Medulla Externa Research Articles

Fine structure of neurosecretory cells and sinus gland in the eyestalk of the freshwater crab Travancoriana schirnerae Bott, 1969 (Decapoda: Gecarcinucidae)

This study elucidated the fine structure of neurosecretory cells and sinus gland in the optic ganglia of the freshwater crab Travancoriana schirnerae Bott, 1969 (Decapoda: Gecarcinucidae). The eyestalk ganglion showed the presence of four well defined ganglia arranged below the ommatidium: lamina ganglionaris, medulla externa, medulla interna and medulla terminalis of which the lamina ganglionaris, was devoid of neurosecretory cells. Groups of neurosecretory cells seen distributed along the medulla externa, interna and terminalis regions constitute the X-organs. Electron microscopic observations of the eyestalk ganglia revealed ten types of neurosecretory cells, mostly apolar with a few unipolar and bipolar cells classified according to the size, shape and density of the cell and nucleus, cell organelles/inclusions, together with the arrangement and properties of chromatin. These cells were characterized by the presence of large nuclei with unusually condensed chromatin, inclusions like vacuoles and vesicles of varying size, shape and density and organelles like Golgi, endoplasmic reticulum, ribosomes and mitochondria and neurosecretory material. The sinus gland of T. schirnerae was positioned laterally between the externa and interna regions, composed of axonal endings of the neurosecretory cells of the optic ganglia with interspersed glial cells. The axon terminals were enclosed with several small to large membrane bound homogenously dense neurosecretory granules which also occur in the preterminal areas of the axons. Based on size, shape and density of granules and axoplasmic matrix, seven terminal types could be distinguished in the sinus gland of T. schirnerae. Mostly, the granules contained in a terminal were of the same type; rarely, the same terminal enclosed granules of varying size, shape and density. The neurosecretory cell types and axon terminal types represent the types of neurohormones they contained. A precise knowledge of the morphology and cytology of neurosecretory cells in the XO-SG complex of the eyestalk that secrete neurohormones controlling major physiological processes such as growth and reproduction is imperative for successful captive breeding of a species of aquaculture potential.

CDNA cloning and in situ localization of a crustacean female sex hormone-like molecule in the kuruma prawn Marsupenaeus japonicus

Recently, a crustacean female sex hormone (CFSH) was purified from the female eyestalk of the blue crab Callinectes sapidus. This report suggested that CFSH plays an essential role in the development of female secondary sexual characteristics. In order to broaden current knowledge of CFSH, here we report the cloning of the cDNA encoding a putative crustacean female sex hormone in Marsupenaeus japonicus (Maj-CFSH). The deduced Maj-CFSH precursor showed identical composition with other CFSHs, including a signal peptide, a CFSH-precursor-related peptide (CPRP), a dibasic processing signal and a mature Maj-CFSH. Although the amino acid sequence identities between Maj-CFSH and C. sapidus CFSH was low (38.9%), positions of eight cysteine residues and the N-glycosylation motif were found to be conserved between the two sequences. Tissue distribution showed that Maj-CFSH was expressed in both female and male eyestalks with roughly the same level. Analysis of in situ hybridization showed that Maj-CFSH was expressed in neurosecretory cells in the medulla terminalis ganglionic X-organ (MTGX), medulla interna ganglionic X-organ (MIGX), as well as neural cells around the medulla externa in both female and male eyestalks. Our results suggest that Maj-CFSH is not a female-specific molecule and, therefore, is a CFSH homolog, which may have various, as yet undetermined functions in males, as well as females of M. japonicus.

An eclosion hormone-like gene participates in the molting process of Palaemonid shrimp Exopalaemon carinicauda

