An ultrastructural analysis of tissue surrounding a microdialysis probe.

Microdialysis is a widely used in vivo sampling technique commonly used to monitor extracellular levels of a variety of molecules including neurotransmitters and metabolites. To facilitate interpretation of microdialysis results, this study critically examines changes in synaptic morphology induced by microdialysis. Tissue surrounding microdialysis probes was examined using light and electron microscopy at three distances from the probe tract. Microdialysis probes were implanted into rat striatum, and after 40 h of post-operative recovery were perfused with a modified Ringer's solution. Light microscope analysis revealed tissue disruption up to 1.4 mm from the probe site. Axonal damage indicative of non-excitotoxic insult was also seen as far away from the probe as was examined. The presence of dark-degenerating neurons was also noted and estimates of neuronal densities revealed loss up to 400 microm from the probe tract. This study, the first qualitative ultrastructural investigation of neuropil surrounding the probe site, indicated swollen processes up to 1.4 mm from the probe tract. Swollen mitochondria and bloated endoplasmic reticulum suggest intracellular chemical disruption. Tissue damage resulting in synaptic and neuronal disruption may affect neurotransmitter efflux or extracellular concentrations of metabolites.

Peritrophic envelopes of Lutzomyia spinicrassa (Diptera: Psychodidae).

The secretion, morphology, and chemical composition of the peritrophic envelope were studied in the phlebotomine sand fly, Lutzomyia spinicrassa Morales, OsornoMesa, Osorno & Hoyos, a suspected vector of Leishmania braziliensis in Colombia and Venezuela. Viewed under light microscopy, the envelope matured rapidly and could be dissected from the blood bolus as early as 12 h and until 36 h after feeding; subsequently it began to degrade. The envelope was initially a closed sac around the blood meal, but opened posteriorly in most flies by 6 h. The posterior opening may facilitate the migration and establishment of Le. braziliensis in the hindgut. Secretion of envelope precursors was from the entire midgut epithelium. Electron microscopy revealed that electron-dense precursor material (possibly chitin) was present, bathing the microvilli during the first 12 h after blood feeding. This secretion appeared to originate from the bases of the microvilli. From 1 to 36 h, an electron-lucid precursor material (possibly protein) was secreted from the entire length of microvilli and from their bases. Both precursors appeared to be formed at the epithelial surface, not associated with secretory vesicles. The envelope developed rapidly from precursor material, and by 6 h a defined electron-lucid structure was present above the microvilli. Most mature envelopes (12-36 h) were 0.5-2.1 microns thick, multilayered, wholly electron-lucid, and composed of microfibrils and granules. Electron-dense components were seen in some envelopes at 24-36 h. An anterior hyaline plug was present from 12 to 36 h. Envelopes were composed of chitin, protein, and glycoprotein, based on chemical and histochemical tests. The likely presence of several amino acids (lysine, aspartic acid, and glutamic acid) that may cross-link chitin and protein was demonstrated by a positive ninhydrin-Schiff test. This study constitutes the first ultrastructural investigation of peritrophic envelope development by a New World sand fly.

Life cycle of Leishmania major (Kinetoplastida: Trypanosomatidae) in the neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae).

The development of Leishmania major Yakimoff & Schokhor in the New World sand fly Lutzomyia longipalpis (Lutz & Neiva) was examined by light and electron microscopy. In this unnatural host, parasites differentiated into 10 typical morphological forms, multiplied at three sites, migrated anteriorly and established in the foregut, and attached to gut surfaces. In the blood meal, amastigotes divided and transformed into two successive dividing, stumpy promastigote stages. Elongate nectomonad promastigotes developed from stumpy forms and subsequently rounded up in some flies into paramastigotes and opisthomastigotes. Differentiation into round opisthomastigotes and the apparent fusion of paramastigotes in the blood meal were novel observations in this study. Three nectomonad promastigotes--elongate, short, and metacyclic--were free-swimming in the midgut lumen. Elongate nectomonad promastigotes were highly oriented in the midgut, with their flagella embedded between the epithelial microvilli. Short haptomonad promastigotes were the predominant form attached to the intima of the stomodeal valve, whereas pear-shaped haptomonad promastigotes and paramastigotes colonized surfaces of the esophagus and pharynx. Peripylarian attachment of promastigotes and paramastigotes in the pylorus, ileum, and colon was noted in 21% of flies, suggesting that suprapylarian leishmanias have not lost the ability to colonize the hindgut. L. longipalpis was a successful biological host for L. major, allowing complete development of the parasite.

