The development of an ethanol model using social insects I: behavior studies of the honey bee (Apis mellifera L.).

BACKGROUND: The purpose of this experiment was to test the feasibility of creating an animal model of ethanol consumption using social insects. Honey bees were selected as the model social insect because much is known about their natural history, physiology, genetics, and behavior. They are also inexpensive to procure and maintain. Of special interest is their use of communication and social organization. METHODS: Using both between- and within-experiment designs, studies were conducted with harnessed foragers to determine whether honey bees would consume ethanol mixed with sucrose (and, in some cases, water). Shuttle-box and running-wheel studies were conducted to examine the effect of ethanol on locomotion. The effect of ethanol on stinging behavior in harnessed foragers was investigated. The effect of ethanol on Pavlovian conditioning of proboscis extension was also investigated. Finally, in a self-administration study, foraging honey bees were trained to fly to an artificial flower containing ethanol. RESULTS: (1) Harnessed honey bees readily consume 1%, 5%, 10%, and 20% ethanol solutions; (2) 95% ethanol will also be consumed as long as the antennae do not make contact with the solution; (3) with the exception of 95% ethanol, consumption as measured by contact time or amount consumed does not differ in animals that consume 1%, 5%, 10%, and 20% ethanol solutions; (4) exposure to a lesser (or greater) concentration of ethanol does not influence consumption of a greater (or lesser) concentration; (5) consumption of 10% and 20% ethanol solutions decreases locomotion when tested in both a shuttle-box and running-wheel situation; (6) consumption of 1%, 5%, 10%, and 20% ethanol does not influence stinging behavior in harnessed foragers; (7) ethanol solutions greater than 5% significantly impair Pavlovian conditioning of proboscis extension; and (8) free-flying honey bee foragers will readily drink from an artificial flower containing 5% ethanol. CONCLUSIONS: The experiments on consumption, locomotion, and learning suggest that exposure to ethanol influences behavior of honey bees similar to that observed in experiments with analogous vertebrates. The honey bee model presents unique research opportunities regarding the influence of ethanol in the areas of language, social interaction, development, and learning. Although the behavioral results are interesting, similarity between the physiologic effects of ethanol on honey bees and vertebrates has not yet been determined.

Detection of a light-sensitive pool of cGMP in goldfish cone color receptors by immunocytochemistry.

A procedure for the immunocytochemical localization of cGMP in the goldfish retina is described. The procedure is then used to evaluate the presence of cGMP in cone color receptors in retinas from dark-adapted and light stimulate fish. The results show that cone color receptors in dark-adapted retinas contain large amounts of immunocytochemically defined cGMP in the ellipsoid region, which are greatly reduced after light exposure.

Cyclic AMP-dependent protein kinase content of murine T and B lymphocytes.

Murine splenic lymphocytes have been separated by non-activating procedures into enriched T and B cell populations. Cyclic AMP-dependent protein kinase (cAPK) activity has been measured in those separated cell types by histone phosphorylation. As in other tissues, lymphocyte cAPK activity was found to be predominantly a soluble enzyme. By an analysis of cyclic AMP (cAMP) activation of enzyme activity it was shown that: (1) the basal activities were indistinguishable between these major lymphocyte subpopulations, (2) the Ka values for cAMP activation were identical for the two cell types (24 nM cAMP), and (3) the Vmax for enzyme activity was higher in T (5.47 pmole/min/10(6) cells) than in B (3.58 pmole/min/10(6) cells) cells. DEAE-cellulose chromatography experiments with mixed and enriched splenic lymphocyte populations extended and amplified this quantitative difference between T and B cells.

Cyclic AMP-dependent protein kinase content of murine splenic T and B lymphocytes and non-lymphoid tissues.

Cyclic AMP-dependent protein kinase activity has been measured in DEAE-cellulose elution profiles of highly enriched murine splenic T and B Lymphocytes. B Lymphocytes were found to contain about 10% of the amount of enzyme activity associated with T lymphocytes and several non-lymphoid tissues. Cyclic AMP-independent casein kinase activities were equivalent in T and B lymphocytes. Lymphocytes contained predominantly type I isozyme as opposed to several non-lymphoid tissues which all contained predominantly the type II form.

Cyclic nucleotide alterations in mixed leukocyte reaction: a preliminary report.

Endogenous cyclic adenosine and guanosine monophosphate (cAMP, cGMP) levels were studied in human peripheral blood lymphocytes during mixed leukocyte reactions (MLR). cAMP level was consistently elevated in one-way MLR, with good correlation to 3H-thymidine uptake in these reactions. In contrast, cGMP level was practically unchanged. Irradiation of reacting cell populations resulted in inhibition of cyclic nucleotide phosphodiesterase (PDE) activity. These results suggest that metabolic alterations in cAMP may be associated with immune reactions of cellular recognition.

Immunohistochemical localization of cyclic GMP in goldfish retina.

