Reintroduction of the PLB1 gene into Candida albicans restores virulence in vivo.

Phospholipases have been proposed to contribute to the virulence of Candida albicans. Recently, a candidal strain deleted for PLB1, the gene encoding the predominant phospholipase B (Plb1) secreted by C. albicans, was constructed and its virulence in an intravenous murine model of disseminated candidiasis was evaluated. In the present study, the PLB1 gene was reintroduced back into the plb1 null mutant to generate the revertant strain, which showed similar growth and morphology to its isogenic parent strain. Virulence of the revertant strain was found to be comparable to that of the parent strain in an intravenous murine model of disseminated candidiasis. To compare the abilities of the plb1 null mutant, the revertant and the isogenic parent strains to cross the gastrointestinal (GI) tract and cause systemic infection, an oral-intragastric infant mouse model of candidiasis was used. Histological examinations and analysis of c.f.u. of the pathogen in liver homogenates revealed that the parental and revertant strains were able to invade and traverse the GI mucosa to a significantly greater extent than the plb1 null mutant. Immunofluorescence and immunoelectron microscopic studies of infected host tissue using anti-Plb1 antibody showed that Plb1 is secreted during invasion of the gastric mucosa by the parental and revertant strains. In contrast, little or no labelling was observed in the null mutant strain. The results indicate that the Plb1 secreted by C. albicans enhances the ability of this organism to cross the GI tract and disseminate haematogenously. These studies provide unequivocal evidence supporting a role for Plb1 during the course of infection by C. albicans.

Vital staining of fungal elements in deep-seated mycotic lesions during experimental murine mycoses using the parenterally applied optical brightener Blankophor.

Optical brighteners of the diaminostilbene type are fluorescent dyes which are popular diagnostic tools in the mycology laboratory. While these dyes are conventionally used for the in vitro diagnosis of mycoses, their low toxicity and chemical reactivity have led us to investigate their potential use for in vivo staining of fungal elements in mycotic tissue. In mice we have established deep-seated candidiasis, cryptococcosis, aspergillosis and zygomycosis, as well as coccidioidomycosis, histoplasmosis and blastomycosis. After establishment of infection, which mostly required immunosuppression, a single dose of 100 microl of an aqueous solution (2.2 x 10(-4) M) of the optical brightener Blankophor P fluessig (4,4'-Bis [(4-anilino-6-substituted-1,3,5-triazine-2-yl) amino] stilbene-2,2'-disulfonic acid) was injected by the tail vein and the animals were sacrificed 1 h later. Sections of freshly prepared target organs were directly subjected to epifluorescence microscopy using an appropriate filter kit. In most cases, fluorescent fungal elements could be detected in the murine tissue. There was little evidence for uptake of the dye by non-infected tissues. It is suggested that radioactive labeling may render parenteral Blankophor suitable for radiographic localization of deep-seated mycotic foci in the host.

A major cell surface antigen of Coccidioides immitis which elicits both humoral and cellular immune responses.

Multinucleate parasitic cells (spherules) of Coccidioides immitis isolates produce a membranous outer wall component (SOW) in vitro which has been reported to be reactive with antibody from patients with coccidioidal infection, elicits a potent proliferative response of murine immune T cells, and has immunoprotective capacity in a murine model of coccidioidomycosis. To identify the antigenic components of SOW, the crude wall material was first subjected to Triton X-114 extraction, and a water-soluble fraction derived from this treatment was examined for protein composition and reactivity in humoral and cellular immunoassays. Protein electrophoresis revealed that the aqueous fraction of three different isolates of C. immitis each contained one or two major glycoproteins (SOWgps), distinguished by their molecular sizes, which ranged from 58 to 82 kDa. The SOWgps, however, showed identical N-terminal amino acid sequences, and each was recognized by sera from patients with C. immitis infection. Antibody raised against the purified 58-kDa glycoprotein (SOWgp58) of the Silveira isolate was used for Western blot and immunolocalization analyses. Expression of SOWgp was shown to be parasitic phase specific, and the antigen was localized to the membranous SOW. The water-soluble fraction of SOW and the purified SOWgp58 were tested for the ability to stimulate proliferation of human peripheral monocytic cells (PBMC). The latter were obtained from healthy volunteers with positive skin test reaction to spherulin, a parasitic-phase antigen of C. immitis, and from volunteers who showed no skin test reaction to the same antigen. The SOW preparations stimulated proliferation of PBMC from skin test-positive but not skin test-negative donors, and the activated cells secreted gamma interferon, which is indicative of a T helper 1 pathway of immune response. Results of this study suggest that SOWgp is a major parasitic cell surface-expressed antigen that elicits both humoral and cellular immune responses in patients with coccidioidal infection.

