Steroids Modulate the Excitability of Crayfish (Procambarus digueti) Photoreceptors: Long- and Short-Term Actions

General and Comparative Endocrinology 105, 255–269 (1997) Article No. GC966827

Steroids Modulate the Excitability of Crayfish (Procambarus digueti) Photoreceptors: Long- and Short-Term Actions Jesu´s Herna´ndez-Falco´n, Oliver Schneider-Ehrenberg, Araceli De la O Martı´nez, Vanessa Campos-Lozada, and Beatriz Fuentes-Pardo1 Departamento de Fisiologı´a, Facultad de Medicina, U.N.A.M., A. Postal 70-250, 04510 Me´xico, D.F., Mexico Accepted September 19, 1996

Short- and long-term actions of the steroids corticosterone, progesterone, and ecdysterone were analyzed upon excitability of crayfish retinular cells. The three hormones showed short-term actions, particularly during photoreceptor dark adaptation; effects were found to be dependent on the hour of the day. Corticosterone and progesterone had similar effects, i.e., resembled darkadapted conditions; ecdysterone actions were opposite and resembled light adaptation. Long-term effects were opposed to short-term effects in all cases. Possible mechanisms underlying these effects are discussed. r 1997 Academic Press

Steroid hormones are widely distributed among animal species. Two main groups of actions of these hormones can be distinguished. Genomic actions occur when hormones cross the cell membrane, bind to a nuclear receptor, and induce changes in protein synthesis (McEwen, 1991; Jo¨els and De Kloet, 1992). Steroid nongenomic actions involve the cell membrane by one or both of the following mechanisms: (1) Interaction with membrane proteins which results in the blockade or facilitation of their activity and can be reflected in the cell’s excitability changes (Ffrench-Mullen and Spence, 1991; Wehling et al., 1991; Gee et al., 1992; Hales et al., 1992; Paul and Purdy, 1992; Purdy et al., 1992): (2) membrane fluidity changes that would induce changes 1

To whom correspondence should be addressed.

0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

in the excitability of the stimulated cell (Kupferberg et al., 1991). Nongenomic steroid actions have been studied in many tissues from diverse species, i.e., 17-b-estradiol in neural cells (Nabekura et al., 1986; Hua and Chen, 1989), progesterone in human sperm (Blackmore, 1993), aldosterone in kidney and vascular smooth muscle (Christ et al., 1994a,b), progesterone in hamster neural tissue (De Bold and Frye, 1994a,b), and have been characterized as short-term effects (from milliseconds to less than 1 hr), with very short or nonexistent latency, generally reversible when the steroid is withdrawn (Fuentes-Pardo et al., 1990). Fink et al. (1992) have classified nongenomic steroid actions as immediate (from milliseconds to seconds), intermediate (hours to days), and long-term type actions (from 1 day to weeks). Some steroids such as progesterone, ponasterone A, and ecdysterone have been isolated from invertebrates such as crayfish (Horn et al., 1968; Beltz, 1988; Cassier et al., 1988; Bradbrook et al., 1990; Bidmon, 1991; Dauphin-Villemant et al., 1995). Ecdysterone has been implicated in the molting cycle (Hopkins, 1986; Kato and Riddiford, 1987; Durliat et al., 1988; Walgrave et al., 1988; Ueno et al., 1992), in development and programmed neuron death (Fahrbach, 1992; Miles and Weeks, 1991; Okazaki and Cheng, 1991; Fahrbach et al., 1994), and in circadian rhythms (Vafopoulu and Steel, 1991, 1992). Ecdysterone release is stress related (Matt-

255

256

son and Spaziani, 1985; Luo et al., 1991). Despite the many known actions of ecdysterone on crayfish, there are no reports of nongenomic effects of this and other steroid hormones upon cellular excitability in this animal. Visual photoreceptors can be used to study steroid actions upon cellular excitability because these cells have a high degree of sensitivity to environmental changes such as light or extracellular ions (Herna´ndezFalco´n and Fuentes-Pardo, 1991). Retinular cells can be modulated in vitro by neurotransmitters such as 5-hydroxytriptamine (Are´chiga et al., 1990) or hormones such as the distal pigment dispersing hormone (Fuentes-Pardo et al., 1984). These cells have been studied extensively, in particular with respect to their role in circadian rhythmicity (FuentesPardo et al., 1984). The aim of this work was to characterize the actions of ecdysterone, progesterone, and corticosterone upon crayfish retinular cell excitability, as well as to evaluate the differences between short-term and long-term effects. We found that short-term effects of corticosterone resembled night–day differences in control animals, and effects of progesterone resembled dark-adapted conditions in control animals. The ecdysterone actions resembled light adaptation. Long-term effects of each hormone were opposite to the short-term effects.

MATERIALS AND METHODS Biological Material Adult crayfish (6–8 cm long) Procambarus digueti in intermolt stage (between A and C molt stages; Durliat et al., 1988) were employed. Animals were obtained from a local supplier and kept in an aerated aquarium with temperature controlled at 17°. Light–dark cycles (LD 12:12) (light turned on at 0700 hr and light turned off at 1900 hr) were applied from the moment the animals arrived at the laboratory. Animals were maintained under these conditions for at least 1 week prior to usage. Experimental preparation consisted of the isolated crayfish eyestalk. Animals received cold anesthesia and the optic tract was cut at the base of the eyestalk

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

Herna´ndez-Falco´n et al.

with fine-tip scissors. Half the cornea was excised with a razor blade, and the preparation was immediately immersed in Van Harreveld solution (VH; Van Harreveld, 1936) and kept in a darkened Faraday’s cage for electrical recording. For short-term experiments, eyestalks were obtained as indicated from intact animals. For long-term experiments, they were obtained from animals that had been injected 24 hr before, in the sinus vein of the base of the sixth meropodite, with one of the hormones under study. In all the cases eyestalks were perfused with saline solution.

Solutions Corticosterone, progesterone, 20-OH-ecdysterone, and DMSO were acquired from Sigma Chemical Co. Corticosterone was prepared by dissolving a 150 mM concentration of the hormone in 1 ml of DMSO and then in a VH solution, to a final concentration of 150 µM. Progesterone and 20-OH ecdysterone were dissolved in a VH solution directly to a final concentration of 150 µM. The eyestalks for the short-term experiments were immersed in these solutions. VH solution was employed for perfusing and dissolving hormones when possible. VH solution composition was as follows (mmol/liter): NaCl, 205; KCl, 5; CaCl2, 13.5; MgCl2, 1.2; NaOH, 2.38; pH 7.2–7.4. For long-term experiments 150 nmol of corticosterone, progesterone, or 20-OH ecdysterone in 0.1 ml was injected into intact crayfish. In this species of crayfish, the hemolymph volume ranges between 3 and 5 ml; therefore, the final doses of the hormones ranged between 30 and 50 nmol/ml. Hormones were tested in a minimum of five animals for both shortterm and long-term conditions.

