2. The dopaminergic reward system
The mesolimbic dopamine reward pathway is composed of dopamine cell bodies in the VTA that project to the NAc. The VTA also projects to parts of the PFC and the amygdala.
The dopaminergic system is thought to be involved the rewarding aspects of motivated behavior.
Consequently, addiction to drugs is thought to involve this system (some drugs directly-cocaine, and some indirectly-nicotine).
The firing patterns of dopaminergic neurons are thought to encode reward prediction: transition from low-frequency tonic to burst firing. Accordingly, their firing is inhibited by aversive stimuli.

3. What’s wrong with the traditional methods?
Electrical stimulation:
- not confined to a specific subpopulation of neurons.
Lesions:
no temporal control.
not confined to a specific subpopulation of neurons.
Pharmacology:
- no temporal control.

4. Optogenetics A conceptually “simple” trick:
Express light-activated channels in neurons, which upon activation with light will de/hyper-polarize the neurons.
Algae/Archae to the rescue:
Channelrhodopsins/halorhodopsins are light-activated sensors in algae/archae.
Channelrhodopsins are non-specific cation channels (depolarization).
Halorhodopsins are chloride transporters (hyperpolarizatiom).
Implementation in neurons:
Remote control of neuronal activity in genetically-identified subpopulations.
Millisecond temporal resolution.

5. Now we can have remote control of a genetically-identified subpopulation of neurons with fine temporal resolution.
Will this bring us closer to the “holy grail” of neuroscience – linking specific activity of specific neurons to specific behaviors?

7. Step 1: targeting a specific subpopulation of neurons
TH-Cre mice express cre recombinase under the tyrosine-hydroxylase (TH) promoter, which is expressed specifically in dopaminergic (DA) neurons.
Channelrhodopsin2 (Chr2) expression is cre-inducible in the virus.
TH-Cre transgenic mouse
Cre-induced channelrhodopsin2 virus
VTA (source)
When the virus is injected into the VTA, Chr2 (tagged with YFP) will only be expressed in DA neurons.
Verification:
>90% of TH+ cells were YFP+.
98% of YFP+ cells were TH+.
NAc (target)

8. Step 2: verifying the functionality of Chr2 in DA neurons
In vitro:
Whole-cell recordings from live slices.
Transduced neurons had typical VTA neuron resting potential and resistance – Chr2 expression does not affect basic physiology.
Flashes of blue light cause inward currents leading to action-potentials.
VTA neurons can reliably follow light flashes only up to ~10 Hz.
In vivo:
Extracellular recordings reveal light-evoked typical VTA neuron spike waveform.
VTA neurons reliably follow low frequency light flashes in vivo.
1 Hz flashes
50 Hz flashes

9. Now we can have remote control of a genetically-identified subpopulation of neurons (VTA DA neurons) with fine temporal resolution (1 to 50 Hz).
Will this bring us closer to the “holy grail” of neuroscience – linking specific activity (tonic vs. phasic) of specific neurons (VTA DA neurons) to specific behaviors?

10. Step 3: testing the causal link between VTA DA neurons to behavior (1)
Conditioned place preference (CPP):
Day 1: pre-test to determine default preference
(15 min).
Day 2: optogenetic conditioning in one chamber
(30 min, 25 flashes at 50 Hz every min).
Day 3: optogenetic conditioning in opposite chamber
(30 min, 25 flashes at 1 Hz every min).
Day 4: post-test to determine conditioned preference

11. Step 3: testing the causal link between VTA DA neurons to behavior (2)
- Optogenetic conditioning with 50 Hz but not 1Hz produces place preference.
Control 1: mice receiving no light flashes were not conditioned (it’s not the virus alone).
Control 2: non-transgenic littermates were not conditioned by flashes of light (it’s not the light alone).
Control 3: the experimental procedure did not affect anxiety or locomotion.

12. Is this effect due to:
1. A positive response to the 50 Hz stimulation OR
2. A negative response to the 1 Hz stimulation
???

13. Step 3: testing the causal link between VTA DA neurons to behavior (3)
To differentiate between a positive response to 50 Hz and a negative response to 1 Hz, each mouse was conditioned only with one of the stimulation patterns.
50 Hz
1 Hz
Preference to 50Hz.
No aversion to 1 Hz.
A trend to prefer 1 Hz.
VTA DA neuron activation at 50 Hz, but not at 1 Hz conditions place preference due to a positive response.

14. Is this effect actually due to more dopamine release???

15. Step 3: testing the causal link between VTA DA neurons to behavior (4)
Dopamine concentration can be measured in vivo using fast-scan cyclic voltammetry (oxidation with a carbon electrode).
The phasic and tonic stimuli have the same illumination duration and number of light flashes (25 x 15 ms light flashes at 1 Hz or 50 Hz).
- The tonic (50-Hz) light stimulus elicited larger DA transients.
Phasic activation causes more DA release than tonic activation

16. Summary and conclusions
Identity: dopamine, but not other neurotransmitters, is sufficient to cause place preference (although VTA DA neurons can also release glutamate…).
Activation pattern: phasic activity, but not tonic activity, of dopamine neurons is sufficient to cause place preference.
Behavioral effect: phasic firing of VTA DA neurons causes a positive response, but not a negative one.
These conclusions could not have been reached without optogenetics

17. Question?