Pharmacogenetic Regulation of Acetylcholinesterase Activity in Drosophila Reveals the Regulatory Mechanisms of AChE Inhibitors in Synaptic Plasticity

We conducted experiments in Drosophila to investigate the consequences of altered acetylcholinesterase (AChE) activity in the nervous system. In ace hypomorphic mutant larvae, the amount of ace mRNA and the activity of AChE both in vivo and in vitro were significantly reduced compared with those of controls. Reduced Ace in Drosophila larvae resulted in significant down-regulation of branch length and the number of boutons in Type 1 glutamatergic neuromuscular junctions (NMJs). These defects in ace hypomorphic mutant larvae were suppressed when Musca domestica AChE was transgenically expressed. Because AChE inhibitors are utilized for medications for Alzheimer’s disease, we investigated whether pharmacological inhibition of AChE activity induced any synaptic defects. We found that controls exposed to a sublethal dose of DDVP phenocopied the synaptic structural defects of the ace hypomorphic mutant. These results suggest that down-regulation of AChE activity, regardless of whether it is due to genetic or pharmacological manipulations, results in altered synaptic architecture. Our study suggests that exposure to AChE inhibitors for 6–12 months may induce altered synaptic architectures in human brains with Alzheimer’s diseases, similar to those reported here. These changes may underlie or contribute to the loss of efficacy of AChE inhibitors after prolonged treatment.

     

Localization and Mobility of Synaptic Vesicles in Myosin VI Mutants of Drosophila

Background

At the Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; however, the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. Myosin VI has been shown to be important for proper synaptic physiology and morphology at the NMJ, likely by functioning as a vesicle tether. Here we investigate vesicle dynamics in Myosin VI mutants of Drosophila.

Results

In Drosophila, Myosin VI is encoded by the gene, jaguar (jar). To visualize active vesicle cycling we used FM dye loading and compared loss of function alleles of jar with controls. These studies revealed a differential distribution of vesicles at the jar mutant nerve terminal, with the newly endocytosed vesicles observed throughout the mutant boutons in contrast to the peripheral localization visualized at control NMJs. This finding is consistent with a role for Myosin VI in restraining vesicle mobility at the synapse to ensure proper localization. To further investigate regulation of vesicle dynamics by Myosin VI, FRAP analysis was used to analyze movement of GFP-labeled synaptic vesicles within individual boutons. FRAP revealed that synaptic vesicles are moving more freely in the jar mutant boutons, indicated by changes in initial bleach depth and rapid recovery of fluorescence following photobleaching.

Conclusion

This data provides insights into the role for Myosin VI in mediating synaptic vesicle dynamics at the nerve terminal. We observed mislocalization of actively cycling vesicles and an apparent increase in vesicle mobility when Myosin VI levels are reduced. These observations support the notion that a major function of Myosin VI in the nerve terminal is tethering synaptic vesicles to proper sub-cellular location within the bouton.     

Effect of reduced impulse activity on the development of identified motor terminals in Drosophila larvae

In Drosophila larvae, motoneurons show distinctive differences in the size of their synaptic boutons; that is, axon 1 has type Ib ("big" boutons) terminals and axon 2 has type Is ("small" boutons) terminals on muscle fibers 6 and 7. To determine whether axon 1 develops large boutons due to its high impulse activity, we reduced impulse activity and examined the motor terminals formed by axon 1. The number of functional Na(+) channels was reduced either with the nap(ts) mutation or by adding tetrodotoxin (TTX) to the media (0.1 microg/g). In both cases, the rate of locomotion was decreased by approximately 40%, presumably reflecting a decrease in impulse activity. Locomotor activity was restored to above wild-type (Canton-S) levels when nap(ts) was combined with a duplication of para, the Na(+)-channel gene. Lucifer yellow was injected into the axon 1 motor terminals, and we measured motor terminal area, length, the number of branches, and the number and width of synaptic boutons. Although all parameters were smaller in nap(ts) and TTX-treated larvae compared to wild-type, most of these differences were not significant when the differences in muscle fiber size were factored out. Only bouton width was significantly smaller in both different nap(ts) and TTX-treated larvae: boutons were about 20% smaller in nap(ts) and TTX-treated larvae, and 20% larger in nap(ts); Dp para(+) compared to wild-type. In addition, terminal area was significantly smaller in nap(ts) compared to wild-type. Bouton size at Ib terminals with reduced impulse activity was similar to that normally seen at Is terminals. Thus, differences in impulse activity play a major role in the differentiation of bouton size at Drosophila motor terminals.     