Electrical Recordings The technique for recording receptor potential (RP) from retinular cells has been described elsewhere (see Herna´ndez-Falco´n and Fuentes-Pardo, 1991). In brief, the eyestalk was immersed in a perfusing chamber in a darkened Faraday cage. Light stimuli were applied parallel to the longitudinal axis of the eyestalk. The photoreceptors were impaled with glass micropipettes with 30–50 MV resistance, filled with 2.7 M KCl. The criteria to consider that the microelectrode had pen-

257

Action of Steroids on Photoreceptors

etrated the membrane of a retinular cell were a sudden voltage drop (around 230 mV), corresponding to the resting membrane potential (RMP), and a depolarizing RP which appeared when the preparation was illuminated with a test light (Grass, PS 22). Light test stimuli were flashes 1800 Ix, 10 µsec, applied at 2-min intervals. Electrical responses were amplified by a high impedance recorder (WPI M 707), visualized in a CRT (Tektronix, 502), and stored in floppy disk after digitizing (Lab Master TMA-1). Each response was sent to a differentiator (Grass, 7P2O) from which the voltage change rate regarding time (first derivative) was obtained and stored (Fig. 1).

Experimental Procedure Experiments were conducted only if resting membrane potential was maintained constant during the recording time. The employed light intensity produced

FIG. 1. Method employed to obtain the receptor potential parameters (upper trace) and the derivative values (lower trace). The voltage change from the resting membrane potential to the maximal peak (a) is the value of the transient phase TP. The time elapsed from the beginning of the response to the time at which the amplitude has been reduced to 80% of its maximum corresponds to RP duration (b). The RP derivative showed two main components, the rate of depolarization, DdV/dt (c), and the rate of repolarization, RdV/dt (d).

a maximum response (a saturating one) when the photoreceptor was maintained in constant darkness for at least 10 min. The sensitivity of the retinular cells was evaluated during adaptation to darkness. Once a receptor potential was obtained, the eyestalk was kept for 10 min in constant darkness. After this time, the eyestalk was stimulated with a dim background light (20 Ix) for 10 min and then kept again in darkness for the rest of the experiment (60 min). During the darkness periods, the photoreceptors were stimulated with test flashes applied at 2-min intervals. These procedures permit studying hormone actions on photoresponse recovery rate after the photoreceptor cell had been exposed to background light (dynamical state of dark adaptation) and once the photo response had attained stable values (steady state of dark adaptation) (Fuentes-Pardo et al., 1984; Herna´ndez-Falco´n and Fuentes Pardo, 1991). For the short-term experiments with corticosterone and progesterone, the eyestalk was perfused with steroid–saline solution for the first 40 min of recording; for ecdysterone experiments the eyestalk was immersed in the steroid–saline solution; after 40 min in steroid solution, the bathing solution was substituted by normal VH solution, perfusing for at least 40 min more. For control experiments, the eyestalk was bathed in VH–saline solution for the first 40 min of recording and then the bathing solution was changed for a fresh VH–saline one. A second control group was conducted to discard any possible action of DMSO on the photoresponse. In this, the eyestalk was immersed in VH saline plus DMSO, 1% solution. Experiments were done between 2200 and 2400 hr (nighttime recordings) and between 1000 and 1200 hr (daytime recordings). The long-term experiments were carried out in normal VH. Twenty-four hours before, crayfish were injected with 0.1 ml of DMSO (1%) in control animals or 0.1 ml of corticosterone, progesterone, or ecdysterone in experimental animals. The experiments were done between 1500 and 1800 hr. In both short- and long-term experiments, results were categorized and analyzed according to daylight hour, sex of donor animal, and hormone employed.

Analysis of Results From each receptor potential recorded, the amplitude of the transient phase (TP) taken as the response

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

258

amplitude in millivolts and the response duration (D) in milliseconds were measured by us. From the derivative of the RP, we measured the rate of depolarization (DdV/dt) and the rate of repolarization (RdV/dt) in volts/second. For comparative purposes the values of TP, DdV/dt, and RdV/dt were normalized. Mean values from at least five experiments (6SE) were plotted for TP and D. Figure 1 shows the criteria used to measure TP, D, DdV/dt, and RdV/dt of the photoresponse. In each experimental group, the statistical differences were calculated for TP, D, DdV/dt, and RdV/dt, once the photoreceptors immersed in VH solution had been recovered after exposure to background light, namely, 40 min after the experiment was initiated (R-40). First, the ANOVA test was applied to the groups. When differences (P # 0.05) were detected, groups were identified by Fisher’s least significant difference test.

RESULTS Short-Term Experiments Controls. When a photoreceptor cell has been impaled a sudden voltage change can be measured. This

Herna´ndez-Falco´n et al.

change corresponds to the RMP which attained a mean value of 235.7 6 3.2 mV (n 5 21). Stimulation with a test flash (1800 Ix; 10 µsec) produced a depolarization in the photoreceptor cell, the RP, whose mean values were TP, 9.5 6 0.5 mV; D, 201.0 6 13.3 msec; DdV/dt, 30.2 6 0.5 V/sec; RdV/dt, 3.8 6 0.7 V/sec (n 5 21). Controls with VH saline plus 1% DMSO showed similar results. Night–day differences. The RP of the retinular cells was recorded in VH solution in order to obtain the reference pattern of response at two extreme times of the day, i.e., during the day, between 1000 and 1200 hr, and during the night, between 2200 and 2400 hr. In the two extreme hours recorded, neither the mean value of the resting membrane potential nor the retinular cell amplitude of the RP changed significantly. However, the retinular cell sensitivity, measured as the ability of the RP to recover values similar to those shown at the beginning of the experiment (namely, before the 10 min of background light), depended on the day hour. Figure 2 shows darkness adaptation curves for TP (Fig. 2a) and for D (Fig. 2b) of responses recorded between 1000 and 1200 hr (open symbols) and between 2200 and 2400 hr (solid symbols) when the eyestalk was perfused with normal VH solution. During the first 10 min of recording TP maintained the same

FIG. 2. Changes in the amplitude of the transient phase TP (a) and D (b) of the RP recorded from eyestalks immersed in VH solution. Responses were recorded between 1000 to 1200 hr (open symbols, n 5 11) and between 2200 and 2400 hours (solid symbols, n 5 10). Triangles corresponded to eyestalks obtained from female crayfish (n 5 10), and circles to eyestalks from male animals (n 5 11). 100% of TP corresponded to the RP amplitude after 10 min in darkness. Arrow indicates the time when perfusate was substituted by VH solution. After 10 min in the test solution the eyestalks were submitted to a dim background illumination for 10 min and then the eyestalks were returned to darkness for the rest of the experiment. Responses were obtained at 2-min intervals except during sustained illumination. Data shown were each recorded 4 min. Values obtained during the daytime were higher than those obtained at night. Each point corresponds to the average of the indicated experiments. Bars correspond to standard error.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

259

Action of Steroids on Photoreceptors

TABLE 1 Mean Values of R-40 in Normal Van Harreveld Solution

Female–night (n 5 5) Male–night (n 5 6) Female–day (n 5 6) Male–day (n 5 5)

Transient phase (%)

Duration (msec)

DdV/dt (%)

RdV/dt (%)

63.30 6 7.56 75.55 6 8.29 112.62 6 12.75*,** 114.25 6 7.78*,**

159.71 6 13.70 158.00 6 19.90 247.60 6 13.45*,** 262.00 6 29.07*,**

78.00 6 4.16 82.39 6 4.52 100.30 6 3.53*,** 101.24 6 2.52*,**

113.03 6 4.17 97.77 6 5.09 97.01 6 2.25 117.48 6 10.41

* P , 0.05 for comparison with female–night group. ** P , 0.05 for comparison with male–night group.