Molting behavior is an important physiological process related to metamorphosis, growth, and reproduction in crustaceans. Previous studies indicated that the molting process was controlled by 20-hydroxyecdysone (20E) and upstream hormones, peptides, and environmental factors, which regulate 20E function. Eclosion hormone (EH) in insect is a kind of neuropeptide that is regulated by 20E and triggers ecdysis behavior at the end of molting process. However, the function of eclosion hormone gene during the molting process in crustaceans is still largely unknown. In the present study, an eclosion hormone-like gene EcEHL was identified from Exopalaemon carinicauda. The deduced amino acid sequence of EcEHL contained a signal peptide, a typical eclosion domain, and six conserved cysteine residues forming three putative disulfide bonds. EcEHL was predominantly expressed in the epidermis, gill, and eyestalk of shrimp. In situ hybridization analysis showed that EcEHL transcripts were localized in gill cells and in medulla externa X-organ, medulla terminalis X-organ, sinus gland, and lamina ganglionaris of eyestalks. During the molting process of shrimp, EcEHL showed the highest expression level in shrimp at the premolt stage. The expression level of EcEHL in shrimp at mid premolt stage was up-regulated by injection of exogenous 20E. Silencing of EcEHL using double-stranded RNA delayed both the molting process and ecdysis rate of E. carinicauda. Furthermore, injection of exogenous 20E to shrimp at mid premolt stage (D2) could remarkably speed up the molting process and also raise the ecdysis rate of E. carinicauda. The results revealed that EcEHL might participate in the molting process of shrimp and its expression was regulated by 20E. These data will help us to understand the molecular mechanism of molting in crustacean.

Prediction of a neuropeptidome for the eyestalk ganglia of the lobster Homarus americanus using a tissue-specific de novo assembled transcriptome

In silico transcriptome mining is a powerful tool for crustacean peptidome prediction. Using homology-based BLAST searches and a simple bioinformatics workflow, large peptidomes have recently been predicted for a variety of crustaceans, including the lobster, Homarus americanus. Interestingly, no in silico studies have been conducted on the eyestalk ganglia (lamina ganglionaris, medulla externa, medulla interna and medulla terminalis) of the lobster, although the eyestalk is the location of a major neuroendocrine complex, i.e., the X-organ-sinus gland system. Here, an H. americanus eyestalk ganglia-specific transcriptome was produced using the de novo assembler Trinity. This transcriptome was generated from 130,973,220 Illumina reads and consists of 147,542 unique contigs. Eighty-nine neuropeptide-encoding transcripts were identified from this dataset, allowing for the deduction of 62 distinct pre/preprohormones. Two hundred sixty-two neuropeptides were predicted from this set of precursors; the peptides include members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon α, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone α2, glycoprotein hormone β5, GSEFLamide, intocin, leucokinin, molt-inhibiting hormone, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pigment dispersing hormone, proctolin, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide, sulfakinin, tachykinin-related peptide and trissin families. The predicted peptides expand the H. americanus eyestalk ganglia neuropeptidome approximately 7-fold, and include 78 peptides new to the lobster. The transcriptome and predicted neuropeptidome described here provide new resources for investigating peptidergic signaling within/from the lobster eyestalk ganglia.

Localization and identification of crustacean hyperglycemic hormone producing neurosecretory cells in the eyestalk of blue swimmer crab, Portunus pelagicus.

This study intensely focuses on to the localization and identification of crustacean hyperglycemic hormone (CHH) producing neurosecretory cells in the eyestalk of the blue swimmer crab Portunus pelagicus. Anti-Carcinus maenas-CHH was used to identify the location of CHH neurosecretory cells by immunohistochemistry. Ten pairs of eyestalks were collected from intact adult intermoult female crab and fixed in Bouin's fixative. Eyestalks were serially sectioned and stained with chrome-hematoxylin-phloxine stain. Histological studies show the presence of different types of neurosecretory cells namely A (multipolar), B (tripolar), C (bipolar), D (unipolar), E (oval), and F (spherical) in the medulla interna, externa, and terminalis regions based on their size, shape, and tinctorial properties. The neurohemal organ, sinus gland (SG) was observed laterally between medulla interna and terminalis regions. Immunohistochemical studies showed the presence of distinct CHH-like immunoreactivity in the optic ganglia. Divergent group of neurosecretory cells with varying degree of immunoreactivity with Anti-Carcinus maenas-CHH (low, moderate, and intense reactivity) were identified in medulla terminalis, medulla interna, medulla externa, and sinus gland. The present study maps the various types of neurosecretory cells in the optic ganglia and also shows the presence of CHH-like immunoreactivity in various regions of optic ganglia in P. pelagicus. The presence of these unique neurosecretory cell types with larger cell diameter in medulla terminalis, a region that bears the neurosecretory cell bodies, suggest high secretory activity.

