Leonardo Rodríguez-Sosa

Circadian clock function in isolated eyestalk tissue of crayfish

Electrical mass response of crayfish photoreceptors (electroretinogram) was recorded continuously for up to seven days in isolated preparations that consisted of the retina and lamina ganglionaris. Electroretinogram amplitude varied in a circadian manner with a nocturnal acrophase and a period of 22–23h in preparations kept in darkness. Acclimatization of animals to reversed light/dark cycles resulted in a phase reversal of the rhythm in vitro. The per (period) gene of Drosophila has been implicated in the genesis of rhythms in insects and in vertebrates. Immunocytochemical staining with an antibody against the PER gene product revealed immunoreactivity in the retinal photoreceptors, as well as in cell bodies in the lamina ganglionaris. Labelled axons run distally towards the photoreceptors and proximally to other areas of the lamina.

Coupling of Environmental and Endogenous Factors in the Control of Rhythmic Behaviour in Decapod Crustaceans

Behavioural patterns of crustaceans are known to vary within the 24 hour cycle and in relation to environmental signals. Light and chemical stimuli induce specific behavioural responses. Retinal and extra-retinal photoreceptors use different motor responses to illumination selectively. Light responsiveness is modulated at various levels, from the light admittance to the retina, up to the integration in higher order interneurones and motorneurones. An endogenous circadian rhythmicity contributes to the various elements of the system.

Distributed Circadian Rhythmicity In The Crustacean Nervous System

Circadian rhythmicity has long been documented in crustaceans (De Coursey 1983; Arechiga and Rodriguez-Sosa 1997). It is expressed in physiological functions as diverse as locomotion, sensory input, tegumentary and retinal pigment position, heart rate, blood sugar and calcium concentration. Other time domains, such as tidal (Naylor 1996), circaseptan and seasonal cycles (Rodriguez-Sosa et al. 1997) have also been documented but, as in other animal groups, the 24-h cycle appears to be the basic unit of time computation.

Range of modulation of light sensitivity by accessory pigments in the crayfish compound eye

The compound eye of the crayfish during dark adaptation undergoes an enhancement of light sensitivity within a range of 3 log units. Only 1 log unit can be explained by the increase in responsiveness of the retinula cells. The rest can be accounted for by the migration of the proximal and distal accessory pigments. In isolated retinas, with the distal pigment paralysed in light-adapted position and the proximal pigment only partially responsive, the sensitivity enhancement in darkness is reduced in more than 1 log unit. By hormonally inducing the expansion of the distal pigment while the rest of the system remains dark-adapted, there is a shift of one log unit in the V-log 1 curve. In a crayfish mutant devoid of the two dark accessory pigments, the sensitivity enhancement in dark adaptation only covers one log unit.

Circadian and ultradian rhythms in the crayfish caudal photoreceptor

The study of circadian clocks in crustaceans has led to the hypothesis of a distributed circadian system of pacemakers. In this review, we investigate the role of the crayfish caudal photoreceptor (CPR) as a candidate to form part of this pacemaking circadian system. Two circadian rhythms are documented for CPR electrical activity. These rhythms correspond to the spontaneous and light‐induced discharge of action potentials. The intrinsic characterization of the rhythms is made through the analysis of the firing rate of the corresponding action potentials. The discharges were extracellularly recorded in the isolated 6th abdominal ganglion (AG) in an organ culture kept at constant temperature for up to 5 days. For preparations kept in the dark, spontaneous activity varies in a circadian manner, with a period of 24.7 h and the acrophase at subjective nighttime (2140). For light‐induced activity, pulses of constant intensity applied regularly throughout the 24‐h cycle show that the firing rate at peak and latency vary rhythmically. The period for this rhythm is 24.24 h and the acrophase is at subjective dawn (0326). Additionally, an ultradian rhythm of a ∼12‐h period was observed for both rhythms. When tested with light pulses of different intensities, the CPR responsiveness at night is almost one log unit greater than in daytime. The effect of temperature on both activities is also described. The phase‐shift caused by temperature for these circadian rhythms depends on the application time. These results show that the 6th AG is capable of generating a circadian rhythm of electrical activity in the CPR, which in turn is likely to be part of the crayfish circadian system. A possible interaction of different pacemakers forming the distributed circadian system is also discussed. The role of serotonin as a possible modulator of the CPR electrical activity is documented. In addition, the level of the 5‐HT1A receptors displays a diurnal rhythm in the 6th AG, with the acrophase at twilight (1849). We suggest that the 5‐HT1A receptor does participate in this modulation. Finally, the hypothesis of the expression of two circadian oscillators in a single identified neuron is presented. Synapse 62:643–652, 2008.