average value in the four groups. Continuous dim light stimulation for 10 min reduced the RP. The reduction was greater in the nighttime recordings than in the daytime recordings. TP reduction was the same in males (n 5 11) and females (n 5 10). During the dynamical phase of dark adaptation, TP amplitude attained a higher level than the initial level in the daytime, and only about 70% of the initial amplitude in the nighttime. During the steady state of adaptation to darkness, both daytime and nighttime TP values reached a slight increment (Fig. 2a). During the first 10 min of recording in darkness, RP duration was longer in the daytime (around 250 msec) than in the nighttime experiments (around 150 msec) (Fig. 2b). After continuous illumination, the duration of the RP was reduced and the original values were recovered during the dynamical state of the dark adaptation. In Figs. 2a and 2b the arrows point to the time when a perfusing solution was changed for fresh VH solution. This change did not modify the RP values. Statistical comparison of these responses showed differences (P , 0.05) only between nighttime and

daytime groups. These differences were present for all the variables except for the rate of repolarization (RdV/dt) which was the same in the four groups. Sex differences could not be detected under control or under any experimental conditions. Therefore, we decided to combine the female and male groups. Under VH control conditions, the pooling of nighttime groups showed statistical differences when compared with the pooled daytime group (Table 2, first row). General short-term hormone actions. None of the hormones tested affected the resting membrane potentials or the mean TP amplitude during the first 10 min in darkness. The values recorded were in the same range as the control values, regardless of the hour. RP duration, however, changed frequently from the first 10 min of the experiment. Depending on the hormone employed, 10 min of background light affected both TP and D. In the recuperation rate of RP parameters to initial values, differences also occurred during both the dynamical range and the stable range of the adaptationto-darkness processes. Corticosterone actions. Resting membrane potential of retinular cells perfused with corticosterone

TABLE 2 Mean Values of R-40 Transient phase (%)

VH Co Pg Ec

RP duration (msec)

DdV/dt (%)

RdV/dt (%)

Night

Day

Night

Day

Night

Day

Night

Day

73.68 6 7.03* 94.31 6 4.31*,**,*** 98.50 6 6.23** 76.44 6 7.34***

113.53 6 8.12 115.21 6 5.45** 106.20 6 8.61** 83.43 6 9.21***

159.70 6 10.60* 196.00 6 7.60*** 440.70 6 40.00*,**,*** 179.40 6 13.10***

254.80 6 15.30 218.30 6 9.90** 244.50 6 17.60** 215.40 6 17.20***

80.00 6 3.00* 93.90 6 2.20** 91.70 6 2.50** 78.90 6 2.60***

100.90 6 3.00 101.20 6 1.60** 99.20 6 2.90** 80.90 6 7.40***

106.10 6 3.90 106.10 6 3.60 105.70 6 3.50 108.80 6 4.90

108.70 6 6.00 101.70 6 3.50 96.90 6 6.00 98.08 6 6.70

Note. VH–night, n 5 10; VH–day, n 5 11; Co–night, n 5 9; Co–day, n 5 9; Pg–night, n 5 8; Pg–day, n 5 7; Ec–night, n 5 6; Ec–day, n 5 6. * P , 0.05 for comparison between nighttime and daytime groups. ** P , 0.05 for comparison with VH-N group. *** P , 0.05 for comparison with VH-D group.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

260

solution was 235.2 6 0.5 mV (n 5 18). TP amplitude and D remained in the same range as cells in VH solution. Figure 3a shows the normalized values of TP obtained from experiments with corticosterone (150 µM) compared with the pooled VH recordings. During the first 10 min in darkness and 10 min in light, corticosterone did not produce changes in the transient phase of the receptor potential (n 5 9). Corticosterone actions on TP were evident in the nighttime TP recovery rate which was greater than that in the VH solution. TP amplitude remained fairly constant once the preparation was returned to VH solution. Night–day differences were also observed. Figure 3b shows the duration of the receptor potential in corticosterone and in VH solution, in both nightand daytime experiments. In corticosterone solution the mean duration of the receptor potential was in the range of 200 msec, regardless of the hour in which the experiment was performed. Continuous light stimulation reduced this value (to about 120 msec), and during dark adaptation the initial value was recovered in 20 min. The change of perfusing solution, to VH solution (arrow in Fig. 3b), slightly reduced the RP duration in both nighttime and daytime recordings. A statistical comparison of RPs after 40 min in corticosterone solution and in VH control solution is shown in Table 2. In the corticosterone solution only the transient phase maintained the night–day differences. Corticosterone increased the D values in the

Herna´ndez-Falco´n et al.

nighttime and slightly decreased those of the daytime experiments, and this is the main reason for the disappearance of night–day differences in duration. Progesterone actions. The resting membrane potential of retinular cells perfused with progesterone solution was 233.5 6 0.2 mV (n 5 15). The RP parameters did not show significant changes with respect to those of controls. In the first 10 min of the experiment, eyestalk perfusion with progesterone (150 µM) did not produce changes in the transient phase of the RP (Fig. 5a), either in daytime (n 5 7) or nighttime (n 5 8) experiments. During the 10 min in light there was a similar drop in all TP recorded. The dark adaptation curve showed a recovery rate of TP amplitude equivalent to those of VH solution. During the stable phase of the dark adaptation curve, just a slight decrease could be observed after the progesterone was washed in VH saline during the daytime. Progesterone effects were more conspicuous in the RP duration of nighttime experiments. In the first 10 min of hormone perfusion, RP duration was twice that of controls (Fig. 4b, solid circles). This value (around 400 msec) was maintained for the rest of the experiment, regardless of the continuous illumination, the darkness, or the changing of the perfusing solution (arrow in Fig. 4b). In regard to controls, progesterone did not produce any change in the duration of the receptor potential in the daytime experiments (Fig. 4b, open circles).

FIG. 3. Changes in TP (a) and D (b) of the RP recorded from eyestalks immersed in VH solution (triangles) and in corticosterone (150 µM) added solution (circles). Responses were recorded between 1000 to 1200 hr (open symbols, n 5 9) and between 2200 and 2400 hr (solid symbols, n 5 9). Procedures were the same as those in Fig. 2. Data from female and male animals were pooled together. The amplitude of the transient phase in nighttime corticosterone experiments was higher than that of controls. In corticosterone, duration of RP diminished in the day and increased at night. Effects were partially reversed after the withdrawal of the corticosterone (arrow).

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

Action of Steroids on Photoreceptors

261

FIG. 4. Changes in TP (a) and D (b) of the RP recorded from eyestalks immersed in VH solution (triangles) and progesterone (150 µM) solution (circles, n 5 15). Responses were recorded between 1000 to 1200 hr (open symbols, n 5 7) and between 2200 and 2400 hr (solid symbols, n 5 8). Procedures were the same as those in Fig. 2. Data from female and male animals were pooled together. In progesterone experiments the amplitude of TP increased during the night and decreased during the day. The duration of RP increased strongly in the night and persisted after the withdrawal of the experimental solution (arrow).