Biochemical Analysis and lmmunohistochemical Examination of a GnRH-like Immunoreactive Peptide in the Central Nervous System of a Decapod Crustacean, the Kuruma Prawn (Marsupenaeus japonicus)

We examined whether a gonadotropin-releasing hormone (GnRH)-like peptide exists in the central nervous system (CNS) of the kuruma prawn, Marsupenaeus japonicus, by reverse-phase high performance liquid chromatography (rpHPLC) combined with time-resolved fluoroimmunoassay (TR-FIA) analysis and by immunohistochemistry. The displacement curve obtained for serially diluted extracts of the kuruma prawn brain paralleled the chicken GnRH-II (cGnRH-II) standard curve obtained by cGnRH-II TR-FIA using the anti-cGnRH-II antibody, which cross-reacts not only with cGnRH-II but also with lamprey GnRH-II (lGnRH-II) and octopus GnRH (octGnRH). Extracts of kuruma prawn brains and eyestalks showed a similar retention time to synthetic lGnRH-II and octGnRH in rpHPLC combined with TR-FIA analysis. Using this antibody, we detected GnRH-like-immunoreactive (ir) cell bodies in the anterior-most part of the supraesophageal ganglion (brain), the protocerebrum. Furthermore, GnRH-like-ir fibers were observed in the protocerebrum and deutocerebrum. In the eyestalk, GnRH-like-ir cell bodies were detected in the medulla interna, and GnRH-like-ir fibers were distributed in the medulla interna, medulla externa, and lamina ganglionalis. In the thoracic ganglion, GnRH-like-ir fibers, but not GnRH-like-ir cell bodies, were detected. No GnRH-like-ir cell bodies or fibers were detected in the abdominal ganglion or ovary. Thus, we have shown the existence and distribution of a GnRH-like peptide in the CNS of the kuruma prawn.

Photoreceptor projection and termination pattern in the lamina of gonodactyloid stomatopods (mantis shrimp)

The apposition compound eyes of gonodactyloid stomatopods are divided into a ventral and a dorsal hemisphere by six equatorial rows of enlarged ommatidia, the mid-band (MB). Whereas the hemispheres are specialized for spatial vision, the MB consists of four dorsal rows of ommatidia specialized for colour vision and two ventral rows specialized for polarization vision. The eight retinula cell axons (RCAs) from each ommatidium project retinotopically onto one corresponding lamina cartridge, so that the three retinal data streams (spatial, colour and polarization) remain anatomically separated. This study investigates whether the retinal specializations are reflected in differences in the RCA arrangement within the corresponding lamina cartridges. We have found that, in all three eye regions, the seven short visual fibres (svfs) formed by retinula cells 1-7 (R1-R7) terminate at two distinct lamina levels, geometrically separating the terminals of photoreceptors sensitive to either orthogonal e-vector directions or different wavelengths of light. This arrangement is required for the establishment of spectral and polarization opponency mechanisms. The long visual fibres (lvfs) of the eighth retinula cells (R8) pass through the lamina and project retinotopically to the distal medulla externa. Differences between the three eye regions exist in the packing of svf terminals and in the branching patterns of the lvfs within the lamina. We hypothesize that the R8 cells of MB rows 1-4 are incorporated into the colour vision system formed by R1-R7, whereas the R8 cells of MB rows 5 and 6 form a separate neural channel from R1 to R7 for polarization processing.

Neuroarchitecture of the color and polarization vision system of the stomatopod Haptosquilla.

The apposition compound eyes of stomatopod crustaceans contain a morphologically distinct eye region specialized for color and polarization vision, called the mid-band. In two stomatopod superfamilies, the mid-band is constructed from six rows of enlarged ommatidia containing multiple photoreceptor classes for spectral and polarization vision. The aim of this study was to begin to analyze the underlying neuroarchitecture, the design of which might reveal clues how the visual system interprets and communicates to deeper levels of the brain the multiple channels of information supplied by the retina. Reduced silver methods were used to investigate the axon pathways from different retinal regions to the lamina ganglionaris and from there to the medulla externa, the medulla interna, and the medulla terminalis. A swollen band of neuropil-here termed the accessory lobe-projects across the equator of the lamina ganglionaris, the medulla externa, and the medulla interna and represents, structurally, the retina's mid-band. Serial semithin and ultrathin resin sections were used to reconstruct the projection of photoreceptor axons from the retina to the lamina ganglionaris. The eight axons originating from one ommatidium project to the same lamina cartridge. Seven short visual fibers end at two distinct levels in each lamina cartridge, thus geometrically separating the two channels of polarization and spectral information. The eighth visual fiber runs axially through the cartridge and terminates in the medulla externa. We conclude that spatial, color, and polarization information is divided into three parallel data streams from the retina to the central nervous system.