Retrovirus-induced immunodeficiency in mice exacerbates gastrointestinal candidiasis.

Dysfunction of neutrophils in patients infected with human immunodeficiency virus is at least partly responsible for secondary microbial diseases in these individuals, including invasive gastrointestinal (GI) candidiasis. Immunoregulatory disturbances associated with the development of AIDS in human immunodeficiency virus-infected patients exacerbates Candida albicans infection of the upper GI tract and frequently leads to oropharyngeal and esophageal candidiasis. In this article, we present the first report of a murine model of invasive GI candidiasis associated with an AIDS-related murine immunodeficiency syndrome that results from infection of C57BL/6 mice with a previously described retrovirus complex (LP-BM5). Mice of the inbred strain were infected with C. albicans by oral-intragastric inoculation as infants and with the retrovirus by the intraperitoneal route 30 days later. Control mice of the same strain were infected with C. albicans as above and subsequently infected with the avirulent, ecotropic helper virus (MBI-5). Animals were killed 90 days after retroviral challenge. Total and differential blood cell counts, CD4+ T-cell counts in the spleen, and the histopathology of the gastric mucosa of experimental and control animals were determined. The virulent LP-BM5-infected animals developed murine AIDS and showed eruptive and suppurative lesions, with associated C. albicans mainly in regions of the cardial-atrium fold of the stomach. Well-defined abscesses with entrapped C. albicans hyphae were observed in the region of the cardial-atrium fold of control mice. A significant increase in the number of C. albicans CFU in homogenized and plated segments of the GI tract was recognized in mice with murine AIDS versus the control animals. The murine model of GI candidiasis reported here permits examination of the nature of C. albicans interaction with the gastric mucosa both in the immunocompetent host under conditions in which the yeast exists predominantly as a commensal organism and in the immunosuppressed host during progressive stages of AIDS induced by a retroviral infection.

Isolation and expression of a gene which encodes a wall-associated proteinase of Coccidioides immitis.

A chymotrypsinlike serine proteinase of Coccidioides immitis with an estimated molecular size of 34 kDa has been shown by immunoelectron microscopy to be associated with the walls of the parasitic cells of this human respiratory pathogen. The proteinase has been suggested to play a role in spherule development. We report the isolation of a 1.2-kb cDNA from an expression library of C. immitis constructed in the lambda ZAP II phage vector. The cDNA is suggested to encode the 34-kDa protein. We demonstrate identity between segments of the deduced amino acid sequence of the open reading frame of the 1.2-kb cDNA and three distinct sequences obtained from cyanogen bromide cleavage peptides of the purified proteinase. The occurrence of N-glycosyl linkage sites in the deduced sequence of 309 amino acids of the open reading frame (ORF) correlates with our identification of such linkage sites in the native glycosylated proteinase. A protein encoded by an 800-bp fragment of the 1.2-kb cDNA, which was produced by transformed Escherichia coli XL1-Blue, was recognized by the anti-34-kDa protein antibody in a Western blot (immunoblot). Northern (RNA) hybridization of total poly(A)-containing RNA of C. immitis with the labeled 1.2-kb cDNA clone revealed a single band of approximately 1.75 kb. Partial homology was demonstrated between the deduced amino acid sequence of the ORF (927 bp) and reported sequences of alpha-chymotrypsin and chymotrypsinogens. Expression of the proteinase gene was examined by Northern dot blot analysis of total RNA from different stages of parasitic cell development in C. immitis. Maximum levels of specific mRNA were detected during early endospore wall differentiation. The 34-kDa proteinase appears to be concentrated in walls of the parasitic cells at stages of active growth. We suggest that the enzyme may participate in wall plasticization and/or intussusception or in cell wall turnover.