Table 2 shows a comparison of RP after 40 min in progesterone solution with those obtained at the same time in VH solution. Differences between control and progesterone recordings reside mainly in the RP duration of nighttime experiments. Ecdysterone actions. Resting membrane potential of retinular cells immersed in ecdysterone solution and measured during the first 10 min of the experiment was 235.7 6 0.7 mV; the parameters of the RP remained substantially the same as that in the controls. Figure 5a shows the actions of 20-OH-ecdysterone

(150 µM) bathing the eyestalk. A behavior similar to that in VH experiments was observed in both the first 10 min of darkness and the first 10 min of light exposure. The TP recovery rate during both dynamical state and stable state of dark adaptation was lower in both daytime (n 5 6) and nighttime (n 5 6) recordings than that in VH controls. After the withdrawal of the hormone and up to 40 min later nighttime and daytime experiments had TP amplitudes lower than those of nighttime controls. Initially, ecdysterone changed the duration of the

FIG. 5. Changes in TP (a) and D (b) of the RP recorded from eyestalks immersed in VH solution (triangles) and in ecdysterone (150 µM) solution (circles, n 5 12). Responses were recorded between 1000 to 1200 hr (open symbols, n 5 6) and between 2200 and 2400 hr (solid symbols, n 5 6). Procedures were the same as those in Fig. 2. Data from female and male animals were pooled together. The amplitude of the transient phase decreased in ecdysterone and that was stronger during the day than that at night. The duration of the RP decreased during the day, and the effect was reverted after the return to VH solution (arrow).

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

262

receptor potential according the day hour, showing values similar to that in controls in VH. Ten minutes of light also induced a drop of D similar to that in controls. The recovery rate of D during the dark adaptation and the D values during 20 min after the withdrawal of the hormone showed that only slight differences between daytime and nighttime could be observed (Fig. 5b), although these differences were smaller than those in VH solution. A comparison between RP after 40 min in an ecdysterone solution and that obtained at the same time in a VH solution is shown in Table 2. RP amplitude decreased when compared with that of controls. A decrease in the differences between nighttime and daytime experiments in ecdysterone recordings also appears clearly.

Long-Term Experiments Controls. Long-term experiments were performed between 1500 and 1800 hr. Typical resting potentials upon electrode penetration were found at 235.6 6 2.3 mV (n 5 18). Stimulation with the test flashes (1800 Ix, 10 µsec) produced receptor potentials, whose mean

Herna´ndez-Falco´n et al.

values, measured after 10 min in darkness, were TP amplitude, 9.5 6 0.8 mV; RP duration, 197 6 5.67 msec; DdV/dt, 29.5 6 7 V/sec; and RdV/dt 4 6 0.1, V/sec (n 5 18). There were no differences between females and males; therefore, female and male data were pooled together. Figure 6a shows, in open triangles, the mean dark adaptation curves for the TP of the pooled data recorded in a VH solution. The duration of the receptor potential in a control solution is shown by the triangles appearing in Fig. 6b. Recordings done in eyestalks from animals injected with 0.1 ml of DMSO 1% solution 24 hr before were similar to those of controls (solid triangles in Figs. 6a and 6b). General long-term hormone actions. Injection of 0.1 ml corticosterone, progesterone, or 20-OH-ecdysterone (150 nM) solution in the adult crayfish (6–8 cm long, 10–20 g wet mass) did not produce any noticeable change in receptor potential, as appeared in recordings taken 24 hr after the treatment. No changes occurred in the RP values when they were evaluated after the first 10 min in darkness. However, changes associated with the application of 10 min of light, the recovery rate during the dynamical phase of dark

FIG. 6. Long-term changes in TP (a) and D (b) of the RP recorded from eyestalks immersed in VH solution. Data correspond to control animals (triangles, n 5 18) and corticosterone (circles, n 5 18)-, progesterone (crosses, n 5 15)-, ecdysterone (asterisks, n 5 18)-, and DMSO (solid triangles, n 5 3)-injected animals. Responses were recorded between 1600 to 1800 hr. Procedures were the same as those in Fig. 2 except that the eyestalks were perfused in VH along the recordings. Data from female and male animals were pooled together. The amplitude of the transient phase had lower values in the experimental than in the control solutions. The value was lower in corticosterone-injected animals, with intermediate values in progesterone-, ecdysterone-, and DMSO-injected animals. The duration of the RP increased substantially in ecdysterone-injected animals.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

263

Action of Steroids on Photoreceptors

TABLE 3 R-40 Mean Values for Long-Term Experiments in Normal Van Harreveld Solution

Female (n 5 9) Male (n 5 9)

Transient phase (%)

Duration (msec)

DdV/dt (%)

RdV/dt (%)

134.02 6 21.73 110.50 6 7.42

204.22 6 8.59 189.78 6 7.06

109.10 6 4.79 100.58 6 6.61

95.96 6 2.45 96.18 6 3.57

adaptation, and the final value reached during the stable phase of dark adaptation were dependent on the hormone considered. Hemolymph volume in this species ranges between 3 and 5 ml and we injected 150 nmol of each of the tested hormones, an increase of the tested hormones ranging between 30 and 50 nmol/ml or between 7.5 and 15 nmol/g of wet mass. This implies an increase between 30 and 50 times the intermolt concentration of ecdysterone reported in Astacus leptodactylus (Durliat et al., 1988). Corticosterone actions. The resting membrane potential of retinular cells from corticosterone-injected animals recorded in the first 10 min of experiment was 235.5 6 0.8 mV (n 5 18). During the first 10 min in darkness, and 10 min in light, the TP showed an amplitude similar to that recorded in VH solution. The recovery rate of the TP was, however, reduced when the eyestalk was returned to darkness. Then, it decreased continuously for the rest of the experiment (Fig. 6a, circles). In the first 10 min in darkness the duration of the receptor potential in corticosterone-treated crayfish was the same as that in controls; 10 min of light reduced the duration to about 100 msec. The recovery of D was complete after 10 min in darkness and remained stable until the experiment ended, although its values were slightly lower than those in controls (Fig. 6b; circles). Table 4 shows mean values of RP measured 40 min after the beginning of the experiments. The first row

shows control values, and the second row corresponds to corticosterone-treated animals (n 5 18). Statistical differences (P , 0.05) were present in the transient phase which were lower in corticosterone-treated animals than in the controls. Reduction in the depolarization rate (DdV/dt) was concomitant with this. RP duration was lower in experimental than in control animals and seems to be reflected in the increase in the rate of repolarization RdV/dt. Progesterone actions. The resting membrane potential of retinular cells from progesterone-treated animals recorded in the first 10 min of the experiment was 234.2 6 0.7 mV (n 5 15). During the first 10 min in darkness the TP was slightly lower in treated than in control eyestalks; with 10 min of light, it showed a reduction similar to those of the control. The dynamical phase of dark adaptation, however, showed a recovery rate of TP different than that of controls: during the first 20 min in darkness there was a recovery to about 80% of the initial values, i.e., lower than that in VH solution and maintained until the end of the experiments (Fig. 6a, crosses). The duration of the receptor potential in progesterone-injected animals had the same values as the controls. The temporal course of reduction by light and recovery rate in darkness in both dynamical and stable phases overlapped with those of the controls (Fig. 6b, crosses). Table 4 shows the comparison between control and progesterone-treated animals (n 5 15). Forty minutes

TABLE 4 R-40 Mean Values for Long-Term Experiments

VH (n 5 18) Co (n 5 18) Pg (n 5 15) Ec (n 5 18)

Transient phase (%)

Duration (msec)

DdV/dt (%)

RdV/dt (%)