Immunolocalization of allatostatin-like neuropeptides and their putative receptor in eyestalks of the tiger prawn, Penaeus monodon

Allatostatin (AST)-like immunoreactivity (IR) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using four anti-AST antibodies. Depending on the antisera, AST-like immunoreactivity was detected in neuronal bodies of the lamina ganglionalis, cell bodies anterior to the medulla externa and cell bodies on the anterior and posterior of the medulla terminalis. Neuronal processes in neuropiles of the medulla externa, medulla terminalis, sinus gland and nerve fibers in the optic nerve were also recognized. No IR in cell bodies or in nerve fibers was found in the medulla interna. Strong AST-like immunoreactivity was found in hundreds of cells of the X organ. The localization of AST-like peptides suggests that they function as neurotransmitters and/or neuromodulators. Antiserum to the Drosophila AST receptor (Dar-2) recognized a single protein in P. monodon eyestalk protein extracts that was identical in size to that found in Drosophila protein extracts. Using this antiserum the putative P. monodon AST receptor was localized to the sinus gland in both juvenile and adult eyestalks. To our knowledge this is the first demonstration of a neuropeptide receptor localized to the crustacean sinus gland. This suggests that ASTs may function directly on the sinus gland as a neuromodulator. In juvenile eyestalks, the putative AST receptor was also localized to neuronal X organ cells of the medulla terminalis in males but not in females. The significance of this sex-specific receptor localization is unclear but emphasizes that ASTs function within the nervous system of the eyestalk.

Corazonin promotes tegumentary pigment migration in the crayfish Procambarus clarkii

The undecapeptide corazonin (pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-AsnNH 2) elicits a retraction of erythrophore pigment granules and dispersion of leucophore pigment granules in the crayfish Procambarus clarkii. The effects are dose-dependent from 10 −10 to 10 −5 M. Influence on erythrophores is lower than that of Red Pigment Concentrating Hormone (RPCH), which is inactive on leucophores. Corazonin effects are partly blocked by an anti-corazonin antibody, and even less by an anti-RPCH antibody. Corazonin effects are completely suppressed by the calcium chelator BAPTA. Immunoreactive somata and fibers were identified in various regions of the eyestalk (medulla terminalis, medulla interna and medulla externa) with the anti-corazonin antibody. These results suggest the possible existence of a corazonin-like peptide in crustaceans.

Histomorphology of the Neurosecretory Cells of Eyestalk, Brain and Thoracic Ganglia of Female Giant Freshwater Prawn, Macrobrachium RosenbergII

Histomorphology of the neurosecretory cells of eyestalk, brain and thoracic ganglia of Macrobrachium rosenbergii were recorded. There were five types of neurosecretory cells (NSCs) in eyestalk having size range of 5 - 35 um, shape varied from round to oval. The cells were distributed in medulla externa, medulla interna and medulla terminalis. Sinus gland measuring 30-35 um was located in medulla interna. Axonal terminals of the neurosecretory cells were found to terminate in this structure. Brain and thoracic ganglia possessed five types of neurosecretory cells such as giant neuron (>80 um), A (60-80 um), B (40-60 um), C (20-40 um) and D (<20 um). They were seen arranged in several groups in different parts of brain. While B, C and D cells were localized in anterior region, giant neurons and A cells dominated in posterior region. In lateral regions A, B, C and D cells were distributed. The thoracic ganglia could be divided into anterior, middle and posterior regions. NSCs were distributed in anterior and posterior portions but were lacking in middle part. A and B cells were present in anterior regions followed by C and D cells. In posterior region, giant neurons and A cells were present. Histochemical tests demonstrated the neurosecretory cells of the prawn to be strongly positive for acid fuchsin, aldehyde fuchsin and paraldehyde fuchsin and also exhibited feeble reaction to Sudan black B and periodic acid-Schiff (PAS) reagent.