Coccidioides immitis fractions which are antigenic for immune T lymphocytes.

The principal mechanism of resistance to coccidioidomycosis in experimental animals has been reported to be T-cell-mediated immunity. We have generated a Coccidioides immitis antigen-specific murine T-cell line to identify specific macromolecules capable of eliciting an immune mouse T-cell proliferative response. The murine T cells were stimulated in vitro with a soluble conidial wall fraction (SCWF), which has been previously characterized by humoral and cellular immunoassays. The SCWF was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane, and the stained blot was cut into seven pieces based on the molecular size of the SCWF components. The nitrocellulose membrane strips were converted into antigen-bearing particles and tested in a T-cell proliferation assay. Antigenic components of the SCWF in the molecular size range of 43 to 66 kDa were identified as the most immunoreactive. In a parallel study, we used a cDNA expression library derived from mRNA of the mycelial phase of C. immitis, which was constructed in lambda gt11 to identify clones that encoded T-cell-reactive fusion proteins (FPs). The cDNA library was screened by using anti-SCWF rabbit serum, and the FPs expressed in Escherichia coli were isolated and tested for T-cell response in the same manner as the SCWF components. The nucleotide sequence of a 0.2-kb cDNA insert encoding a protein which elicited vigorous T-cell response was determined. The isolated cDNA insert hybridized to a single 1.9-kb mRNA band in a Northern blot of the total RNA fraction of the mycelial phase of C. immitis. Antibody with affinity for the T-cell-reactive FP was isolated from anti-SCWF rabbit serum by solid-phase immunoadsorption. The FP-specific antibody reacted with a 47-kDa polypeptide in Western blots (immunoblots) of the SCWF. The same antibody preparation was used for immunoelectron microscopy to show that the FP was localized in the walls of arthroconidia and spherules of C. immitis. Attempts to clone and sequence the entire gene which encodes the T-cell-reactive protein are under way. The results of this study should lead to the determination of the complete structure of an important T-cell-stimulating antigen of C. immitis.

Chlamydospore-like cells of Candida albicans in the gastrointestinal tract of infected, immunocompromised mice.

We have demonstrated in a previously described murine model of gastrointestinal (GI) and systemic candidiasis that the antifungal angent cilofungin was efficacious in clearing infection of body organs when administered subcutaneously by infusion, but permitted large numbers of Candida albicans in the GI tract to persist. Yeast and hyphae in these animals were associated primarily with the stratified squamous epithelium of the stomach. Administration of immunocompromising drugs (cyclophosphamide plus cortisone acetate) to animals with persistent GI infection resulted in relapse of systemic candidiasis. Histological examination of the gastric mucosa revealed invasive hyphal elements and yeast as well as multiple chlamydospore-like cells. Comparative histochemical and electron-microscopic examinations of these latter cells produced in host tissue and chlamydospores formed in vitro were conducted. The results suggested that similarities in wall and cytoplasmic composition and ultrastructure exist between these in vivo and in vitro produced C. albicans cells. Exposure of C. albicans to cyclophosphamide during in vitro growth resulted in stimulation of chlamydospore production. No significant effect of cortisone acetate on C. albicans morphogenesis was detected. The murine model used in this study permits investigation of the formation of chlamydospore-like cells of C. albicans during early stages of fungal invasion of cyclophosphamide-treated mice, and of the possible influence of these cells on immunological response of the host to persistent candidiasis of the GI tract.

Antigen complex of Coccidioides immitis which elicits a precipitin antibody response in patients.