112.08 6 5.73 78.55 6 6.32* 95.63 6 5.57* 109.77 6 5.91

197.00 6 5.67 173.50 6 5.60* 213.58 6 15.18 458.23 6 35.01*

104.28 6 3.87 87.43 6 3.87* 98.71 6 3.50 104.45 6 3.87

96.89 6 2.51 110.39 6 2.72* 104.73 6 2.50* 94.57 6 2.61

* P , 0.05 for comparison with VH.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

264

after starting the experiment the transient phase in treated animals was significantly lower (P , 0.05) than that in controls. The RP duration and the rate of depolarization (DdV/dt) were in the same range in the experimental and the control groups. The rate of repolarization (RdV/dt) in the progesterone-treated group was higher (P , 0.05) than that in the control group. Ecdysterone actions. The resting membrane potential of retinular cells from ecdysterone-injected animals was 236.4 6 0.4 mV (n 5 18), measured in the first 10 min of the experiment. Response curves for ecdysterone-treated animals are shown in Fig. 6 (asterisks). The values of TP of experimental and control groups followed the same profile during each stage of the experiments, including the dynamical and stable phases of dark adaptation (Fig. 6a). The mean RP duration values obtained during the first 10 min in darkness were more than twice those of controls. Continuous light stimulation reduced the RP duration 50%. Twenty minutes after the end of the light stimulus, the mean value of D recovered its initial value which was the same until the end of the experiment (Fig. 6b, asterisks). Table 4 shows a comparison between mean values in controls and ecdysterone-treated animals (n 5 18) in the parameters of RP 40 minutes after the beginning of the experiment. Note that only the RP duration was different between the control and the experimental groups (P , 0.05).

DISCUSSION In many invertebrate photoreceptors the electrical response to light (the RP) is a graded depolarization with two main components, a fast depolarization or transient phase, followed by a slow depolarization which precedes repolarization. The transient phase has been associated with light intensity and the slow depolarization to stimulus duration (Naka and Kuwabara, 1959). Owing to the short duration of the light stimulus we employed (10 µsec), it was not possible to separate both components and only the transient phase was measured in this work. Under our experimental conditions the amplitude of the transient phase was equivalent to the amplitude of the receptor poten-

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

Herna´ndez-Falco´n et al.

tial. While the preparation was in a normal saline solution, the magnitude of the mean values of resting membrane potential, transient phase, duration, rates of depolarization, and repolarization, as well as their temporal course for each stage of the experiment, confirms results reported in previous works (Eguchi, 1965; Fuentes-Pardo et al., 1984; Cummins and Goldsmith, 1986; Herna´ndez-Falco´n and Fuentes-Pardo, 1991). We previously described diurnal changes in visual photoreceptor sensitivity (Fuentes-Pardo et al., 1984) by means of dark adaptation curves. This form allowed us to explore visual response dynamic conditions which mainly involve the excitability of the retinular cells and exclude the role of the shielding pigments that show minimum changes in the isolated eyestalk (Sa´nchez and Fuentes-Pardo, 1977).

Night–Day Differences The night–day variations in the recovery rate of dark adaptation curves express changes in the light sensitivity of the photoreceptor cells that can be associated with a circadian rhythm. This proposition is supported by previous work in intact crayfish, isolated eyestalk, and individual retinular cells (for a review see FuentesPardo and Herna´ndez-Falco´n, 1993). It is noteworthy that night–day differences are the most clear when the eyestalk is immersed in a saline solution. In a corticosterone solution, for example, night–day differences were detected to a lesser degree, only in the recovery rate of the TP, while in progesterone and ecdysterone solutions, they only appeared in the recovery rate of D. For progesterone, night–day differences in TP seem to disappear due to a greater increase in the nighttime response than in the daytime response. For ecdysterone there is a greater reduction in the recovery rate of TP during the nighttime experiments than during those made at day. On the basis of the role of calcium on the ERG circadian rhythm recorded from the eyestalk of the crayfish (Fuentes-Pardo et al., 1985) it is plausible to propose that the reduction in the night–day differences detected in the experimental solutions can be attributable to changes in the calcium level related to hormonal actions on the photoreceptor cell (see next paragraphs).

Action of Steroids on Photoreceptors

In the vertebrate, steroid receptor levels change along the 24-hr cycle (Spencer et al., 1993) and this may be related to circadian changes in hormone levels. Although this has not been proved in crayfish (Bidmon, 1991; Bidmon et al., 1991; Fahrbach, 1992; Ueno et al., 1992; Elhaj et al., 1994), the neural tissue of other arthropods has steroid receptors in many places (Bidmon, 1991; Bidmon et al., 1991; Fahrbach, 1992; Ueno et al., 1992; Elhaj et al., 1994), and the production of ecdysterone displays a circadian rhythm (Vafopoulu and Steel, 1991, 1992). These results point in the same direction: a change in the number or the affinity of steroid receptors in the retinular cells. Binding experiments must be done to test this hypothesis.

Short-Term Actions of Steroids Steroid hormones have both genomic and nongenomic actions upon diverse cell types. A common criterion to differentiate between these two actions is the duration of interaction of the hormone and the studied cell. It is accepted that for short-term effects this time must not exceed 1 hr (Fuentes-Pardo et al., 1990; Fink et al., 1992; McEwen, 1994). For the shortterm experiments we kept the eyestalk immersed in the hormone solution for a maximum of 40 min. After this time the eyestalk was perfused with fresh VH saline. High doses of progesterone and ecdysterone, and equivalent doses of corticosterone, were employed to obtain the maximum effect in the minimum time (Horn et al., 1968; Hopkins, 1986; Durliat et al., 1988; Walgrave et al., 1988). Minimal short-term actions of corticosterone could be detected. There was a discrete increase in the amplitude recovery rate of the nighttime response which, however, did not reduce the night–day differences. In a certain way, we employed the corticosterone as a hormonal control, i.e., a hormone with which we did not expect any effect due to its absence in crayfish. The small effect could be due to receptor capability for binding the hormone as it occurs in some insects (Bradbrook et al., 1990). Short-term actions of progesterone upon the response of retinular cells can be described as a discrete increase in the transient phase recovery rate, and a high increase on the RP duration during the nighttime.