Neurosecretory Role of Crustacean Eyestalk in the Control of Neuronal Activity

From early histological work with methylene blue staining, the existence of neurosecretory cells was postulated in different regions of the eyestalk. Histochemical work rendered similar results (see Gabe, 1966). The most conspicuous system is that composed by the sinus gland, a neurohemal organ located in the distal part of the eyestalk, between the medulla externa and the medulla interna in many species (Fig. 1A). Its basic structure, as seen in Fig. 1B, is that of a bunch of neurosecretory endings, which are the dilated terminals of axons coming from other regions of the eyestalk, to end in apposition to a blood sinus. From morphological and physiological work, the notion was evolved of the sinus gland as the common end of secretory neurons all over the eyestalk and even of incoming fibers from other central ganglia. However, more recently, from experiments with cobalt backfills, a more restricted origin has been advocated, limiting the source of neurosecretory fibers to the sinus gland, to a group of 100–150 cell bodies clustered in the medulla terminalis and known since long ago as the X organ, or Hanstrom’s organ (Andrew et al., 1978; Jaros, 1978). Only a small number of cells outside this cluster were backfilled from the sinus gland.

SEASONAL RHYTHM OF RED PIGMENT CONCENTRATING HORMONE IN THE CRAYFISH

The content of red pigment concentrating hormone (RPCH) in the eye-stalk of the crayfish Procambarus clarkii varies seasonally, with maximum values during the summer months and the lowest values in winter. The responsiveness of tegumentary chromatophores to synthetic RPCH varies concurrently.

Octopamine cyclic release and its modulation of visual sensitivity in crayfish

The biogenic amine octopamine (OA) modulates invertebrate behavior by changing neuronal responses from sensory inputs to motor outputs. However, the OA modulation of visual sensitivity and its possible coupling to diurnal cycles remains unexplored. Here we studied the diurnal variations in the OA levels in the hemolymph of the crayfish Procambarus clarkii, its release from the structures in the eyestalk and its modulation of the retinal light sensitivity. The hemolymph concentration of OA and its amino acid precursor tyrosine was measured by high-resolution liquid chromatography; OA varied along the 24-hcycle. The peak value appeared about 2h before the light offset which preceded the peak locomotor activity. OA was found in every structure of the eyestalk but displayed higher levels in the retina-lamina ganglionaris. Moreover, OA was released from isolated eyestalks at a rate of 92nmol/eyestalk/min and a calcium-dependent release was evoked by incubation in a high potassium solution. OA injected into dark-adapted crayfish or applied to the isolated retina at concentrations of 1, 10 and 100μM produced a proportionally increasing reduction in the amplitude of the photoreceptor light responses. These OA concentrations did not affect the position of the visual accessory pigments. Our results suggest that OA release in the crayfish eyestalk is coupled to the 24-hcycle to regulate the diurnal reduction of the photoreceptor sensitivity and to favor the expression of exploratory locomotion during the dark phase of the circadian cycle.

Dopaminergic modulation of the caudal photoreceptor in crayfish.

In our study we investigated the influence of dopamine (DA) on the caudal photoreceptor (CPR) in crayfish. Here we report the following: (a) the chromatographic determination of DA in the sixth abdominal ganglion (6th AG) shows a variation in the content during a 24‐h cycle with the maximum value at dawn. (b) There are possibly dopaminergic neurons in the 6th AG with antityrosine hydroxylase antibodies. Immunopositive neurons (164) were located in the anterior and posterior regions of the 6th AG with the mean (± SE) diameter of their somata 23 ± 1 μm. In addition, there is immunopositive staining in axons, neuropilar fibers, and varicosities. (c) We also identified, using immunohistochemistry, 108 neurons in the sixth AG that contain dopamine D1‐like receptors, with the mean (±SE) diameter of their somata 18 ± 1 μm. (d) We examined the exogenous action of DA on the electrical activity of the CPR in the isolated sixth AG by conventional extracellular‐recording methods. This CPR displays spontaneous activity and phasic‐tonic responses to light pulses. Topical application of dopamine to ganglia kept in the dark increased the spontaneous firing rate of the CPR, whereas the photoresponse of the CPR remained unchanged. The effect on the spontaneous activity is dose‐dependent with an ED50 of 33 μM, and is blocked by the dopamine D1‐like antagonist SCH23390. These observations suggested that the DA is playing the role of a neurotransmitter or a neuromodulator of the CPR in the 6th AG in both species of crayfish, Procambarus clarkii and Cherax quadricarinatus. Synapse, 2011.

Seasonal Rhythm of Serotonin Content in the Crayfish Eyestalk

5-Hydroxytryptamine (5-HT) is a common neurotransmitter and modulator in crustaceans. It has been shown to participate in a wide variety of functions, such as a) the facilitation of neuromuscular transmission (Glusman and Kravitz 1982, Dixon and Atwood 1985), b) the enhancement of visual input (Arechiga et al. 1990) and of mechanoreception (El Manira et al. 1991, Rossi-Durand 1993, Pasztor and Golas 1993), c) the induction of the complete behavioral pattern of aggressiveness and social dominance (Kravitz 2000) and the modulation of the escape behavior (Glanzman and Krasne 1986, Yeh et al. 1996), d) the regulation of heart activity (Battelle and Kravitz 1978, Listerman et al. 2000), e) neurohormone release, facilitating that of the crustacean hyperglycemic hormone (Keller and Beyer 1968, Lee et al. 2000), and the molt-inhibit-ing hormone (Mattson and Spaziani 1985) and f) modulating the electrical activity of neurosecretory cells (Saenzetal. 1997, Glowiketal. 1997, Alvarado-Alvarez et al. 2000), and of neurons at various levels of the crustacean central nervous system (Zhang and Harris-Warrick 1994).