Formation and composition of the peritrophic membrane in the sand fly, Phlebotomus perniciosus (Diptera: Psychodidae).

The secretion, morphology, and chemical composition of the peritrophic membrane was studied in the sand fly, Phlebotomus perniciosus Newstead. The membrane was secreted from the entire midgut epithelium. An electron-dense fine granular secretion, possibly chitin, was present along the length of the microvilli immediately until 24 h after feeding. From 12-48 h, an electron-lucid coarse granular component, possibly protein, was also secreted from the microvillar surface. By light microscopy, the mature 36-h membrane characteristically consisted of a dark anterior cap and posterior open ring, with a transparent intervening membrane and anterior plug. Ultrastructure of the fully formed membrane at 24-48 h was highly variable. Undifferentiated membranes appeared as a single electron-lucid layer; differentiated membranes were more complex, sometimes two-layered, containing electron-lucid and -dense fibers and granules. Results of binding to succinylated wheat germ agglutinin, histochemistry, and amino acid analysis indicated that the membrane was composed of chitin, glycoprotein, and protein. Eighteen amino acids were identified in membrane proteins; aspartic-glutamic acids (and amides), serine, glycine, and lysine (45% by weight) may be important in cross-linking membrane components.

Refractory barriers in the sand fly Phlebotomus papatasi (Diptera: Psychodidae) to infection with Leishmania panamensis.

The life cycle of Leishmania panamensis in Phlebotomus papatasi was studied to characterize barriers limiting parasite colonization, differentiation, migration, and attachment in an unnatural sand fly host. The insects were fed a suspension of L. panamensis-infected macrophages and human erythrocytes, and were examined up to 16 days post-infection by light and electron microscopy. Histologic examination of 401 flies showed the peritrophic membrane to be the first important barrier to parasite establishment in the gut lumen. In most flies, parasites were unable to escape from the closed peritrophic sac, which was either excreted or retained intact in the midgut. After five days, only 31% of the flies were infected; attached parasites colonized the pylorus-ileum and/or colon regions of the hindgut. Anterior migration into the cardia region of the midgut occurred in less than 1% of infected flies; no parasites colonized the foregut. In the bloodmeal and residual bloodmeal, five morphologic forms developed from ingested amastigotes: stumpy, spatulate, elongate, short nectomonad promastigotes, and paramastigotes. Abnormal retention of amastigotes in macrophages and delayed development of promastigote stages was observed. The primary form attached in the hindgut was a pear-shaped haptomonad promastigote. Differentiation of L. panamensis in Ph. papatasi appeared to be similar to that described in natural hosts, except that metacyclic infective forms were not observed, and some forms developed in unusual locations. Phlebotomus papatasi was a partly refractory biological host for L. panamensis. The peritrophic membrane adversely affected the infection rate; rare anterior migration and a lack of metacyclic promastigotes may preclude transmission by bite.

Abnormal neuromuscular junctions in the lateral rectus muscle of wobbler mice.

We have studied the lateral rectus muscles and neuromuscular junctions (NMJs) of abducens motoneurons in wobbler (wr/wr) mutant mice from 26 to 58 days of age. The muscles of wr/wr weighed about 70% of the weight of littermate controls and were composed of fiber types comparable to those of controls, as assayed by succinate dehydrogenase activity. The most obvious difference between wr/wr and control NMJs was a reduction in the length of the postjunctional membrane of wr/wr mice. The mutant muscle endplate membrane was only about 70% (6.58 micron) the length of control muscle regions (9.44 micron). There were no obvious differences at the light microscopic level in the distribution of acetylcholine (ACh) receptors at junctional regions or staining of acetylcholinesterase, as assayed with alpha-bungarotoxin binding or enzyme histochemistry. Indirect immunocytochemical studies using antibodies directed against the subunits of the ACh receptor failed to indicate an abnormal presence of immature receptors clustered at the NMJs of wr/wr mice. Our findings suggest that the formation or maintenance of normal postjunctional folds and the differentiation of receptors at the junctions are under independent control during development. Furthermore, the wobbler mutation may affect muscle cell differentiation as well as neuronal differentiation. This mutant mouse should prove a useful model for study of postjunctional fold formation and function.