Native opioid-like peptides in Squilla mantis ganglia.

To detect the presence of a mammalian-like enkephalin precursor in suboesophageal ganglia of Squilla mantis, an arthropod shown to be sensitive in vivo to opiates [8], protein acid extracts were fractionated by gel filtration into three large pools: A(Mr greater than 65,000), B(10,000 less than Mr less than 65,000) and C(Mr less than 10,000). Only the low molecular weight pool, pool C, showed opioid-like activity when assayed by displacing labeled D-Ala2-D-Leu5-enkephalin from rat brain membranes. After trypsin and carboxypeptidase B proteolysis, pool A remained inactive, while pool B turned out to be active and was shown to inhibit the twitch response of electrically stimulated guinea-pig ileum. After HPLC fractionation of proteolyzed pool B, most of the opioid-like activity was found to be associated with a fraction showing an elution volume different from that of opioid peptide standards. Furthermore, no fraction showed immunoreactivity with anti-Met-enkephalin antibodies. The results suggest that native opioid-like peptides are present in Squilla mantis and are most likely released from higher molecular weight precursor(s).

Role of the GABA chloride ion channel component on aggressive behavior in the male crab.

Picrotoxin blocking effects on GABA-induced inhibition of lateral merus display in the male crab Carcinus mediterraneus suggest that the chloride ion channel component of the GABA molecule is involved in the control of this defensive behavior. The low GABA doses (3 and 5 micrograms/g body weight) inhibited lateral merus display as early as 10 min after drug treatment while the administration of higher GABA concentrations (greater than 5 micrograms/g) prolonged the duration of the behavioral effects. The administration of 1 microgram/g picrotoxin to the animals treated with the effective GABA doses (5, 10 and 20 micrograms/g) restored lateral merus display. The antagonizing activity of picrotoxin on the inhibitory GABA effects, in a temporal manner, demonstrates that GABAergic sites other than peripheral ones are probably participating with the regulation of this agonistic posture. Quantitative autoradiography results revealed interesting receptor levels changes of the specific chloride ion channel antagonist [35S] t-butylbicyclophosphorothionate in the different brain areas of the male crab displaying lateral merus with respect to normal nonreactive animals. Elevated receptor binding levels were encountered in the middle and posterior brain area whereas low levels were obtained at the anterior level. Both the behavioral and autoradiographic results suggest that the defensive type of aggression behavior in crustacean may not only be mediated by a peripherally controlled GABA-gated chloride ion flux but also by a central GABAergic mechanism.

Opiate effect on the threat display in the crab Carcinus mediterraneus.

The well-known defense response of a crab (laterus merus display, LMD) was easily evoked in Carcinus mediterraneus by striking the cephalothoraxic protogastric region between the eyestalks. Following a program aimed at investigating the regulatory action of diverse neuromodulators on the LMD of this crab, a study on the role of opioids was started by testing the effect of morphine administration. Injection of morphine HC1 (MP) (40, 50, 60, 70, or 100 micrograms/g) produced a dose-dependent reduction of the LMD so elicited that dissipated with the postinjection time. Only MP doses higher than 50 micrograms/g were effective 30 min after drug administration. The MP-induced inhibition of LMD was blocked by a 4.8-micrograms naloxone HC1/g dose injected 10 min before MP. These results and those previously obtained as the action of GABA on the LMD of this crab are discussed in connection with results reporting a similar effect of these drugs on another agonistic item of behavior in the crab Chasmagnatus granulatus. The possibility of demonstrating habituation of the LMD to an iterated stimulation in C. mediterraneus and of using such a process to elucidate the acting paths of the drugs is discussed.

The stinging response of the honeybee: effects of morphine, naloxone and some opioid peptides.

