The paper that layed the foundation of my staining protocal

During my background research, my supervisor sent me a paper for me to read, he said this paper will help me with my immunostaining protocol. The paper he sent me was conducted by researchers from Canada, Israel and Switzerland on locust foraging gene.

The background for this paper is that animals, including locusts display a astonishing genetic interaction with their environment to generate behavioral plasticity. In this study, the model animal researchers used were migratory locusts, Locusta migratoria, and desert locusts, Schistocerca gregaria. During their solitary-phase, those animals actively avoid contact with conspecifics and are relatively sedentary. However, they become more active, and start marching in bands of hoppers or flying in swarms in their gregarious phase, when their population density increases.


So far, numerous neurobiology studies have characterized the differences in locusts' sensory interneurons that faciliated this behavioral change. In contrast, little has been done to study the molecular and genetic contribution to this phenomenon.

Recently, there are a handful of other well-studied animals that have shed light on the genetic and  pathways that mediate environmental and behavior variations and plasticity. Recent studies have found that those foraging-related behaviors have been linked to the foraging gene (for) and a cGMP-dependent protein kinase (PKG). For example, it has been found in Drosophila melanogaster that allelic variation in for, for mRNA and PKG have been associated with a similar foraging phenomenon. Interesting, those with a rover allele (for^R) exhibit a greater moving distance while feeding comparing to those with the sitter allele (for^S).


In their study, researchers demonstrated that different allele variations were favoured under different environments; in a crowded population, the environment favours those with for^R, while uncrowded population favours those with for^S.

The researchers in this study cloned the locust for gene, placed it in an insect phylogeny of PKG, examined for expression in locust brain and measured the PKG activity. In this paper, the researchers confirmed that locusts grew up in different population-density colonies illustrated differences in development, coloration and pattern, neurophysiologies and behavior... ...

Researchers also found that the two species of locusts, L. migratoria, and S. gregaria, shared 90% pairwire identity and a 70% pairwise similarity in for gene when comparied with D. melanogaster. Further more, the for gene in locusts had the typical PKG domains, and two cGMP-binding domains. The researchers also compared locust's for sequences with all currently known insect PKG-encoding genes. They found that within the type I PKG cluster, a group formed by the dipterans and a group with all the social insects except the wasp Vespula vulgaris can be distinguished. The two locust sequences are the locust foraging genes and are part of the PKG type I cluster. 

This phylogenetic tree illustrated the phylogenetic analysis (Neighbour joining method) of the relationship of 44 PKG sequences spanning 30 insect species (variant sequences were discarded). Pairwise comparisons using 5,000 bootstrap replications were used to build the tree. The 2 locust species are in bold.

During the PKG activity assay between the two phases of locusts, researchers concluded that the PKG activity in gregarious locusts was significantly higher than that in solitary locusts. The differences in PKG activity are also sex specific, with higher PKG activity found in males. However, no interaction was found between sex and phases. This sex difference in PKG activity was found for both solitary gregarious locusts.

Through neruonal staining, the researchers also identified the locust for gene expression location within the locust brain.

This group of images illustrates the FOR expression patterns in the locust brain. A: Merged confocal image of frontal 3-mm optical sections of a locust brain showing the major brain neuropils (in green, mouse antibody). B: A distinct PKG-IR region is seen as a cluster of cell bodies in the anterior midline of the brain (double staining with anti-FOR in red and mouse antibody in green). C: Similar FOR expression patterns is seen in gregarious (Gre) or solitary-reared animals (Sol), in adult male (M), female (F), and larvae. Each scale bar represents 250 mm.

What really spiked my interested in this paper was the section of their method on immunocytochemistry and neuronal staining.

'Immunohistochemistry was as in Belay et al. (2007). Briefly, whole brains were dissected in PBS (0.1 M, pH 7.4), fixed in 4% paraformaldehyde, and blocked in 4% normal goat serum (Jackson ImmunoResearch, West Grove, PA) in 0.5% Triton X-100/PBS. The specific guinea pig antibody called anti-FOR, described in Belay et al. (2007), was used at 1:150; the neuropile marker mouse mAb nc82 was used at 1:20 (22, 23). Incubation was for 48 h at 41C. After incubation, brains were washed several times in 0.5% Triton X-100/PBS before adding a goat Cy2-conjugated anti-mouse and a Cy5-conjugated anti-guinea pig Ig (1:100, Jackson ImmunoResearch) for 24 h at 41C. For negative controls, brains were incubated in only secondary antibody, in the absence of primary antibody or in pre-absorbed anti-FOR serum. The specificity of the primary antibody, anti-FOR antibody generated in guinea pig, was measured by using Western blot immunodetection.'

What I really liked about this method section was that the steps for immunostaining was very clearly layed out, which I can copy and adjust for my experiment. The researchers also thoroughly tested the specificity of the primary antibody through Western blot immunodetection. In addition, they ensured the staining they were observing were not just background by omitting the primary antibodies in the negative control. However, the researchers did not indicate how long they fixed their tissues for, nor did they indicate the length for blocking in normal goat serum.

None the less, I got my immunohistochemistry sorted out in this paper, now it's time to fine tune the details to get everything work for my experiment!