Four novel PYFs: members of NPY/PP peptide superfamily from the eyestalk of the giant tiger prawn Penaeus monodon

An immunocytochemical method was used for localization of pancreatic polypeptide (PP) immunoreactive substances in the eyestalk of Penaeus monodon using anti-C-terminal hexapeptide of PP (anti-PP6) antiserum. Approximately 200 neuronal cell bodies were recognized in the ganglia between the medulla interna (MI) and medulla terminalis (MT) and surrounding MT in conjunction with the neuronal processes in medulla externa (ME), MI, MT and sinus gland. About half of the PP immunoreactive neurons were also recognized by a combination of three monoclonal antibodies raised against FMRFamide-like peptides. Isolation of the PP immunoreactive substances from the eyestalk was performed using 7500 eyestalks extracted in methanol/acetic acid/water (90/1/9) followed by five to six steps of RP-HPLC separation. Dot-ELISA with anti-PP6 antiserum was used to monitor PP-like substances in various fractions during the purification processes. Four new sequences of one hexapeptide; RARPRFamide, and three nonapeptides; YSQVSRPRFamide, YAIAGRPRFamide and YSLRARPRFamide were identified, and named as Pem-PYF1–4 due to their structural similarity to the PYF found in squid Loligo vulgaris. Each of the new peptides shares four to seven common residues with the C-terminus of the squid PYF and with the NPFs found in other invertebrates. The NPY/PP superfamily as well as the FMRFamide peptide family may be present throughout vertebrates and invertebrates.

A new look at an old visual system: structure and development of the compound eyes and optic ganglia of the brine shrimp Artemia salina Linnaeus, 1758 (Branchiopoda, anostraca).

Compared to research carried out on decapod crustaceans, the development of the visual system in representatives of the entomostracan crustaceans is poorly understood. However, the structural evolution of the arthropod visual system is an important topic in the new debate on arthropod relationships, and entomostracan crustaceans play a key role in this discussion. Hence, data on structure and ontogeny of the entomostracan visual system are likely to contribute new aspects to our understanding of arthropod phylogeny. Therefore, we explored the proliferation of neuronal stem cells (in vivo incorporation of bromodeoxyuridine) and the developmental expression of synaptic proteins (immunohistochemistry against synapsins) in the developing optic neuropils of the brine shrimp Artemia salina Linnaeus, 1758 (Crustacea, Entomostraca, Branchiopoda, Anostraca) from hatching to adulthood. The morphology of the adult visual system was examined in serial sections of plastic embedded specimens. Our results indicate that the cellular material that gives rise to the visual system (compound eyes and two optic ganglia) is contributed by the mitotic activity of neuronal stem cells that are arranged in three band-shaped proliferation zones. Synapsin-like immunoreactivity in the lamina ganglionaris and the medulla externa initiated only after the anlagen of the compound eyes had already formed, suggesting that the emergence of the two optic neuropils lags behind the proliferative action of these stem cells. Neurogenesis in A. salina is compared to similar processes in malacostracan crustaceans and possible phylogenetic implications are discussed.

Seven novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant tiger prawn Penaeus monodon

FMRFamide-like immunoreactivity (FLI) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using a combination of three anti-FMRFamide-like peptide (FLPs) monoclonal antibodies. Approximately 3000 small neuronal cell bodies in the lamina ganglionalis; 100 medium to large size at the ganglion between the medulla interna and the medulla terminalis; and 250 medium size around the medulla terminalis were stained intensely. The neuronal processes in neuropils of the medulla externa, medulla interna, medulla terminalis, sinus gland and some nerve fibers in the optic nerve were also recognized. The small cell bodies, approximately 1500 cells, anterior to the medulla externa were stained inconsistently and the neuronal processes were not observed from these cells. Isolation of FLPs from 9000 eyestalks was performed using methanol/acetic/water (90:1:9) extraction. After the extract was partially purified using C18 cartridges, it was further purified by five to seven steps of RP-HPLC using three kinds of columns: C18; C8; and cyano, and three solvent systems: acetonitrile/trifluoro acetic acid; aceonitrile/heptafluoro butyric acid; and acetonitrile/triethyl ammonium acetate. Dot-ELISA using the combination of the same antibodies was used to monitor FLPs in the fractions during purification processes. Seven new sequences of FLPs were identified which can be divided into four subgroups according to the primary structure of the C-terminus: (1) GDRNFLRFamide; (2) AYSNLNYLRFamide; (3) AQPSMRLRFamide, SQPSMRLRFamide, SMPSLRLRFamide and DGRTPALRLRFamide; and (4) GYRKPPFNGSIFamide. These data indicate the high complexity of this peptide family in which multiple forms are usually exist.

