Effects of naloxone on c-jun/AP-1 in met-enkephalin-and FMRFamide-immunreactive neurons of a gastropod snail.

1. Opioid- and FMRFamide (FMRFa)-ergic systems are believed to play antagonistic behavioral roles in both higher and lower animals. In our previous experiments on a snail, behavioral choice has been demonstrated to be dependent on a balance between FMRFa and enkephalins [7]. Here, we examined if the disturbance of the balance causes changes in the activity of both systems. Opiate receptor blocker naloxone was applied and its effect on c-jun expression of met-enkephalin (MEnk)- and FMRFa-ergic neurons was examined immunocytochemically in terrestrial gastropod snail Cepaea nemoralis. 2. In control, untreated snails, central neurons with c-jun/AP-1-like-immunoreactivity were found to occur. These included MEnk-, FMRFa- and 5HT-immunoreactive (-ir) neurons, as was revealed by double-labelling. 3. After treatment with naloxone for 4 h, the following changes were observed: (i) increase in the number of MEnk-ir neurons; increase in the number of neurons showing c-jun/AP-1 and MEnk double-labeling; (ii) disappearance of c-jun/AP-1-immunoreactivity from some FMRFa-ir neurons. 4. It is suggested that immediate early genes are involved in the mechanisms responsible for the reciprocal regulation of the opioid and antiopioid neuropeptide systems.

Anatomical basis for interactions of enkephalins with other transmitters in the CNS of a snail.

Immunocytochemical techniques for double staining were employed to investigate the morphological basis for interactions between enkephalins and other neuroactive compounds in the behavior of the gastropod mollusc Cepaea nemoralis. Coexistence of each of the two enkephalins with FMRFamide, serotonin or GABA-like immunoreactivity was found in certain neurons in cerebral, parietal, and pedal ganglia. Tyrosine hydroxylase-immunoreactive neurons were occasionally seen in close apposition to, but never colocalized with, the enkephalins. A comparison between these anatomical observations and previous behavioral studies suggests that in gastropod molluscs cotransmission of enkephalins with classical transmitters may, at least partly, reflect synergism of these substances in the control of definite behavioral programs.

Complex avoidance behaviour and its neurochemical regulation in the land snail Cepaea nemoralis.

1. In hot plate experiments, the pulmonate land snail Cepaea nemoralis displays a biphasic passive/active avoidance behaviour composed of retraction and subsequent searching mediated by antagonistic muscular systems. The switch, between the behaviours, is under neuronal control. 2. Leu- and met-enkephalin, as well as FMRFamide-antiserum, attenuated the retraction response and potentiated the searching behaviour. Opposite effects were achieved by injection of antisera to the enkephalins. 3. Both retraction and searching behaviours were potentiated by 5-HT. Methysergide antagonized the effects of the enkephalins on the searching behaviour. 4. We conclude that endogenous opioids act antagonistic to FMRFamide in the neuronally controlled switch between passive and active avoidance behaviour.

Transmitter-Specific Subsets of Sensory Elements in the Prosobranch Osphradium.

The osphradium is a putative chemosensory organ of aquatic molluscs. Previously, we identified two distinct types of primary sensory neurons in the osphradial ganglion of freshwater pulmonates, one immunoreactive to leucine-enkephalin (LEnk-ir) and another to FMRFamide (FMRFa-ir). In addition, NADPH diaphorase (NADPHd)-positive elements apparently producing nitric oxide (NO) were demonstrated in the organ. In the present study, prosobranch molluscs, which have retained the osphradial sensory neurons within the epithelium, were studied. Both types of peptidergic neurons, as well as NADPHd-positive cells, were found within the epithelium or in a basiepithelial position in the relatively simple osphradium of the mesogastropod Littorina littorea and in the complex, bipectinate osphradium of the neogastropod Buccinum undatum. Similar evidence was also obtained for another mesogastropod, Ampullarius sp. Transmitter-specific sensory cell types like those discovered in the osphradium are also present as single neuroepithelial cells in other organs of the mantle complex in prosobranchs and in the pelecypod Anodonta cygnea. We suggest that evolutionarily conservative, transmitter-specific types of epithelial and neuroepithelial sensory cells predated the osphradium, which developed as the site of their concentration, while retaining characteristic subsets of sensory neurons.

Patterns of enkephalin immunolabeling in the pulmonate snail Cepaea nemoralis and related molluscs.

