Figure 2. VTA neurons, and among them dopamine neurons, respond to footshocks and acquire CS responsiveness. A, Scheme of the 3 d auditory-cued fear learning protocol (top), and freezing data (bottom) of the example mouse of C–I. B, Schematic drawing of an optrode with 16 recording channels implanted in the VTA of a DATCre x ChR2 mouse. C, Post hoc histologic image showing the placement of an optrode (dashed line) in the VTA of the example mouse (FT7612) of C–I. The tracks of two tetrodes are visible (arrows). Bottom, Scheme of the arrangement of four tetrodes (T1-T4) around the optical fiber. D, Illustration of optogenetic identification of putative DAT+ units. Raster plot for four electrodes (EI-EIV) of one tetrode, showing unsorted spikes aligned to the onsets of n = 100, 2-ms-long laser light pulses (blue shading). Spikes at 2-8 ms after the light pulse were collected and subjected to spike clustering (see Materials and Methods). E, Spiking activity of a single opto-tagged putative DAT+ unit during day 2 (training day). AP frequency (top) and z score (bottom) are shown in response to the CS (averages over the n = 30 tone presentations for each tone block), as well as in response to footshocks (averages over the n = 6 footshock presentations; right). F, Spiking activity of the same unit as in E, in response to CS presentations on day 3 (fear memory retrieval). Right, AP waveforms for the unit shown in E, F. G, H, Responses of all units in mouse FT7612 to tone presentations during days 1-3 (G), and to footshocks on day 2 (H). The peak z score responses were plotted as a function of the tone-block number (CS) or paired CS-US presentation. Pink represents units classified as CS-entrained (G) or as US responders (H). Gray represents the others. Thick red and black lines indicate average ± SEM across these groups, respectively. Square symbols connected by lines represent DAT+ units identified by optotagging. I–K, Venn-type diagrams showing the number of units in the different response classes and their overlap. I, The data are from the example mouse shown in A–H. Note the overall similar distribution and overlap of response types across the N = 3 mice. Two US-responsive units in mouse FT6963 showed reduced AP firing frequencies on the footshock (J, dashed areas). L, AP firing response of unit 17 of mouse FT6963 during day 2 of the fear learning protocol. This unit responded with a decreased AP firing frequency to footshock stimulation, and corresponds to the red dashed area in J.