Polarization analysis in the crayfish visual system

It is proposed that polarization sensitivity at the most peripheral stages of the crayfish visual system (lamina ganglionaris and medulla externa) is used to enhance contrast and thus may contribute to motion detection in low contrast environments. The four classes of visual interneurons that exhibit polarization sensitivity (lamina monopolar cells, tangential cells, sustaining fibers and dimming fibers) are not sensitive exclusively to polarized light but also respond to unpolarized contrast stimuli. Furthermore, many of these cells and the sustaining fibers in particular exhibit a greater differential e-vector responsiveness to a changing e-vector than to e-vector variations among steady-state stimuli. While all four cell types respond modestly to light flashes at an e-vector of 90 degrees to the preferred orientation, the dynamic response to a changing e-vector is small or absent at this orientation. Because the sustaining fibers exhibit polarization sensitivity, and they provide afferent input to a subset of optomotor neurons, the latter were also tested for polarization sensitivity. The optomotor neurons involved in compensatory reflexes for body pitch were differentially sensitive to the e-vector angle of a flash of light, with maximum responses for e-vectors near the vertical. The motor neurons also exhibited a maximum response near the vertical e-vector to a continuously rotating polarizer. Two scenarios are described in which the sensitivity to a changing e-vector can produce motion responses in the absence of intensity contrast.

Two-channel polarization analyzer in the sustaining fiber-dimming fiber ensemble of crayfish visual system.

Polarization sensitivity (PS) was examined in two classes of neurons, sustaining fibers and dimming fibers, in the medulla externa (second optic neuropile) of the crayfish, Pacifasticus leniusculus. Visual responses were recorded intracellularly and extracellularly. The influence of e-vector orientation (theta) was probed in steady-state responses, with brief flashes and with a rotating polarizer. The results indicate that the entire sustaining fiber population appears to be maximally sensitive to vertically polarized light. Although the evidence is less complete for dimming fibers, they appear to be maximally inhibited by vertically polarized light and excited by horizontally polarized light. Thus the sustaining fibers and dimming fibers form a two-channel polarization analyzer that captures the main features of the polarization system established in photoreceptors and lamina monopolar cells. The available evidence suggests that this two-channel system has the same characteristics across most or all of the retinula. Lateral inhibition in sustaining fibers is differentially sensitive to theta. Inhibition is substantial at theta = 90 degrees (horizontal) and essentially absent at theta = 0 degrees. The details of the sustaining fiber polarization response closely follow features established in more peripheral neurons, including the magnitude of PS, enhanced responsiveness to a changing e-vector, and modest directionality to a changing e-vector in approximately 40% of the cells.

Regional Distribution and Immunocytological Localization of Red Pigment Concentrating Hormone in the Crayfish Eyestalk

A polyclonal antibody was raised against synthetic tyrosinated crustacean red pigment concentrating hormone (RPCH-Tyr) with the sequence Tyr-Leu-Asn-Phe-Ser-Pro-Gly- Trp-NH 2 with a tryptophan amide at the carboxyl terminal end. Its specificity was tested in comparison with peptides of similar structure. It appears to recognize the three to five residues near the carboxyl terminal. Native RPCH in the crayfish eyestalk was determined by two methods: (a) immunoenzymatic assay (ELISA) using the aforementioned antibody; and (b) bioassay on segments of isolated crayfish tegumentary epithelium. The unitary content in whole eyestalks was 5.5 ± 1.0 nmol for samples ( n = 18) taken at night. The regional distribution of RPCH content in the eyestalk was determined. The greatest proportion (40%) was found in the sinus gland, and the lowest in the retina plus lamina ganglionaris (6%). The medulla interna, medulla externa, and medulla terminalis contained similar proportions (about 16% each). The highest specific content was in the sinus gland (65.0 vs 24.4 pmol/μg protein for the whole eyestalk). Immunopositive neurons were identified in the various regions of the eyestalk. In 22 preparations, an average of 7 cells were identified in the ventromedial rim of the medulla terminalis, sending axons to the sinus gland, after branching in the neuropil of the medulla terminalis. Dorsally, 2 cells were identified in the medulla interna and 4 large cells and 11 small cells were located in the medulla externa in close proximity to the lamina ganglionaris: none of these cells appeared to project to the sinus gland. Profuse immunopositive fibers were found in the lamina ganglionaris projecting distally toward the base of the retina. Immunopositive axons were also found in the optic nerve.