Aph‐1 is required to regulate Presenilin‐mediated γ‐secretase activity and cell survival in Drosophila wing development

Aph‐1 is a multipass transmembrane protein and an essential component of the Presenilin (Psn)‐mediated γ‐secretase complex. During protease assembly, Aph‐1 stabilizes the newly synthesized Psn holoprotein to facilitate generation of the active form of Psn, which is a Psn‐NTF/Psn‐CTF heterodimer produced through a Presenilinase‐initiated endoproteolytic cleavage of the Psn holoprotein. Although it is clear that loss of Aph‐1 activity leads to failure of Psn heterodimer formation, little is understood about whether Aph‐1 plays a role in regulating γ‐secretase activity in addition to assisting Psn maturation. Using various modified Psn forms that do not require endoproteolysis or have a large deletion of the cytosolic loop, we show that in Drosophila Aph‐1 is still required for γ‐secretase activity independent of its role in promoting Psn endoproteolysis. In addition, our results indicate that Aph‐1 is required to promote cell survival in the wing imaginal disc; aph‐1 mutant cells are lost either through cell death or because of a defect in cell proliferation. This function of Aph‐1 is independent of its role in regulating γ‐secretase activity, but possibly involves downregulating the activity of uncleaved Psn holoprotein. genesis 47:169–174, 2009. © 2009 Wiley‐Liss, Inc.     

Nervous wreck, an SH3 adaptor protein that interacts with Wsp, regulates synaptic growth in Drosophila

We describe the isolation and characterization of nwk (nervous wreck), a temperature-sensitive paralytic mutant that causes excessive growth of larval neuromuscular junctions (NMJs), resulting in increased synaptic bouton number and branch formation. Ultrastructurally, mutant boutons have reduced size and fewer active zones, associated with a reduction in synaptic transmission. nwk encodes an FCH and SH3 domain-containing adaptor protein that localizes to the periactive zone of presynaptic terminals and binds to the Drosophila ortholog of Wasp (Wsp), a key regulator of actin polymerization. wsp null mutants display synaptic overgrowth similar to nwk and enhance the nwk morphological phenotype in a dose-dependent manner. Evolutionarily, Nwk belongs to a previously undescribed family of adaptor proteins that includes the human srGAPs, which regulate Rho activity downstream of Robo receptors. We propose that Nwk controls synapse morphology by regulating actin dynamics downstream of growth signals in presynaptic terminals.     

The hangover gene negatively regulates bouton addition at the Drosophila neuromuscular junction

The synaptic growth of neurons during the development and adult life of an animal is a very dynamic and highly regulated process. During larval development in Drosophila new boutons and branches are added at the glutamatergic neuromuscular junction (NMJ) until a balance between neuronal activity and morphological structures is reached. Analysis of several Drosophila mutants suggest that bouton number and size might be regulated by separate signaling processes [Budnik, V., 1996. Synapse maturation and structural plasticity at Drosophila neuromuscular junctions. Curr. Opin. Neurobiol. 6, 858-867.]. Here we show a new role for Hangover as a negative regulator of bouton number at the NMJ. The hangover gene (hang) encodes a nuclear zinc finger protein. It has a function in neuronal plasticity mediating ethanol tolerance, a behavior that develops upon previous experience with ethanol. hang^AE10 mutants have more boutons and an extended synaptic span. Moreover, Hang expression in the motoneuron is required for the regulation of bouton number and the overall length of muscle innervation. However, the increase in bouton number does not correlate with a change in synaptic transmission, suggesting a mechanism independent from neuronal activity leads to the surplus of synaptic boutons. In contrast, we find that expression levels of the cell adhesion molecule Fasciclin II (FASII) are reduced in the hang mutant. This finding suggests that the increase in bouton number in hang mutants is caused by a reduction in FASII expression, thus, linking the regulation of nuclear gene expression with the addition of boutons at the NMJ regulated by cell adhesion molecules.     