265

Similar effects are found when extracellular calcium is reduced or the retinular cell is strongly dark adapted (Fuentes-Pardo et al., 1984; Herna´ndez-Falco´n and Fuentes-Pardo, 1991). A similar effect can be produced when intracellular pH is increased (Brown and Meech, 1976). Progesterone actions cannot be explained by a direct effect of the hormone with the GABAA receptor if one takes into account the apparent lack of GABAA receptor in crustaceans, and therefore in crayfish, which has been proposed by Oliver et al. (1991). However, there are GABA-ergic neurons in diverse regions of the crayfish nervous system (Mulloney and Hall, 1990) and GABA-gated Cl2 channels in neural and muscle tissue of crayfish (Pasternack et al., 1992). Then, progesterone actions could be due to a GABA potentiation by its d-4-3-ceto configuration (Wu et al., 1990) or by a metabolic product with 3-a configuration in the A ring as it occurs in vertebrates (Harrison et al., 1987). If progesterone effects upon the retinular cell are produced by an increase in gCl2 the result should be a hyperpolarization, and a higher RP with long duration caused by a decrease in the Ca21 entrance induced by the hyperpolarization (O’Day et al., 1982; Payne and Fein, 1986; O’Day and Gray-Keller, 1989). Although we did not find changes in resting membrane potential, the effects found include the increase in transient phase and in RP duration. Therefore, we can hypothesize that the short-term effects of progesterone, and to a lesser degree of corticosterone, could be due to an indirect decrease in the intracellular levels of Ca21. More experiments are necessary to test this hypothesis. Short-term ecdysterone actions reduce TP recovery rate and, to a lesser degree, recovery rate of D. To our knowledge, there are no previous reports about this kind of actions of ecdysterone. As in the case of progesterone, ecdysterone does not seem to act through GABAA receptors. The molting hormone 20-OHecdysterone has an OH2 in the a position in C3 (Horn et al., 1968; Bradbrook et al., 1990) and this condition can explain the actions of the hormone upon the photoreceptor via a GABA potentiation, as it occurs in some neurons (Majewska et al., 1986, 1990; Harrison et al., 1987; MacIver and Roth, 1987). GABA induces depolarization and an inward current of some neurons in crayfish (MacIver and Roth, 1987; Kaila et al., 1992) but we did not detect a sustained depolarization while

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

266

ecdysterone was perfusing the eyestalk. Another explanation of the short-term actions of ecdysterone involves changes in intracellular Ca21; the observed changes in retinular cell response resemble those observed during light adaptation (reduced TP and duration) or those occurring when extracellular Ca21 is increased (Herna´ndez-Falco´n and Fuentes-Pardo, 1991).

Long-Term Effects of Steroids Genomic actions of steroids include increases in number and size of dendrites (McEwen and Woolley, 1994; Kawashima and Takagi, 1994; Jo¨els and Karst, 1995; Landgren and Selstam, 1995), changes in cell position (Tobet et al., 1994), expression or repression of genes or functions (Kupferberg et al., 1991; Loi and Tublitz, 1993; Seong et al., 1993; Tublitz and Loi, 1993; Jindra et al., 1994; Matsumoto et al., 1994), and changes in membrane conductance to some ions (Irwin et al., 1992; Jo¨els and Karst, 1995). Morphological effects of progesterone (or corticosterone) upon retinular cells are possible although we have no evidence suggesting an increase in cell size or a change in connectivity. Other changes, however, could be detected in the photoreceptor cells when the steroids were injected 24 hr before the electrical recording. Corticosterone has an irreversible effect in the recovery rate of TP, and progesterone modified the receptor potential in the same direction although it has a lesser effect. The reduction in TP amplitude observed in eyestalks from corticosterone- and progesteroneinjected crayfish could be due to a change in the processes of adapting to darkness, which never attains the same recovery rate as that in VH, rather than to changes in the number or quality of the membrane channels participating in responses to light and/or in photopigment synthesis and storage. Although there are no reports on long-term actions of corticosterone in crayfish, our results are in line with the possibility of receptor-mediated binding of this hormone in some insects (Bradbrook et al., 1990). In hippocampus, corticosterone inhibits neuronal excitability and one of the mechanisms proposed involves changes in intracellular Ca21 concentration (Jo¨els and de Kloet, 1992). During light adaptation in crayfish retinular cells, the amplitude of TP and the duration of the RP are reduced. The mechanisms proposed include an increase in intracellular Ca21 via second messen-

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

Herna´ndez-Falco´n et al.

gers, similar to that described for other depolarizing photoreceptors. Opposite changes seem to occur during dark adaptation (Payne and Fein, 1986; Herna´ndezFalco´n and Fuentes-Pardo, 1991). The observed changes in long-term experiments could be due to an increase in intracellular Ca21 that would resemble a fictitious light adaptation state and a decrease in retinular cell excitability. This effect could be similar for both corticosterone and progesterone. Injection of a single dose of ecdysterone has intense effects on the duration of the RP, even after 24 hr. The increase in RP duration resembles a strong dark adaptation state or a decrease in extracellular calcium concentration. Although there are descriptions of longterm actions of ecdysterone on diverse cell types, there is a lack of information concerning the actions on excitability of neural cells (Bidmon, 1991; Fahrbach, 1992; Ueno et al., 1992; Tublitz and Loi, 1993; Elhaj et al., 1994; Fahrbach et al., 1994). One way to explain the increase in RP duration without any other noticeable change could be a reduction in calcium entry to the retinular cell, which could be linked to an increase in the systemic calcium sequestering mechanisms activated by the hormone. However, we do not have evidence for this hypothesis. Summarizing, our results show that corticosterone, progesterone, and ecdysterone have both short- and long-term actions on the electrical response to light of crayfish photoreceptor cells; these effects are enhanced when the retinular cells are studied during the process of dark adaptation. Short-term effects and long-term effects are different in magnitude and direction; shortterm effects depend on the time of day. Progesterone and corticosterone have similar actions in short-term and long-term experiments. The effects of ecdysterone were opposite to those of the other hormones. These findings raise the possibility of steroids affecting excitability at the membrane, as well as at the intracellular level, on crayfish retinular cells. The mechanisms involved resemble the effects of modifying intracellular levels of calcium ions.

ACKNOWLEDGMENTS The authors are indebted to Dr. M. Hiriart and Dr. F. Ferna´ndez-deMiguel for their helpful criticism and discussion in the elaboration of

Action of Steroids on Photoreceptors

the paper. We thank Mrs. M. Cerrilla for technical assistance. This work was partially supported by Grants IN213394 DGAPA and 2153-P-M CONACYT.

REFERENCES Are´chiga, H., Ban˜uelos, E., Frixiones, E., Picones, A., and Rodrı´guezSosa, L. (1990). Modulation of crayfish retinal sensitivity by 5-hydroxytriptamine. J. Exp. Biol. 150, 123–143. Beltz, B. S. (1988). Crustacean neuro-hormones. In ‘‘Endocrinology of Selected Invertebrate Types,’’ pp. 235–258. A. R. Liss, New York. Bidmon, H. J. (1991). Developmental changes in the presence of ecdysteroid receptors in the central nervous system of third instar larvae of Sarcophaga bullata. Dev. Brain Res. 63, 121–133. Bidmon, H. J., Stumpf, W. E., and Granger, N. A. (1991). Ecdysteroid binding sites localized by autoradiography in the central nervous system of precommitment fifth-stadium Manduca sexta larvae. Cell Tissue Res. 263(1), 183–194. Blackmore, P. F. (1993). Rapid non-genomic actions of progesterone stimulate Ca21 influx and the acrosome reaction in human sperm. Cell Signal 5(5), 531–538. Bradbrook, D. A., Clement, C. Y., Cook, B., and Dinan, L. (1990). The occurrence of vertebrate-type steroids in insects and a comparison with ecdysteroid levels. Comp. Biochem. Physiol. B. 95(2), 365–374. Brown, H. M., and Meech, R. W. (1976). Intracellular pH and light adaptation in barnacle photoreceptors. J. Physiol. 263, 218P. Cassier, P., Baghdassarian-Chalaye, D., de Besse, N., Papillon, M., Baldaia, L., and Porcheron, P. (1988). Ecdysteroids and activation of epidermal cells in the locust, Locusta migratoria. J. Insect. Physiol. 34(7), 669–677. Christ, M., Dowes, K., Eisen, C., Bechtner, G., Theissen, K., and Wehling, M. (1994a). Rapid effects of aldosterone on sodium transport in vascular smooth muscle cells. Hypertension 25(1), 117–123. Christ, M., Sippel, K., Eisen, C., and Wehling, M. (1994b). Nonclassical receptors for aldosterone in plasma membranes from pig kidneys. Mol. Cell. Endocrinol. 99(2), R31–R34. Cummins, D. R., and Goldsmith, T. (1986). Responses of crayfish photoreceptor cells following intense light adaptation. J. Comp. Physiol. A. 158, 35–42. Dauphin-Villemant, C., Bocking, D., and Sedlmeier, D. (1995). Regulation of steroidogenesis in crayfish moulting glands: Involvement of protein synthesis. Mol. Cell. Endocrinol. 109(1), 97–103. De Bold, J. F., and Frye, C. A., (1994a). Genomic and non-genomic actions of progesterone in the control of hamster sexual behavior. Horm. Behav. 28(4), 445–453. De Bold, J. F., and Frye, C. A. (1994b). Progesterone and the neural mechanisms of hamster sexual behavior. Psychoneuroendocrinology 19(5–7), 563–579. Durliat, M., Morniere, M., and Porcheron, P. (1988). Changes in ecdysteroids in Astacus leptodactylus during the molting cycle. Comp. Biochem. Physiol. A 89(2), 223–229. Eguchi, E. (1965). Rhabdom structure and receptor potentials in single crayfish retinular cells. J. Cell. Comp. Physiol. 66, 411–429.