The occurrence in patients of elevated levels of immunoglobulin M (IgM) precipitin antibody to Coccidioides immitis antigens, which are commonly detected by the immunodiffusion-tube precipitin (TP) assay, is suggestive of primary nondisseminating coccidioidomycosis. We previously demonstrated that the concanavalin A-bound mycelial culture filtrate plus lysate preparation is a source of at least two TP antibody-reactive antigens (TP-Ags), which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 120- and 110-kDa fractions. Evidence is presented here that the crude filtrate plus lysate preparation contains additional lectin-bound, TP antibody-reactive fractions as well as a component which elicits a complement fixation antibody response in patients. The 120- and 110-kDa fractions were isolated from the antigen complex and further characterized in this paper. Both TP-Ags are glycoproteins and have been shown by immunoelectron microscopy to be colocalized within cytoplasmic vesicles and the wall of spherules. Deglycosylation of these TP-Ags by sodium periodate treatment resulted in a loss in patients of 82 to 95% of IgM adsorption to the antigens as detected by the enzyme-linked immunosorbent assay (ELISA). Comparison of their carbohydrate compositions revealed that mannose and glucose are the predominant monosaccharides of both TP-Ags but only the 120-kDa fraction contained 3-O-methylmannose, a sugar which appears to be unique to C. immitis among the systemic fungal pathogens. We previously showed that 3-O-methylmannose is at least partly responsible for the reactivity of IgM antibody with the 120-kDa TP-Ag. Good correlation was shown between results of immunodiffusion-TP assays and ELISAs of IgM response to both the 120- and 110-kDa fractions by using 70 serum samples from patients with proved coccidioidomycosis. However, only 2.8% (3 of 109) of the serum samples from patients with other mycoses and nonmycotic infections showed IgM adsorption to the 120-kDa TP-Ag as detected by the ELISA, while 21.1% (23 of 109) showed IgM adsorption to the 110-kDa TP-Ag. The 120-kDa TP-Ag is a potentially valuable serodiagnostic reagent for detection of specific IgM by ELISA in patients with primary coccidioidomycosis.

Evaluation of a murine model of hepatic candidiasis.

A murine model of focal hepatic candidiasis which we suggest simulates certain conditions of this clinical variant of systemic candidiasis in leukemic patients is described. We have shown that outbred mice inoculated with Candida albicans by the oral-intragastric route as infants (6 days old) and then immunocompromised by cyclophosphamide and cortisone acetate treatment 2 weeks later demonstrate systemic spread of the opportunistic pathogen to the liver, lungs, spleen, and kidneys. Treatment with the immunosuppressive drugs cyclophosphamide and cortisone acetate resulted in alteration of the normal integrity of the mucosal epithelium of the gut as well as in granulocytopenia. Approximately 55% of the animals with C. albicans infections in the liver demonstrated hepatic abscesses. After these same infected, immunocompromised animals were treated with suboptimal dosages of antifungal agents (cilofungin or amphotericin B), either by intraperitoneal or subcutaneous (s.c.) routes, persistent hepatic abscesses were fewer in number and delimited by a distinct outer layer of host tissue but still contained large numbers of the viable pathogen. Blood cell counts indicated that these antifungal drug-treated animals had reestablished approximately the same number of leukocytes per microliter of blood as estimated prior to the immunocompromising drug treatment. Similar conditions in leukemic patients who were in remission and who were undergoing antifungal drug therapy for systemic candidiasis have been reported. Clearance of hepatic infections in mice was accomplished by using appropriate concentrations of amphotericin B administered by daily intraperitoneal or s.c. injection for 5 to 7 days or cilofungin by continuous s.c. infusion for 7 days. However, systemic antifungal therapy did not significantly reduce numbers of C. albicans cells in the stomach and esophagus. Persistent foci of gastrointestinal colonization by C. albicans, especially in the region of the cardial-atrium fold of the stomach of these mice, are reservoirs of the opportunistic pathogen from which reinfection may occur, leading to relapse of systemic candidiasis.

An animal model for oropharyngeal, esophageal and gastric candidosis.