Ultrastructure of neuromuscular junctions in Drosophilal: Comparison of wild type and mutants with increased excitability

The ventral longitudinal muscles of the Drosphila larval body wall are innervated by at least four types of synaptic terminals that can be distinguished on morphological grounds at the light microscopical level. The innervation of these muscles has been previously shown to be regulated by neuronal activity. In this report we investigate the ultrastructural basis for synaptic bouton differences by using serial sections, and examine the structure of synaptic terminals in mutants with increased excitability. We report that individual identifiable muscle fibers are innervated by terminals containing two to three types of synaptic boutons that can be distinguished in terms of synaptic vesicle population, presynaptic and postsynaptic specialization, and general shape. We propose a model to account for the bouton types observed at the light microscopical level. We find that in the hyperexcitable mutant eag Sh, there are dramatic ultrastructural alterations at synaptic boutons. These alterations include a partial depletion of two types of synaptic vesicles and a change in appearance of a third type, changes in number and appearance of synaptic densities, and the presence of multivesicular bodies. Our results show that an increase in neuronal excitability produces profound effects in synaptic terminal structure. © 1993 John Wiley & Sons, Inc.     

Localization and Function of the Cannabinoid CB1 Receptor in the Anterolateral Bed Nucleus of the Stria Terminalis

Background

The bed nucleus of the stria terminalis (BNST) is involved in behaviors related to natural reward, drug addiction and stress. In spite of the emerging role of the endogenous cannabinoid (eCB) system in these behaviors, little is known about the anatomy and function of this system in the anterolateral BNST (alBNST). The aim of this study was to provide a detailed morphological characterization of the localization of the cannabinoid 1 (CB1) receptor a necessary step toward a better understanding of the physiological roles of the eCB system in this region of the brain.

Methodology/Principal Findings

We have combined anatomical approaches at the confocal and electron microscopy level to ex-vivo electrophysiological techniques. Here, we report that CB1 is localized on presynaptic membranes of about 55% of immunopositive synaptic terminals for the vesicular glutamate transporter 1 (vGluT1), which contain abundant spherical, clear synaptic vesicles and make asymmetrical synapses with alBNST neurons. About 64% of vGluT1 immunonegative synaptic terminals show CB1 immunolabeling. Furthermore, 30% and 35% of presynaptic boutons localize CB1 in alBNST of conditional mutant mice lacking CB1 mainly from GABAergic neurons (GABA-CB1-KO mice) and mainly from cortical glutamatergic neurons (Glu-CB1-KO mice), respectively. Extracellular field recordings and whole cell patch clamp in the alBNST rat brain slice preparation revealed that activation of CB1 strongly inhibits excitatory and inhibitory synaptic transmission.

Conclusions/Significance

This study supports the anterolateral BNST as a potential neuronal substrate of the effects of cannabinoids on stress-related behaviors.     

latheo, a Drosophila gene involved in learning, regulates functional synaptic plasticity.

Mutations in the latheo (lat) gene disrupt associative learning in Drosophila , but a role for LAT in regulating neuronal function has not been demonstrated. Here, we report that LAT plays a central role in regulating Ca2(+)- and activity-dependent synaptic plasticity. Immunological localization of the LAT protein indicates it is present at synaptic connections of the larval neuromuscular junction (NMJ) and is enriched in presynaptic boutons. Basal synaptic transmission amplitude at the lat mutant NMJ is elevated 3- to 4-fold, and Ca2+ dependence of transmission is significantly reduced. Multiple forms of synaptic facilitation and posttetanic potentiation (PTP) are strongly depressed or absent at the mutant synapse. Our results suggest that LAT is a novel presynaptic protein with a role in the Ca2(+)-dependent synaptic modulation mechanisms necessary for behavioral plasticity.     

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

NAD⁺-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD⁺ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD⁺ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.     