Changes in responsiveness for the stinging reaction of honeybees fixed in a holder after receiving 3 electrical shocks delivered with 1 min interval, was registered and used as measurement for the effect of 2 microliter of different solutions injected. Every shock consisted of a train of pulses of 1 msec each, delivered for 2 sec at a frequency of 100 Hz. Injection of morphine-HCl (50 to 200 n-moles/bee) produced a dose dependent reduction of the honeybee stinging response to the electrical shocks. The morphine dose that produced a 50% inhibition of the response (D50) was 148 n-moles/bee (927 micrograms/g), i.e., a value far greater than that reported for vertebrates in behavioral test of analgesia. Naloxone 1.1 micrograms/g produces a significant reduction of morphine D50 effect and at 4-5 micrograms/g, a full disinhibition. Thus, whereas the D50 of morphine for honeybees is far greater than that for vertebrates, the doses of naloxone that antagonize morphine are similar for bees and vertebrates. Possible explanations of this difference are mentioned. Injections of met-enkephalin, leu-enkephalin, kyotorphin and (D-Ala2) methionine-enkephalinamide, given in doses of 200 n-moles/bee, an amount greater than that of the morphine D50, exhibited no effect on the stinging response.

Opiate receptor in praying mantis: effect of morphine and naloxone.

A praying mantis displays a "frightening reaction" called deimatic reaction (DR), any time that it is faced with a patterned visual stimulus that represents a potential damage for the insect. Results of the present paper show that the DR could be also elicited by an actual noxious (an electrical shock) and that this response is similar to that elicited by a potential nociceptive stimulus (a patterned visual stimulus). The DR elicited by the electric shock was used as a model for studying the analgesic effect of opiates. The mantis was placed in an apparatus that allowed us to give the insect an electrical shock and to measure the strength of its DR. During a first session the voltage threshold necessary to induce a full DR was determined, and then, the insect was injected with a certain solution. The voltage threshold was tested one, two and four hours after injection. Mantises that were injected with only distilled water showed no changes in their voltage threshold during the three tests. Injections of 300, 350 and 400 micrograms/g of morphine-HCl increased the voltage threshold in both a time-dependent and a dose related manner. A dose of 350 micrograms/g of morphine-HCl produced 50% of response inhibition after two hours of injections and is referred to as the median antinoxious dose ( AD50 ). Sixteen micrograms/g of naloxone given in conjunction with an AD50 of morphine, partially blocked the effect of morphine during the first hour and fully blocked it during the second hour. Thirty-two micrograms/g of naloxone fully blocked the morphine effect during the first and the second hour. However, more than 48 micrograms/g of naloxone alone also increased the voltage threshold in insects, similar to those described for vertebrates.

Light- and electron-microscopic structure of cells protruding into the mesencephalic ventricle of Scyllium stellare (Elasmobranchii, Selachii).

In the elasmobranch fish, Scyllium stellare, a complex group of cells protrudes into the cavity of the mesencephalic ventricle of the optic tectum. It consists of six to seven large spherical perikarya which resemble neurons of the mesencephalic nucleus of the Vth cranial nerve. The bundled processes of these cells form a stalk connecting the protrusion with the brain tissue. The protrusion is located in the region where the mesencephalic ventricle joins the cerebral aqueduct. This complex was not found in all specimens examined in the present study. The functional role of this peculiar group of cells, which contain dense core granules and are bathed in the cerebrospinal fluid, is open to discussion.

The habenular nuclei of the elasmobranch "Scyllium stellare": myelinated perikarya.

Some habenular cells of the elasmobranch, Scyllium stellare, have a perikaryon completely enveloped by myelin of the compact type. They are found only in the left habenula and not in the right. The myelin sheath accompanies the proximal segment of the process arising from the perikaryon, in a node of Ranvier-like pattern. The identity of these myelinated cells and the functional significance of their myelination are unknown.

Asymmetry of the habenulae in the elasmobranch "Scyllium stellare". II. Electron microscopy.

The structure of the habenular nuclei of the elasmobranch Scyllium stellare has been examined with the electron microscope. There is asymmetry of the left and right habenulae; difference is manifest in their cell types and myelin distribution. The presence in only the left habenula of cells bearing large granules - probably secretory - and of cells with myelinated perikarya suggest a functional diversification of the habenulae. A large variety of synaptic vesicles occur in the habenulae of both sides but we could not demonstrate a preferential localization of one particular type in specific habenular subnuclei. A feature common to all portions of the habenular complex is the presence of multilamellar astroglial formations wrapping cell bodies and synapses.