Conventional mice inoculated with Candida albicans by the oral-intragastric route as infants (6-day-old) have previously been shown to develop gastrointestinal (GI) candidosis which persists for at least 30-60 days post-challenge without the use of compromising procedures. Histological preparations of the stomachs of these animals reveal hyphae which have crossed the mucin barrier and are associated with the stratified squamous epithelium of the gastric mucosa primarily in the region of the cardial-atrium fold. Host inflammatory cells are frequently observed adjacent to these filaments and yeast cells. In this study, groups of neonates were challenged oral-intragastrically with either C. albicans strain CA30, or strain CA87. The two strains showed marked differences in the numbers of cells associated with tissue of the tongue, esophagus and stomach of non-immunocompromised mice at 20 days post-inoculation. After immunocompromising treatment by intraperitoneal administration of cyclosphosphamide and cortisone acetate, both groups of mice showed extensive colonization and tissue invasion of the tongue, proximal and distal portions of the esophagus, and cardial-atrium fold of the stomach. C. albicans-containing abscesses were occasionally observed on the tongue of these animals. Histological preparations of the cardiac antrum, located at the junction of esophagus and stomach, frequently revealed concentrations of hyphae and yeast cells associated with the stratified squamous epithelium. We suggest that these non-immunocompromised and immunocompromised mice with persistent C. albicans infections of oropharyngeal, esophageal and gastric tissue, initiated by oral-intragastric challenge at infancy, simulates conditions in different groups of chronically infected humans, and serves as a useful model for testing the efficacy of anti-Candida drugs in clearance of candidosis from the alimentary canal.

Composition, serologic reactivity, and immunolocalization of a 120-kilodalton tube precipitin antigen of Coccidioides immitis.

Diagnosis of coccidioidomycosis largely depends on serologic tests. In this investigation, the enzyme-linked immunosorbent assay (ELISA) was used to detect patient immunoglobulin M (IgM) precipitin antibody binding to a 120-kilodalton (kDa) fraction previously isolated from an alkali-soluble, water-soluble extract of the arthroconidial wall and mycelial culture filtrate plus toluene lysate of Coccidioides immitis. Results of the serologic response to this tube precipitin antigen (TP-Ag) in the ELISA correlated well with results of immunodiffusion assays of 30 serum samples from patients. Immunoelectron microscopic examinations of arthroconidia and spherules were performed with patient IgM precipitin antibodies isolated from sera eluted over a solid-phase immunosorbent column containing the purified 120-kDa TP-Ag. The antibody probe located the 120-kDa TP-Ag on the walls of in vitro-grown arthroconidia and spherules. Pronase digestion and heating (100 degrees C, 5 min) had no apparent effect on the activity of the 120-kDa TP-Ag, while periodate oxidation resulted in total loss of its immunodiffusion-TP activity. Analysis of the carbohydrate composition of the TP-Ag revealed xylose, 3-O-methylmannose (3-O-MM), mannose, galactose, and glucose. Competitive inhibition ELISAs were used to demonstrate that 3-O-MM is largely responsible for the reactivity of IgM precipitin antibodies with the 120-kDa TP-Ag. Synthetic 3-O-MM may be a useful probe for detection of anti-Coccidioides precipitin antibodies in the ELISA.

Gastrointestinal and systemic candidosis in immunocompromised mice.