Altered GPI modification of insect AChE improves tolerance to organophosphate insecticides

The olive fruit fly Bactrocera oleae is the most destructive and intractable pest of olives. The management of B. oleae has been based on the use of organophosphate (OP) insecticides, a practice that induced resistance. OP-resistance in the olive fly was previously shown to be associated with two mutations in the acetylcholinesterase (AChE) enzyme that, apparently, hinder the entrance of the OP into the active site. The search for additional mutations in the ace gene that encodes AChE revealed a short deletion of three glutamines (??3Q) from a stretch of five glutamines, in the C-terminal peptide that is normally cleaved and substituted by a GPI anchor. We verified that AChEs from B. oleae and other Dipterans are actually GPI-anchored, although this is not predicted by the “big-PI” algorithm. The ??3Q mutation shortens the unusually long hydrophilic spacer that follows the predicted GPI attachment site and may thus improve the efficiency of GPI anchor addition. We expressed the wild type B. oleae AChE, the natural mutant ??3Q and a constructed mutant lacking all 5 consecutive glutamines (??5Q) in COS cells and compared their kinetic properties. All constructs presented identical K_m and k_cat values, in agreement with the fact that the mutations did not affect the catalytic domain of the enzyme. In contrast, the mutants produced higher AChE activity, suggesting that a higher proportion of the precursor protein becomes GPI-anchored. An increase in the number of GPI-anchored molecules in the synaptic cleft may reduce the sensitivity to insecticides.     

Proteomic Analysis of Exosomes from Mutant KRAS Colon Cancer Cells Identifies Intercellular Transfer of Mutant KRAS

Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior. Herein, we show the results of a comprehensive proteomic analysis of exosomes from parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only). Mutant KRAS status dramatically affects the composition of the exosome proteome. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including KRAS, EGFR, SRC family kinases, and integrins. DKs-8 cells internalize DKO-1 exosomes, and, notably, DKO-1 exosomes transfer mutant KRAS to DKs-8 cells, leading to enhanced three-dimensional growth of these wild-type KRAS-expressing non-transformed cells. These results have important implications for non-cell autonomous effects of mutant KRAS, such as field effect and tumor progression.K-RAS (KRAS) is a small, monomeric GTPase whose biological activity is specified by its nucleotide binding state. Multiple lines of evidence highlight the importance of KRAS in colorectal cancer (CRC).¹ For example, activating missense mutations in KRAS, which lock the protein into the GTP-bound state, occur in 30% to 40% of CRCs and are strongly associated with poor prognosis (1, 2). Also, mutant KRAS negatively predicts responsiveness to anti-EGF receptor (EGFR) therapy (3).Early attempts to decipher the neoplastic consequences of mutant KRAS relied on overexpression studies. A drawback of these studies is their failure to simulate the genetic conditions present in human tumors, where there is often one wild-type (WT) and one mutant KRAS allele (1). More recently, KRAS mutant CRC cell lines have been engineered to selectively contain either the wild-type or the mutant KRAS allele (4), and a single mutant Kras allele has been activated in the intestine using genetically engineered mice (5). Detailed studies using these complementary approaches demonstrate a wide range of tumor-promoting effects of mutant KRAS (reviewed in Ref. 6). Much of what is known about mutant KRAS pertains to its ability to alter the behavior of a transformed cell in a cell autonomous manner. With the exception of increased tumor vascularity via increased tumor-derived VEGF expression (7, 8), non-cell autonomous effects of mutant KRAS have been much less studied.Exosomes are 30- to 100-nm secreted vesicles that have emerged as a novel mode of intercellular communication (9). We recently reported that exosomes purified from conditioned medium of mutant KRAS CRC cells contained higher levels of the EGFR ligand amphiregulin (AREG) and enhanced invasiveness of recipient cancer cells relative to exosomes from isogenically matched wild-type KRAS cells (10). These results prompted us to perform a comprehensive analysis of exosomes purified from these cells. Herein, we show that mutant KRAS induces many changes in exosomal protein composition. Notably, we show that (i) KRAS is contained within exosomes, (ii) exosomes can transfer mutant KRAS to cells expressing only wild-type KRAS, and (iii) mutant KRAS-containing exosomes enhance wild-type KRAS cell growth in collagen matrix and soft agar. These results have important implications for the progression of CRC tumors by providing a mechanism by which the tumor microenvironment may be influenced by non-cell autonomous signals released by mutant KRAS-expressing tumor cells.     