In the land pulmonate snail Cepaea nemoralis, immunocytochemical localization of methionine- and leucine-enkephalin-like substances was demonstrated in specific populations of brain neurons. Methionine-enkephalin reactivity is also present in the peripheral nervous system as (i) abundant axonal projections of central neurons to the sole of the foot, and (ii) sparse local neurons of the head and mantle wall. Similar peripheral methionine-enkephalinergic elements were found immunocytochemically in all pulmonates (Helix aspersa, Lymnaea stagnalis) and prosobranchs (Littorina littorea, Acmaea testudinalis) examined and seem therefore conserved among gastropods. Their distribution was different from those of nervous elements labeled by antisera against serotonin and FMRFamide. The chiton Lepidopleurus asellus considered to be a more primitive mollusc, had both types of enkephalin-immunoreactive elements in the central nervous system whereas peripheral methionine-enkephalin-immunoreactive elements were lacking. It is suggested on the morphological evidence that enkephalinergic (especially methionine-enkephalinergic) neurons are part of a sensory system mediating analgetic effects in molluscs.

Is nitric oxide (NO) produced by invertebrate neurones?

NADPH-diaphorase (NADPHd) is known to be identical to nitric oxide (NO) synthase in the mammalian nervous system, and is therefore used as a marker of NO-producing neurones. Using the histochemical reaction for NADPHd, we searched for such neurones in a selection of invertebrates. Special emphasis was given to molluscs. No selective neuronal staining was found in representatives of coelenterates, turbellarians, nematodes and urochordates. In all annelids, arthropods and molluscs examined, with the exception of a chiton, specific neurones were selectively stained. The reaction was particularly strong in pulmonate molluscs where scattered positive neurones were found in various ganglia and clustered symmetrically in the paired buccal ganglia. Biochemical assay of NO synthase in osphradia of the gastropod mollusc Lymnaea stagnalis revealed a formation of citrullin that was inhibited by the specific NO synthase N omega-nitro-L-arginine (NO2Arg). Both histochemical and biochemical methods indicate that NO can be used as a signal molecule by specific neurones in advanced invertebrates.

The aesthetasc concept: structural variations of putative olfactory receptor cell complexes in Crustacea.

The structure of the aesthetascs has been investigated in the prawn Macrobrachium rosenbergii (larvae and juveniles), the opossum shrimp Neomysis integer, the euphausid Meganyctiphanes, and in the water-fleas Daphnia magna and D. longispina. The aesthetascs, that are thought to represent olfactory receptors, exhibit a considerable structural variation, ranging from the well known aesthetascs of higher crustaceans (lobster, crab, crayfish) to the corresponding sensilla found in the water-fleas and the males of opossum shrimps. The two following morphological characteristics of the aesthetascs are thought to indicate an olfactory function: the shape of the cuticular hair that is long and essentially hose-shaped, and the thin, loosely arranged cuticle of at least the outer part of the cuticular hair. The presence of other structural elements such as sensory cells, cilia, and enveloping cells are vital for the olfactory function, but the development is variable, which makes their use in the morphological definition of aesthetascs problematic.

Single modified cilia displayed by cells of human internal stratified epithelia (oral cavity, vagina).

Serial sections of human vaginal and keratinized oral-gingival epithelia were investigated for ciliary structures. Most melanocytes of the gingival epithelium lacked cilia, whereas almost all basal keratinocytes of the deeper portion of the epithelial ridges possessed one cilium each. In the suprabasal layers of the ridges only a few keratinocytes exhibited a single cilium. In the basal layer, at the top of the connective tissue papillae, approximately every second keratinocyte displayed a single cilium. In the suprabasal layers above the ridges no ciliated keratinocytes were observed. The basal cells of the vaginal epithelium were endowed with cilia, while cilia were absent from the suprabasal cells. In the human forearm epidermis most melanocytes and keratinocytes are supplied with a single cilium; it has been suggested that they may play a role in light reception. However, the widespread occurrence of 9 + 0 cilia in epithelial cells of internal epithelia and their coincidence with the sites of renewal of keratinocytes suggests that a relationship may exist between solitary cilia and mitotic activity.

Presence of 3,4-dihydroxyphenylalanine and 3,4,5-trihydroxyphenylalanine in a coelenterate nervous system: Possible tyrosinase-mediated accumulation.

