Hello friends, we meet again.
I took a brief hiatus while I focused on my internship but I’m back to share some news with you and to give you an idea of what I worked on this summer.
First of all, we use THCre transgenic rats which contain a tyrosine hydroxylase (TH) promoter. This promoter directs expression of Cre recombinase to catecholaminergic cells. During adolescence, these animals were exposed to the synthetic cannabinoid drug WIN or received a control Vehicle injection. In adulthood, some animals were inserted the excitatory DREADD (Gi) virus surgery using the Cre-dependent virus. This gives us the ability to use the DREADD ligand CNO to activate the Gi DREADD receptors in TH dopamine neurons, and force them to fire and release dopamine for us. We want to know whether the brain response to dopamine is different in WIN-exposed animals, because brains that respond differently to dopamine may be more (or less) prone to addiction behavior. We waited 3 weeks for the brains to express the DREADD receptors in the TH dopamine neurons, so now we can inject animals with CNO and collect brain tissue.
1) VEH+ no surgery+CNO = This shows us the baseline levels in a regular rat. CNO should not change brain chemistry in animals that don’t express DREADDs, but we use it in our control group just in case there is some small effect of CNO or getting injected.
2) VEH+Gi DREADD+CNO = this shows us how a normal rat responds to dopamine release. CNO here causes dopamine release.
3) WIN+no surgery+CNO = this shows us baseline levels in a WIN-exposed rat. Baseline levels in a WIN rat may be different than a regular control rat. CNO should not do anything here.
4) WIN+Gi DREADD+CNO= this is our exciting group. This will show us how a WIN-exposed brain responds to dopamine release. CNO here causes dopamine release.
We collect brain slices from prefrontal cortex, nucleus accumbens, and basolateral amygdala because these brain regions receive dopamine projections, and thus should produce an endocannabinoid response to dopamine. We stain for all tissue for mCherry which shows us the entire cell body and axons of dopamine neurons in TH Cre rats with Cre-dependent DREADD virus. In this case, we are more focused on seeing the axons of the DA neurons rather than the fact that they contain DREADDs. We know that dopamine neurons can release dopamine, glutamate (excitatory neurotransmitter) and GABA (inhibitory neurotransmitter). What we don’t know is how the dopamine cell does this.
It’s not very logical to release both excitatory glutamate and inhibitory GABA onto the same target cell. It would be telling the cell to both fire and not to fire which doesn’t make much sense. It could be releasing dopamine and GABA or dopamine and glutamate from a single terminal because dopamine is modulatory and tends to have a helper role rather than working alone. What we want to do is look at the axon terminals and see if there are differences in what the terminals are making. Is there TH (making dopamine) at all of the axon terminals, or if some terminals don’t make dopamine, could they be making something else like glutamate or GABA?
All the tissue is stained for mCherry but we also stain half the tissue for TH which is an enzyme that makes dopamine. If an axon terminal is releasing dopamine, it should have TH at the terminal producing that dopamine. The other half of the issue is stained for VGlut2. Vesicular glutamate transporter 2 is a protein that loads glutamate into vesicles. The vesicles of glutamate can be docked at the membrane next to the synapse where they wait to be released onto the target cell’s dendrite. If an axon is releasing glutamate, we should find vGlut2 packing up the vesicles.
Our next steps is to use confocal fluorescence microscopy to take images of these regions at different magnifications and run them through Volocity, an image analysis software, to look for cases of co-localization. Co-localization, composed of co-occurrence and correlation, tells us the degree of overlap between two or more channels (fluorescent labels) in an image. This is important because in our case, it can give us statistical information on the co-expression of TH and glutamate.