depression reaches a 49.2% of the control value. The light intensities of 0.06 and 0.125 Cd/f? produced depression of the response of 0.5 and 17.3% respectively. The latency time (12-25 min) and the slope of the falling phase depended strongly on the intensity of the fight heteroiateraly applied. Circadian hlanifkstations latera& Induced f,IG. I. Photomicrograph of’ a transversal section ofcrayfish cerebral ganglion. The site of surgical bisection is signaled by upper and lower arrows. 0.t.. optic tract; elf., olfactory lobe: opt., optic lobe; act.. accessory tobe: tr.c., trito cerebrum; g.c., globuli cells; cc.. circumoesopl~a~eai connectives.

RESULTS

As it is well known the test light stimuli applied to the recorded eye evoke an ERG wave, whose value increases during dark adaptation, the maximum amplitude is reached in 60 min. The ERG magnitude after 80 min of dark adaptation is shown in the first recording in Fig. 2, which illustrates the results of a typical experiment. The contralateral 60 min illumination was initiated at zero time. Note that the ERG voltage gradually decreases, but it takes 12-25 min to begin with. The responses started increasing again only 1O--20 min after the end of the heterolateral stimulus. The relationship between the intensity of the heterolateral stimulation and the decrease in ERG voltage thus

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In some experiments the course of the above described ERG depression suggested that a circadian component could be present. The ERG voltage decreased less at night (19-20 hr) as compared with that obtained during the early morning (8- I2 hrj in the same animal. Ail of the preparations showed changes similar to those depicted by Fig. 4. In these experiments, light was applied for 60 min at three hr intervals for two days, and the ERG was continuously recorded. As it can be seen the ERG depression is notably different along a circadian cycie. A very small decrease of the ERG potentials was recorded during the active phase of the rhythm, In contrast, the major depression of ERG was observed during the rest phase. In all these long-term experiments the simuitaneously recorded motor activity {Fig. 4, lower traces) also showed a circadian rhythm, i.e. the activity was higher during the night-phase of the rhythm. As the latency and the magnitude of the ERG depression, the relationship between the initial ERG value and ERG depression were found inversely related. it is interesting to note that the initiation of the recovery after the end of the heterolateral stimulation residual action time depended on the circadian phase. Thus, during the active or nocturnal phase the recovery was initiated within 3&4 min after the light was

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FIG. 3. Effects of light intensity on the heterolaterally induced ERG depression. Notice the progressive depression in the amplitude of the ERG from an almost unnoticeable value (0.5%) when the light intensity was very low (0.06 Cd/ft’) up to almost half the control value (49.2%) when the intensity was higher (0.3 Cd/ft’ ). Points and vertical lines represent the mean and standard deviation respective from the experiments (N). These experiments were made at the same hour of the day (lo-16 hr). Right (0) and left (a) ERG activities modified by the contralateral illumination.

turned off, while in the diurnal phase lo-20 before the initiation of the recovery.

min elapsed

Ejyects of the Bisection of the Cerebral tiangliotl on the Heterolateral ERG Depression

The possibility of neural connections between both eyestalks through the cerebral ganglion, mediating the above described responses to the contralateral illumination was tested. In fact, those responses were completely abolished immediately after the bisection of the cerebral ganglion (split brain). The effects of the partial and complete bisection are illustrated in Fig. 5. Note that the ERG depression persists after both circumoesophageal connectives were severed (upper trace A) and also after the partial bisection of the cerebral ganglion (Fig. SB). Contrariwise, in splitbrain animals the ERG depression (Fig. SC) was not present. In some of these animals, recorded 24-48 hr after the bisection, it was found that the magnitude of ERG increased after the heterolateral light stimulation. DISCUSSION

The elactrophysiological evidence offered in this paper shows that a mutual modulatory influence on the crayfish

retinal activity is present. Both the long latency of the ERG depression and its residual action time suggest that a neuroendocrine mechanism would be involved in the ERG depression heterolaterally induced. In fact, its time course rules out some alternative explanations such as, an heterolateral nervous inhibition of the excitability of the retinal photoreceptors, or the direct action of light upon the recorded eye due to its inadequate confinement to the contralateral eye. The relatively long latency of the depression in the intact animal (12-25 min), is probably explained by the diffusion time of the hormonal substances (light adapting hormone, LAH) that once liberated reach the pigmentary effector (retinal shielding pigment, RSP) in the recorded eyestalk (see Fig. 6). That latency would be absent if a direct neural effect on the light adapting photoreceptor response is involved. If indeed, an endocrine mechanism is present in the above described ERG depression, the humoral liberation probably takes place in the sinus gland which is known to store several neurohumoral substances [ 12, 33, 34 I. This liberation would be due to the heterolateral activation conveyed by specific neural pathways as suggested in Fig. 6. Of particular interest are the sustained response neurones (SRNs) described by Wiersma and co-workers [36.37] in the optic tract of the crayfish. Since some SRNs transmit

BARRERA-MERA

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AND ABASTA

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FIG. 4. Heterolateral ERG depression at different hours ut‘ the day (right nul~~bers~. FIven though the stimulus was constant in all cases (0.3 Cd/fP), the depression was maximum during the day time and it was minimum during the night. The black triangles signal the onset (A) and the suppression (v) of the light stimulus ( - ). The lower traces illustrate the locomotor activity. Cal. 1 mV. 15 min.

visual information, it is likely that they play a major role in the above described ERG depression. In spite of electrophysiological evidences of heterolateral ERG modulation a humoral action on the RSP, i.e., specific action of the LAH [ 16, 20, 21, 23. 241 as the main factor involved in the ERG modification is far from being conclusive. An associated depressing hormonal action on photoreceptors can not be discarded. In fact, some experiments on crayfish [30] and on other crustaceans 15,281 have provided behavioral and also electrophysiological evidence of the presence of an inhibitory hormonal action on the nervous system. The circadian manifestations of the ERG depressing h~terolateral

influences may be intiInately related to the position of RSP. These changes [9, 17, 32 1 involved in the ERG sensitivity [4] are interesting features related to the daily pattern here reported. An increase in the level of the hormone, i.e. the light adapting hormone, during the rest phase of the ERG rhythm is the probable explanation of our results. However, a rise of some other hormonal level during the nocturnal phase of the rhythm may also play a role. Finally, the supression of the bilateral influences on the ERG modulation in the split brain preparation provides good evidence for the existence of a mutual neuroendocrine modulation in the visual system of these animals.

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MINUTES FIG. 5. Illustrates the effect of cerebral ganglion bisection on the heterolateral ERG depression. In contrast with circumoesophageal connective transection (A) and also with partial bisection of the ganglion (B), the complete separation of both cerebral hemiganglia immediately abolished the ERG depression heterolaterally induced (C). Points represent the mean of experiments (N). The time (min) for both figures in abscissa. ERG voltage at ordinate (in percent). Right figures shows the corresponding cerebral section. l ++ Control before the surgical operation and after the section o---o. LEFT d

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FIG. 6. Schematic illustration of some elements of crayfish visual system. The contralateral connections between photoreceptors (r) and sinus gland (SG) is probably mediated through sustaining response neurones (S). Neural connection of S to SG through cerebral ganglion (cg) led us to postulate that an ipsi and a contralateral neuroendocrine reflexes initiated in photoreceptors after light stimulation probably promote the liberation (from SG) of the light adapting hormone (LAH). The target of LAH, the distal (d) accessory pigments (RSP) shield the cristalline cones reducing the input of light. The proximal (P) retinal shielding pigment apparently is under neural control of retinular photoreceptors (r) (Ludolph ef al. 1973).

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