3,4-dihydroxyphenylalanine (DOPA), 3,4,5-trihydroxyphenylalanine (5-OH-DOPA), 5-S-cysteinylDOPA (5-SC.D) and 2-S-cysteinylDOPA (2-SC.D) in the tentacles of the sea anemone, Metridium senile, were studied by the combined use of differential centrifugation of tissue homogenates, ultracentrifugation on sucrose density gradients, HPLC and electron microscopy. DOPA, 5-OH-DOPA and o-diphenol:O(2) oxidoreductase (Tyrosinase) were enriched in fractions containing membranes and subcellular particles, and the cysteinylDOPAs in the cytosol fractions. Ultrastructurally studied fractions rich in DOPA and 5-OH-DOPA contained large numbers of highly osmium-reducing vesicles. Identical structures were localized in ectodermal nerves and epidermal sensory cells. The results suggest that previous findings of catecholderivatives in the tentacles of Metridium and other sea anemone species by histochemical methods, are explained by a tyrosinase-based accumulation of DOPA and 5-OH-DOPA in the ectodermal nerve net. These substances are confined in specific compartments (vesicles) in the neurons and sensory cells.

Retinal S-antigen: immunocytochemical and immunochemical studies on distribution in animal photoreceptors and pineal organs.

Antiserum against bovine retinal S-antigen, a soluble protein (MW = 50 kDa) thought to be involved in phototransduction, was used in an immunohistochemical and immunochemical study of vertebrate eyes and pineal systems and invertebrate photoreceptor organs. Positive reactions, not seen with antiserum preabsorbed with highly purified S-antigen, were observed in planarian and starfish ocelli; scallop eyes; polychaete eye; crayfish compound eye; lamprey, salmon, frog, turtle, quail and hamster eyes. A specific reaction was also seen in the pineal organ of all the vertebrates examined, albeit weak in turtle and quail. In addition, several structures associated with photoreceptor organs, including the reduced frontal eyes of crayfish, the organ of Bellonci in crayfish eyestalk, and bipolar cells resembling those giving rise to Landolt's clubs in quail and golden hamster retinae, were immunopositive. Immunochemical studies revealed the presence of a single immunopositive band of protein which was similar but not identical in size in all vertebrate eyes and pineal organs (except that of chicken pineal) and invertebrate tissue examined. The wide distribution of positive reaction in photoreceptive tissue indicates that the retinal S-antigen determinant has been highly conserved during evolution.

The ontogenetic development of refracting superposition eyes in crustaceans: Transformation of optical design.

We have investigated, comparatively, the ontogenetic development of the compound eye in larvae of a mysid (Neomysis) and a euphausiid (Thysanoessa) species and found it to be close to identical in the two species. The larval eye is of apposition type with special adaptations for planktonic life. The elongated dioptric apparatus is devoid of screening pigment and instead has a proximal lens optically isolating the ommatidium. The pigmented retina is extremely compressed making the eye largely transparent and presumably suitable for a planktonic life. The presence of this specialized type of eye in the planktonic larvae of euphausiids was known before but it is intriguing to find exactly the same type in mysids, spending their entire larval life as embryos in the female marsupium. A possible explanation is offered if mysids earlier in evolution had planktonic larvae. Upon reduction of free-living larvae, the transparent type of eye may have been preserved because there is no selection pressure on the larva to change it. In late larval life, both species transform their eyes to a refracting superposition type typical for adult mysids and euphausiids. The process of transformation and the functional connection between transparent apposition and superposition is described.

The ciliated human keratinocyte.

Human keratinocytes were investigated for the presence of single cilia. Almost all basal keratinocytes were found to carry a single cilium in normal, occluded, and psoriatic skin. The ciliary structure was progressively reduced in keratinocytes approaching the surface. No remnants of the ciliary apparatus were found in the granular layer. In one case of nickel-allergic dermatitis (patch test), the keratinocytes had lost their cilia; the significance of this surprising finding remains to be elucidated.

5-HT-like immunoreactivity in the central nervous system of the crayfish, Pacifastacus leniusculus.

An immunocytochemical technique with the use of three different antibodies raised against serotonin was applied to localize the immunoreactive neurons in the central nervous system of the crayfish, Pacifastacus leniusculus. Immunoreactive neurons were found in three optic ganglia (medulla externa, interna and terminalis). They appeared in three layers of the medulla externa and interna. The medulla terminalis displayed three prominent groups of immunoreactive perikarya and mainly marginal immunoreactive fibres. Immunoreactive areas of the brain comprised the protocerebral bridge, central body, paracentral lobes and two loci in the anterior portion of the protocerebrum, i.e., the terminal areas for immunoreactive fibres from the optic centres. The olfactory lobes showed a specific immunoreactive pattern. In addition, diffusely and sparsely distributed immunoreactive fibres were found throughout the brain. The immunoreactive neurons are largely localized in the same areas of the central nervous system as the catecholaminergic neurons although some distinct differences occur.