267 Elhaj, A. J., Harrison, P., and Chang, E. S. (1994). Localization of ecdysteroid receptor immunoreactivity in eyestalk and muscle tissue of the American lobster Homarus americanus. J. Exp. Zool. 270(4), 343–349. Fahrbach, S. E. (1992). Developmental regulation of ecdysteroid receptors in the nervous system of Manduca sexta. J. Exp. Zool. 216(3), 245–253. Fahrbach, S. E., Choi, M. K., and Truman, J. W. (1994). Inhibitory effects of actinomycin-D and cycloheximide on neurone death in adult Manduca sexta. J. Neurobiol. 25(1), 59–69. Ffrench-Mullen, J. M., and Spence, K. T. (1991). Neurosteroids block Ca21 channel current in freshly isolated hippocampal CA1 neurons. Eur. J. Pharmacol. 202(2), 269–272. Fink, G., Rosie, R., Sheward, W. J., Thomson, E., and Wilson, H. (1992). Steroid control of central neural interactions and function. J. Steroid Biochem. Mol. Biol. 40(1–3), 123–132. Fuentes-Pardo, B., Herna´ndez-Falco´n, J., and Noguero´n, I. (1984). Effect of external level of calcium upon the photoreceptor potential of crayfish along the twenty-four hour cycle. Comp. Biochem. Physiol. A 78, 723–727. Fuentes-Pardo, B., Verdugo-Dı´az, L., and Incla´n-Rubio, V. (1985). Effect of external level of calcium on ERG circadian rhythm in isolated eyestalk of crayfish. Comp. Biochem. Physiol. A 82, 385–389. Fuentes-Pardo, B., Herna´ndez-Falco´n, J., Vela´zquez, P. N., and Romano, M. C. (1990). Role of corticosterone on the development of passive electrical properties of chick embryo neurons. J. Dev. Physiol. 13(1), 67–73. Fuentes-Pardo, B., and Herna´ndez-Falco´n, J. (1993). Neurobiology of the circadian clock of crayfish. Trends Comp. Biochem. Physiol. 1, 635–673. Gee, K. W., Lan, N. C., Bolzer, M. B., Wieland, S., Belelli, D., and Chen, J. S. (1992). Pharmacology of a GABAA receptor coupled steroid recognition site. Adv. Biochem. Psychopharmacol. 47, 111–117. Hales, T. G., Kim, H., Longoni, B., Olse, R. W., and Tobin, A. J. (1992). Immortalized hypothalamic GT1–7 neurons express functional gamma-aminobutyric acid type A receptors. Mol. Pharmacol. 42(2), 197–202. Harrison, N. L., Majewska, M. D., Harrington, J. W., and Barker, J. L. (1987). Structure–activity relationships for steroid interaction with the gamma aminobutyric acidA receptor complex. J. Pharmacol. Exp. Ther. 241, 346–353. Herna´ndez-Falco´n, J., and Fuentes-Pardo, B. (1991). Crayfish retinular cells: Influence of extracellular sodium and calcium upon the receptor potential. Comp. Biochem. Physiol. A 100, 823–832. Hopkins, P. M. (1986). Ecdysteroid titers and Y-organ activity during late anecdysis and precdysis in the fiddler crab Uca pugilator. Gen. Comp. Endocrinol. 63, 362–373. Horn, D. H. S., Fabbri, S., Hampshire, F., and Lowe, M. E. (1968). Isolation of crustecdysone (20R-hydroxyecdysone) from a crayfish (Jasus Ialandei H. Milne-Edwards). Biochem. J. 109, 399–406. Hua, S. Y., and Chen, Y. Z. (1989). Membrane receptor-mediated electrophysiological effects of glucocorticoid on mammalian neurons. Endocrinology 124(2), 687–691. Irwin, R. P., Maragakis, N. J., Rogawski, M. A., Purdy, R. H., Farb, D. H., and Paul, S. M. (1992). Pregnenolone sulfate augments

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

268 NMDA receptor medianted increases in intracellular Ca21 in cultured rat hippocampal neurons. Neurosci. Lett. 141(1), 30–34. Jindra, M., Sehnal, F., and Riddford, L. M. (1994). Isolation, characterization and development expression of the ecdysteroid-induced E75 gene of the wax moth Galleria mellonella. Eur. J. Biochem. 221(2): 665–675. Jo¨els, M., and de Kloet, E. R. (1992). Control of neuronal excitability by corticosteroid hormones. Trends Neurosci. 15(1), 25–30. Jo¨els, M., and Karst, H. (1995). Effects of estradiol and progesterone on voltage-gated calcium and potassium conductances in rat CA1 hippocampal neurons. J. Neurosci. 15(6), 4289–4297. Kaila, K., Rydqvist, B., Pasternack, M., and Voipio, J. (1992). Inward current caused by sodium dependent uptake of gaba in the crayfish strecht receptor neurone. J. Physiol. 453, 627–645. Kato, Y., and Riddiford, L. (1987). The role of 20-hydroxyecdysone in stimulating epidermal mitoses during the larval–pupal transformation of the tobacco hornworm, Manduca sexta. Development 100, 227–236. Kawashima, S., and Takagi, K. (1994). Role of sexsteroids on the survival, neuritic outgrowth of neurons and dopamine neurons in cultured preoptic area and hypothalamus. Horm. Behav. 28, 305– 312. Kupferberg, A., Cremel, G., Behr, P., Van Dorselaer, A., Luu, B., and Mersel, M. (1991). Differential sensitivity of astrocyte primary cultures and spontaneous transformed cell lines to 7 betahydroxycholesterol: Effect on plasma membrane lipid composition and fluidity, and on cell surface protein expression. Mol. Cell. Biochem. 101(1), 11–22. Landgren, S., and Selstam, G. (1995). Interaction of 17b-oestradiol and 3a-hydroxy-5a-pregnane-20-one in the control of neuronal excitability in slices from the CA1 hippocampus in vitro of guinea-pigs and rats. Acta Physiol. Scand. 154, 165–176. Loi, P. K., and Tublitz, N. J. (1993). Hormonal control of transmitter plasticity in insect peptidergic neurons. I. Steroid regulation of the decline in cardioacceleratory peptide 2 (CAP2) expression. J. Exp. Biol. 181, 175–194. Luo, Y., Amin, J., and Voullmy, R. (1991). Ecdysterone receptor is a sequence-specific transcription factor involved in the developmental regulation of heat-shock genes. Mol. Cell. Biol. 11(7), 3660–3675. MacIver, M. B., and Roth, S. H. (1987). Anesthetics produce differential actions on membrane responses of the crayfish stretch receptor neuron. Eur. J. Pharmacol. 141(1), 67–78. Majewska, M. D., Harrison, N. L., Schwatz, R. D., Barker, J. L., and Paul, S. M. (1986). Steroid hormone metabolites are barbituratelike modulators of the GABA receptor. Science 232, 1004–1007. Majewska, M. D., Demirgo¨ren, S., Spivak, C. E., and London, E. D. (1990). The nerosteroid dehydroepiandosterone sulfate is an allosteric antagonist of the GABAA receptor. Brain Res. 526, 143–146. Matsumoto, A., Arai, Y., Urano, A., and Hyodo, S. (1994). Androgen regulates gene expression of cytoskeletal proteins in adult rat motoneurones. Horm. Behav. 28, 357–366. Mattson, M. P., and Spaziani, E. (1985). Stress reduces hemolymph ecdysteroid levels in the crab: Mediation by the eyestalks. J. Exp. Zool. 234, 319–323. McEwen, B. S. (1991). Non-genomic and genomic effects of steroids on neural activity. Trends Pharmacol. Sci. 12, 141–157.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