The immunocytochemical procedure for cyclic GMP (cGMP) localization has been modified by the development of: 1) an anti-cGMP antibody depleted control serum and 2) a tissue preadsorption procedure that greatly reduces nonspecific fluorescence. cGMP remaining associated with tissue sections following prolonged exposure to aqueous buffer was determined, by extraction and measurement by radioimmunoassay, to be approximately 38% of the initial cGMP content. Application of this improved immunohistochemical procedure to goldfish retinas reveals the highest concentrations of cGMP to be associated with the cone inner segments and nuclei of the photoreceptors. This retinal distribution of cGMP is in good agreement with that independently determined by microdissection studies. The validity of this procedure is therefore strengthened and cautions to its application and interpretation are outlined.

Residual cyclic nucleotide associated with tissues after exposure to aqueous buffer analogous to that used in immunocytochemistry.

A question concerning cyclic nucleotide immunocytochemical localization has been how much nucleotide remains associated with the tissue section. To answer that question cryostat sections of goldfish eye and mouse spleen, liver and lung were mounted on microscope slides and air dried. Following fixation by a variety of procedures employing heat, paraformaldehyde, glutaraldehyde, acetone, or ethanol, the sections were exposed to buffer (20 microM NaHPO4, 154 microM NaCl, pH 6.1) for 4 hours. The tissues were then scraped from the slides into 0.4N Perchloric Acid and cAMP and cGMP extracted and measured. The results show that fractions of both nucleotides are retained during buffer exposure. However, the amount retained varied with the: i) neucleotide, ii) fixation procedure, and iii) tissue type. Cyclic GMP retention was consistently higher (20-70%) than cAMP (6-30%). Glutaraldehyde was consistently more efficient in fixing cAMP, while cGMP retention was more variable with different fixation procedures. Tissue variability is seen in the example that spleen and liver retained more cGmp (71.4 and 70.6% respectively) than lung and eye (22.8 and 37.7% respectively). Maximum nucleotide loss occured during the first 5-30 minutes of buffer exposure with no additional loss accuring after another 20 hours. Collectively, these results demonstrate that cyclic nucleotides are retained during immunocytochemical staining procedures but that the degree of retention is dependent on several variables.

Immunotherapy with levamisole: early decrease of cAMP levels in lymphocytes from treated cancer patients.

Lymphocyte cyclic nucleotide content was studied before and after Levamisole administration to cancer patients. Twelve patients with disseminated cancer received 100 mg/m2 on two consecutive days; nine comparable patients with disseminated cancer served as controls. Endogenous cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured in lymphocytes before, 24, and 48 hours after ingestion of the first dose of Levamisole. A statistically significant decline in lymphocyte cAMP level was observed after drug administration and no significant changes were noted in cGMP levels. Further studies will be necessary to correlate this biochemical change in cyclic nucleotides with modulation of the functional level of cellular immune mechanisms.

A semi quantitative method for cyclic nucleotide localization by immunocytochemistry and its application in determining the distribution of cyclic AMP in lungs of normal and pertussis-vaccinated mice following histamine or epinephrine challenge.

A method is described which allows quantitative comparison of cAMP content determined by immunocytochemical procedures. This technique was then employed to localize cAMP in lungs of normal and pertussis-vaccinated mice following saline, histamine, or epinephrine challenge. All primary pulmonary compartments were shown to contain some immunoreactive (cAMP) material. However, epinephrine and histamine challenge selectively increased the cAMP content of the vasculature. No effect of epinephrine or histamine was detected in bronchial smooth muscle or interstitial tissue. This increased cAMP accumulation was observed in both normal and pertussis-vaccinated mice following epinephrine challenge but only in pertussis-vaccinated mice after histamine administration. These results demonstrate that histamine and epinephrine stimulate cAMP accumulation in the same pulmonary compartments supporting earlier speculation that histamine acts indirectly through epinephrine release. Further, primary involvement of the vasculature supports a more prominent role for this tissue in pertussis mediated histamine hypersensitivity.

Loss of lymphocyte cyclic AMP dependent protein kinase activity in malignant melanoma.

Cyclic AMP dependent protein kinase activity was depressed in whole thymus and spleen as well as isolated splenic lymphocytes from B16 melanoma bearing C57B1/6J mice as compared to control animals. A similar loss of enzyme activity was observed in human peripheral blood lymphocytes from melanoma bearing patients as compared to normal subjects. An unaltered level of activity in the heart of tumor bearing mice suggested some specificity for the lymphoid system. This depressed enzyme activity was the result of a diminished Vmax for cAMP stimulated calf histone phosphorylation. The tumor bearing state in the mouse was also accompanied by a depletion of small lymphocytes from both thymus and spleen and it is hypothesized that the losses of lymphocytes and cAMP dependent protein kinase activity are related.

On the relationship between inflammation and altered cAMP metabolism in lungs of B pertussis-vaccinated mice.

Bordetella pertussis-vaccinated mice were examined for evidence of inflammation. Using polymorphonuclear leukocyte and fluid accumulation as markers, inflammation was evidenced in the lungs and to a lesser extent in the livers of such mice. Both heart and kidney showed no evidence of inflammation. Development of the inflammatory lesion followed a time course similar to that previously reported for increased sensitivity to histamine-mediated cAMP accumulation. This close parrallelism between inflammation and altered cAMP metabolism supports the hypothesis that the increased cAMP accumulation might be related to a feedback mechanism regulating inflammatory mediator release.