The compound eye of Leptodora kindtii (Cladocera). An adaptation to planktonic life.

Each of the approximately 500 ommatidia in the compound eye of the cladoceran crustacean Leptodora kindtii has a crystalline cone consisting of five cells. Five retinula cells are also present, one of which contributes to the distal 1-2 micrometer of the rhabdom only; the other four retinula cells from a continuous rhabdom. Throughout the rhabdom its cross section displays two separate halves with the axis of the microvilli in one half perpendicular to that in the other (orthogonal pattern). Interferometric analysis of the refractive index of the crystalline cone revealed an inhomogeneous system with one distal and one proximal gradient. The gradient system was found to exclude rays entering from adjacent facets, thus maintaining the optical isolation. Consequently, these optics replace distal screening pigment, which is absent in the eye. The long and unscreened crystalline cones give rise to an almost transparent eye in conformity with the overall transparency of this plank-tonic animal. The morphological characteristics of the eye of this species deviate from other cladoceran eyes, but the optical design closely resembles that of some pelagic marine amphipod crustaceans.

The basement membrane of the insect and crustacean compound eye: definition, fine structure, and comparative morphology.

The basement membrane of the compound eye of four insect species and three crustacean species was investigated employing electron microscopy. The basement membrane consists of an extracellular (basal lamina) and a cellular portion, the latter being composed of the flattened terminal extensions of cone cells and accessory pigment cells in insects and distal pigment cells in crustaceans. Other cells can also contribute to the basement membrane. It is thus a complex structure in all well--developed compound eyes. The cellular contributions vary in different species and were found to correlate to specific taxonomic units.

Neuronal connectivity patterns in the compound eyes of Artemia salina and Daphnia magna (Crustacea: Branchiopoda).

The neuronal types and patterns in the visual system of the species Artemia salina and Daphina magna have been studied with the Golgi method and electron microscopy. The lamina contains five classes of neurons: photoreceptor axons, monopolar, centrifugal, tangential and amacrine neurons. The terminals of the receptor axons are distributed in two (A. salina) or three (D. magna) layers. The dilated terminals have an extensive and wide array of fine branches. One axon from each ommatidium bypasses the lamina and terminates in the medula in A. salina. A. salina has four types of monopolar neurons, two of which are stratified, whereas in D. magna only two types are found, one of which is bistratified. Tangential T-neurons connect the lamina with the protocerebrum. D. magna has in addition one tangential T-neuron connecting both the lamina and the medulla with the protocerebrum. In both species monopolar-type centrifugal neurons connect the medulla and the lamina, whereas that of A. salina has a wide laminar distribution. Both species also have amacrine cells in the lamina. The medulla contains, besides those shared with the lamina, transmedullary neurons (two types in A. salina), amacrine cells and neurons originating in the protocerebrum. "Cartridge"-type synaptic compartments are lacking in the investigated species, although a periodic arrangement is discernible in the distal portion of the lamina of A. salina. The receptors from three types of specialized contacts in Artemia, one of which involves a dyad. D. magna has only one-to-one synapses. Neurosecretory fibres are absent in A. salina.

Catecholaminergic innervation of muscles in the hindgut of crustaceans. An ultrastructural study.

The crustacean species Pacifastacus leniusculus and Gammarus pulex were investigated by electron microscopy in a search for possible neuromuscular junctions in the hindgut, which has a rich supply of catecholaminergic fibres. True neuromuscular synapses were found in both species between nerve terminals containing dense-core vesicles (80-110 nm in diam.) and muscle fibres. We suggest that the dense-core vesicle terminals contain a catecholamine, and this is supported by ultrahistochemical tests for monoamines. Two types of junctions are found: one in which the nerve terminal is embedded in the muscle cell (both species) and one in which protrusions from the muscle cell meet nerve terminals (Pacifastacus). Gammarus pulex, which has only circular muscles in the hindgut, has only catecholaminergic innervation, whereas Pacifastacus leniusculus has circular and longitudinal muscles both with at least two types of innervation.

In vitro uptake of L-dopa and catecholamines into the epidermal Langerhans cell.

The Langerhans cells are capable of taking up L-dopa and the catecholamines dopamine and noradrenaline when exposed to these substances in vitro. Within the cell L-dopa is found in the cytoplasm as well as in the nucleus, whereas the catecholamines are confined to cytoplasmic granules. The L-dopa uptake is most probably carrier-mediated and the hypothesis is brought forward that L-dopa enters the cell by exchange diffusion. At present little is known about the nature of the amine uptake mechanism.