Herna´ndez-Falco´n et al.

McEwen, B. S. (1994). How do sex and stress hormones affect nerve cells? Ann. NY Acad. Sci. 743, 1–18. McEwen, B. S., and Woolley, C. S. (1994). Estradiol and progesterone regulate neuron structure and synaptic connectivity in adult as well as developing brain. Exp. Gerontol. 29(3–4), 431–436. Miles, C. I., and Weeks, J. C. (1991). Developmental attenuation of the pre-ecdysis motor pattern in the tobacco hornworm, Manduca sexta. J. Comp. Physiol. A 168(2), 179–190. Mulloney, B., and Hall, W. M. (1990). GABA-ergic neurons in the crayfish nervous system: an immunocytochemical census of the segmental ganglia and stomatogastric system. J. Comp. Neurol. 291(3), 383–394. Nabekura, J., Oomura, Y., Minami, T., Mizuno, Y., and Fukuda, A. (1986). Mechanism of the rapid effect of 17 beta-estradiol on medial amygdala neurons. Science 233, 226–228. Naka, K. I., and Kuwabara, M. (1959). Two components from the compound eye of the crayfish. J. Exp. Biol. 36, 51–61. O’Day, P. M., Lisman, J. E., and Goldring, M. (1982). Functional significance of voltage-dependent conductances in Limulus ventral photoreceptors. J. Gen. Physiol. 79, 211–232. O’Day, P. M., and Gray-Keller, M. P. (1989). Evidence for electrogenic Na1/Ca21 exchange in Limulus ventral photoreceptors. J. Gen. Physiol. 93, 473–494. Okazaki, R. K., and Cheng, E. S. (1991). Ecdysteroids in the embryos and sera of the crabs, Cancer magister and C. anthongi. Gen. Comp. Endocrinol. 81, 174–186. Oliver, A. E., Deamer, D. W., and Akeson, M. (1991). Evidence that sensitivity to steroid anesthetics appears late in evolution. Brain Res. 557, 298–302. Pasternack, M., Rydqvist, B., and Kaila, K. (1992). GABA-gated anion channels in intact crayfish opener muscle fibres and strechreceptor neurons are neither activated nor desensitized by glutamate. J. Comp. Physiol. A 170(4), 521–524. Paul, S. M., and Purdy, R. H. (1992). Neuroactive steroids. FASEB J. 6(6), 2311–2322. Payne, R., and Fein, A. (1986). The initial response of Limulus ventral photoreceptors to bright flashes. released calcium as a synergist to excitation. J. Gen. Physiol. 87, 243–269. Purdy, R. H., Moore, P. H., Jr., Morrow, A. L., and Paul, S. M. (1992). Neurosteroids and GABAA receptor function. Adv. Biochem. Psychopharmacol. 47, 87–92. Sa´nchez, J. A., and Fuentes-Pardo, B. (1977). Circadian rhythm in the amplitude of the electroretinogram in the isolated eyestalk of the crayfish. Comp. Biochem. Physiol. A. 56, 601–605. Seong, J. Y., Lee, Y. K., Lee, C. C., and Kim, K. (1993). NMDA receptor antagonist decreases the progesterone-induced increase in GnRH gene expression in the rat hypothalamus. Neuroendocrinology 58(2), 234–239. Spencer, R. L., Miller, A. H., Moday, H., Stein, M., and McEwen, B. S. (1993). Diurnal differences in basal and acute stress levels of type I and type II adrenal steroid receptor activation in neural and immune tissues. Endocrinology 133(5), 1941–1950. Tobet, S. A., Chickering, T. W., Hanna, I., Crandall, J. E., and Schwartriz, G. A. (1994). Can gonadal steroids influence cell position in the developing brain? Horm. Behav. 28, 320–328.

Action of Steroids on Photoreceptors

Tublitz, N. J., and Loi, P. K. (1993). Hormonal control of transmitter plasticity in insect peptidergir neurons. II. Steroid control of the up-regulation of bursicon expression. J. Exp. Biol. 181, 195–212. Ueno, M., Bidmon, H. J., and Stumpf, W. E. (1992). Ecdysteroid binding sites in gastrolith forming tissue and stomach during the molting cycle in crayfish Procambarus clarkii. Histochemistry 98(1), 1–6. Vafopoulu, X., and Steel, C. G. (1991). Circadian regulation of synthesis of ecdysteroids by prothoracic glands of the insect Rhodnius prolixus: Evidence of a dual oscillator system. Gen. Comp. Endocrinol. 83, 27–34. Vafopoulu, X., and Steel, C. G. (1992). In vitro photosensitivity of

269 ecdysteroid synthesis by prothoracic glands of Rhodnius prolixus (Hemiptera). Gen. Comp. Endocrinol. 86, 1–9. Van Harreveld, A. (1936). A physiological solution for freshwater crustaceans. Proc. Soc. Exp. Biol. Med. B 34, 428–432. Walgrave, H. R., Criel, G. R., Sorgeloos, P., and De Leenheer, P. (1988). Determination of ecdysteroids during the moult cycle of adult Artemia. J. Insect Physiol. 34(7), 597–602. Wehling, M., Kasmayr, J., and Theisien, K. (1991). Rapid effects of mineralocorticoids on sodium–proton exchanger: Genomic or non-genomic pathway? Am. J. Physiol. 260, E719–E726. Wu, F. S., Gibbs, T. T., and Farb, H. A. (1990). Inverse modulation of GABA- and glicine-induced currents by progesterone. Mol. Pharmacol. 37, 597–602.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.