Zachary Hopkins and Jeffrey G. Edwards, Department of Physiology and Developmental Biology
Introduction
The ventral tegmental area or VTA is considered to be one of the foremost centers involved in motivation and reward processing in the brain [1]. This area is also heavily implicated in addiction. However, little is known in regards to the exact cell types and subtypes found there, or the types of signaling that occur. We were interested in investigating the possibility of retrograde signaling among the two predominant cell groups in the VTA: GABA and dopamine neurons. Both cell types can be acted on by drugs of abuse, either to directly affect DA release or indirectly through GABA cells also leading to increased release of dopamine [2]. We chose to investigate a form of retrograde signaling that occurs via endocannabinoids. Thus, the focus of our research was two-pronged: to accurately identify subtypes of neurons and also investigate if the neurons are capable of participating in endocannabinoid signaling.
Methods
Each sample came from single cells extracted from rat or mouse brain slices. All animal use was in accordance with IACUC protocols. Unlabeled VTA neurons were pulled from rat brain slices, while GABA neurons were specifically targeted using a green fluorescent protein label in mouse brain slices. During the extraction process an electrode was utilized to inject current into the cell and monitor its subsequent firing activities. Afterwards, mRNA, taken from the cell, was converted to cDNA using reverse transcriptase PCR.
Following the extraction, quantitative real-time PCR (qRT-PCR) was used to probe the cDNA samples for markers indicating which neuron type had been collected (GABA or dopamine). Further, the samples were tested for the presence of enzymes involved in the production of endocannabinoids and receptors thought to be necessary for their activation.
Results
The principle focus of our study was to identify endocannabinoid producing and receiving proteins in VTA neurons, particularly GABA neurons. In order to successfully do this the cells needed to be classified accurately. For this we used electrophysiological and molecular data.
The electrophysiology demonstrated some notable differences between GABA and dopamine neurons. Namely, the GABA neurons tended to have higher firing frequencies in response to injection of current than the dopamine neurons. Also, dopamine cells responded differently to a hyper-polarizing protocol than GABA cells, however there was overlap. We also ran these protocols on our marked mouse GABA cells as a positive control. The results seen with these paralleled those seen in rat. To evaluate cell types using RT-PCR each sample was probed for enzymes involved in the production of GABA and dopamine. When compared with the results of experiments done in the marked mouse GABA cells, we found this method could accurately identify cell type, especially when teamed with our physiological data.
In order to evaluate the presence of endocannabinoid enzymes and receptors between GABAergic and dopamine neurons we probed for these targets using qRT-PCR. We found expression of each target (both enzyme for producing endocannabinoids and receptor) to some degree in both GABA and dopamine cells. It is also notable that the expression was almost identical between the two types of neurons. We also evaluated the presence of these markers in our mice whose GABA neurons were marked. We again found evidence of endocannabinoid production enzymes and receptors, confirming our previous data from rat.
Discussion
Since long-term synaptic changes, especially in dopamine transmission, within the VTA can be induced by endocannabinoids, understanding their location and role in neurotransmission within this area is important for efforts to help those with addiction. Our data shows expression of endocannabinoid machinery in both neuron subtypes thus suggesting that both could be actively involved in endocannabinoid signaling. This further suggests that endocannabinoid-mediated changes in synaptic function could play an important role in addiction.
In regards to cell classification, our physiological data shows that while certain characteristics can be seen between dopamine and GABA cells, there are sufficient exceptions that the use of physiology alone would provide an insufficient level of accuracy. However, when used in conjunction with our qRT-PCR we found that cell types could be accurately identified.
Conclusion
In conclusion, our data provide new evidence for the distribution of endocannabinoid producing enzymes and receptors within GABA and dopamine neurons in the VTA. Notably we found similar expression patterns between the two cell types thus suggesting that both cell types are possibly involved in modulating dopamine transmission via endocannabinoid signaling. Past thought implicated mostly dopamine neurons as being involved in endocannabinoid signaling whereas our studies indicate that this may not be the case. Finding functional elements of endocannabinoid in multiple cell subtypes offers insight into both the functioning and complexity of the reward and motivational pathway circuitry and may provide future targets for clinical intervention, important in helping those suffering from addiction.
Sources
- Luscher, Christian, & Malenka, Robert C. (2011). Drug-Evoked Synaptic Plasticity in Addiction: From Molecular Changes to Circuit Remodeling. Neuron, 69 (650-663).
- van Zessen, R., Phillips, J.L., Budygin, E.A. and Stuber, G.D. (2012) Activation of VTA gaba neurons disrupts reward consumption. Neuron, 73, 1184-1194