Oral-intragastric inoculation of 6-day-old outbred Crl:CFW(SW) BR mice with Candida albicans can lead to colonization of the gastrointestinal (GI) tract. We have shown that in the absence of an immunocompromising treatment, Candida is primarily localized in the stomach and intestines of mice at 20 days post-inoculation. Cultures of homogenates of the esophagus of most animals tested, and homogenates of the liver, lungs, spleen and kidneys of all animals tested, proved negative for C. albicans. Previous histological examinations of the GI tract of these colonized, non-immunocompromised mice showed hyphal elements associated with the stratified, squamous epithelium of the stomach in the region of the cardial-atrium fold. In this study, mice were immunocompromised by intraperitoneal injection of cyclophosphamide and cortisone acetate 11-14 days after oral-intragastric challenge with C. albicans and then sacrificed 20 days post-challenge. A high density of invasive hyphae was observed in the same, cardial-atrium region of the stomach of these animals. Cultures of the homogenized stomach showed a 100-fold increase in colony forming units (c.f.u.) of C. albicans compared with stomach homogenates of infected but non-immunocompromised controls. In addition, homogenates of the esophagus and selected body organs of most immunocompromised mice examined were positive for C. albicans by plate culture. When the immunocompromising drug treatment was delayed 3-5 weeks after oral-intragastric challenge, proliferation of C. albicans in the stomach and intestines was still evident, although fewer mice showed systemic spread and lower numbers of c.f.u. were recovered from body organ homogenates. Abscesses which contained both C. albicans hyphae and yeast cells were frequently observed in the liver and occasionally in the lungs and kidneys of immunocompromised mice sacrificed 20 days post-inoculation. The frequent occurrence of abscesses in the liver simulates a clinical variant of this mycosis, referred to as focal hepatic candidosis, which has been recognized with increasing frequency in immunocompromised patients. We suggest that the animal model described here may be particularly useful both for exploring methods which may prevent dissemination of C. albicans from localized foci of colonization in the GI tract after exposure of the host to immunocompromising drugs, and for testing the efficacy of anti-Candida drugs in clearance of the pathogen from body organs with established fungal abscesses.

Isolation and morphology of an immunoreactive outer wall fraction produced by spherules of Coccidioides immitis.

A previously undescribed, immunoreactive, membranous spherule outer wall (SOW) fraction produced by Coccidioides immitis (strains 634 and 735) grown in culture was isolated. Both this fraction and intact spherules were reactive with sera from coccidioidomycosis patients, as demonstrated by immunofluorescence microscopy. The serological activity of SOW was also demonstrated by its reactivity with human anti-C. immitis tube precipitin in a standardized immunodiffusion assay. Extraction of SOW with the nonionic detergent N-octyl-beta-D-glucopyranoside (OG) permitted the isolation of an OG-soluble fraction which was reactive in the immunodiffusion assay. Rabbit antisera raised against the OG-soluble fraction were used in immunofluorescence and immunoelectron-microscopic studies of the parasitic cycle to confirm that the immunoreactive components of the solubilized fraction of SOW were associated with the inner and outer layers of the spherule wall as well as with distinct cytoplasmic organelles observed in thin sections of spherules. The immunoreactivity of SOW with sera from patients suggested that infected individuals are exposed to this surface wall material isolated from in vitro-grown spherules.

Morphological aspects of gastrointestinal tract invasion by Candida albicans in the infant mouse.

The infant mouse has proved to be a useful model for examination of various aspects of gastrointestinal and systemic candidosis. Oral-intragastric inoculation of 5-6-day-old mice with yeast of a virulent strain of Candida albicans (CA30) resulted in systemic spread within 30 min after challenge. Histological examinations of the gastrointestinal (GI) tract have shown that the highest frequency of invasion of the mucosa by yeast cells occurred in the region of the jejunum 1-3 h after inoculation. Results of ultrastructural examinations of sites where the fungus invaded the bowel wall suggested that C. albicans yeast cells are capable of progressive extracellular digestion of the intestinal mucus barrier and microvillus layer, followed by intracellular invasion of columnar epithelial cells. Minimal disruption of cytoplasmic contents of the host epithelial cells appears to result from invasion and transmigration of the pathogen. The infant mouse model is suggested to be well suited for localization of extracellular products of C. albicans yeast in vivo which may play pivotal roles in the invasion of host tissue during GI candidosis.

Protein transport between crayfish lateral giant axons.

Segmental lateral giant axons (SLGAs) in crayfish were used to determine whether functionally intact proteins can move between axons under physiological conditions. Horseradish peroxidase (HRP) was chosen as the tracer protein because its localization requires intact enzymatic activity and because it can be localized in living cells using a non-cytotoxic procedure. Following iontophoretic injection of HRP in a single SLGA, HRP often transferred to adjacent SLGAs. HRP transferred from an injected SLGA to a caudal SLGA with greater frequency than HRP transferred to a rostral SLGA. When HRP transferred between SLGAs, it was ultrastructurally associated with vesicles on both sides of septate junctions between adjacent SLGAs and was also seen in the perijunctional extracellular space. There was no difference between the electrical resistance of synapses at which HRP transferred and those synapses where HRP did not transfer. HRP transfer was significantly reduced when axons were bathed in reduced calcium saline. These and other observations indicate that axon-to-axon transport in this system is accomplished by exocytosis of HRP from injected axons followed by its endocytotic uptake by adjacent, non-injected axons. Similar transfer of endogenous proteins may contribute to the long-term survival for months to years of distal stumps of severed SLGAs.

