Behavioral assays

Most studies of bacterial chemotaxis have used some form of the capillary tube assay originally described by Pfeffer (1884) and later modified by Adler (1973). This method, often referred to as the Adler method, measures the movement of bacteria into a capillary that has been filled with a test chemical. While the original method examined chemotaxis in Escherichia coli, this method, with minor modifications, has proven effective for measuring chemotaxis in marine microorganisms. 

In a generalized Adler method, one end of a capillary tube (1 Xl disposable micropipette, 3 cm long with an internal diameter of 0.2 mm) is flame sealed. The entire capillary tube is then quickly passed through a flame, and while warm, the open end is plunged into a reservoir containing the test chemical dissolved in chemotaxis medium. The liquid is drawn up into the capillary as it cools and the filled capillary is then withdrawn from the reservoir and inserted into a chemotaxis chamber, which is constructed by placing a U-shaped melting point capillary tube between a microscope slide and a coverslip (Fig. 1.2). The chamber is filled with an appropriate chemotaxis medium and inoculated with bacteria so that the final concentration is approximately 6 x 10 cells/ml. After a 1-h incubation, the capillary is removed from the chamber and the exterior rinsed with sterile water. The sealed end of the capillary is then broken and the contents are 

The advantages of the capillary assay are its simplicity, quantitative nature, and high sensitivity. Alternative methods for studying chemotaxis such as the swarm plate method of Adler (1966) require that the chemoattractant be metabolized. This is not necessary in the standard capillary assay. In addition, due to the small size of the chemotaxis chamber, only small amounts of compound are required to perform the experiments. The main disadvantage of this method is that the compound tested must be soluble in the chemotaxis medium. 

Willey and Waterbury (1989) used a substantially different method to study chemotaxis in the marine cyanobacterium Synechococcus sp. This experiment involved the use of blind-well chemotaxis chambers (Neuroprobe, Inc., Cabin John, MD) that consisted of an upper (800- il) and lower (200- li1) acrylic chamber separated by a polycarbonate filter (3.0 pm). A cell suspension (165 pi) of the cyanobacterium was placed in the lower chamber over which the polycarbonate filter was placed. An air space was left between the cell suspension and the filter to control the starting time of the experiment. The upper chamber was filled with sterile seawater containing the compound to be tested and then inverted, allowing the cell suspension to contact the polycarbonate filter and the seawater/compound solution. The experiments were run for 65 min, after which time the chambers were inverted to stop the experiment. The number of cells crossing the filter into the seawater chamber was determined by direct cell counts using epifluorescence microscopy. The motile strain of Synechococcus sp. tested in this assay elicited positive chemotaxis to compounds such as ammonia, nitrate, urea, glycine, and P-alanine. Control chambers with the same concentration of chemoattractant in both the upper and lower chambers failed to elicit a chemotactic response. While the compounds tested in this study were relatively simple metabolites, one could 

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Following from the above, behavioral assays, which can be relatively simple and cost-effective, can be very useful as primary screens when testing chemicals for their neurotoxicity in the context of medical toxicology (see Dewar 1983, Atterwill et al. 1991, and Tilson 1993). Where disturbances of behavior are identified, subsequent more specific tests, including in vitro assays, may then be performed to establish... 

Because the psychotomimetic benzomorphans, classed as sigma opioids, inhibit binding of 3H-PCP and show PCP-like actions in several behavioral assays, it has been suggested that PCP and sigma opioids act through the same binding sites. However, recent work by a number of investigators (Su 1982 Tam 1983 Martin et al. 

Kuhn, D. M., White, F. J., and Appel, J. B. (1977) Discriminative stimulus properties of hallucinogens Behavioral assay of drug action. In Discriminative Stimulus Properties of Drugs, edited by H. Lai, pp. 137-154. Plenum Press, New York. 

Perhaps the most studied constituent of passionflower is the flavonoid chrysin. Chiysin was isolated from P. coeruiea, a species closely related to P. incarnata. It binds to benzodiazepine receptors with micromolar affinity (Ki = 3 pM) and competes for binding with the benzodiazepine flunitrazepam (Medina et al. 1990). Behavioral assays (see below) suggest that chrysin acts as a partial agonist at central benzodiazepine receptors (Wolfman et al. 1994). 

The sedative and anxiolytic effects of passionflower were examined in two other animal behavioral assays (staircase test, light/dark box choice test). Both anxiolytic and sedative effects occur, as well as potentiation... 

Table II. Potency of Octopamine and DCDM in the Crayfish Behavioral Assay and the Effect of Selected Octopaminergic Antagonists on their Action.