Ultrastructural Features of Neurosecretory Cells in the Medulla Externa of the Crayfish Eyestalk

A conglomerate of 8-12 neurons in the medulla externa of the crayfish eyestalk was explored in their reaction to a polyclonal antibody against the tyrosinated octapeptide Red Pigment Concentrating Hormone (Tyr-RPCH). These are large neurons with diameters within a range of 33-43 μm and they were all positively stained with neutral red. By intracellular staining with lucifer yellow, the neurons were found to branch extensively within the medulla externa and the lamina ganglionaris of the eyestalk. Each neurite bifurcates at about 40 μm from the soma. Both branches run to the medial edge of the eyestalk; one proceeds distally to the lamina ganglionaris, while the other runs proximally to the medulla interna. Both end freely in multiple arborizations, covering from the medial to the lateral edges of the eyestalk. No branches were found to the sinus gland, the main neurohaemal organ of the eyestalk. A group of 4 neurons in the conglomerate consistently rendered positive reaction to the anti-Tyr-RPCH antibody (A-RPCH). They are superficially located in the cluster, and at the electron microscope, they showed the usual features of a secretory cell, i.e., clear and dense granules, an active and well-developed Golgi apparatus, and rough endoplasmic reticulum. The dense granules were larger (mean diameter: 101.5 nm) than the clear granules (mean diameter: 90.3 nm). The immunopositive reaction at the electron microscope was found to be largely confined to the dense-cored granules.

Directional selectivity in a nonspiking interneuron of the crayfish optic lobe: evaluation of a linear model

1. Intracellular recordings, sine wave gratings, and paired flashes were used to characterize the directional selectivity (DS) of the peripheral neurons of the crayfish visual pathway. DS was observed in nonspiking tangential (Tan1) neurons of the distal medulla externa and it is expressed by the amplitude of the modulated synaptic potential elicited with drifting gratings. 2. The directional mechanism was characterized by variations in the grating contrast, spatial frequency, and temporal frequency. DS is both contrast and velocity dependent. 3. The velocity dependence of DS for fixed stimulus contrast can be described by a linear model including a delay and subtractive compare operation. This mechanism operates over the entire useful range of spatial and temporal frequencies. 4. The parameters of the linear model can be estimated from the spatiotemporal structure of the Tan1 cell receptive field. The receptive field exhibits a spatially asymmetric inhibitory subfield that is offset from the excitatory subfield by 3-5 degrees (1-2 ommatidia). The inhibition is delayed relative to excitation by 50-100 ms. 5. The contrast dependence of DS reflects an apparent nonlinearity in the mechanism that determines the null response amplitude. The preferred response magnitude is approximately linear with variations in contrast. 6. The nonlinearity observed in the null direction can in principle be attributed to either a tonic excitation at 0 contrast or a threshold for inhibition. There is evidence for both processes in the Tan1 cell visual response.

GABA-mediated inhibition of visual interneurons in the crayfish medulla

1. The actions of GABA on three classes of visual interneurons in crayfish, Procambarus clarkii, medulla externa are examined. The effect of GABA on the visual response is compared to GABA's action on agonist-elicited responses purported to mediate the visual response. 2. GABA produces a shunting type of inhibition in medullary amacrine cells which is associated with a small depolarization (Figs. 2, 3), a large increase in input conductance (Gn) and a reversal potential close to rest (Fig. 4). GABA is a potent antagonist to the depolarizing action of acetylcholine (ACh) (Fig. 5). 3. GABA depolarizes dimming fibers (Fig. 2), and the response is mediated by an increase in Gn (Fig. 6). GABA antagonizes the light-elicited IPSP and the hyperpolarizing action of ACh (Fig. 7). 4. Sustaining fibers (SF) do not appear to have GABA receptors but GABA inhibits the excitatory visual input pathway to the SFs (Fig. 8). Conversely, the GABA antagonist, bicuculline, potentiates the SF light response (Fig. 9). 5. GABA has at least three different modes of antagonist action in the medulla: i) Increased conductance and depolarization in dimming fibers and medullary amacrine neurons; ii) Decreased chloride conductance in tangential cells; and iii) An inhibitory action on the visual pathway which drives SFs.