Most motile animals exhibit foraging behavior and are able to respond to a stimulus. A stimulus may be harmless or aversive, and the animal may respond with approaching or avoidance. Reward learning and reward seeking behaviors are actions that represent fundamental aspects of animal behavior, and are universal across animals (Barron et al., 2010).
Neuroanatomical structures that are involved in reward pathway are a part of mesolimbic dopaminergic pathway, which is located ‘deep in the brain’. Main structures are ventral tegmental area (VTA) that projects its axons to nucleus accumbens (NAc), and further make connections with neurons of prefrontal cortex (PFC), which represents the conscious part of this pathway, and it evolved later (Adinoff, 2004) (Figure 1A).
Neurotransmitter dopamine is a key modulator of this circuit and also of the behavioral responses to rewards (Berridge and Robinson, 1998) and tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis. Dopamine is synthesized in VTA and then signaling cascade is established through efferent VTA axon projections towards NAc and further to PFC. Different receptors and signaling molecules are involved in dopaminergic mesolimbic pathway besides tyrosine hydroxylase. Besides tyrosine hydroxylase, different dopamine receptors are present on the presynaptic and postsynaptic neuron (Figure 1B).
Tyrosine hydroxylase is a molecular marker for dopaminergic neurons, and is only found in VTA in higher levels, and since a new protocol for these brain regions isolations was being developed in the lab, a molecular confirmation of the accurate brain region isolation was needed.
Materials and Methods
3 month-old wild-type C57BL/6J male mice were sacrificed by cervical dislocation and brains were immediately taken and frozen in hexane to preserve the shape and structure organization, after which they were kept on the dry ice and stored at -80°C until following dissections took place.
Brain region isolation
Brains were placed on the dry ice and taken to cryostat were temperature was at -20°C constantly, so that the brains did not thaw too quickly, and the dissections were performed. Nucleus accumbens and ventral tegmental area were dissected from each brain using acrylic brain matrix for coronal sections that gave slices of 1mm in depth. With guidance of the Mouse Brain Map Atlas, cuts were made and nucleus accumbens was punched first with 1mm punchers, afterwards slices for cutting out ventral tegmental area were made and it was punched with 1.5 mm diameter puncher. For each animal, each structure was dissected bilaterally and pulled. Immediately after that step, structures were snap frozen in liquid nitrogen until further RNA extraction steps.
RNA Extraction and purification
Both tissues were homogenized using light weight tissue homogenizer (Fisherbrand 150 Handheld Homogenizer Motor and Soft Tissue Omni Tip Plastic Homogenizing Probes) with addition of lysis buffer RLT lysis buffer containing β-mercaptoethanol. Then, from all of the lysates DNA and RNA were extracted using All Prep Mini Kit from Qiagen. Further, only the RNA was used for the subsequent analyses. The RNA levels of purity and concentration were checked using UV spectrometry and 5 ng/μg dilutions stocks were made and stored at -20°C for further use.
RT-PCR (reverse transcription polymerase chain reaction)
Reverse transcription and PCR reaction were performed in one-step using Qiagen One-Step RT-PCR Kit. 10μl volume reactions were made with 10ng of RNA in each reaction and primer concentrations were always 0.5 uM.
Specific primers were designed for Th (Figure 2).
Visualizations of the products were done in a 1% agarose gel with Ethidium bromide.
Results were quantified using densitometric quantification method via ImageJ.
Products were then gel purified using the QIAquick Gel Extraction Kit or PCR product purified using QIAquick PCR Purification Kit and sent out for Sanger sequencing by Genewiz.
Sequencing results were further analyzed using NCBI BLAST.
RT-PCR was performed with RNA from NAc and VTA brain regions (Figure 3A). Based off of the RT-PCR results, it appears there is more Th expression in the VTA as compared to NAc in the same animal. This was later confirmed using second pair of primers designed to a different region of the Th transcript. To ensure that this difference was not due to differences in the amount of total RNA in the RT-PCR reaction, I amplified CypA (cyclophilin A – a housekeeping gene), which showed no difference in expression between the two regions.
To quantify this difference I averaged my duplicates and took the ratio of the Th to CypA in NAc and compared that to the average ratio of the Th to CypA in the VTA. This analysis was done for both primer pairs using samples isolated from two different animals (Figure 3B).
In Figure 3A, left and right panel, results acquired via RT-PCR that were later quantified are depicted, and those clearly showed corroboration of literature data and the fact that indeed in ventral tegmental area there is far more tyrosine hydroxylase mRNA expressed in comparison to nucleus accumbens.
In Figure 3B, if we look at the individual graphs, showing data for individual animals, as well as results acquired with different primer pairs, there is an overall trend showing that in all animals with both primer pairs there is more tyrosine hydroxylase in ventral tegmental area than in the nucleus accumbens.
This data is in agreement with the literature, which reports that there is higher expression of tyrosine hydroxylase in ventral tegmental area as compared to expression in the nucleus accumbens.
These results also confirmed that the brain regions I harvested for the study were indeed ventral tegmental area and nucleus accumbens, since tyrosine hydroxylase is used as a marker for determination of dopaminergic neuron
bodies, only present in ventral tegmental area.
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