Phenylethylamines. The properties of phenylethylamines have been investigated in some detail as stimulators of cAMP synthesis in homogenates of the CNS of the American cockroach (54 55), in the crayfish behavioral assay (31) and as inhibitors of the myogenic contraction of the locust ETi muscle (56) as shown in Table III. 

It is very important to note that none of these assays measures the interaction with the receptor directly and each may be subject to interferences from such factors as competition from amine uptake and metabolic systems the presence of penetration barriers, and possible indirect effects on the receptor by release of natural amines from presynaptic storage sites. This is particularly true for the behavioral assay with the crayfish. The results therefore cannot be taken as a completely reliable guide to the relative activity of these compounds at the OA receptor. However, the three studies show a number of similarities as well as some differences. 

Table VI. Comparative Activities of Several Phenyl Imidazolines on Locust Extensor Tibiae (ETi) Muscle and in the Crayfish Behavioral Assay.

Cupilure or (S j-TT-dimethyl citrate was identified as the contact sex pheromone of the female spider, Cupiennius salei. Chemical analysis, electrophysiology and behavioral assay demonstrated that it was indeed the contact sex pheromone." ... 

Blanchard DC, Griebel G, Blanchard RJ (2001a) Mouse defensive behavior pharmacological and behavioral assays for anxiety and panic. Neurosci Biobehav Rev 25 205-218 Blanchard DC, Hynd AL, Minke KA, Minemoto T, Blanchard RJ (2001b) Human defense behaviors to threat scenarios show parallels to fear- and anxiety-related defense patterns of non-human mammals. Neurosci Biobehav Rev 25 761-770 Blanchard DC, Griebel G, Blanchard RJ (2003) The mouse defense test battery pharmacological and behavioral assays for anxiety and panic. Eur J Pharmacol 463 97-116 Blanchard RJ, Blanchard DC (1989) Anti-predator defense behaviors in a visible burrow system. J Comp Psychol 103 70-82... 

In sum, both rigorous analyses of floral odors and olfactory/behavioral assays will be required to tease apart the critical factors that underlie pollinator-flower interactions. Recent work from independent sources indicates progress in this area. For example, studies using GC-EAD and conditioned proboscis extension (Wadhams et al., 1994 Blight et al., 1997 Pham-Delegue et al., 1997) have... 

Le Mdtayer, M., Marion-Poll, F., Sandoz, J. C. el al. (1997). Effect of conditioning on discrimination of oilseed rape volatiles by the honeybee use of a combined gas chromatography-proboscis extension behavioral assay. Chemical Senses 22 ... 

Wadhams, L. J., Blight, M. M., Kerguelen, V. et al. (1994). Discrimination of oilseed rape volatiles by honey bee novel combined gas chromatographic-electrophysiological behavioral assay. Journal of Chemical Ecology 20 3221-3231. 

Females begin to emit pheromone 9 or more days after the adult molt (Bodenstein, 1970 Takahashi et al., 1976 Hawkins and Rust, 1977), but clearly, this is variable and temperature dependent. The attractancy of gut extracts made on the first day after the imaginal molt corresponds to that of 0.1 ng ( )-periplanone-B (Sass, 1983). During the next 20 days, the effectiveness of both fractions of the sex pheromone (periplanone-A and periplanone-B) in behavioral assays increases 100-fold and remains high for at least the next 45 days. Collection of airborne pheromone with Tenax followed by behavioral assays showed that periplanone-A and periplanone-B were released by 10-25-day-old females in equal amounts, equivalent to 0.6 ng periplanone-B per female per day (Sass, 1983). Yang et al. (1998) confirmed an increase in pheromone activity in the early adult but showed a decline in pheromone between days 20 and 30. 

Other female pheromone components Behavioral assays have shown that males are highly responsive to isolated antennae fromteneral (i.e., newly molted) females ... 

Analytical studies of the tergal secretions of male B. germanica have identified a number of volatile compounds, none of which has so far been subjected to behavioral assays on females. Brossut et al. (1975) found p-hydroxybenzyl alcohol, o-hydroxybenzyl alcohol, di- and tri-methylnaphthalene, benzothiazole, two isomers of nonyl phenol, and myristic, palmitic, and oleic acids. The fatty acids constituted > 92% of the volatile fraction given their abundance in feces and frass, and their role as putative aggregation pheromones (Wileyto and Boush, 1983 Fuchs et al., 1985 Wendler and Vlatten, 1993 Scherkenbeck et al., 1999),... 

Laboratory observations remain essential because they can delineate the timing of mate finding and mating in relation to other activities, and observations can provide important information about interactions between the sexes and the role of sex pheromones. The identification of sex pheromones also requires standard laboratory behavioral assays. We urge students of cockroach behavior and chemical ecology to develop more realistic behavioral assays that will facilitate the identification of sex pheromones as well as a better understanding of their role in cockroach mating systems. 

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