Developmental and other factors affecting regeneration of crayfish CNS axons.

According to histological and ultrastructural criteria, nongiant CNS axons in newly hatched crayfish regenerate more rapidly and with greater frequency than do similar axons in adult crayfish. Regenerative ability is greater in one species (Procambarus clarkii) than in another species (Procambarus simulans), is greater at 20-25 degrees C than at 15-16 degrees C, and is greater in nongiant axons than in giant axons. In contrast to axonal regeneration, nerve cell bodies do not regenerate in newly hatched or adult crayfish of either species. While the ability to regenerate CNS axons differs between newly hatched and adult crayfish, the ultrastructural appearance of the CNS is very similar at any age it is examined.

Organization of axoplasm in crayfish giant axons.

Distributions of subcellular organelles in medial giant axons (MGAs) and segmental lateral giant axons (SLGAs) of crayfish were evaluated as part of an ongoing effort to understand and explain differences in distal stump survival following axonotomy. Both axons were able to endocytose tracer proteins placed extracellularly, and horseradish peroxidase injected by cannulation into MGAs could transfer into adaxonal glial cells. Concentrations of tubulovesicular organelles near axonal cell membranes were measured as a possible index of the relative level of axonal endo- and exocytosis, and concentrations in MGAs were found to be twice those in SLGAs. In both axons, microtubule concentrations were highest near the axolemma and lowest in the central core of axoplasm. In thoracic and abdominal regions of MGAs, microtubules and other organelles were located only in a thin layer of subaxolemmal axoplasm. Overall, MGAs contained fewer microtubules per cross-section than did SLGAs, although MGAs are five to ten times as long as SLGAs and support more synapses. Total numbers of microtubules per cross-section varied with distance from the cell body of an MGA, whereas microtubule numbers were similar in proximal and distal cross-sections of SLGAs. In addition to a layer of subaxolemmal mitochondria which was observed in MGAs and SLGAs and which is characteristic of crayfish axons, mitochondria were also concentrated in the central core of SLGA axoplasm.

Formation of cockroach interganglionic connectives: an in vitro analysis.

Individual ganglia of the cockroach embryo (Periplaneta americana) were explanted on clean glass coverslips immersed in a chemically defined liquid medium and incubated for periods up to eight weeks. Substantial, straight interganglionic connections were formed between: (1) rows of ganglia arranged in the normal in vivo configuration; (2) rows of ganglia placed in abnormal orders; (3) rows of ganglia which never form connections in vivo because they occur singly in the embryo; and (4) rows of ganglia in natural sequences but which have had their rostro-caudal axes rotated 90 degrees in relation to the line of the row. Therefore fascicles and interganglionic connectives were formed without regard to normal in vivo relationships. Daily observations with a Nomarski microscope indicated that several processes are involved in connective formation. (1) Initial outgrowth is in a random, radial pattern. (2) Intersecting fibers from adjacent ganglia are deflected toward each others' perikarya. (3) Initially bowed fiber connectives are straightened, perhaps by increases in fiber tension or by fiber shortening which may be brought about by neuronal or extraneuronal (glial) processes. (4) Outgrowing fibers follow already established fiber pathways. The present results indicate that fiber-fiber and fiber-target interactions play a significant role in the formation of interganglionic connectives. In this system, the spatial relationships between ganglia determine the patterns and varieties of permissible neuronal connections. Thus, major, straight nerve trunks may be formed between adjacent ganglia which are growing out fibers on a glass surface submerged in a liquid medium which offers minimal orientation cues and provides a growth substrate vastly different and simpler than that encountered by outgrowing fibers in vivo.