Non-canonical role of wild-type SEC23B in the cellular stress response pathway

While germline recessive loss-of-function mutations in SEC23B in humans cause a rare form of anaemia, heterozygous change-of-function mutations result in increased predisposition to cancer. SEC23B encodes SEC23 homologue B, a component of coat protein complex II (COPII), which canonically transports proteins from the endoplasmic reticulum (ER) to the Golgi. Despite the association of SEC23B with anaemia and cancer, the precise pathophysiology of these phenotypic outcomes remains unknown. Recently, we reported that mutant SEC23B has non-canonical COPII-independent function, particularly within the ER stress and ribosome biogenesis pathways, and that may contribute to the pathobiology of cancer predisposition. In this study, we hypothesized that wild-type SEC23B has a baseline function within such cellular stress response pathways, with the mutant protein reflecting exaggerated effects. Here, we show that the wild-type SEC23B protein localizes to the nucleus in addition to classical distribution at the ER/Golgi interface and identify multiple putative nuclear localization and export signals regulating nuclear–cytoplasmic transport. Unexpectedly, we show that, independently of COPII, wild-type SEC23B can also localize to cell nucleoli under proteasome inhibition conditions, with distinct distribution patterns compared to mutant cells. Unbiased proteomic analyses through mass spectrometry further revealed that wild-type SEC23B interacts with a subset of nuclear proteins, in addition to central proteins in the ER stress, protein ubiquitination, and EIF2 signalling pathways. We validate the genotype-specific differential SEC23B–UBA52 (ribosomal protein RPL40) interaction. Finally, utilizing patient-derived lymphoblastoid cell lines harbouring either wild-type or mutant SEC23B, we show that SEC23B levels increase in response to ER stress, further corroborating its role as a cellular stress response sensor and/or effector. Overall, these observations suggest that SEC23B, irrespective of mutation status, has unexplored roles in the cellular stress response pathway, with implications relevant to cancer and beyond that, CDAII and normal cell biology.Subject terms: Cell biology, Cancer genetics     

Terminal Axonal Arborization and Synaptic Bouton Formation Critically Rely on Abp1 and the Arp2/3 Complex

Neuronal network formation depends on properly timed and localized generation of presynaptic as well as postsynaptic structures. Although of utmost importance for understanding development and plasticity of the nervous system and neurodegenerative diseases, the molecular mechanisms that ensure the fine-control needed for coordinated establishment of pre- and postsynapses are still largely unknown. We show that the F-actin-binding protein Abp1 is prominently expressed in the Drosophila nervous system and reveal that Abp1 is an important regulator in shaping glutamatergic neuromuscular junctions (NMJs) of flies. STED microscopy shows that Abp1 accumulations can be found in close proximity of synaptic vesicles and at the cell cortex in nerve terminals. Abp1 knock-out larvae have locomotion defects and underdeveloped NMJs that are characterized by a reduced number of both type Ib synaptic boutons and branches of motornerve terminals. Abp1 is able to indirectly trigger Arp2/3 complex-mediated actin nucleation and interacts with both WASP and Scar. Consistently, Arp2 and Arp3 loss-of-function also resulted in impairments of bouton formation and arborization at NMJs, i.e. fully phenocopied abp1 knock-out. Interestingly, neuron- and muscle-specific rescue experiments revealed that synaptic bouton formation critically depends on presynaptic Abp1, whereas the NMJ branching defects can be compensated for by restoring Abp1 functions at either side. In line with this presynaptic importance of Abp1, also presynaptic Arp2 and Arp3 are crucial for the formation of type Ib synaptic boutons. Interestingly, presynaptic Abp1 functions in NMJ formation were fully dependent on the Arp2/3 complex, as revealed by suppression of Abp1-induced synaptic bouton formation and branching of axon terminals upon presynaptic Arp2 RNAi. These data reveal that Abp1 and Arp2/3 complex-mediated actin cytoskeletal dynamics drive both synaptic bouton formation and NMJ branching. Our data furthermore shed light on an intense bidirectional functional crosstalk between pre- and postsynapses during the development of synaptic contacts.     