Ref:
Lucas, C., Kornfein, R., Chakaborty, M., Schonfeld, J., Geva, N., Sokolowski, M.B., Ayali, A., 2010. The locust foraging gene. Archives of Insect Biochemistry and Physiology. 74:52-66.

The origin of my project

For every research project, there will always be a predecessor that led to the current project. Hopefully, there will also be a successor that follows the it. The paper that started my project was a paper published from our lab. In this paper, the researchers used the locust ventilatory central pattern generator (CPG) to study spread depression(SD)-like events. SD is closely associated with pathologies such as stroke, seizures, migraine and epilepsy. It is been characterized by a wave of electrohphysiological hyperactivity in the neurons followed by a wave inhibition.

In this experiment, neuronal hyperactivity is stimulated in locusts' ventilatory CPG by several stressors such as anoxia, ATP depletion, and hyperthermia. The neuronal acitivty is then monitored using  K+ sensitive microelectrodes to measured the extracellular K+ concentration in the metathoracic neuropile of the CPG. In addition, CPG output is monitored electromyographically from muscle 161 to investigate the role K+ in failure of neural circuit operation induced by those stressors.

In this study, through using K+ sensitive microelectrodes and electromyographic electrodes, researchers found the stress-induced ventilatory pattern failure is tightly correlated with a abrupt increase in the extracellular potassium concentration. This suggested that the failure of ventilation in response to different stressors (hyperthermia, anoxia, ATP depletion, Na+/K+ ATPase impairment, K+ injection) was associated with a disturbance of CNS ion homeostasis. This disturbance in CNS ion homeostasis also shares the characteristics of cortical SD and SD-like events in vertebrates.

These figured illustrated the tight correlation between the failure in ventilatory CPG and the abrupt increase in the extracellular potassium concentration. Noticeably, locusts were able to recovery their ventilatory motor pattern once they have restored their extracellular potassium homeostasis. Vent: ventilatory motor pattern monitored using and EMG electrode, [K+]o: extracellular potassium concentration monitored using the K+-sensitive microelectrode.

In addition, researchers found that heatshock delayed the onset of ventilatory pattern failure and shortened the time to restore Na+/K+ homeostasis and recovery of ventilatory pattern failure. The researchers found that this phenomenon is archived through an increased rate of extracellular potassium clearance by heatshock.

These two figure illustrated that HS decreased the rate of extracellular potassium accumulation and increased the rate of extracellular potassium clearance. This phenomenon hinted that after a HS pretreatment, locusts were better at restoring their potassium homeostasis.

However, when researchers studied the mechanism behind this increasesd clearance rate, they found that it was not due an increase in Na+/K+-ATPase enzymatic activity in heatshocked locusts. They measured the production of NADH spectrophotometrically at 340 nm. The Na+/K+-ATPase activity was assessed using a pyruvate kinase/lactate dehydrogenase assay. In this experiment, both Na+/K+-ATPase and pyruvate kinase/lactate dehydrogenase assay required ATP. The pyruvate kinase/lactate dehydrogenase assay produces NADH, and will compete for ATP with Na+/K+-ATPase. If the ATPase had a higher enzymatic activity, then it would utilize ATP at a faster rate, minimizing the amount of ATP is used for the pyruvate kinase/lactate dehydrogenase, in return, decreases NADH production

In addition, researches examined the cellular ATP level in heatshocked and control animals. The ATP level in heatshocked and control animals was quantified using a luciferin-luciferase reaction. In this reaction, ATP is consumed and light is emitted when firefly luciferase catalyzes the oxidation of D-luciferin. Since ATP is the limiting reagent, the the amount of ATP present is proportional to the light emitted, which is measured using an Lmax luminometer. Each measurement of light intensity was compared to a standard curve generated using known quantities of ATP prepared in water. The end result illustrated that there was not a significant difference in cellular ATP concentration between control and heatshocked animals. This indicated that the lower clearance rate observed in control locusts was not due to a lower level ATP, which could hinder their Na+/K+-ATPase function.

This figure illustrated that there was no significant difference in cellular ATP level between control and heatshocked locusts at the same time. However, a significance was observed during anoxic period for both control and heatshocked locusts.

This study concluded that when hyperthermic failure that was preconditioned by prior heat shock, it resulted in an induced-thermotolerance which was associated with an increase in the rate of clearance of extracellular K+. However, this increase in clearance rate was not linked to changes in ATP levels or total Na+/K+ ATPase activity. As a result, the mechanism behind this increased clearance remains unclear.

My project as one of the successors to this paper, is aimed to elucidate the underlying mechanism of this rate increase. The aim of my research project is to use fluorescence antibody to study whether the location/trafficking of the Na+/K+-ATPases differ between a control and heat-shocked locusts, which might have allowed heatshocked locusts to increase the amount of surface-bound, active ATPase on their membranes.

Reference:
Rodgers, C.I., Armstrong, G.A.B., Shoemaker, K.L., LaBrie, J.D., Moyes, C.D., Robertson, R.M., 2007. Stress preconditioning of spreading depression in the locust CNS. Public Library of Science. 12:e1366.