Asymmetry of the habenulae in the elasmobranch "Scyllium stellare". I. Light microscopy.

The habenular nuclei of the elasmobranch "Scyllium stellare" have been studied with conventional histological techniques for light microscopy. Their left-right asymmetry has been defined by the observation of serially cut sections. In addition, their cytoarchitectonic organization has been defined quantitatively. In all specimens observed, the left habenula was larger, longer and richer in myelin than the right. However, it appears that the right habenula is subdivided into more subnuclei than the left. We hypothesize that the morphologic asymmetry of the habenulae might reflect a functional specialization of the two sides of the epithalamus.

Central myelin in the shark Scyllium stellare (Elasmobranchii, Selachii).

The central myelin of the phylogenetically ancient shark Scyllium stellare (Elasmobranchii, Selachii) was examined at the electron microscope. The known different chemical compositions of the myelin in higher and lower vertebrates is not associated in the Scyllium with a lack of any basic structural myelin elements, but is probably linked to variations in the myelin compactness and periodicity. Several unusual myelin structures and irregular patterns of unknown origin have been observed. The relationship between myelin-forming cells and the most external layers of myelinated structures suggest the possibility that the cytoplasmic glial wrapping is not the only mechanism responsible for central myelination in this vertebrate.

Electrophysiological study of the effects of cardiodilatin 1-16 on the frog spinal cord in vitro.

Using the isolated spinal cord of the frog, hemisected and further divided into two distinct quadrants, we studied electrophysiological changes produced by peptides present in the atrial natriuretic factor (ANF) preprohormone. ANF and related peptides (atriopeptin I and atriopeptin III) did not affect the frog spinal cord. The 1-16 fragment from cardiodilatin (10(-5) M) induced slow depolarization in ventral and dorsal nerve stumps. The depolarization was associated with an increase of the evoked dorsal root potentials and depression of the fast component of the reflex responses. When depolarization approached its maximum value, spontaneous slow potentials appeared progressively similar to the evoked potentials, and became rhythmic until they reached a frequency of one potential every 15-20 seconds. The effects of cardiodilatin 1-16 are localized at dorsal horn level. It is suggested that this substance exerts a modulatory effect on frog cord physiology.

Chronotropic and inotropic effects of atrial peptides on the isolated systemic heart of Octopus vulgaris.

The chronotropic and inotropic effects of four atrial peptides (cardiodilatin 1-16, atrial natriuretic factor 8-33 and atriopeptin I and III) on the isolated systemic heart of Octopus vulgaris were studied. Using a preparation that produces a physiological stroke volume at physiological input pressures, it was found that ANF, atriopeptin I and atriopeptin III exerted both negative chronotropic and inotropic effects. In contrast, cardiodilatin produced a positive inotropic effect. A dose-response curve of ANF is reported, showing a threshold concentration of about 10(-12) M. The pharmacological and physiological implications of these results are discussed in relation to some characteristics of the cephalopod systemic heart.

Structural organization of the normal and anoxic heart of Scyllium stellare.

The general and ultrastructural organization of the heart of the elasmobranch, Scyllium stellare, was studied in normal and in anoxic animals. The rich coronary supply was revealed three-dimensionally by the use of corrosion casts, showing a thebesian system of coronary arterioles and capillaries in the thin, outer compact layer as well as in the predominant, inner spongy layer of trabeculae. Only the sinus venosus received a neuronal input of large bundles of granule-containing axons terminating at fenestrated regions of the endocardium and suggesting a neurohormonal function. A simple, tubular sarcoplasmic reticulum with flattened junctional cisternae was present in myocardial cells of 1-5 microns diameter, which contained one or two bundles of myofibrils. The latter were closely apposed to the inner aspect of the plasmalemma. Mitochondria were located centrally in the cells, which were joined by unfolded desmosomes involving Z-band material. Long periods of anoxia were tolerated without loss of heart function, but at the expense of cytoplasmic glycogen. Lipid granules were abundant in all layers and chambers, notably in animals prepared in the summer. The lipid granules displayed a marked increased in electron density when the heart was incubated in a buffered oxalate solution prior to fixation. A glycogen-sparing effect of the lipids during anoxia was observed.