Substrate activation in acetylcholinesterase induced by low pH or mutation in the π-cation subsite

Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the π-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the π-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.     

Acetylcholinesterase activity at the synapses of presynaptic boutons with presumed alpha-motoneurons in chicken ventral horn. Light- and electron-microscopic studies

Acetylcholinesterase (AChE) activity at the synapses of presynaptic boutons on presumed alpha-motoneurons in the chicken ventral horn was studied histochemically at the light- and electron-microscope levels. At the light-microscope level, many dot-like AChE-active sites were observed on the soma and dendrites of presumed alpha-motoneurons. On electron microscopy, reaction products for AChE activity were observed mainly in the synaptic clefts of the four kinds of presynaptic boutons: (1) S type boutons, (2) boutons containing small, spherical, dense cored vesicles (diameter range, 60-105 nm) and spherical, clear vesicles, (3) boutons containing medium-sized, spherical, dense cored vesicles (65-115 nm) and spherical, clear vesicles, and (4) boutons containing large, spherical, dense cored vesicles (80-130 nm) and spherical, clear vesicles. In the light of previous physiological and biochemical studies, the present results suggest the possibility that each of these presynaptic boutons which are AChE-active in their synaptic clefts may contain acetylcholine, substance P, or enkephalins which acts as a neurotransmitter or modulator.     

The pineal gland in wild-type and two zebrafish mutants with retinal defects

Light and transmission electron microscopy were used to characterize the ultrastructural features of the pineal glands of wild-type and two mutant zebrafish strains that have retinal defects. Particular attention was given to the pineal photoreceptors. Photoreceptors in the pineal gland appear quite similar to retinal cone photoreceptors, having many of the same structural characteristics including outer segment disk membranes often confluent with the plasma membrane, calycal processes surrounding the outer segments, and classic connecting cilia. The pineal photoreceptor terminals differ from photoreceptor terminals in the retina in that they have short synaptic ribbons and make dyad synapses which may or may not be invaginated. Pineal photoreceptors in two zebrafish mutants with abnormal retinal photoreceptors were also studied. Pineal photoreceptors in the niezerka (nie) mutant degenerate, as they do in the retina, indicating that pineal and retinal photoreceptors share at least some genes. However, the synaptic terminals of no optokinetic response c (nrc) pineal photoreceptors are normal, suggesting that this mutation is specific to the retina.     

Neuronal and synaptic organization of the lateral geniculate nucleus of the tree shrew,Tupaia glis

Summary The ultrastructural study of the lateral geniculate nucleus (LGN) of the tree shrew (Tupaia glis) revealed two types of neurons: (1) a large thalamocortical relay cell (TCR), which may bear cilia, and (2) a small Golgi type-II interneuron (IN) with an invaginated nucleus. The narrow rim of pale cytoplasm of the IN contains fewer lysosomes and fewer Nissl bodies than the cytoplasm of the TCR. The IN perikarya, which in some cases establish somatosomatic contacts, frequently contain flattened or pleomorphic synaptic vesicles. The ratio of TCR to IN is 31.Three types of axon terminals were observed in the LGN. Two of them contain round synaptic vesicles but differ in size. The large RL boutons undergo dark degeneration after enucleation; they are the terminals of retino-geniculate fibers. The smaller RS boutons show dark degeneration after ablation of the visual cortex; they are the terminals of the cortico-geniculate fibers. The third type of bouton (F₁ does not degenerate after either intervention. The boutons of this type are filled with flattened vesicles and are believed to be intrageniculate terminals. F₂-profiles were interpreted as presynaptic dendrites of the IN. The characteristic synaptic glomeruli found in the LGN contain in their center an optic terminal. These optic terminals establish synaptic contacts with dendrites or spine-like dendritic protrusions of TCRs as well as with presynaptic dendrites. Synaptic triads were also seen. The distribution of the individual types of synaptic contacts in layers 3 and 4 was determined. Layer 4 contains only one third of the retino-geniculate synapses and of the synaptic contacts of F₁-terminals.