Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. This study used optogenetic tools to selectively stimulate VTA dopaminergic neurons in freely behaving mammals. Phasic activation of these neurons was sufficient to drive behavioral conditioning and elicited dopamine transients with magnitudes not achieved by longer, lower-frequency spiking. These results demonstrate that phasic dopaminergic activity is sufficient to mediate mammalian behavioral conditioning. Dopaminergic (DA) neurons are involved in the cognitive and hedonic underpinnings of motivated behaviors. Changes in the firing pattern of DA neurons between low-frequency tonic activity and phasic bursts of action potentials could encode reward prediction errors and incentive salience. However, it remains unclear whether DA neuron activation alone is sufficient to elicit reward-related behaviors. Lesion, electrical stimulation, and psychopharmacology studies have been important but have not allowed causal and temporally precise control of DA neurons in freely moving mammals. To control DA neurons selectively, the researchers used a Cre-inducible adeno-associated virus (AAV) vector carrying the gene encoding the light-activated cation channel channel rhodopsin-2 (ChR2) in-frame fused to enhanced yellow fluorescent protein (ChR2-EYFP). This vector was designed with a double-floxed inverted open reading frame (ORF), wherein the ChR2-EYFP sequence is present in the anti-sense orientation. Stereotactic delivery of this vector into the VTA of tyrosine hydroxylase (TH)::internal ribosomal entry site (IRES)–Cre transgenic mice enabled DA neuron-specific expression of ChR2-EYFP. The researchers validated the specificity and efficacy of this targeting strategy in vivo. In coronal VTA sections of transduced TH::IRES-Cre brains, ChR2-EYFP specifically co-localized with endogenous TH. Greater than 90% of TH-immunopositive cells were positive for ChR2-EYFP near virus injection sites, and more than 50% were positive overall, demonstrating highly efficacious transduction of the TH cells. The researchers used whole-cell patch clamps to measure light-induced membrane currents in ChR2-expressing DA neurons. ChR2-EYFP-expressing cells in the VTA displayed typical electrophysiological properties of DA neurons. Under blue light illumination, all patched cells exhibited prominent inward photocurrents. Trains of light flashes drove action potential firing in ChR2-EYFP neurons; with use of low- and high-frequency light trains, they evoked tonic and phasic DA neuron firing, respectively. The researchers tested the behavioral conditioning effects of phasic DA neuron activity via the conditioned place preference (CPP) paradigm. Mice developed a clear place preference for the chamber associated with phasic optical stimulation. Control experiments showed that nontransgenic littermates and TH::IRESPhasic firing in dopaminergic neurons is sufficient for behavioral conditioning. This study used optogenetic tools to selectively stimulate VTA dopaminergic neurons in freely behaving mammals. Phasic activation of these neurons was sufficient to drive behavioral conditioning and elicited dopamine transients with magnitudes not achieved by longer, lower-frequency spiking. These results demonstrate that phasic dopaminergic activity is sufficient to mediate mammalian behavioral conditioning. Dopaminergic (DA) neurons are involved in the cognitive and hedonic underpinnings of motivated behaviors. Changes in the firing pattern of DA neurons between low-frequency tonic activity and phasic bursts of action potentials could encode reward prediction errors and incentive salience. However, it remains unclear whether DA neuron activation alone is sufficient to elicit reward-related behaviors. Lesion, electrical stimulation, and psychopharmacology studies have been important but have not allowed causal and temporally precise control of DA neurons in freely moving mammals. To control DA neurons selectively, the researchers used a Cre-inducible adeno-associated virus (AAV) vector carrying the gene encoding the light-activated cation channel channel rhodopsin-2 (ChR2) in-frame fused to enhanced yellow fluorescent protein (ChR2-EYFP). This vector was designed with a double-floxed inverted open reading frame (ORF), wherein the ChR2-EYFP sequence is present in the anti-sense orientation. Stereotactic delivery of this vector into the VTA of tyrosine hydroxylase (TH)::internal ribosomal entry site (IRES)–Cre transgenic mice enabled DA neuron-specific expression of ChR2-EYFP. The researchers validated the specificity and efficacy of this targeting strategy in vivo. In coronal VTA sections of transduced TH::IRES-Cre brains, ChR2-EYFP specifically co-localized with endogenous TH. Greater than 90% of TH-immunopositive cells were positive for ChR2-EYFP near virus injection sites, and more than 50% were positive overall, demonstrating highly efficacious transduction of the TH cells. The researchers used whole-cell patch clamps to measure light-induced membrane currents in ChR2-expressing DA neurons. ChR2-EYFP-expressing cells in the VTA displayed typical electrophysiological properties of DA neurons. Under blue light illumination, all patched cells exhibited prominent inward photocurrents. Trains of light flashes drove action potential firing in ChR2-EYFP neurons; with use of low- and high-frequency light trains, they evoked tonic and phasic DA neuron firing, respectively. The researchers tested the behavioral conditioning effects of phasic DA neuron activity via the conditioned place preference (CPP) paradigm. Mice developed a clear place preference for the chamber associated with phasic optical stimulation. Control experiments showed that nontransgenic littermates and TH::IRES