Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Summary

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Abstract

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.

We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein ^1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA ^6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.

We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Protocol

1. Dissection

1. On a bed of wet-ice, chill a rodent brain matrix (coronal sections segregated 1 mm apart), a Petri dish containing 5 razors, and a #11 scalpel. In a separate container, place labeled 2 ml size microfuge tubes into dry ice.
2. The investigator will choose the method of euthanasia. We have reproducible results conducting a very brief anesthesia with isoflurane, Ideally, a vaporizer should be used. However, if not available, we use a large battery jar containing a platform. With the battery jar located in an approved ventilating hood, place an isoflurane-saturated gauze pad below the platform and wait 3-5 minutes to allow the isoflura....

Discussion

As outlined in Figure 1, the methods detailed above should yield multiple readouts of dopamine and its regulating proteins TH, DAT, and VMAT2 from one sample of SN or VTA obtained from either the rat or mouse. Again, the benefits of carrying out this protocol are that the investigator can obtain operationally-matched readouts of how dopamine is regulated in vivo under virtually any experimental paradigm and, in doing so, save significant experimental resources by reducing the number of animals required in any ex.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HPLC system:
The basic system consists of a Shimadzu LC10-ADvp HPLC pump, a Waters WISP 717 automatic sample injector, a 250 X 4.4 mm 5 micron Spherosorb ODS-1 C18 reverse-phase column (Waters), a Bioanalytical Systems (BAS) TL12 dual glassy carbon electrode, two BAS LC4B electrochemical detectors, and a Waters Empower 2 data collection and integration system.
The column is maintained at 30-45 °C (BAS LC22A column heater). The mobile phase is 0.1 M sodium phosphate (pH 3.0), 0.1 mM EDTA, 0.2-0.4 mM 1-Octane Sulfonic Acid (Eastman-Kodak), and 0.35% acetonitrile (v/v), filtered through a 0.45 micron filter. Flow rate is of 1.2 ml/min. Four liter batches of mobile phase are optimized for separations by adjusting the pH, Octane Sulfonic Acid and column temperature. The mobile phase is recycled, and is continuously purged with helium gas to remove dissolved oxygen. Recycling of the mobile phase is almost essential to maintain good resolution for a reasonable period of time. The mobile phase shelf-life is maintained by using a flow switch (controlled by the integrator) to divert to waste the first 2-7 min of each run.
The electrodes are maintained at potentials of approximately 0.78 and 0.95V with respect to a Ag/AgC1 reference electrode. The electrode at the higher potential is used exclusively for the determination of tryptophan (and the NMDA internal standard). The 0.78 V potential provides a superior signal to noise ratio for detection of the monoamines and compounds, other than tryptophan. The chromatograms are stored on the hard drive of the Empower workstation, and subsequently processed and the data transferred directly into an Excel spreadsheet for computation of metabolite amounts and compilation of group data.
Pump: Shimadzu LC-10AD Cell: BAS Cross Flow. Glassy carbon working electrode at 0.780 and 0.950 V potential. Detector: BAS LC-4B operated in dual channel mode. Data Acq. System: Waters Empower Pro 2. Injector: Waters WISP 717 Column: Waters Spherosorb ODS-1, 5 μM particle, 4.4 mm X 250 mm.
Sodium Dodecyl Sulfate (SDS)	J.T. Baker	4095-02
Trizma Base	Sigma	T1503-1KG
Trizma HCl	Sigma	T3253-1KG
Glycerol	Sigma	G8773-500 mL
PVP-40	Sigma	PVP40-1KG
dPBS	Gibco	21600-069
Tween20	Sigma	P1379-500 mL
Glycine	Sigma	G8898-1KG
Ponceau S	Fluka	81460
Bromophenol Blue	Sigma	B8026-5G
Dithiothreitol	Sigma	D-9163
Protein Standard 2 mg BSA	Sigma	P5619-25VL
Pierce BCA Protein Assay Reagent A	Thermo-Fisher Scientific	23223
Precision Plus Protein Standard	Bio Rad	161-0373
[125I]-protein A, specific activity	Perkin-Elmer
Table 2. Specific reagents.
10% SDS			10 g SDS, 100 mL DI H20
1% SDS (pH to 8.2)			10 mL 10%SDS 60.5 mg Trizma Base 37.22 mg EDTA 90 mL DI H20
Copper II Sulfate Solution			1 g Copper II sulfate 25 mL DI H20
3X Sample Buffer			Trizma Base 2.27 g SDS 6 g Dithiothreitol 0.463 g Glycerol 30 g Bromophenol Blue 10 mg D I H20 (initially add above reagents to 40 mL of H20 in a graduated cylinder; then add H20 until volume reaches 100 mL) HCl (add as needed to reach pH of 6.85) Freeze solution in 50 2.0 mL tubes. (makes 100 mL): Volume of 3X Sample Buffer needed = ½ volume of SDS used in sample.
1X Sample Buffer			Dilute 3X Sample Buffer down to 1X sample buffer using DI H20
10X Running Buffer (Makes 4 L)			Trizma Base 121.1 g Glycine 577 g SDS 40 g
10X Transfer Buffer: (Makes 4 L)			Glycine 360 g Trizma Base 96 g
Ponceau			Ponceau S. 0.5 g Acetic Acid 5 mL DI H20 95 mL
.2% HCl Solution			5.2 mL HCl in 500mL of DI H20
PVP-T20 Blocking Soln. (Makes 4 L)			PVP-40 40 g dPBS 38.2 g Tween20 2 g Thimerisol 0.4 g 1M Tris pH 7.6 (60.6 g Tris HCl + 13.9 g Tris Base in 500 mL DI H20)- 50 mL
10X Blot Buffer (Makes 4 L)			Tween 20 20 g Tris Base 14 g Tris HCl 61 g
Table 3. Protein Assay and Western Blotting Formulas.
Tyrosine hydroxylase standards: The calibrated TH protein and phosphorylation standards used by this laboratory are derived from PC12 cell extracts, which were analyzed for TH protein content and phosphorylation stoichiometries against a previously calibrated TH standards that ultimately originated from the laboratory of Dr. John Haycock 11.